1. health and fitness
2. disease
3. cholesterol

Europaisches Patentamt
J
»
European Patent Office
©
Publication number:
Office europeen des brevets
EUROPEAN
0 253
A1
0 2 2
PATENT A P P L I C A T I O N
intci- A 61 K 37/22, A 61 K 3 5 / 3 7
Application number: 86117698.0
Date of filing: 18.12.86
Priority: 20.12.85 US 811375
Applicant: THE TRUSTEES OF BOSTON UNIVERSITY,
881 Commonwealth Avenue, Boston, MA02215 (US)
Applicant: Anglo-Medical Corporation, 1345 Avenue of
the Americas, New York New York 10105 (US)
Inventor: Catsimpoolas, Nicholas, 65 Montvale Road,
Newton Centre, Massachusetts 02159 (US)
Inventor: Gavras, Haralambos, 11 Coolidge Road,
Wayland, Massachusetts 01778 (US)
Inventor: Haudenschild, Christian C, 106 Harvard Street,
Newtonville, Massachusetts 02160 (US)
Inventor: Klibaner, Michael I., 76 Egmont Street, Apt. 5,
Brookline, Massachusetts 02146 (US)
Date of publication of application : 20.01 .88
Bulletin 88/3
Designated Contracting States: AT BE CH DE ES FR GB
GRITLILUNLSE
®
Representative: Patentanwalte Schulze Horn und
Hoffmeister, Goldstrasse 36, D-4400 Miinster (DE)
Method for treatment of angina and myocardia! infarctions with omental lipids.
© Angiogenesis healing factors residing in omentumderived lipid fractions with or without gangliosides or their
synthetic equivalents can be used to treat myocardial
ischemic conditions including but not limited to myocardial
infarction, angina, as well as in heart transplant, vascular
grafts, and re-opened vessels leading to improved vascularization, perfusion, collagenization and organization of
said lesions of the involved and adjacent tissues.
M
<M
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CO
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CM
o
Q.
Ill
ACTORUMAG
A
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#5
AMED 2 0 6
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Summary
Angiogenesis
omentum-derived
ischemic
vascular
purpose
(MI) ,
grafts
of
angina,
but
heart
used
not
involved
to
myocardial
to
and
myocardial
hearts
arteries
coronary
and
an
treat
limited
vascularization,
the
in
residing
transplants
recanalized
or
of
(s)
are
including
improving
organization
fraction
lipid
conditions
infarction
factor
healing
with
per f u s i o n
adjacent
with
the
and
tissues.
Description
Heart
overall
attacks
mortality
averages
30%.
are
a major
rate
Most
the
death
and
the
the
first
months
after
MI
deaths
occur
in
first
12
during
of
of
cause
the
hours.
Myocardial
infarction
occlusion
or
substantial
thrombus,
or
by
atherosclerotic
combination
of
is
ischemic
atherosclerosis,
plaque,
these
o.f
narrowing
or
by
or
spastic
mechanisms.
-
2 -
by
necrosis
due
a coronary
to
artery
hemorrhage
constriction,
by
into
an
or
by
a
0 2 5 3 0 2 2
#5
Treatment
one
of
myocardial
containment
Symptoms
the
damaged
discussions
al.
in
and
Current
Los
treatment
(analgesics)
thrombolytic
agents
activator);
local
diuretics;
balloon
reopening
catheters
and
surgery.
No t h e r a p y
that
only
similar
in
Sulfinpyrazone
aggregation
in
the
drugs
has
drugs
months
include
also
limolol,
Lange
typical,
sedation;
for
is
effect
to
acute
the
as
such
and/or
(.tissue
nitroprusside,
as
digitalis;
assist
occlusion
cardiac
leak
shown
to
by
the
is
to
metoprolol.
3 -
bypass
save
surgery
platelet
recurrent
months.
Beta-adrenergic
likelihood
propranolol,
and,
slow.
counter
myocardial
aortic
balloon
, or
rupture
prevent
seven
-
TPA
or
(angioplasty)
decrease
following
Krupp
vasopressors;
acute
where
up
rest;
a circulatory
exists
an
as
anticoagulants
such
devices
cases
1985
Treatment,
streptokinase
as
(Anturane)
and
of
agents
the
infarctions
myocardial
blocking
of
Various
example,
California,
systemic
inotropic
recently,
and
as
counterpulsation
for
help
seq.
vasodilators
nitroglycerine,
to
system.
As
and
; oxygen;
such
available
cited.
consists
pain-killers
plasminogen
be
Altos,
et
is
been
largely
a patient.
myocardial
Diagnosis
P. 121
on
or
has
for
therapy
can
Medical
this
no
vascular
Publications,
discusses
infarction
stabilization
medicine
Current
Medical
and
treated
are
rebuild
et
for
AMED 2 0 6
12/17/85
of
reinfarction
infarction;
alprenolol
and
such
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Rehabilitation
physical
and
life
possibly
and
Most
not
address
the
reperfuse
increase
capillary
collateralization.
appropriate
known
materials
are
healing
and
as
invention
to
collagen
wherein
the
neovascularization
as
some
of
quality
most
well
an
damage".
and/or
are
undesirable
and
in
basic
neovascularizat.ion
is
the
method
mechanisms
seen
do
without
accelerate
This
healing
bypass
but
Methods
perfusion,
do
or
inflow
to
or
vessels,
outflow
leads
formation.
above
or
and
modern
angioplasty
increased
enhance
promote
symptomatic
large
vessels
drainage
to
the
the
"reperfusion
needed
mechanisms
scarring,
i.e.
Sometimes,
capillary
condition
Even
are
(thrombolysis,
inflow,
the
to
acceptable
treatments
damage.
approaches
return
employment.
current
basic
early
activities,
gainful
these
interventive
not
include
progressive
of
surgery)
goals
of
the
including
mammalian
myocardial
infarctions.
The
application
their
the
invention
present
bio-
of
or
uses
omentum-derived
organic-
acceleration
systemic
fractions
lipid
synthetic
or
purified
of. v a s c u l a r i z a t i o n ,
vascular
collateralization,
collagen
formation
or
and
-
4 -
local)
the
or
use
analogues
of
for
neovascularization,
promotion
scarring,
(and/or
of
perfusion,
organization
and
(cellular
and
0 2 5 3 0 2 2
AMED 2 0 6
12/17/85
#5
of
collagenous)
result
surprising
atherosclerotic
October
fibroblast
into
the
effect
one
agents.
U.S.
and
heart,
the
see
an
the
and
vivo
as
especially
response
The
to
treatment
Treatment
of
and
rapid
of
of
and
adjacent
of
partially
of
living
accelerated
discussion
is
a local
In
of
which
at
injection
wound
healing,
after
to
which
see
may
material
both
promotes
effect
a protein
agents
surprising
in
organization
the
as
a preventive
healing
cellular/collagenous
systemic
following
MI.
by
a
issued
on
be
is
regarded
direct
by
to
appears
It
invention
present
vascularization
1.
is
more
reports
promotes
vascularization.
promote
for
which
are
used
affected
there
or
is
This
4,296,100
Franco
what
area
agent
Patent
which
has
lesions.
materials
P.
Wayne
factor
growth
regarding
can
in
lipid
to
vascularization
as
since
1981
20,
ischemic
myocardiac
lesion
site
in
application.
4 applications
heart
cited
as
examples
conditions:
acute
infarction
myocardial
effective
border
neovascularization
salvage
zones,
heart
injured
repaired
be
can
cells
formation
of
muscle
of
with
of
the
the
a maximal
goals
lesion
number
cells,
rapid
damaged
tissue
and
a mechanically
stable
scar.
to
-
the
5 -
access
0 2 5 3 0 2 2
#5
2.
Treatment
of
permanent
increase
general
myocardial
of
collateralization
overall
allowing
AMED 2 0 6
12/17/85
ischemia
(angina)
vascularization
improved
by
and
myocardial
perfusion.
3.
The
improvement
transplanted
4.
The
of
of
coronary,
aortic
reopened
coronary
thrombolysis
to
and
minimize
increase
and
of
perfusion
hearts.
improvement
order
vascularization
the
the
and
vascular
peripheral
arteries
similar
supplied
vascular
(by
by
and
grafts
or
angioplasty,
interventive
outflow
graft
chance
beds
procedures)
obstruction
of - s a l v a g i n g
the
and
affected
in
to
limbs
or
organs.
"Increased
"increased
vessels,
number
can
to,
be
through
new
of
including
"Increased
flow
vascularization"
through
achieved
existing
vessels
effective
(or
capillaries,
perfusion"
and
by
from
better
vessels,
(angiogenesis)
be
-unit
defined
a specific
opening
or
by
the
-
6 -
by
and
large
volume
by
vascular
and
both.
, or
defined
open)
per
can
be
can
tissue".
"improved
bed".
accelerated
formation
of
small
of
blood
This
flow
additional
0 2 5 3 0 2 2
#5
Increased
therapeutic
blood
since
goal
vessel
it
and
growth
hearts
1)
caused
supply
vascularization
myocardial
with
by
typically
to
per f u s i o n
(ischemic
complications
of
insufficient
major
arteriosclerotic
by
a desirable
small
in:
of
narrowing
their
or
is
stimulation
focally
severe
arteries
coronary
leads
AMED 2 0 6
12/17/85
one
blood
or
more
branches,
lesions
myocardium,
and
their
angina
patients)
and,
2)
sufficient
coronary
myocardial
cell
3)
vessels
may
death
be
with
mentioned
before)
restored
and
,
blood
limbs
to
the
has
supply
hearts
lack
led
of
focal
to
where
existing
blood
and,
vascular
arteries
coronary
where
and,
damaged
hearts
reopened
organs
blood
transplanted
4)
the
infarcts
myocardial
(by
(similar
supplied
by
-
or
procedures
distribution
argument
vascular
7 -
grafts
interventive
optimal
assure
flow
by-pass
also
grafts)
in
.
of
other
0 2 5 3 0 2 2
AMED 2 0 6
12/17/85
#5
The
spontaneous
associated
of
with
a number
site
increased
of
of
sequence
healing
living
Such
with
vesssels
include:
macrophages,
vascular
cells
involved
The
in
by
processes
vascularization
and
its
border
the
access
of
early
tissue
cells
of
the
lipid
incorporated
U.S.
by
an
can
be
of
in
the
accelerate
the
leukocytes,
these
smooth
vascular
of
novel
order
to
diseased
and
and
collagenous
improve
myocardium
in
this
cells,
the
promote
contractible
organization
organized
the
fibroblasts..
factors,
possible
in
affected
and
repair,
replacement
strength.
obtained
by
with
area
local,
systemic
or
omentum-derived
fractions.
extraction
co-pending
most
tensile
results
angiogenic
The
of
to
to
brought
application
of
in
pericytes,
enhancement
perfusion
involved
adequate
These
perfusion
of
salvage
mainly
, and
local)
and
involvement
polymorphonuclear
growth
and
areas,
formation
of
(or
and
is
functions
(endothelium,
the
vascular
the
manner
is
systemic
omentum-derived
are
angiogenesis)
aim
specific
infarct
perfusion
specific
cells
through
muscle;
a myocardial.
vascular
cells
repair.
of
of
cat
omentum
application
S.N.
reference.
There
-
is
described
642,624
is
8 -
also
which
in
is
a
hereby
a publication
0 2 5 3 0 2 2
#5
Angiogenesis
al.
(1984)
1985
of
potent
extraction
The
Nicholas
in
five
provide
lipid
Filed
of
animals
one
this
contains
will
cause
in
The
lipid
was
the
at
the
living
1,
solvent.
organic
from
process
and
with
October
reference.
the
a copending
note
in
and
most
the
our
is
potent
least
the
one
development
tissue.
of
The
use
been
patent
which
wound
Robert
by
found
filed
to
interact
angiogenic
in
used
S.N.
by
has
subtractions
material
incorporated
healing
which
patent
782,724,
by
composition
Catsimpoolas
We a l s o
supply,
hereby
Molecules
least
at
is
Lipid
omentum
connections
in
angiogenic
hereby
bone
in
which
et
itself
least
experiments
the
Goldsmith
further
a
by
obtained
factor
heterogeneous
to
S.N.
using
material
abundant
another
Containing
from
neovascular
at
is
"Lipid
S.
Harry
^5_2:2034
incorporated
obtained
omentum
by
There
Activity"
hereby
preparation
contain
in
reference.
angiogenic
lipid
and
Ass'n.
is
an
is
numerous
one
Med.
angiogenic
omentum
is
Amer.
"Composition
preparation
The
Omentum"
Angiogenic
which
result
From
by
application
from
Factor
J.
incorporated
Enhanced
factor
angiogenesis
concerning
AMED 2 0 6
12/17/85
with
effect.
healing
Sinn
and
December
4,
19 85
reference.
of
the
copending
-
angiogenic
application
9 -
omentum
S.N.
material
0 2 5 3 0 2 2
AMED 2 0 6
12/17/85
♦5
filed
and
'
hereby
in
skin
incorporated
conditions
by
Obtaining
(CMFr)
female
anesthetized
by
was
to
according
were
plastic
an
in
held
a manner
approximately
weight
as
Waring
buffered
The
through
4°C
for
and
omenta
cut
omental
procedures.
into
placed
was
also
to
the
omental
were
omental
pieces,
(hereinafter
pieces
300
were
"PBS")
-
10
size
-
using
proper
out
onto
ml
for
of
surgical
ranging
in
placed
in
were
which
blended
spread
use
pieces
individual
containing
for
Using
weighed,
grams,
and
tissue
control.
individual
66
sterile
removed
in
7 to
a
processing.
fat
lipid
at
a
surgical
and
into
Ketamine
incision
centimeters
blender
saline
of
square
from
were
anesthetized,
immediate
identical
kg.
a mid-line
known
removed
the
four
These
Once
a non-omental
individually
sterile
Containing
injection
mg/kg.
subcutaneous
surface
scissors.
7.0
at
technique,
a plastic
4°O.
of
conventionally
procedures
aseptic
Lipid
2.4-3.2
intramuscular
perfqrmed
Simultaneously,
in
Methanol
weighing
surgically
bags
treated
cats
dosage
laparotomy
Omenta
Chloroform
I
Fraction
Adult
preferred
the
filed
reference.
Example
I#
S.N.
a
phosphate
was
five
precooled
minutes
to
at
0 2 5 3 0 2 2
♦5
20,500
subsequently
an
omental
homogenate
placed
in
sterile
250
at
centrifuged
1600
at
centrifuge
three
yield
to
rpm
4°C
distinct
bottles:
fractions
of
pellet
The
(i.e.
precipitate
protein
precipitant
revealed"
the
The
to
contain
a turbid
cake.
Each
the
in
visible
homogenate
and
material,
of
these
composition
the
aqueous
total
fraction
was
ammonium
sulfate
in
protein
in
(resuspended
these
this
fraction
homogenate
of
discarded
was
PBS)
and
by
preparations
fully
which
fraction.
the
total
the
cornea
had
good
assay
activity.
fraction
lipid
composition
color
centrifugation,
proteinaceous
homogenate
neither
of
composed
in
were
lipid
with
turbid
that
angiogenic
quantity.
mixed
100%)
of
fractions
the
and
bottles
individually.
turbid
Testing
After
composition;
all
colored,
isolated
was
saturated
was
mixed
was
a refrigerated
minutes.
separable
of
cream
completely.
to
and
twenty
which
plastic
in
gravity
substantially
a floating,
acted
for
a pellet
containing
The
times
ml
AMED 2 0 6
12/17/85
and
than
two
distinct
a more
the
isolated
an
active
For
this
compact
Each
layer
each
-
11
lipid
an
foamy
was
of
in
these
-
cake
floating
upper
layer
factor
angiogenic
reason,
a
layers:
dense,
upper.
as
which
evaluated
was
and
substantial
lipid
layers
darker
found
0 2 5 3 0 2 2
#5
individually
and
fraction.
The
layers
was
found
of
omentum
the
cake
lipid
extract
lipid
and
to
from
comprising
quantities
combination
weight
which
Active
in
is
be
of
the
used
the
lipid
it
to
was
active
fractions
proportions
were
of
both
it
total
is
factor
the
in
Table
Lipid
Cake*
P o r t i o n of L i p i d
Cake E x t r a c t e d *
NR
3.6
2
65.7
56.0
3.2.
3
50.1
48.3
3.1
4
61.0
NR
7.1
5
38.0
37.0
3.5
6
39.9
NR •
3.0
7
7.8
6.2
2.4
8
22.3
21.4
3.4
(gm)
Reported
-
I:
I
31.2
Not
organic
composition.
using
given
weight
this
1
♦Weight
NR -
cake
lipid
concentrated
extracted
lipid
the
and
angiogenic
Total
Omentum*
No.
of
derived
the
active
comprising
93%
prepare
TABLE
Exp.
cake
approximately
which
the
comprise
AMED 2 0 6
12/17/85
12
-
0 2 5 3 0 2 2
#5
The
indicated
combined
with
8575
microblender
and
lipid/organic
solvent
times
in
for
gravity
minutes
ten
and
yield
a clear,
a particulate
supernatant
was
isolated
completely
for
used
place
obtained
mately
II.
which
4 ml
of
was
then
PBS
for
preferably
use
in
cornea
obtained
supra,
hexane
and
phases
separate.
0.66
The
95% e t h a n o l ,
re-extraction
combined
60
ml
volumes
of
95%
upper
and
was
the
phase
(hexane)
lower
resulting
with
fractions
the
13
for
then
-
to
Other
and
art
in
was
assays.
a
every
then
allowed
10
g of
added.
to
re-extracted
of
be
approxi-
cam
was
may
liquid
in
and
phase
first
were
-
the
were
, was
and
vacuum
solvent.
ethanol
and
thoroughly,
combined
ethanol
hexane
200
The
procedures
dissolved
was
(approx.
mixed
were
in
at
colored,
suspended
the
The
temperature
A viscous
evaporation.
The
extract),
The
rotary
known
are
Fractionates
of
room
37 °C u n d e r
Purified
CMFr,
at
chloroform/methanol
removal
Eberbach
an
precipitate.
Obtaining
mixture
The
solvent
of
at
in
minutes.
coventional
using
solvent
centrifuged
golden
matter
evaporation
the
remove
methods
in
rotary
v/v)
then
was
were
organic
two
centrifuge
and
to
an
for
homogenate
cake
(2:1,
homogenized
supernatant
subjected
of
methanol
a clinical
to
lipid
ml
21
approximately
chloroform
comprising
of
quantities
AMED 2 0 6
12/17/85
with
the
ethanol
fraction.
re-extracted
with
0 2 5 3 0 2 2
#5
and
hexane,
first
the
hexane
"hexane
fraction.
upper
material"
following
(i.e.,
the
water
were
allowed
to
partition
with
0.4
upper
phase,
as
was
then
Unisil
removed,
and
the
dried
UP."
The
as
(1967).
20
volumes
and
methanol.
lipids
of
This
v/wt)
of
0.2
volumes
and
mixed,
the
Folch
was
washed
This
produces
combined
with
the
the
is
also
Folch
known
portion
dried,
an
upper
hereafter
and
is
LP."
was
dissolved
by
column
J.Lipid
Res.
successively
with
separates
(9:1),
neutral
(acetone /methanol)
.
-
al.,
14
-
and
acid,
ace t o n e / m e t h a n o l ,
elution
glycolipids
chloroform,
a silicic
et
eluted
was
successive
in
on
Vance,
chloroform,
(methanol)
226;497-509
, and
partition
chromatography
The
Folch
in
thoroughly
portion
described
(chloroform),
phospholipids
lower
to
is
obtain
(1:1).
obtain
LP p o r t i o n
8:621-630
column
to
"Folch
as
subjected
column,
which
phase,
to
J.Biol.Chem.
lower
the
phase
subjected
phase
methanol/water
then
Folch
were
upper
hereafter
The
Phases
lower
.
then
volumes,
The
methanol
and
20
(2:1,
to
hereafter)
dissolved
was
with
dried,
"ethanol
al.,
.
volumes
"Folch
known
was
et
fraction
separate)
were
and
was
Folch,
added.
combined
ethanol-LP,
fraction
chloroform/methanol
of
and
layer
phases
material,"
ethanol-LP
partition,
(1957)
phase
Both
(hexane-UP,
The
hexane
resulting
AMED 2 0 6
12/17/85
, and
0 2 5 3 0 2 2
-
p\
|
S
f A G - e
S
i 5
i
f
j
i
0 2 5 3 0 2 2
#5
centrifuged
The
top
2000
at
then
removed.
with
an
The
to
phase
was
obtain
separation
of
yielded
performed
with
were
pooled.
the
"acetic
UP and
and
(approximately
3 gm e a c h )
water
in
a sintered
water
chroma t o g r a p h y
buffer,
performed
of
100
2 ml
ml
elution
of
performed
as
Each
phases.
solvent
and
all
the
fraction.
gelatin
fractions
Excess
Folch-UP)
and
applied
with
the
same
minute)
water
the
with
to
.
binding)
were
50
ml
or
of
to
the
16
-
ml
by
the
acid.
off
0.01
Elution
binding
acetic
-
in
100
followed
ml
beads
drained
column.
gelatin
450
were
x 9 cm
was
from
with
sepharose
a 2.5
(total
was
0.5%
washed
suspended
salts
remove
heparin
was
Gelatin-Sepharose
The
water
buffer
This
or
were
into
packed
7.0
per
of
and
beads
filter.
glass
column.
(e.g.,
pH
was
combined
was
of
(heparin
beads
sample
two
soluble"
CL-6B
dry
the
opposite
J*PBS h o m o g e n a t e "
fraction
centrifuged
acid
Heparin-Sepharose
in
bottles.
follows:
as
suspended
the
was
Evaporation
chromotography
ml
fraction
a clean
chloroform
Folch
soluble
and
phases
the
acid
twice
200
acid-insoluble
chloroform
chloroform
of
in
of
backwashed
Affinity
5 minutes
acetic
acetic
volume
equal
above
the
i.e.,
layer,
for
rpm
AMED 2 0 6
12/17/85
at
was
a flow
column.
material
the
phosphate
a washing
After
and
rate
with
Final
was
evaporation
0 2 5 3 0 2 2
#5
of
acetic
buffer
acid
for
The
further
the
c h l o r o form/me t h a n o l
characterized
For
B_^ (E.
Heftmann,
used.
In
our
extract
the
was
was
5.0
gel
100
ethyl
acetate;
ethyl
(4:1)
followed
by
ml
elution
LH-20
methanol
extract,
column
methanol
solvent
and
of
200
ml
(1:1)
was
the
Chromatography
a chromatography
mesh
the
Sigma
with
was
of
was
solvent.
evaporated
acid/water
was
ethanol-LP
corned
out
Fractions
were
testing.
-
17
-
sequence
chloroform;
methanol/water
comprising
Five
(I-V) .
performed
mg of
with
Using
(25:15:4:2).
obtained
the
Company)
in
solvents:
mixture
hundred
were
Chemical
(3:1);
One
for
Part
column
performed
a solvent
were
1983,
chloroform.
following
chromatography
of
fractionations
chloroform/methanol
of
fractions
or
liquid
ml
elutions
column.
elution
Kuksin,
acetate/methanol
permeation
Sephadex
A.
or
New Y o r k ,
chloroform/methanol/-acetic
individual
iatrobead
or
equilibrated
aliquots
parts
was
Elsevier,
in
columns,
using
Gel
phosphate
fraction
lipid
component
(100-200
gel
previously
silica
in
placed
v/v)
chroma t o g r a p h i c
editor),
silica
containing
these
method,
its
gel
described
procedures
which
in
resuspended
(2:1
to
as
silica
using
chromatography.
lipid
was
testing.
subtractions
the
material
AMED 2 0 6
12/17/85
the
on
a
chloroform-
were . p l a c e d
on
the
a chloroformcollected
and
the
0 2 5 3 0 2 2
#5
III.
The
with
lower
and
described
were
Fractions
phase
glycolipids
visualized
by
also
with
Svennerholm,
analyzed
previously
(60:35:8)
the
their
as
Ullman,
by
(1978).
The
upper
glycolipid
fraction
was
examined
major
component
of
the
fraction
glycolipid
or
by
chromatography
60
u)
eluted
taught
by
with
an
et
purified
Hakomori,
et
by
TLC
preparative
(1
x
50
mixtures
237(24)
J.Biol.Chem.
al.,
The
neutral
column
J.Biol.Chem.
al.r
by
antibodies.
complex
Iatrobead
Res.
neutral
and
hexane/isopropanol/water
Kannagi,
and
(1982);
on
They
derivatives
J.Lipid
HPTLC
as
(1956).
complex
phase
further
1:42
SSEA-1
developing
reagent
spray
al.,
by
and
upper
was
et
HPTLC
by
the
perbenzoyl
phase
Forsmann
as
orcinol
1^9:910-913
with
examined
were
J.Neurochem.
HPLC
by
reported
immunoblotting
v
the
chloroform/methanol/water
solvent
as
of
Analysis
AMED 2 0 6
12/17/85
cm,
as
14865-14874
259(7)
4672-4680
(1984).
The
in
hydroxide
subjected
to
with
methanol
fractions,
acid
The
cartridge.
eluted
methanol
acetic
glacial
fraction
ganglioside
to
for
and
alkali
2 hrs
treated
0.0 8M and
obtain
mono-,
with
0.05M
and
a C18
fraction
polysialo-
-
18
-
et
N sodium
with
reversed-phase
ammonium
Ledeen,
0.25
neutralized
a DEAE-Sephadex
di-,
Sefe
37 °C,
with
ganglioside
on
0.02M,
respectively.
at
desalted
chromatography
treated
was
al.,
was
column
acetate
then
and
in
ganglioside
Methods
in
K\
h
/
y
i S-S
&
£
i
\H
-
^
O
0 2 5 3 0 2 2
0 2 5 3 0 2 2
AMED 20 6
12/17/85
#5
For
were
esters
analyzed
their
as
of
fraction
was
perbenzoylated
pyridine
and
the
the
lower
quantitated
elution
gradient
described
by
spectrometry,
were
with
HPLC
and
on
230
Ullman,
For
or
in
of
the
was
derivative
heated
spectra
mass
from
were
It
and
an
was
150 °C.
to
950
was
ionizing
m/e
was
100
operated
voltage
Repetitive
were
in
acquired
with
70
scans
at
with
mass
(5-50
samples
a sample
a rate
with
of
probe
(where
ug)
cup
of
and
the
30°/min.
4500
56K
an
ionizing
current
of
eV.
The
the
5 sec
-
20
mass
range
intervals.
-
mass
quadrupole
model
ionizer
probe
The
Teknivent,
of
luoro
* microgram)
ug
model
a Finnigan
equipped
separated
pyridine /hexamethyldi-
5 ug
350 °C a t
with
of
in
,O-bistrimethylsilyltrif
1 to
placed
to
obtained
spectrometer
system.
from
Anywhere
direct
ul
silane/trimethylchlorosilane/N
acetamide.
the
previously
ganglioside
25
same
column
Zipax
as
were
HPTLC
chloride
benzoyl
uncoated
supra.
trimethylsilylated
For
glycosphingolipids
an
methyl
the
glycolipids,
nm d e t e c t i o n
glycolipid
acid
on
in
al.,
glycosides
supra.
neutral
phase
et
fatty
derivatives
Kozulec,
benzoylated
by
The
N HCl
0.75
Kozulec,
methyl
trimethylsilyl
analysis
and
and
The
by
glycolipids
anhydrous
(1979).
TLC.
by
described
as
in
supra,
94:36-39
analyzed
column
OV-l
Ledeen,
the
analysis,
methanolysis
Biochem
were
acid
fatty
following
Analytical
>
to
subjected
methanol
y
'
and
sugar
data
0.5
ma
temperature
from
100
m/e
0 2 5 3 0 2 2
#5
Glycolipids
backed
HPTLC
(60:35:8),
were
chromatographed
with
plates
then
dried,
methacrylate
in
J.Biol.Chem.
256:13223-13225
in
soaked
albumin
serum
for
antibody
neutral
for
2 hours
was
antibody
plates
to
washing
were
IV.
the
with
and
in
0.025%
floating
lipid
in
to
0.02
phase
upper
monoclonal
Culture
the
GD3 m o n o c l o n a l
After
anti-mouse
goat
for
were
IgM
2 hours
buffer
4°C.
at
with
developed
M Tris-HCl
in
washing
33 mN
containing
20%
H_O2.
of
was
cake
hexane
the
the
of
with
inventors.
plates
PBS,
omentum
The
the
by
1% b o v i n e
to
Type
of
then
were
exposure
Forssman
treated
was
in
Feline
material
treated
plate
peroxidase
Characteristics
2.
The
exposed
chloroform/methanol
Fig.
similar
al,
et
plates
containing
horseradish
4-chloro-naphthol
methanol
saline
40°C.
fraction
IgM p r e p a r e d
conjugated
After
The
(1981).
before
Brockhause
by
p u r c h a s e d from A m e r i c a n
a,
(TIB 121) .
The TLC p l a t e s
disialoganglioside
the
polyisobutyl
IgM,
Collection
PBS
at
0.05%
described
as
2 hours
glycolipid
antibody
in
buffered
phosphate
aluminum-
on
chloroform-methanol-water
dipped
hexane
AMED 2 0 6
12/17/85
CMFr
and
the
homogenized,
was
centrifuged
extracted
further
phase
and
Fractions
was
with
fractionated
contained
shown
-
21
as
approximately
to
-
and
consist
shown
in
9 8% of
primarily
the
of
0 2 5 3 0 2 2
#5
triglycerides,
and
GC/MS
number
the
that
the
14:0,
major
second
The
the
number
partition
and
recovered
80% of
from
acids
and
as
the
The
column
also
by
TLC
examined
was
hexosylceramide,
of
analysis
infra.
TLC
The
and
phosphatidylserine,
were
column.
to
Folch
were
neural
lipid
consisted
TLC
fraction
primarily
and
The
and
acid,
which
cholesterol
by
first
fatty
lipids,
analysis.
18:2
.
lipids,
phase
detected
the
subjected
phase
of
of
and
the
(i.e.,
bonds)
was
lower
amounts
Quantitative
by
length
small
and
examined
chain
ethanol
globotriaosylceramide
described
acids
esters
methyl
18:1
18:0,
fatty
methanolysis
acid
fatty
unsaturated
Unisil
fraction
Alkaline
17:0,
material
the
the
were
glycolipid
of
a Unisil
on
triglycerides
16:1,
carbon
phase
fractionated
migrating
the
TLC.
by
resulting
16:0,
ethanol
constituted
fatty
the
triglyceride
indicates
solvent
of
analysis
revealed
were
determined
as
AMED 2 0 6
12/17/85
free
acetone
components
lactosylceramide,
globoside
these
were
present.
glucolipids
methanol
by
phospholipid
components
migrating
phosphatidylcholine
HPTLC
is
fraction
was
as
and
sphingomyelin
present.
Approximately
material
was
20% by
recovered
in
weight
the
of
the
Folch-UP.
-
22
-
ethanol-phase
This
Folch-UP
of
0 2 5 3 0 2 2
l
i
/
H \
i
S
S
/ i-J &
P / \ O 6
/
0 2 5 3 0 2 2
#5
material
contained
indicated
a GC/MS
this
material
acids,
peptides
distribution
is
extract
of
the
in
are
shown
percent
distribution
The
HPTLC
and
positive
globoside
polar
of
mixture
the
of
sugars,
Weight
omentum
fraction
and
pryidine
HPLC w i t h
3.
These
(Nfa) ,
12%;
were
the
230nm
crude
lipid
9.6%;
LacCer,
benzoylated
perbenzoylated
detection.
data
in
glycolipids
(Nfa),
positive
II.
Figure
found
to
material
consist
show
this
The
that
the
fraction
GlcCer
11%;
as
(Hfa)
+ GalCer
GbOse3Cer,
The
as
positive
of
major
about
well
more
small
as
no
component
10%;
SSEA-1
-
24
examined
than
slowly
the
of
3 to
major
positive
further
-
by
orcinol
Immunoblotting
indicated
was
an
amounts
components.
and
was
90% of
slightly
SSEA-1 ' a n t i b o d y
positive
fraction
lipid
migrating
standard,
and
neutral
phase
upper
Forsmann
present.
a complex
from
material
trimethylsilylation,
glycopeptides.
GalCer
26%;
after
soluble
26%.
orcinol
Forsmann
was
in
+ GaOse2Cer
GbOse4Cer,
ninhydrin
glycolipid
results
(Hfa)
and
was
a n a l y z e d " by
(Nfa) ,
orcinol
analysis,
derivatives
GlcCer
methanol
Table
cloride
benzoyl
The
fractions
in
of
salt.
and
the
shown
Aliquots
with
six
and
components
amino
least
at
be
to
appeared
AMED 2 0 6
12/17/85
the
4 more
with
component
components
purified
by
were
0 2 5 3 0 2 2
#5
chromatography
found
to
were
present
glycolipid
mass
the
also
of
the
Forsmann
antibody
The
to
and
of
show
spectra,
of
C-18
indicate
linkages
have
and
reactivity
any
ester
linkages
that
into
mono,
di
components
by
migrating
of
mobility
material
migrating
fraction
was
Iatrobead
components
as
and
migrating
acids.
fatty
glycolipid
position
directly
is
and
determined,
data
analytical
may
The
purified
as
have
mild
been
of
The
and
primarily
bands
and
fractions
of
corresponding
a small
to
of
amount
chromatography
fractions
-
present
monosialoganglioside
by
GM3 w e r e
alkali
monosialoganglioside
consist
to
a triplet
GM1.
the
with
polysialoganglioside
GM3 s t a n d a r d
further
column
and
HPTLC
as
the
and
treated
chromatography.
shown
indicated
3,
structure.
was
was
probe
hexose
Although
glycolipid
fraction
DEAE-Sephadex
direct
Figure
the
been
not
intact
internal
that
acids
fatty
by
sphingosine
ratios
Hexose
The
in
hexosamine,
data
this
The
examined
ganglioside
destroy
fraction
the
The
terminal
support
separated
by
1:2:2.
pentaglycosylceramide.
configuration
GC/MS ..
by
to
subjected
.
silylated
these
and
'
presence
together
strongly
analysis
primarily
presence
column
Glc/Gal/NAcGal
spectrometry.
Taken
the
component
be
was
residues,
the
Iatrobead
an
and
methanolysis
were
on
AMED 2 0 6
12/17/85
containing
pooled.
25
-
• This
on
an
only
material
was
0 2 5 3 0 2 2
#5
treated
with
neuraminidase
characterized
as
The
probe-MS.
analysis
to
liberated
intact
ganglioside
sugars
and
of
primarily
24:1.
The
of
spectrometry
mass
spectra
16:0,
(sialyl
ceramide)
.
was
the
to
of
further
column
component
migrating
subjected
to
acid
was
components
also
that
treated
identified
and
methyl
with
as
the
20:0,
22:0,
examined
by
given
by
to
16:0>
ether
fractions
be
and
the
18:0,
and
shown
was
by
-
26
The
methyl
by
major
on
only
A
24:1.
lipid
HPTLC
and
to
This
an
a single
preparation
The
fatty
the
-
HPTLC
by
glycosides
GC/MS.
24:0,
and
lactosylceramide
and
GM3
GD3.
as
containing
the
18:1.
neuraminidase
24:0
probe
ganglioside
pooled.
examined
1:1
consisted
derivative.
chromatography
GD3 w e r e
ratio
direct
purified
as
The
23:0,
migrating
the
the
[ l-4]glucosy [1-1]
fraction
by
The
and
acids
fatty
a component
esters
found
acid.
GC a n a l y s i s .
and
GC
by
methanolysis
by
18:1,
was
direct
shown
was
trimethylsilyl
methanolysis
were
to
[ 2-3]galalctosyl
Iatrobead
was
18:0r
as
primarily
material
Glc/Gal
1:1
was
and
N-acetylneuraminic
disialoganglioside
consist
fatty
be
product
HPTLC
acid
examined
to
similar
standard
of
lipid
by
subjected
preparation
mass
The
was
found
was
sialic
only
acids
fatty
Glc/Gal
the
lactosylceramide
consist
of
and
AMED 2 0 6
12/17/85
was
and
ratio
of
acid
The
material
product
direct
probe
MS
0 2 5 3 0 2 2
#5
The
analysis.
only
of
of
the
sialic
N-Acetylneuraminic
TMS d e r i v a t i v e
material
also
was
monoclonal
The
contain
in
with
with
GD3.
The
to
react
with
a
with
laboratory
fraction
migrating
insufficient
MS
GD3.
polysialoganglioside
but
probe
consistent
this
consist
to
Direct
immunoblotting
prepared
components
shown
was
GC a n a l y s i s .
spectra
by
reactivity
acid
by
gave
shown
antibody
demonstrated
GTlb,
liberated
AMED 2 0 6
12/17/85
HPTLC
on
quantities
shown
was
as
ganglioside
obtained
were
to
for
GDla,
further
analysis.
V.
Angiogenesis
The
angiogenic
described
in
fractions
I-V
following
manner:
Figure
anesthetized
From
each
the
cornea
second,
ocular
any
of
each
Silica
Gel
tested
by
the
rabbit
cornea
series
fourth,
New
edema
the
in
eye.
The
I,
an
vessel
growth
-
27
the
microscope
-
and
was
were
50
animals
day
the
mg./Kg).
suspension
tenth
inflammation
(30
intrastromally
operating
and
in
rabbits
a single
of
corneas
test
white
lipid
placed
eighth,
Blood
and/or
Table
aqueous
needle
with
Zealand
chromatography
pentobarbitol
gauge
sixth,
injection.
corneal
of
shown
of
and
grossly
preparations
the
intravenous
a 25
lipid
of
injection
through
examined
I
the
and
a
with
of
preparation
microliter
made
were
activity
was
into
were
on
the
following
the
noted.
presence
On t h e
of
0 2 5 3 0 2 2
#5
tenth
after
day
sacrificed
individually
with
examination
hematoxylin
obtained
from
formalin
fixed
rabbit
six
no
identified
dilation
vessels
the
the
from
10-20%
the
the
involving
at
4 ml
gram
of
PBS
microblender
and
for
sections
stained
were
manner,
from
cut
records
4+,
from
of
the
positive
to
fat
cornea;
vessels
with
the
of
an
the
homogenizing
two
minutes
site
dense
cornea.
an
aqueous
also
by
subcutaneous
this
at
-
mixture
4°C.
28
-
vessels
vessel
suspension
of
prepared
made
was
to
way
injection
preparation
were
the
blood
involving
blood
the
the
fatty
of
blood
injection
aqueous
fat
thirds
to
coloration
individual
multiple
limbus
preparation
of
two
0,
1+,
red
several
identifies
the
follows:
as
a clear
limbus
purposes,
and
graded
identified
non-omental
portion
was
and
the
30-40%
cake
non-omental
conventional
identified
limbus
comparison
subcutaneous
three
least
lipid
were
slides,
Photo
scleral
3+,
cornea;
extending
The
of
from
formation
omental
the
thick
response
site;
extending
For
in
eyes.
limbus ;*2+,
migrating
of
eosin
angiogenesis
injection
rabbits
recorded.
angiogenic
at
the
histological
micrometer
identified
noted
and
enucleated
were
eyes
The
and
visually,
AMED 2 0 6
12/17/85
site
of
the
and
tested.
combining
tissue
using
Similarly,
the
the
an
a
with
Eberbach
aqueous
0 2 5 3 0 2 2
#5
of
suspension
four
homogenizing
of
PBS
in
the
the
of
homogenate
proteinaceous
evaluated.
omental
gram
whole
fractions
The
results
of
portions
microblender
the
cake
lipid
for
two
omentum
was
the
and
lipid
are
as
at
in
with
4°C.
The
centrifugation
was
Table
II
4 ml
into
also
below.
II
Test
Sample
Angiogenic A c t i v i t y
( p e r 50 m i c r o l i t e r )
3
4
5
6
7
8
9
10
11
12
13
Extracted
lipid preparatipn
in a q u e o u s
medium -
14
15
1'6
17
PBS h o m o g e n a t e
of l i p i d c a k e
18
19
20
21
PBS h o m o g e n a t e
of w h o l e o m e n t u m
22
23
24
25
26
PBS h o m o g e n a t e
of n o n - o m e n t a l
fatty tissue
27
28
29
PBS
•
"
alone
to
by
cake
lipid
fractions
shown
TABLE
prepared
minutes
prior
AMED 2 0 6
12/17/85
No.
No.
No.
No.
No.
No.
No.
No.
1
2
3
4
5
6
7
8
4+
4+
3+
4+
3+
4+
4+
4+
No.
No.
No.
1
2
3
+1
+1
+1
No.
No.
No.
1
2
3
+1
+1
+1
No.
No.
No.
1
2
3
0
0
0
No.
No.
uo
1
2
^
0
0
0
-
29
-
(inflammation)
(inflammation)
(inflammation)
0 2 5 3 0 2 2
#5
The
data
activity
In
noted
with
PBS
of
homogenate
that
affinity
showed
38) .
a heparin
non-omental
in
the
subcutaneous
fat
corneal
The
PBS
total
omentum
the
all
data
of
fat
taken
Table
of
the
was
in
that
was
the
PBS
Note,
CAM a s s a y
those
which
(see
page
instances
subcutaneous
the
within
cornea
with
concentrated
however,
injected
abdominal
cat
was
and
the
homogenate
the
or
with
A complication
II,
from
and
the
occurred
alone
activity
extraction.
PBS
with
extract
omentum
component
from
PBS
to
prior
activity
at
total
central
lipid
angiogenic
the
homogenate.
two
wall
days
caused
after
injection.
of
course
injected
the
aqueous
preparation
followed
development
and
the
of
binding
of
inflammation
severe
minimal
cake
lipid
injection
microliter
chloroform/methanol
the
angiogenic
angiogenic
50
single
homogenate
No a n g i o g e n e s i s
noted
a
only
of
the
excellent
chromatography
good
following
the
PBS
homogenate
however,
the
comparison,
the
that
after
of
injection
CMFr.
by
observed
was
corneal
the
indicates
AMED 2 0 6
12/17/85
extracted
reaction
was
subsided
within
a consistent
observed
pattern
activity.
'fraction,
within
forty-eight
in
response
the
chloroform/methanol
suspended
intense
lipid
angiogenic
Following
a mild
This
hours.
30
-
lipid
rapid
injection
corneal
twenty-four
-
of
cornea
hours
of
inflammatory
which
initial
to
0 2 5 3 0 2 2
AMED 2 0 6
12/17/85
#5
inflammation
with
cornea
often
minimal
the
formation
became
injection.
is
Histological
tenth
stromay
day
a photograph
in
It
such
of
fraction
lipid
and
process
that
the
omentum
which
in
blood
the
capillaries
histological
A
eye.
around
vessel
blood
network
enucleated
was
the
following
vessels
within
in
photograph
the
3.
Fig.
harvested
eyes
within
the
on
corneal
section
within
capillaries
only
the
response
is
injection
initiates
of
and
in
that
medium
the
the
50
mechanism
presently
seven
of
to
this
unknown,
lipid
new
blood
dense,
well
develops
ten
-
31
and
microliter
extracted
into
solvent
initiates
a single
the
organized
networks
note
aqueous
Although
becomes
vascular
particular
after
angiogenesis
injection.
the
vessels
4 days
day,
the
the
the
blood
structured
multiple
multiple
tenth
by
of
of
which
area
from
the
stroma
is
4.
Fig.
is
to
richly
of
scleral
3 to
of
clouding
interstitial
evident
illustrated
showed
the
a curtain
seventh
and
slight
discharge
with
examination
illustrating
shown
of
grossly
a dense
This
in
a slight
cornea,
By t h e
formed
cornea.
the
with
the
by
erythema
appearance
of
margin
had
characterized
accompanied
pannus,
the
is
days.
-
extracted
sustains
dose
angiogenic
it
is
apparent
fraction
vessel
from
the
formation
structured,
0 2 5 3 0 2 2
#5
As
was
shown
in
Figure
3 further
performed
by
silica
gel
testing
of
cornea
assay
in
each
of
the
the
showed
subfraction
any
of
V however,
chloroform/methanol
obtained.
It
although
having
when
the
was
combined
overall
found
angiogenic
activity
subfraction
of
potency
that
V act
the
to
only
effect
from
of
than
the
originally
subf a c t o r s
in
CMFr
the
present
preparation
subsequently
with
with
activity
less
measurably
extraction
lipid
no
and
activity
was
be
angiogenic
The
the
Subsequent
to
activity
I-IV.
of
subtractions,
a noticeable
subfractions
subfraction
lipid
angiogenic
V with
fractionation
chroma t o g r a p h y .
five
12/17/85
and
of
enhance
angiogenic
I-IV,
themselves,
and
increase
composition
as
a
whole.
The
experiments
exhibits
angiogenic
performed,
using
outline
Figure
of
additional
I.
CAM a s s a y s ,
derivation
of
the
the
additional
derived.
The
omental
the
of
fractions
the
in
these
are
explained
described
obtained
by
the
32
is
-
to
the
a measure
An
is
confined
experiments
i
-
leads
also
infra.
not
were
the
of
CAM a s s a y s .
Unit",
then
were
following
This
which
"Discrimination
CMFr
experiments
infra.
the
the
consisted
Index",
had
that
prepared
experiments
"Angiogenic
experiments
fractions
Further
described
fractions
value,
Both
The
show
supra
activity.
performing
effect
forth
set
to
the
described
of
0 2 5 3 0 2 2
#5
the
Once
supra.
effective
fractions
containing
It
materials
was
are
unexpectedly
found
that
Again,
available
unexpected
interest
greater
of
different
the
of
these
properties
fact
that
had
angiogenic
performed
found.
were
than
using
combinations.
new
Of
with
compositions
greater
CAM a s s a y
the
of
shell
incubation.
"window" .
outside
incubator
the
is
even
mixtures
additive
Eggs:
these
subjecting
Membrane
By u s i n g
removed
from
The . o p e n i n g
another
fractionates,
("CAM
Assays
chicken
eggs,
a power
drill,
to
assays")
.
and
steps:
tape
environment.
for
by
fertilized
uses
Cellophane
extracts,
determined
following
Preparing
square
were
Chorioallantoic
Embryo
involves
of
properties
compositions
The
the
is
many
were
art,
CAM A s s a y s
Chick
of
angiogenic
the
in
gangliosides,
additional
to
were
more
lipid-
known
unexpected
experiments
gangliosides
Angiogenic
and
strong,
the
effect.
angiogenic
VI.
possessed
contain
gangliosides,
which
Additionally,
commercially
to
molecules
also
activity.
especially
of
composition
determined
was
molecules,
lipid-containing
used.
molecular
general
AMED 2 0 6
12/17/85
is
the
now
tightly
The
eggs
fertilized
referred
seals
are
4 days.
-
33
-
off
then
a
on
egg
to
as
the
put
2 cm
day
4
a
window
in
the
to
37°
C
0 2 5 3 0 2 2
#5
the
Making
g of
agarose
in
small
a
Discs:
and
10 ml
vial
glass
2% BSA s o l u t i o n
BSA
in
40 ml
to
(in
is
PBS)
the
fractionate,
or
is
hardened
are
The
in
warm
8th
after
mixed
and
a water
constant
bath.
mixed
is
to
50°
at
Using
0.4
100"
C
C with
a
1%
plus
20
a pipet
extract,
with
subdivided
of
a drop
After
stirring.
it
heated
(2% a g a r o s e
(i.e.,
is
incubation,
mixed
mixture
solution
gelation,
day
subsequently
composition)
by
by
PBS
and
testing
mixture
agarose
of
PBS) .
kept
of
On t h e
AMED 2 0 6
12/17/85
the
into
the
disc
large
4 smaller
discs.
Placing
after
the
the
Discs
on
the
discs
incubation,
CAM;
blood
choosing
vessel
that
inside
shell
4 days.
All
inside
placed
various
The
selected
area
disc
will
lie
wall.
The
the
used
the
CAM or
stick
incubated
previously
of
is
but
are
on
degrees
embryo
then
are
day
eggs
chick
beyond
eggs
8th
the
CAM w i t h
from
instruments
On t h e
the
1 cm away
the
Membrane:
are
on
development.
approximately
away
areas
the
not
for
soaked
so
to
far
the
another
in
98%
ethanol.
Plastic
Discs:
hole
puncher.
each
disc,
Additional
to
the
disc
After
the
discs
placing
2.5
solution
2 . 5 ml
and
Plastic
is
aliquots
dried
were
ml
allowed
of
between
the
of
to
test
prepared
the
dry
34
-
over
solution
applications.
-
test
using
a
solution
a warm
may
After
be
the
on
plate.
added
disc
0 2 5 3 0 2 2
#5
is
it
prepared,
the
Rating
the
made
discs
is
by
the
on
Effects;
located
are
larger
placed
the
Upon
inside
the
off
breaking
CAM as
bits
described
12th
of
of
day
and
eggs
AMED 2 0 6
12/17/85
the
supra.
incubation,
windows
the
shell
with
are
of
a pair
forceps.
The
The
that
vascularization
surrounding
the
direction
the
effects
disc
then
are
eggs
is
of
of
rated
1 = one
around
the
disc;
2 = three
disc.
the
disc
other
a scale
of
1 to
or
the
disc;
of
compared
"wheel
the
and
as
compared
The
in
with
effects
of
each
follows:
increased
branching
negative.
areas
a weak
"wheel
increased
of
increased
response.
spoke
effect,"
branching
around
which
is
the
disc;
disc,
spoke
to
effect"
a degree
with
increased
a
greater
than
in
with
extensive
branching
"3";
a strong
response.
5 = "wheel
around
to
neovascularization
eggs.
of
areas
small
more
of
5,
is
microscope.
response.
4 *
around
small
light
egg
determined
in
explanatory;
moderate
is
the
the
Degrees
discs
3 = formation
self
of
rest
essentially
around
branching
the
the
two
or
under
in
the
on
examined
the
disc;
spoke
effect"
a very
strong
response.
-
35
-
branching
0 2 5 3 0 2 2
#5
Plus
and
response
Based
INDEX
The
The
in
are
7 are
"weak,"
1 is
"negative"
III
and
The
and
canine
the
for
comparative
is
as:
defined
"moderate,"
+ 1(3)
x 5
sets
forth
data
samples
omenta,
would
in
'the
same
as
be
x
•
are
Figures
obtained
I,
that
by
feline,
indicated.
36
II
used
from
-
of
"strong,"
and
4
follows:
as
.iUU = 2 8 . 3 3
components
as
analysis
CAM a s s a y s ,
calculated
obtained
partition
forth
were
none
ANGIOGENIC
assays
12
containing
7(2)
12
is
(no
is
tables)
activity.
A=
used
1-
response,
the
system,
angiogenic
A Index
set
from
strong
following
allows
the
procedures
terminology
scoring
a sample
solvent
numerical
ranging
x T o t a l of s c o r e s on i n d i v i d u a l
Maximum s c o r e p o s s i b l e .
in
extracts,
the
of
a value
(exceptionally
in
A Index
example,
Table
have
each
.
"A"
Index
Angiogenic
For
5
with
used,
foregoing
or
terms
100
if
the
Index"
determined.
to
branching)
upon
("A"
samples,
could
whatsoever)
extensive
with
also
are
a CAM a s s a y
so
value,
minuses
AMED 2 0 6
12/17/85
-
analyzing
obtained
and
in
III.
the
using
The
Figures.
bovine,
porcine,
0 2 5 3 0 2 2
#5
TABLE
AMED 2 0 6
12/17/85
III
EXTRACTION
COMPOUND
Cat
CMFr
"
Bovine
*"
m
Porcine
"
"
Ovine
«"
@
"
Canine
"
"
n
CatPBS
supernatant
1% HAc
sol,
1% HAc
insol,
Cat
subcut.
Porcine
Cat
CMFr
lipid
UP
LP
EGGS
A
%NEG
41
28.98
46.34
14
35.27
18
M
S
17
4
1
35.71
5
4
0
24.9
61
7
0
0
4
23.35
50.00
2
0
0
4
45.05
3
1
15
19.12
73.33
4
0
0
18
28.17
44.44
6
4
0
10
28.72
70.00
1
2
0
20.03
83.33
0
1
33.43
37.50
5
0
0
100
0
0
0
38
9
1
fat
subcut.
fat
8
cake
supernatant
13.36
(cat)
W
16
34.7
-
37 -
0
0
0
0
0 2 5 3 0 2 2
#5
CMFr
pellet
CMFr
supernatant
(cat)
19
39.0
32
9
4
7
32.4
43
4
0
8
40.1
25
5
1
awirr.
w
M
16.7
75
6
0
15.05
75
3
1
23
35.4
30
16
0
0
18
21.9
72
5
0
0
100
0
0
0
(porcine)
CMFr
pellet
AMED 2 0 6
12/17/85
(porcine)
0
0
SOLVENT P A R T I T I O N
COMPOUND
EGGS
Cat-Hexane
UP
Bovine-Hexane
, 2 4
UP
Porcine-Hexane
Cat-Ethanol
UP
LP
Bovine-Ethanol
Porcine
LP
Ethanol
Cat-Folch
4
LP
UP
Porcine-Folch
Ovine-Folch
UP
UP
5
A
13.36
22
19.4
64
7
1
44
28.8
50
13
9
80
1
10
'
18.7
1
0
0
0
0
0
4
18.40
100
0
0
0
23.40
100
0
0
0
Dog-Folch
UP
4
Cat
LP
44
33.2
41
16
8
2
15
22.7
60
6
0
0
Folch
Porcine
Ovine
Dog
Folch
Folch
F o l c h 'LP
LP
LP
4
10.00
100
0
0
0
4
38.40
25
3
0
0
-
38
-
0 2 5 3 0 2 2
#5
C18
UUMFOUND
Cat,
Lipid
Porcine,
Cat
C18
Porcine,
C18
Lipid
trt
Cat,
Lipid
Cat,
C18
C18
35.05
50
1
25.8
64
5
8
15.88
75
1 1
o
5
18.72
80
1 0
0
10
38.70
20
8
0
0
4 6 ..7700
18
9
0
0
M
1
1
0
0
DP,
nonlipid
C18
w
14
UP b a s e
P o r c i n e , C18
Cat,
Lipid
A
»M^r.
4
nonlipid
base
Porcine,
COLUMN ELUATES
EGGS
C18
AMED 2 0 6
12/17/85
trt
MeOH s o l
nonlipid
nonlipid
nonlipid
11
MeOH
11
4 0 ..77
18
9
0
0
E^O
12
27.8
42
7
0
0
10
40.1
10
8
1
(sol.)
-
39
-
0
0 2 5 3 0 2 2
#5
AMED 2 0 6
12/17/85
DEAE COLUMN
COMPOUND
Cat,
Cat
Total
Monosialogang.
disialogang.
Porcine,
Cat,
Gangs.
monosialgang.
Porcine,
Cat,
EGGS
disialogang.
trisialogang.
Porcine,
trisialogang,
Cat, N e u t r a l
Forssman
Porcine,
gang.
Neutral
Gang,
A
%NEG
W
M
S
12
27.8
50
3
3
0
18
34.1
39
10
1
0
29
23.2
62
11
2
2
15
15.1
100
0
0
0
28
25.8
57
10
2
0
17
21.6
82
3
0
0
35
30.2
49
12
0
0
49
29.0
49
25
0
0
14
33.4
50
0
0
0
Cat, N o n - l i p i d ,
neutral
DEAE
15
23.6
67
5
0
0
Cat, N o n - l i p i d ,
H2Osol.
neutral,
14
24.3
57
6
0
0
14
33.8
36
8
1
Cat. N o n l i p i d ,
MeOH s o l .
neut.
-
40
-
0
0 2 5 3 0 2 2
#5
AMED 2 0 6
12/17/85
DEAE -COLUMN CONT'D
COMPOUND
C a t , Mono,
gang.
EGGS
%NEG
23
38.9
13
Porcine,
Mono,di,
tri-sialo
26
21.8
65
Cat,
9
34.1
P o r c i n e , mono ,
disialogang.
18
Cat,
mono,
mono,
di,tri-sial
A
di-sialogang
trisialogang.
Porcine,
mono,
trisialogang.
Cat,
di,
Porcine,
trisialogang.
di, t r i s i a l o g a n g .
Cat, m o n o s i a l o g a n g l .
Forssman
W
M
19
1
0
33
6
0
0
28.6
44
10
0
0
16
37.6
19
1.3
0
0
26
27.0
50
13
0
0
8
30.9
37
3
2
0
27
27.0
56
12
0
0
16
20.9
87
2
0
-
41
-
0 2 5 3 0 2 2
#5
UNISIL
COMPOUND
CAT,
LP,
Porcine
CAT,
LP,
Porcine
Cat,
LP,
Porcine,
COLUMN
EGGS
A
%NEG
W
M
S
22
30.0
36
13
1
0
9
34.1
22
7
0
0
39
39
35.1
23
29
1
0
12
12
26.7
58
5
0
0
Methanol
26
33.1
23
20
0
0
LP,
H
34.0
27
8
0
0
W
M
S_
CHC13
LP,
CHC1.
A c e t o n e :MeOH
LP,
A c e t o n e :MeOH
Methanol
IATROBEADS
FRACTION
AMED 2 0 6
12/17/85
NO
(CHROMATOGRAPHY)
EGGS .
A
%NEG
I
4
45
0
3
1
0
II
4
15
75
1
0
0
III
4
41.75
25
2
1
0
IV
4
43.4
0
4
0
0
V
4
33.35
25
2
1
0
-
42
-
0 2 5 3 0 2 2
#5
AMED 2 0 6
12/17/85
GEL PERMEATION
FRACTION
NO
EGGS
A
%NEG
W
M
S
0
I
5
32.08
40
4
0
II
7
24.77
43
3
1
III
6
33.40
33
2
2
IV
4
29.44
80
1 0
V
5
25.36
60
2
0
0
VI
5
29.40
40
3
0
0
VII
5
22.72
60
2
0
0
VIII
6
32.27
33
3
1
IX
3
17.87
100
0
0
X
3
33.40
33
1
XI
4
40.05
25
1 2
AFFINITY
FRACTION
Heparin-binding
Folch
UP
Heparin-binding
PBS
1
14
29.1
1)
%NEG
W
M
50
6
1
3
1
1
1
from
(cat)
Gelatin-binding
Folch
UP
46.8
from
(cat)
31.7
-
43
50
-
0
0
homogenate
(Fig.
0
0
from
(cat)
0
0
CHROMATOGRAPHY
EGGS
0
0 2 5 3 0 2 2
#5
Table
that
in
IV,
which
follows,
Tables
I-III.
The
materials.
ganglioside
obtained
from
into
separated
were
also
were
performed
gangliosides
This
"DU"
and
Similar
analyses
glycosides.
for
analysis
known,
(entries
new
1-4
of
this
of
compositions
to
determine
compounds
of
a class
These
of
numbers,
and
w2
the
"DU"
for
the
the
materials,
and
and
I,
sJf
of
SJf
XlT,
X2T;
-
and
class
44
-
A Index
Means
and
s
s^
each
the
value,
negative
percent
distribution
V^
a value
Value."
the
values
were
components,
represent
presents
as
II.
all
are
ganglioside
omenta-derived
however,
to
ganglioside
ratios.
presents
well
as
centroids
this,
also
order
the
class
table
In
Sj
1:1:1
similar
.
Discriminator
taken
The
gangliosides,
5-8,
is
group
tri-sialyated
or
group
or
is
and
porcine
available
Entries
.
first
data
however,
extracts.
1:1,
with
"Supelco"
commercially
group)
in
mixed,
The
di
mono,
samples,
The
omental
cat
contains
AMED 2 0 6
12/17/85
of
S
S^
values
s
and
values
of
a
determine
compounds.
"weight
value
Using
coefficients"
0 2 5 3 0 2 2
#5
are
determined
via
Wl = S I I
-
SI
X1T =
(SS11
W2 = S I I
-
Si
X2T =
(SH
s
The
+ S
(wI-+w2*-)^s
1
T = Xlmn
n
{
,,.
+w2~)Js
i»x1 -w2
and
smaller
properties
the
of
from
compound,
DU v a l u e ,
the
best
sample.
to
Major
Ccnponent(s)
Catponent(s)
+ Si>/2
= Si> / 2
2
2 h
-,
(WI +W2 }
+ X2T
2 . - 2 ^
Tw1-+w2*-)35
the
greater
A ranking
worst
is
the
of
presented
TABLE
Cai1Pound
AMED 2 0 6
12/17/85
angiogenic
DU v a l u e s
in
by
Table
V.
IV
Eggs
DU
A
%
Neg
Neg
W
M
S
Cat Ctnentum
Acidic
DEAE gangl
12
39.17
39>17
27.8
^^
50%
5Q%
3
3
0
Monoslalogangl.
QQ
OG
18
i8
26.77
34.1
39%
39%
10
1
0
Disialogangl.
GD3
0,3
15
94.59
15.1
100%
100%
0
0
0
17
75.63
75>63
21.6
n>6
82%
82%
3
0
0
23
0.54
Q54
38.9
38>9
13%
u%
19
1
0
Trisialogangl
Mono, Di,
Tri,
MDnO' °i
(M^)
9
21.06
34.1
33%
33%
6
0
0
MOn°' f c l
( ^
16
6.66
6-66
37.5
14%
13
0
0
(Mix)
8g
25<84
25.84
30>9
30.9
3?%
37%
3
2
0
49
37>85
37.85
29>0
29.0
4g%
49%
25
0
0
16
80#67
80.67
2Q9
20.9
g?%
87%
2
0
0
8
23.56
38.4
37%
37%
2
3
0
14
38.77
2g>1
29.1
5Q%
50%
6
1
0
Di,
Tri
Neutral
(Mix)
gangl Forssman
^
Mono, Forssman
(Mix)
•BralnGMl
G"!
GM1
Brains
q^ q
-
45
-
0 2 5 3 0 2 2
#5
AMED 20 6
12/17/85
TABLE IV OONT'D
Major
Conponent(s)
Corrpound
Supelco
(Brain)
Purified
Mix G a n g l .
Monosialogangl.
GM1
Disialogangl.
GDla
Trisialogangl.
GTlb + GDlb
Mono/Di
(Mix)
Mono/Tri
Di/Tri
(Mix)
(Mix)
Mono/Di/Tri
Porcine
(Mix)
DU
Eggs
%
Neg
W
M
S
12
21.45
32.8
33%
7
1
0
12
47.28
26.2
58%
5
0
0
9
21.51
32.6
33%
6
0
0
18
33.67
27.1
44%
10
0
0
6
19.90
37.9
33%
4
0
0
5
93.27
21.4
100%
0
0
0
6
4.36
38.9
17%
5
0
0
6
10.56
41.2
17%
4
1
0
Ctnentum
Monosialogangl
29
52.0
23.2
62%
11
2
2
Disialogangl
28
46.44
25.8
57%
10
2
0
Trisialogangl
25
40.74
28.9
52%
12
0
0
26
55.28
21.8
65%
9
0
0
18
33.21
28.6
44%
10
0
0
26
34.41
27.0
50%
13
0
0
27
45.13
27.0
56%
12
0
0
Mono, Di,
Tri
Mono, Di
(Mix)
Mono, Tri
Di,
Tri
(Mix)
(Mix)
(Mix)
-
46
-
0 2 5 3 0 2 2
#5
AMED 2 0 6
12/17/85
TABLE V
GANGLIOSIDES
ANGIOGENIC POTENCY
RANK
DU
1
-10.56
2
COMPOUND
Supelco,
Mono/Di/Tri
Mixture
0.54
Cat
Mono/Di/Tri
Mixture
3
4.36
Supelco,
Di/Tri
4
6.66
Cat
Mono/Tri
5
19.90
Supelco,
Mono/Di
Mixture
6
21.06
Cat
Mono/Di
Mixture
7
21.45
Supelco
Purified
8
21.51
Supelco
Disialo
9
23.56
GM1
10
25.84
Cat
Om.,
Di/Tri
11
26.77
Cat
Om.,
Monosialo
12
33.21
Porcine
Om.
13
33.67
Supelco
Trisialo
14
37.85
Cat
15
38.77
GM3
16
39.17
Cat
17
40.74
Porcine
Om. , t r i s i a l o g a n g l
18
45.13
Porcine
Om. , D i / T r i
19
46.44
Porcine
Om,
Om.,
Om.,
Om.,
Om.,
Mixture
mixed
gangliosides
Mixture
(GM3)
Mono/Di
Neutral
Om. , A c i d i c
-
Mixture
Mixture
Gangl.
DEAF
gangl
mixture
Disialogangl
47
-
Forssman
0 2 5 3 0 2 2
AMED 2 0 6
12/17/85
#5
TABLE V CONT'D
GANGLIOSIDES
ANGIOGENIC
POTENCY
RANK
DU
COMPOUND
20
47.28
Supelco
monosialogangl.
21
52.00
Porcine
Om. , d i s i a l o g a n g l
22
55.28
Porcine
Om.,
23
75.63
Cat
Om. , T r i s i a l o g a n g l .
24
80.67
Cat
Om./Mono/Forrsman
25
93.27
Supelco
26
94.59
Cat
Om.,
show
that,
These
possess
results
angiogenic
additional
outlined
in
Figure
properties.
CMFr
of
the
sides
of
(the
first
tests
negative
sides
-
a drop,
comparison,
from
Cat
a vis
The
a mix
omentum
spite
of
of
the
of
and
di,
values
of
negative.
-
48
-
but
Cat
46.34%
monosialoganglioshow
contrast,
show
III,
28.98,
contrast,
In
the
process
Table
23.6
purification.
mono,
shows
CAM a s s a y ,
to
purer,
in
does
Angiogenic
A value
an
DEAE c o l u m n s ,
in
the
reference
by
CMFr
following
39% n e g a t i v e .
from
the
greater
DEAE c o l u m n ,
only
also
vis
negative.
on
Mixture
Mixture
while
possess
has
mixture
Disialogangl
obtained
example,
were
with
fractions,
II,
entry)
obtained
34.1,
this
For
Mono/Tri
activity
fractionates
Mono/Di/Tri
tri
38.9
an
A value
non-lipid
and
67%
Finally,
for
sialoganglioand
only
13%
0 2 5 3 0 2 2
#5
Additional
in
Table
is
a useful
it
takes
III.
effective
it
The
showing
account
not
only
The
lower
the
material
be
(Supelco
the. f r a c t i o n
seen
by
results
categorization
to
as
platted
the
against
point
platted,
and
every
two
index
to
Figure
all
the
which
III
groups
are
to
left
composition.
is
T2
are
these
against
shows
Figure
16
group
obtained
shows
the
-
or
of
materials
49
an
best
-
so
a comparison
effective
as
linear
A "centroid"
more
in
In
is
then
than
of
an
others.
for
T values,
Figures
7-16
Anything
be
linear
are
is
T value
T values.
promise
Table
will
Index
from
guidelines
the
to
as
Figures
This'
Subsequently,
plotted
These
negative.
compounds.
more
and
graphically,
Angiogenic
establishes
of
shown
each
the
compositions.
herein,
for
as
known
di-,
applied
percent
V,
tri-sialogangliosides),
substances.
compositions
tested.
of
effective
6-16..
and
referring
various
T value
of
be
by
mono-,
most
IV
the
for
obtained
groups
samples
the
is
the
but
DU v a l u e ,
and
data
effectiveness,
mixture
feline
Figures
graphs)
categorization,
"mean"
di-
also
may
referring
the
the
Tables
A value,
novel
containing
are
in
Hence,
mono-,
tri-sialogangliosides,
These
the
from
actual
the
tested.
that
seen
drawn
displayed
for
gangliosides
and
DU v a l u e ,
negative.
can
be
can
shorthand
into
percentage
VII,
comparisons
AMED 20 6
12/17/85
using
different
plotting
angiogenic
compositions.
0 2 5 3 0 2 2
#5
17
Figure
index
plotted
standard,
is
included
against
mixtures
The
trisialogangliosides.
mixture
This
is
diis
graph
is
complexing
reactions
taking
unlike
This
of
the
di-
and
in
and
the
that
a 1:2
best
ratio.
obtained
curves
when
are
antigen-antibody
that
suggests
Angiogenic
percentage
demonstrates
obtained,
precipitant
following
a complexing
agglutination
antigen-antibody
III-VII,
the
Discrimination
mixtures
One
assay).
be
the
in
vivo
in
reported
19 84.
20,
August
are
show
experiments
has
supra,
experiments
with
known
graph
those
characteristic
described
would
of
the
type
is
systems
place.
The
and
to
plotted.
not
negative
because
interesting
of
a graph
tri-sialogangliosides,
similar
strinkingly
reaction
and
inverted
an
novel
using
show
to
AMED 2 0 6
12/17/85
As w i l l
CMFr
has
Unit
tested
in
skilled
expected
additional
Serial
be
in
that
by
similar
will
(i.e.,
see,
experiments
with
fractions,
-
50
-
a higher
fractions
the
CAM
therefore,
may
expected
results.
and
additional
fashion
art
these
the
be
The
Tables
to
Index,
.
filed
642,624,
reference
Angiogenic
do
CMFr,
angiogenesis
Number
seen,
than
the
the
in
efficacy
a lower
value
that
that
repeated
superior
it
0 2 5 3 0 2 2
#5
Other
from
primarily
supplied
the
to
induce
presented
in
available
Supelco
individual
by
ability
are
commercially
and
lipid
Sigma
investigators
in
angiogenesis
Table
compounds
Chemical
were
the
AMED 2 0 6
12/17/85
CAM.
purchased
companies,
tested
These
for
or
the
results
VI.
TABLE V I
GLYCOLI-PIDS
COMPOUND
AND GANGLIOSIDES
EGGS
%NEG
W
M
£
Cerebrosides
(Supelco)
Gangliosides
(Fidia
16
36.3
38
9
1
18
38.6
17
14
1
0
15
34.3
33
8
2
0
17
34.6
29
10
2
0
16
43.0
19
11
2
0
17
32.2
41
8
2
0
14
37.2
29
10
0
0
4
27.6
43
4
0
0
20
27.0
60
7
1
0
.
Cronassial
20)
Gangliosides
(Supelco)
Globoside
Steryl
(Supelco)
glucoside
(Supelco)
Ceramides
(Supelco)
Digalactosyl-diglyceride
(Supelco)
Monogalactosyl
diglyceride
Ceramide
(Supelco)
(Supelco)
galactoside
-
51
-
0
0 2 5 3 0 2 2
#5
Purified
AMED 2 0 6
12/17/85
mixed
gangliosides
{Supelco)
COMPOUND
Ceramides,
Type
Type
;
EGGS
A
%NEG
18
31.9
44
10
0
,
19
30.2
53
8
1
0
16
30.5
37
10
0
0
13
30.3
38
8
0
0
10
W
1
0
M
$3
0
IV-
(Sigma)
Cerabrosides,
42
I
Type
(Sigma)
Ceramides,
28.8
III
(Sigma)
Cerebrosides,
19
Type
II
(Sigma)
Sulfatides
(Sigma)
7
28.7
57
3
0
0
Sulfatides
(Supelco)
6
30.0
50
3
0
0
7
26.7
43
4
0
0
38.4
25
3
0
5
1
Glucocerebrosides
Ceramide
(Sigma)
trihexoside
(Supelco)
Steryl
glucoside
(Supelco)
46.8
-
52
-
0 2 5 3 0 2 2
#5
AMED 2 0 6
12/17/85
GANGLIOSIDES
COMPOUND
EGGS
A
%NEG
W
M
S
8
38.4
37
2
3
0
GM1 p u r i f i e d
15
32.5
27
11
0
0
GM3
14
29.1
50
6
1
0
17
29.1
47
8
1
0
18
36.7
33
11
1
GM1
GM3 P u r i f i e d
Made-up
-
mixture
GM1:GM3
(1:1)
purified
*
0
PHOSPHOLIPIDS
COMPOUND
EGGS
A
%NEG
W
M
10
28.7
60
4
0
8
20.9
100
0
0
10
33.4
40
6
0
6
29.0
67
2
0
0
3
31.1
33
2
0
0
Phosphatidylinositol
(Sigma)
Sphingomyelin
(brain)
(Sigma)
Phosphatidylcholine
(Sigma)
P h o s p h a t i d y l i n o s i to 1
(Supelco)
Phosphoinositides
•
(Sigma)
-
53
-
0 2 5 3 0 2 2
#5
PHOSPHOLIPIDS
COMPOUND
(CONT'D.)
EGGS
Phosphatidylinositol
diphosphate
%NEG
W
M
4,5
(Sigma)
7
31.5
43
4
0
(Sigma)
6
28.9
50
3
0
0
(egg . y o l k )
6
22.2
83
1
0
0
W
M
inositol
Phosphatidyl
-4-monophosphate
Sphingomyelin
Lysophosphatidyl
stearoyl
AMED 2 0 6
12/17/85
choline
(Sigma)
100%
NEUTRAL
death
rate
LIPIDS
NEUTRO L I P I D S
COMPOUND
EGGS
%NEG
Mono , Di , a n d
Tristearin
(1:1:1)
(Sigma)
Mono,
(Sigma)
Tristearin
Triolein
(Sigma)
(Sigma)
Monostearin
(Sigma)
Monoolein
Distearin
67
0
100
0
0
0
36.5
43
8
3
1
4
45
25
0
3
0
4
50.0
0
2
2
0
4
50.0
0
2
2
0
3
26.7
67
0
1
Triolein
Di,
(1:1:1)
20
(Sigma)
2
21
-
54
-
0
0 2 5 3 0 2 2
#5
NEUTRAL
LIPIDS
NEUTRO L I P I D S
COMPOUND
Diolein
EGGS
AMED 20 6
12/17/85
(CONT'D.)
A
%NEG
W
M
S
'.
3
13.3
100
0
0
0
Tripalmitin
(Sigma)
4
5
100
0
0
0
Cholesterol
palmitate
3
0
100
0
0
0
(Sigma)
(Sigma)
Triarachidin
(Sigma)
4
41.7
0
4
0
0
Paraffin
(Fisher)
4
40
0
4
0
0
W
M
S
3
0
oil
STEROIDS
COMPOUND
Ergos'terol
%NEG
14
37.7
36
4
26.7
50
2
0
0
8
28.4
38
5
0
0
6
37.9
33
4
0
0
(Supelco)
(Supelco)
Stigmasterol
(Supelco)
One
skilled
tissues,
in
the
characterized
ganglioside
fashion
A
(Supelco)
Desmosterol
Lanosterol
EGGS
containing
art
by
will
the
see
presence
molecules,
may
to
obtain
potentially
active
containing
and/or
ganglioside
-
tissues,
such
as
the
liver,
of
be
55
additional
analyzed
in
or
this
lipid
mammalian
epithelial
-
and
lipid
fractions
containing
brain,
-
that
'6
tissue,
and
so
0 2 5 3 0 2 2
#5
forth,
are
well
as
known
active
as
bio-
as
good
or
produced
omental
be
used
A set
of
experiments
tissue
was
anesthetized
using
position
and
inguinal
crease
the
first
incision
deep
intravenous
attaches
cells
in
the
radionuclide
In
this
radioactively
made
removed
femoral
branch
and
and
or
each
of
cat
blood
red
Ketamine
was
placed
the
knee
in
and
femoifal
between
were
a supine
the
arteries
the
groin
canal).
The
subjected
and
immediately
to
chloride /Technitium-9 9
labels
and
using
and
visualized.
56
the
quantity
a Gamma
capillaries
cells
-
in
of
location
blood
lipid
injection
The
stannous
vessels
the
cats
radioactively
The
demonstrate
female
(Hunter's
be . i d e n t i f i e d
tagged
ovine
vascularization
between
and
can
Other
Adult
cat
ligated,
tissue.
matter,
Each
legs.
to
found.
bovine,
to
normal
hind
injection
which
be
also.
non-aqueous
intramuscular
both
closed
was
the
the
7 ml/Kg.
of
may
porcine,
destroyed.
incision
an
isolated,
were
where
an
of
a dosage
used
performed
of
purposely
with
be
as
was
effects
a site
at
preparation
lecithins
may
such
angiogenically
well.
as
neovascularization
the
lipids
sources
and
lecithins,
Plants
seeds.
especially
synthesized
can
equine
of
sources
organo
Synthetically
mammalian
tissues,
plant
AMED 2 0 6
12/17/85
are
red
of
camera
blood
this
scan.
carrying
specifically
the
an
0 2 5 3 0 2 2
#5
The
10
stannous
mg of
sodium
chloride/phosphate
arid
trimetaphosphate,
preparation
1.0
ml
0.95
of
this
solution
bronchial
vein
of
dose
Nuclear
area.
blood
observed
After
the
cats
ml
scan
sites
of
viscous
the
amounts
right
leg
in
into
the
A placebo
injection
similarly
identified
body
normal
to
this
collateral
forming
the
new
femoral
digital
by
kind
blood
of
circulation
capillaries
artery
the
was
and
was
that
tissue
of
PBS
the
marked
to
be
the
blood
severed.
-
the
on
to
vessels
57
was
in
and
removed.
two
of
the
establish
tissues
in
the
leg.
response
try
on
into
left
following
-
evaporated
made
injured
By
between
sites
artery
recognized
will
of
and
PBS was
sites
the
intramuscularly
and
only
a
of
injection
femoral
marked
surgery
an
prepared,
extracted
in
containing
circumstances,
the
integration
radioactivity
preselected
and
in
and
into
later,
and
"followed
where
area
PBS
Technisium-99m
surgically
suspended
two
of
intravenously
cells
background
lipid
ml
This
followed.
been
made,
2.0
blood
chloroform/methanol
liquid
equal
was
and
had
for
red
chloride.
minutes
of
scanning
was
surgical
Under
the
imaging
sodium
adding
Twenty
10m C u r r i e s
flow
"base-line"
6-7
of
mg of
injected
cat.
radiolabel
to
injected
the
was
by
contained
preparation
mg s t a n n o u s
reconstituted
was
intravenous
30
pyrophosphate,
AMED 2 0 6
12/17/85
the
and
by
area
where
comparing
0 2 5 3 0 2 2
#5
the
and
rate
the
following
the
of
degree
a direct
surgery,
angiogenic
chloride/Technitium-99m
preselected
sites
on
at
three,
scan
The
operation.
18-20
for
The
shows
in
the
lipid
seventy-two
of
and
hours
was
increase
over
18-20
differences
and
of
these
each
leg
of
assessment
the
preparation
in
was
that
provide
in
new
in
left
at
in
the
29.6%
six
days
visual
blood
evidence
vessel
of
nine
showed
-
58
-
in
days
a
time,
the
right
the
65.8%
photographs
the
formation
in
operative
post
The
leg.
At
leg.
difference
after
leg
omental
integrated
observed
was
cat.
formation
the
higher
in
lipid
vessel
with
a
right
left
shown
are
the
(control)
and
left;
the
scans
blood
to
the
a representative
in
surgery,
the
subjected
after
of
(injected
the
into
was
days
substantially
the
leg
effects
in
stannous
made
nuclear
increase
cat
the
was
each
radioactivity
to
of
nine
the
observed;
neovascularization
Figs.
six,
then
post
comparison
increase
and
leg
this
counts
radioactivity
of
each
the
preparation)
in
of
injection
exemplify
that
leg
radioactivity
leg
lipid
neovascularization
right
a 38.2%
in
verifiable
preparation
results
which
fraction
data
and
potency
extracted
intravenous
Subsequent
Figs.
circulation
made.
accurately
nuclear
and
properties
c h l o r o form/me t h a n o l
blood
new
AMED 2 0 6
12/17/85
of
substantial
using
the
rate
0 2 5 3 0 2 2
#5
chloroforra/methanol
a linear
illustrating
the
comparing
lipid
vascularization
21.
Fig.
of
extracted
The
of
the
data
reveals
fraction
blood
vessel
in
the
given.
Gamma
scan
which
of
of
a -common
to
this
camera
scans
operatively
right
and
23
shows
post
surgery
same
day.
an
and
The
the
no
made
left
is
leg
in
in
is
new
after
anterior
view
one
24
shows
scan
indicated
of
the
no
-
cat
three
visible
Fig.
new
and
the
using
be
in
effect
by
of
blood
on
posterior
-
in
was
day
22-24.
The
22
Fig.
in
vessel
three
leg
kind
any
six
Figs.
shown
surgically
described
as
and
difference
59
was
arteries
shown'
are
in
overlooks
by
injection
at
difference
circulation
Fig.
another
femoral
instance,
lipid
however,
to
left.
experiment.
control,
the
the
increase
shown
was
control
the
effect
systemic
in
increase
to
organization
data
the
hour
per
of
counts)
provided
compared
substantial
which
remove
0.25%
effect
This
discernable
collateral
the
by
A graph
is
leg
leg
leg.
post
no
right
angiogenic
right
in
the
of
the
upon
intervals
the
additional
But,
to
injected
the
additional
above.
vascularization
vascular
following
operated
(in
a rate
preparation
this
radioactivity
and
extract
In
of
formation
possibility
lipid
in
evidenced
as
structure
the
shows
clearly
fraction.
leg
saline
neovascularization
This
lipid
increase
injected
AMED 2 0 6
12/17/85
days
the
view
in
duration.
sixth
on
counts
day
the
0 2 5 3 0 2 2
AMED 2 0 6
12/17/85
#5
between
the
lesser
two
earlier
left
less
the
In
fact
the
that
cat
of
degree
post
in
leg
in
the
fact,
this
in
(control)
time
any
neovascularization
experiment.
noticeably
of
of
degree
at
legs
in
work.
In
the
previous
experiment
(injected
control)
exhibited
(although
than
in
the
right
leg)
, there
is
a basis
part
of
the
lipid
preparation
in
the
transferred
to
systemically
the
to
comparison
than
experiment
earlier
neovascularization
a much
the
neovascularization
additional
the
and
surgery
that
in
of
the
the
this
left
and
leg
a relatively
higher
substantially
lesser
for
right
left
view
was
leg
in
leg
that
believing
was
the
probably
earlier
experiments .
with
The
in
the
different
vivo
hexane/ethanol
fractions
and
III-VII,
these
CMFr may
extracts
angiogenesis.
from
angiogenic
Tissues
similar
to
the
in
not
such
will
in
The
-
that
rapid,
and
possess
have
been
lipid
shown
been
in
compositions,
previously
-
Tables
more
molecules
have
60
in
these
conclude
even
which
as
repeated
between
data
containing
a way
omentum,
the
result-
amounts,
be
following
art
tissues.
effective
activity
from
in
lipid
mammalian
therapeutically
made
would
may
a comparison
Compositions
active
angiogenically
obtained
be
CMFr,
obtained
As
skilled
one
supra,
using
materials
extraction.
purified
better
experiments,
to
expected.
containing
affect
0 2 5 3 0 2 2
#5
mammaliam
tissue
plant
angiogenically
active
based
structures
upon
the
angiogenically
Other
/
and
as
active,
organic
tissue,
molecules
are
solvents
of
may
as
the
well.
be
well.
used
to
)
-
61
shown
as
well.
-
to
expected
Synthetic
molecules
foreseen
can
be
AMED 2 0 6
12/17/85
extract
to
have
lipids,
be
omentum
0 2 5 3 0 2 2
#5
It
is
the
note
of
use
October
omentum
in
co-pending
October
31,
1985
1985
and
hereby
31,
fractions.
and
We
for
extraction
gas
our
other
using
angiogenic
supercritical
filed
incorporated
active
described
as
S.N.
extract
obtain
to
factors
omentum
to
possible
solvents
organic
also
also
AMED 2 0 6
12/17/85
application
hereby
incorporated
by
reference.
Here
a supercritical
omentum.
An SCF
above
temperatures
in
the
non-polar
example,
3500
materialsof
Thus
from
these
and
is
also
omentum.
membranes,
Lipids
or
amphipathic
other
are
use
detergent
"soluble"
in
aqueous
recovered
by
flotation
from
involving
molecules
media.
after
which
The
with
and
to
released
62
-
the
lipid
or
use
materials.
homogenized
render
are
toxic
isolate
proteins,
more
For
extraction
centrifugation.
-
the
pressures
avoid
detergents
displaced
complexes
gangliosides
extractions
time-consuming
to
can
inefficient
or
critical
triglycerides.
and
extract
at
contains
3 8 - 3 9 °C
are
conditions
possible
as
to
and
(Pc)
extract
as
used
power
such
such
toxic
extraction,
expensive
It
used
temperatures
psig.
the
materials
lipid
is
pressure
materials
while
CO-
solvation
critical
Polar
residue
or
(SCF)
increased
the
(Tc) .
temperature
remain
has
fluid
lipids
cell
by
proteins
material
is
0 2 5 3 0 2 2
#5
of
A list
B below/
to
2.0%
possible
These
and
(w/v)
Cryogenic
subdivision
use
of
the
the
subject
are
of
in
from
7.0
to
also
can
omentum
liquid
or
our
in
other
be
nitrogen
such
with
techniques.
filed
63
Tables
ranging
used
application
-
in
A and
from
0.1
8.0.
extraction
copending
given
concentrations
techniques
above
of
used
a pH
is
detergents
AMED 2 0 6
12/17/85
-
S.N.
as
subsequent
This
is
0 2 5 3 0 2 2
#5
AMED 2 0 6
12/17/85
TAIIX A
DETERGENTS WITH FLEXOLE HYDROPHOBE REGIONS
D«ttri«at xrv
Stnminl fonwk
Fonrul (ind trivial)
Stm*
Sodium dodtcyUulphaii
VWWVo-j-O'Nr
Crtyltrimnhyhnaioefaa
kraaida
VVWVVvVr-o<, tr
OS
)
"W«Jdy"
task
ZvtttcdoGftc
»»
Sodium <od«eyUV.urtoin»» .
(tukmtf)
W W W /
Solfotetakt
(2»Utm»nt)*
ftlniwyOriolKhUB
VV/WVWr-CH^CHj-CHj-I-O"
o
en,
-oh •
J
W 1 / s A / W * c - o - c h , - c m 1 - c h , - o f-O-CH.-CH.-ir-CH,
0
0CM,
tHawthykOcrlvniM axiin
(A»«oay» LO)»
W W V W W
CM,
P»lyo>yt<i>ytaM aleobel
(Irlsrin.
Utuotw.AL.PBrics) PetyexrtthyUn* aotiytphnoi
(Trito* K icriu t|*ptl CO Mrict
Sanbaic N»ritt Emulfta »nei)
VVW^-o-lCM1-CHa-o^
)
fetymcytthyhnt prorcyl phenol
(TrfeoaXMrin
Iftpd CA Khu
N«ud*t P40)
ftpon
aA«vliblt from Calbwditm-lchrini. Scrva.
»AnU»bta from Onyx Cfcmidl Co, IM w m Snt, Jtmy Cby NJ 07032
U3^.
-
64
-
J »
0 2 5 3 0 2 2
#5
AMED 2 0 6
12/17/85
TABU B
DETERGENTS WITH RIGID HYDROfHOBIC REGIONS
D«uxjtat tjy*
Formal (t
Sincntnl tomah
SedJus
chohii
SueeftriMic
fc«OM
-NH-CHt-CHj-l-OUT
I
O
•Vmttr'
SodtaM
ttvocboka
HO*
-«r
CHAM"
Zvixurkssic
J
v
U
*
tto»4oek
DiihaKbi
)
*H(^<i«>I>"udopfopyn tfintthylimineaio]-l'fTopu(iulphoutt.aviiliblt freraCiIbioch«»-»«hrin»,
ttTTX.
-
65
-
0 2 5 3 0 2 2
#5
In
any
of
with
the
practice,
the
standard
and
amount,
or
like.
the
intravenously,
known
ways
carriers,
appropriate
solvents
compositions
to
These
will
of
the
methods
of
substances,
include
and
in
pharmacology
inert
orally,
course
administered
art
combinations,
intramuscularly,
dose,
be
can
AMED 2 0 6
12/17/85
intradermally,
The
topically.
vary
from
patient
which
have
been
to
patient.
The
used
as
there
terms
terms
is
no
that
the
and
of
expressions
description
intention
of
expressions
shown
and
not
of
the
use
excluding
any
equivalents
or
portions
modifications
test
the
are
possibility
and
promote
healing
infarct
(MI) , t h e
Twenty- four
produced
the
left
angiogenic
solution
the
terms
of
the
it
being
within
possible
and
features
recognized
the
of
scope
invention.
To
of
such
are
and
limitation,
thereof,
EXAMPLE
was
of
in
described
various
and
employed
MI and
by
adult
anterior
(n=12)
of
occlusion
feline
continuing
injected
for
(n=12)
or
-
of
66
-
ligation
days.
MI
(n=21)
The
a control
starting
10
acute
artery.
or
IM d a i l y ,
a total
done.
experimental
(n=3)
can
myocardial
were
coronary
CMFr
extract
a recent
whom
on
descending
was
angiogenic
experiments
dogs
balloon
extract
that
angiogenesis
following
II
on
saline
day
1 of
0 2 5 3 0 2 2
AMED 20 6
12/17/85
#5
After
was
4-5
exposed
by
by
the
pericardium
respiratory
Jones)
was
branch.
pumps.
was
placed
descending
chest
exteriorized.
The
the
1 g/day
to
hours
saline
induce
the
In
induced
by
descending
balloon
and
first
all
acute
were
tablets
On t h e
of
morning
extract
Two
controls
and
of
or
three
to
inflated
to
animals
were
the
on
-
67
clamp
the
-
artery
was
proximal
and
was
distal
Harris
two-stage
The
the
was
anterior
the
.
at
placed
from
left
to
(1950)]
A small
minutes
infarction
myocardial
according
1^1318
vessel.
ten
by
fully
was
placed
artery
ligatures
the
diagonal
convalescence
angiogenic
both
anterior
extract.
dogs,
and
in
the
(R.E.
catheters
intramuscularly.
infarction
21
of
room
after
dogs,
2 weeks
allowed
occluder
ligatures
for
closed
injected
coronary
hanging
the
to
with
occluder
portion
CMFr
dissected
left
mm b a l l o o n
feline
next
of
3-5
2 cc
[Circulation
end
a
depletion.
of
IV
three
sodium
technique
distal
first
distal
heart
ventilated
was
dog
the
were
angiogenic
silk
with
prevent
the
the
anesthesia
supplemented
myocardial
receiving
dogs
were
later,
was
diet
experiment,
normal
The
In
then
was
a normal
receiving
conditions:
a dissected
artery
coronary
procedure
the
opened,
along
The
NaCl
and
75 m g / k g ,
air
following
after
thoracotomy
pentobarbital
while
sterile
under
performed
the
acclimatization,
days
attached
end
to
and
narrow
0 2 5 3 0 2 2
15
the
vessel
the
ligatures
were
tied
The
chest
then
closed
without
was
exteriorized.
In
2-week
period,
waiting
it
occluding
to
these
the
vessel
above
and
all
animals
there
that
so
After
totally.
occlude
as
the
AMED 2 0 6
12/17/85
was
acute
10
minutes
completely.
catheters
were
need
a
no
for
experiment
began
immediately.
The
extract
or
salne
intramuscular
injection
end
day
of
the
10
of
injection
pathologic
sections
and
1 cm t h i c k .
to
record
at
35CC
nitro-blue
All
areas.
in
fresh
the.
a phosphate
tetrazolium
slices
buffered
stain
in
undamaged
areas
of
ischemic
injury.
These
fixed
buffered
formalin
and
then
The
processing.
projected
• analyzer,
traced
in
onto
where
and
a percentage
the
of
of
the
'infarcted
of
total
the
then
were
for
ventricular
68
-
muscle
the
rephotographed
further
was
for
blue
outlining
a Zeiss
tissue
infarcted
intense
an
histological
were
MOP I I
non-infarcted
infarcted
and
of
macrophotographs
of
for
incubated
clearly
slices
vs.
-
of
produced
an
examined
solution
heart,
tablet
magnetic
volume
the
the
color
developed
the
were
with
the
transverse
were
appearance
(NBT) , w h i c h
parts
into
Slices
At
removed
were
cut
by
days.
sacrificed
were
were
daily
10
next
hearts
Hearts
approximately
given
the
dogs
and
pentobarbital,
was
for
cc)
the
period,
non-infarcted
15 min
(2
examination.
photographed
injection
areas
image
were
calculated
volume.
as
0 2 5 3 0 2 2
#5
In
with
those
ventricular
sections
(P)
were
signs
of
areas
with
injury,
formation
of
the
(LB,
zones
and
myocardium
SB)
to
permit
criteria
(A)
the
penetration
of
the
and
lateral
microscopic
cellular
cells,
other
of
of
eosin-stained
NBT-stained
angiogenesis,
living
and
from
more
and
hematoxylin-
prepared
of
9% or
involving
myocardium,
regions
border
infarcts
AMED 2 0 6
12/17/85
and
septal
of
assessment
of
infarcted
organization,
the
unstained
collagen
and
progression
degree
healing.
Histologic
stains
used
include;
Hematoxilin/Eosin
Staining
cells
intense
collagen
viable
red
light
cell
nuclei
dead
cells
collagen
blue,
, red
("eosinophilic")
cytoplasm
cells
red,
dead
pink,
and
pink.
Fibrin-Trichrome
Staining
(HE) ;
viable
grey,
red
nuclei
cells
, m o d i f i e d (F) :
viable
purple,
yellow,
fibrin
green.
-
69
-
cytoplasm
bright
red
red,
and
0 2 5 3 0 2 2
#5
Below
for
each
in
Table
VII
is
seen
the
estimate
AMED 20
12/17
of
infarct
size
dog.
TABLE V I I
ESTIMATE
OF PERCENT
DOG #
ID
1
2
3*
4*
I
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
*Dogs
no
3 and
tissue
+ Infarct
4 died
was
size
INFARCT
#
% INFARCT
9638
10585
19%
4% +
10597
10594
10598
10595
514
333
683
633
323
71098
11024
10824
75356
11378
11653
10852
10557
10665
77745
11045
7% +
9%
23%
16%
13%
18%
21%
19%
20%
12%
6% +
16%
15%
2% +
31%
13%
21%
19%
9%
19%
spontaneously
•
before
taken.
less
than
VOLUME
9%.
-
70
-
the
10
day
period,
0 2 5 3 0 2 2
AMED 2 0 6
12/17/85
#5
Observations
Histological
hearts
All
wound
which
criteria
histologic
ligation)
coronary
artery
collagen
organization:
non-hemolized,
Their
area.
of
important
located
within
the
viable
criterium
pattern
(as
since
living
such
vascular
of
amounts
Large
with
along
absence
vascular
necrotic
area
regenerating
for
optimal
or
(10
days
for
channels
if
had
considered
71
-
been
died
A vascular
bleeding)
were
are
with
penetration
accelerated
-
they
old
had
supply
cells
filled
was
and
infarct
diffuse
plain
channels,
even
the
flow.
regenerating
cells
along
definition)
to
deep
after
fresh,
of
blood
blood
of
of
vascularization,
that
(by
specific
assessment
center
opposed
and
The
distributed
blood
which
tissue
histologic
the
made
was
indicated
a temporary
distribution
was
in
hearts
necrotic
the
presence
re-established
because
in
channels
vascular
in
: perfusion,
non-clotted
blind
control.
vs.
dog
A search
PERFUSION:
1)
for
sent
used
was
infarcted
in
healing
and
experimental
determine
to
study
coded
were
seen
perfused.
fresh
of
this
blood
area
a positive
wound
healing.
0 2 5 3 0 2 2
#5
2)
VASCULARIZATION:
vessels
in
necrotic
the
and
of
density
heart
living
vessels
multilayered
v
granulation
with
vessel
wall
indicators
vascular
the
at
cells
foci
border
of
component
blood
between
assessed.
,A h i g h
showing
capillaries
considered
were
zone
profiles
of
of
maturity
was
vascular
and
pattern
the
tissue
many
and
density
muscle
a hemangioma-like
of
The
AMED 2 0 6
12/17/85
a
displaying
positive
wound
healing,
or
angiogenesis.
3)
COLLAGEN
the
infarcts,
cells
than
) *
of
healing
acquire
highly
in
ones
indicator
an
part
and
the
of
advanced
the
very
cellular;
become
more
fibrilar
in
strength
fibrillar
of
periphery
of
the
areas
infarct
of
component
this
way.
over
was
the
as
evaluated
wound
process.
These
criteria
of
overall
healing
tensile
border
appeared
areas
organized
an
the
At
tissue
these
collagen,
cellular
cellular
as
regenerating
produce
Predominance
ORGANIZATION:
an
process
by
used
were
only
in
the
pathologic-anantomic
two
experienced
context
and
evaluation
of
as
a
the
cardiovascular
specialists.
The
reports
with
non-edited
are
some
as
follows
description
of
transcripts
including
of
the
a gross
photos
gross
-
original
72
-
of
pathology
description
of
same
0 2 5 3 0 2 2
#5
the
together
hearts,
1-8) , m i c r o s c o p i c
Each
for
degree
of
a series
of
microscopic
to
a series
of
micrographs,
descriptions
section
individual
hearts
entire
the
with
referring
descriptions
(1)
poorest
(2)
no
(3)-
some
were
healing,
convincing
changes
(4)
advanced
(5)
be
but
probably
based
and
upon
collagenization:
untreated
most
untreated
likely
well
could
impression
be
within
usual
untreated
cannot
tell
whether
treated
untreated
(3)+
be
follows
as
must
equivocal
completely
or
rated
overall
the
then
change,
change,
variations,
(3)
and
(dogs
9-24) .
(dogs
neovascularization
perfusion,
AMED 2 0 6
12/17/85
allow
educated
healing
by
guess
serveral
only,
maybe
criteria,
likely
treated
impressive
must
acceleration,
-
73
-
be
treated
treated
to
0 2 5 3 0 2 2
AMED 206
#5
12/17/85
Pathologic-anatomical
Dog
•valuation
dog
hearts
'
Dog _ £ 6 3 8
4
Groas D e s c r i p t i o n ;
TFesE h e a r t
in s i t u
In I A D .
with balloon
Surface
s m o o t h and g l i s t e n i n g .
F a i t a r e a in a n t e r i o r
IV and a p e x .
On a e c t i o n
demarcated
there
is a v e i l
anteri
w h i c h is u n u s u a l l y
does not s t a i n v i t h
red,
tetrazoliua
( N I B ) and i n v o l v e s
an e s t i m a t e d
( i -. e .
volumt.
19* of t o t a l
U
cross-sectional
19>
"\ s l i c e s
through both v e n t r i c l e s
non-blut
vert
or-lateral
infarct
nitro blue
19* of t h e m u s c u l a r
a r e a on a l l 6 1 ce m
@ non v i a b l e . )
*
Gross P h o t o s :
•.
Tl
Front viev,
fresh heart
.
2*
Tresh s l i c e s
through both v e n t r i c l e s
a
f
t
e
r
5TB r e a c t i o n
(blut
J.
brovniih-red
Same,
@ non-infarcted,
• infarcted)
fixation
and r e m o v a l
4«
of s p e c i m e n !
for h i s t o l o g y :
Same, a f t e r
P @ non-infarcted
(posterior)
area
(lateral)
1 @ border
tone
A @ ri idle
of i n f a r e t
(anterior)
)
Microscopic D e s c r i p t i o n ;
Specimen ? ( p o s t e r i o r )
P h o t o 5: l o n g i t u d i n a l
sections
shov S l i g h t l y
but
congested,
othervise
normal m y o c a r d i u m .
P h o t o 6: c r o s s
sections
shov s a m e .
P h o t o 7: h i g h p o w e r
of RIB p o s i t i v e
viable ayocardiua.
Tht
KTB s t a i n s
cells.
only t h t most s u p e r f i c i a l
Specimen I ( l a t e r a l )
P h o t o 8: f e w f j . b r o c e l l u l a r
patches
present.
8pecimen A ( a n t e r i o r )
P h o t o 9: l o v p o w e r : n e e r o t i e
Infarct
@yoeardiua,
htaorrhagie
and p e r i p h e r a l
border,
fibrocellular
organisation.
P h o t o 10:
center
of i n f a r c t
ausclt
vith
shoving nccrotic
extensive
hemorrhage,
no c e l l u l a r
organization.
P h o t o 11: h e m o r r h a g i c
and c o n g e s t e d
between dead
interface
@ u s c l e and o r g a n i s i n g
fibroblasts.
@ P h o t o 12: d e t a i l ,
center
of I n f a r c t ,
bo v i a b l e t i s s u e ,
no
a u c h h e m o r r h a g e and h e m o l y s i s .
organization,
P h o t o 13:- d e t a i l
of f i b r o b l a s t i c
organization.
' P h o t o 14:
arterioles,
neerotie ayocardium
aicxohemorrhage,
and o r g a n i z i n g
fibroblasts.
P h o t o 15: l o n g i t u d i n a l
sections
through p a r t i a l l y
vavy,
aecrotic
ryocardial
vith
cells,
inflammatory c e l l s ,
txtravasated
and o c c a s i o n a l
blood,
close
fibroblasts,
to c e n t e r
o
f
i
n
f
a
r
c
t
.
•
..
-
74
-
v
0 2 5 3 0 2 2
AMED 2 0 6
12/17/85
#5
Pathological-anatomical
tvaluation
dog
hearts
.
p_p_£ 2,
10585
Dog 2
Gross D e s c r i p t i o n :
in I A D .
in s i t u
balloon
Fresh h e a r t ,
epicarTTal
changes. •
Ko s i g n i f i c a n t
show s n a i l
Slices
patchy. areas which art only p a r t i a l l y
HTB
mid-muscular
in a c i r c u l a r ,
pattern
a n d on a p a p i l l a r j
@n e g a t i v e ,
4£ o f a l l
Busclt.
Istimated
cross-sectional
areas
art filfi
non-viable).
(i.t.
negative
x
*
•
Gross P h o t o s ;
V,
Frontal
v i e w of f r e s h h e a r t .
• '
2.
Presh slices,
h o m o g e n e o u s l y brown ( v h i t i s h
p a t c h on l a r g e s t
slice
is p a r t
of v a l v e
r i n g a t b a s e of h e a r t ) .
3*
Kon-stained
8 a m e , a f t e r KTB s t a i n .
a r e a s b a r e l y vi t i t l e .
and r e m o v a l
fixation
of s a m p l e s
4.
for h i s t o l o g y
Same, a f t e r
(from s l i c e
J u s t above blue l a b e l s ) .
V e a l c e r STB s t a i n i n g
visible
at fev p l a c e s .
*
"
Microscopic Description;
•
'
Specimen P ( p o s t e r i o r ;
P h o t o 5s c u t s u r f a c e
of e s s e n t i a l l y n o r m a l m y o c a r d i u m
KTB r e a c t i o n
(viable
bluish
• s h o v i n g the p o s i t i v e
cells,
reaction
product)
in the u p p e r m o s t
cell l a y e r .
P h o t o 6: c r o s s ' s e c t i o n
normal
through
essentially
Bed " d o t s " r e p r e s e n t
ayocardium.
vhich a r t
capillaries
' •
and o c c a s i o n a l l y
-filled
overfilled
w i t h red c e l l s x
.
mild c o n g e s t i o n .
indicating
'
• P h o t o 7: same as 6, l o n g i t u d i n a l
sections.
Specimen L ( l a t e r a l )
P h o t o 8: l o w p o v e r o v e r v i e w s h o v i n g l a r g e a r e a o f s l i g h t l y
but o t h e r w i s e
)
normal m y o c a r d i u m .
congested
Specimen A ( a n t e r i o r )
P h o t o 9: l o v p o w e r o v e r v i e w o f e s s e n t i a l l y
normal
@yocardiua.
P h o t o 10: h i g h p o w e r s h o w s o c c a s i o n a l
(pinker)
eosinophilic
'@yocardial
i
n
d
i
v
i
d
u
a
l
c
e
l
l
cells,
indicating.
damage.
-
75
-
0 2 5 3 0 2 2
#5
Pathologic-anatomical
50g
5
-
(7% NTB)
tvaluation
of
hearts
dog
AMED 2 0 6
12/17/85
Dog
£
10597
»
•
QroBB D e s c r i p t i o n :
T T e s S dog h e a r t v i t h l i g a t e d I A D .
eitt.
. Email s c a r at l i g a t i o n
of a n t e r i o r
apical
surfact,
with dull surface)
. * Patchy paleness
•
a n u . . .
•
• • "
'
' ~
" ?rO85, P h o t o s :
Fresh heart,
").- "H
front
viev,
Hots
ligature
scar v i s i b l e
In LAD.
d
u
l
l
p
a
l
e
,
a
n
t
e
r
i
o
r
and
p
a
t
c
h
e
s
on
a
p
i
c
a
l
v
e
n
t
r
i
c
u
l
a
r
s
u
r
f
a
c
e.
•„
'
2.
R e a r v i e v of f r e s h h e a r t .
Trash
slices
5»
through myocardium.
Hott that
(A) a n d
anterior
..
(P) t i d e s
••'posterior
a r t not a l w a y s o r i t n t t d
in t h t s a m e
direction..
.'
NTB r e a c t i o n
4*
on f r e s h s l i c e s .
A @ front
P @
anterior;
.
posterior.
5.
fixation
and r e m o v a l o f h i s t o l o g i c
Same, a f t e r
specimens.
1 m posterior
L - lateral
A @ anterior
J
Microscopic D e s c r i p t i o n :
Specimen P ( p o s t e r i o r . /
, ,
P h o t o 6: t s s e n t i a l l y
nornal ayocardiuV l o n g i t u d i n a l l r
arteriolts
and c a p i l l a r i e s .
. sectioned vith
P h o t o 7: h i g h p o v e r c r o s s - s e c t i o n
of s a m e .
P h o t o 8: h i g h p o v e r l o n g i t u d i n a l
section
of s a n e .
l o t t
»ito8i8
@
(?endothelial)
at a r r o v .
Specimen L ( l a t e r a l )
.
P h o t o 9: a c u t e and c h r o n i c p e r i c a r d i t i s ,
•picardial
fat,
and v e s s e l ,
unaffected
ntrve,
tubepicardial
nyoeardium.
P h o t o 10: o"blio.ue s e c t i o n t h r o u g h t s s e n t i a l l y
normal
@yocardiua.
Hote t h t c a p i l l a r y
pattern.
P h o t o 11: t s s e n t i a l l y
normal »ybcardium,
longitudinal
Specimen A ( a n t e r i o r )
P h o t o 12: a c u t e a n d c h r o n i c p e r i c a r d i t i s
vith extensive
vascularization.
Hormal c o r o n a r y
Organizing
artery.
@yocardial
infarct.
P h o t o 13: n e c r o t i c
(red p a t c h e s ) ,
cellular
ayocardium
organisation
vith
and s o m e
blood v e s s e l s ,
fibrpblasta,
collagen
(pink).
Calcification
(blue p a t c h e s ) .
Z n d o c a r d i u o at a r r o v .
P h o t o 14:
detail
(fibroblastic)
of s o s t l y
cellular
organisation.
» y o e a r d i u m at a r r o v .
Remaining necrotic
Kicrohemorrhages
eztravascular
in c e n t e r
(individual
red
cells).
-
76
-
Pathologies-anatomical
Vog
0 2 5 3 0 2 2
..
«
&
evaluation
of
flog
hearts
)
10594
tj
(9% NTB)
. Oroas D e s c r i p t i o n :
l i g a t u r e Jji s i t u
TFeaS,
s m a l l flog h e a r t ,
relatively
a l o n g LAO.
Petedinal
hemorrhages
epicardial
surface
toward a p e x .
dull patches
Pale,
on a n t e r i o r
"^
Tog'S'
gross P h o t o n
? r o n t v i e v of f r e s h h e a r t ,
T.
v i t h IAD l i g a t t d .
R e a r v i e v of s a n e .
2.
Fresh myocardial
slices.
J.
A @ anterior,
? - posterior
SIB r e a c t i o n
4.
slices.
on f r e s h
Hote c l e a r
d e m o n s t r a t i o n of i n f a r c t .
m
at
"
.
I A D . . •
'"•
* '
Microscopic D e s c r i p t i o n ;
Specimen P ( p o s t e r i o r ;
• •
P h o t o 5: e s s e n t i a l l y
normal myocardium,
l o n g i t u d i n a l . .
P h o t o 6: s i n g l e
s m a l l f o c u s of f i b r o s i s
and n e c r o t i e
• .
cells.
@yoeardial
Specimen I ( l a t e r a l )
P h o t o 7: c r o s s - s e c t i o n
t h r o u g h v i a b l e m y o c a r d i u m of l a t e r a l
left
ventricular
vail.
Positive
HTB r e a c t i o n v i s i b l e
as
blue c e l l u l a r
stain
in c o r n e r .
Capillaries
axe p r o m i n e n t
and f i l l e d
vith
red c e l l s
(red d o t s ) .
or ' o v e r f i l l e d
show t h i c k e n e d
Arteriols
(hyperplastic)
vails.
Photo 8:Markedly t h i c k e n e d
arteriolar
vail
and c a p i l l a r y
congestion.
s m o o t h m u s c l e h y p e r p l a s i a of a r t e r i a l
P h o t o 9: e x t e n s i v e
and c o n g e s t e d c a p i l l a r i e s .
Cross-sectioned
muscle
vail,
cells
normal.
essentially
"
•
. Specimen A ( a n t e r i o r )
P h o t o 10: a c u t e and c h r o n i c p e r i c a r d i t i s ,
normal c o r o n a r y
infarcted
totally
artery,
myocardium.
of i n f a r c t .
P h o t o 11: c e n t e r
thin necrotie
fibres
ayocardial
(Without nuclear
m
i
n
i
m
a
l
r
e
a
c
t
i
o
n
staining),
except f o r
few d i s i n t e g r a t e d
in b l u e
polymorphonuclear leukocytes
Ho
t
h
i
s
c
o
n
t
r
o
l
v
a
s
c
u
l
a
r
i
s
a
t
i
o
n
in
periphery.
area.
P h o t o 12: p e r i p h e r a l
vith n e c r o t i e
a r e a of I n f a r c t
infiltrated
@yocardium p a r t i a l l y
by p o l y m o r p h s .
tissue
is m o s t l y c e l l u l a r
Organising granulation
(fibroblasts)
vith
c o l l a g e n and u s u a l e x t e n t
of
some t a r l y
vascularitation.
P h o t o 13: n e c r o t i e
fev
B y o c a r d i u m n e a r c e n t e r of I n f a r c t ,
r
e
d
e
x
t
r
a
v
a
s
c
u
l
a
r
disintegrating
cells,
polymorphs,
some
no new v e s s e l s .
Photo U:
and c e l l u l a r
organising area a t
very a c t i v e
Infarct
a
uscle
cells,
7
e
v
n
e
c
r
o
t
i
c
periphery.
r
e
d cells
predominance
f
i
b
r
o
b
l
a
s
t
s
,
inside
a
c
t
i
v
a
t
e
d
of
and o u t s i d e
of c a p i l l a r i e s ,
few p o l y m o r p h s .
P h o t o 15: v e r y f i b r o u s
»ostly
a r e a of o r g a n i s a t i o n ,
.
a
c
t
i
v
a
t
e
d
collagen.
fibroblasts.
Predominant
cells
are
and v e n u l e s ,
Tessels
are t h i c k v a i l e d a r t e r i o l e s
vithout
» u c h c o n g e s t i o n and v i t h o u t
capillaries.
prominent
-
77
-
•
0 2 5 3 0 2 2
AMED 206
Pathologic-anatomical
Dog
7
(231
tvaluation
of
dog
hearts
Dog
7
l'05*»
NTB)
'
Gross D e s c r i p t i o n *
•
vith ligated
LAD.
superficial
f r e s h dog h e a r t
Kicrohemorrhages,
and d u l l n e s s
a l o n g LAD.
Apical yellow
paleness
area d e m a r c a t e d
vith red, hemorrhagic border z o n t . . . . .
• •
•
Cross P h o t o s :
Front view, f r e s h
'TZ
•*"«.of IAD a n d
specimen vith l i g a t u r e
"
and
a
n
t
e
r
i
o
r
tpicardial,
changes.
apical,
Rear v i e v ,
2.
cloudiness
Vhitish
toot l a t e r a l
apical paleness.
o
f
pericardium.
•.
fresh
slices.
Hote the e x t e n t
of t h t B o t t l e d
J.
)
myocardial
infarcted
yellov-red
• .
area.
•
.
HB1 r e a c t i o n .
infarction
(yellov-red),
4*
Kote t o t a l
mildly
ischemic
(?) c o n e s ( b r o w n i s h - b l u e ) ,
and n o r m a l
zones ( b l u e ) .
•
t
o
t
a
l
i
n
f
a
r
c
t
m
e
a
s
u
r
e
d
.
Only
vas
r
e
m
o
v
a
l
o
f
Same a f t e r
fixation
and
5*
specimens.
histologic
. }
'
Microscopic Description;
•
.
Specimen P ( p o s t e r i o r )
P h o t o 6: u n a f f e c t e d
longitudinal.
myocardium,
Specimen L ( l a t e r a l )
normal
P h o t o 7: a c u t e and s l i d
chronic p e r i c o n d i t i s ,
fibrous
and c e l l u l a r
organleation,'
coronary vessel,
and n e c r o t i o
b
o
r
d
e
r
f
e
v
b
e
m
o
r
r
h
a
g
i
c
p
o
l
y
m
o
r
p
h
s
,
z
o
n
e
,
.
m y o c a r d i u m in t y p i c a l
layering.
P h o t o 8: t y p i c a l v i e v of b o r d e r t o n e b e t v e e n d e a d m u s c l e
( r e d ) and r e a c t i v e
tissue.
A fev
granulation
organizing
( a r r o v ) but n o t
vessels
i n t o the dead t i s s u e
penetrate
into
its full
d e p t h . - T h e r e is l i t t l e
polymorph r e a c t i o n
(i«e.,
r
e
m
a
r
kable
bluish
border
z o n e ) and a t t h i s
no
v a s c u l a r ! e a t ion o f g r a n u l a t i o n
is n o t
tissue
place,
T
h
e
r
e
i
s
l
i
t
t
l
e
of
red c e l l s
e
x
t
r
a
v
a
s
a
t
i
o
n
overwhelming.
b
o
r
d
e
r
and no h e m o r r h a g i c
or c o n g e s t e d
zone.
P h o t o 9: d i f f e r e n t
o r g a n i z i n g area shows l e s s n e c r o t i c
more v a s c u l a r i z a t i o n ,
muscle,
more h e m o r r h a g e t h a n a r e a
t
h
e
r
e
i
s
a
l
s
o
f
o
c
u s of c a l c i f i c a t i o n
(blue).
6;
on p h o t o
a
necrotic
o
f
c
e
n
t
e
r
P h o t o 10: t o t a l l y
near
myocardium,
v i t h a fev d i s i n t e g r a t i n g
infarct,
polymorphs,
sparse
extravascular
and t h e m o s t a d v a n c e d
red c e l l s ,
round c e l l s ,
blood v e s s e l s .
viable
m u s c l e at o r g a n i z i n g cone, p a r t i a l
P h o t o 11: n e c r o t i c
into
flbroblastic
dead
hemorrhages
organization,
partial
tissue.
P h o t o 12: s i m i l a r
t o p h o t o 11, v i t h f o c u s of c a l c i f i c a t i o n .
Bed c e l l s
of b l o o d v e s s e l s .
i
n
and o u t s i d e
are
P h o t o 13: t y p i c a l
at b o r d e r
active
cellular
organization
of
of i n d i v i d u a l
mecrotic myocardium.
red
Extravasation
cells
is c l e a r l y
muscle
Remaining necrotic
visible.
cells
are s u r r o u n d e d
f
i
b
r
o
b
l
a
s
t
s
.
by a c t i v e
P h o t o 14: d e t a i l
and b l e e d i n g v i t h i n
of c a l c i f i c a t i o n
organiring
tissue.
P h o t o 15: t y p i c a l
and
organizing pattern vith f i b r o b l a s t s
-
78
-
0 2 5 3 0 2 2
♦5
*
•
pathologic-anatomical
»Og 8
(16%
AMED 2 0 6
12/17/85
NTB)
evaluation
of
hearts
dog
Dog £
'
10595
*
.
Gross D e s c r i p t i o n :
Epicardial
F r e s h d o g h e a r t w i t h IAD l i g a t u r t .
pal«
hemorrhages;
and a p i c a l .
anterior
and d u l l s u r f a c s
•
gross Photos:
.
.
Front viev,
fresh
T*
specimen^
v i t h IAD l i g a t u r t ,
pal*
dull,
and r e d s u b e p i c a r d i a l
@urface a n t e r i o r - a p i c a l ,
bleeding.
Posterior
2.
lateral
viev,
palt
apex.
?resh myocardial
slices.
A @ anterior
tide
(vith
infarct).
~) ?•
HTB r e a c t i o n
4*
A @ anterior
on f r e s h
(vith
myocardium.
Infarct).
fixation
and r e m o v a l o f h i s t o l o g i c
5*
Same, a f t e r
specimens!
• A @ anterior
L - lateral
1 » posterior
•
Microscopic Description
.
• Sptcimen P ( p o s t e r i o r ;
P h o t o 6: e s s e n t i a l l y
normal oyocardium c r o s s - s e c t i o n e d .
Specimen I ( l a t e r a l )
P h o t o 7: e s s e n t i a l l y n o r m a l m y o c a r d i u m , l o v p o v t r ,
vith
and c a p i l l a r i e s .
penetrating
artery,
arterioles,
Specimen A ( a n t e r i o r )
and o r g a n i s i n g t i s s u e
P h o t o 8: b o r d e r of s e c r o t i c
vithin
infarcted
f
ibrocellular
area.
Typical
organisation,
1
aoderately vascular,
no b l e e d i n g ;
•
very fev p o l y m o r p h s
vith
P h o t o 9: p p r i r a r a i u p .
a.nd
acute
chronic p e r i c a r d i t i s .
Hott round c e l l
narked vascular ity,
and
)
infiltrate,
underlying necrotic myocardium.
P h o t o 10:
organising tissue vith unusual prominence o f
arterioles
and o t h e r b l o o d v e s s e l s .
P h o t o 11: P o c u s of c a l c i f i c a t i o n
in a d v a n c e d ,
collagenous
and c e l l u l a r
fibrotic
consistent
vith
organising tissue,
•arly tear f o r m a t i o n .
-
79
-
0 2 5 3 0 2 2
. .
SOP
"
g
.
control
Posterior
segment
—unremarkable
oyocardium
—no e p e c i a l v a s c u l a r
pattern
Slide
93 2 1
infarct
center
Anterior,
v
i
t
h
—total
infarct,
good p e r f u s i o n
and no r e a c t i o n
"but a t y p i c a l
pattern
equivocal
Slide
-«v
V *
934;
*.
Zy_alt
Overall
•
#5
AMED 2 0 6
12/17/85
Dog
933 %
Evalt
.
•
Elide
*
'
'
•
of
by
514
old c h a n n e l s ,
cell!
living
border
lateral
v i t h g o o d i n g r o w t h of v i a b l *
reaction,
— very c e l l u l a r
cells
i n t o dead a r e a
areas shov n a t u r e
— perivascular
collagen
@
•
—many polys p r e s e n t
fitroblastie
p a t c h e s of r e a c t i v e
— large
cells
also
embedded v i t h i n a d j a c e n t
noninfarcted ayocardius
increased
to bt t r e a t e d
activity,
likely
•
Evaluation;
l i k e l y t o be t r e a t e d .
b o r d e r cone;
c e n t e r of i n f a r c t
reviev.)
Rating @ 4
)
-
80
-
(Rote:
» o s t l y based
less convincing i n
on
0 2 5 3 0 2 2
#5
Dog
BOO 1 0
without
Slide
935 t
control
Posterior,
— normal m y o c a r d i u a
Slide
936 i
border
lateral
p a r t n o t on e l i d e
— infarcted
—myocardium u n r e m a r k a b l e
not a p p l i c a b l e
Ival:
Slide
937;
Ivali
Overall
segment
infaret
anterior
Center i n f a r c t ,
— dead a r e a p o o r l y p e r f u s e d
i n g r o w t h not nuch a d v a n c e d
— cellular
immature
vith collagen
rare,
—areas
tissuue varied
o
f
g
r
a
n
u
l
a
t
i
o
n
v
a
s
c
u
l
a
r
i
t
y
—
a u s t be c o n t r o l
evaluation;
Untreated;
Rating
nust
be
control.
@ 1
y
-
81
-
AMED 2 0 6
12/17/85
333
0 2 5 3 0 2 2
AMED 2 0 6
12/17/85
#5
Dog
683
DOG 11
Blldo
938:
Slidt
939;
Xval;
^
Blide
•
940;
lyal:
Slide
956;
gval;
Slide
)
957;
gval;
Slide
^5S:
Zval;
Overall
"Posterior control segment unremarkable,'
vascular
Ho s p e c i a l
ayocardiua.
patttrn
Intact
•
*
lateral
border tont*
vail,
—active
perfusion
—dead
nuscle
e n t i r e l y p e n e t r a t e d by l i v i n g c e l l !
@following c h a n n e l s
and v a s c u l a r i t y
—high
cellularity
of r e g e n e r a t i n g
tin s u t
— most advanced c o l l a g e n
is in t h i c k b u n d l e s v i t h
few r e m a i n i n g c e l l s ,
relatively
b u t a r e a s In b e t v e e n
are s t i l l
s m a l l f o c u s of c a l c i f i c a t i o n
very c e l l u l a r ;
treated
definitely
infarct
center
Anterior,
— extensive
of o l d c h a n n e l ! "
pattern
reperfusion
*
cells
— living
d e e p l y i n t o dead a r e a
penetrating
book" i n t e n s i v e
— "picture
regeneration
pattern
— s o m e , b u t -not a l l ,
regenerating
areas vith h i g h
and m a t u r e v e s s e l s
vascularity
a d v a n c e d o n l y in p e r i p h e r y ;
—collagen
s o o t of t h t
dead m u s c l e has a l s o a g r e e n i s h t i n t
stain
on t h i s
treated
definitely
v
Beeut;
s e p t a l border «one,
a
c
tive
regeneration,
—very
939
treated
slice #4
qualitatively
*@
•
siailar
to
8 1 i c e #5, c e n t e r
infarct
(anterior)
rscut
a
c
t
i
v
e
breaking p a t t e r n
—very
—has
a r e a s of v a s c u l a r i t y
r e s e m b l i n g the p a t t e r n
hemangiomas
-treated
of
Additional
slice
cut,
—similar
to 939, v i t h
treated
(center
#6, a n t e r i o r
Infarct)
some B o r e d e a d s a t e r i a l
evaluation:
treated
(aoBt active
vithin
Definitely
of
f
i
v
e
Rating « 5
d o g s #s 9 - 1 3 ) .
group
-
82
-
this
0 2 5 3 0 2 2
DOS
12
Dog 6 3 3
Slid*
941 1
Slide
2i2*
Xvalx
Slide
942:
Bval:
Slide
959:
Zval:
81ide
960:
2val:
Slide
)
#5
"
Posterior
control
segment:
AWED 2 0 6
12/17/85
unremarlcablt
center
infarct
Anterior,
— some a r e a s w i t h v e r y g o o d p e r f u s i o n
of old c h a n n e l ! .
But o t h e r s i n s u f f i c i e n t ;
dust
m o r e p o l y s and u n c l e a r
t h a n u s u a l b o t h in c h a n n e l s
aand p e r i p h e r y
cells
but not f o l l o w i n g
— regenerating
present,
@ •
channels very f a r
—new v e s s e l s
present
of c o l l a g e n c l o s t to d e a d t i s s u t
— soae t h i c k s t r a n d s
equivocal
*
•
lateral
border
— s a a l l area vith e x t e n s i v e
infiltrate
of
round c e l l s
and h i g h v a s c u l a r i t y ,
but not
not t y p i c a l ,
ojaestionablt.
Inflammation?
-
.
inflammatory
auch r e p a i r
•
.
lateral
b o r d e r of i n f a r c t ,
slice #4
— extensive
a c u t e and c h r o n i c p e r i c a r d i t i s
—advanced healing,
and v e r y v a s c u l a r
very c e l l u l a r
— yet n e c r o t i c ,
p a r t i a l l y perfused areas i m m e d i a t e l y
adjacent
—collagen
r e a s o n a b l y a d v a n c e d , 'but g r e e n I s
overstained
at p l a c t s
be
t
r
e
a
t
e
d
say
slict
Septal border,
4
—florid
pericarditis
—active
but s t i l l
healing pattern,
relatively
large
necrotic
areas.
but I n f l a m m a t i o n obscures p i c t u r e .
n a y be t r e a t e d ,
961:
Center i n f a r c t ,
a n t e r i o r , -.slice 2
' -—large
dead a r e a ,
often poorly p e r f u s e d
and u n c l e a r
dust
— nany p o l y s ,
i
n
s
m
a l l rim, not very a c t i v e
-regeneration
only
a
t
h
r
o
m
b
o
s
e
d
v
e
s
s
e
l
s
—large
in c e n t e r
Zval:
of t r e a t m e n t r e c o g n i t e d
inflammatory,
no t f f e c t
Slide
962:
Zval:
Overall
Center i n f a r c t ,
slice
1
anterior,
littlt
recognizable
—very necrotic,
p r o b a b l y not t r e a t e d
evaluation;
yote;
probably
not
activity
Rating
treated.
»-2
•
•• •
(after
sections
©n r e v i e w of a d d i t i o n a l
disclosure
of
eode) the a c t i v i t y
s a y have been u n d e r e s t i m a t e d
the s p e c i m e n
i s u n u s u a l and d i f f i c u l t
originally;
to
r e a d b e c a u s e of t h e p r e s e n c e of a a n y s o r e a c u t e
Sections
cells
inflammatory
than usual.
943 a n d 9 6 0
v o u l d p r o b a b l y be e v a l u a t e d
as s h o v i n g t r e a t m e n t
•ffccts
i f r e v i e w e d a g a i n as u n k n o w n s .
-
83
-
0 2 5 3 0 2 2
#5
Dog
SLIPS
Slide
left
ventriclt:
Posterior
section
.
— unrenarkabls
944:
945:
Ival;
963:
gvalt
Slide
964 t
'
)
tval;
Slide
*
.
Slide
control
.
slice #4
lateral
border,
—moderate,
average regenerative
equivocal
reaction
slice 4 "
Beptal border,
and v a s c u l a r
reaction
cellular
—intensive
h
o
w
e
v
e
r
not a d v a n c e d ,
—collagen
but many
of dead m a t e r i a l
—perfusion
average,
c e l l s are p r e s e n t
of v i a b l e
Individual
islets
»aybe
slice 5
center
Infarct,
Anterior,
•-some areas vith impressive penetration
cells
•
.
.
.
.
• —otherwise
tquivocal
Tval;
average
965:
966;
Evalt
Overall
*
of I n f a r c t
center
Anterior,
in a l l
c
o
n
s
i
s
tent
perfusion
—relatively
pattern
t
o
o
d
etail
necrotie
b
u
t
c
o
t
In
i
m
p
r
e
s
s
i
v
e
areas,
cells
—good f o l l o v - u p vith sickle
and c o l l a g e n s a t u r a t i o n
— 'Vascularity
average
effect
but l i t t l e
t
r
e
a
t
e
d,
possibly
946x
Zvalt
Slide
!323
lateral
left
ventriclt
border,
often band-like
— patchy,
of n e c r o t i e
interruption
tissue
a r e a s by r e g e n e r a t i n g
. . .
of old c h a n n e l s
— perfusion
so-so
of r e g e n e r a t i n g
tissue
and c o l l a g e n
—maturity
average
— vascularity
high
n a y be t r e a t e d
^
J
Slide
non-infarcted
AMED 2 0 6
12/17/85
slice
#6
Anterior,
infarcted,
cells
— g o o d p e n e t r a t i o n by v i a b l e
—good p e r f u s i o n
w i t h o u t »uch
repair,
^-very cellular
— vaccularity
average
a a y be t r e a t e d
svalur.tlon;
considered treated
"•
Rating »
guess).
(educated
-
84
but
3+
-
vith
by
siature
veak*
viable
collagen
tffect
0 2 5 3 0 2 2 "
A
S
SB
IB
(and n u m b e r of s l i c e ) "
anterior
septal
'
septal border
lateral
border .
x photo
12* I n f a r c t
•
71098,
O05 l i
Dog:
*
•
1001
A3
Hot a l l o f s e c t i o n
infarcttd
Ho d e a d t i s s u e ,
channels;
no r e v i t a l i z e d
polys s c a r e s
sot i m p r e s s i v e .
. Loose e a r l y f i b r o s i s ,
vascularity
1002
lateral
13
Essentially
normal
myocardium;
—see
?
003
1004
no
infarct,
LB l a t e r a l
no
• .
*
• •
reaction.
border
stptal
83
Ho l n f a r e t
A2
Snail areas
*«
of t o t a l ,
non-removed i n f a r c t
Channels
utilized
partially
Kostly c e l l u l a r
vith maerophages
reaction,
of r e a c t i o n
Tascularity
is m o d e r a t e o v e r a l l
Collagenization
moderately advanced
Perfusion
relatively
poor
lateral
b o r d e r 1B2
dead m a t e r i a l
Very l i t t l e
left
little
utilization
of c h a n n e l s
in dead a a t e r i a l
Good c e l l u l a r
g
r
a
n
u
l
a
t
i
o
n
.
b u t "not i m p r e s s i v e l y
t
i
s
s
u
e
,
.
vascular
P o o r f o l l o w i n g of c h a n n e l s by f i b r o b l a s t s ,
but t h e r e is
.
in t h e v i a b l e m u s c l e a r e a s
interlacing
Collagenization
not far a d v a n c e d .
1005
.
)
1006
1007
1008
1009
Apex A1
lot infarcted
liver
Congestion
Hild f o c a l
'
fatty
change
or
Muscle i n j e c t i o n
site
Perfectly
normal s k e l e t a l
Kuscle c o n t r o l
formal s k e l e t a l
smscle,
Overall
Impression
of
1
oog
'smscle
poor
U:
'
fclyeogen
lection
» u s t be a c o n t r o l .
Rating » 1.
-
85
-
•
•
some
0 2 5 3 0 2 2
#5
£°£ii
•
1010
Dog:
11024.
shovs
signs
1011 IB l a t e r a l
border
• S m a l l r i m of c e l l u l a r
and l o o s e
Eome m a e r o p h a g e s
Vascularieation
pot i m p r e s s i v e
Adjacent muscle u n r e m a r k a b l e
T e r y few i n d i v i d u a l
dead f i b e r s
Xralt
)
not
impressiTS
In
overall
1013
treataent
collagenous
"effect"
but
too
organisation
\
small
an
area
equivocal
* •
A n t e r i o r A2
M o s t l y dead m u s c l e w i t h s m a l l ( w h i t e )
ria p e r i c a r d i a l .
Prominent channel
reperfusion
Vigorous
cellular
cut not too « u c h
regeneration,
interdigitation
Advanced,
mature c o l l a g e n o u s
subepicardial
organisation
"
Prominent v a e c u l a r i t a t i o n
in one c o r n e r
Xvalt
1014
of
S e p t a l b o r d e r 3,
BB3
Good l a y e r i n g f r o m d e a d a u s c l e t o c e l l u l a r t o f i b r o u s
to n o r m a l m u s c l e
regeneration
Channel r e - u t i l i z a t i o n
p r o m i n e n t in dead a r e a
Cellular
reaction
but not f a r e x t e n d i n g
vigorous,
R e l a t i v e l y well formed c o l l a g e n ,
not o v e r w h e l m i n g l y h o w e v e r
not i m p r e s s i v e ,
Vascularity
about average •
Green o v e r a t a i n e d
Xval:
.
Infarct
A n t t r i o r A3
Bitable
aVovt a v e r a g e u s a g e of o l d
dead a r e a ,
vith
slightly
channels
Advanced c o l l a g e n o u s
o r g a n i s a t i o n from the e p i c a r d i u m
( g r o s s l y white rim)
interdigitating
w i t h l i v e and d e a d
muscle f i b e r s
Scattered
foci
of m y x o m a t o u s l o o s e e a r l y c o n n e c t i v e
tissue
with occasional a i c r o h e a o r r h a g e
vay w i t h i n dead a r e a ,
Into
and f a i n t
it,
green early c o l l a g e n .
Heovasculariration
in t h e s e a r e a s p r o m i n e n t ,
vith c o l l a g e n
a r o u n d each a r t e r i o l t
Xvalx
1012
16*
AMED 2 0 6
12/17/85
lilcely
Tery
treated
A n t e r i o r A1
dead f i b e r s
Jew s c a t t e r e d
left,
no o l d c h a n n e l s
Tery dense e e l l u l a r i t y
a r o u n d dead m a t e r i a l
A d v a n c e d and h i g h l y
organized
fibrocellular
regeneration
vith »ature c o l l a g e n
-
86
-
0 2 5 3 0 2 2
#5
Dog
1_£ c o n t i n u e d
*
@
Small r i a v i t h o r i g i n a l v i a b l e n u s e l t
hut
areas i m p r e s s i v e ,
In c e l l u l a r
Yascularity
to d i s a p p e a r
in t h e n o s t c o l l a g e n o u s
parti
scar.
advanced collagenous
Zpicardial
Sval:
1015
)
.
liver
Uniformly fatty
congestion
livtr
(or
adraneed
healing,
glycogen?)
vith
site
Muscle,
injection
Part s k e l e t a l
part f a t
ausclt,
So r e a c t i o n
*- *
control
1017
Kusclt,
Identical
to a b o v e , a l s o v i t h f a t
AMED 2 0 6
12/17/85
* •
•
seems
already
likely
treated
much
actrt*
1016
Overall
eral
Bog
15:
likely
Ratine
.*
t o "be t r e a t e d
@ 3+
)
-
87
-
•
0 2 5 3 0 2 2
#5
£0£i£
1018
Dog:
•
1OB24,
ICjClnfaret,
A3 a n t e r i o r
Much d e a d a a t e r i a l
Moderate channel
reperfusion
Interdigitation
cellt
of r e g e n e r a t i n g
along c h a n n e l !
but not o v e r w h e l m i n g
present,
Regenerating
tissue
than f i b r o t i e
»ors c e l l u l a r
Yascularity
a
t
b
e
l
t
average
It alt
»aj
or
Truli
with
be
•' •*• "
treated
average
anount
of
insufficient
BB3
.
Impressive
of s m a l l d e a d a r e a
reperfusion
loose
to a e d i u a
organised
tiaaut
regenerating
Baall foci
of a b o v e a v e r a g e v a s c u l a r i t y
Maturation
of c o l l a g e n
not i s p r t i t i v t
Sral:
1021
sot
may
1019
1B3
P
o
o
r section!,
•
re c u t
.
Iffect
does not teem e x t r a o r d i n a r y
Littlt
dead m a t e r i a l
Much f i b r o s i s ,
rtlativtly
loose,
vessels
and c e l l i
1020
AMED 2 0 6
12/17/85
«ob»
of
tigns
treatment,
\
but
veak
A2 a n t e r i o r
.
large
necrotic
tone
by l i v i n g
cella.
Ctllular
erganiting
v i t h good r e p e r f u s i o n
(of dead c h a n n e l s )
?ev p o l y s .
tissue
vith
increased
slightly
vascularity;
eone v e r y p r o m i n e n t t h i c k - v a l l e d
arterioles
*C o l l a g e n
development aoderats
to a d v a n c e d
1022
A1 A n t e r i o r
Ctllular
tissue
regenerating
vith norsal
interlacing
fibers.
Ho d e a d m a t e r i a l
Ovtrall
organization
advanced,
v i t h aany prominent t h i c k valled
arterioles
of v e r y e a t u r e
Joci
collagen
and d e c r e a s e d
cellularity
* o o d f o r p h o t o on " f l f c r i n " " t a i n
Ytii5}S
and s o m e w h a t l e s s
on
?TAH
photo
Sval:
1023
1024
liver
Sxtensive
All c e l l s
Muscle
Negative
congestion
ballooning
injection
•
both
(fat
Sjosie
of
signs
treatment
and f r e s h b l o o d
hemolytic
or g l y c o g e n
or b o t h )
site
'
-
88
-
a
Mm
0 2 5 3 0 2 2
#5
Dog
16
•
AMED 2 0 6
12/17/85
• i
control
Huiclt
alio t n t i r t l j
noraal.
A largt
Eegitivt.
a r t e r y and a n e r v t
•@
>
.
.
I m t - n o t v l t h @axlnua e f f e c t
O r e r a l l « T a l Vog 1 6 : l l l c e l j t r e a t e d ,
R a t i n g - 3+
1025
(
-
89
-
0 2 5 3 0 2 2
#5
.
12J|!1'
"
15*
75356,
Dogi
AMED 2 0 6
12/17/85
Infarct
A3 a n t e r i o r
Snail areas
of d e a d m u s c l t v e r y a c t i v e l y p e r f u s e d ;
subdivided
i n t o p a t c h e s by a c t i v e l y
growing regenerative
also
tissue;
@
appears loosened.
.
• A c t i v e and v e r y v a s c u l a r
regenerating
vith f a r
tissue,
'
advanced fibrous
at s e v e r a l
organisation
and v e r y
places,
cellular
and v a s c u l a r
spots e l s e v h e r t
l
o
o
ks like a hemangioaa.
One p l a c s
025
•
z
It alt
1027
treated
definitely
lateral
b o r d e r 133 ( t h i r d
sllet)
S i m i l a r to 1026,
v i t h soae i n t a c t B u s c l t vhlch shove n o
—
altered
n u m b e r and d i s t r i b u t i o n
of v e s s e l s .
It's
all
in the r e g e n e r a t i n g t i s s u e ,
f r o a the p r e starting
and t h e e p i c a r d i u *
existing vessels
Iral:
1028
photo
treated
definitely
*
.
•
border
BB3 t e p t a l
Interesting
of dead a a t e r i a l
a r e a of f r a g m e n t a t i o n
by
and
perfused
channels
nonperfused
Yigorous active vascular
tissue vith s e v e r a l
regeneration
collagenited
a r e a s and v e r y p r o m i n e n t v e s s e l s
Ihick-valled
arterioles
prominent
Again n o n i n v o l v e d muscle a b s o l u t e l y u n r e m a r k a b l e
vfaols
EX f i b r i n
Sral:
as
stain
N
good
above
1029
Posterior
?3
Some s u b e p i c a r d i a l
of f i b r o b l a s t s
patches
(age? g e n e r a l
a c t i v a t i o n by t r e a t m e n t
ischemia?,
of s p o n t a n e o u s
ischemic
lesions?
1030
A2
Large i n f a r c t e d
and g o o d
a r e a in t h e a c t i v e
reperfusion
of l i v e r e g e n e g r a t i n g
interdigitation
cells
Hierohemorrhage
present
photo *
. . .
of s u r r o u n d i n g a d v a n c e d
Tascularity
tissue
regenerating
Increased
P a t c h e s of a a t u r e
collagen very small,
peripheral
Xvalt
1031
treated
iefinitely
A1
•
P a t c h y dead t o n e s ;
perfusion
regular
slides
as o t h e r
Oood i n t e r d i g i t a t i o n
between
of
channels
breaking
-
90
present,
necrotic
-
but
aaterial
not
as
and
r
0 2 5 3 0 2 2
#5
flog 1J[ c o n t i n u e d
AMED 20 6
12/17/85
, i••
livt
and v e s s e l s
cells
regenerating
n
a
t
u
r
e
b
u
t
Saa*
collagen,
iioitly c e l l u l a r
Svalt
1032
1033
llvtr
Pronounced
Ho
clearing
treated
and
of
ballooning
cells
Kuiele
s i t t
injection
reaction
significant
Overall
evaluation
of
•f f ect , Rating
B o g 17:
'@ 5.
definitely
-
91
treated,
-
vith
cood
0 2 5 3 0 2 2
...
Dogifi
1034
.
Dog »
„
•
vith
advanced
trtattd
lateral
border third
•lict
l
i
ttlt
Siailarj
dead t i s s u e ;
(
AMEEf 2 0 6
12/17/85
2% I n f a r c t
11 3 7 8
W
P e r ' u s l ,o n
of old c h a n n e l s - p o s i t i v e
Kecrotlc n a t e r i a l
breaking up
Active, vascular
regenerating tissut
organitation
Xvalt
1035
#5
LB3
vessels
treated
It alt
not
so
iapressivt
(probablj)
•
1036
Beptal b o r d e r
Looks l i k e a h e a a n g i o a a ,
although lost Bight be
Bieroheaorrhage
One s p o t
(arrow):
denst a e e u a u l a t i o n
of Y t s s e l i v i t h o u t
photo
'
A2
1037
»
At In A3, a l l c r i t e r i a
of t r e a t a e n t
art p r e s e n t
Xval:
1038
*
1040
" 1041
photo
A1
l o o k s as I f t h e v e s s e l s
really
b r e a k tip t h e l i t t l t
r e a a i n i n g dead a a t e T I i l ;
neither
p o l y s nor B a c r o p h a g e s
proainent
Xnzyaes s t a i n s
a Bust
It
Xval:
1039
'
treattd
Mood
treated,
definitely
liver
Ttry congested
and p a r t i a l l y
henorrhagic,
clear
ballooning
cell phenoaena
Muscle,
site
Injection
Soaevhat widened i n t e r s t i t i u a ,
Kusclt
control
sase
•
otherwise
but
little
of
noraal
• —
O v e r a l l t v a l u a t l o n Dog IB: t r e a t e d
vith
g o o d , b u t . n o t g r e a t ,'
•
• .
«ffect,
R a t i n g « 5.
«ote:
Gross
Infarct
s i t e too s n a i l ,
b u t c o u l d t h i s be a n
additional
tffect
of t r e t t a e n t ?
(Must knov i n f a r c t
•i«t
a t b e g i n n i n g of t x p e r i a e n t )
-
92
art
-
the
0 2 5 3 0 2 2
AMED 206"
#5' 1 2 / 1 7 / 8 5
*
^2
p
A1
11*53,
Dog:
31*
Infarct!
1
«
Anterior,
slics
out
infaret)
of
(centtr
. .
and s u b e p i c a r d i a l
subendo car d i a l
vith
Hecrotie
tons
•
•
tissue
granulation
but not c a p i l l a r i e s
channels perfused,
- Deadt l a r g e
not o v e r l y v a s c u l a r
not a d v a n c e d ,
tissue
- Granulation
into
nercrosis
along
f
a
r
not p e n e t r a t i n g
- libroblasts
channel
veil
not p e a c h i n g n e c r o s i s
subepicardial,
- P o l y s few,
only p e r i v a s c u l a r
developed,
- Collagen" reasonably veil
*
and s u b e n d o c a r d i a l
n
u
c
h
n
o
t
f
i
b
r
i
n
o
u
s
p
e
r
i
c
a
r
d
i
t
i
s
,
PTAH)
l
i
b
r
i
n
:
(on
s
o
m
e
intravascular
-
(
Xvslt
12
-
Xral:
slice"
(center
of
tvo
healing,
poor
slice
Anterior,
-
like*
Sralt
8S3
pattern,
poor
perfusion
infarct)
v i t h very small r i «
t o JI1; M o s t l y n e e r o t i c
Very e i n i l i a r
tissue.
of g r a n u l a t i o n
Bone c a l c i f i c a t i o n
aueh
neither
o f d e a d t i s s u e by c e l l s ;
Poor p e n e t r a t i o n
p o l y s nor s u c h f i b r o b l a s t s
fibrin
present
some i n t r a v a s c u l a r
pattern;
lo good p e r f u s i o n
•
-
(
healing
Anterior,
-
A3
xinrenarkabls
• Beptal
A1
and
insufficient
reaction,
»ust
he
control
three
A2.
Boae
interstitial
.fibrin.
control
border,
third
slice
sone
few
polys,
necrosis,
non-perfused
Extensive,
d
u
s
t
"
.
"
n
u
c
l
e
a
r
t
o
disintegrating
p^uite d e n s e ,
tissue vhich contain
of g r a n u l a t i o n
- patches
h
o
w
e
v
e
r
.
n
a
t
u
r
e
v
e
s
s
e
l
s
,
relatively
normal n u s c l e ,
no
to t o t a l l y
inert,
demarcation
- 8harp
of
l
i
v
e
i
n
p
a
r
t
v
a
s
c
u
l
a
r
i
t
a
t
i
o
n
nor p e r f u s i o n
increased
on'
PXAH)
(
b
e
s
t
border
sone
in dead s i d e v e s s e l s
not advanced;
some f i b r i n
- Collagen
of b o r d e r
-
Ivalt
poor
healing,
despite
soae
-
dense
93
-
nature
vessels
0 2 5 3 0 2 2
#5- 1 2 / 1 7 / 8 5
I*tT*l
13
•
"
free
wall
vith
border
infarct
'
tone
-.A
singlt
field
dead
increased*
within
tissut
shows
perfusion,rest
is p o o r l y
and
perfuseff
poorly
penttrattd
'
by l i v t
ftv poly«.
cells;
especially
is s o a e
- Iher»
between neerotic
interdigitation
'sateritl
and g r a n u l a t i o n
but not i o p r e s s i v t
tissue,
- Tascularity
of g r a n u l a t i o n
tissue
is c o d t r a t e
* '. '•
- Collagen
is
maturs
around
only
a large
preexisting
vessel,
reaction
overall
la a t i l l
with
very c e l l u l a r ,
little
collagen
- S h a r p b o r d e r to l i v i n g M u s d t ,
which looks n o r m a l
•
It all
little
effect
"
(
u T t r a l l Pog 19:
control
(even c o n s i d e r i n g
the l a r g e s t
infarct
so f a r )
Rating - l
-
94
-
that
this
aniaal
baft
0 2 5 3 0 2 2
AMED 2 0 5
#5 ' 1 2 / 1 7 / 8 5
]O£ 20
>.
Dog
Anterior,
1
.
-
Much
Anterior,
-
A3
infarct
.
ont
little
neerotic
tissue,
coopletely
interdigitating
vith
living
but l i t t l t
cells,
vessel!,
blood
(good for p h o t o
or t h i s
'
aspect)
...
Quite v a s c u l a r
granulation
tissue,
sort
cellular
than
collagenous
A
vascular
of
loost
very
spot
fibrosis
t o t a l l y
'
s u r r o u n d e d by n o r m a l t i s s u e
(aicroinfarct?)
•
far
Collagen
advanced
and d e n s e
in c l o s e v i n c i n i t y
tven
of t h e few r e m a i n i n g dead f i b e r s
— > p h o t o @•
It alt
12
slics
13*
1 10852,
activity
slice
and
advanced
healing
tvo
Perfused
necrotic
tissue
vith
good
ingrovth
of
individual
live
c e l l c o r d s and v e s s e l s
cells,
Advanced c o l l a g e n a t i o n
Tery
vascular
with
varied
of
stages
vascular
v a i l
@aturation;
no u n u s u a l d e n s e a c c u a u l a ^ i o n
spots h o v e v e r
Anterior,
slice
three
CtaTs
level
to
the
corresponds
used
in
one
e a r l i e r
tvaluations)
- Totally
dead a r e a s
hard to f i n d ;
almost
all
is e i t h e r
perfused
or i n t e r u p t e d
vith r e g e n e r a t i n g
cells
- Active looking granulation
tissue
- Collagen
is
dense
at
s u b e n d o c o r d ial
only
and
perovascular
but f a i n t p o s i t i v e
places,
stains
penetrate
• •
throughout necrotic
area
- Occasional
perivascular
fibrin
around v a s c u l a r
channels
p a s s i n g t h r o u g h dead m a t e r i a l
SB3
Xval:
sot
8eptal
border
• .
"
•
Ivali
likely
treated
Vide
of
extensive
area
of
dead
interdigitation
and
regenerating
tissue,
very v a s c u l a r
Remainders
of d e a d
tissue
look
perfused
although
no
capillary
pattern
c a n be r e c o g n i z e d
• *
Selected
show " c r o w d i n g "
areas
t h i n - a'nd
of v e s s e l s ,
thick vailed
("heBangiomatous"?)
ones
*8r7 q u e s t i o n a b l e
increase
of l i v e « u s c l e
in p e r f u s i o n
Areas vith » a t u r e
c o l l a g e n e a s y to f i n d
treated
@
-
QS _
0 2 5 3 0 2 2
#5**12/17/85
Dog 2 0
Itttral
133
•
,,.
-
tlict
thrsa
Siniliar
t o BB3
lWt
in t h t
Every
hai
capillary
profilt
tissue
a rtd
cell
in i t ;
i
n
c
r
e
a
s
e
d
increased
maybe
perfusion?
without
vessels
there?
Collagen
at
s u r r o u n d !
luite
places
nature;
also
individual
nuscle
fibers!
living
folIovY've'ssels
and
—
cells
Xar i n t o n e c r o i l a "
Sralx
Overall
border,
Dog
adtaaced
20:
responaa
very l i k e l y
Rating @ 4
#<
treated
-
9& -
0 2 5 3 0 2 2
AMED 2 0 6
#5-12/17/85
Dog
At
Gavra's
21.
Anterior,
*
S
Infarct
one
•
(mural) vith organising f i b r i n
- large thrombus
in n e c r o t i c
- Ho c a p i l l a r y
pattern
areas
of g r a n u l a t i o n
tissue
- Average a c t i v i t y
- C o l l a g e n not very dense
but not very m a t u r e ,
in s e v e r a l
- Yascularity
dense,
Bval:
A2
slice
21*
10557,
. . . .
area*
equivocal
infarct
Anterior,
center
material partially
Kecrotic
but l a r g e
perfused,
areas
cut o f f
totally
amounts
of p o l y s
- Unnusual
much more t h a n
penetrating
fibroblasts
do
but not o v e r l y v a s c u l a r
- Granulation tissue
active,
fibrin
i n some l a r g e
dead
- Perivaacular
channels
vithin
n
e
c
r
o
t
i
c
t
i
s
s
u
e
.
-
Jval:
A3
-
poor
Septal
border,
-
infarct
slice
no
vhat
so
border,
mud
healing
organisation
ilice
Dog 2 1 :
and
ever
three
not
much
and
nuclear
poorly
dust
vascular
advanced
three
of g r a n u l a t i o n t i s s u e ,
maturation
loose
fibrosis
vith
organisation
• unimpressive,
Rating
(>.
regenerating
center
effect
Impressive
cellularity
and l i t t l e
vascularity
Several
of v e r y
spots
capillaries
Jtval:
Overall
response;
healing
lateral
-
than
dissolving
Poor p e r f u s i o n
of n e c r o t i c
material,
from p o l y s
instead
S m a l l r i m s of p o o r l y i n t e r d i p i t a t i n g
tissue
granulation
Xval:
-
three,
sore
necrotic
vith
large
poorly perfused,
mlnnlmal r i n
area,
of s u b e n d o c a r d i a l
granulaton t i s s u e
cells
Tascularity
penetration
minimal.
by v e s s e l s ,
poor;
Ho i n t e r d i d p i t a t i o n s .
vealc.
One
clotted
Collagen
ateriole.
Zval:
-
LB3
reaction;
slice
Anterior,
-
SB3
veak
not
probably
@ 2
-.97
-
advanced
control
but
l i t t l e
very
small
0 2 5 3 0 2 2
#5
£ofi22
A1
Dog
Anterior,
ctnttr
infarct,
19f
1O665t
ilict
12/17/85
Infarct
ont
•* E m a i l s p e c i m e n ,
not such n e c r o t i o
tissue
- Reutilitation
of o l d c h a n n e l s n o t i m p r e s s i v e
— -> b u t f i b r o b l a s t i c
ptnetration
a l o n g old c h a n n t l t
i n t o fit ad t i s s u e
it n o t i c e a b l t
""""
•
#
Xvalt
c o u l d be t r t a t t d
\2
Much i n t e r s t i t i a l
b u t n o t in
blood,
fresh,
More p o l j s
than u s u a l
'
Tibroblastie
i n g r o v t h only f o c a l l y
Boat l a r g e r
vtnels
have p o l y s
in t h e v a i l
•
Xralt
A3
(vasculitis)
- Relatively
large necrotic
tont vithout p e r f u i i o n
"
« ?ibroblastic
i n g r o v t h at b o r d e r
g o o d '«
vith vaseulitis
• Kany v e s s e l s
and t h r o s b o s i s ,
fresh
tissue
cellular
and r e a s o n a b l y v a s c u l a r
- Granulation
- Collagen noflerately veil developed along v e s s e l s
• 8ubepicardial
bleeding p r e s e n t
•
•^uivocal
•
•
and
distorted
by
N
Tinftction
D e a d a r e a s v i t h good r e p e r f u s i o n
Active
of r e a c t i v e
live
interdigitation
tissue
and d e a d
'
area
Granulation
t i s s u e very v a s c u l a r
at p l a c e s
Areas v i t h
advanced collagen
are p r e s e n t
organisation
but not p r e d o m i n a n t
Xval:
1B3
channtlt
•q.uiTocal
Xral:
8B3
d t t p
advanced
healing
8ooe r e u t i l i t a t i o n
of c h a n n e l s
present
Tibroblast
not d e e p
penetration
tissue
vith
Bealing
aoderate,
organisation
average
vascularieation
not advanced;
aost
Collagen
regenerating
areas
s t i l l
very c e l l u l a r
Svals
veak
change
if
any
O v e r a l l t o g 22x
vealc c h a n g e ;
affect.
R a t i n g @ 3+
treated
but
probably
vith
l i t t l e
j |
.
QQ
0 2 5 3 0 2 2
AMED 2 0 6
#5
Voj
22
Dog
Anttrlor,
A1
•
slice
ont,
centir
7 7 7 4 5 » 9*
infarct
12/17/85
Infarct
'
•
of n e c r o t i o
islands
Ysry fev,
snail
tissue l i f t
Vascular
vithin
channels
dead
hav*
tissue
do n o t
tut
reperfusion
c
e l l *
patterns,
Infiltrating
living
instead
( e n d o t h e l i u m and f i b r o b l a s t s ,
no p o l y s )
* Granulation
tissue
vascular,
vith
very
n a t u r e ,
aany
arteriole
- type v e s s e l *
between
- Eev:
tissue
granulation
and p r e e x i s t i n g
l i r e
suscle
t
h
e
r
e
i
s
cells,
a layer
of b a l l o o n i n g
n u i c l e
cells
(hydropic change)
is very n a t u r e ,
- Collagen
highly organised
-
•
• Xvals
A2
advanced
-
Svalt
very
-
Svalt
1B3
•
-
oa .v e r a l, l,
Infarct!)
advanced
definitely
treated
f o c u s of g r a n u l a t i o n
Only s n a i l
tissue
looking a c t i v e
Questionable
increase
of a d j a c e n t
in v a s c u l a r i t y
intact
@uscle
Xval:
BB3
small
Sonevhat
Islands'
larger
necrotic
vith
extensive
old- '
channel-perfusion-pattern,
followed
closely
by l i v s
cells
regenerating
.
The p e n e t r a t i o n
of d e a d t i s s u e
vith
live r e g e n e r a t i n g
tissue
i s one of t h e b e s t
In a l l d o g s so f a r
teen
> Photo;
{
r e c u t HE » i n c e
some of t h e s e p e n e t r a t i o n
c o u l d be c u t b e t t e r )
areas
•.
very a c t i v e ,
very v a s c u l a r
tissue
granulation
one f o c u s
of c a l c i f i c a t i o n
U3)
is t r u e l y u n u s u a l !
vascularity
-
•
(but
Taseular
c h a n n e l s in n e c r o t i e
l i r e
area veil
reutillted;
cells
but
following
there
closely,
is
d
ead
still
a
c e n t e r v i t h only a fev p o l y s
In t v o a r e a s , v a s c u l a r i t a t i o n
in t h e r e g e n e r a t i n g t i s s u e is a l s o s t
but also vith o i c r o h t n o r r h a g e .
henangiomatous,
Granulation
tissue
auch c e l l u l a r ,
but
activity
high,
a l s o v i t h a r e a s of x a t u r s
"«
s
collagen
-
A3
healing
'aot
tnough I n f a r c t e d
area
( o r h e a l i n g so a d v a n c e d
reduced
infarct
site?)
that
m
and p h o t o !
- lecut
Can't l a a g i n e
• In a d v a n c e d h e a l i n g t h a n t h i s .
» o g 23:
revised.
if
t h i s vas not
Rating « 5
treated,
nn
a
it's
reflected
in
»ore
desireable
effect-
JHOTO
ay c r i t e r i a
have
to
th»
^«
amed
#5
0 2 5 3 0 2 2
12/17/85
195^ I n f a r c t
11045,
'
•
@
slice
center
infarct
@
•• Anterior,
one,
•
Large n e c r o t i c
rone, poorly p e r f u s e d
.
- Only o c c a s i o n a l
dust
p o l y s and n u c l e a r
' - Only few red b l o o d c e l l s b e t w e e n f i b e r s
rest
fresh,
art
Is old
(-ghosts")
- Cellular
i n g r o v t h very l i m i t e d
- Many v e s s e l s
in g r a n u l a t i o n
tissue
are t h r o o b o s e d
- New
tissue
cellular
vith
mostly
c o l l a g e n
poor
and m u l t i p l e
f o c i of c a l c i f i c a t i o n
organisation
- Poorly organising fibrinous
pericarditis
also p r e s e n t
fibrin
in b o r d e r
- Much
and
e
v
e
r
y
w
h
e
r
e
(recent
s
o
n
e
.
photo)
UTAH o v e r s t a i n e d ,
repeat)
Vo£ 2£
A1
Gavra's:
Xralt
-
12.
-
-
.
•
@
' -
-
healing;
poor
Lateral
-
delayed
possibly
perfusion
blockage
Same as A1 .and A2 or v o r s e ,
vith early c a l c i f i c a t i o n
Good
HE
for
quality
of
photo
rim
spicardial
of
tissue
granulation
and c a l c i u m
Thrombosed
vessels
t h r o m b i l e s s t h a n 10, b u t
present,
more t h a n 2 days o l d
Snail
island
of v a s c u l a r
c e l l s smd f i b r o b l a s t s
close
to
center
of n e c r o s i s ,
i
m
m
a
t
u
r
e
very
Zval:
133
healing,
c o m p l i c a t i o n s by c l o t t i n g
*
*
Similar
t o A1; r i m v i t h g r a n u l a t i o n t i s s u e
small and
immature,
only s u b e p i c a r d i a l
Cellular
ingrowth
o n l y 0.5mm and o n l y
along
larger
vessels
not well i n t e r d i g i t a t i n g
(photo)
Much f i b r i n
in l a r g e r " c h a n n e l s
.,
v
.
PI AH s h o w s i m p r e s s i v e
fibrin
clot
in a l a r g e r
artery
Xval:
A3
delayed
healing;
border,
third
thrombotic
complications
slice
Partial
fresh perfusion
of n e c r o t i c m a t e r i a l
I n g r o w t h p r e s e n t but not i m p r e s s i v e
Most a d v a n c e d
areas
than c o l l a g e n o u s ;
are more c e l l u l a r
@uch c a l c i f i c a t i o n
Preserved
muscle shows h y d r o p i c
c h a n g e s in b o r d e r t o n e
and a l s o
subepicardial;
otherwise
unremarkable
and n o t
overly vasculariced
,
Harked
chronic
and a c u t e p e r i c a r d i t i s ,
o f a few m u s c l e l a y e r s u n d e r n e a t h
Ival:
delayed
healing;
infection
complication
-
100
-
vith
involvement
possibly
due
to
0 2 5 3 0 2 2
AMED 2 0 6
#5
SB3
Septal
-
third
*
»lic»
-
.
like
Essentially
previous
but
slides,
no e p i c a r d i u *
and n o t as s u c h f i b r i n present,
Ria
vith
and i n f i l t r a t i n g
tissue
iivt
granulation
cells
b
e
t
v
e
e
n
a
n
d
r a n g^^e^ s — —'^ ^' ^ ^^ ^^»^ O
auch s m a l l e r
la
than
@ O.7«an,
^^ ".^J^jm^^^a^^^m ^^^@
other
dogs
Bral:
Oterall
border,
12/17/85
delayed,
Dog
24:
healing
poor
vith
control,
(throabotio
Rating
-
1
additional
and p o s s i b l y
-
101
-
c o n p l i c a t i o n s
infection)
0 2 5 3 0 2 2
AMED 2 0 6
12/17/85
#5
above
The
1 and
grades
~\
done
by
School
Gavros
by
with
actual
the
" c o r r e c t " vif
considered
and
control
the
opposite
dogs
were
and
happened.
1 or
3-
of
matched
dogs
of
the
by
was
graded
"mismatched"
or
3+
from
" According
to
this
3-
3,
was
afterward
were
and
graded
3+
or
Haudenschild
Matching
2,
"equivocal".
of
rating
unaware
experimental
Those
or
Christian
treatment.
(i.e.,
5 indicating
3,
grades
was
with
healing
4 and
grading
and
dog,
5,
suggestive
Medicine)
graded
considered
were
group
the
(i.e.,
(Dr.
of
received
treatment
H.
(CH)
a pathologist
minimal
, grades
The
1 to
graded
or
treatment)
results.
equivocal
University
Boston
of
healing
experimental
indicating
lack
treatment)
accelerated
or
effective
control
were
reports
2 indicating
of
suggestive
advanced
pathology
4 or
5
when
either
procedure :
J
Dogs
were
#10,
were
were
#6,
graded
21
7,
#1,
#14
dog
(experimental
(control)
and
#12
dog
(experimental)
22
13,
(experimental)
and
dog
#16
(control)
3.
graded
Dogs
and
2.
graded
Dogs
(control
1.
graded
Dogs
24
19,
#8
and
20 > ( e x p e r i m e n t a l )
and
dog
#9
(control)
4.
Dogs
#11,
17,
23
were
(experimental)
-
102
-
graded
5.
were
0 2 5 3 0 2 2
#5
\1,
Dogs
infarcts
4 died
and
Of
5,
and
15,
were
too
small
before
the
end
In
total,
four
were
the
of
the
discarded
Out
of
these
dogs
were
and
6 controls),
experimental
and
1 control)
experimental
and
onek c o n t r o l )
the
above)
infarct
three
, and
6
1
(2)
or
(4)
5
2
(3)-
or
(3)+
2
1
Michael
seen
Klibaner
(2
Can't
Tell
_
_
I
13
4
1
also
(MK)
for
of
(3
equivocal
Incorrect
(5)
are
(5
.
or
Results
8 were
correctly
were
died,
small.
too
mismatched
four
Two
on.
was
and
matched
(1)
Totals
and
#3
Dogs
.
operated
experimental
Correct
3
Dr.
were
eleven
18,
experimental
Results:
VII
their
study.
because
ten
18,
because
discarded
table
(see
twenty- four
remaining
controls.
18 w e r e
AMED 2 0 6
12/17/85
rating
Boston
by
another
University
pathologist
in
the
same
manner.
These
are
not
examples
meant
to
are
limit
for
the
illustrative
invention.
-
103
-
purposes
only
and
0 2 5 3 0 2 2
#5
III
Example
OF A HUMAN MYOCARDIAL
VASCULARIZATION
Four
40x
of
the
several
^
healing
Fig.
of
old
all
not
of
in
a 78
(figures
humans
and
of
25A)
treated
Human
not
In
present
cells
dead
show
d)
the
mechanisms
omental
infarct.
the
infarct
angiogenesis
lacking
healing
had
the
of
part
such
This
collagenization.
with
who
that
extract
surviving
for
a
long
time.
Without
which
would
dissolve
muscle
cells
arrive
cells
Human
also
areas
or
as
patient
center
have
can
zone,
not
or
remain
vascularization,
these
phagocytize
late
little
at
all;
access
to
areas.
myocardial
Necrotic
infarct.
vascularized.
-
104
-
also
(CMFr) .
Necrotic
such
patients,
at
zones
vascularized.
nonvascularized
25B)
different
patient
indeed
regions
myocardial
regenerating
Fig.
and
from
c,
are
subsequent
INFARCT:
human
b,
25a,
that
organization
been
year
vascularization
signs
cellular
has
sections
infarcts
mechanisms
lack
Hematoxilin/Eosin
heart
AMED 20 6
12/17/85
zone,
0 2 5 3 0 2 2
#5
the
In
round
cells
Fig.
form
blood
seen
blue
as
surviving
later
by
regenerating
a scar
of
sufficient
25C)
Human
abundant
vessels,
In
followed
to
be
can
cells.
muscle
of
vicinity
dots
which
mechanical
infarct.
myocardial
would
down
lay
heart
dead
cells
such
patients,
blood-borne
the
between
cells
AMED 20 6
12/17/85
be
collagen
strength.
Vascular
reactive
epicardium.
The
reacts
muscle,
to
growth
such
as
filled
with
red
new
represent
source
of
muscle.
heart,
similar
The
shares
blood
blood
this
cells
this
as
organs
react
the
can
space
channels
be
around
layer
lungs
and
-
around
quickly
the
heart
around
peritoneal
neovascularization.
105
vessel
new
the
vascularization
strongly
-
heart
tissue
infarcted
spaces
serosa
the
tortuous
vessels
the
for
latter,
the
as
the
lining
other
lining
well
of
with
loose
Such
potential
the
infarct
within
of
layer
micrograph:
vessels.
epicardium,
In
covering
underlying
in
seen
layers
peritoneal
an
revascularization
intestines.
omentum
a thin
epicardium,
with
or
space,
the
with
the
"@! -; k:JS1U@
:
0 2 5 3 0 2 2
AMED 2 0 6
#5 1 2 / 1 7 / 8 5
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few
vascularization
Spontaneous
of
microscopic
In
the
heart
which
muscle
to
enough
round
be
in
area
cells
only
and
spaced
the
reaction.
Vascularization
effectively
infarct
myocardial
of
lost.
this
micrograph,
This
focus
filled
with
but
densely,
this
focus
incidentally
epicardium
which
also
spontaneous
healing
often
are
been
more
underneath
that
center
entirely
Note
however,
the
have
perfused.
the
human
size.
rapidly
not
are
in
is
a normal
vascularized
by
cells
living
new
small
vessels,
also
better
is
located
a vascular
and
essential
necrotic
Large
process.
poorly
populated
shows
is
a
and
zones,
therefore
not
for
necessary
in
step
the
healing.
The
invention
claims
to
reduce
such
zones.
IV
Example
DOG MI
26:
Figure
heart
ventricles
infarcted
reaction
remain
spots.
Gross
and
of
areas
and
in
all
standard
The
dogs.
muscle
stains
unstained
Some
two
normal
which
of
photos
live
is
difference
enhanced
muscle
by
blueish;
show
their
whitish
the
first
slice
-
slices
106
an
color
the
between
enzyme
infarcted
(upper
-
through
with
left)
areas
red
blood
are
white
0 2 5 3 0 2 2
#5
of
because
the
of
beginning
the
AMED 2 0 6
12/17/85
not
valve
area;
these
are
usually
taken
from
slice
#4
center
of
infarcted.
sections
Microscopic
infarct,
of
top
unaffected
left
in
middle
(lower
the
control
ventricle,
normal
these
photos)
slice
needs
were
These
infarcts
are
for
at
#10
dogs
this
time
supplied
or
the
evaluation,
the
by
infarct
size
should
area
of
at
it
basic
that
be
which
at
between
least
is
is
only
ligated
(This
#11
Due
to
measured
the
for
artery
-
dogs
to
107
-
the
and
assure
all
at
since
area
anatomical
extent
and
ligations,
criterium
for
that
experiments,
ventricular
represents
coronary
by
evaluation
a poor
exist
the
by
time
similar
(treated),
not
artery,
be
the
the
at
should
individual
time
9% of
later,
determined
healing.
of
ligation
days
and
and
test.
infarcts
coronary
conditions
infarcted.
the
the
mainly
the
this
Ten
of
These
sections).
total
by
wall
infarct
between
a blind
control),
ligated
effects;
reasonable
of
is
variability
treatment
meaning
size
free
(laterial
several
as
P- ( p o s t e r i o r ,
photos),
zone
artery.
size
effectiveness
technical
border
done
(untreated
the
L
produced
coronary
A- ( a n t e r i o r ,
these
sometimes
readings
of
at:
, and
important
tissue,
anterior
in
muscle)
pathologic
left
are
muscle
least
usually
half
mass
of
the
supplies,
0 2 5 3 0 2 2
#5
within
the
size
scar
assessing
the
healing
process
first
set
of
8 were
the
5 experimental
)
the
of
determination
and
recognized
dogs
one
was
and
least
unusual
27;
Figure
of
prominence
Necrotic
with
vessels
Area
infarcts:
4 were
the
3 controls
In
experimental.
2
at
16% MI q u a l i t a t i v e l y
was
(2A:
of
From
point.
adequate
and
of
way
experiment,
considered
was
animal
experimental
one
the
indifferent,
one
time
with
Of
morphometric
a meaningful
a later
usable
3 controls.
the
be
may
at
and
recognized
were
known,
Once
variations.
anatomic
is
variation
this
of
extent
individual
of
limits
the
AMED 2 0 6
12/17/85
seen.
dog
#10
2B dog
control;
#11
treated)
Low p o w e r
infarct.
All
and
represent
lines
clotted
or
follows
channels,
is
more
controls
is
such
.
unusual
as
dog
be
likely
to
and
treated
of
and
#10.
muscle.
in
is
the
the.
heart
must
have
died
not
seen
to
Diffuse
and
expected,
animals,
small
other
-
108
treated
dog
in
-
#11
cells
this
the
area.
in
extent
into
not
vessel
larger
the
places,
hemorrhages
occur.
dots
(i.e.
too
this
red
fresh
muscle
bleeding
at
the
The
and
capillary
with
of
center
which
pattern
original
cells
the
dead
some
The
together
vascular
pattern
controls
blood,
the
although
respective
is
background
hemolyzed)
apparently
The
pink
of
(16x)
micrographs
do
infarct
both
indeed
in
0 2 5 3 0 2 2
AMED 2 0 6
12/17/85
#5
Border
28;
Figure
Similar
untreated
fibrin
the
#10)
the
red
red)
the
represent
either
white
cells
larger
tissue
cells,
can
and
more
the
faster
greater
is
connect
with
healing
step,
channels
more
chance
the
is
the
but
29;
cellular
purple
dots
or
regenerating
vascular
are
controls
the
These
which
left
is
but
(A)
imagine
the
arise,
possibly
a desired
of
perfusion
it
far,
so
old
was
animals.
(4 Ox)
Of
which
ellipsoids
cells.
and
The
be.
living
Infarct
micrograph
in
not
to
vessels
Perfusion
of
regenerating
follow
can
regeneration.
channels
control
in
may
micrographs
power
active
they
first
easy
new
and
periphery
the
or
is
the
channels.
of
blood
numerous
treated
Border
Higher
cells
in
the
It
that
seen
where
more
lumina.
the
at
channels,
seen.
bleeding
also
impressive
Figure
the
old
be
vascular
the
positions
from
is
blood
fresh
Both
#12.
in
and
#12)
(Dog
vascularity
seen:
present
cells,
that
Dog
vascular
or
treated
are
from
are
lines
infarct,
between
samples
sample
(bright
The
infarct
differences
(Dog
in
intense
of
are
(dog
lower
at
the
interest
seen
follow
nuclei
here
in
#10) , m o s t
corner;
-
109
the
in
-
the
the
the
most
larger
of
living
meshwork
both
of
of
the
are
represent
cells
edge
of
old
In
pictures.
regenerating
micrograph
of
the
0 2 5 3 0 2 2
AMED 2 0 6
12/17/85
#5
animal
treated
throughout
all
channels
and
are
intermixed
cells,
but
details,
since
differences,
position,
there
specimen
showing
overall
The
is
30:
These
from
an
treated)
of
the
rim
by
in
the
represent
also
shows
left
corner.
pinker
pink
from
photo
of
rest
blood
control,
the
muscle
cells
muscle
both
cells
at
the
at
the
infarct
the
8
dog
border
infarcted
line
is
area
top
of
is
right
contain
micrographs
side
of
living,
structures
and
some
elongated
which
are
more
prominent
vessels,
treated
a preexisting
exactly
the
taken
are
7 control,
(dog
specimen,
Circular
the
either
experienced
show
to
the
In
a few
new
in
sections
experiments
treated
cells.
this
cellularity.
several
HE s t a i n e d
the
The
picture.
regenerating
of
slightly
in
of
importance.
selected
are
of
and
important
cells
living
or
of
pale
(40x) .
of
one
cells
from
away
gradient
muscle
Border
series
They
represented
slits
of
by
relatively
picture;
the
a kind
round
evidence
as
are
(gray),
illustrate
more
many
Regenerating
infarct
a small
the
.
be
therefore
earlier
this
a millimeter
only
may
dead
smaller
suitable
too
evaluation
pathologists
Figure
not
are
the
with
like
cells
regenerating
between
Pictures
neutrophils.
^v
#11) , t h e
(dog
animal.
coronary
-
This
artery
110
-
photo
branch
incidently
in
the
upper
0 2 5 3 0 2 2
AHED 2 0 6
12/17/85
15
large
may
specimen
treated
trained
most
impressive
pictures
living
are
in
the
section
vessels
can
also
areas
When
reduced.
U
of
the
cells
The
can
such
more
higher
safely
is
the
is
seen.
In
and
within
the
the
lower
the
treatment
be
tensile
to
nor
heart
muscle
ris*
is
Thus
will
organized
strength
of
achieve
is
collagen
of
is
It
Ill
also
rcost
organizat.on.
the
present
only
strength
"
most
treated
soar
developing
the
tensile
-
cellularity
and
the
advanced
rupture.
such
in
green
completed,
collagen
as
interpreted
be
area
this
the
areas
the
produced
the
(treated,,
these
and
cellular,
very
.12
do,
greenest
The
green.
organization
have
highly
the
cells,
♦ 10
disappear.
will
vessels
animal
which
in
from
prominent,
are
cells
stains
be
these
All
Collagen
Bo,
provided
(40x, .
regenerating
present.
are
area
differences
most
section
each
in
organization
cdlagenous
results.
absolutely
the
with
area
the
for
several
preferentially
or
healing
advanced
Most
many
from
impressions
conclusive
yield
(green)
collagen
more
one
by
The
seen.
be
oan
combined
the
may
advanced
cells
a little
pathologist*
Searching
the
show
sections
32:
Figure
differences
multiple
in
£ieWs
no
Only
control.
the
than
virtually
areas
over
that
demonstrate
to
(1«.)
micrographs
power
Low
reaction
Cellular
31:
Figure
one
and
goal
and
of
advanced
0 2 5 3 0 2 2
AMED 2 0 6
12/17/85
#5
organization
few
Relatively
advanced
must
scar
and
that
all
have
been
but
preceding
not
so
easily
the
as
as
for
the
also
point
this
to
leading
of
degree
the
success
of
measurable
healing
events,
function
well
event
thus
accelerated;
time
this
at
this
However,
rupture.
healing
indicator
hearts
surviving
organization
rupture.
prevent
from
an
determine
which
to
die
is
collagenization
desirable,
thus
MI p a t i e n t s
healing
indicates
and
early
very
and
the
of
performance
a possibly
lower
and
healing
previous
rish
of
reinfarction.
i
33;
Figure
These
treated.
of
Advanced
micrographs
shows
One
regenerating
gradient
ranging
to
right)
vessels
dead
from
heart
represent
relatively
muscle
organized
high
a
final
Haudenschild
Cardiovascular
analysis
(C.H.)
'and
Research
by
Dr.
dogs,
the
other
, with
active
two
a
abundant
patterns
processes.
V
pathologists
Michael
Laboratory,
-
(B)
(green,
Both
healing
both
distribution
even
cellularity.
desirable,
(40x)
collagen
left)
(gray,
Example
In
(A) ,
collagen
highly
and
different
two
of
area
and
active
from
are
an
cells
(Everywhere)
to
appear
healing
112
Klibaner
Boston
-
Dr.
Christian
(M.K.)
University
of
the
School
0 2 5 3 0 2 2
#5
of
Medicine,
blind
done
evaluations
were,
both
by
Mass,
of
the
dog
myocardial
experimental
and
control
reviewers
For
this
second
(1-5)
were
not
VIII.
dogs
Boston,
independently
but
the
size
of
the
animals
See
following
infarct,
were
table
balloon
early,
all
infarcts
above.
as
the
analysis,
included,
of
regardless
of
AMED 2 0 6
12/17/85
occluded
(6-24)
areas
in
resulting
19
evaluations.
between
Agreement
animals
both
and
were
between
7,
of
these
CH and
CH was
CH w e r e
13/19
incorrect
animals
were
called
totalled
treated
This
these
4 and
MK was
4 of
for
is
19
dogs
as
10
are
evaluated
as
to
9 dogs
16).
19
in
animals
6 i.e.
and
10
animals
the
10
and
were
-
compared.
113
-
19
dogs
by
in
12
of
19
The
9 control
treated
calls
following
these
experiments.
3 of
were
of
7/10
MK w e r e
incorrect
and
3.
of
and
3 of
animals
and
for
analysis
8 of
19
Disagreement
correct
and
The
in
correct
for
9 control
summarized
of
calls
treated
treated
12
controls
CH t o t a l l e d
5 of
2 of
6/9
correct
controls.
7 i.e.
data
of
9 controls
called
as
and
the
7 of
calls
correct
in
13,
for
calls
were
(#9,
in
and
of
consisting
were
3 dogs
consisting
experimental
both
found
the
found
MK was
MK was
correct
Therefore
12,
to
as
wrong
CH and
for
MK
called
called
Table
Also
animals
as
as
controls.
IX w h e r e
included
in
0 2 5 3 0 2 2
#5
the
following
(MK)
and
Thus
these
one
-tables
CH c o r r e c t
dog
#5) .
dog
non-evaluation
tables
the
is
total
calls
20
were
for
#5,
(i.e.
which
3,
and
14/20
without
From
dogs.
for
>
-
114
one
-
Table
AMED 2 0 6
12/17/85
evaluation
+ or-).
guess
IX
MK 1 3 / 2 0
it
is
seen
(including
0 2 5 3 0 2 2
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#5 1 2 / 1 7 / 8 5
TABLE
VIII
MATCHED EVALUATION OF
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LEGEND t o
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or c o n t r o l .
E/C - e x p e r i m e n t a l
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Thert
right
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CM o f
and
which
•
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-
116
-
vtre
0 2 5 3 0 2 2
AMED 2 0 6
#5 1 2 / 1 7 / 8 5
TABLE
COMPARISON
R
1
3
2
1
IX
OF PATHOLOGISTS1
UK
RATINGS
CH
W
It
1
3
1
3
1
2
2
1
_W_
33
.
2
3+
3
4
2
5
4
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T h t 1 ft 5 a r « ▼ • r y s i g n i f i c a n t
for
l o o k r a n d o m f o r HK b u t s i g n i f i c a n t
-
117
b o t h RK ft CB.
for CB.
-
Tht
2 ft 4
0 2 5 3 0 2 2
#5
What
is
1.
Compositions
Claimed;
ischemic
humans
for
the
lesions,
treatment
or
consisting
omentum-derived
2.
AMED 20 6
12/17/85
of
and
lipids
in
infarcts
myocardial
isolated
gangliosides.
of
Claim
1 consisting
essentially
Composition
of
Claim
1 consisting
essentially
essentially
of
ganglioside
Composition
in
general
of
mixtures
of
essentially
myocardial
angina,
of
lipid
material.
3.
4.
Compositions
ischemic
x
5.
for
lesions
or
humans
consisting
lipids
and
of
Compositions
lipid
mixtures
Composition
of
physiologically
ganglioside
of
treatment
infarcts
myocardial
essentially
angina,
of
myocardial
in
purified
general
in
mixtures
of
gangliosides.
physiologically
6.
the
material.
Claim
4 wherein
active
or
material
a single
Claim
or
the
consists
a single
-
118
purified
consists
purified
4 wherein
material
mixtures
the
of
lipid.
purified
essentially
purified
-
essentially
of
ganglioside.
0 2 5 3 0 2 2
#5
7.
Claim
of
Composition
active
physiologically
of
extraction
8.
Composition
omentum
of
Claim
least
at
mixture
is
a physiologically
extract
of
omentum.
one
active
obtained
organic
3 wherein
2 or
1,
is
mixture
omentum
by
the
3 wherein
2 or
1,
AMED 2 0 6
12/17/85
the
by
solvent.
omentum
methanol
chloroform
s
>
a
9.
Composition
mixture
is
of
Claim
3 wherein
2 or
1,
active
a physiologically
the
omentum
hexane-ethanol
extract.
10.
Composition
extract
is
of
Claim
derived
11.
12.
Composition
of
Claim
a physiologically
of
omentum.
Claim
active
of
essentially
ganglioside,
to
collateralization
lesions
the
or
2,
1,
myocardial
the
supercritical
4,
3,
lipid
the
or
5 or
composition
angina,
6 wherein
infarctions.
-
119
-
the
ganglioside-lipid
myocardial
infarctions
extract
gas
vascularization
improved
in
3 wherein
2 or
1,
Composition
leads
of
extract.
is
material
hexane-ethanol
extraction
further
from
chloroform-methanol
the
9 wherein
and
and
ischimic
tissue
bordering
3
,
0 2 5 3 0 2 2
#5
13.
of
Composition
the
Claim
essentially
material
leads
in
14.
to
.
essentially
ganglioside,
claims
1,
myocardial
tissues
Composition
of
Claim
essentially
ganglioside,
leads
to
collagenation
lesions,
or
of
essentially
ganglioside,
material
perfusion
leads
in
to
or
ganglioside-lipid
of
myocardial
lesions,
the
2,
3,
lipid
or
or
perfusion
myocardial
infarction.
5 or
4,
the
6 wherein
the
ganglioside-lipid
accelerated
myocardial
organization
ischemic
infarctions.
Composition
Claim
6 wherein
lipid
angina,
myocardial
the
5 or
increased
in
bordering
4,
bordering
1,
lesions,
3f
2,
ischemic
or
and
myocardial
tissues
improvement
infarctions
and
16.
and
of
material
ganglioside-lipid
ischemic
myocardial
Composition
angina,
or
improved
infarctions
in
lipid
allowing
infarctions
to
6 wherein
5 or
4,
vascularization
angina,
leads
3,
improved
myocardial
material
15.
2,
ganglioside,
collateralization
perfusion
1,
AMED 2 0 6
12/17/85
1,
2,
3,
lipid
improvement
4,
or
of
hearts.
transplanted
-
120
-
5 or
6 wherein
the
ganglioside-lipid
vascularization
and
0 2 5 3 0 2 2
#5
17.
Composition
of
essentially
ganglioside,
material
by
but
18.
leads
aortic
not
in
1,
to
essentially
ganglioside,
in
Claims
infarcts
Method
for
lesions
which
of
enhanced
using
beds
supplied
cardiac
coronary
techniques
3,
4,
5 or
lipid
or
ganglioside-lipid
2,
such
6 wherein
perfusion,
ischemic
of
as
the
vascularization
and
lesions,
angina,
infarction
collagenation
and
myocardial
conditions
in
isolated
amounts
vascularization
and
ischemic
myocardial
in
active
physiologically
omen t u m - d e r i v e d
materials
ganglioside
and
organization
treatment
myocardial
comprises
mixtures
vascular
humans.
the
or
the
by
increased
myocardial
in
of
6 wherein
or
ganglioside-lipid
or
vascular
1,
collateralization,
angina,
5,
angioplasty.
of
and
4,
reopened
Composition
to
3,
improvement
vessels
leads
2,
lipid,
peripheral
limited
material
19.
to
and
and
grafts
Claims
AMED 2 0 6
12/17/85
lipid
sufficient
humans
amounts
of
and
to
collateralization
observe
in
said
conditions.
20.
Method
consists
of
Claim
19 w h e r e i n
essentially
of
the
omentum
lipid.
-
121
-
derived
material
0 2 5 3 0 2 2
#5
21.
Method
of
Claim
consists
22.
Method
for
23.
Method
of
Method
Claim
Method
22
consists
Claim
of
material
25.
using
purified
Claims
extraction
26.
Method
extract
27.
Method
extract
of
is
of
is
of
ischemic
conditions
and
lipids
wherein
the
22 w h e r e i n
20
19,
physiologically
of
21
at
25 w h e r e i n
25
wherein
is
least
the
wherein
the
a hexane-ethanol
-
active
active
the
obtained
one
by
organic
physiologically
solvent.
active
extract.
physiologically
extract.
122
of
ganglioside.
a chloroform-methanol
Claim
amounts
lipid.
material
by
active
humans
gangliosides.
of
the
or
in
physiologically
essentially
omentum
Claim
angina , myocardial
physiologically
active
physiologically
of
essentially
consists
of
material
omentum
infarction
myocardial
of
derived
the
ganglioside.
treatment
comprises
material
24.
the
or
mixtures
).
of
essentially
lesions
which
wherein
19
AMED 2 0 6
12/17/85
-
active
0 2 5 3 0 2 2
#5
28.
Method
of
extract
Claim
is
27 w h e r e i n
a hexane-ethanol
chloroform-methanol
29.
Method
of
Claims
essentially
material
30.
Method
of
essentially
31.
Method
essentially
material
32.
19,
to
conditions
.
Method
Claims
essentially
material
20,
19,
to
collateralization
23
22,
lipid
or
in
21,
from
the
21,
or
lipid
20,
or
lipid
improvement
and
123
24
or
the
or
24 w h e r e i n
the
or
24
in
said
wherein
the
lipid-ganglioside
vascularization,
in
-
wherein
lipid-ganglioside
23
of
the
conditions.
formation
perfusion
-
or
said
23
22,
wherein
lipid-ganglioside
in
scar
21,
24
conditions.
23
22,
or
lipid-ganglioside
said
22,
lipid
improved
ganglioside,
reads
21,
collagenation
ganglioside,
leads
of
20,
ganglioside,
Claims
derived
extract
perfusion
19,
improves
of
20,
ganglioside,
claims
active
physiologically
extract.
19,
enhances
material
the
AMED 2 0 6
12/17/85
transplanted
hearts
0 2 5 3 0 2 2
#5
33.
Method
Claims
of
essentially
material
by
grafts
but
not
ganglioside,
reads
aortic,
and
20,
19,
to
limited
to
lipid
of
124
wherein
beds
cardiac
techniques
by
angioplasty.
-
24
vascular
grafts,
reopened
or
the
lipid-ganglioside
or
vascular
vessels
23
22,
improvement
perpheral
in
21,
AMED 2 0 6
12/17/85
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supplied
coronary
such
as
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J
)
Cate9ory
X,D
European. Patent PARTIAL EUROPEAN SEARCH REPORT
which under Rule 45 of the European Patent Convention
Office
shall be considered, for the purposes of subsequent
proceedings, as the European search report
DOCUMENTS CONSIDERED TO BE RELEVANT
Citation of document with indication, where appropriate.
of relevant passages
Y
Y
article
*
* Abstract
*
EP-A-0
682
095
* Claims 1 , 7 , 9 ;
Hn*
p3rp ^
86
11
A 61- K 3 7 / 2 2
,,
, 01
A
1-18
CHEMICAL ABSTRACTS, v o l .
89, n o .
13, S e p t e m b e r 25, 1 9 7 8 , p a g e 5 2 3 ,
ref.no.
US
Ohio,
104765g;
Columbus,
J.I.
RABINOWITZ e t a l . :
"Lipid
of
and b i o s y n t h e s i s
composition
human o m e n t a l t i s s u e . "
& LIPIDS 1978,
13(5),
3
1
7
2
EP
„*
:oic0//o*7'
-
@.
.
2
.
.
.
:
.
1-18
(SOLCO
page
m *
BASEL
1,
line
AG)
32
-
1-18
technical fields
SEARCHED (.nt. CM,
A 61
K
./.
INCOMPLETE SEARCH
The Search Division considers that the present European patent application does not comply with
the provisions of the European Patent Convention to such an extent that it is not possible to carry
out a meaningful search into the state of the art on the basis of some of the claims.
1- 18
Claims searched completely:
Claims searched incompletely:
19 —33
Claims not searched:
Reason for the limitation of the search:
of t h e human or a n i m a l
Method for t r e a t m e n t
(see a r t .
52(4)
b o d y by s u r g e r y or t h e r a p y
of t h e E u r o p e a n P a t e n t
Convention) .
Place of search
The
Date of completion of the search
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19-10-1987
CATEGORY OF CITED DOCUMENTS
X: particularly relevant if taken alone
Y: particularly relevant if combined with another
document of the same category
A : technological background
O: non-written disclosure
P : intermediate document
7698
CLASSIFICATION OF THE
APPLICATION (Int. CI.4)
Relevant
to claim
JOURNAL OF THE AMERICAN MEDICAL.
15,
2 5 2 , no.
ASSOCIATION, v o l .
O c t o b e r 19, 1 9 8 4 , p a g e s 2 0 3 4 - 2 0 3 6 ;
H.S.
GOLDSMITH e t a l . :
"Lipid a n g i o from o m e n t u m . "
genie factor
* Whole
Application number
Examiner
RIJCKEBOSCH
T: theory or principle underlying the invention
E: earlier patent document, but published on, or
after the filing date
.
D: document cited in the application
L : document cited for other reasons
_
_
&: member of the same patent family,
corresponding
document
J
»
European Patent
Office
PARTIAL EUROPEAN SEARCH R E P O R T
EP
86
11
-
DOCUMENTS CONSIDERED TO BE RELEVANT
Category
Citation of document with indication, where appropriate, of relevant
passages
EP-A-0
038
* Claims
P,X
18-26;
{CHEM.
INST.
example
2 -
CLASSIFICATION OF THE
APPLICATION (Int. CI.4)
SCHAFER
2 *
1-18
BIOLOGICAL ABSTRACTS, v o l .
82, n o .
ref.no.
56680
6, 1 9 8 6 , p a g e A B - 7 7 0 ,
US
Philadelphia,
"Increased
H . S . GOLDSMITH e t a l . :
vascuclar
adminiper f u s i o n a f t e r
stration
of an o m e n t a l l i p i d
fraction."
& SURG. GYNECOL. OBSTET. , 1 6 2 ( 6 ) ,
1986.
579-584,
* Abstract
P,X,
D
511
Relevant
to claim
7698
*
WO-A-86 01 111
UNIVERSITY)
1-18
(TRUSTEES
OF BOSTON
* Claims;
15-26;
p a g e 5, l i n e s
page
15 - p a g e 18, l i n e
13 *
15, l i n e
EPO Form 1505.3 06.78'-
1-18
TECHNICAL FIELDS
SEARCHED (Int. CM)