801

Collagenase-Induced Intracerebral
Hemorrhage in Rats
Gary A. Rosenberg, MD, Sheila Mun-Bryce, BS, Mary Wesley, BS, and Mario Kornfeld, MD

Intracranial bleeding is an important cause of brain masses and edema. To study the
pathophysiology of intracerebral hemorrhage, we produced experimental hemorrhages in 53
rats and characterized the lesion by histology, brain water content, and behavior. Adult rats
had 2 fil saline containing 0.5 unit bacterial collagenase infused into the left caudate nucleus.
Histologically, erythrocytes were seen around blood vessels at the needle puncture site within
the first hour. By 4 hours there were hematomas, the size of which depended on the amount of
collagenase injected. Necrotic masses containing fluid, blood cells, and fibrin were seen at 24
hours. Lipid-filled macrophages were observed at 7 days and cysts at 3 weeks. Water content
was significantly increased 4,24, and 48 hours after infusion at the needle puncture site and for
24 hours in posterior brain sections. Behavioral abnormalities were present for 48 hours, with
recovery of function occurring during the first week. Brain tissue contains Type IV collagen in
the basal lamina. Collagenase, which occurs in an inactive form in cells, is released and
activated during injury, leading to disruption of the extracellular matrix. Collagenase-induced
intracerebral hemorrhage is a reproducible animal model for the study of the effects of the
hematoma and brain edema. (Stroke 1990:21:801-807)
Downloaded from http://ahajournals.org by on March 8, 2020

I ntracerebral hemorrhage occurs frequently in

patients with cerebrovascular disease, brain
tumors, or head trauma.1"3 The effects of a
hematoma on brain tissue have been studied experi-
mentally using the direct infusion of blood4-8 and by
inducing a hemorrhage after infarction.9"13 During a
study of the effect of proteolytic enzymes on the
extracellular matrix in rats, we observed that the
metalloproteinase collagenase injected directly into
the caudate nucleus caused an intracerebral hema-
toma. Earlier investigators had shown an increase in
blood-brain barrier permeability following intraven-
tricular injection of this enzyme.14 Collagenases are
proteolytic enzymes that are present within cells in an
inactive form and that are secreted at sites of inflam-
mation by mononuclear cells and by metastatic
tumors.15-18 Brain tissue contains collagen in the basal
lamina of blood vessels.19"21 We describe the effects of
From the Neurology Service (G.A.R., M.W.), Veterans Affairs
Medical Center and the Departments of Neurology (G.A.R.),
Physiology (G.A.R., S.M-B.), and Pathology (Neuropathology)
(M.K.), University of New Mexico School of Medicine, Albuquer-
que, New Mexico.
A preliminary report of this work was presented in New
Orleans, Louisiana, at the September 1989 meeting of the Amer-
ican Neurological Association.
Supported by a Veterans Administration Merit Review and
National Institutes of Health grant RO1 NS21169-04 (to G.A.R.).
Address for correspondence: Gary A. Rosenberg, MD, Depart-
ment of Neurology, University of New Mexico School of Medicine,
Albuquerque, NM 87131.
Received November 13, 1989; accepted January 26, 1990.
collagenase-induced intracranial bleeding on histol-
ogy, brain water content, and behavior over 3 weeks.
Materials and Methods
We anesthetized 53 adult male Sprague-Dawley
rats weighing 200-300 g with 50 mg/kg i.p. pentobar-
bital. Rats were placed in a stereotactic apparatus
(David Kopf Instruments, Tujunga, California), and
a 23-gauge needle was implanted into the caudate
nucleus at coordinates (A5.8, L3.0, H1.0).22 The 40
experimental rats had 2 ^,1 of saline containing 0.01-1
unit bacterial collagenase (Type XI or Type VII,
Sigma Chemical Co., St. Louis, Missouri) infused by
a microinfusion pump (Harvard Apparatus, South
Natick, Massachusetts) over 9 minutes. Initially,
Type XI collagenase with some protease contamina-
tion was used. Subsequent studies were done with
Type VII collagenase that was essentially free of
proteases. The 13 control rats had similar infusions of
2 n\ saline. After infusion, the needle was removed
and the wound was sutured. The rats recovered from
surgery in a warm place with access to food. They
were killed by the intracardiac injection of KC1. The
experimental protocols were approved by the Animal
Research Committee and adhered to National Insti-
tutes of Health guidelines.
For histology, nine experimental rats were infused
with 0.01 (n=3), 0.1 (n=2), or 1 (n=4) unit (1,680
units/mg) Type XI collagenase; 4 hours later they
were anesthetized again and killed. The brains were
 802 Stroke Vol 21, No 5, May 1990
removed, placed in phosphate-buffered 10% forma-
lin for 24 hours, and embedded in paraffin for
sectioning on a microtome into 4-^irn slices. Slides
were stained with hematoxylin and eosin, cresyl violet
(Nissl), or Luxol fast blue. Four control rats were
infused with saline, two with heat-inactivated colla-
genase, and two with 250 units hyaluronidase (Sigma)
and killed 4 hours later to test for nonspecific effects
of the infusion protocol. Three experimental rats
Downloaded from http://ahajournals.org by on March 8, 2020
infused with 0.5 unit Type VII collagenase were
killed 10, 20, or 45 minutes later to determine the
early histologic changes caused by collagenase. Six
experimental rats infused with 0.5 unit Type VII
collagenase were killed 7 days or 3 weeks later to
determine late histologic changes.
For the determination of brain water content, 13
experimental and control rats were infused with 0
(*=2), 0.1 («=3), 0.5 (n=5), or 1 («=3) unit Type XI
collagenase; 4 hours later they were killed, the brains
were removed and divided into hemispheres, and
each hemisphere was sectioned into four parts from
front to back. Each section was weighed before and
after drying for 24 hours in an oven at 100° C. Water
content was expressed as the percentage change
between wet weight (WW) and dry weight (DW):
[ ( W W - D W ) H - W W ] X 1 0 0 % . Another six experimen-
tal rats had 0.5 (n=3) or 1.0 (n=3) unit Type XI
collagenase infused to study the effect on brain water
after 24 hours. Thirteen experimental rats were
infused with 0.5 unit (1,920 units/mg) Type VII
collagenase and killed 4, 24, or 48 hours later. The
brains were prepared as above. These 13 rats were
compared with five control rats injected with saline
and killed 4 hours later.
A behavioral rating scale to grade the extent of
injury was modified from published scales.11-23"26
Three behaviors were tested: ipsilateral circling,
bilateral grasp, and beam walking. The extent of
FIGURE 1. Photomicrograph
of histologic changes at needle
puncture site in caudate nucleus
4 hours after infusion of 2 \d
saline in control rat. There is
damage at needle puncture site,
with preservation of adjacent
cells. Hematoxylin and eosin
stain.
circling to the side of the infusion was graded from 0
(no circling) to 4 (always circled). Grasp was tested
by placing the rat's paws on the edge of a box 14
inches high; strength of the hemiparetic paw was
graded from 0 (grasped well) to 4 (unable to grasp
with forepaw). Beam walking was graded by placing
each rat on a beam; a grade of 0 indicated that it
easily traversed the beam, while a grade of 4 was
given those unable to walk on the beam.24 A total
injury score was calculated as the sum of the grades
on the three tests.
Data were analyzed using SAS.27 Statistical sig-
nificance (p<0.05) was determined using one-way
analysis of variance with the Bonferroni correction
for multiple tests.
Results
Infusion of 2 fil saline into the caudate nucleus
caused a small focal area of damage around the
needle in the control rats (Figure 1). Rats infused
with heat-inactivated collagenase or hyaluronidase
showed needle damage similar to controls. Infusion
of 0.01 unit collagenase resulted in a lesion slightly
larger than that in controls, and increasingly larger
lesions were produced by infusions of 0.1 and 1 unit
collagenase. The extent of the lesion depended on
the amount of enzyme injected. Bleeding was seen 10
minutes after infusion around a large, thin-walled
vessel in the caudate nucleus (Figure 2, top). Rats
studied 20 and 45 minutes after infusion had intact
erythrocytes dissecting between normal brain cells in
the caudate nucleus (Figure 2, bottom). By 4 hours
there was extensive bleeding without evidence of
tissue necrosis. Erythrocytes dissected into the tissue,
and brain cells remained intact.
At 24 hours after infusion the lesion consisted of
two zones. The large central zone had a mosaic-like
appearance, with numerous areas composed of pale-

Downloaded from http://ahajournals.org by on March 8, 2020
staining, extravasated erythrocytes separated by frag-
ments of necrotic parenchyma. The sharp border
between these two elements was in places obscured
by extravasated fibrin found near necrotic blood
vessels containing fibrin thrombi. The peripheral
zone of the lesion consisted of a narrow band of
poorly staining parenchyma containing either viable
or necrotic glial and neuronal cells. There were foci
of hemorrhage composed of well-preserved erythro-
cytes and occasional polymorphonuclear leukocytes.
This zone also contained intact blood vessels and
some with necrosis and fibrin thrombi within their
lumina. After 48 hours, the changes were similar to
those observed at 24 hours (Figure 3, left), except
that in the central zone erythrocytes were in a more
advanced stage of decay, while the peripheral zone
Rosenberg et al Collagenase-Induced Hemorrhage 803
FIGURE 2. Photomicrographs
of histologic changes in caudate
nucleus of rat soon after infusion
of 0.5 unit collagenase. Top: At
10 minutes, large, thin-walled
vessel surrounded by intact eryth-
rocytes is seen (hemataxylin and
eosin stain, x30). Bottom: At 45
minutes, erythrocytes are seen to
dissect between intact cells (hema-
toxylin and eosin stain, x30).
contained an intense infiltrate of polymorphonuclear
leukocytes (Figure 3, right). The peripheral zone
contained extravasates of preserved erythrocytes,
blood vessels with hypertrophied endothelium, and
sparse mononuclear cells in the perivascular spaces.
Seven days after infusion, the mosaic-like pattern
was still seen in the central zone, where fragments of
the necTotic parenchyma were recognizable only by
the presence of ghost cells (Figure 4). The erythro-
cytes were extremely pale and often had ill-defined
contours. There were also rare macrophages and
small vascular profiles with swollen endothelium,
mostly at the edges of the central zone. The periph-
eral zone consisted of a broad band of densely
packed macrophages, many containing multiple lipid
droplets. A few macrophages containing hemosiderin
 804 Stroke Vol 21, No 5, May 1990

FIGURE 3. Photomicrographs ofhistologic changes in caudate nucleus of rat 48 hours after infusion of 0.5 unit collagenase. Left:
Large lesion protrudes into lateral ventricle. Central white matter of each hemisphere behind basal ganglia exhibits some pallor.
Right: Infiltrate of potymorphonuclear leukocytes at periphery of lesion (hematoxylin and eosin stain, X229).
Downloaded from http://ahajournals.org by on March 8, 2020

were found at the outer limits of the peripheral zone.

In places, small fragments of necrotic parenchyma
were discernable among the macrophages. There was
an increase in vascularity, with small blood vessels
characterized by plump endothelium.
Twenty-one days after infusion, the two zones were
no longer apparent. The lesion was transformed into
a cyst traversed by delicate gliovascular trabeculae,
the meshes of which contained variable numbers of
hemosiderin and lipid-laden macrophages. At the
border of the cyst there were variable numbers of
reactive astrocytes.
Edema present 4 hours after infusion depended on
the amount of collagenase infused (Figure 5). Water
content at the needle puncture site was significantly
greater than that in the contralateral hemisphere of
rats infused with 0.5 or 1.0 unit collagenase (p<0.05).
No rat injected with 1.0 unit collagenase survived for
24 hours, while those given 0.5 unit did. Therefore, 0.5
unit was selected for long-term experiments.
Water contents in the four sections of each hemi-
sphere 4,24, and 48 hours after the infusion of 0.5 unit
collagenase are shown in Figure 6. By 4 hours a
significant increase was seen at the needle puncture

FIGURE 4. Photomicrograph of histologic changes in cau-

date nucleus of rat 7 days after infusion of 0.5 unit collagen-
ase. Band of macrophages surrounds central zone, which
exhibits mosaic pattern (hematoxylin and eosin stain, x36).
 Rosenberg et al Collagenase-Induced Hemorrhage 805

* *

T
2 .H

T
T
T
i
1
T • FIGURE 5. Bar graph of effect of
different doses of collagenase in 2
yl saline on water content in con-
tralateral (open bars) and ipsilateral

•
(shaded bars) caudate nucleus of

I
T 13 rats 4 hours after infusion.
78-

1 1
CONTROL 0.1
COLLAOEHASe DOSE (UMTS)
as 1.0
*p<0.05 different from contralat-
eral by analysis of variance.

site and in both posterior sections. Rats killed at 24 disruption by the dissecting erythrocytes. Brain
hours had significantly increased water contents in a edema and behavioral abnormalities began to resolve
similar pattern, except the contralateral caudate by 48 hours. Histologically, collagenase caused a
nucleus was also edematous. At 48 hours water con- hemorrhagic lesion, with erythrocytes seen in the
tent was increased only in the infused hemisphere. perivascular spaces and in white matter fiber tracts.
Total injury scores over 3 weeks are shown in Consolidation of the necrotic mass resulted in a cyst
Figure 7. Before infusion the rats performed all tests by 3 weeks. There was diffuse brain edema, maximal
without difficulty, as shown by the score of 0. Some at the needle puncture site, but also present in both
variability, most likely related to the anesthetic, was posterior brain sections and in the contralateral
seen at 4 hours. Performance remained significantly caudate nucleus.
impaired for up to 48 hours. Recovery began by 72 Earlier investigators, who simulated an intracere-
hours and continued for the 3 weeks of testing, with bral hemorrhage by injecting 0.5-1.5 ml blood into
most of the improvement seen during the first week. the brains of dogs, observed dissection of tissue, with
slit-like lesions through the white matter.4 More
Discussion recently, investigators showed that the intracerebral
Downloaded from http://ahajournals.org by on March 8, 2020

injection of 0.05-0.25 ml autologous blood into the

We found that an intracerebral hemorrhage was brains of cats reduced cerebral blood flow in the
produced by the infusion of bacterial collagenase into tissue around the hematoma.5"7 Hemorrhages have
the caudate nucleus. Ten minutes after injection, been observed9 to occur sporadically after experimen-
erythrocytes were seen around large caudate blood tal infarction. -13
vessels. There was extensive bleeding 4 hours after We found that rats injected with ^0.5 unit bacte-
the infusion of ^0.5 unit collagenase, with tissue rial collagenase had extensive edema at multiple

* 4
*
83 -I 1
82-
T
4

• 81- T 4
a
9- T

n
\ •"-**'
$ 80- T T ^ . T
FIGURE 6. Bar graphs of water
79- •
content in four brain sections of
78- 13 rats 4, 24, and 48 hours after
1 infusion of 0.5 unit collagenase
\ into caudate nucleus. Small
drawing in center shown regions
- ^
1 sampled and needle puncture
83- 3. 4. site. *p<0.05 different from con-
*
82- trol by analysis of variance.
T

o 81"
*
4 * 4
4 4 *
J 80- X • * -
x T

S« 7 9 - Si
T
0 4 24 46 0 4 24 48 0 4 24 48 0 4 24 48
Hours Hours Hours Hours
 806 Stroke Vol 21, No 5, May 1990
TIME
sites. All rats injected with 1 unit collagenase died of
massive edema and brain herniation by 24 hours,
while most of those injected with 0.5 unit survived.
The greatest increase in water content was seen at
the site of the hemorrhage, probably due to the large
hematoma and the surrounding swollen tissue. At 4
and 24 hours, both posterior brain sections had
edema, which had resolved by 48 hours. Behavioral
testing showed abnormal total injury scores at 4, 24,
Downloaded from http://ahajournals.org by on March 8, 2020
and 48 hours, with improvement beginning at 48
hours.
The pathologic changes that were observed in the
collagenase lesion are most consistent with those
described for an intracerebral hemorrhage.28 The
earliest change seen was an accumulation of eryth-
rocytes around blood vessels. The blood cells were
subsequently seen to dissect away from the blood
vessels into intact brain tissue. Some necrotic vessels
were seen in the lesion, but the erythrocytes
appeared to move between brain cells. Collagenase-
induced intracranial bleeding may be important in
clinical conditions with leukocyte infiltration, tissue
necrosis with release of intracellular proteolytic
enzymes, and rapidly growing infiltrative malignan-
cies. Enzymatic disruption of the extracellular matrix
may occur in hemorrhagic infarctions, traumatic
hemorrhages (early and late), and in brain tumors
that bleed, such as metastatic melanomas. 18>29'30
Collagenases are a group of metalloproteinases
that degrade interstitial and basement membrane
collagen.16 The enzymes are released by macro-
phages and other mononuclear cells in response to
injury and are able to act on the extracellular matrix
at neutral pH.17 Immunocytochemical studies indi-
cate that brain collagenase surrounds blood vessels.31
Type IV collagen is found in the basal lamina sur-
rounding brain blood vessels and in the pial lining of
the Virchow-Robin spaces. 19~21- 32
Collagenase-induced bleeding is a reproducible
method to induce an intracranial hemorrhage. This
F I G U R E 7. Graph of total
injury (sum of three tests) (behav-
ioral) scores for rats infused with
0.5 unit collagenase into caudate
nucleus. Increased numbers of
rats at earlier times reflect use of
rats from histologic and water
content studies. *p<0.05 dif-
ferent from controls by analysis of
variance.
aid
model has features seen in intracerebral hemorrhage
in humans, but on a compressed time scale. It should
provide information on the response of brain tissue
to bleeding and on the role of the basal lamina in the
blood-brain barrier.
Acknowledgments
Hakim Zamir prepared the histological slides.
Pamela Livingston and Alisa Sherwood expertly
edited and typed the manuscript.
References
1. McKissock W, Richardson A, Taylor J: Primary intracerebral
haemorrhage: A controlled trial of surgical and conservative
treatment in 180 unselected cases. Lancet 1961;2:221-226
2. Wheelock B, Weir B, Watts R, Mohr G, Khan M, Hunter M,
Fewer D, Ferguson G, Durity F, Cochrane D, Benoit B:
Timing of surgery for intracerebral hematomas due to aneu-
rysm rupture. / Neurosurg 1983^8:476-481
3. Cordobes F, de la Fuente M, Lobato RD, Roger R, Perez C,
Millan JM, Barcena A, Lamas E: Intraventricular hemorrhage
in severe head injury. J Neurosurg 1983^8:217-222
4. Whisnant JP, Sayre GP, Millikan CH: Experimental intrace-
rebral hematoma. Arch Neurol 1963;9:586-592
5. Ropper AH, Zervas NT: Cerebral blood flow after experimen-
tal basal ganglia hemorrhage. Ann Neurol 1982;11:266-271
6. Nath FP, Kelly PT, Jenkins A, Mendelow AD, Graham DI,
Teasdale GM: Effects of experimental intracerebral hemor-
rhage on blood flow, capillary permeability, and histochemis-
try. / Neurosurg 1987;66:555-562
7. Sinar EJ, Mendelow AD, Graham DI, Teasdale GM: Exper-
imental intracerebral hemorrhage: Effects of a temporary
mass lesion. / Neurosurg 1987;66:568-576
8. Sinar EJ, Mendelow AD, Graham DI, Teasdale GM: Exper-
imental intracerebral haemorrhage: The effect of nimodipine
pretreatment. / Neurol Neurosurg Psychiatry 1988^51:651-662
9. Hain RF, Westhaysen PU, Swank RL: Hemorrhagic cerebral
infarction by arterial occlusion. / Neuropathol Exp Neurol
1952;11:34-43
10. Globus JH, Epstein JA: Massive cerebral hemorrhage: Spon-
taneous and experimentally induced. / Neuropathol Exp Neurol
1953;12:107-131
11. McGraw CP, Pashayan AG, Wendel OT: Cerebral infarction
in the Mongolian gerbil exacerbated by phenoxybenzamine
treatment. Stroke 1976;7:485-488
 Rosenberg et al
12. Laurent JP, Molinari GF, Oakley JC: Primate model of
cerebral hematoma. J Neuropathol Exp Neurol 1976;35:
560-568 24.
13. Yamori Y, Horie R, Handa H, Sato M, Fukase M: Pathoge-
netic similarity of strokes in stroke-prone spontaneously
hypertensive rats and humans. Stroke 1976;7:46-53 25.
14. Robert AM, Godeau G: Action of proteolytic and glycolytic
enzymes on the permeability of the blood-brain barrier.
Biomedicine 1974;21:36-39
15. Janoff A, Zeligs JD: Vascular injury and lysis of basement 26.
membrane in vitro by neutral protease of human leukocytes.
Science 1968;161:702-704
16. Harris ED Jr, Krane SM: Collagenases (third of three parts).
N EnglJMed 1974;291:652-661 27.
17. Weiss SJ: Tissue destruction by neutrophils. N Engl J Med
1989;320:365-376 28.
18. Liotta LA, Tryggvason K, Garbisa S, Hart I, Foltz CM, Shane
S: Metastatic potential correlates with enzymatic degradation
of basement membrane collagen. Nature 1980;284:67-68 29.
19. McArdle JP, Muller HK, Roff BT, Murphy WH: Basal lamina
redevelopment in tumors metastatic to brain: An immuno-
peroxidase study using an antibody to Type IV collagen. Int J
Cancer 1984;34:633-638 30.
20. Sapsford I, Buontempo J, Weller RO: Basement membrane
surfaces and perivascular compartments in normal human 31.
brain and glial tumours: A scanning electron microscope
study. Neuropathol Appl Neurobiol 1983;9:181-194 32.
21. Alcolado R, Weller RO, Parrish EP, Garrod D: The cranial
arachnoid and pia mater in man: Anatomical and ultrastruc-
tural observations. Neuropathol Appl Neurobiol 1988;14:1—17
22. Pellegrino LJ, Pellegrino AS, Cushman AJ:A Stereotaxic Atlas
of the Rat Brain. New York, Plenum Publishing Corp, 1979
23. Marshall JF, Ungerstedt U: Supersensitivity to apomorphine KEY WORDS
following destruction of the ascending dopamine neurons: brain edema
Downloaded from http://ahajournals.org by on March 8, 2020
Collagenase-Induced Hemorrhage 807
Quantification using the rotational model. Eur J Pharmacol
1977;41:361-367
Feeney DM, Boyeson MG, Linn RT, Murray HM, Dail WG:
Responses to cortical injury: I. Methodology and local effects
of contusions in the rat. Brain Res 1981;211:67—77
Bederson JB, Pitts LH, Tsuji M, Nishimura MC, Davis RL,
Bartkowski H: Rat middle cerebral artery occlusion: Evalua-
tion of the model and development of a neurologic examina-
tion. Stroke 1986;17:472-476
Wolfson LI, Brown LL: Intrastriatal injection of 3H dopamine
through a chronic cannula to produce rotation: Distribution
and concentration of the tracer in specific brain regions. Brain
Res 1983;261:205-212
SAS Institute, Inc: SAS User's Guide: Statistics, Version 5. Cary,
NC, SAS Institute, Inc, 1980
Zulch KJ: Hemorrhage, thrombosis, embolism, in Minckler J
(ed): Pathology of the Nervous System. New York, McGraw-Hill
Book Co, 1971, pp 1499-1535
Yates PO: Vascular disease of the central nervous system, in
Blackwood N, Corsellis JAN (eds): Greenfield's Neuropathol-
ogy. Chicago, Year Book Medical Publishers, Inc, 1971, pp
86-147
Morin MA, Pitts FW: Delayed apoplexy following head injury
("traumatische spat-apop\zxis"). J Neurosurg 1970;33:542-547
Montfort I, Perez Tamayo R: The distribution of collagenase
in normal rat tissues. / Histochem Cytochem 1975;23:910-920
Williams SK, Gillis JF, Matthews MA, Wagner RC, Bitensky
MW: Isolation and characterization of brain endothelial cells:
Morphology and enzyme activity. J Neurochem 1980;35:
374-381
blood-brain barrier cerebral hemorrhage
rats