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(Stroke. 1996;27:2312-2320.)
© 1996 American Heart Association, Inc.

Articles

Experimental Intracerebral Hemorrhage in Rats

Magnetic Resonance Imaging and Histopathological Correlates

Marc R. Del Bigio, MD, PhD; Hui-Jin Yan, MD; Richard Buist, PhD James Peeling, PhD

the Departments of Pathology (M.R. Del B.) and Radiology and Pharmacology (H.-J.Y., R.B., J.P.), The University of Manitoba and Health Sciences Centre (Winnipeg).

Correspondence to Dr J. Peeling, Department of Pharmacology and Therapeutics, University of Manitoba, 770 Bannatyne Ave, Winnipeg, Manitoba, R3E 0W3, Canada. E-mail jim@bionmr.mrrl.umanitoba.ca.

	Abstract

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Background and Purpose Intracerebral hemorrhage is associated with a considerable proportion of strokes and head injuries. The mechanism of brain cell injury associated with hemorrhage may be different from that due to pure ischemia. Therefore, it is essential that models of intracerebral hemorrhage be developed and well characterized. The purpose of this study was to obtain high-field MR images of rat brain at progressive times after induction of intracerebral hemorrhage and to correlate the images with behavior and histological evolution.

Methods Intracerebral hemorrhage was induced in rats by injection of bacterial collagenase and heparin into the caudate nucleus. Histopathological changes and corresponding MR images were studied from 30 minutes to 3 weeks after injection. Behavioral changes were also followed for 3 weeks.

Results Histological correlation showed that MR is capable of resolving the accumulation and degeneration of the hematoma, a centripetal wave of neutrophils infiltrating from the surrounding tissue beginning at 12 hours, and centripetal invasion of macrophages beginning at 48 hours. Widespread white matter edema was clearly evident on MR images for 1 week after the hemorrhage. Medium-sized striatal neurons were lost in the tissue surrounding the hematoma. Behavioral improvement was rapid during resolution of the edema but incomplete at 3 weeks.

Conclusions MR images correlate very well with histological changes in this experimental model of intracerebral hemorrhage and can therefore be used to follow changes due to drug treatments in vivo. The intense neutrophilic response to this lesion may contribute to neuronal injury at the periphery of the hematoma.

Key Words: brain edema • hematoma • intracerebral hemorrhage • magnetic resonance imaging • neutrophils • rats

	Introduction

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Considerable effort is being devoted to developing treatments for ischemic stroke. Much less effort is being directed toward hemorrhagic stroke, although it represents a significant clinical problem. Primary intracerebral hemorrhage (ICH) accounts for approximately 10% of all strokes in Western populations¹ and a considerably higher proportion in Oriental populations.² Approximately half of the clinical cases of ICH are associated with hypertension. The most common sites are basal ganglia (putamen), thalamus, cerebellum, and pons.³ Acute neurological deficits are due to direct tissue destruction, space-occupying effect of the hematoma with potential ischemic damage to adjacent tissue, and cerebral edema. Mortality in the first month after primary ICH is 32% to 55%,¹ and recovery after ICH is poor, with most survivors left with a considerable functional handicap.^{4 5} Few pharmaceutical therapies have been evaluated for treating clinical ICH; neither glycerol nor dexamethasone for edema management was found to be of any benefit.^{6 7}

The development of therapies relies on the use of animal models. Several experimental models of ICH have been described.^{8 9 10 11 12 13} Rosenberg and coworkers^{14 15} have developed a particularly elegant rat model in which intrastriatal injection of bacterial collagenase disrupts the basal lamina of cerebral capillaries and causes bleeding into the brain tissue. The evolution of the hematoma has been characterized by histological analysis at various times from 10 minutes to 70 days after injection. Brain edema accompanying the hematoma for the first few days can be reduced by treatment with the calcium channel blocker (S)-emopamil, the neuropeptide atrial natriuretic peptide, and an antagonist to the V1 receptor of arginine vasopressin.^{16 17 18} Impairment of blood clotting by intravenous administration of heparin can increase the size of ICH in rats.¹⁹ A low-field (1.5 T) MR imaging study of the evolution of the hematoma in this model has been described.²⁰ The changing appearance of the hematoma correlates with lysis of red blood cells and degradation of hemoglobin.

A highly reproducible brain injury due to ICH and the ability to follow changes in vivo before animal death are necessary to properly evaluate treatments. We have slightly modified the procedure developed by Rosenberg et al by adding heparin to the collagenase infusion. This allows us to use smaller quantities of injected collagenase and produce a hematoma that becomes contiguous more rapidly. We have obtained MR images at high field (7 T) several times up to 3 weeks after the collagenase/heparin injection. Features not apparent in MR images obtained at lower field strength provide additional information about the evolution of the hematoma. The correlation of MR images with histological and behavioral data is described here.

	Materials and Methods

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Intracerebral Hemorrhage
The experiments used 61 male Sprague-Dawley rats weighing from 280 to 350 g. The animals were cared for in accordance with the guidelines of the Canadian Council on Animal Care. Four rats served as unoperated controls. Each of the other 57 rats was anesthetized with pentobarbital (50 mg/kg IP) and placed in a stereotactic frame (David Kopf Instruments). A 30-gauge needle was introduced through a burr hole into the caudate nucleus (3 mm lateral to midline, 0.2 mm posterior to bregma, depth 6 mm below the surface of the skull). A microinfusion pump (Sage Instruments) was used to infuse 0.7 µL saline containing 0.14 U collagenase (Type IV, Sigma Chemical Co) and 1.4 U heparin (Sigma) over 5 minutes (36 rats). Three rats received heparin (1.4 U) alone, 3 rats received collagenase (0.14 U) alone, and 3 rats received a collagenase/heparin combination, which had been inactivated by microwaving for 30 seconds, in the same total volume of infusate. Twelve sham-operated control rats received a similar 0.7-µL infusion of saline without collagenase or heparin. Brain temperature was monitored and maintained (37.6°C) throughout the procedure using a tympanic membrane thermocouple probe and a thermostatically controlled water blanket. After the infusion, the needle was removed, the burr hole was sealed with bone wax, the wound was sutured, and the animal was placed in a warm box with free access to food and water.

MR Imaging Studies
MR imaging was performed on 32 rats using a Bruker MSL-X Biospec 7/21 spectrometer. For each imaging study, the rat was anesthetized with pentobarbital (50 mg/kg IP) and placed in a holder with the head positioned in a 3-cm-diameter saddle coil with an incisor bar. Spin-echo scout images were acquired first in the coronal plane and then in the sagittal plane to select reproducible coronal slice positions. Eleven contiguous coronal slices, centered 1.5 mm posterior to bregma, were imaged in two interleaved sets to minimize interslice excitation. For all coronal images, the matrix size was 256x256. Four averages were accumulated using a field of view of 3.5x3.5 cm² and a slice thickness of 1 mm. A field of view extension factor of 2 was used in the read direction, and 256 phase-encoding steps were acquired. Multislice multiecho T₂-weighted spin-echo images were acquired with echo times of 20, 40, and 60 milliseconds and repetition time of 1500 milliseconds. Total acquisition time was 51 minutes.

Three groups of rats underwent sequential examinations after collagenase/heparin injection. For early periods, the time point was defined when the acquisition was half completed. Individual rats did not undergo imaging at all 10 time points because of the risk associated with repeated anesthesia within a short period. Table 1 specifies the times at which MR images were acquired.

View this table: [in this window] [in a new window]   Table 1. Experimental Groups

Changes in white matter hyperintensity were quantified by calculating the ratio of the MR image intensity in the corpus callosum to the intensity in the lateral neocortex contralateral to the hematoma in a slice 4 mm posterior to bregma, using the image obtained with an echo time of 60 milliseconds. There was no difference in this ratio between intact controls and sham-injected controls.

Behavioral Evaluation
Behavior was evaluated by an observer blinded to the identity of the rats beginning at 12 hours after collagenase/heparin injection and repeated daily. The tests included (1) spontaneous ipsilateral circling behavior graded from 0 (no circling) to 4 (continuous); (2) contralateral hindlimb retraction after displacement of the limb 2 to 3 cm laterally, graded from 0 (immediate replacement) to 4 (no replacement); (3) ability to walk a 70-cm-longx2.4-cm-wide wood beam, graded from 0 for a rat that readily traversed the beam to 4 for a rat that was unable to move or fell off the beam; and (4) bilateral grasp, which measures the ability to hold onto a 2-mm steel rail with forepaws, graded from 0 for a rat with normal forepaw grasping behavior to 4 for a rat unable to grasp with the forepaw. The neurological deficit score was the sum of the scores from all four tests (maximum deficit score, 16).

Histological Examinations
After the final MR examination, each rat was anesthetized and killed by cardiac perfusion with 4% paraformaldehyde in 0.1 mol/L PBS (pH 7.2). Unimaged rats were killed in the same way. Table 1 specifies the times at which rats were killed for histological examination. Fixed brains were cut coronally through the needle entry site (identifiable on the brain surface), as well as 2 mm anterior and 2 mm posterior to that plane. Brain slices were dehydrated and embedded in paraffin. Sections (6 µm) were cut and stained with hematoxylin and eosin. Selected sections were stained with modified Martius scarlet blue for fibrin, Perls' Prussian blue stain for iron, and Leder's stain for chloracetate esterase activity.²¹ Selected sections were also stained by routine immunohistological methods using primary antibodies against glial fibrillary acidic protein and neurofilament. Secondary biotinylated conjugates were demonstrated using the Vector ABC kit and diaminobenzidine. Anti-rat IgG was revealed with secondary antibodies conjugated to Cy3 and examined by epifluorescence microscopy.

Medium-sized striatal neurons were quantified at the coronal level of the maximum hematoma diameter in animals surviving 1 day, 2 weeks, and 3 weeks after collagenase injection. With a square ocular graticule and x250 ocular magnification (objective magnification x20), neurons were counted in three fields (each area 400x400 µm) immediately adjacent to the hematoma site, attempting to avoid areas with large blood vessels. The hematoma margin was defined as the interface between macrophage clusters or cavity and neuroglial tissue. Three anatomically comparable fields in the contralateral caudate nucleus were assessed in the same manner. The difference between the sums of the three fields on the side of the hematoma and the contralateral side was used as an index of relative neuronal depletion in striatal tissue adjacent to the hematoma.

Statistics
Statistical analysis of the behavior scores and MR signal intensity data was carried out with ANOVA. In both cases, the posthemorrhage groups had variances significantly different than control and sham groups. Therefore, further analysis was performed with pairwise comparison using the Mann-Whitney U test for nonparametric data. Neuronal counts were analyzed with ANOVA with post hoc Bonferroni-Dunn correction for intergroup comparisons.

	Results

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There was no unexpected mortality in this set of experiments. There was a mild lag in weight gain during the first week after the induction of striatal hemorrhage. Neurological deficit scores at times from 12 hours to 21 days after collagenase/heparin injection are shown in Fig 1. Sham-operated rats had a score of 0 (no deficit) at all times after sham injection. Rats were significantly impaired during the first day after collagenase injection. Improvement was noted beginning on the second day after the hemorrhage and was maximal by 1 to 2 weeks. Even after 3 weeks there was some residual neurological deficit (P<.005), which consisted of subtle disabilities in all tasks.

View larger version (13K): [in this window] [in a new window]   Figure 1. Neurological deficit score (maximum total score is 16) of rats after intrastriatal injection of collagenase/heparin (mean±SD). All time points are significantly different from control values (12 hours to 2 weeks, P<.0001; 21 days, P<.005).

Features of the MR images (Fig 2) correlated well with gross histopathology (Fig 3). MR images from the control rats exhibited clear anatomic definition of neocortex, hippocampus, thalamus, striatum, and white matter structures including external capsule, internal capsule, corpus callosum, and fimbria. MR images from sham-operated rats showed mild hyperintensity along the needle tract in brain parenchyma at 24 and 72 hours after injection but were indistinguishable from images from control rats at later times. Histological assessment of sham injections showed small (50 µm in diameter) collections of blood alongside striatal white matter bundles and a few neutrophils at 4 hours. At 24 and 72 hours, rare macrophages and neutrophils were present along the needle tract, and at 1 week there was focal reactive astrogliosis and a few macrophages. Rats that received heparin alone and those that received microwave-inactivated collagenase/heparin had slightly larger collections of blood (100 to 150 µm) than those with sham injections, but the neutrophilic infiltrate was minimal.

View larger version (197K): [in this window] [in a new window]   Figure 2. Representative T2-weighted MR images of intracerebral hematoma in rat brain at the level of the collagenase/heparin injection. The images, taken at the times indicated, are not from a single animal. Note the excellent correlation with the photographs of comparable histological sections in Fig 3. C indicates intact control; S, sham saline injection.

View larger version (138K): [in this window] [in a new window]   Figure 3. Photographs of representative hematoxylin and eosin–stained coronal sections of rat brain at the level of the collagenase/heparin injection. Except in the case of the last three time points, these are not from the same rats whose images are shown in Fig 2; they are intended to demonstrate that the MR image appearance corresponds with the histological changes. The correlation is described in Table 2. C indicates intact control; S, sham saline injection.

Detailed correlation of the MR image features and histopathological findings are presented in Table 2. Within 30 minutes of collagenase/heparin injections, erythrocytes aggregated (50 to 100 µm) in multiple sites at the periphery of white matter bundles in the striatum and to a lesser extent along the needle tract. At 1 hour, the blood collections were still discontinuous, although overall the hematoma took on an ovoid shape with blood at the periphery restricted to margins of the white matter bundles (Fig 4). Small fibrin deposits in the striatum were demonstrable with Martius scarlet blue stain. Blood dissected through otherwise normal brain tissue, presumably along a pressure gradient following the path of least resistance along blood vessels or along axons. However, by 2 hours, the blood collection was almost contiguous and ignored microanatomic boundaries. It occupied approximately two thirds of the cross-sectional area of the striatum, extending to the ventricle wall medially and corpus callosum superiorly. Swollen glial cells with watery cytoplasm and some with eosinophilic cytoplasm were evident in the immediate vicinity. At 4 hours, there was a single roughly spherical hematoma (2.5 to 4 mm in diameter) with extensions along white matter dorsal to the striatum. In two of three rats, blood was present in the lateral ventricles. At 12 hours, fragmented brain tissue at the hematoma site contained eosinophilic neurons and some karyorrhectic nuclei. Around the periphery of the hematoma were scattered neutrophils and many swollen glial cells with pale cytoplasm. Immunohistochemical labeling for IgG (not shown) showed a diffuse halo around the hematoma and in white matter, indicating the presence of plasma-derived edema fluid. This labeling, which was less widespread than the hyperintensity evident on MR images, persisted until 72 hours. At 24 hours, viable neutrophils were concentrated in a compact band, corresponding to the MR-visible hypointense ring at the periphery of the hematoma. There was extensive neutrophil infiltrate in all major white matter structures bilaterally. Attempts to aspirate the hematoma at 4 and 24 hours were unsuccessful because the blood was coagulated.

View this table: [in this window] [in a new window]   Table 2. Summary of MRI Features and Histopathological Correlates After Intrastriatal Injection of Collagenase/Heparin

View larger version (140K): [in this window] [in a new window]   Figure 4. Photomicrograph showing the edge of the hematoma 1 hour after injection of collagenase/heparin. The blood collection is discontinuous and tends to follow the periphery of striatal white matter bundles (arrow). Hematoxylin and eosin; bar=100 µm.

At 48 hours, the hematoma developed a "targetoid" appearance on MR images and histologically. The central debris was surrounded by a compact band of neutrophils that lacked strong esterase activity, and this in turn was surrounded by viable neutrophils, some cell debris, a few macrophages, and rare clusters of intact erythrocytes (Fig 5). Rats injected with collagenase alone had hematomas of roughly the same overall area, but there was greater inhomogeneity with irregular islands of necrotic brain tissue. Neutrophils were common but tended to be more diffusely distributed, not forming the compact band seen in the collagenase/heparin-injected rats. At 72 hours, the targetoid appearance of the collagenase/heparin-induced hematoma was altered. The neutrophil layer was less compact and had moved closer to the center of the hematoma. The outer narrow hypointense rim was composed of a solid band of macrophages (Fig 6). Beginning at this stage and to a much greater extent in all subsequent stages, macrophages stained for ferric iron by Perls' Prussian blue method. At 1 week, the hematoma was resolving and there was enlargement of the lateral ventricles, often bilateral but more pronounced on the side ipsilateral to the resolving hematoma. At 2 weeks, the hematoma site consisted of a small fluid-filled cavity and some macrophages. Neutrophils were no longer evident. At 3 weeks, there were no significant changes. The surrounding tissue contained many reactive astrocytes as demonstrated by immunohistochemical staining for glial fibrillary acidic protein (Fig 7).

View larger version (153K): [in this window] [in a new window]   Figure 5. Photomicrograph showing the hematoma 48 hours after injection of collagenase/heparin. The center (arrow) consists of degenerating cells. This is surrounded by a dense collection of neutrophils (between arrowheads). Striatal tissue surrounding the periphery of the hematoma appears hypercellular. Hematoxylin and eosin; bar=250 µm.

View larger version (153K): [in this window] [in a new window]   Figure 6. Photomicrograph showing the peripheral margin of the hematoma 72 hours after injection of collagenase/heparin. Some intact erythrocytes remain (arrowhead) among the macrophages. Reactive capillaries are prominent in the surrounding neuropil (arrow). Hematoxylin and eosin; bar=100 µm.

View larger version (139K): [in this window] [in a new window]   Figure 7. Photomicrograph showing a (fluid-filled) cavity that persists at the site of degraded hematoma 3 weeks after injection of collagenase/heparin. Reactive astrocytes (dark stained cells) and dilated blood vessels surround the lesion. Immunohistochemical stain for glial fibrillary acidic protein with hematoxylin counterstain; bar=100 µm.

Quantitative analysis demonstrated a total of 270±32 (mean±SD) medium-sized neurons in the counted areas of control striatum (n=3). Striatal tissue surrounding the hematoma at 2 and 3 weeks had significantly fewer neurons than were on the contralateral control side (P<.04). This loss was not apparent at 24 hours (Table 3). The width of the band depleted of neurons measured up to 0.5 mm.

View this table: [in this window] [in a new window]   Table 3. Relative Neuronal Loss Defined as Difference Between Neuron Density Around the Hematoma and Corresponding Locations in Contralateral Striatum*

Changes in the white matter signal on T₂-weighted MR images at coronal levels posterior to the hematoma are shown in Fig 8. With the exception of the 4-hour time point, which exhibited a wide variance in signal, all time points from 2 to 72 hours inclusive were statistically significantly different from both intact controls and pooled intact and sham controls. Quantitative analysis showed that edema was maximal at 24 hours (P<.01) and almost completely resolved by 1 week (Fig 9).

View larger version (193K): [in this window] [in a new window]   Figure 8. MR images (T2-weighted) of rat brain at the level of the hippocampus 3 mm posterior to the intracerebral hematoma. The increased signal intensity (bright) represents edema in the white matter (external capsule, internal capsule, alveus, fimbria, and corpus callosum).

View larger version (41K): [in this window] [in a new window]   Figure 9. Ratio of the MR image intensity (echo time, 60 milliseconds) in the corpus callosum to that in the neocortex after collagenase/heparin injection (mean±SD). In comparison with intact control (C) and sham-injected (S) rats, rats with hematomas have increased signal intensity beginning at 2 hours and resolving by 1 week. **P<.01; *P<.05.

	Discussion

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This study shows that injection of a combination of heparin and bacterial collagenase into the rat striatum gives rise to a rapidly forming hematoma of uniform shape and reproducible size. This minor modification to the model described by Rosenberg et al¹⁴ has the benefit of minimizing the quantity of collagenase and volume of injected liquid required to produce a hematoma of a given size (0.5 U collagenase in 2 µL used by Rosenberg et al versus 0.14 U in 0.7 µL in the present study). Possible confounding effects due to tissue compression and "infusion edema" after solution injection are thus minimized. The marked behavioral abnormalities observed immediately after collagenase injection begin to resolve by 48 hours. This corresponds well with the resolution of generalized white matter edema that was found, by wet-dry weight determinations, to peak at 24 to 48 hours in collagenase-induced ICH.¹⁴ The T₂-weighted images obtained in the present study indicate that much of the edema is confined to white matter, the greatly increased signal intensity being due to increased free water.²² Edema fluid readily spreads through white matter.²³

The evolving appearance of human ICH on T₂-weighted MR images has been attributed to the effects of hemoglobin degeneration together with changes in tissue water (edema).²⁴ Briefly, early hypointensity in the hemorrhage is attributed to dephasing of proton spins as water moves into erythrocytes that contain paramagnetic deoxyhemoglobin and methemoglobin. As the erythrocytes lyse, release of methemoglobin causes increased T₂, yielding isointensity or hyperintensity on the MR images. As macrophages ingest extracellular iron and form hemosiderin, T₂ in the surrounding tissue decreases. The evolving appearance of collagenase-induced hemorrhage in rat brain on low-field (1.5 T) MR imaging is consistent with this explanation. In our present study, the progression seen from early (1 to 12 hours) hypointensity to later hyperintensity (1 to 2 days) at the center of the hematoma is also in accord with this model; so is the development of the strongly hypointense rim around the resolving hematoma after 72 hours, corresponding histologically to aggregation of hemosiderin-containing macrophages. However, the high-field (7 T) MR images obtained in the present study identify features that are not apparent on MR images obtained at lower magnetic fields. In particular, image contrast was influenced by the accumulation of neutrophils and macrophages.

Aggregating extravascular erythrocytes yield a hypointense region on T₂-weighted MR images through the effects of compartmentalized paramagnetic hemoglobin derivatives. Scattered regions of isointensity or slight hyperintensity correspond to islands of edematous brain tissue within the developing hematoma. Increasing hyperintensity at the center of the hematoma on T₂ images correlates with degenerating erythrocytes. Neutrophil infiltration into brain surrounding the hematoma begins at approximately 4 hours. Clotting blood and damaged brain tissue liberate chemotactic factors, including thrombin, that prompt the movement of neutrophils from blood into intact brain and toward the hematoma.^{25 26} Many neutrophils invade intracerebral hematomas in humans and in rats after collagenase injection alone; however, these tend to be somewhat dispersed.^{14 27} The fact that neutrophils are more concentrated in rats with collagenase/heparin injections suggests that the heparin may be acting as a potentiator of chemoattractant agents, as has been previously shown in culture.^{28 29} The aggregation of neutrophils into a compact band at the periphery of the hematoma peaks at 48 hours and is clearly seen on high-field T₂ MR images as a pronounced hypointense band. The dense aggregate of neutrophils results in high local concentrations of paramagnetic oxygen radicals,^{30 31} which may be the origin of the observed low signal on T₂ images.³² At 48 hours, as the neutrophils move further into the hematoma and become dispersed, the hypointense band becomes less well defined. Invasion of the hematoma by monocytes, which subsequently transform to macrophages, begins at 48 hours. As macrophages move into the hematoma, phagocytosed iron compounds are degraded, and the iron is sequestered in the form of hemosiderin and ferritin,³³ appearing hypointense on MR images because of the relaxation effects of these paramagnetic iron compounds. A detailed correlation of MR images and histology has not been attempted in humans and would be difficult because it would be critical to obtain tissue immediately after imaging.

The histological progression of this experimental hematoma resembles that previously reported in rats with autologous blood injections³⁴ and hematomas in dogs and humans.^{27 35} The time for resolution of the hematoma is shorter in the rat than in larger species. Differences in the rate at which a hematoma evolves may simply reflect its size. The hematoma is largely degraded by inflammatory cells that must migrate from the periphery, and it is therefore possible that the migratory speed of the cells could affect the rate of hematoma resolution. The time course of neutrophil and subsequent monocyte invasion is identical to that observed when rabbit ears are subjected to microscopic focal heat injury.^{36 37} Nevertheless, bacterial collagenase and heparin may both affect inflammatory cell migration and tissue degradation either directly or indirectly. In this regard, the model reported here differs from the human situation and models using autologous blood injections.

There is considerable interest in the possibility that activated neutrophils can cause secondary tissue injury and contribute to edema formation through the release of reactive oxygen species and a variety of proteases.^{30 38} Depletion of circulating neutrophils reduces brain injury after middle cerebral artery occlusion.³⁹ The loss of neurons from tissue peripheral to the hematoma site proper may be multifactorial. Passing neutrophils may release neurotoxic substances as they migrate into the hematoma. In addition, as in brain injury due to focal ischemia, a penumbra of partially ischemic tissue may surround the hematoma as a result of focal compression and distortion.^{9 40 41} In human ICH, the space-occupying effect remains the major acute problem. However, subsequent management of patients depends on controlling edema, and late recovery is determined by loss of neurons and connections at and around the site of bleeding. This experimental model of ICH in the rat can therefore be used to test therapies directed at three separate processes that may contribute to brain injury: edema, inflammation, and penumbral ischemia.

	Acknowledgments

 
This work was funded by the Canadian Heart and Stroke Foundation and by the Canada/Fisons Fight Stroke program. We thank Dr Shang-Gong Chen for his valuable assistance.

Received March 18, 1996; revision received July 8, 1996; accepted August 28, 1996.

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Editorial Comment

Magnetic Resonance Imaging and Histopathological Correlates

Gregory J. del Zoppo, MD, Guest Editor

Department of Molecular and Experimental MedicineThe Scripps Research InstituteLa Jolla, Calif

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References Introduction  References

 
Intracerebral hemorrhage is an important cause of neuron injury. Whether from transformation of ischemic tissue after vascular occlusion or secondary to aneurysmal rupture, the results may be devastating. Hemorrhagic transformation is a common accompaniment of ischemic cerebral vascular disease manifest as stroke and is likely to involve microvascular processes.^{1R 2R 3R 4R 5R} In particular, hemorrhage that follows rupture of a lentriculostriatal artery may have devastating consequences as the blood pool expands into this delicate tissue. Contact with tissue factor, found mostly within the gray matter of the striatum,^{6R 7R} initiates thrombin formation by activation of the extrinsic coagulation systems,^{8R 9R} which converts the blood to a coagulum. The cellular and mechanical events that follow are poorly understood.

There are few models with which to study hemorrhagic transformation. Del Bigio et al, in this elegant study, describe cellular changes in the surrounding tissue as hemorrhage induced in the rat caudate nucleus evolves. According to the authors, their model, derived from a collagenase-based system,^10R takes advantage of the use of heparin intended to reduce collagenase concentration and infusion volume and thereby produces relatively consistent lesions. Both this model and its forerunner depend on the degradation of collagen IV fibrils, which contribute to the microvascular extracellular matrix and basal lamina.^3R The addition of heparin provides a questionable contribution, although it may have maintained the liquidity of the leaked blood until its antithrombin effect was overwhelmed by tissue factor.

Nonetheless, a relatively consistent lesion could be produced with a predictable evolution monitored by MR, and a predictable resolution of the neurological deficits, leaving only minor residue by 21 days. The cellular changes are of interest and the major focus of this article. Much of the early invasion of polymorphonuclear leukocytes is owed to cytokine release and endothelial adhesion receptor responses. Polymorphonuclear leukocytes also release MMP-8, a unique collagenase capable of cleaving collagens I through III^11R and other enzymes adept at dissolving matrix boundaries. Here, the injected bacterial collagenase would be expected to facilitate leukocyte transmigration and may have accelerated the appearance of those cells. The attendant edema seemed to contribute to the neurological deficit, since resolution of edema on the MR images was related to behavioral improvement. The cause of the white matter hyperintensity attributed to edema was not clarified. This may be an important avenue for future investigation. To what degree the edema may have been due to microvascular leakage of plasma, or the response of surrounding neuropil to the presence of the hemorrhagic mass, was not explored. Given the time course of events, the delay in resolution of the hyperintense band compared with neutrophil disappearance and involution suggests that the two events were not entirely related. How cytokines might be generated in the evolution of the hemorrhage is also of considerable interest. Further investigation into these and other provocative questions is allowed by the availability of a relatively simple model of intracerebral hemorrhage.

	References

Top Abstract Introduction Materials and Methods Results Discussion References Introduction  References

 

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