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(Stroke. 1996;27:490-497.)
© 1996 American Heart Association, Inc.

Articles

Lobar Intracerebral Hemorrhage Model in Pigs

Rapid Edema Development in Perihematomal White Matter

Kenneth R. Wagner, PhD; Guohua Xi, MD; Ya Hua, MD; Marla Kleinholz, BS, BSN; Gabrielle M. de Courten-Myers, MD; Ronald E. Myers, MD, PhD; Joseph P. Broderick, MD Thomas G. Brott, MD

From the Departments of Neurology (K.R.W., Y.H., R.E.M., J.P.B., T.G.B.) and Pathology and Laboratory Medicine (G.X., G.M. de C.-M.), University of Cincinnati College of Medicine, and Medical Research Service (K.R.W., M.K., R.E.M.), Department of Veterans Affairs Medical Center, Cincinnati, Ohio.

	Abstract

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Background and Purpose The mechanisms underlying brain injury from intracerebral hemorrhage (ICH) are complex and poorly understood. To comprehensively examine pathophysiological and pathochemical alterations after ICH and to examine the effects of hematoma removal on these processes, we developed a physiologically controlled, reproducible, large-animal model of ICH in pigs (weight, 6 to 8 kg).

Methods We produced lobar hematomas by pressure-controlled infusions of 1.7 mL of autologous blood into the right frontal hemispheric white matter over 15 minutes. We froze brains in situ at 1, 3, 5, and 8 hours after hematoma induction and cut coronal sections for hematoma assessment, morphological brain examination, and immunohistochemical and water content determinations.

Results At 1 hour after blood infusion, "translucent" white matter areas were present directly adjacent to the hematoma. These markedly edematous regions had a greater than 10% increase in water content (>85%) compared with the contralateral white matter (73%), and this increased water content persisted through 8 hours. In addition, these areas were strongly immunoreactive for serum proteins. Intravascular Evans blue dye failed to penetrate into the brain tissue at all time points, demonstrating that this serum protein accumulation and edema development were not due to increased blood-brain barrier permeability.

Conclusions Experimental lobar ICH in pigs models a prominent pathological feature of human ICH, ie, early perihematomal edema. Our findings suggest that serum proteins, originating from the hematoma, accumulate in adjacent white matter and result in rapid and prolonged edema after ICH. This interstitial edema likely corresponds to the low densities on CT scans and the hyperintensities on T₂-weighted MR images that surround intracerebral hematomas acutely after human ICH.

Key Words: brain edema • cerebral hemorrhage • white matter • pigs

	Introduction

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It has been demonstrated that ICH exhibits a higher mortality rate and produces more severe neurological deficits in survivors than any other stroke subtype.¹ This fact is highlighted in the recent epidemiological studies by Broderick et al² and Lisk et al,³ who reported that 89% (n=171) and 79% (n=75) of ICH patients, respectively, either died or were moderately to severely disabled at discharge or after 30-day follow-up. At present, there is no effective surgical or medical treatment that improves ICH outcome.⁴

To effectively treat ICH, a better understanding of the pathophysiological and pathochemical mechanisms that lead to brain injury from ICH is necessary. To study these mechanisms, we have developed a large-animal model of ICH in pigs. To avoid unphysiological conditions and to have well-contained, reproducible hematomas for pathophysiological, biochemical, and surgical removal studies, we chose to produce lobar hematomas and to infuse blood over 15 minutes rather than by rapid injections.^{5 6 7 8} This method reduces the likelihood of ventricular rupture or reflux of blood along the needle track that can lead to subarachnoid and subdural blood accumulations,^{6 7 8} and it also avoids severe mechanical trauma. Furthermore, slower blood infusion protocols compared with rapid infusions at high pressures more closely model human brain ICH. Such bleeds generally originate from small intraparenchymal arteries and occur at lower rates and pressures than those from ruptured aneurysms that bleed into the subarachnoid space at arterial pressures.⁴ This conclusion is also supported by the "gradual or smooth" time course of signs and symptoms that develop in the majority of ICH cases.⁴ Recently, slower blood infusions with improved hematoma productions were also described in rats by Hoff and coworkers.⁹

We chose to use the pig as an animal model and to produce lobar hematomas for several reasons. The pig's large, gyrated brain, well-developed white matter, uniform health status, and relatively low cost make it an excellent animal model for lobar ICH studies. The ability to use 20- to 30-fold larger ICH volumes in the pig compared with rodent species^{5 7} (recent studies use 2.5-mL infusion volumes^{10 11} ) enables examination of surgical hematoma removal. Infusions into the subcortical white matter rather than the gray matter of the basal ganglia result in more reproducible hematoma volumes, since blood more easily dissects between myelinated fibers¹² than through the tight neuropil of gray matter structures. In addition, the greater white matter volume in large animals enables serum to accumulate and edema to develop adjacent to hematomas. This model is clinically relevant, since the frequency of lobar ICH is second only to putaminal ICH and is as common as or more common than thalamic hemorrhage.⁴ In younger patients, aged 15 to 45 years, lobar ICH is reported to be the most frequent site.^{4 13}

In the present report we describe our initial findings with a model of lobar ICH in pigs. Preliminary reports of these findings have been presented.^{10 11 14 15}

	Materials and Methods

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The animal protocol for these studies was approved by the Institutional Animal Care and Use Committees of the University of Cincinnati College of Medicine and the Research Service of the Department of Veterans Affairs Medical Center in Cincinnati.

Animals
Pigs (weight, 6 to 8 kg) were obtained from a local farm (John Erickson Company, Dillsboro, Ind). Preoperatively, all pigs were allowed food and water ad libitum. Pigs were not deprived of food overnight before the experimental procedure because preliminary results showed their strong tendency to develop marked hypoglycemia during longer procedures (>5 hours).

Animal Surgical Preparation
The surgical procedures for physiological monitoring (tracheotomy, femoral vessel catheterization) were similar to those described previously by our laboratory for other large-animal species.^{16 17 18 19 20} All surgical procedures were performed with the use of aseptic techniques. Pigs were initially anesthetized with ketamine (25 to 30 mg/kg) IM. After sedation, pentobarbital (35 mg/kg) was administered through an ear vein to achieve a deep surgical level of anesthesia. After placement of the femoral vein catheter, pentobarbital was infused at a rate of 10 mg/kg per hour throughout the remainder of the experiment.

After surgical anesthesia was achieved, a tracheotomy was quickly performed and a cuffed endotracheal tube inserted and the cuff inflated. Pigs were then mechanically ventilated with the use of a pediatric respirator, and the respirator rate and tidal volume were adjusted to achieve arterial blood gases and pH within physiological limits (PO₂, 100 to 150; PCO₂, 35 to 40 mm Hg; pH, 7.40 to 7.50). The right femoral artery was catheterized for continuous blood pressure monitoring and to permit withdrawal of blood samples (0.3 mL) for determinations of respiratory gas and acid-base status and glucose concentrations (0.3 mL). A right femoral vein catheter was also placed to infuse saline solutions and to inject pharmacological agents. Core temperature was measured with a rectal thermistor probe and was maintained at 38.5±0.5°C with the use of a warm water blanket. Arterial blood samples were withdrawn during the control period, and hourly after ICH production up to the time of brain freezing. They were analyzed for PO₂, PCO₂, pH, and base deficit with the use of a Corning model 168 acid-base and respiratory gas analyzer. Serum was separated by centrifugation in a Beckman microfuge, and serum glucose concentrations were determined with the use of a Beckman Glucose Analyzer II.

Intracerebral Blood Infusion
Before blood infusion, the animal's head was shaved and disinfected, and full aseptic techniques were used. A cranial burr hole (1.5 mm) was then drilled 10 mm to the left of the sagittal and 10 mm anterior to the coronal suture. A 20-gauge sterile plastic catheter cut to a length of 14 mm was then placed stereotaxically (guided by the pig brain atlas of Yoshikawa²¹ ) into the center of the left frontal cerebral white matter (centrum semiovale) at the level of the caudate nucleus and cemented in place. A 300-mm-long silicone elastomer tubing (Sil-Med) connected to the arterial catheter was filled with 10 mL of arterial blood by opening a three-way stopcock. A pressure-controlled IVAC infusion pump was then connected, and 1.7 mL of blood was infused into the brain tissue over 15 minutes.

Evans Blue Infusions
To test BBB permeability to albumin-bound Evans blue dye, we infused Evans blue (1 mL/kg IV, 2% wt/vol solution; Clasen et al²² ) immediately after the completion of the intracerebral blood infusion. Three animals were infused at each time point. After in situ brain freezing (described below), brain sections were examined grossly for Evans blue staining (Fig 5). To determine whether albumin-bound Evans blue was extravasated from the clot and permeated the extracellular space, we infused Evans blue IV (1 mL/kg) at 30 minutes before removing the arterial blood sample for intracerebral injection. Brains of these animals (n=2) were then frozen at 8 hours after blood infusion (Fig 6).

View larger version (159K): [in this window] [in a new window]   Figure 5. Coronal section through a hematoma in the subcortical white matter of a single gyrus at 8 hours after 1.7-mL blood infusion. In this animal, Evans blue dye was administered intravenously immediately after hematoma induction. Although a rim of edema surrounds the hematoma, no blue staining of this edematous white matter was observed.

View larger version (157K): [in this window] [in a new window]   Figure 6. Coronal section through a hematoma at the level of the thalamus at 8 hours after blood infusion shows diffuse Evans blue staining of subcortical white matter adjacent to the hematoma. In this animal, Evans blue dye was given intravenously 30 minutes before the blood was removed for infusion into brain. This result demonstrates the diffusion of serum albumin with bound Evans blue from the hematoma into the surrounding white matter.

CTP Recording
A Millar microtip pressure transducer was used to measure CTP. The catheter tip was inserted into the brain to a depth of 2 mm through a 1-mm burr hole in the skull placed 10 mm posterior to the ICH injection site. An 18-gauge stainless steel needle guide was cemented in place through the burr hole to direct the catheter tip for brain penetration. The CTP was continuously recorded along with blood pressure and heart rate. All CTP recordings showed complex waveforms that reflected the superimposition of the pressure waves of both blood pressure and respiration (Fig 1). These recordings remained drift-free throughout the periods of recording. Previous brain pathology studies showed only a minute penetrating cortical lesion corresponding to catheter tip penetration.

View larger version (10K): [in this window] [in a new window]   Figure 1. Example of CTP and MABP recordings during and for 45 minutes after blood infusion. The arrows indicate the beginning and end of the 15 minutes of infusion. The average CTP value at the end of infusion was 15.7±12.8 mm Hg (mean±SD, n=21).

Brain Tissue Fixation and Sampling
One, 3, 5, or 8 hours (n=5 each except n=6 at 8 hours) after ICH production, brains were frozen in situ by decanting liquid nitrogen into a stainless steel cap adhered to the skull with Dow-Corning silicone grease as described previously by us for several large-animal species.^{16 17 18 19} The frozen heads were then cut with a band saw into 5-mm-thick coronal sections in a low-temperature room (-10°C). Coronal slices were examined for hematoma location and volume measurements and for Evans blue penetration. Tissue was then sampled for water content determinations or immunohistochemical studies in a refrigerated glove box (-20°C) from the coronal sections at specific white matter sites identified by their relation to the hematoma. These sites included "translucent" and "normal-appearing" white matter directly adjoining the hematoma (Fig 2), posterior ipsilateral white matter (centrum semiovale) located on the first coronal slice posterior to the hematoma and/or edema, and corresponding contralateral gyral white matter.

View larger version (137K): [in this window] [in a new window]   Figure 2. Coronal section at the level of the caudate nucleus at 1 hour after blood infusion shows a hematoma extending into the subcortical white matter of two gyri. The asterisk indicates white matter regions adjacent to hematomas (referred to as translucent) that were visibly changed in appearance due to marked edema. The cross indicates white matter regions adjacent but medial to hematomas that failed to show physical changes (referred to as normal-appearing) but were mildly edematous compared with posterior or contralateral subcortical white matter.

Immunohistochemistry
Direct immunofluorescence for fibrinogen and complement or immunoperoxidase for albumin was performed on cryostat sections by standard methods^{23 24} on in situ frozen brain tissue samples (n=3) at 1 hour after hematoma induction.

Brain Tissue Water Contents
Edema was quantitated by determining the water content of white matter samples listed above by weighing and then drying the samples at 100°C for more than 24 hours to a constant weight. Water contents were expressed as a percentage of wet weight.

Statistical Analysis
Data from different animal groups and brain sites were analyzed with a one-way ANOVA (Statgraphics, Manugistics, Inc). Post hoc comparisons between groups were made with Duncan's multiple range test. Differences were considered significant at P<.05.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
In preliminary studies, we found that large-gauge stainless steel needles could not be used to slowly infuse blood because of clotting within the needle shaft. By using plastic catheters and silicone elastomer tubing to contain the blood during the infusion, we successfully infused 1.7 mL of blood over 15 minutes without anticoagulating agents.

Physiological parameters were recorded at the end of the control period and then hourly after infusion (Table). No significant differences were observed for arterial blood gases and pH at any time points. No differences were present between postinfusion values for core temperature; however, all values were slightly but significantly higher than control. Cerebral perfusion pressure, calculated as MABP-CTP, was maintained greater than 80 mm Hg at all time points after infusion.

View this table: [in this window] [in a new window]   Table 1. Physiological Parameters at the End of the Control Period and Hourly After Intracerebral Blood Infusion

Fig 1 shows an example of the CTP and MABP tracings during blood infusion. In this animal, CTP rose rapidly within the first 3 minutes to 15 mm Hg and then gradually increased to a mean value of 28 mm Hg by 7.5 minutes of infusion. CTP remained at this level until the end of the 15-minute infusion. Blood pressure in the animal in Fig 1 also increased gradually during infusion, from a mean value of 90 at the start to 105 mm Hg by the end of the infusion.

Mean values (±SD) for the change in CTP from control values (CTP) after infusion are presented in the Table. When the infusion was stopped, the mean value for CTP fell to 3.8±2.3 mm Hg at 1 hour after infusion. Thereafter, CTP averaged approximately 6 mm Hg through 8 hours after infusion.

Size and Appearance of Hematomas, Immunofluorescence, and Evans Blue Staining
We successfully infused 1.7 mL of autologous blood into the centrum semiovale near the caudate nucleus in 21 of 23 animals. In 2 animals that were excluded, hematoma sizes were substantially smaller (<0.25 mL), apparently as a result of blood loss along the needle track and into the subarachnoid space. Hematoma sizes were similar (no significant differences) at the four time points; average hematoma volumes were as follows (mean±SD): 1.26±0.36 mL (n=5) at 1 hour, 1.57±0.47 mL (n=5) at 3 hours, 1.60±0.58 mL (n=5) at 5 hours, and 1.19±0.37 mL (n=6) at 8 hours after infusion.

Hematomas were consistently located in the white matter of the centrum semiovale at the level of the caudate nucleus and anterior thalamus (Figs 2, 5, 6) and extended into the gyral white matter to the frontal pole (Fig 3). In all animals studied, we observed white matter regions adjacent to hematomas (referred to as translucent) that were visibly changed in appearance (Fig 2) because of marked edema (demonstrated by water content measurements in Fig 8). Other white matter regions, also located adjacent but medial to hematomas, appeared normal but were mildly edematous by water content determinations compared with posterior or contralateral subcortical white matter (referred to as normal-appearing). In coronal sections through the ipsilateral anterior pole white matter, we also observed substantial edema development (Fig 3). This edema accumulation presumably resulted from fluid movement in the extracellular space along white matter fiber tracts toward the frontal pole from the posterior extent of the hematoma. These 1.7-mL blood infusions resulted in a mass effect sufficient to cause bilateral herniation of cerebral hemispheric tissue and produce lateral compression against the superior colliculi (Fig 4).

View larger version (137K): [in this window] [in a new window]   Figure 3. Coronal section at the anterior pole at 3 hours after blood infusion demonstrates substantial edema development at this level in white matter surrounding the hematoma.

View larger version (27K): [in this window] [in a new window]   Figure 8. Water contents in translucent, normal-appearing, and posterior ipsilateral and corresponding contralateral white matter samples (regions defined in Fig 2 and in "Materials and Methods") in experimental animals at 1, 3, 5, and 8 hours after blood infusion. Values indicated by the bars and vertical lines are mean±SD for n=5 animals at 1, 3, and 5 hours and for 6 animals at 8 hours after infusion. *P<.001 vs ipsilateral-posterior and corresponding contralateral white matter. **P<.05 vs ipsilateral-posterior and corresponding contralateral white matter.

View larger version (181K): [in this window] [in a new window]   Figure 4. Coronal section at the level of superior colliculus at 8 hours after blood infusion shows bilateral herniation of cerebral hemispheral tissue (arrows) and lateral compression against the superior colliculi. On frozen sections, a slight pink discoloration was observed in the tips and along the compressed edges of the herniated tissue. Unpublished data (K.R.W. et al, 1995) show these areas to have fivefold increases in lactate and 50% to 75% reductions in high-energy phosphate concentrations, suggestive of ischemia.

Fig 5 illustrates a hematoma at 8 hours after 1.7-mL intracerebral blood infusion. In this animal, Evans blue was administered intravenously immediately after hematoma induction. No blue staining of the edematous white matter surrounding the hematoma was observed, suggesting that the BBB remained intact to plasma proteins. In contrast, when Evans blue was administered intravenously before the arterial blood for intracerebral injection was removed, diffuse blue staining of the white matter resulted as albumin with bound Evans blue was extravasated from the hematoma into the surrounding white matter (Fig 6). The presence of plasma proteins in the surrounding white matter was further demonstrated by immunopositive reactions for fibrinogen (Fig 7), complement, and albumin (not shown). A striking immunofluorescence for fibrinogen was observed in translucent white matter adjacent to the hematoma at 1 hour after blood infusion (Fig 7). This appearance is contrasted with the corresponding white matter region contralateral to the hematoma in which immunofluorescence for fibrinogen was only present within blood vessels, which was an expected result in unperfused in situ frozen brains.

View larger version (128K): [in this window] [in a new window]   Figure 7. Left, Immunofluorescence for fibrinogen in samples from translucent white matter at 1 hour after hematoma induction. Right, White matter region contralateral to the hematoma in which immunofluorescence for fibrinogen was only present within blood vessels.

Water Contents
The two white matter regions adjoining hematomas that differed in their physical appearance also differed markedly in their water contents (Fig 8). Water content in translucent white matter was significantly increased at all time points compared with the water content in all normal-appearing ipsilateral and corresponding contralateral white matter samples in experimental and control animals (P<.001). These increases in water content averaged 10% to 15% above posterior ipsilateral and contralateral white matter values. The water content of the translucent white matter was the same for all time points. The water content of normal-appearing white matter directly adjacent to hematomas was also significantly increased compared with the water content of other normal white matter regions in experimental animals at 3, 5, and 8 hours. This 6% increase in water content was less than that observed in translucent white matter.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
Several mechanisms have been suggested to be responsible for perihematomal edema development, including mechanical trauma,²⁵ increased BBB permeability,^{9 26} and ischemia (see below). However, our present results suggest a different mechanism. The demonstration of rapid serum protein accumulation along with markedly increased water contents in translucent white matter adjacent to hematomas strongly supports the roles of hydrostatic pressure during hematoma formation^{27 28} and clot retraction^{29 30 31 32 33} in edema development in the white matter extracellular space. This conclusion is also supported by our observation that this early edema develops in our model despite an intact BBB. Extensive white matter edema and an acellular zone of serum separating hematomas from the surrounding brain tissue have also been described after experimental ICH in dogs.³⁴

This development of perihematomal edema as a result of interstitial serum protein accumulation is similar pathophysiologically to vasogenic³⁵ (or open barrier²⁷ ) edema, in which increased BBB permeability results in the influx of protein molecules into the ECS. Since the plasma protein content of the ECS is normally very low, its accumulation in the ECS causes the osmotic pressure of the interstitial fluid to approach that of plasma, resulting in water movement from blood to brain that produces edema.²⁷ Indeed, a linear correlation between protein and water contents in the ECS after experimental BBB injury has been reported.³⁶

This accumulation of serum proteins in the ECS also provides an explanation for the prolongation of perihematomal edema after ICH. Groger and Marmarou³⁷ demonstrated a significant relationship between the protein concentration of edema fluid and the rate of edema clearance. In their studies, edema resolution was minimal within the first 3 days after infusion of a high concentration of albumin (65 mg/mL), while in contrast, edema was completely resolved within 72 hours when mock cerebrospinal fluid was infused. In preliminary neuropathology studies, we observed that white matter near the hematoma in our model continued to be markedly edematous at 3 days after blood infusion.^{10 11} Similarly, in collagenase-induced ICH and blood infusion models into rat basal ganglia, water contents that were increased by 4 and 24 hours remained at this level through 72 hours³⁸ and 4 days,⁹ respectively. In human ICH, perihematomal edema was observed for 5 to 7 days after a bleed. This edema can be substantial with volumes equal to or greater than the hematoma volume itself (Reference 39, Table 1).

In human ICH, clot retraction is generally considered to contribute to early perihematomal edema development. Brott et al³² have suggested this mechanism to explain the low densities they observed on CT scans as early as 3 hours after symptom onset. Similarly, others have also implicated clot retraction for the hyperintensities indicative of edema they observed surrounding hyperacute hematomas on T₂-weighted MR images.^{12 33}

Several results also suggest that mechanisms other than mass effect are involved in the contribution of blood and/or its serum components to perihematomal edema formation. The strongest evidence is from the demonstration that inflation of a microballoon, to model mass effect alone, failed to produce perihematomal edema at 4 hours even though it markedly reduced CBF to less than 20 mL/100 g per minute.⁴⁰ In addition, blood produces larger lesions than would be expected from its space-occupying effects alone,⁴¹ and Suzuki and Ebina⁴² reported that blood injections into the caudate nucleus in dogs produced significantly more progressive perilesional sponginess and necrosis compared with inert mass lesions (oil-wax mixture).

At present, the role of ischemia in perihematomal edema development is controversial. Early CBF reductions apparently reflect the immediate and maximal hemodynamic adjustments in brain after an ICH. Nath et al²⁶ described that ischemia persisted only to a marginal degree beyond 10 minutes and had returned to normal within 3 hours, and Yang et al⁹ found that 50% reductions in CBF at 1 hour had returned to control levels by 4 hours. Similar 50% reductions in CBF in both hemispheres were reported in the cat⁴³ at 5 minutes after basal ganglia hemorrhage. Ropper and Zervas⁴⁴ reported that their lowest CBF values were 25 to 30 mL/100 g per minute even when large volumes (240 to 280 µL) of blood were injected within 1 second into the caudate nucleus, causing coma in awake rats. These values are well above the accepted threshold blood flow levels for ischemic injury of 15 to 20 mL/100 g per minute.⁴⁵ Furthermore, these reduced flows were transitory and recovered to baseline by 4 hours. Thus, it appears unlikely that blood flow reduction or "local ischemia" is sufficient in degree and duration to produce the perihematomal edema that we observe.

Our recent brain metabolite studies also support the conclusion that blood flow reductions surrounding hematomas during the early hours after ICH are unlikely to be either severe or prolonged and are unlikely to contribute to the edema we observe. In markedly edematous perihematomal white matter in our ICH model, we observed essentially unchanged ATP at 3 to 8 hours, increased phosphocreatine at 3 and 5 hours, and a fourfold increase in glycogen concentrations by 8 hours after hematoma induction (References 15 and 46 and K.R.W. et al, unpublished data, 1995). These results are suggestive of a reduced metabolic rate in this edematous white matter region. This conclusion is supported by the findings of Nath et al,²⁶ who reported early decreases in the activity of the glycogenolytic enzyme phosphorylase after blood injections in rat caudate.

Finally, in our ongoing studies we observe a continued presence of white matter edema ipsilateral to hematomas and the development of reactive astrocytosis and myelin pallor at 3 days after infusion.^{10 11} In these white matter regions, eventual glial scar, cyst formation, and atrophy develop,¹¹ similar to the white matter pathology in human ICH.⁴⁷ Thus, we observe a wide spectrum of pathophysiological and neuropathologic changes in this large-animal lobar ICH model that appear similar to those that occur after ICH in humans. This model should be useful for examination of therapeutic interventions that reduce edema development,⁴⁸ prevent white matter injury, and improve brain outcome after ICH.

	Selected Abbreviations and Acronyms

 

BBB = blood-brain barrier CBF = cerebral blood flow CTP = cerebral tissue pressure ECS = extracellular space ICH = intracerebral hemorrhage MABP = mean arterial blood pressure

	Acknowledgments

 
This study was supported by grant R01-NS-30652 from the National Institutes of Health.

	Footnotes

 
Reprint requests to Kenneth R. Wagner, PhD, Research Service (151), Department of Veterans Affairs Medical Center, 3200 Vine St, Cincinnati, OH 45220.

Received July 21, 1995; revision received November 21, 1995; accepted December 6, 1995.

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