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the Cerebral Vascular Disease Research Center, Department of Neurology, University of Miami School of Medicine, Miami, Fla.
Abstract
Background and Purpose— Acute intracerebral hemorrhage (ICH) is a common and severe form of stroke. To date, medical management of ICH has had scant impact on morbidity and mortality. Because albumin therapy is markedly neuroprotective in preclinical models of ischemic stroke, and because ischemic and hemorrhagic stroke share several common injury mechanisms, we hypothesized that albumin therapy might also benefit ICH.
Methods— Acute intracortical hematoma was produced in anesthetized, normothermic rats by the single stereotaxic injection of 50 μL of autologous, nonheparinized whole blood over 5 minutes. Separate animal groups were treated either with 25% human albumin, 1.25 g/kg, or with intravenous saline vehicle at 60 minutes after ICH. Neurobehavior was quantified sequentially over the next 2 to 7 days. Damage to the blood–brain barrier was assessed at 2 days after ICH by fluorometric measurement of Evans blue extravasation in dissected brain regions.
Results— High-grade neurological deficits were present in all rats at 50 minutes after ICH (score 10.3±0.2, mean±SEM [maximal score 12]). Albumin-treated rats showed improved neuroscores relative to saline-treated animals beginning within hours of treatment and persisting throughout the 7-day survival period. At 3 and 7 days, mean total neuroscores of the albumin group were 38% to 43% lower than in saline-treated animals. Perihematomal Evans blue discoloration was readily evident in saline-treated ICH rats but was reduced by albumin treatment. Hemispheric Evans blue content ipsilateral to the hematoma was reduced by 49% by albumin treatment (albumin 93.9±13.3 versus saline 184.7±33.7 mg/g, P<0.05). Hematoma volume and brain swelling were not affected by albumin treatment.
Conclusions— Prompt albumin therapy improves neurological function and blood–brain barrier integrity after acute intracortical hematoma. These observations have important potential clinical implications.
Key Words: acute therapy albumin basic science blood–brain barrier neuroprotection hemorrhage, intracranial neuroprotective agents neuroprotectants
Introduction
Intracerebral hemorrhage (ICH) constitutes 10% to 15% of all strokes and is associated with high morbidity and mortality. Thirty-day case fatality rates of 35% have been reported in hospital-based studies and up to 52% in community-based studies,1 rates that are 2- to 6-fold higher than in ischemic stroke. Among chronic ICH survivors, nearly half are dependent on outside help in their activities of daily living, and only 20% of patients become independent at 6 months.2 To date, the medical management of ICH has had only a minimal impact on the disease, and the preclinical development of protective strategies has lagged markedly behind ischemic stroke.
Human albumin therapy, in moderate-to-high doses, has proved markedly neuroprotective in experimental models of focal3–6 and global7 cerebral ischemia and in traumatic brain injury.8 In the present study, we have extended the therapeutic applications of albumin therapy to the setting of ICH. The rationale for this approach is 2-fold: (1) ICH and ischemic stroke share a number of common injury mechanisms, which include perilesional edema and blood–brain barrier (BBB) disruption,9 oxidative injury and inflammation,10,11 excitotoxicity,12 and apoptosis.13 (2) Albumin therapy is known to antagonize many of these mechanisms by directly protecting both parenchymal and vascular elements of the brain, diminishing brain edema, maintaining microvascular integrity, inhibiting endothelial cell apoptosis, and exerting antioxidant effects.3–5,14 These considerations provide a strong rationale for suspecting that albumin therapy might also be beneficial in acute intracerebral hematoma. In the present study, we tested this hypothesis in a model of acute intracortical hematoma that gives rise to a consistent neurobehavioral deficit and BBB dysfunction. We present evidence that albumin therapy exerts a substantial protective effect in this setting.
Materials and Methods
Animal Preparation
Twenty-nine male Sprague-Dawley rats (weight 289 to 378 g; Charles River Laboratories, Wilmington, Mass) were fasted overnight but allowed free access to water. Animal protocols for these studies were approved by the University of Miami Animal Care and Use Committee. Anesthesia was induced with 3.5% halothane in a mixture of 70% nitrous oxide and 30% oxygen. Rats were orally intubated and mechanically ventilated on 2% halothane and 70% nitrous oxide. Blood pressure arterial blood gases and pH were monitored, and left temporalis muscle temperature and rectal temperature were monitored with probes and maintained at 37.0°C to 37.5°C as described previously.5,15 Rectal temperature and body weight were monitored periodically after ICH.
Production of Intracortical Hematoma
Acute intracortical hemorrhage was produced by the single-injection method as described by Xue et al.16 In brief, the rat was placed in a stereotaxic frame (David Kopf Instruments), and a specially fabricated 27-gauge stainless steel cannula with 30° bevel was introduced through a burr hole into the midcerebral cortex (3 mm lateral to midline, 0.2 mm anterior to the coronal suture, and 2.5 mm below the surface of the skull). Each rat received a 50-μL injection of autologous whole blood over a period of 5 minutes with a microinfusion pump. (Blood was taken from the femoral artery with a 250-μL syringe, which was filled from the back of the barrel; no heparin was used.) After infusion, the injection cannula was left in place for 3 minutes and then removed slowly. The burr hole was filled with bone wax, the scalp incision closed, and the animal placed in a cage with free access to food and water.
Neurobehavioral Evaluation
A standardized quantitative neurobehavioral battery was used to assess sensorimotor function at 50 minutes after ICH and at sequential intervals after albumin or saline treatment. In 16 animals (albumin-treated, n=8; saline-treated, n=8), these observations were made at 2 hours, 4 hours, 1 day, and 2 days after treatment. Another 13 rats (albumin treated, n=6; saline-treated, n=7) were monitored from 1 hour through 7 days after treatment. The neurobehavioral battery consisted of postural reflex17 and placing tests,18 as described previously. Neurological function was graded on a scale of 0 to 11 (normal score=0, maximal score=12).15 Tests were conducted by an observer blinded to the treatment group.
Albumin Treatment
At 60 minutes after production of ICH, 14 rats received human serum albumin (25% solution, Baxter Healthcare Corp) 1.25 g/kg IV. Another 15 rats received a comparable volume of 0.9% saline vehicle.
Evaluation of BBB Integrity
We investigated the integrity of the BBB in 16 rats by measuring the extravasation of Evans blue.19,20 Evans blue dye (2% in saline, 4 mL/kg) was injected intravenously at 48 hours after ICH. Two hours later, the chest was opened under halothane anesthesia, and the brain was perfused with normal saline through the left ventricle at 110 mm Hg pressure until colorless perfusion fluid was obtained from the right atrium. After decapitation, the brain surface was photographed. The brain was then sectioned into 3 coronal blocks (2 mm in thickness) that included bregma levels +1.2, –0.3, and –1.3 mm, and the surfaces of each block were photographed.
In 8 brains, coronal blocks were next divided into right and left hemispheres and dissected into 8 regions for measurement of regional Evans blue (Figure 1). The dissected samples were weighed and extracted in 50% trichloroacetic acid solution. After homogenization and centrifugation, the extract was diluted with ethanol (1:3), and its fluorescence was determined (excitation at 620 nm and emission at 680 nm) with a Perkin-Elmer LS-5B luminescence spectrometer. Calculations were based on external standards in the same solvent (100 to 500 ng/mL). The tissue content of Evans blue was quantified from a linear standard curve derived from known amounts of the dye and was expressed per gram of tissue. We have described these methods previously.20
Histopathology
Two separate groups of animals (saline [n=6] and albumin [n=7]) were allowed to survive for 7 days. Brains were then perfusion fixed, paraffin embedded, coronally sectioned, and stained with hematoxylin and eosin as described previously.5,15 Hematoma area was measured at 9 standardized coronal levels by a blinded observer, and hematoma volume was computed by numerical integration with image-analysis methods reported previously.5 Brain swelling was determined from the difference in ipsilateral versus contralateral hemispheric volumes.
Statistical Analysis
Repeated-measures ANOVA was used for intergroup comparisons of neurobehavioral data and tissue Evans blue content.
Results
Physiological Variables
Rectal and cranial (temporalis muscle) temperatures, arterial blood pressure and blood gases, and plasma glucose levels in the 29 animals of this study showed no significant differences between groups. After treatment, modest hemodilution was present in the albumin group (36±1.2 versus 42±0.9 for the saline group).
Neurobehavior
Severe neurological deficits contralateral to ICH were evident in all rats when tested at 50 minutes after ICH (Figure 2). The neuroscores of albumin-treated rats tended to improve beyond those of the saline-treated group within 1 to 2 hours of treatment, and this intergroup difference was highly significant by 4 hours (P<0.01; Figure 2). Although progressive neurological improvement occurred in both groups over the 2- to 7-day survival period, the degree of recovery in albumin-treated animals exceeded that of the saline group at each time point (treatmentxtime interaction, P<0.01 by repeated-measures ANOVA; Figure 2). At 3 and 7 days, mean total neuroscores of the albumin group were 38% to 43% lower than in saline-treated animals.
Evans Blue Extravasation
Representative brains from each group are shown in Figure 3. A round hemorrhagic lesion was evident on the dorsolateral brain surface of all animals. Coronal sections revealed a cylindrical or triangular hemorrhagic lesion that extended through the neocortex with its epicenter at the coronal level of midstriatum (Figure 3). Brains of the saline-treated ICH group showed a halo of distinct Evans blue discoloration surrounding the hematoma, evident both on the brain surface and on coronal sections. This discoloration was fainter and less extensive in the albumin group (Figure 3). Spectrofluorometric measurement of tissue Evans blue content revealed marked left-hemisphere elevations, ipsilateral to the hematoma, which were highest in the cortex at the coronal level of the lesion itself (Figure 3; region L1 in Figure 4) and in the adjacent slice (region L2 in Figure 4). The albumin-treated group exhibited a marked reduction of overall left-hemisphere Evans blue extravasation, amounting to 49%, compared with saline-treated controls (Figure 4; P<0.05).
Histopathology
Histological examination after 7-day survival revealed a localized hematoma in the left cerebral cortex. Three animals showed hemorrhage in the adjacent white matter. Total hematoma volume was identical in the saline- and albumin-treated groups (2.11±0.7 and 2.07±0.25 mm3, respectively). Brain swelling was not affected by albumin treatment (saline-treated animals 0.5% and albumin-treated animals 1.1%, P=0.5, NS).
Discussion
We have shown in this study that the prompt administration of moderate-dose human albumin to rats with acute cortical intracerebral hematoma leads to substantially improved neurological function throughout a 7-day survival period and to markedly improved BBB integrity as evidenced by diminished Evans blue permeability.
Vascular damage during cerebral ischemia occurs early and in a progressive fashion and contributes to edema, hemorrhagic transformation, and worsened clinical outcome in stroke patients.21 Although the beneficial effect of vascular protection has been shown in studies of cerebral ischemia, relatively few studies have addressed this topic in ICH models. Increased BBB permeability occurs after ICH and contributes to brain edema formation. During the early hours after ICH, the BBB remains intact to large molecules; however, BBB permeability increases markedly 8 to 12 hours later and even more so by 48 hours.22,23 Because edema is nearly maximal by 24 hours after ICH,23 therapy directed at reducing edema formation must be instituted within the first day. We have shown in the present study that prompt albumin therapy instituted at 60 minutes after production of ICH improves BBB integrity.
In cerebral ischemia, albumin-neuroprotection is mediated via multiple mechanisms. These include its major antioxidant actions,24 binding of transition metals,25 improvement of tissue perfusion,6,26 reversal of microvascular blood-element aggregation and sludging in pathological states,14 reduction of brain edema,3,4 normalization of brain water homeostasis,4 provision of essential fatty acids to the injured brain,27 and its role in maintaining normal endothelial28 and astrocytic function.29 In subjects with acute ischemic stroke, albumin therapy is currently being evaluated in a National Institutes of Health–supported phase I clinical trial (NS 40406).
In addition, albumin therapy might exert other beneficial effects specific to ICH itself. The pathophysiology of ICH involves not only mass effect and compressive injury to tissue but also potential toxicity from blood degradation products, which leads to secondary neuronal and parenchymal damage. A central event in ICH is the massive release of heme from red cells into the extracellular space9 and the metabolism of heme by heme oxygenase (HO).9 Free heme is both very hydrophobic and strongly pro-oxidant; it readily penetrates cell membranes, where it increases the susceptibility to oxidant-mediated killing. Heme also acts as a catalyst for the oxidation of LDL, which generates products that are toxic to endothelium.30 The release of heme also provides catalytically active iron to neighboring tissues.31 Albumin, together with hemopexin, is a major heme-binding protein. Albumin in the extracellular space is capable of binding free heme that results from hemorrhage or that is released from dying cells.32 The peroxidase- and catalase-like activities of heme are 50% to 60% inhibited by albumin. Once heme is complexed with albumin, it exhibits much lower reactivity toward hydrogen peroxide and other peroxides than nonprotein heme. Thus, by complexing heme, albumin helps to prevent the toxic effects of extracellular heme.32
Biliverdin is the initial product of HO-mediated hemoglobin cleavage and is subsequently reduced to bilirubin by biliverdin reductase.33 This enzyme is stabilized by serum albumin. Although heme itself is a potentially toxic pro-oxidant, biliverdin and bilirubin are both potent antioxidants.34,35 In its natural conformation, bilirubin is folded to yield a surface that contains an array of hydrophobic residues and hence is insoluble in aqueous media.36 Albumin binds to bilirubin with high affinity (2 moles per mole of albumin). Once bound to albumin, bilirubin protects albumin-bound fatty acids from peroxyl radical–induced oxidation.37 These mechanisms may contribute to the albumin-induced neuroprotection observed in the present study.
In the present study, there were no differences among treatment groups with respect to hematoma size or brain swelling, but neurobehavioral outcome was significantly improved by albumin therapy. ICH differs from ischemic stroke in that the volume of tissue into which bleeding occurs may be irretrievably lost, even with early intervention. The results described here are consistent with other studies in suggesting that behavioral measures may be more responsive to therapy than histopathology. For example, treatment with the free radical inhibitors alpha-phenyl-N-tert-butyl nitrone38 and NXY-05939 significantly improved behavioral function in rats after ICH but did not affect hematoma size or edema.
The model of acute intracortical hematoma used in the present study gives rise to a severe neurobehavioral deficit together with consistent BBB dysfunction. The model is relevant to the clinical condition of lobar hematoma.40 Thrombin, a serine protease essential to the coagulation cascade, plays a major role in inducing early edema and BBB disruption, and the use of heparinized blood to induce ICH may impede this injury mechanism.41 Therefore, in the present study, we used nonheparinized blood. Of relevance in this respect, in stroke patients treated with tissue plasminogen activator who develop thrombolysis-related ICH, the degree of perihematomal edema is much less than in patients with spontaneous ICH.42 This observation implicates intrahematomal blood clotting as a major pathogenetic factor in hyperacute perihematomal edema.42
Conclusions
We have shown that prompt therapy with moderate-dose human albumin reduces neurological deficits and improves BBB integrity in the setting of acute intracortical hematoma in the rat. These findings, once confirmed and extended, have important potential clinical implications. One intriguing implication of the fact that human albumin therapy is neuroprotective in both acute ischemic stroke and acute ICH is that this therapy, if validated in controlled clinical trials, could be instituted in acute stroke patients without the need for a prior CT scan to distinguish ischemic from hemorrhagic stroke.
Acknowledgments
This study was supported by National Institutes of Health program-project grant NS 05820. The authors thank Guillermo Fernandez, BS, for his technical assistance.
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