Weizhao Zhao

Middle Cerebral Artery Occlusion in the Rat by Intraluminal Suture: Neurological and Pathological Evaluation of an Improved Model

BACKGROUND AND PURPOSE The purpose of the present study was to evaluate a modified method of intraluminal suture occlusion of the middle cerebral artery (MCA) on the volume of brain infarction and on neurobehavioral function in rats subjected to a temporary focal ischemic insult. METHODS Male Sprague-Dawley rats were anesthetized with halothane and subjected to 60 minutes or 2 hours of temporary MCA occlusion (MCAo) by an intraluminal thread. In one group of rats, the suture was coated with poly-L-lysine, while in a second group, a conventional uncoated suture was used. Behavioral function was evaluated at 50 to 60 minutes after occlusion and during a 3-day period after MCAo. Three days after MCAo brains were perfusion-fixed and infarct volumes were measured. RESULTS In rats with 60-minute MCAo, only 3 of 7 animals with uncoated sutures had infarcts, whereas in the group with poly-L-lysine-coated sutures, all rats (n = 7) exhibited infarction (P = .009, Fishers exact test). With 2 hours of MCAo, total infarct volume (corrected for brain edema) was significantly larger in rats with poly-L-lysine-coated sutures than in the group with uncoated sutures (mean +/- SEM, 122.1 +/- 4.8 versus 67.0 +/- 18.2 mm3, respectively; P = .03; n = 4 in each group). In the 2-hour MCAo study, infarct volumes in the uncoated-suture group tended to be variable and inconsistent (coefficient of variation, 54%) compared with the group in which sutures were coated with poly-L-lysine, in which a highly consistent infarct was produced (coefficient of variation of infarct volume, 8%). CONCLUSIONS Reversible MCAo in which a poly-L-lysine-coated intraluminal suture was used proved to be a reliable and effective modification of this technique, yielding consistently larger infarcts and greatly reduced interanimal variability.

Quantitative evaluation of blood-brain barrier permeability following middle cerebral artery occlusion in rats

A sensitive quantitative fluorescence method was used to explore the time course and regional pattern of blood-brain barrier (BBB) opening after transient middle cerebral artery occlusion (MCAo). Male Sprague-Dawley rats were anesthetized with halothane and subjected to 2 h of temporary MCAo by retrograde insertion of an intraluminal nylon suture, coated with poly-L-lysine, through the external carotid artery into the internal carotid artery and MCA. Damage to the BBB was judged by extravasation of Evans Blue (EB) dye, which was administered either 2, 3, 24 or 48 h after onset of MCAo. Fluorometric quantitation of EB was performed 1 or 2 h later in six brain regions. Cerebral infarction volumes were quantitated from histopathological material at 72 h. EB extravasation first became grossly visible in the ipsilateral caudoputamen and neocortex following 3 h of MCAo, was grossly unapparent at 24-26 h, and was maximal at 48-50 h. Fluorescence quantitation confirmed that BBB opening was absent at 2-3 h but present at all later times. In the hemisphere ipsilateral to MCAo, a 179% mean increase in extravasation of EB (compared to sham rats) was measured at 4 h, 407% at 5 h, 311% at 26 h and 264% at 50 h. (in each case, P < 0.05 vs. sham). The volume of infarcted tissue at 72 h in this model was 163.6 +/- 7.7 mm3. Our results indicate that an initial, acute disruption of the BBB occurs between 3 and 5 h following MCAo, and that a later, more widespread increase in regional BBB permeability is present at 48 h. Regional measurement of Evans Blue extravasation offers a precise means of quantitating BBB disruption in focal cerebral ischemia; this method will be of considerable utility in assessing the BBB-protective properties of pharmacological agents.

Human Albumin Therapy of Acute Ischemic Stroke Marked Neuroprotective Efficacy at Moderate Doses and With a Broad Therapeutic Window

Background and Purpose — We examined the neuroprotective efficacy of moderate-dose human albumin therapy in acute focal ischemic stroke and defined the therapeutic window after stroke onset, within which this therapy would confer neurobehavioral and histopathological neuroprotection. Methods — Sprague-Dawley rats were anesthetized with halothane/nitrous oxide and received 2-hour middle cerebral artery occlusion (MCAo) by a poly-l-lysine–coated intraluminal suture. Neurological status was evaluated during occlusion (60 minutes) and daily for 3 days after MCAo. In the dose-response study, human albumin doses of either of 0.63 or 1.25 g/kg or saline vehicle (5 mL/kg) were given intravenously immediately after suture removal. In the therapeutic window study, a human albumin dose of 1.25 g/kg was administered intravenously at 2 hours, 3 hours, 4 hours, or 5 hours after onset of MCAo. Three days after MCAo, brains were perfusion-fixed, and infarct volumes and brain swelling were determined. Results — Moderate-dose albumin therapy significantly improved the neurological score at 24 hours, 48 hours, and 72 hours and significantly reduced total infarct volume (by 67% and 58%, respectively, at the 1.25- and 0.63-g/kg doses). Cortical and striatal infarct volumes were also significantly reduced by both doses. Brain swelling was virtually eliminated by albumin treatment. Even when albumin therapy (1.25 g/kg) was initiated as late as 4 hours after onset of MCAo, it improved the neurological score and markedly reduced infarct volumes in cortex (by 68%), subcortical regions (by 52%), and total infarct (by 61%). Conclusions — Moderate-dose albumin therapy markedly improves neurological function and reduces infarction volume and brain swelling, even when treatment is delayed up to 4 hours after onset of ischemia.

Diffusion-Weighted Magnetic Resonance Imaging Confirms Marked Neuroprotective Efficacy of Albumin Therapy in Focal Cerebral Ischemia

BACKGROUND AND PURPOSE We have recently shown high-dose human serum albumin therapy to confer marked histological protection in experimental middle cerebral artery occlusion (MCAo). We have now used diffusion-weighted magnetic resonance imaging (DWI) in conjunction with morphological methods to expand our understanding of this therapeutic approach. METHODS Physiologically controlled Sprague-Dawley rats received 2-hour MCAo by the modified intraluminal suture method. Treated rats received 25% human serum albumin solution (1% by body weight) immediately after the MCA was reopened. Vehicle-treated rats received saline. Computer-based image averaging was used to analyze DWI data obtained 24 hours after MCAo and light-microscopic histopathology obtained at 3 days. In a matched series, plasma osmolality and colloid oncotic pressure, as well as brain water content, were determined. RESULTS Albumin therapy, which lowered the hematocrit on average by 37% and raised plasma colloid oncotic pressure by 56%, improved the neurological score throughout the 3-day survival period. Within the ischemic focus, the apparent diffusion coefficient (ADC) computed from DWI data declined by 40% in vehicle-treated rats but was preserved at near-normal levels (8% decline) in albumin-treated rats (P<0.001). Albumin also led to higher ADC values within unlesioned brain regions. Histology revealed large consistent cortical and subcortical infarcts in vehicle-treated rats, while albumin therapy reduced infarct volume at these sites, on average, by 84% and 33%, respectively. Total infarct volume was reduced by 66% and brain swelling was virtually eliminated by albumin treatment. Microscopically, while infarcted regions of vehicle-treated rats had the typical changes of pannecrosis, infarcted zones of albumin-treated brains showed persistence of vascular endothelium and prominent microglial activation, suggesting that albumin therapy may help to preserve the neuropil within zones of residual infarction. CONCLUSIONS These findings confirm the striking neuroprotective efficacy of albumin therapy in focal cerebral ischemia and reveal that this effect is associated with DWI normalization and a mitigation of pannecrotic changes within zones of residual injury.

Middle cerebral artery occlusion in the mouse by intraluminal suture coated with poly-L-lysine : neurological and histological validation

The present study was conducted to validate a modified method of temporary focal cerebral ischemia in the mouse; neurobehavioral function and histopathological infarction were quantitated following various periods of middle cerebral artery occlusion (MCAo). Male C57BL/6 mice were anesthetized with 3% halothane in a mixture of 30%O2/70%N2O delivered by face mask and were subjected to 30- to 180-min of temporary middle cerebral artery occlusion (MCAo) by an intraluminal suture coated with poly-l-lysine. Twenty-eight of 40 mice showed an initial high-grade neurological deficit (30-min MCAo, n=7; 60-min, n=8; 120-min, n=8; 180-min, n=5) when examined during MCAo; these were used for subsequent study. One day after MCAo, behavioral function was re-evaluated, and brains were perfusion-fixed and infarct volumes were measured. The initial neurological deficit improved at 24 h in mice with 30- or 60-min of prior MCAo but tended to persist in mice with 120- or 180-min insults. Following each duration of ischemia, mice exhibited ipsilateral infarcts. Small, inconsistent predominantly subcortical infarcts were present after 30-min MCAo, while longer occlusion periods gave rise to consistent foci of subcortical infarction involving striatum, septum, thalamus, and hippocampus, as well as areas of frontoparietal cortical infarction. The major advantages of the improved intraluminal MCAo model reported here, incorporating sutures coated with poly-l-lysine, include: a 100% incidence of infarction of predictable location and size in mice having an initial neurological deficit. Periods of 60- to 180-min MCA occlusion in this model yield sufficiently reproducible sequelae to permit the effects of various therapeutic agents on neurological outcome and size of infarction to be readily studied.

Transient middle cerebral artery occlusion by intraluminal suture: I. Three-dimensional autoradiographic image-analysis of local cerebral glucose metabolism-blood flow interrelationships during ischemia and early recirculation.

Using autoradiographic image-averaging strategies, we studied the relationship between local glucose utilization (LCMRglc) and blood flow (LCBF) in a highly reproducible model of transient (2-hour) middle cerebral artery occlusion (MCAO) produced in Sprague-Dawley rats by insertion of an intraluminal suture coated with poly-L-lysine. Neurobehavioral examination at 60 minutes after occlusion substantiated a high-grade deficit in all animals. In two subgroups, LCBF was measured with 14C-iodoantipyrine at either 1.5 hours of MCAO, or at 1 hour of recirculation after suture removal. In two other matched subgroups, LCMRglc was measured with 14C-2-deoxyglucose at 1.5 to 2.25 hours of MCAO, and at 0.75 to 1.5 hours of recirculation after 2 hours of MCAO. Average image data sets were generated for LCBF, LCMRglc, and the LCMRglc/LCBF ratio for each study time. Middle cerebral artery occlusion for 2 hours induced graded LCBF decrements affecting ipsilateral cortical and basal ganglionic regions. After 1 hour of recirculation, LCBF in previously ischemic neocortical regions increased by 40% to 200% above ischemic levels, but remained depressed, on average, at about 40% of control. By contrast, frank hyperemia was noted in the previously ischemic caudoputamen. Mean cortical LCBF values during MCAO correlated highly with their respective LCBF values after 1 hour of recirculation (R = 0.93), suggesting that postischemic LCBF recovery is related to the depth of ischemia. Despite focal ischemia, LCMRglc during ~2 hours of MCAO was preserved, on average, at near-normal levels; but following ~1 h of recirculation, LCMRglc became markedly depressed (on average, 55% of control in previously densely ischemic cortical regions). Regression analysis indicated that this depressed glucose utilization was determined largely by the intensity of antecedent ischemia. By pixel analysis, the ischemic core (defined as LCBF 0% to 20% of control) comprised 33% of the ischemic hemisphere, and the penumbra (LCBF 20% to 40%) accounted for 26%. The penumbra was concentrated at the coronal poles of the ischemic lesion and formed a thin shell around the central ischemic core. During 2 hours of MCAO, the LCMRglc/LCBF ratio within the ischemic penumbra was increased four-fold above normal (average, 179 umol/100 mL). In marked contrast, after ~1 h recirculation, this uncoupling had almost completely subsided. The companion study (Zhao et al., 1997) further analyzes these findings in relation to patterns of infarctive histopathology.

Neuroprotective Effect of Darbepoetin Alfa, a Novel Recombinant Erythropoietic Protein, in Focal Cerebral Ischemia in Rats

Background and Purpose— Darbepoetin alfa is a novel erythropoiesis-stimulating protein developed for treating anemia. In animal models, exogenous recombinant human erythropoietin has been reported to be beneficial in treating experimental cerebral ischemia. In this study, we determined whether darbepoetin alfa would protect in a rat model of transient focal cerebral ischemia. Methods— Rats received 2-hour middle cerebral artery suture-occlusion. The drug (darbepoetin alfa, 10 &mgr;g/kg) or vehicle was administered intraperitoneally 2 hours after onset of middle cerebral artery occlusion. Animals were allowed to survive for 3 or 14 days. Behavioral tests were performed sequentially. Infarct volumes and brain swelling were determined. Results— Darbepoetin alfa-treated rats showed improved neuroscores relative to vehicle-treated animals beginning within 1 hour of treatment and persisting throughout the 14-day survival period. Darbepoetin alfa significantly reduced corrected total (cortical + subcortical) infarct volume (56.3±20.6 and 110.8±6.8 mm3, respectively) and total infarct areas at multiple levels compared with vehicle in the 14-day survival group. Brain swelling was not affected by treatment. Conclusion— Darbepoetin alfa confers behavioral and histological neuroprotection after focal ischemia in rats.

Posttraumatic Cerebral Ischemia after Fluid Percussion Brain Injury: An Autoradiographic and Histopathological Study in Rats

OBJECTIVES Mild-to-moderate reductions in local cerebral blood flow (ICBF) have been reported to occur in rats after moderate (1.7-2.2 atm) fluid percussion brain injury. The purpose of this study was to determine whether evidence for severe ischemia (i.e., mean ICBF < 0.25 ml/g/min) could be demonstrated after severe brain injury. In addition, patterns of indium-labeled platelet accumulation and histopathological outcome were correlated with the hemodynamic alterations. METHODS Sprague-Dawley rats (n = 23), anesthetized with halothane and maintained on a 70:30 mixture of nitrous oxide:oxygen and 0.5% halothane, underwent normothermic (37 degrees C) parasagittal fluid percussion brain injury (2.4-2.6 atm). Indium-111-tropolone-labeled platelets were injected 30 minutes before traumatic brain injury (TBI), while 14C-iodoantipyrine was infused 30 minutes after trauma for ICBF determination. Sham-operated animals (n = 8) underwent similar surgical procedures but were not injured. For histopathological analysis, traumatized rats (n = 5) were perfusion-fixed 3 days after TBI. RESULTS In autoradiographic images of indium-labeled platelets, abnormal platelet accumulation that was most pronounced overlying the pial surface was commonly associated with severe reductions in ICBF within underlying cortical regions 30 minutes after TBI. For example, within the lateral parietal cortex, ICBF was significantly reduced from 1.67 +/- 0.11 ml/g per minute (mean +/- standard error of the mean) in sham-operated animals to 0.23 +/- 0.03 ml/g per minute within the traumatized group. In addition to focal severe ischemia, moderate reductions in ICBF were detected throughout the traumatized hemisphere, including the frontal and occipital cortices, hippocampus, thalamus, and striatum. Mild decreases in ICBF were also observed throughout the contralateral cerebral cortex. At 3 days after severe TBI, histopathology demonstrated intracerebral and subarachnoid hemorrhage associated with cerebral contusion and selective neuronal necrosis. CONCLUSION These data indicate that multiple cerebrovascular abnormalities, including subarachnoid hemorrhage, focal platelet accumulation, and severe ischemia, are important early events in the pathogenesis of cortical contusion formation after TBI. Injury severity is expected to be a critical factor in determining what therapeutic strategies are attempted in the clinical setting.

HU-211, a Novel Noncompetitive N-Methyl-d-Aspartate Antagonist, Improves Neurological Deficit and Reduces Infarct Volume After Reversible Focal Cerebral Ischemia in the Rat

BACKGROUND AND PURPOSE HU-211 is a nonpsychotropic cannabinoid analogue that has been shown to act as a functional N-methyl-D-aspartate receptor blocker. We investigated the neuroprotective efficacy of HU-211 in a model of reversible middle cerebral artery occlusion (MCAo) in rats. METHODS Male Wistar rats were anesthetized with halothane and subjected to 90 minutes of temporary MCAo by retrograde insertion of an intraluminal nylon suture, coated with poly-L-lysine, through the external carotid artery into the internal carotid artery and MCA. The drug (HU-211 in cosolvent, 4 mg/kg IV) or vehicle was administered in a blinded fashion 70 minutes after onset of MCAo. Behavioral tests were evaluated during occlusion (60 minutes) and for a 3-day period after MCAo. Three days after MCAo, brains were perfusion-fixed, and infarct volumes were determined. RESULTS HU-211 significantly improved the neurological score compared with vehicle during the 3 days after MCAo. Treatment with HU-211 also significantly reduced both infarct volume (mean +/- SEM, 66.6 +/- 12.5 versus 149.8 +/- 36.3 mm3) and brain swelling (2.61 +/- 1.33% versus 6.66 +/- 1.24%) compared with vehicle-treated rats (n = 17 in each group). CONCLUSIONS These results demonstrate the neuroprotective ability of HU-211 in focal cerebral ischemia as judged by neurological score, infarct size, and brain swelling. Reversible MCAo with the use of a poly-L-lysine-coated intraluminal suture proved to be a reliable and effective modification of this technique, yielding consistent results.

Experimental Intracerebral Hemorrhage in the Mouse. Histological, Behavioral, and Hemodynamic Characterization of a Double-Injection Model

BACKGROUND AND PURPOSE A major limitation of intracerebral hemorrhage (ICH) research is the lack of reproducible animal models. The present study was conducted to validate in the mouse the double-injection method of ICH initially developed in the rat. We investigated the effect of intrastriatal injection of blood or cerebrospinal fluid (CSF) on cerebral blood flow (CBF), neurological score, hematoma volume, and brain swelling. METHODS Male C57BL/6 mice were anesthetized with halothane/nitrous oxide delivered by face mask. Rectal and cranial temperatures were regulated at 37 degrees C to 37.5 degrees C. Mice were placed in a stereotactic frame, and a 30-gauge stainless steel cannula was introduced through a burr hole into the left striatum. Each mouse received a 5-microL injection of either whole blood or CSF (over 3 minutes), followed 7 minutes later by 10 microL injected over 5 minutes. The injection cannula was slowly withdrawn 10 minutes after the second injection. Control mice had only cannula insertion. CBF was studied by laser Doppler perfusion imaging. Neurological status was evaluated on days 1 and 2. After 2 days, hematoma volume and brain swelling were calculated. RESULTS Physiological values were stable. Mice with ICH but not those with CSF or cannula alone had a marked, persistent neurological deficit and a highly reproducible hematoma, whose mean+/-SEM volume was 2.0+/-0.2 mm3 compared with a lesion size of 0.2+/-0.1 mm3 in mice with CSF. Residual swelling of the ipsilateral hemisphere at 48 hours was 5.7% in the hematoma and 1.5% in the CSF groups. Relative CBF in the neocortex ipsilateral to the injection site declined by approximately 45% to 60% during the first 20 minutes after cannula insertion/injection in all groups but began to renormalize at approximately 25 to 30 minutes in the CSF and cannula-only groups; in the hematoma group, cortical hypoperfusion of approximately 35% to 50% persisted during the 90-minute measurement period. CONCLUSIONS The present ICH model in mice produces a consistent neurological deficit, hypoperfusion, hematoma volume, and brain swelling. This model closely mimics human hypertensive basal ganglionic ICH and should be useful for the evaluation of pharmaceutical therapies. Laser Doppler perfusion imaging is a useful new technique to quantify relative CBF changes and can be used for studies of dynamic changes of CBF in this in vivo model of ICH in mice.