Ludmila Belayev

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.

Robust Docosahexaenoic Acid–Mediated Neuroprotection in a Rat Model of Transient, Focal Cerebral Ischemia

Background and Purpose— Docosahexaenoic acid (DHA; 22:6n-3), an &ohgr;-3 essential fatty acid family member, is the precursor of neuroprotectin D1, which downregulates apoptosis and, in turn, promotes cell survival. This study was conducted to assess whether DHA would show neuroprotective efficacy when systemically administered in different doses after middle cerebral artery occlusion (MCAo) in rats. Methods— Sprague-Dawley rats were anesthetized with isoflurane and subjected to 2 hour of MCAo. Animals were treated with either DHA (low doses=3.5 or 7 mg/kg; medium doses=16 or 35 mg/kg; and high dose=70 mg/kg) or an equivalent volume of saline intravenously 3 hours after MCAo onset. Neurologic status was evaluated during occlusion (60 minutes) and on days 1, 2, 3, and 7 after MCAo. Seven days after MCAo, brains were perfusion-fixed, and infarct areas and volumes were determined. Results— Only the low and medium doses of DHA significantly improved the neurologic score compared with vehicle-treated rats at 24 hours, 48 hours, 72 hours, and 7 days. DHA markedly reduced total corrected infarct volume in all treated groups compared with vehicle-treated rats (3.5 mg/kg, 26±9 mm3; 7 mg/kg, 46±12 mm3; 16 mg/kg, 37±5 mm3; and 35 mg/kg, 34±15 mm3 vs vehicle, 94±12 mm3). Cortical and striatal infarct volumes were also significantly reduced by treatment with DHA. No neuroprotective effects were observed with 70 mg/kg DHA. Conclusions— We conclude that DHA experimental therapy at low and medium doses improves neurologic and histologic outcomes after focal cerebral ischemia and might provide benefits in patients after ischemic stroke.

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.

Docosahexaenoic Acid Complexed to Albumin Elicits High-Grade Ischemic Neuroprotection

Background and Purpose— High-dose human albumin therapy is strongly neuroprotective in models of brain ischemia and trauma and is currently being studied in a pilot-phase clinical stroke trial. Among its actions in ischemia, albumin induces the systemic mobilization of n-3 polyunsaturated fatty acids and may help to replenish polyunsaturated fatty acids lost from neural membranes. Methods— We complexed 25% human albumin to docosahexaenoic acid (DHA; 22:6n-3) and compared its neuroprotective efficacy with that of native albumin in rats with 2-hour focal ischemia produced by intraluminal suture-occlusion of the middle cerebral artery. Results— In animals treated with DHA–albumin, 0.63 g/kg, the improvement in neurobehavioral scores at 72 hours significantly exceeded that of other treatment groups, and the extent of histological protection (86% reduction in cortical infarction) was highly significant and tended to surpass the degree of cortical protection produced by native albumin at 1.25 g/kg (65%). DHA–albumin 0.63 g/kg, but not native albumin, also significantly reduced subcortical infarction and markedly diminished brain swelling. Lipidomic analysis of DHA–albumin-treated postischemic brains revealed a large accumulation of the neuroprotective DHA metabolite, 10,17S-docosatriene, in the ipsilateral hemisphere. Conclusions— The high-grade neuroprotection afforded by the DHA–albumin complex at relatively low albumin doses is clinically advantageous in that it might reduce the likelihood of acute intravascular volume overload and congestive heart failure sometimes induced when patients with compromised cardiovascular function are treated with high-dose albumin.

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.

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.