A. Lorris Betz

Middle cerebral artery occlusion in rats: a neurological and pathological evaluation of a reproducible model.

Middle cerebral artery occlusion (MCAO) in rats produces an infarct of varying size. We examined three factors that may influence this variability: animal weight, vascular anatomy, and extent of occlusion in rats undergoing MCAO. We also developed a four-point neurological evaluation scale and validated its usefulness by comparing it with a four-grade pathological determination of the size of the infarct. Of 82 animals subjected to a standard MCAO, 34 developed small cortical infarcts (pathological grades I-II; infarct size less than 25 mm2, 6-17% of the ipsilateral cortex surface area), and 48 large infarcts (pathological grades III-IV, infarct size greater than 25 mm2, 20-56% of surface area). We were able to predict the size of infarction from the neurological evaluation in 83% of the animals, and this accuracy reached 91% when grades I and II and III and IV were considered together (P less than 0.001). In 41 animals subjected to a more extensive vascular occlusion, 89% exhibited large infarcts. Four vascular patterns were identified but none played a significant role in the incidence or size of the cortical stroke. However, rats weighing less than 300 g showed a smaller lesion size than did rats greater than 300 g. Our proposed new MCAO technique appears useful in reproducing large-sized infarcts of the frontoparietal cortex.

Reduced Ischemic Brain Injury in Interleukin-1β Converting Enzyme—Deficient Mice

A variety of recent studies suggest a role for both inflammatory cytokines such as interleukin-1 beta (IL-1β), and apoptosis in ischemic brain injury. Because IL-1β converting enzyme (ICE) is required for the conversion of proIL-1β to its biologically active form, and has homology with proteins that regulate apoptosis in invertebrates, we studied the effect of cerebral ischemia on brain injury in mutant mice deficient in the ICE gene (ICE knockout [KO] mice). Focal cerebral ischemia, produced by occlusion of the middle cerebral artery, resulted in brain edema (increased water and sodium content) at 4 hours and a histologically defined brain lesion at 24 hours. Both of these markers of brain injury were significantly reduced in the ICE KO mice as compared to wild-type C57BL/6 mice. Regional cerebral blood flow, determined using the flow tracer, N-isopropyl [methyl 1,3-14C] p-iodoamphetamine (14C-IMP), was similar in the two strains of mice, indicating that the reduced brain injury in the KO mice was not a result of a lesser degree of ischemia. These data show that ICE contributes to the development of ischemic brain damage, and that it plays a role at an early time in the pathologic process. Although the mechanism of this effect is uncertain, our results suggest that pharmacologic inhibition of ICE may be a useful treatment for stroke.

Attenuation of Stroke Size in Rats Using an Adenoviral Vector to Induce Overexpression of Interleukin-1 Receptor Antagonist in Brain

Adenoviruses have been proposed as potential vectors for gene therapy in the central nervous system, but there are no reports of their use in the treatment of a brain disease. Because central administration of interleukin-1 receptor antagonist protein (IL-1ra) reduces ischemic brain damage, we determined whether a recombinant adenovirus vector carrying the human IL-1ra cDNA (Ad.RSVIL-1ra) could be used to ameliorate brain injury in permanent focal ischemia. Groups of six rats received intraventricular injections of Ad.RSVIL-1ra or a control adenovirus containing the Escherichia coli β-galactosidase gene (Ad.RSVlacZ). Histochemical staining for β-galactosidase 5 days after virus injection indicated that transgene expression was confined primarily to the cells lining the ventricle. The concentrations of IL-1ra were fivefold to 50-fold higher in the Ad.RSVIL-1ra-injected animals, achieving levels of 9.1 ± 3.3 ng/g in brain and 23.7 ± 22.5 ng/ml in CSF. In these animals, cerebral infarct volume resulting from 24 h of permanent middle cerebral artery occlusion was reduced 64%. These studies demonstrate that adenoviral vectors can be used to deliver genes that attenuate brain injury.

Identification of Hypoxanthine Transport and Xanthine Oxidase Activity in Brain Capillaries

Abstract: Microvessel segments were isolated from rat brain and used for studies of hypoxanthine transport and metabolism. Compared to an homogenate of cerebral cortex, the isolated microvessels were 3.7‐fold enriched in xanthine oxidase. Incubation of the isolated microvessels with labeled hypoxanthine resulted in its rapid uptake followed by the slower accumulation of hypoxanthine metabolites including xanthine and uric acid. The intracellular accumulation of these metabolites was inhibited by the xanthine oxidase inhibitor allopurinol. Hypoxanthine transport into isolated capillaries was inhibited by adenine but not by representative pyrimidines or nucleosides. Similar results were obtained when blood to brain transport of hypoxanthine in vivo was measured using the intracarotid bolus injection technique. Thus, hypoxanthine is transported into brain capillaries by a transport system shared with adenine. Once inside the cell, hypoxanthine can be metabolized to xanthine and uric acid by xanthine oxidase. Since this reaction leads to the release of oxygen radicals, it is suggested that brain capillaries may be susceptible to free radical mediated damage. This would be most likely to occur in conditions where the brain hypoxanthine concentration is increased as following ischemia.

Primary culture of capillary endothelium from rat brain.

SummaryTo provide an in vitro system for studies of brain capillary function we developed a method for culture of brain capillary endothelial cells. Capillaries were isolated from rat brain and enzymatically treated to remove the basement membrane and contaminating pericytes. Subsequent Percoll gradient centrifugation resulted in a homogeneous population of capillary endothelial cells that attached to a collagen substrate and incorporated [3H]thymidine. Evidence for the endothelial nature of these cells was provided by the presence of Factor VIII antigen and angiotensin converting enzyme activity and by the failure of platelets to adhere to the cell surface. In addition, the cells were joined together by tight junctions. Thus, primary cultures of these cells retained both endothelial and blood-brain barrier features.

Overexpression of interleukin-1 receptor antagonist in the mouse brain reduces ischemic brain injury.

It has been reported that middle cerebral artery occlusion in rats causes overexpression of interleukin-1, and that administration of the interleukin-1 receptor antagonist protein (IL-1ra) reduces ischemic brain injury. The aim of the present study is to determine whether a recombinant adenovirus vector carrying human interleukin-1 receptor antagonist cDNA (Ad.RSVIL-1ra) could be used to overexpress IL-1ra in mouse brain and to evaluate its effect on brain edema formation and infarction after permanent focal ischemia in mice. Ad.RSVIL-1ra, control adenovirus containing the lacZ gene (Ad.RSVlacZ), or saline was injected into the right cerebral ventricle in mice. Brain IL-1ra concentrations were measured 1 to 13 days later. On the fifth day after virus injection, the middle cerebral artery was occluded for 24 h. Brain water content was determined and a histological technique was used to measure the infarction size. Overexpression of human IL-1ra protein in whole brain was confirmed by immunoassay in the Ad.RSVIL-1ra injected mice. It began on the first day, peaked at 5-7 days, and was sustained for 13 days. Brain edema and cerebral infarct volume were significantly reduced following 24 h of permanent middle cerebral artery occlusion in mice transfected with Ad.RSVIL-1ra compared to Ad.RSVlacZ or saline 5 days earlier. These studies demonstrate that adenoviral vectors can be used to deliver genes to small animals such as mice and also suggest the feasibility of gene therapy for stroke and other neurological diseases. Overexpression of human IL-1ra attenuated ischemic brain injury, suggesting that IL-1 may play an important role in cerebral ischemia.

Hyperglycemia and the Vascular Effects of Cerebral Ischemia

Hyperglycemia generally enhances cerebral ischemic injury. Most research has focused on the adverse effect of increased lactate production (acidosis) leading to neuronal injury. The effects of hyperglycemia on another possible primary target, the cerebral microvasculature, is examined in this study. Focal cerebral ischemia was achieved by thread occlusion of the middle cerebral artery (MCA). Preischemic hyperglycemia was induced by intra peritoneal administration of 50% of D-glucose solution. In contrast to normoglycemic controls, glucose-injected rats showed a well demarcated pale infarct after 2 or 4 hours of ischemia reflecting a reduction in cerebral plasma volume (CPV) to 73 +/- 9 and 55 +/- 6% of the contralateral hemisphere by 2 and 4 hours respectively. Cerebral blood flow (CBF) measured by laser Doppler flowmetry indicated that after the initial decline in CBF with MCA occlusion, hyperglycemia led to a further progressive reduction during ischemia. On reperfusion, hyperglycemia resulted in poor restoration of CBF, increased occurrence of hemorrhagic infarction (12 of 12) and a large infarct volume. Hyperglycemia induces progressive cerebrovascular changes during ischemia and affects hemodynamic recovery on reperfusion. These changes may contribute to the adverse effects of hyperglycemia in stroke. A reduction in CPV may be a useful indicator of an increased incidence of hemorrhagic infarction after thrombolytic therapy for ischemic stroke.

Blood-brain barrier permeability and brain concentration of sodium, potassium, and chloride during focal ischemia

Brain edema formation during the early stages of focal cerebral ischemia is associated with an increase in both sodium content and blood–brain barrier (BBB) sodium transport. The goals of this study were to determine whether chloride is the principal anion that accumulates in ischemic brain, how the rate of BBB transport of chloride compares with its rate of accumulation, and whether the stimulation seen in BBB sodium transport is also seen with other cations. Focal ischemia was produced by occlusion of the middle cerebral artery (MCAO) in anesthetized rats. Over the first 6 h after MCAO, the amount of brain water in the center of the ischemic cortex increased progressively at a rate of 0.15 ± 0.02 (SE) g/g dry wt/h. This was accompanied by a net increase in brain sodium (48 ± 12 μmol/g dry wt/h) and a loss of potassium (34 ± 7 μmol/g dry wt/h). The net rate of chloride accumulation (16 ± 1 μmol/g dry wt/h) approximated the net rate of increase of cations. Three hours after MCAO, the BBB permeability to three ions (22Na, 36Cl, and 86Rb) and two passive permeability tracers {[3H]α-aminoisobutyric acid (3H]AIB) and [14C]urea} was determined. Permeability to either passive tracer was not increased, indicating that the BBB was intact. The rate of 36Cl influx was 3 times greater and the rate of 22Na influx 1.8 times greater than their respective net rates of accumulation in ischemic brain. The BBB permeability to 22Na relative to that of [3H]AIB was significantly increased in the ischemic cortex, the relative permeability to 86Rb was significantly decreased, and the relative permeability to 36Cl was unchanged. These results indicate that the stimulation in BBB sodium transport is specific for sodium. Further, chloride accumulates with sodium in brain during the early stages of ischemia; however, its rate of accumulation is low compared with its rate of transport from blood to brain. Therefore, inhibition of BBB sodium transport is more likely to reduce edema formation than is inhibition of BBB chloride transport. This study demonstrates that chloride is the principal anion that accompanies the accumulation of sodium in ischemic brain, but its rate of accumulation in brain is much less than its rate of movement into brain, and therefore inhibition of chloride uptake would have little effect on brain edema formation. There is a specific acceleration of blood-to-brain sodium transport during ischemia that is not seen with another positively charged ion, 86Rb. This is consistent with stimulation of brain capillary Na,K-ATPase activity in response to the elevated extracellular potassium concentration. Inhibition of potassium influx across the BBB would probably be more successful in lessening edema formation than accelerating potassium efflux. However, inhibition of blood-to-brain sodium transport is likely to be a more effective approach to reducing brain edema formation during the early stages of cerebral ischemia.

Tumor necrosis factor alpha expression produces increased blood–brain barrier permeability following temporary focal cerebral ischemia in mice

Alteration of blood-brain barrier (BBB) function occurs in both permanent and temporary cerebral ischemia. Studies in vivo and in vitro have shown that tumor necrosis factor-alpha (TNFalpha) is involved in changes of BBB permeability. However, the relationship between TNFalpha expression and BBB disruption during reperfusion is unclear. The aim of this study is to find the cell source of TNFalpha and to determine the relationship between TNFalpha expression and BBB disruption following temporary focal cerebral ischemia in mice. Adult CD-1 mice received 1 h middle cerebral artery occlusion (MCAO) followed by 2 h, 6 h, 12 h, 24 h, and 48 h of reperfusion. MCAO was achieved using an intraluminal suture technique and reperfusion was performed by the suture withdrawal. Neutralizing monoclonal anti-mouse TNFalpha antibody was administrated intraventricularly immediately after reperfusion. TNFalpha expression was determined by double labeling immunohistochemistry. BBB permeability was determined by albumin immunostaining. TNFalpha immunoreactivity (IR) was observed in the ipsilateral hemisphere from 1 h MCAO with 2 h reperfusion. TNFalpha positive cells included neurons, astrocytes, and ependymal cells. BBB disruption was detected beginning at 6 h reperfusion but was not present at 2 h of reperfusion. The areas of BBB disruption were significantly enlarged at 12 h reperfusion and plateaued at 24 h to 48 h reperfusion. BBB disruptions were significantly attenuated in the anti-TNFalpha antibody treated mice (p<0.05). Our results demonstrate that TNFalpha IR existed in neurons, astrocytes, and ependymal cells during reperfusion. TNFalpha IR following temporary focal cerebral ischemia precedes increased BBB permeability. Treatment with TNFalpha antibody reduces BBB disruption, suggesting TNFalpha may be an important mediator in altering BBB permeability during reperfusion.

Sodium Transport in Capillaries Isolated from Rat Brain

Abstract: Brain capillary endothelial cells form a bloodbrain barrier (BBB) that appears to play a role in fluid and ion homeostasis in brain. One important transport system that may be involved in this regulatory function is the Na+,K+‐ATPase that was previously demonstrated to be present in isolated brain capillaries. The goal of the present study was to identify additional Na+ transport systems in brain capillaries that might contribute to BBB function. Microvessels were isolated from rat brains and 22Na + uptake by and efflux from the cells were studied. Total 22Na + uptake was increased and the rate of 22Na + efflux was decreased by ouabain, confirming the presence of Na+,K+‐ATPase in capillary cells. After inhibition of Na+,K+‐ATPase activity, another saturable Na + transport mechanism became apparent. Capillary uptake of 22Na + was stimulated by an elevated concentration of Na +or H+ inside the cells and inhibited by extracellular Na+, H+, Li+, and NH4+. Amiloride inhibited 22Na + uptake with a Ki between 10−5 and 10−6M but there was no effect of 1 mM furosemide on 22Na+ uptake by the isolated microvessels. These results indicate the presence in brain capillaries of a transport system capable of mediating Na +/ Na + and Na +/H + exchange. As a similar transport system does not appear to be present on the luminal membrane of the brain capillary endothelial cell, it is proposed that Na +/H + exchange occurs primarily across the antiluminal membrane.