Baowan Lin

Detection of free radical activity during transient global ischemia and recirculation : effects of intraischemic brain temperature modulation

Abstract: To obtain direct evidence of oxygen radical activity in the course of cerebral ischemia under different intraischemic temperatures, we used a method based on the chemical trapping of hydroxyl radical in the form of the stable adducts 2,3‐ and 2,5‐dihydroxybenzoic acid (DHBA) following salicylate administration. Wistar rats were subjected to 20 min of global forebrain ischemia by two‐vessel occlusion plus systemic hypotension (50 mm Hg). Intraischemic striatal temperature was maintained as normothermic (37°C), hypothermic (30°C), or hyperthermic (39°C) but was held at 37°C before and following ischemia. Salicylate was administered either systemically (200 mg/kg, i.p.) or by continuous infusion (5 mM) through a microdialysis probe implanted in the striatum. Striatal extracellular fluid was sampled at regular intervals before, during, and after ischemia, and levels of 2,3‐ and 2,5‐DHBA were assayed by HPLC with electrochemical detection. Following systemic administration of salicylate, stable baseline levels of 2,3‐ and 2,5‐DHBA were observed before ischemia. During 20 min of normothermic ischemia, a 50% reduction in mean levels of both DHBAs was documented, suggesting a baseline level of hydroxyl radical that was diminished during ischemia, presumably owing to oxygen restriction to tissue at that time. During recirculation, 2,3‐ and 2,5‐DHBA levels increased by 2.5‐ and 2.8‐fold, respectively. Levels of 2,3‐DHBA remained elevated during 1 h of reperfusion, whereas the increase in 2,5‐DHBA levels persisted for 2 h. The increases in 2,3‐ and 2,5‐DHBA levels observed following hyperthermic ischemia were significantly higher (3.8‐ and fivefold, respectively). In contrast, no significant changes in DHBA levels were observed following hypothermic ischemia. The postischemic changes in DHBA content observed following local administration of salicylate were comparable to the results obtained with systemic administration, thus confirming that the hydroxyl radicals arose within brain parenchyma itself. These results provide evidence that hydroxyl radical levels are increased during postischemic recirculation, and this process is modulated by intraischemic brain temperature. Hence, these data suggest a possible mechanism for the effects of temperature on ischemic outcome and support a key role for free radical‐induced injury in the development of ischemic damage.

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.

Effect of delayed MK-801 (dizocilpine) treatment with or without immediate postischemic hypothermia on chronic neuronal survival after global forebrain ischemia in rats

In contrast to intraischemic hypothermia, immediate postischemic hypothermia (30°C) has been shown to delay but not chronically protect the CA1 hippocampus from transient global forebrain ischemia. The inability of a relatively short postischemic hypothermic period to protect chronically might involve a delayed or secondary injury mechanism. We determined whether delayed treatment with the noncompetitive N-methyl-d-aspartate receptor antagonist MK-801 (dizocilpine), alone or in combination with immediate postischemic hypothermia, would chronically protect histopathologically. Wistar rats underwent 10 min of normothermic forebrain ischemia induced by bilateral common carotid artery occlusion plus hypotension (50 mg Hg). Four ischemia groups were studied after normothermic (37°C) ischemia: no treatment; 3 h of immediate postischemic hypothermia (30°C); delayed MK-801 treatment (4 mg/kg) on postischemic days 3, 5, and 7; and postischemic hypothermia combined with multiple MK-801 treatments. Two months after the ischemic insult, rats were perfusion-fixed for quantitative histopathological assessment. Postischemic hypothermia alone or MK-801 treatment alone failed to protect the CA, hippocampus chronically. However, immediate postischemic hypothermia combined with delayed MK-801 treatment led to significant increases in normal CA1 neuron counts per microscopic field compared with normothermic ischemia. For example, neuronal counts within the hippocampal CA1 areas were 58 ± 39 (mean ± SD) in normothermic ischemic rats compared with 395 ± 198 in rats treated with postischemic hypothermia and MK-801. Chronic survival also led to pronounced striatal damage. Within the dorsolateral striatum, significant protection was documented with either postischemic hypothermia alone or delayed MK-801 treatment alone. In the striatum, neuronal counts were 8 ± 5, 47 ± 29, and 63 ± 30 in normothermic, MK-801-treated, and postischemic hypothermic rats, respectively. Significant protection within the somatosensory cortex was not observed with any of the treatment protocols. These findings indicate that the postischemic hippocampus and striatum undergo a delayed excitotoxic insult as late as postischemic day 3. Based on chronic histopathological assessment, the therapeutic window for striatal neuroprotection after a brief global ischemic insult appears to be longer than previously appreciated.

Combined postischemic hypothermia and delayed MK-801 treatment attenuates neurobehavioral deficits associated with transient global ischemia in rats

The present study was designed to determine whether postischemic hypothermia, delayed MK-801 (dizocilpine) administration, or a combination of these treatments can provide lasting neurobehavioral protection following transient global ischemia in rats. Rats were subjected to 10 min of normothermic (37 degrees C) ischemia induced by 2-vessel occlusion and hypotension (50 mmHg) or sham procedures. Ischemia was followed by either: (a) 3 h at normothermic brain temperatures, (b) 3 h of postischemic brain hypothermia at 30 degrees C, (c) hypothermia coupled with MK-801 (4 mg/kg, i.p.) on postischemic days 3, 5 and 7, or (d) postischemic MK-801 treatment alone. Neurobehavioral evaluation 6-8 weeks following surgery showed that normothermic ischemia (NI) was associated with water maze navigational deficits, including performance on a simple place task involving finding a hidden platform maintained in one position for 6 days, and a learning set task in which the platform was moved to a different location each day (both Ps < 0.02 vs. sham). NI was also associated with increased locomotion in an open field (P < 0.01 vs. sham). A combination of postischemic hypothermia and delayed MK-801 injections provided partial protection from ischemic-associated hyperactivity in the open field (P < 0.02 vs. NI), and robust protection from simple place task deficits (P < 0.02 vs. NI). Evidence for significant protective effects of MK-801 or hypothermia alone was observed in the learning set, during the final trial blocks each day. These results provide further evidence for neuroprotective effects of these treatments at chronic survival intervals, and indicate that the therapeutic window for attenuating ischemic damage is considerably longer than has heretofore been appreciated.

Hyperglycemic exacerbation of neuronal damage following forebrain ischemia: Microglial, astrocytic and endothelial alterations

Abstract We undertook a detailed characterization of the cellular responses to acute global cerebral ischemia complicated by hyperglycemia. Anesthetized, physiologically monitored male Wistar rats received 12.5 min of global forebrain ischemia by bilateral common carotid artery occlusions plus hemorrhagic hypotension to 45 mm Hg. Cranial temperature was maintained at normothermic levels. Hyperglycemic animals received dextrose (2.5 ml of a 25% solution, intraperitoneally) prior to ischemia; this doubled the mean plasma glucose concentration to 296 mg/100 ml. At 3 days (n = 10) or 24 h (n = 4) after ischemia, brains were perfusion-fixed and paraffin-embedded for light microscopic histopathology and for the histochemical visualization of activated microglia and the immunocytochemical visualization of glial fibrillary acid protein. Normal-neuron counts in the vulnerable hippocampal CA1 sector of hyperglycemic-ischemic (HI) rats were reduced to one-third the number observed in normoglycemic-ischemic (NI) animals. Ischemic cell counts in the striatum were increased fivefold or more in HI compared to NI rats, and normal small-neuron counts were reduced by two-thirds. The neocortex and striatum of NI rats showed only mild damage, while the majority of HI rats had extensive lesions, and several showed large cortical, striatal or thalamic infarcts. In addition, widespread cortical ischemic neuronal changes were evident in HI animals. No endothelial alterations were present in NI rats. By contrast, HI rats showed prominent peri- and intravascular polymorphonuclear and monocytic accumulation evident at 24 h; frequent white cell thrombi in pial arterioles on day 3; and thickening of vascular endothelium, with foci of parenchymal rarefaction or microinfarction adjacent to occluded vessels. Prominent microglial activation, often along the course of penetrating blood vessels, was common in the striatum and neocortex of HI animals but was much less extensive in the NI group. Activated microglia in HI rats were typically hypertrophic and amoeboid. These results suggest that the detrimental influence of hyperglycemia in ischemia is initially mediated by an action on vascular endothelium, which in turn leads to widespread foci of infarction and neuronal loss.

Differing Neurochemical and Morphological Sequelae of Global Ischemia: Comparison of Single- and Multiple-Insult Paradigms

Abstract: The purpose of this investigation was to investigate pathomechanisms responsible for the deleterious effects of repeated episodes of brief forebrain ischemia. Halothane‐anesthetized male Wistar rats were subjected to either (a) a single 15‐min period or (b) three 5‐min periods (separated by 1 h) of global forebrain ischemia by bilateral carotid artery occlusions plus hypotension (50 mm Hg), followed by various periods of recirculation. Brain temperature was normothermic throughout. In one series of rats, extracellular levels of glutamate, glycine, and γ‐aminobutyric acid (GABA) were measured in the dorsolateral striatum (n = 6–8 per group) and lateral thalamus (n = 4–6 per group) by microdialysis and HPLC before and during ischemia and during 3–5 h of recirculation. In a parallel series of rats (n = 6 per group), ischemic cell change was quantified at 2 (dark neurons), 24, or 72 h following either single or multiple ischemic insults. A single 15‐min ischemic period led to massive glutamate release (13‐fold increase; p= 0.001), which returned to normal by 20–30 min of recirculation and remained normal thereafter. By contrast, in rats with three 5‐min periods of ischemia, the glutamate level rise with each repeated insult (four‐ to 4.5‐fold; p 0.02) was smaller than that observed during the single 15‐min insult, but a late sustained rise (five‐ to six‐fold; p < 0.05) occurred at 2–3 h of recirculation. Brief ischemia‐induced elevations of glycine and GABA levels were detected in both the single‐ and multiple‐insult groups, with normalization during recirculation. In contrast, the excitotoxic index, a composite measure of neurotransmitter release ([glutamate] X [glycine]/[GABA]), differed markedly following single versus multiple insults (p= 0.002 by repeated‐measures analysis of variance) and increased by seven‐ to 12‐fold (p < 0.05) at 1–3 h following the third insult. The total amount of glutamate released was 3.3‐fold higher in the multiple‐insult than in the single‐insult group (p < 0.02). At 2 h of recirculation, histopathological analysis of dorsolateral striatum showed a significantly greater frequency of dark neurons in the multiple‐ than in the single‐insult group (p < 0.05 by analysis of variance). In the thalamus, a higher frequency of ischemic neurons was seen in the multiple‐ than in the single‐insult group at all intervals studied. Thus, in rats with multiple ischemic insults, accelerated ischemic damage was found in the striatum, and severe ischemic injury was documented in the thalamus. These results demonstrate that multiple ischemic insults lead to a massive, sustained glutamate accumulation and to a major increase in the excitotoxic index during early recirculation, which is not seen following a single brief ischemic episode. These neurochemical changes correlate with our histopathological data showing an accelerated evolution of striatal pathology at 2 h following multiple ischemic insults.

Sequential analysis of subacute and chronic neuronal, astrocytic and microglial alterations after transient global ischemia in rats

Abstract Recent experimental investigations have emphasized the importance of assessing both acute and chronic histopathological changes occurring after cerebral ischemia. The purpose of this study was to evaluate the temporal profile of neuronal, astrocytic and microglial alterations within vulnerable regions (striatum and CA1 sector of hippocampus) following transient global ischemia. Anesthetized Wistar rats underwent 10 min of normothermic (37° C) ischemia induced by bilateral carotid ligations plus hypotension (45–50 mm Hg) and were allowed to survive for periods ranging from 1 to 10 weeks (n = 4–6/ group) prior to quantitative histopathological analysis. Adjacent sections were examined by hematoxylin-and-eosin histopathology, immunostaining for glial fibrillary acidic protein, and B4-isolectin immunochemistry for microglia. In the striatum, normal-neuron counts were first decreased significantly at 2 weeks after the ischemic insult. Neuronal loss was associated with the proliferation of reactive microglia, which peaked at 1 week. By contrast, reactive astrocytosis displayed a more protracted pattern, with peak activation at 2 weeks. In the CA1 hippocampus, a decreased number of normal neurons was seen at 1 week post ischemia, together with a significant increase in immunoreactive microglia at that time; the latter normalized after 2 weeks. Reactive astrocytes in the CA1 hippocampus were significantly increased at 1–2 weeks after ischemia. In a subgroup of severely injured animals, foci of frank striatal infarction were associated with early and severe microglial and astrocytic proliferation at week 4 or later. Finally, cerebrovascular changes included endothelial disruption within affected areas. These observations document a subacute and chronic sequence of cellular responses following brief periods of global ischemia, involving both neurons, glia and vascular endothelium.

Hyperglycemia triggers massive neutrophil deposition in brain following transient ischemia in rats.

Myeloperoxidase (MPO) immunohistochemistry was used to ascertain the role of polymorphonuclear leukocytes in hyperglycemia-induced accentuation of brain injury after transient ischemia. Rats received 12.5 min of normothermic global cerebral ischemia by bilateral carotid occlusion plus hypotension. Hyperglycemia was induced before ischemia by intraperitoneal dextrose administration. Quantitative MPO immunohistochemistry was performed at 24 h and 3 days postischemia. Brains of normoglycemic-ischemic animals contained almost no MPO activity. By contrast, striking numbers of MPO-positive cells were present in brains studied 24 h after hyperglycemic ischemia, both within pial and parenchymal vessels and within the parenchyma. MPO deposition tended to subside at 3 days. These results indicate that hyperglycemia triggers the early, massive deposition of neutrophils in the postischemic brain--an event that may contribute to exacerbation of injury.

Experimental intracerebral hematoma in the rat : Characterization by sequential magnetic resonance imaging, behavior, and histopathology. Effect of albumin therapy

We characterized acute intracerebral hemorrhage (ICH) in the rat by sequential magnetic resonance imaging (MRI) and correlated MRI findings with neurobehavior and histopathology. In addition, we investigated whether albumin treatment would reduce ICH-induced brain injury. ICH was produced in rats by a double-injection method in which 45 microl of fresh arterial blood was injected into the right striatum. Susceptibility-weighted (SWI) and T2-weighted (T2WI) MRI was carried out on a 4.7T magnet at 0-1 h, 6 h, 24 h, 72 h, and 7 days after ICH. Animals were treated with either 25% human albumin, 1.25 g/kg, or saline vehicle i.v. at 90 min after ICH. Neurological status was evaluated before ICH and after treatment (at 4 h, 24 h, 48 h, 72 h, and 7 days). Brains were then perfusion-fixed, re-imaged on an 11.7T magnet, and studied by histopathology and immunochemistry. MRI revealed a consistent hematoma involving the striatum and overlying corpus callosum, with significant volume changes over time. Lesion volumes computed from T2WI images and by histopathology agreed closely with one another and were highly correlated (p=0.002). SWI lesion volumes were also highly correlated to histological volumes (p<0.001) but overestimated histological hematoma volume by approximately 5-fold. Albumin treatment significantly improved neurological scores compared to saline at 72 h (3.8+/-0.6 vs. 1.5+/-0.7) and 7 days (3.8+/-0.4 vs. 1.3+/-0.5, respectively, p<0.05), but did not affect histological or MRI lesion volumes. Taken together, sequential MRI plus histopathology provides a comprehensive characterization of experimental ICH. Albumin treatment improves neurological deficit after ICH but does not affect MRI or histological hematoma size.

Transient changes of brain-derived neurotrophic factor (BDNF) mRNA expression in hippocampus during moderate ischemia induced by chronic bilateral common carotid artery occlusions in the rat

Chronic bilateral common carotid artery occlusion (BCCAO) induces moderate ischemia (oligemia) in the rat forebrain in the absence of overt neuronal damage. In situ hybridization for brain-derived neurotrophic factor (BDNF) mRNA was used to search for a molecular response to moderate ischemia. BDNF mRNA was significantly increased in the hippocampal granule cells at 6 h of occlusion (ANOVA, Tukey test P<0.05). At 1, 7 and 14 days BDNF mRNA levels returned to control levels. The frequency of BDNF gene expression at 6 h was 83%, which was significantly higher than the 7% incidence of histological injury in the hippocampus (Fishers exact test, P<0.002). Cerebral blood flow was reduced to 75% of control levels in the hippocampus after 1 week of BCCAO when measured with the autoradiographic method. Measurements of tissue flow with a microprobe for laser Doppler flow excluded decreases into the ischemic range during the period when elevated gene expression was observed. Prolonged moderate ischemia (oligemia) is a sufficient stimulus for BDNF gene expression in the hippocampus. These molecular studies provide direct evidence for an involvement of the hippocampus in the BCCAO model.