Cathepsin B and middle cerebral artery occlusion in the rat

Lysosomal proteases, although tightly regulated under physiological conditions, are known to contribute to cell injury after various forms of tissue ischemia have occurred. Because cathepsin B is a prominent lysosomal protease found in brain parenchyma, the authors hypothesized that it may contribute to neuronal cell death after focal cerebral ischemia. The authors measured the expression and spatial distribution of cathepsin B within the ischemic brain in 43 animals by means of immunohistochemical analysis in a rat model of transient middle cerebral artery (MCA) occlusion. Cathepsin B activity was also measured within specific ischemic brain regions by using an in vitro assay (22 animals). In addition, the authors tested the therapeutic effect of preischemic intraventricular administration of stefin A, a cysteine protease inhibitor, on the volume of cerebral infarction after transient MCA occlusion (15 animals). Increased cathepsin B immunoreactivity was detected exclusively within the ischemic neurons after 2 hours of reperfusion following a 2-hour MCA occlusion. Cathepsin B immunolocalization in the ischemic region decreased by 24 hours of reperfusion, but then increased by 48 hours of reperfusion because the infarct was infiltrated by inflammatory cells. Increased immunolocalization of cathepsin B in the inflammatory cells located in the necrotic infarct core continued through 7 days of reperfusion. Cathepsin B enzymatic activity was significantly increased in the ischemic tissue at 2, 8, and 48 hours, but not at 24 hours of reperfusion after 2 hours of MCA occlusion. Continuous intraventricular infusion of stefin A, before 2 hours of MCA occlusion (15 animals), significantly reduced infarct volume compared with control animals (12 animals): the percentage of hemispheric infarct volume was 20+/-3.9 compared with 33+/-3.5 (standard error of the mean; p = 0.025). These data indicate that neuronal cathepsin B undergoes increased expression and activation within 2 hours of reperfusion after a 2-hour MCA occlusion and may be a mechanism contributing to neuronal cell death. Intraventricular infusion of stefin A, an inhibitor of cathepsin B, significantly reduces cerebral infarct volume in rats.     

A novel endothelin antagonist, A-127722, attenuates ischemic lesion size in rats with temporary middle cerebral artery occlusion: a diffusion and perfusion MRI study

BACKGROUND AND PURPOSE: Endothelins (ETs) are potent vasoconstrictors. Plasma ET levels increase during acute brain ischemia and may worsen the ischemic damage. Diffusion-weighted MRI (DWI) and perfusion imaging (PI) are powerful tools for evaluation of acute cerebral ischemia. We studied the effects of A-127722, a novel ET(A)-selective ET antagonist, on cerebral ischemic lesion size using 2,3,5-triphenyltetrazolium chloride (TTC) staining postmortem, on acute ischemic lesion development with DWI, and on the cerebral circulation using PI. METHODS: Twenty male Sprague-Dawley rats received either 5 mg/kg of A-127722 or vehicle (n=10 per group) intravenously 30 minutes and subcutaneously 4 hours after middle cerebral artery occlusion (MCAO). Whole-brain DWI and single-slice PI were done before initiation of treatment and repeated frequently thereafter up to 4 hours after MCAO. The animals were reperfused in the MRI scanner 90 minutes after the onset of MCAO. At 24 hours the animals were killed, and the brains were cut into six 2-mm-thick slices and stained with 2% TTC. Percent hemispheric lesion volume (%HLV) was calculated for each animal. RESULTS: Physiological parameters, body weight, neurological scores, and premature mortality (2 versus 2) did not differ between the two groups. No hypotension, abnormal behavior, or other adverse effects were seen. TTC-derived %HLV was 25.3+/-5.6% for controls and 16.2+/-9.6% for treated animals (36% reduction, P<.02). Six animals in each group had successful reperfusion as shown by PI. Among these animals, %HLV was 23.2+/-3.1% for controls and 9.3+/-4.4% for treated animals (60% reduction, P=.0001). The beneficial effect of A-127722 was limited to animals in which successful reperfusion was demonstrated. No difference in PI-detected perfusion deficit size was observed between the groups. DWI did not demonstrate significant in vivo lesion size differences. CONCLUSIONS: A-127722 significantly reduced ischemic lesion size in rats without observable adverse effects. It is not clear whether the effect was due to vasodilatation of collateral arterioles not detectable by PI or whether A-127722 has neuroprotective properties that are independent of vascular effects.     

Pretreatment with a competitive NMDA antagonist D-CPPene attenuates focal cerebral infarction and brain swelling in awake rats

The purpose of the study was to assess effects of the competitive N-methyl-D-aspartate (NMDA) receptor antagonist D-(E)-4-(3-phosphonoprop-2-enyl)piperazine-2-carboxylic acid (D-CPPene) upon focal cerebral infarction and brain oedema in the rat. Focal cerebral ischaemia was produced by permanent occlusion of the middle cerebral artery under halothane anaesthesia. The anaesthetic gas was discontinued immediately after the occlusion and the rats were killed 24 hours later. Cerebral infarction and brain swelling were each assessed on the frozen brain sections at 8 predetermined coronal planes. Pretreatment with D-CPPene (4.5 mg/kg i.v. followed by continuous infusion at 3 mg/kg/h until sacrifice) 15 minutes prior to MCA occlusion, significantly reduced the volume of infarction in the cerebral hemisphere by 29% (p < 0.05). Brain swelling, obtained by subtracting the nonischaemic hemispheric volume from the ischaemic hemispheric volume, was significantly reduced with D-CPPene treatment and the mean reduction in swelling (34% less than the controls: p < 0.001) proportionately similar to the decrease in infarct volume in the same animals. These data indicate that systemic administration of the competitive NMDA receptor antagonist D-CPPene has neuroprotective effects against ischaemic brain damage, and the reduction in brain swelling occurs in parallel with the reduction in ischaemic damage.     

Reduction of ischemic brain injury by topical application of glial cell line-derived neurotrophic factor after permanent middle cerebral artery occlusion in rats

BACKGROUND AND PURPOSE: Glial cell line-derived neurotrophic factor (GDNF) plays important roles in the survival and recovery of some mature neurons under pathological conditions. However, the effect of GDNF in ameliorating ischemic brain injury has not been well documented. Therefore, we investigated a possible effect of GDNF on the changes of infarct size, brain edema, DNA fragmentation, and immunoreactivities for caspases after permanent middle cerebral artery occlusion (MCAO) in rats. METHODS: For the estimation of ischemic brain injury, we calculated the infarct size of MCA region and also measured the brain water content as edema formation at 24 hours after the MCAO. Terminal deoxynucleotidyl transferase-mediated dUTP-biotin in situ nick labeling (TUNEL) staining was performed for the detection of DNA fragmentation. Immunoreactivities for caspase-1 (ICE), caspase-2 (Nedd-2), and caspase-3 (CPP32) were stained. RESULTS: Both infarct size and brain edema after permanent MCAO were significantly reduced by topical application of GDNF (48% and 30% decreases, P=0.01). TUNEL staining and immunoreactivities for caspases were markedly induced at 12 hours after permanent MCAO in the vehicle-treated animals. However, the spatial distribution of those immunohistochemically positive cells was dissociative in each caspase. Induction of TUNEL staining and immunoreactivities for caspases-1 and -3 was greatly reduced with GDNF treatment, whereas the reduction of caspase-2 staining was only minimum. CONCLUSIONS: These data suggest that the reduction of infarct size and brain edema by GDNF was greatly associated with the reduction of DNA fragmentation and apoptotic signals predominantly through caspases-1 and -3 cascades.     

Intraventricular brain-derived neurotrophic factor reduces infarct size after focal cerebral ischemia in rats

Brain-derived neurotrophic factor (BDNF), acting through the high-affinity receptor tyrosine kinase (TrkB), is widely distributed throughout the central nervous system and displays in vitro trophic effects on a wide range of neuronal cells, including hippocampal, cerebellar, and cortical neurons. In vivo, BDNF rescues motorneurons, hippocampal, and substantia nigral dopaminergic cells from traumatic and toxic brain injury. After transient middle cerebral artery occlusion (MCAO), upregulation of BDNF-mRNA in cortical neurons suggests that BDNF potentially plays a neuroprotective role in focal cerebral ischemia. In the current study, BDNF (2.1 micrograms/d) in vehicle or vehicle alone (controls) was delivered intraventricularly for 8 days, beginning 24 hours before permanent middle cerebral artery occlusion by intraluminal suture in Wistar rats (n = 13 per group). There were no differences in physiological variables recorded during surgery for the two groups. Neurological deficit (0 to 4 scale), which was assessed on a daily basis, improved in BDNF-treated animals compared with controls (P < 0.05; analysis of variance and Scheffe's test). There were no significant differences in weight in BDNF-treated animals and controls during the experiment. After elective killing on day 7 after MCAO, brains underwent 2,3,5-triphenyltetrazolium chloride staining for calculation of the infarct volume and for histology (hematoxylin and eosin and glial fibrillary acid protein). The mean total infarct volume was 83.1 +/- 27.1 mm3 in BDNF-treated animals and 139.2 +/- 56.4 mm3 in controls (mean +/- SD; P < 0.01, unpaired, two-tailed t-test). The cortical infarct volume was 10.8 +/- 7.1 mm3 in BDNF-treated animals and 37.9 +/- 19.8 mm3 in controls (mean +/- SD; P < 0.05; unpaired, two-tailed t-test), whereas ischemic lesion volume in caudoputaminal infarction was not significantly different. These results show that pretreatment with intraventricular BDNF reduces infarct size after focal cerebral ischemia in rats and support the hypothesis of a neuroprotective role for BDNF in stoke.     

The effects of a butanediol treatment on acute focal cerebral ischemia assessed by quantitative diffusion and T2 MR imaging

Increased water T2 values indicates the presence of vasogenic edema. Decreased apparent diffusion coefficient (ADC) maps reveal ischemic areas displaying cytotoxic edema. ADC and T2 abnormalities spread through the middle cerebral artery (MCA) territory up to 24 h after middle cerebral artery occlusion (MCAO). Also, it was found that ADC and T2 contours closely match at 3.5 and 24 h. Since butanediol reduces vasogenic edema and improves energy status in various models of ischemia, we used these two techniques to investigate putative improvements in cytotoxic and vasogenic edema after permanent MCAO performed on rats. Rats were given no treatment (n = 8), or a treatment with 25 mmol/kg intraperitoneal (i.p.) butanediol (n = 5), 30 min before and 2.5 h after MCAO. Quantitative ADC and T2 maps of brain water were obtained, from which the volumes presenting abnormalities were calculated at various time points up to 24 h. Effects of butanediol on the ADC and T2 values in these areas were determined. Butanediol reduced neither the ADC volume nor the initial ADC decline. However, it reduced T2 volumes by 32% at 3.5 h and 15% at 24 h (p < 0.05), and reduced T2 increase in the striatum at 3.5 h post-MCAO. Therefore, our results show for the first time that a pharmacological agent such as butanediol can delay the development of vasogenic edema but does not limit the development of vasogenic edema but does not limit the development of cytotoxic edema. ADC imaging detects areas of severe metabolic disturbance but not moderately ischemic peripheral areas where butanediol is presumed to be more efficacious.     

Monitoring aortic root effluent during retrograde cardioplegia delivery

Early signs of brain infarction can be detected by modern CCT technology even within the first 6 h after stroke. Little is known about the prognostic significance of early infarction signs in CCT. We prospectively evaluated clinical and CCT findings of 95 consecutive patients with an acute ischemia in the territory of the middle cerebral artery. All patients were admitted to our stroke unit within 6 h after stroke. In 55 patients CCT was performed within 3 h, and in 40 cases between 3 and 6 h. In all patients the clinical findings were assessed by the Scandinavian Stroke Scale (SSS). The disability due to stroke was evaluated after 4 weeks by use of the modified Rankin Scale. We could demonstrate the following early signs of cerebral infarction: focal hypodensity (23.2%), obscuration of basal ganglia (12.6%), focal brain swelling (22.1%), hyperdense middle cerebral artery sign (HMCA; 11.5%). In 3 patients early edema led to ventricular compression, in 1 patient to midline shift. The occurrence of early infarction signs did not depend on the etiology of ischemia but was significantly associated with a severe neurological deficit at admission and an unfavourable disability status 4 weeks after stroke. Focal brain swelling and HMCA were often followed by extensive infarction lesions on the follow-up CCT. In conclusion, early signs of hemispheric brain infarction visible on CCT scans performed within 6 h after stroke are correlated with severe stroke and an unfavourable functional outcome. However, a substantial part of our patients had a benign course of the disease in spite of early CCT pathology. Decisions on therapy in individual patients therefore should not depend on early CCT findings exclusively.     

Matrix metalloproteinases and TIMPs are associated with blood-brain barrier opening after reperfusion in rat brain

BACKGROUND and PURPOSE: Reperfusion disrupts cerebral capillaries, causing cerebral edema and hemorrhage. Middle cerebral artery occlusion (MCAO) induces the matrix-degrading metalloproteinases, but their role in capillary injury after reperfusion is unknown. Matrix metalloproteinases (MMPs) and tissue inhibitors to metalloproteinases (TIMPs) modulate capillary permeability. Therefore, we measured blood-brain barrier (BBB) permeability, brain water and electrolytes, MMPs, and TIMPs at multiple times after reperfusion. METHODS: Adult rats underwent MCAO for 2 hours by the suture method. Brain uptake of 14C-sucrose was measured from 3 hours to 14 days after reperfusion. Levels of MMPs and TIMPs were measured by zymography and reverse zymography, respectively, in contiguous tissues. Other rats had water and electrolytes measured at 3, 24, or 48 hours after reperfusion. Treatment with a synthetic MMP inhibitor, BB-1101, on BBB permeability and cerebral edema was studied. RESULTS: Brain sucrose uptake increased after 3 and 48 hours of reperfusion, with maximal opening at 48 hours and return to normal by 14 days. There was a correlation between the levels of gelatinase A at 3 hours and the sucrose uptake (P<0.05). Gelatinase A (MMP-2) was maximally increased at 5 days, and TIMP-2 was highest at 5 days. Gelatinase B and TIMP-1 were maximally elevated at 48 hours. The inhibitor of gelatinase B, TIMP-1, was also increased at 48 hours. Treatment with BB-1101 reduced BBB opening at 3 hours and brain edema at 24 hours, but neither was affected at 48 hours. CONCLUSIONS: The initial opening at 3 hours correlated with gelatinase A levels and was blocked by a synthetic MMP inhibitor. The delayed opening, which was associated with elevated levels of gelatinase B, failed to respond to the MMP inhibitor, suggesting different mechanisms of injury for the biphasic BBB injury.     

Transient middle cerebral artery occlusion by intraluminal suture: II. Neurological deficits, and pixel-based correlation of histopathology with local blood flow and glucose utilization

We conducted a pixel-based analysis of the acute hemodynamic and metabolic determinants of infarctive histopathology in a reproducible model of temporary (2-hour) middle cerebral artery occlusion (MCAO) produced in rats by an intraluminal suture. Three-dimensional averaged image data sets of local cerebral blood flow (LCBF) and glucose utilization (LCMRglc) acquired in the companion study (Belayev et al., 1997) either at the end of a 2-hour period of MCAO or after 1 hour of recirculation were comapped (using digitized atlas-templates) with data sets depicting the frequency of histological infarction in a matched animal group (n = 8) in which 2 hours of MCAO was followed by 3-day survival, sequential neuro behavioral examinations, and perfusion-fixation and paraffin-embedding of brains for light-microscopic analysis. All rats developed marked postural-reflex and forelimb-placing deficits at 60 minutes of MCAO, signifying high-grade ischemia. Tactile placing deficits persisted during the 72-hour observation period while visual placing and postural-reflex abnormalities variably improved. Comapping of LCBF and histopathology showed that in those pixels destined to undergo infarction, LCBF measured at 2 hours of MCAO showed a sharp distributional peak centered at 0.14 mL/g/min. In 70% of pixels destined to infarct, LCBF at 2 hours of MCAO was 0.24 mL/g/min or below, and in 89% LCBF was below 0.47 mL/g/min (the upper limits of the ischemic core and penumbra, respectively, as defined in the companion study [Belayev et al., 1997]). Local cerebral glucose utilization measured at approximately 1 hour after 2 hours of MCAO was distributed bimodally in the previously ischemic hemisphere. The major peak, at 22 mumol/100g/min, coincided exactly with the distribution peak of pixels destined to undergo infarction, while in pixels with a zero probability of infarction, LCMRglc was higher by 12 to 13 mumol/100g/min. These results indicate that local blood flow at 2 hours of MCAO is a robust predictor of eventual infarction. Pixels with ischemic-core levels of LCBF (0% to 20% of control) have a 96% probability of infarction, while the fate of the penumbra is more heterogeneous: below LCBF of 0.35 mL/g/min, the probability of infarction is 92%, while approximately 20% pixels in the upper-penumbral LCBF range (30% to 40% of control) escape infarction. Our data strongly support the view that the likelihood of infarction within the ischemic penumbra is highly influenced by very subtle differences in early perfusion.     

Immunologic tolerance to myelin basic protein decreases stroke size after transient focal cerebral ischemia

Immune mechanisms contribute to cerebral ischemic injury. Therapeutic immunosuppressive options are limited due to systemic side effects. We attempted to achieve immunosuppression in the brain through oral tolerance to myelin basic protein (MBP). Lewis rats were fed low-dose bovine MBP or ovalbumin (1 mg, five times) before 3 h of middle cerebral artery occlusion (MCAO). A third group of animals was sensitized to MBP but did not survive the post-stroke period. Infarct size at 24 and 96 h after ischemia was significantly less in tolerized animals. Tolerance to MBP was confirmed in vivo by a decrease in delayed-type hypersensitivity to MBP. Systemic immune responses, characterized in vitro by spleen cell proliferation to Con A, lipopolysaccharide, and MBP, again confirmed antigen-specific immunologic tolerance. Immunohistochemistry revealed transforming growth factor beta1 production by T cells in the brains of tolerized but not control animals. Systemic transforming growth factor beta1 levels were equivalent in both groups. Corticosterone levels 24 h after surgery were elevated in all sham-operated animals and ischemic control animals but not in ischemic tolerized animals. These results demonstrate that antigen-specific modulation of the immune response decreases infarct size after focal cerebral ischemia and that sensitization to the same antigen may actually worsen outcome.     

Cytosolic redistribution of cytochrome c after transient focal cerebral ischemia in rats

Recent in vitro cell-free studies have shown that cytochrome c release from mitochondria is a critical step in the apoptotic process. The present study examined the expression of cytochrome c protein after transient focal cerebral ischemia in rats, in which apoptosis was assumed to contribute to the expansion of the ischemic lesion. In situ labeling of DNA breaks in frozen sections after 90 minutes of middle cerebral artery (MCA) occlusion showed a significant number of striatal and cortical neurons, which were maximized at 24 hours after ischemia, exhibiting chromatin condensation, nuclear segmentation, and apoptotic bodies. Cytosolic localization of cytochrome c was detected immunohistochemically in the ischemic area as early as 4 hours after 90 minutes of MCA occlusion. Western blot analysis of the cytosolic fraction revealed a strong single 15-kDa band, characteristic of cytochrome c, only in the samples from the ischemic hemisphere. Western blot analysis of the mitochondrial fraction showed a significant amount of mitochondrial cytochrome c in nonischemic brain, which was decreased in ischemic brain 24 hours after ischemia. These results provide the first evidence that cytochrome c is being released from mitochondria to the cytosol after transient focal ischemia. Although further evaluation is necessary to elucidate its correlation with DNA fragmentation, our results suggest the possibility that cytochrome c release may play a role in DNA-damaged neuronal cell death after transient focal cerebral ischemia in rats.     

Rapid disappearance of zinc positive terminals in focal brain ischemia

The study was undertaken to determine if the levels of vesicular zinc in neuronal terminals would decrease in response to focal brain ischemia. The middle cerebral artery was occluded distal to the striatal branches in male spontaneously hypertensive rats. At 7, 15, 30, 45, 60, 90, 120 min; 3, 6, 12, 24, 48 h and 7 days later the animals were sacrificed and the brains were stained for zinc-sulfides, cell bodies and AChE-positive cholinergic fibers. The density of zinc positive terminals significantly decreased in the neocortical ischemic zone 7 min after middle cerebral artery occlusion (MCAO). In the neocortical layers II and III most zinc positive neuronal terminals disappeared at 7 min after MCAO whereas the zinc positive terminals in layers V and VI remained positive at least 2 h. Beginning at 1 h after MCAO and progressing to 24 h a significant decrease in the density of zinc positive terminals was observed in the dorsolateral striatum, and ventrobasal thalamic nucleus, both major projection areas of the sensorimotor cortex. The disappearance of zinc positive neuronal terminals in the ischemic neocortex and related areas, is most likely due to a neuronal release of vesicular zinc in response to hypoxia. The high extracellular concentration of zinc is thought to be both neuroprotective by blocking the NMDA receptor and neurotoxic by activating neuronal influx of Ca2+ through voltage gated calcium channels. It seems evident that the latter effect of zinc is contributing to the neuronal death in focal brain ischemia.     

Cell density-dependent mitogenic effect and -independent cellular handling of epidermal growth factor in primary cultured rat hepatocytes

Brain swelling is a serious complication associated with focal ischemia in stroke and severe head injury. Experimentally, reperfusion following focal cerebral ischemia exacerbates the level of brain swelling. In this study, the permeability of the blood-brain barrier has been investigated as a possible cause of reperfusion-related acute brain swelling. Blood-brain barrier disruption was investigated using Evans Blue dye and [14C]aminoisobutyric acid autoradiography in a rodent model of reversible middle cerebral artery (MCA) occlusion. Acute brain swelling and cerebral blood flow (CBF) during ischemia and reperfusion were analyzed from double-label CBF autoradiograms after application of the potent vasoconstrictor peptide endothelin-1 to the MCA. Ischemia was apparent within ipsilateral MCA territory, 5 min after endothelin-1 application to the exposed artery. Reperfusion, examined at 30 min and 1, 2, and 4 h, was gradual but incomplete within this time frame in the core of middle cerebral artery territory and associated with significant brain swelling. Ipsilateral hemispheric swelling increased over time to a maximum (>5%) at 1-2 h after endothelin-1 but was not associated with a significant increase in the ipsilateral transfer constant for [14C]aminoisobutyric acid over this time frame. These results indicate that endothelin-1 induced focal cerebral ischemia is associated with an acute but reversible hemispheric swelling during the early phase of reperfusion which is not associated with a disruption of the blood-brain barrier.     

The use of somatosensory evoked potential monitoring to produce a canine model of uniform, moderately severe stroke with permanent arterial occlusion

To develop a reliable canine model of cerebral infarction of moderate size, we compared infarctions caused by permanent occlusion of the following vessels in 42 dogs: 1) the middle cerebral artery (MCA), 2) the MCA and azygous anterior cerebral artery (ACA), 3) the MCA, azygous ACA, and posterior cerebral artery (PCA), and 4) sham-operated controls. The infarction volume was determined at 6 hours in half the animals and at 6 days in the others. Studies of somatosensory evoked potentials (SSEPs) and regional cerebral blood flow (rCBF) were performed before and after arterial occlusion, and good correlation was observed between the decrease in amplitude of the SSEPs and the decrease in rCBF observed after arterial occlusion. Only the groups in which the MCA and azygous ACA were occluded showed moderate infarctions of relatively consistent size. Analysis involving all groups revealed that the animals with SSEP amplitude preserved after vessel occlusion had only small infarctions; thus, preservation of SSEP amplitude after occlusion of the MCA and azygous ACA could in the future be used prospectively as a rejection criterion to improve the uniformity of infarction size. Conversely, animals with loss of SSEP amplitude after vessel occlusion had infarctions of moderate to large size; thus, loss of SSEP amplitude after MCA occlusion alone could in the future be used prospectively as a rejection criterion. When these rejection criteria were retrospectively applied to the groups in which both the MCA and azygous ACA were occluded, the resulting mean infarction volumes +/- 1 SEM) for the acute and chronic subgroups were 20.3 +/- 2.8% and 38.2 +/- 4.5% of the hemisphere, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Focal cerebral ischemia in the mouse: description of a model and effects of permanent and temporary occlusion

Our study is to demonstrate the advantages and disadvantages of middle cerebral artery occlusion (MCAO) model in the mouse. CD-1 mice had permanent MCAO for 24 h, or temporary occlusion for either 1 h followed by 23 h of reperfusion or 2 h of occlusion with 22 h of reperfusion. The infarct volume and blood-brain barrier disruption were smaller in the 1-h/23-h temporary occlusion than in either the 24-h permanent occlusion group or the 2-h/22-h temporary occlusion group (p<0.05). Our study demonstrates that blood flow, infarct volume, and blood-brain barrier disruption remain important markers of focal cerebral ischemia.     

Regional cerebral blood flow during and after 2 hours of middle cerebral artery occlusion in the rat

In this study we explored if the secondary bioenergetic failure, which occurs a few hours after recirculation, following transient middle cerebral artery occlusion (MCAO) in rats, is caused by a compromised reflow. We induced 2 hours of MCAO and measured CBF at the end of the ischemia, as well as 15 minutes, 1, 2, and 4 hours after the start of recirculation, using autoradiographic or tissue sampling 14C-iodoantipyrine techniques. After 2 hours of MCAO, the autoradiographically measured CBF in the ischemic core areas was reduced to 3 to 5% of contralateral values. The reduction in CBF was less in neighboring, penumbral areas. After recirculation, flow already normalized in core tissues after 15 minutes, and remained close to normal for the 4 hours recirculation period studied. However, in penumbral tissues, recovery CBF values were usually below normal. The results show that tissues that are heavily compromised by the 2-hour period of ischemia and are destined to incur infarction, show a "relative hyperemia" during recirculation. In fact, some areas of the previously densely ischemic tissue showed overt hyperperfusion. This finding raises the question whether the relative or absolute hyperemia reflects events that are pathogenetically important. Because drugs that clearly ameliorate the final damage incurred fail to alter the relative hyperperfusion of previously ischemic tissues, it is concluded that vascular events in the reperfusion period do not play a major role in causing the final damage.     

E-selectin appears in nonischemic tissue during experimental focal cerebral ischemia

BACKGROUND AND PURPOSE: E-selectin participates in leukocyte-endothelial adhesion and the inflammatory processes that follow focal cerebral ischemia and reperfusion. The temporal and topographical patterns of microvascular E-selectin presentation after experimental focal cerebral ischemia are relevant to microvascular reactivity to ischemia. METHODS: The upregulation and fate of E-selectin antigen during 2 hours of middle cerebral artery occlusion (n = 4) and 3 hours of occlusion with reperfusion (1 hour, n = 4; 4 hours, n = 6; 24 hours, n = 6) were evaluated in the nonhuman primate. E-selectin and E:P-selectin immunoreactivities were semiquantitated with the use of computerized light microscopy video imaging and laser confocal microscopy. RESULTS: Three patterns of microvascular E-selectin expression, defined by the antibody E-1E4, were confirmed by complete elimination of E-1E4 binding after incubation with soluble recombinant human E-selectin: (1) Low immunoperoxidase intensity was observed in ischemic microvessels at 2 hours of occlusion extending to 4 hours of reperfusion (E-selectin/laminin = 0.32 +/- 0.10). (2) A significant fraction of ischemic microvessels displayed high-intensity E-selectin signal by 24 hours of reperfusion (0.61 +/- 0.17) compared with control and nonischemic tissues (2P < .003). (3) In the contralateral nonischemic basal ganglia and other nonischemic tissues, low but significant E-selectin levels appeared by 24 hours of reperfusion (2P = .0005). The latter were further confirmed by an E:P-selectin immunoprobe. CONCLUSIONS: E-selectin antigen is distinctively and significantly upregulated in nonhuman primate brain after focal ischemia and reperfusion. The late appearance of E-selectin in nonischemic cerebral tissues suggests stimulation by transferable factors generated during brain injury.     

Estrogen-mediated neuroprotection after experimental stroke in male rats

BACKGROUND AND PURPOSE: We have previously shown that 17beta-estradiol reduces infarction volume in female rats. The present study determined whether single injection or chronic implantation of estrogen confers neuroprotection in male animals with middle cerebral artery occlusion (MCAO) and whether there is an interaction with endogenous testosterone. METHODS: Male Wistar rats were treated with 2 hours of reversible MCAO. In protocol 1, acute versus chronic estrogen administration was examined in groups receiving the following: Premarin (USP) 1 mg/kg IV, immediately before MCAO (Acute, n=13, plasma estradiol=171+/-51 pg/mL); 7 days of 25 microg (E25, n=10, 10+/-3 pg/mL) or 100 microg 17beta-estradiol (E100, n=12, 69+/-20 pg/mL) by subcutaneous implant; or saline (SAL, n=21, 3+/-1 pg/mL). Laser-Doppler flowmetry was used to monitor the ipsilateral parietal cortex throughout the ischemic period and early reperfusion. At 22 hours of reperfusion, infarction volume was determined by 0 2,3,5-triphenyltetrazolium chloride staining and image analysis. In protocol 2, rats were castrated to deplete endogenous testosterone and then treated with estradiol implants: castration only (CAST, n= 13, estradiol=5+/-2 pg/mL), sham-operated (SHAM, n= 10, 4+/-2 pg/mL), estradiol implant 25 microg (CAST+E25, n=16, 7+/-2 pg/mL) or 100 microg (CAST+E100, n=14, 77+/-14 pg/mL). RESULTS: Cortical infarct volumes were reduced in all estrogen-treated groups: Acute (21+/-4% of ipsilateral cortex), E25 (12+/-5%), and E100 (12+/-3%) relative to SAL (38+/-5%). Caudate infarction was similarly decreased: Acute (39+/-7% of ipsilateral striatum), E25 (25+/-7%), and E100 (34+/-6%) relative to SAL (63+/-4%). Castration did not alter ischemic outcome; cortical and caudate infarction (percentage of respective ipsilateral regions) were 37+/-5% and 59+/-5% in CAST and 39+/-7% and 57+/-5% in SHAM, respectively. Estrogen replacement reduced infarction volume in castrated animals in cortex (19+/-4% in CAST+E25 and 12+/-4% in CAST+E100) and in caudate (42+/-6% in CAST+25 and 20+/-7% in CAST + 100). Laser-Doppler flowmetry results during ischemia and reperfusion was not different among groups. CONCLUSIONS: Both acute and chronic 17beta-estradiol treatments protect male brain in experimental stroke. Testosterone availability does not alter estradiol-mediated tissue salvage after MCAO.     

Does an early postnatal check-up improve maternal health: results from a randomised trial in Australian general practice

Nitric oxide from neuronal cells plays detrimental roles in glutamate neurotoxicity and in focal brain ischemia. Nitric oxide directly damages DNA, and breaks in the DNA strands activate poly(ADP-ribose) polymerase (PARP), which brings poly(ADP-ribosyl)ation of the nuclear proteins. The excessive activation of PARP is thought to cause depletion of ATP and the energy failure resulting in cell death. To clarify the involvement of poly(ADP-ribosyl)ation in ischemic insult, we examined poly(ADP ribosyl)ation by immunohistochemical methods and the protective effect of 3-aminobenzamide, which is a PARP inhibitor, on focal brain ischemia using an intraluminal permanent middle cerebral artery occlusion model in rats. Poly(ADP ribosyl)ation was widely and markedly detected 2 hours after the ischemic insult in the cerebral cortex and striatum in which infarction developed 24 hours later. The enhanced immunoreactivity of poly(ADP-ribose) gradually decreased, and 16 hours later, no immunoreactivity was detected. Intraventricular administration of 3-aminobenzamide (1 to 30 mg/kg) 30 minutes before the ischemic insult decreased infarction volume in a dose-dependent manner along with the immunohistochemical reduction of poly(ADP-ribosyl)ation. Pretreatment with 7-nitroindazole (25 mg/kg, intraperitoneally), a selective neuronal nitric oxide synthetase inhibitor, partially reduced poly(ADP-ribosyl)ation. These data suggest the involvement of poly(ADP-ribosyl)ation in the development of cerebral infarction.     

Prolongation and enhancement of postischemic c-fos expression after fasting

BACKGROUND AND PURPOSE: A rapid but transient expression of c-fos after cerebral ischemia has been extensively documented. However, the mechanism of this induction and whether induction of c-fos is neuroprotective or detrimental to the brain after ischemia is presently not clear. Fasting before transient cerebral ischemia has been shown to reduce delayed neuronal necrosis and infarct volume. The purpose of the present study was to examine the effect of preischemic fasting for 24 hours on the expression of c-fos after transient focal cerebral ischemia. METHODS: Focal cerebral ischemia was induced by temporary occlusion of the right middle cerebral artery and both common carotid arteries for 60 minutes. Male Long-Evans rats weighting 250 to 300 g were randomly divided into two groups: fed (control group) and food deprived for 24 hours (fasted group) before ischemic surgery. Infarct volumes were measured on the basis of triphenyltetrazolium chloride-delineated infarct areas, and plasma glucose levels were determined by the glucose oxidase method. Temporal and spatial expression of c-fos was assessed by Northern blot analysis, in situ hybridization, and immunohistochemistry. RESULTS: Fasting for 24 hours before 60 minutes of ischemia resulted in a 26.6% decrease in preischemic plasma glucose levels and a 74.5% reduction in infarct volumes in the fasted group compared with the control group. A rapid but transient induction of c-fos mRNA was observed in the ischemic cortex in control animals after 60 minutes of ischemia. Fasting not only prolonged but also enhanced the intensity of c-fos expression in the ischemic cortex. Regional c-fos expression was also different between these two groups. CONCLUSIONS: The results support the contention that c-fos expression may be compatible with its purported neuroprotective role in selected experimental paradigms. The signaling mechanisms underlying the effect of fasting and subsequent lowering of plasma glucose levels on postischemic c-fos expression remain to be explored.