Expression of a glutamate transporter subtype, EAAT4, in the developing human cerebellum

The effect of tumor necrosis factor binding protein (TNFbp) was studied in mice subjected to a permanent middle cerebral artery occlusion (MCAO). TNFbp is a dimeric form of the type I soluble TNF receptor linked to polyethylene glycol (TNFbp), and binds and inhibits TNF-alpha. TNFbp produced a significant reduction in the cortical infarct volume (22.6 +/- 3.5 mm3 immediately after MCAO; 25.2 +/- 2.4 mm3 1 h after MCAO) compared with vehicle-treated animals (30.3 +/- 3.7 mm3 immediately post MCAO; 31 +/- 3.7 mm3 1 h after MCAO (mean +/- S.D.) when administered intracranially up to 60 min post-occlusion. The neuroprotective effect of TNFbp was sustained in mice for 2 weeks after MCAO. DNA fragmentation at the margin of the cortical infarcts was dramatically reduced in mice treated with TNFbp whereas all control animals showed consistent and obvious DNA fragmentation 2 weeks after MCAO. TNFbp could have therapeutic value for the treatment of ischemic stroke if the problem of delivery to brain can be overcome.     

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.     

Estradiol protects against ischemic injury

Clinical studies demonstrate that estrogen replacement therapy in postmenopausal women may enhance cognitive function and reduce neurodegeneration associated with Alzheimer's disease and stroke. This study assesses whether physiologic levels of estradiol prevent brain injury in an in vivo model of permanent focal ischemia. Sprague-Dawley rats were ovariectomized; they then were implanted, immediately or at the onset of ischemia, with capsules that produced physiologically low or physiologically high 17beta-estradiol levels in serum (10 or 60 pg/mL, respectively). One week after ovariectomy, ischemia was induced. Estradiol pretreatment significantly reduced overall infarct volume compared with oil-pretreated controls (mean+/-SD: oil = 241+/-88; low = 139+/-91; high = 132+/-88 mm3); this protective effect was regionally specific to the cortex, since no protection was observed in the striatum. Baseline and ischemic regional CBF did not differ between oil and estradiol pretreated rats, as measured by laser Doppler flowmetry. Acute estradiol treatment did not protect against ischemic injury. Our finding that estradiol pretreatment reduces injury demonstrates that physiologic levels of estradiol can protect against neurodegeneration.     

Calcitonin gene-related peptide reduces brain injury in a rat model of focal cerebral ischemia

BACKGROUND AND PURPOSE: Calcitonin gene-related peptide is an endogenous vasodilating neuropeptide with a dense concentration in the trigeminocerebrovascular system. It is hypothesized that depletion of this peptide contributes to delayed cerebral ischemia after subarachnoid hemorrhage and that an exogenous supply of calcitonin gene-related peptide will augment ischemic cerebral blood flow and reduce neuronal injury. METHODS: In this study we have investigated the effect of an intravenous infusion of calcitonin gene-related peptide (100 ng/kg per minute), started 1 hour before and continued throughout 4 hours of focal cerebral ischemia, on cerebral blood flow and the volume of brain injury in a rat model of middle cerebral artery occlusion. RESULTS: Calcitonin gene-related peptide produces a significant improvement in ischemic cerebral blood flow (32 +/- 2 compared with 13 +/- 2 mL/100 g per minute in the controls; t = 6.92, P < .0001) with a concomitant reduction in the volume of ischemic brain injury (102 +/- 22 compared with 234 +/- 19 mm3; t = 4.47, P < .001). CONCLUSIONS: These findings lend support for the potential use of this peptide in the prophylactic treatment of delayed cerebral ischemia after aneurysmal subarachnoid hemorrhage.     

Serum levels of tumor necrosis factor alpha and soluble tumor necrosis factor-receptor I in asthmatic patients and patients with chronic respiratory tract infection

In order to investigate the role of tumor necrosis factor alpha (TNF-alpha) in bronchial asthma or chronic respiratory infection, we measured serum levels of TNF-alpha and serum soluble tumor necrosis factor-receptor I (sTNF-RI) in asthmatic patients (n = 11) and patients (n = 10) with chronic respiratory infection by Pseudomonas aeruginosa. We also measured serum levels of eosinophil cationic protein (ECP) in the asthmatic patients. The serum levels of TNF-alpha in the asthmatic patients, patients with chronic respiratory tract infection and control group were 2.864 +/- 0.719 g/ml, 2.564-1.384 pg/ml and 0.681 +/- 0.453 pg/ml respectively. The levels of the former two groups were higher than those of the control group (p < 0.05). The serum levels of sTNF-RI in the asthmatic patients, the patients with chronic respiratory tract infection, and the control group was 758 +/- 268 pg/ml, 999 +/- 242 pg/ml and 909 +/- 268 pg/ml respectively. The levels of the former two groups did not differ significantly from those of the control group. There were significant correlations between TNF-alpha and sTNF-RI in the control group and in the patients with chronic respiratory tract infection, but there was no significant correlation in the asthmatic patients. In the asthmatic patients. TNF-alpha/s TNF-RI correlated with %best of PEF (r = 0.691, n = 9, p 0.0373). The serum levels of ECP correlated significantly with TNF-alpha, but not with sTNF-RI in the asthmatic patients. It is suggested that TNF-alpha plays a significant role in the pathogenesis of bronchial asthma and chronic respiratory tract infection as a factor causing inflammation and that the increase of TNF-alpha/sTNF-RI reflects the activation of eosinophil functions in an asthmatic attack.     

In vivo uptake of [3H]nimodipine in focal cerebral ischemia: modulation by hyperglycemia

Cell membrane depolarization and tissue acidosis occur rapidly in severely ischemic brain. Preischemic hyperglycemia is recognized to increase ischemic tissue acidosis and the present studies were undertaken to correlate depolarization and tissue acidosis during acute focal cerebral ischemia and hyperglycemia. We used a dual-label autoradiography method to simultaneously measure the in vivo distribution of [3H]nimodipine and [14C]DMO (5,5-dimethyl-2,4-oxazolidinedione) in brain to identify regions of ischemic depolarization and measure regional net tissue pH. Regional cerebral blood flow (CBF) was measured in separate studies. Measurements were made 30 minutes after combined middle cerebral artery and ipsilateral common carotid artery occlusion in normoglycemic and hyperglycemic rats. Tissue pH in the ischemic cortex was depressed to 6.76 +/- 0.11 in normoglycemic rats (n = 12) and 6.57 +/- 0.13 in hyperglycemic rats (n = 12), with significantly greater acidosis in the hyperglycemic group (P < 0.001). In contrast the ratio of [3H]nimodipine uptake in the ischemic cortex relative to the contralateral nonischemic cortex was significantly greater in normoglycemic (1.83 +/- 0.45) than hyperglycemic (1.40 +/- 0.50) rats (P < 0.05). Within this region of ischemic cortex CBF was 31 +/- 22 mL/100 g in normoglycemic rats (n = 8) and 33 +/- 22 mL/100 g/min in hyperglycemic rats (n = 9). Cerebral blood flow did not differ between these two groups in any region. Thus hyperglycemia reduced the extent of ischemic depolarization within the cortex during the first 30 minutes of focal cerebral ischemia. This effect may be related to the increased tissue acidosis or to other factors that may lessen calcium influx and preserve cellular energy stores in the ischemic cortex of the hyperglycemic 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.     

HPMA copolymer bound adriamycin overcomes MDR1 gene encoded resistance in a human ovarian carcinoma cell line

Neutrophils are known to mediate injury in acute ischemic stroke especially during reperfusion. Migration of neutrophils into regions of ischemic injury involves binding to the endothelial cell's intercellular adhesion molecule (ICAM-1) through the leukocyte integrin, CD11/CD18. We studied the potential for neuroprotection with a humanized antibody that binds to and blocks the functions of the CD11/CD18 integrin in a rabbit model of transient focal ischemia. Fifteen New Zealand White rabbits underwent transorbital occlusion of the left middle cerebral, anterior cerebral, and internal carotid arteries using aneurysm clips for 2 h, followed by 6 h of reperfusion. Treatment with a maximally saturating dose (4 mg/kg) of a humanized CD11/CD18 monoclonal antibody (Hu23F2G, ICOS Corp., Bothell, WA) (n = 8) or placebo (n = 7) was administered 20 min after occlusion and given as a single intravenous bolus. Hemispheric ischemic neuronal damage (IND) as seen on hematoxylin- and eosin-stained sections was significantly reduced in Hu23F2G-treated animals by 57% (Hu23F2G: 15 +/- 6.9%; placebo: 35 +/- 5%; mean +/- SEM, P < 0.05, t-test). Immunohistochemical staining with neutrophil elastase confirmed the presence of neutrophils within regions of IND in control brains. Treatment with Hu23F2G resulted in marked reduction of neutrophil infiltration. (No. of neutrophils/IND area: Hu23F2G 36.1 +/- 36.7 cm-2, placebo 460.6 +/- 101.8 cm-2, P = 0.001. ) Antagonism of neutrophil migration at the level of the CD11/CD18 integrin reduces ischemic injury in experimental stroke.     

Incomplete infarct and delayed neuronal death after transient middle cerebral artery occlusion in rats

BACKGROUND AND PURPOSE: The clinical syndrome of transient ischemic attacks is accompanied in a significant percentage of patients by brain lesions or neuroimaging abnormalities whose structural counterparts have not been defined. The objective of this study was to analyze, in an experimental model of short-term (< 25 minutes) focal ischemia and long-term (< or = 28 days) reperfusion, the extent and nature of the structural abnormalities affecting neurons and glia located within the territory of the transiently occluded artery. METHODS: Adult Wistar rats (n = 121) had the origin of one middle cerebral artery (MCA) occluded with a nylon monofilament for periods of 10 to 25 minutes. Experiments of transient MCA occlusion were terminated at variable periods ranging from 1 day to 4 weeks. Control experiments consisted of (1) MCA occlusion without reperfusion (n = 7) lasting 7 to 14 days and (2) sham operations (n = 2) followed by 1- to 4-day survival. After in situ fixation, brain specimens were serially sectioned and subjected to detailed morphometric evaluations utilizing light and electron microscopes. The statistical method used to evaluate the results was based on ANOVA followed by Bonferroni's corrected t test and Student's t test comparisons. RESULTS: Brain lesions were not detectable in the sham-operated controls. All brains with permanent MCA occlusion (7 to 14 days) had large infarctions with abundant macrophage infiltration and early cavitation. Forty-five (37%) of the experiments involving transient MCA occlusion had no detectable brain lesions after 4 weeks. Selective neuronal necrosis was found in 76 of 121 rats (63%) with transient MCA occlusion. Neuronal necrosis always involved the striatum, and in 29% of the brains with ischemic injury, necrosis also included a short segment of the cortex. In the striatum, the length of the arterial occlusion was the main determinant of the number of necrotic neurons (20 minutes [22.6 +/- 19] is worse than 10 minutes [4.9 +/- 7]) (P < .0001). In the cortex, the length of reperfusion determined the number of necrotic neurons appearing in layer 3. Experiments with reperfusion of 4 to 7 days' duration yielded more necrotic neurons per microscopic field (2.02 +/- 3) than those lasting fewer days (0.04 +/- 0.1) (P < .05). The histological features of these lesions underwent continuous change until the end of the fourth week, at which time necrotic neurons were still visible both in the striatum and in the cortex. CONCLUSIONS: Arterial occlusions of short duration (< 25 minutes) produced, in 76 of 121 experiments (63%), brain lesions characterized by selective neuronal necrosis and various glial responses (or incomplete infarction). This lesion is entirely different from the pannecrosis/cavitation typical of an infarction that appears 3 to 4 days after a prolonged arterial occlusion. Delayed neuronal necrosis, secondary to a transient arterial occlusion or increasing numbers of necrotic neurons in experiments with variable periods of reperfusion, was a response observed only at a predictable segment of the frontoparietal cortex.     

Amylin-mediated reduction in insulin sensitivity corresponds to reduced insulin receptor kinase activity in the rat in vivo

Studies were undertaken to elucidate further the mechanism whereby the pancreatic peptide amylin induces insulin resistance. Sixteen male Sprague-Dawley rats underwent hyperinsulinemic (14 pmol/kg/min, 0 to 120 minutes) euglycemic clamps in the presence or absence of amylin (500 pmol/kg/min, 60 to 120 minutes). Amylin induced insulin resistance at both the hepatic level (mean +/- SE: hepatic glucose output [HGO] with amylin 1.4 +/- 0.2 v without amylin -1.9 +/- 0.3 mmol/kg/h, P < .001) and peripheral level (glucose disposal [Rd] with amylin 5.0 +/- 0.2 v without amylin 8.5 +/- 0.6 mmol/kg/h, P < .001). Serum insulin levels were similar in the presence or absence of amylin alone (661 +/- 89 v 636 +/- 50 pmol/L, respectively, P = NS), but were significantly less when somatostatin (SRIF) was simultaneously infused (408 +/- 15 pmol/L, P < .02 v the other two groups). This suggests that endogenous insulin production was not suppressed by amylin under these study conditions. Similar findings were obtained in 18 animals in the absence of exogenous insulin infusion. In vitro kinase activity toward histone of skeletal muscle insulin receptors (IRs) activated by insulin in vivo was reduced in the presence of amylin to 6.0 +/- 0.8 versus 9.1 +/- 1.2 fmol phosphate into histone (insulin-infused) and 3.9 +/- 0.7 versus 6.9 +/- 1.4 (non-insulin-infused; P < .03 by ANOVA). Serum calcium was significantly decreased in amylin-treated animals (1.93 +/- 0.04 v 2.30 +/- 0.05 mmol/L, P < .001).(ABSTRACT TRUNCATED AT 250 WORDS)     

Optimal depth and duration of mild hypothermia in a focal model of transient cerebral ischemia: effects on neurologic outcome, infarct size, apoptosis, and inflammation

BACKGROUND and PURPOSE: Mild hypothermia is possibly the single most effective method of cerebroprotection developed to date. However, many questions regarding mild hypothermia remain to be addressed before its potential implementation in the treatment of human stroke. Here we report the results of 2 studies designed to determine the optimal depth and duration of mild hypothermia in focal stroke and its effects on infarct size, neurological outcome, programmed cell death, and inflammation. METHODS: Rats underwent a 2-hour occlusion of the left middle cerebral artery. In the first study (I) animals were kept (intraischemically) at either 37 degreesC (n=8), 33 degreesC (n=8), or 30 degreesC (n=8). Study II consisted of 4 groups: (1) controls (37 degreesC, n=10), (2) 30 minutes of hypothermia started at ischemic onset (33 degreesC, n=9), (3)1 hour (33 degreesC, n=8), and (4) 2 hours (33 degreesC, n=8). Brain temperature was measured by a thermocouple probe placed in the contralateral cortex. After suture removal, all animals were rewarmed and reperfused for 22 hours (I) or 70 hours (II). RESULTS: Mild hypothermia to 33 degreesC or 30 degreesC was neuroprotective (17+/-7% and 27+/-6%, respectively) relative to controls (53+/-8%, P<0.02), but 33 degreesC was better tolerated and recovery from anesthesia was faster. The neurological score of hypothermic animals was significantly better than that of controls (I & II) at both 24 and 72 hours postischemia except for the 30-minute group (II), which showed no improvement. In Study II, 2 hours of hypothermia reduced injury by 59%, 1 hour reduced injury by 84% whereas 30 minutes did not reduce injury. Normalized for infarct size, 2 hours of mild hypothermia decreased neutrophil accumulation by 57% whereas both 1 hour and 30 minutes had no effect. At 72 hours, 1 and 2 hours of mild hypothermia decreased transferase dUTP nick-end labeling (TUNEL) staining by 78% and 99%, respectively, and 30 minutes of hypothermia had no effect. CONCLUSIONS: Intraischemic mild hypothermia must be maintained for 1 to 2 hours to obtain optimal neuroprotection against ischemic cell death due to necrosis and apoptosis.     

Diaspirin cross-linked hemoglobin improves neurological outcome following reversible but not irreversible CNS ischemia in rabbits

BACKGROUND AND PURPOSE: Hemodilution using modified hemoglobin solutions may reduce ischemic central nervous system injury. Purified diaspirin cross-linked hemoglobin (DCLHb) is a cell-free hemoglobin that is intramolecularly cross-linked between the two alpha subunits, resulting in enhanced oxygen offloading to tissues and increased half-life. In the present experiments, we evaluated the ability of DCLHb to reduce neurological damage in two rabbit stroke models. METHODS: In a reversible spinal cord ischemia model, ischemia of the caudal lumbar spinal cord was produced by temporary occlusion of the abdominal aorta. In an irreversible model of cerebral ischemia, plastic microspheres (50 microns) were injected into the internal carotid artery and lodged in the cerebral microvasculature. DCLHb was administered 5 minutes after initiation of ischemia as either a 10-mL/kg infusion, 10-mL/kg exchange transfusion, or a 20-mL/kg infusion. Control animals received human serum albumin that was oncotically matched to the DCLHb. RESULTS: In the spinal cord model, DCLHb significantly increased the duration of ischemia required to produce permanent paralysis from 27.33 +/- 8.71 minutes (mean +/- SD) in controls to 42.59 +/- 10.10 minutes in the 10-mL/kg exchange transfusion group and to 40.82 +/- 18.16 minutes in the 20-mL/kg infusion condition (P < .05). DCLHb did not significantly reduce neurological damage in the microsphere embolization model. CONCLUSIONS: These data suggest that cross-linked hemoglobin reduces neurological damage after reversible central nervous system ischemia and that this is not attributable to hemodilution or hypervolemia only.     

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.     

Spatial stability of extracellular potassium ion and blood flow distribution in rat cerebral cortex after permanent middle cerebral artery occlusion

Extracellular potassium ion activity ([K+]o) increases precipitously during brain ischemia when blood flow falls below threshold values less than approximately 15 mL/100 g/min. This flow threshold for increase of [K+]o occurs also in focal ischemia producing gradient from ischemic core to adjacent normally perfused brain. In this study we investigated the spatial and temporal stability of extracellular potassium ion and blood flow gradients after permanent middle cerebral artery occlusion (MCAO) in rats. [K+]o and regional CBF were measured, respectively, with K+-sensitive and polarographic hydrogen-sensitive microelectrodes at different cortical locations in the middle cerebral artery distribution region. Spatial assessment of [K+]o and regional CBF was conducted at 30, 90, and 180 minutes after MCAO. [K+]o in the more lateral cortex (core) increased from near 3 mmol/L before MCAO to greater than 50 mmol/L and was associated with flow values less than 25% of pre-ischemic levels. Measurements medial to the core (penumbra) indicated progressively decreasing levels of [K+]o and improvement of CBF. There was a tendency for [K+]o in penumbral zones to decrease toward normal levels with time, but there was little dissipation of [K+]o in core regions. In contrast, the spatial CBF profile remained remarkably constant for the entire recording period. Thus, unlike infarction which has been reported to expand with time after focal ischemia, the spatial [K+]o disturbance tends to contract primarily due to decreasing [K+]o with time in the penumbra. Thus, steady state levels of [K+]o after focal ischemia may not be a valuable predictor of cell viability.     

Effects of MDL 72527, a specific inhibitor of polyamine oxidase, on brain edema, ischemic injury volume, and tissue polyamine levels in rats after temporary middle cerebral artery occlusion

The possible effects of the polyamine interconversion pathway on tissue polyamine levels, brain edema formation, and ischemic injury volume were studied by using a selective irreversible inhibitor, MDL 72527, of the interconversion pathway enzyme, polyamine oxidase. In an intraluminal suture occlusion model of middle cerebral artery in spontaneously hypertensive rats, 100 mg/kg MDL 72527 changed the brain edema formation from 85.7 +/- 0.3 to 84.5 +/- 0.9% in cortex (p < 0.05) and from 79.9 +/- 1.7 to 78.4 +/- 2.0% in subcortex (difference not significant). Ischemic injury volume was reduced by 22% in the cortex (p < 0.05) and 17% in the subcortex (p < 0.05) after inhibition of polyamine oxidase by MDL 72527. There was an increase in tissue putrescine levels together with a decrease in spermine and spermidine levels at the ischemic site compared with the nonischemic site after ischemia-reperfusion injury. The increase in putrescine levels at the ischemic cortical and subcortical region was reduced by a mean of 45% with MDL 72527 treatment. These results suggest that the polyamine interconversion pathway has an important role in the postischemic increase in putrescine levels and that blocking of this pathway can be neuroprotective against neuronal cell damage after temporary focal cerebral ischemia.     

Increased susceptibility to ischemic brain damage in transgenic mice overexpressing the amyloid precursor protein

We studied the role of the amyloid precursor protein (APP) in ischemic brain damage using transgenic mice overexpressing APP. The middle cerebral artery (MCA) was occluded in FVB/N mice expressing APP695.SWE (Swedish mutation) and in nontransgenic littermates. Infarct volume (cubic millimeters) was assessed 24 hr later in thionin-stained brain sections. The infarct produced by MCA occlusion was enlarged in the transgenics (+32 +/- 6%; n = 12; p < 0. 05; t test). Measurement of APP by ELISA revealed that, although relatively high levels of Abeta were present in the brain of the transgenics (Abeta1-40 = 80 +/- 19 pmol/g; n = 6), there were no differences between ischemic and nonischemic hemispheres (p > 0.05). The reduction in cerebral blood flow produced by MCA occlusion at the periphery of the ischemic territory was more pronounced in APP transgenics (-42 +/- 8%; n = 9) than in controls (-20 +/- 8%; n = 9). Furthermore, the vasodilatation produced by neocortical application of the endothelium-dependent vasodilator acetylcholine (10 microM) was reduced by 82 +/- 5% (n = 8; p < 0.05) in APP transgenics. The data demonstrate that APP overexpression increases the susceptibility of the brain to ischemic injury. The effect is likely to involve the Abeta-induced disturbance in endothelium-dependent vascular reactivity that leads to more severe ischemia in regions at risk for infarction. The cerebral vascular actions of peptides deriving from APP metabolism may play a role in the pathogenic effects of APP.     

8-epi PGF2 alpha generation during coronary reperfusion. A potential quantitative marker of oxidant stress in vivo

BACKGROUND: Myocardial reperfusion is believed to be associated with free radical injury. However, indexes of oxidative stress in vivo have been limited by their poor specificity and sensitivity. Isoprostanes are stable products of arachidonic acid formed in a nonenzymatic, free radical-catalyzed manner. We have developed a sensitive and specific assay for one of these compounds, 8-epi prostaglandin (PG) F2 alpha. METHODS AND RESULTS: To address its utility as an index of oxidative stress during coronary reperfusion, we measured urinary levels by gas chromatography/mass spectrometry in a canine model of coronary thrombolysis, in patients with acute myocardial infarction treated with thrombolytic therapy, and in patients after elective coronary artery bypass surgery. Urinary 8-epi PGF2 alpha was unchanged after circumflex artery occlusion in a canine model of coronary thrombolysis (n = 13; 437.2 +/- 56.4 versus 432.7 +/- 55.2 pmol/mmol creatinine) but increased significantly (P < .05) immediately after reperfusion (553.8 +/- 64.7 pmol/mmol). Urinary levels were increased (P < .001) in patients (n = 12) with acute myocardial infarction given lytic therapy (265.8 +/- 40.8 pmol/mmol) compared with age-matched control subjects (n = 20; 91.5 +/- 11.8 pmol/mmol) and patients with stable coronary disease (n = 20; 95.7 +/- 6.3 pmol/mmol). Preoperative levels rose from 113.2 +/- 11.8 to 248.2 +/- 86.3 pmol/mmol at 30 minutes into revascularization to 332.2 +/- 82.6 pmol/mmol by 15 minutes after global myocardial reperfusion (P < .05) and dropped to 181.2 +/- 50.4 pmol/mmol at 30 minutes and 120.2 +/- 9.9 pmol/mmol at 24 hours after bypass surgery (n = 5). Corresponding changes in spin adduct formation, found with electron paramagnetic resonance, were noted in 2 patients. CONCLUSIONS: These data support the hypothesis that free radical generation occurs during myocardial reperfusion. Measurement of isoprostane production may serve as a noninvasive index of oxidative stress.