Increasing small conductance Ca2+-activated potassium channel activity reverses ischemia-induced impairment of long-term potentiation.

Global cerebral ischemia following cardiac arrest and cardiopulmonary resuscitation (CA/CPR) causes injury to hippocampal CA1 pyramidal neurons and impairs cognition. Small conductance Ca(2+)-activated potassium channels type 2 (SK2), expressed in CA1 pyramidal neurons, have been implicated as potential protective targets. Here we showed that, in mice, hippocampal long-term potentiation (LTP) was impaired as early as 3 h after recovery from CA/CPR and LTP remained impaired for at least 30 days. Treatment with the SK2 channel agonist 1-Ethyl-2-benzimidazolinone (1-EBIO) at 30 min after CA provided sustained protection from plasticity deficits, with LTP being maintained at control levels at 30 days after recovery from CA/CPR. Minimal changes in glutamate release probability were observed at delayed times after CA/CPR, implicating post-synaptic mechanisms. Real-time quantitative reverse transcriptase-polymerase chain reaction indicated that CA/CPR did not cause a loss of N-methyl-D-aspartate (NMDA) receptor mRNA at 7 or 30 days after CA/CPR. Similarly, no change in synaptic NMDA receptor protein levels was observed at 7 or 30 days after CA/CPR. Further, patch-clamp experiments demonstrated no change in functional synaptic NMDA receptors at 7 or 30 days after CA/CPR. Electrophysiology recordings showed that synaptic SK channel activity was reduced for the duration of experiments performed (up to 30 days) and that, surprisingly, treatment with 1-EBIO did not prevent the CA/CPR-induced loss of synaptic SK channel function. We concluded that CA/CPR caused alterations in post-synaptic signaling that were prevented by treatment with the SK2 agonist 1-EBIO, indicating that activators of SK2 channels may be useful therapeutic agents to prevent ischemic injury and cognitive impairments.

Quantitative assessment of blood flow, blood volume and blood oxygenation effects in functional magnetic resonance imaging.

The ability to measure the effects of local alterations in blood flow, blood volume and oxygenation by nuclear magnetic resonance has stimulated a surge of activity in functional MRI of many organs, particularly in its application to cognitive neuroscience. However, the exact description of these effects in terms of the interrelations between the MRI signal changes and the basic physiological parameters has remained an elusive goal. We here present this fundamental theory for spin-echo signal changes in perfused tissue and validate it in vivo in the cat brain by using the physiological alteration of hypoxic hypoxia. These experiments show that high-resolution absolute blood volume images can be obtained by using hemoglobin as a natural intravascular contrast agent. The theory also correctly predicts the magnitude of spin-echo MRI signal intensity changes on brain activation and thereby provides a sound physiological basis for these types of studies.

Poly(ADP-ribose) polymerase gene disruption renders mice resistant to cerebral ischemia.

Nitric oxide (NO) and peroxynitrite, formed from NO and superoxide anion, have been implicated as mediators of neuronal damage following focal ischemia, but their molecular targets have not been defined. One candidate pathway is DNA damage leading to activation of the nuclear enzyme, poly(ADP-ribose) polymerase (PARP), which catalyzes attachment of ADP ribose units from NAD to nuclear proteins following DNA damage. Excessive activation of PARP can deplete NAD and ATP, which is consumed in regeneration of NAD, leading to cell death by energy depletion. We show that genetic disruption of PARP provides profound protection against glutamate-NO-mediated ischemic insults in vitro and major decreases in infarct volume after reversible middle cerebral artery occlusion. These results provide compelling evidence for a primary involvement of PARP activation in neuronal damage following focal ischemia and suggest that therapies designed towards inhibiting PARP may provide benefit in the treatment of cerebrovascular disease.

Selective inhibition of NAALADase, which converts NAAG to glutamate, reduces ischemic brain injury.

We describe here a new strategy for the treatment of stroke, through the inhibition of NAALADase (N-acetylated-alpha-linked-acidic dipeptidase), an enzyme responsible for the hydrolysis of the neuropeptide NAAG (N-acetyl-aspartyl-glutamate) to N-acetyl-aspartate and glutamate. We demonstrate that the newly described NAALADase inhibitor 2-PMPA (2-(phosphonomethyl)pentanedioic acid) robustly protects against ischemic injury in a neuronal culture model of stroke and in rats after transient middle cerebral artery occlusion. Consistent with inhibition of NAALADase, we show that 2-PMPA increases NAAG and attenuates the ischemia-induced rise in glutamate. Both effects could contribute to neuroprotection. These data indicate that NAALADase inhibition may have use in neurological disorders in which excessive excitatory amino acid transmission is pathogenic.

Oxygen delivery to the brain before and after birth.

We studied the relationship between cerebral oxygen consumption and cerebral oxygen delivery (cerebral blood flow x arterial oxygen content) in fetal, newborn, and adult sheep, Relative to the amount of oxygen consumed, cerebral oxygen delivery in the fetus exceeds that in the lamb and adult by 70 percent. This may represent a protective advantage for the fetus or simply a necessary adaptation to the low arterial oxygen pressure in the intrauterine environment.

Unintended changes in cognition, mood, and behavior arising from cell-based interventions for neurological conditions: ethical challenges.

The prospect of using cell-based interventions (CBIs) to treat neurological conditions raises several important ethical and policy questions. In this target article, we focus on issues related to the unique constellation of traits that characterize CBIs targeted at the central nervous system. In particular, there is at least a theoretical prospect that these cells will alter the recipients' cognition, mood, and behavior-brain functions that are central to our concept of the self. The potential for such changes, although perhaps remote, is cause for concern and careful ethical analysis. Both to enable better informed consent in the future and as an end in itself, we argue that early human trials of CBIs for neurological conditions must monitor subjects for changes in cognition, mood, and behavior; further, we recommend concrete steps for that monitoring. Such steps will help better characterize the potential risks and benefits of CBIs as they are tested and potentially used for treatment.

Single dose of 17ß-estradiol provides transient neuroprotection in female juvenile mice after cardiac-arrest and cardiopulmonary resuscitation.

Each year there are approximately 7000 out of hospital cardiac arrests in the pediatric population, with 30% resuscitation rate and a 6-10% rate of survival to hospital discharge. Survivors of cardiac arrest exhibit learning and memory deficits that are devastating during the school years. Delayed neuronal cell death occurs in the hippocampus following cardiac arrest and likely contributes to memory impairments. Circulating endogenous estrogen in young adult females has been shown to provide protection against ischemic cell death, as does chronic exogenous administration of 17ß-estradiol (E2). Chronic estrogen benefit can have undesirable feminizing effects, particularly in pre-adolescents. Here, we tested if a single-dose of E2 is neuroprotective in our pediatric cardiac arrest mouse model performed in juvenile mice. We subjected P21P25 C57Blk6 male and female mice to 8 min of cardiac arrest followed by cardiopulmonary resuscitation (CA/CPR). This developmental stage preceded the hormonal onset and serum estradiol and testosterone levels were not different in males and females. A single dose of E2 (100µg/kg) or vehicle was administered 30 min after resuscitation. Neuronal cell death measured 3 days after CA/CPR showed reduced hippocampal cell death in E2-treated females, but not males. Benefit of E2 in females was blocked by the P38 MAPK inhibitor, SB203580. Hippocampal-dependent memory function was equally impaired in E2-and vehicle-treated females measured in the contextual fear conditioning task at 7 days. Our findings demonstrate female-specific transient neuroprotection with E2 that does not provide sustained functional benefit.

Physiologic manifestations of human anaphylaxis.

In the course of a controlled study to evaluate different forms of immunotherapy for subjects with insect-sting hypersensitivity, we observed 11 subjects who had systemic cutaneous urticarial reactions and 3 subjects who experienced systemic anaphylaxis. With the exception of tachycardia, there were no cardiopulmonary changes in the subjects with urticaria, whereas the major manifestation of anaphylactic shock in the other three subjects was severe hypotension that was probably secondary to peripheral vasodilation. Significant abnormalities in gas exchange developed in two subjects. In one, bronchospasm precipitated a respiratory arrest followed by endotracheal intubation with mechanical ventilation. Although plasma histamine levels were not related to the development of cutaneous reactions, the plasma histamine levels correlated with the severity and duration of the cardiopulmonary changes observed during anaphylactic shock. The two subjects with the most severe shock showed evidence of intravascular coagulation characterized by a diminution of Factor V, Factor VIII, fibrinogen, and high molecular weight kininogen, as well as changes in components of the complement system. Standard therapy with epinephrine and fluids, usually recommended for the treatment of systemic anaphylaxis, did not immediately reverse either the hemodynamic or the respiratory abnormalities in the two subjects with the most severe anaphylactic shock. Hemodynamic recovery was gradual and did not seem directly related to any specific therapeutic intervention.

Antiangiogenic effects of the quinoline-3-carboxamide linomide.

Linomide (N-phenylmethyl-1,2-dihydro-4-hydroxyl-1-methyl-2-oxoquinoline-3-carboxa mide) has a reproducible in vivo antitumor effect against a series of both androgen responsive and independent Dunning R-3327 rat prostatic cancers. This antitumor effect of linomide is host mediated. One possible mechanism involving the host is that linomide has antiangiogenic activity. An indication that linomide treatment has antiangiogenic activity is the observation that prostatic cancers from linomide treated rats have more focal necrosis than sized matched tumors from untreated rats. To directly test if linomide has antiangiogenic activity, a newly developed Matrigel based quantitative in vivo angiogenic assay was used. These experiments demonstrated that linomide has dose dependent, antiangiogenic activity in vivo in the rat. Additional studies demonstrated that due to its antiangiogenic activity, linomide treatment of rats bearing prostate cancers resulted in a more than 40% decrease in tumor blood flow. Blood flow to a variety of non-tumor bearing organs was not decreased suggesting that linomide selectively inhibits angiogenesis and does not induce loss of established blood vessels. Using as a model the response of human umbilical vein endothelial cells to linomide treatment in a variety of in vitro assays, linomide was demonstrated to have cytostatic but not cytotoxic effect on human umbilical vein endothelial cells at a medium concentration of > or = 100 micrograms/ml. In addition, both endothelial cell chemotactic migration and invasion are steps in angiogenesis inhibited by linomide treatment.

Therapeutic hypothermia protects against ischemia-induced impairment of synaptic plasticity following juvenile cardiac arrest in sex-dependent manner.

Pediatric cardiac arrest (CA) often leads to poor neurologic outcomes, including deficits in learning and memory. The only approved treatment for CA is therapeutic hypothermia, although its utility in the pediatric population remains unclear. This study analyzed the effect of mild therapeutic hypothermia after CA in juvenile mice on hippocampal neuronal injury and the cellular model of learning and memory, termed long-term potentiation (LTP). Juvenile mice were subjected to cardiac arrest and cardiopulmonary resuscitation (CA/CPR) followed by normothermia (37°C) and hypothermia (30°C, 32°C). Histological injury of hippocampal CA1 neurons was performed 3days after resuscitation using hematoxylin and eosin (H&E) staining. Field excitatory post-synaptic potentials (fEPSPs) were recorded from acute hippocampal slices 7days after CA/CPR to determine LTP. Synaptic function was impaired 7days after CA/CPR. Mice exposed to hypothermia showed equivalent neuroprotection, but exhibited sexually dimorphic protection against ischemia-induced impairment of LTP. Hypothermia (32°C) protects synaptic plasticity more effectively in females, with males requiring a deeper level of hypothermia (30°C) for equivalent protection. In conclusion, male and female juvenile mice exhibit equivalent neuronal injury following CA/CPR and hypothermia protects both males and females. We made the surprising finding that juvenile mice have a sexually dimorphic response to mild therapeutic hypothermia protection of synaptic function, where males may need a deeper level of hypothermia for equivalent synaptic protection.

Estrogen and Bcl-2: gene induction and effect of transgene in experimental stroke.

Female rodents producing endogenous estrogens are protected from stroke damage in comparison with male counterparts. This natural protection is lost after ovariectomy or reproductive senescence. The aim of this study is to determine whether estrogen reduces early neuronal injury and cell loss after ischemia by increasing the expression of Bcl-2. Male, intact female, ovariectomized, and estrogen-repleted ovariectomized rats were subjected to middle cerebral artery occlusion, and 22 hr later the level and localization of Bcl-2 mRNA and protein were determined. The levels of post-ischemic bcl-2 mRNA and protein were increased exclusively in neurons within the peri-infarct region. Intact females and estrogen-treated castrates demonstrated increased bcl-2 mRNA and protein expression compared with males and estrogen-deficient females, accompanied by a decrease in infarct size. To test the hypothesis that the neuroprotective mechanism of estrogen functions via Bcl-2, we compared ischemic outcome in male, female, and ovariectomized wild-type mice and mice overexpressing Bcl-2 exclusively in neurons. Wild-type female mice sustained smaller infarcts compared with males. Bcl-2 overexpression reduced infarct size in males, but provided no added protection in the female. Moreover, ovariectomy exacerbated infarction in wild-type females, but had no effect in Bcl-2 overexpressors. These data indicate that overexpression of Bcl-2 simulates the protection against ischemic injury conferred by endogenous female sex steroids. We concluded that estrogen rescues neurons after focal cerebral ischemia by increasing the level of Bcl-2 in peri-infarct regions and that estrogen-induced bcl-2 gene expression is an important downstream component of neuronal protection in female stroke.

Effect of glutamine synthetase inhibition on astrocyte swelling and altered astroglial protein expression during hyperammonemia in rats.

Inhibition of glutamine synthesis reduces astrocyte swelling and associated physiological abnormalities during acute ammonium acetate infusion in anesthetized rats. We tested the hypothesis that inhibition of glutamine accumulation during more prolonged ammonium acetate infusion in unanesthetized rats reduces cortical astrocyte swelling and immunohistochemical changes in astrocytic proteins. Rats received a continuous i.v. infusion of either sodium acetate or ammonium acetate for 24 h to increase plasma ammonia from about 30-400 mumol/l. Cohorts were pretreated with vehicle or l-methionine-S-sulfoximine (MSO; 0.83 mmol/kg). MSO reduced glutamine synthetase activity by 57% and glutamine synthetase immunopositive cell number by 69%, and attenuated cortical glutamine accumulation by 71%. Hyperammonemia increased the number of swollen astrocytes in cortex and MSO reduced this increase to control values. The number of glial fibrillary acidic protein immunopositive cells in cortex was greater in hyperammonemic rats and the increase in superficial cortical layers was attenuated by MSO. Immunoreactivity for the gap junction protein connexin-43 in the neuropil, assessed by optical density, was greater in the hyperammonemic group compared with controls, but this increase was not attenuated by MSO. No changes in the optical density of GLT1 glutamate transporter immunoreactivity in cortex were detected in any group. We conclude that glutamine synthetase inhibition reduces astrocyte swelling and ameliorates some of the reactive astroglial cytoskeletal alterations seen at 24 h of hyperammonemia, but that gap junction changes in astrocytes occur independently of glutamine accumulation and swelling.

Region-specific role for GluN2B-containing NMDA receptors in injury to Purkinje cells and CA1 neurons following global cerebral ischemia.

Motor deficits are present in cardiac arrest survivors and injury to cerebellar Purkinje cells (PCs) likely contribute to impairments in motor coordination and post-hypoxic myoclonus. N-Methyl-D-aspartic acid (NMDA) receptor-mediated excitotoxicity is a well-established mechanism of cell death in several brain regions, but the role of NMDA receptors in PC injury remains understudied. Emerging data in cortical and hippocampal neurons indicate that the GluN2A-containing NMDA receptors signal to improve cell survival and GluN2B-containing receptors contribute to neuronal injury. This study compared neuronal injury in the hippocampal CA1 region to that in PCs and investigated the role of NMDA receptors in PC injury in our mouse model of cardiac arrest and cardiopulmonary resuscitation (CA/CPR). Analysis of cell density demonstrated a 24% loss of PCs within 24 h after 8 min CA/CPR and injury stabilized to 33% by 7 days. The subunit promiscuous NMDA receptor antagonist MK-801 protected both CA1 neurons and PCs from ischemic injury following CA/CPR, demonstrating a role for NMDA receptor activation in injury to both brain regions. In contrast, the GluN2B antagonist, Co 101244, had no effect on PC loss while protecting against injury in the CA1 region. These data indicate that ischemic injury to cerebellar PCs progresses via different cell death mechanisms compared to hippocampal CA1 neurons.

Cognitive and behavioral assessment in experimental stroke research: will it prove useful?

Stroke in humans is associated with deficits in sensorimotor and cognitive function. Consequently, many stroke researchers recently have expanded their techniques to assess cognitive and behavioral correlates of histologically-determined stroke damage in animal models. Although the incorporation of functional outcome assessment represents an important step forward in stroke research, reports of middle cerebral artery occlusion (MCAO) induced behavioral deficits often conflict, and a significant correlation between post-stroke histology and behavior has been reported in few stroke studies. Discrepancies in behavioral outcomes among studies may be due to several factors, such as method of MCAO, duration of occlusion, strain, the timing and method of the behavioral testing and the laboratory environment. Furthermore, proper experimental and control groups, necessary to rule out potential confounding factors during cognitive testing, often are not incorporated. The goal of this review is: (1) to provide a description of the techniques most commonly employed to assess functional outcome after (MCAO) in rodents and (2) to identify potential confounding factors that may interfere with a clear interpretation of the behavioral data.

Anticonvulsant lamotrigine administered on reperfusion fails to improve experimental stroke outcomes.

BACKGROUND AND PURPOSE: Recent results suggest that selective inhibitors of presynaptic neuronal ion channels can diminish glutamate release during cerebral ischemia and modulate excitotoxic cell death. The aim of the present study was to evaluate lamotrigine (LTG), an antiepileptic that inhibits presynaptic sodium and voltage-sensitive calcium channels, as a potential stroke resuscitation agent in the rat. Three dosages of LTG were examined for effect on infarction volume and sensorimotor behavioral recovery after middle cerebral artery (MCA) occlusion. METHODS: Halothane-anesthetized male Wistar rats were subjected to 2 hours of MCA occlusion by the intraluminal occlusion technique. Physiological variables were controlled, and ipsilateral cortical perfusion was monitored by laser Doppler flowmetry throughout ischemia. At onset of reperfusion, rats received intravenous LTG 5, 10, or 20 mg/kg or PBS (n=9 to 11 per group) during 15 minutes. Behavioral assessment was completed at 3 and 7 days after stroke, and the brain was harvested for histology (triphenyltetrazolium chloride staining). RESULTS: Values are mean+/-SE. Cortical infarction volumes were unchanged in LTG-treated animals: 14+/-6% of contralateral cortex at 5 mg/kg LTG, 17+/-7% at 10 mg/kg, and 30+/-6% at 20 mg/kg, versus saline-treated cohorts (12+/-3%; P:=0.19; n=9). Caudate-putamen infarction injury was also unchanged (37+/-11% of contralateral caudate-putamen at 5 mg/kg LTG, 44+/-8% at 10 mg/kg, and 65+/-9% at 20 mg/kg versus saline (38+/-11%; P:=0.18). Total infarction was not different among groups (P:=0.15). Consistent with histology, behavioral outcomes were unimproved by treatment. CONCLUSIONS: Histological damage and behavioral recovery at 7 days after MCA occlusion was not altered by LTG treatment over the dosage range used in the present study.

Postischemic estrogen reduces hypoperfusion and secondary ischemia after experimental stroke.

BACKGROUND AND PURPOSE: Estrogen is a known neuroprotective and vasoprotective agent in experimental cerebral ischemia. Preischemic steroid treatment protects animals of both sexes from focal cerebral ischemia. This study determined whether intravenous estrogen acts as a vasodilator when administered on reperfusion and whether the resulting increase in cerebral blood flow (CBF) provides tissue protection from middle cerebral artery occlusion. METHODS: Adult male Wistar rats were treated with reversible middle cerebral artery occlusion (2 hours), then infused with intravenous estrogen (Premarin; 1 mg/kg) or vehicle during the first minutes of reperfusion (n=15 per group). Cortical laser-Doppler flowmetry was used to assess adequacy of occlusion. Ischemic lesion volume was determined at 22 hours after occlusion by 2,3,5-triphenyltetrazolium chloride staining and image analysis. Cortical and striatal CBF was measured by (14)[C]iodoantipyrine autoradiography at 10 (n=10) or 90 (n=11) minutes of reperfusion. RESULTS: As expected, supraphysiological plasma estrogen levels were achieved during reperfusion (estrogen, 198+/-45 pg/mL; vehicle, 6+/-5; P:=0.001). Physiological variables were controlled and not different between groups. Total hemispheric infarction was reduced in estrogen-treated rats (estrogen, 49+/-4% of ipsilateral structure; vehicle, 33+/-5%; P:=0.02), which was most pronounced in striatum (estrogen, 40+/-6% of ipsilateral striatum; vehicle, 60+/-3%; P:=0.01). CBF recovery was strikingly increased by estrogen infusion at 10 minutes in frontal (estrogen, 102+/-12 mL/100 g per minute; vehicle, 45+/-15; P:=0.01) and parietal cortex (estrogen, 74+/-15 mL/100 g per minute; vehicle, 22+/-13; P:=0.028) and throughout striatum (estrogen, 87+/-13 mL/100 g per minute; vehicle, 25+/-20; P:=0.02). Hemispheric volume with low CBF recovery (eg, <20 mL/100 g per minute) was smaller in estrogen-treated animals (estrogen, 73+/-18 mm(3); vehicle, 257+/-46; P:=0.002). However, differences in CBF recovery could not be appreciated between groups by 90 minutes of reperfusion. CONCLUSIONS: Acute estrogen therapy during reperfusion improves tissue outcome from experimental stroke. The steroid rapidly promotes CBF recovery and reduces hemispheric no-reflow zones. This beneficial effect appears only during early reperfusion and likely complements other known mechanisms by which estrogen salvages brain from focal necrosis.

Estrogen decreases infarct size after temporary focal ischemia in a genetic model of type 1 diabetes mellitus.

BACKGROUND AND PURPOSE: It is unclear how genetic type 1 diabetes mellitus (DM) influences infarct size when blood glucose is tightly controlled. The aim of this study was to determine the effect of genetic type 1 DM, as occurs in BB rats, on infarct size after transient unilateral middle cerebral artery occlusion (MCAO) in male and female rats. In addition, studies suggest that male type 1 DM rats have a higher incidence of end-organ complications than do females. A second aim of this study was to determine the effect of chronic 17beta-estradiol (E(2)) administration on infarct size in male BB rats. METHODS: Diabetic male (MDiab, n=14) and female (FDiab, n=8) BB rats were studied and compared with background strain Wistar rats (MWist, n=16; FWist, n=14). Two additional male cohorts (MWist+E(2), n=15; MDiab+E(2), n=14) received subcutaneous 25 microg E(2) implants 7 to 10 days before MCAO. Rats underwent 1 hour of MCAO followed by 22 hours of reperfusion. Physiological variables were controlled among groups, and the intraischemic laser Doppler flow signal was reduced similarly in all animals. Infarction volume was evaluated by 2,3,5-triphenyltetrazolium chloride staining and image analysis. RESULTS: Preischemic blood glucose was 94+/-5, 127+/-13, 90+/-15, 63+/-18, 122+/-8, and 81+/-14 mg/dL in MWist, FWist, MDiab, FDiab, MWist+E(2), and MDiab+E(2) rats, respectively (mean+/-SE). Intraischemic laser Doppler flow was reduced to 20% to 25% of baseline in all groups. Striatal infarct size (percentage of ipsilateral caudate putamen) was increased in male diabetic rats relative to nondiabetic MWist rats (41+/-3% versus 28+/-3%). Striatal injury was not increased in FDiab rats, and infarction volume was smaller than that in FWist rats (23+/-4% in FWist versus 13+/-3% in FDiab). Chronic estrogen treatment reduced cortical and striatal infarction in MDiab+E(2) rats compared with untreated MDiab rats. CONCLUSIONS: Type 1 DM is associated with increased infarct size after temporary MCAO, despite tight control of blood glucose. The deleterious effect of DM is evident only in males rats; female diabetic BB rats sustain small infarcts. Chronic E(2) treatment reduced injury in the male BB rat, providing neuroprotection even in the presence of DM. These data suggest that genetic diabetes even with mild glucose elevation plays a role in determining neuropathology in experimental stroke. However, factors such as reproductive steroids also determine outcome in DM stroke.

LY353381.HCl, a selective estrogen receptor modulator, and experimental stroke.

BACKGROUND AND PURPOSE: The impact of postmenopausal estrogen replacement therapy on stroke prevention and stroke severity remains controversial. Previously we have shown that cerebral tissue infarction volume sustained after middle cerebral artery (MCA) occlusion is smaller in female than in male animals. This protection is lost after ovariectomy but is restored by 17ss-estradiol replacement. However, the therapeutic range for estradiol is suboptimal, since only doses resulting in a narrow range of plasma levels are protective in brain. The present study tested the hypothesis that a benzothiophene analogue and selective estrogen receptor modulator, LY353381.HCl (LY), reduces tissue infarction after MCA occlusion in estrogen-deficient, ovariectomized female rats. METHODS: Ovariectomized female Wistar rats received LY 10 mg/kg (n=16) or an equivalent volume of vehicle (n=14) by gavage for 5 to 8 days. Subsequently, each animal was anesthetized with halothane (1.2%) and treated with 2 hours of MCA occlusion by the intraluminal filament technique and 22 hours of recovery. Infarction volumes in the cerebral cortex and caudoputamen were determined by 2, 3,5-triphenyltetrazolium chloride staining and digital image analysis. End-ischemic regional cerebral blood flow (CBF) was measured in separate animal cohorts by quantitative [(14)C]iodoantipyrine autoradiography. RESULTS: Caudoputamen infarction was reduced by LY treatment (49+/-6% versus 64+/-4% of ipsilateral caudoputamen in LY and vehicle groups, respectively; P:<0.05). Cerebral cortical infarction was not different in the LY compared with vehicle group (7+/-3% versus 13+/-4% of ipsilateral cerebral cortex, respectively). Intra-ischemic blood pressure, arterial blood gases, and temporalis muscle temperature were controlled and equivalent between treatment groups. Averaged laser-Doppler flow during MCA occlusion was 36+/-3% of baseline in the LY group versus 29%+/-2% in the vehicle group. However, end-ischemic CBF or blood flow distribution within the MCA territory was not altered by LY treatment. Cortical or caudoputamen tissue volumes with end-ischemic CBF <20 mL/100 g per minute were similar in both groups. CONCLUSIONS: We conclude that LY confers neuroprotection from focal cerebral ischemia in caudoputamen in ovariectomized female rats. The mechanism of protection is not linked to preservation of ischemic cerebral blood flow, as determined by end-occlusion quantitative autoradiography.

Stroke outcome in double-mutant antioxidant transgenic mice.

BACKGROUND AND PURPOSE: Both NO and superoxide cytotoxicity are important in experimental stroke; however, it is unclear whether these molecules act within parallel pathological pathways or as coreagents in a common reaction. We examined these alternatives by comparing outcomes after middle cerebral artery occlusion in male and female neuronal NO synthase (nNOS)-deficient (nNOS-/-) or human CuZn superoxide dismutase-overexpressing (hSOD1+/-) mice and a novel strain with both mutations. METHODS: Permanent middle cerebral artery occlusion was performed by use of the intraluminal filament technique (18 hours). Neurological status was scored, and tissue infarction volume was determined by 2,3,5-triphenyltetrazolium staining and image analysis. RESULTS: Hemispheric infarction volume was reduced in each transgenic strain relative to the genetically matched, wild-type, control cohorts (WT mice): nNOS-/- (80+/-6 mm(3)) and double-mutant (49+/-6 mm(3)) mice versus WT mice (114+/-7 mm(3)) and hSOD1+/- mice (52+/-7 mm(3)) versus WT mice (95+/-5 mm(3)). Human CuZn superoxide dismutase had a larger effect on mean infarction volume (30% of contralateral hemisphere) than did nNOS deficiency (46%). Although infarction volume was less in double-mutant mice compared with nNOS-/- mice, injury was not improved relative to hSOD1+/- mice. There was no difference in histological damage by sex within each strain; however, female nNOS-/- mice were not protected from ischemic injury, unlike male mutants. CONCLUSIONS: Superoxide generation contributes to severe ischemic brain injury in vivo to a greater extent than does neuronally derived NO. In vivo, significant superoxide scavenging by CuZn superoxide dismutase occurs within cellular compartments or through biochemical pathways that are not restricted to, and may be distinct from, neuronal NO/superoxide reaction and peroxynitrite synthesis.

Preservation of cerebral blood flow responses to hypoxia and arterial pressure alterations in hyperammonemic rats.

Acute hyperammonemia causes cerebral edema, elevated intracranial pressure and loss of cerebral blood flow (CBF) responsivity to CO2. Inhibition of glutamine synthetase prevents these abnormalities. If the loss of CO2 responsivity is secondary to the mechanical effects of edema, one would anticipate loss of responsivity to other physiological stimuli, such as hypoxia and changes in mean arterial blood pressure (MABP). To test this possibility, pentobarbital-anesthetized rats were subjected to either hypoxic hypoxia (PaO2 approximately 30 mm Hg), hemorrhagic hypotension (MABP approximately 70 and 50 mm Hg), or phenylephrine-induced hypertension (MABP approximately 125 and 145 mm Hg). CBF was measured with radiolabeled microspheres. Experimental groups received intravenous ammonium acetate (approximately 50 mumol min-1 kg-1) for 6 h to increase plasma ammonia to 500-600 microM. Control groups received sodium acetate plus HCl to prevent metabolic alkalosis. The increase in CBF during 10 min of hypoxia after 6 h of ammonium acetate infusion (84 +/- 19 to 259 +/- 52 ml min-1 100 g-1) was similar to that after sodium acetate infusion (105 +/- 20 to 265 +/- 76 ml min-1 100 g-1). Cortical glutamine concentration was elevated equivalently in hyperammonemic rats subjected to normoxia only or to 10 min of hypoxia. With severe hypotension, CBF was unchanged in both the ammonium (80 +/- 20 to 76 +/- 24 ml min-1 100 g-1) and the sodium (80 +/- 14 to 73 +/- 16 ml min-1 100 g-1) acetate groups. With moderate hypertension, CBF was unchanged. With the most severe hypertension, significant increases in CBF occurred in both groups, but there was no difference between groups. We conclude that hypoxic and autoregulatory responses are intact during acute hyperammonemia. The previously observed loss of CO2 responsivity is not the result of a generalized vasoparalysis to all physiological stimuli.