Min Of Bilateral Carotid Occlusion Research Articles

Cilostazol Alleviates NLRP3 Inflammasome–Induced Allodynia/Hyperalgesia in Murine Cerebral Cortex Following Transient Ischemia: Focus on TRPA1/Glutamate and Akt/Dopamine/BDNF/Nrf2 Trajectories

Global cerebral ischemia/reperfusion (I/R) provokes inflammation that augments neuropathic pain. Cilostazol (CLZ) has pleiotropic effects including neuroprotection in several ravaging central disorders; nonetheless, its potential role in transient central ischemic-induced allodynia and hyperalgesia has not been asserted before. Rats were allocated into 4 groups; sham, sham + CLZ, and 45 min-bilateral carotid occlusion followed by a 48 h-reperfusion period either with or without CLZ (50 mg/kg; p.o) post-treatment. CLZ prolonged latency of hindlimb withdrawal following von Frey filaments, 4 °C cold, and noxious mechanical stimulations. Histopathological alterations and the immunoexpression of glial fibrillary acidic protein induced by I/R were reduced by CLZ in the anterior cingulate cortex (ACC) area, while, CLZ enhanced intact neuronal count. Meanwhile, CLZ modulated cerebral cortical glutamate, dopamine neurotransmission, and transient receptor potential ankyrin 1 (TRPA1). CLZ anti-inflammatory potential was mediated by the downregulated p65 NF-κB and sirtuin-1 enhancement to reduce nucleotide-binding domain-like receptor protein 3 (NLRP3), apoptosis-associated speck-like protein (ASC), active caspase-1, and interleukin-1β, indicative of inflammasome deactivation. It also revealed an antioxidant capacity via boosting nuclear factor E2-related factor (Nrf2) enhancing glutathione through forkhead box protein O3a (FOXO3a) reduction. Additionally, CLZ triggered neuronal survival by promoting the p-content of Akt, TrkB, and CREB as well as BDNF content. A novel approach of CLZ in hindering global cerebral I/R–mediated neuropathy is firstly documented herein to forward its adjunct action via deactivating the NLRP3 inflammasome, besides enhancing Nrf2 axis, neuronal survival, and dopamine neurotransmission as well as inhibiting TRPA1 and excitotoxicity.Graphical abstract

Dietary essential oil components in the prevention of hypoperfusion/reperfusion-induced tissue damage in the rat cerebral cortex

To extend our previous observations on the beneficial effect of dietary Pistacia lentiscus L. essential oil during cerebral bilateral common carotid artery occlusioninduced injury, we evaluated the activity of one of its major components, beta-caryophyllene (BCP), already known to possess peculiar biological activities, in Wistar rat cerebral cortex. Cerebral hypoperfusion was produced by a 30 min bilateral common carotid artery occlusion followed by 60 min reperfusion (BCCAO/R). Animals were starved for 12 hours before surgery and, 6 hours prior to hypoperfusion, BCP (40 mg/kg/0, 45 ml of sunflower oil as vehicle) was administered via gavage. Biological samples of brain tissue, plasma and cerebrospinal fluid (CSF) were examined by HPLC, western blot, gel zymography and immunohistochemistry and analyzed for fatty acids, expression of the enzyme ciclooxygenase-2 (COX-2), CB receptors for endocannabinoids (eCBs), and peroxisome proliferator-activated receptor (PPAR)-alpha and enzymatic activity of matrix-metalloprotease-9 (MMP9). Data obtained indicate that BCP appears to influence the outcome of BCCAO/R cerebral injury by modulating changes in levels of polyunsaturated fatty acids, biosynthesis of eCBs and eCB congeners, expression of CB1 and CB2 receptors, COX-2 protein levels and enzymatic activity of MMP9. Brain tissue response to the hypoperfusion/reperfusion-induced cerebral insult is modulated by dietary administration of BCP, suggesting the possible use of this molecule as nutritional treatment in neuroprevention. Work funded by Fondazione Banco di Sardegna.

P47 A novel hydrogen sulfide-releasing NMDA receptor antagonist prevents ischemic neuronal death

Background Hydrogen sulfide (H2S) is an endogenously produced gaseous signaling molecule. A number of studies examined therapeutic potential of H2S-donating compounds and H2S gas itself in a variety of animal models of human disease and injury. In the majority of experimental studies, sodium sulfide (Na2S) and sodium hydrosulfide (NaHS) have been used as H2S donor compounds. However, because the short half-lives of these sulfide salts in biological fluid, plasma sulfide levels rapidly increase after bolus administration of Na2S or NaHS and then return to baseline instantaneously. To sustain “physiological” levels of sulfide in circulation after bolus administration, many slow-releasing H2S donor compounds, including 4-(3-thioxo-3H-1,2-dithiol-4-yl)-benzoic acid (ACS48), have been developed. While it has been reported that low and physiological levels of H2S protect neurons, H2S also exhibits neurotoxicity especially at high concentrations. Some investigators have suggested that H2S-induced neurotoxicity may be mediated via enhancement of N-methyl-D-aspartate receptor (NMDAR) activity, because toxicity of H2S was abolished by NMDAR antagonist in vitro and in vivo. Based on these observations, we hypothesized that a hybrid NMDAR antagonist that is capable of slowly releasing H2S in circulation is more effective in protecting neurons than H2S donor compounds alone. Method To characterize the neuroprotective effects of combination of exogenous H2S and NMDAR antagonism, we synthesized a novel H2S-releasing NMDAR antagonist N-((1r,3R,5S,7r)-3,5-dimethyladamantan-1-yl)-4-(3-thioxo-3H-1,2-dithiol- 4-yl)-benzamide (sulfur-memantine) by chemically combining a slow releasing H2S donor ACS48 with a NMDAR antagonist memantine. We examined protective effects of sulfur-memantine in cultured cells at baseline and after oxygen glucose deprivation (OGD) using a panel of cytotoxicity assays. Neuroprotective effects of sulfur-memantine were validated in a mice model of cerebral ischemia and reperfusion injury. Result While sulfur-memantine and ACS48 increased sulfide levels similarly in cell culture medium, sulfur-memantine augmented intracellular sulfide concentration in human neuroblastoma cells (SH-SY5Y) ten-fold higher than did ACS48 ( Fig. 1 ). Incubation with sulfur-memantine after reoxygenation following OGD protected SH-SY5Y and murine primary cortical neurons more markedly than did ACS48 or memantine. Although Na2S and ACS48 exhibited dose-dependent cytotoxicity to primary cortical neurons measured by lactate dehydrogenase release, memantine and sulfur-memantine showed no cytotoxicity up to 50 μM. Glutamate-induced intracellular calcium accumulation in primary cortical neurons were aggravated by Na2S or ACS48, but suppressed by memantine and sulfur-memantine. Sulfur-memantine prevented glutamate-induced glutathione depletion in SH-SY5Y more markedly than did Na2S or ACS48. Sulfur-memantine prevented caspase-3 activation in SH-SY5Y subjected to OGD. The anti-apoptotic effects of sulfur-memantine were associated with maintenance of extracellular-signal regulated kinase 1/2 (ERK) phosphorylation but not with changes in Akt phosphorylation. Administration of sulfur-memantine 1 min after reperfusion following 45 min of bilateral carotid occlusion reduced cerebral infarct volume and improved survival and neurological function of mice compared to Na2S, ACS48, or memantine up to 60 days after the ischemic insult ( Fig. 2 ). Conclusion These results suggest that a novel H2S-releasing NMDAR antagonist derivative sulfur-memantine prevents cerebral ischemia–reperfusion injury. Hybrid H2S-releasing NMDAR antagonists may provide a novel therapeutic strategy for ischemic brain injury and other forms for neuredegeneration.

Effect of acute administration of dietary Pistacia lentiscus L. essential oil on the ischemia-reperfusion-induced changes in rat frontal cortex and plasma

In this study Pistacia lentiscus L. essential oil (E.O.), a mixture of terpenes and sesquiterpenes, was tested for its protective effects in cerebral ischemia/reperfusion-induced injury in Wistar rat frontal cortex and plasma. Cerebral ischemia was produced by a 20 min bilateral common carotid artery occlusion followed by 30 min reperfusion. Pistacia lentiscus L. essential oil (E.O.) (200 mg/0, 45 ml of sunflower oil as vehicle) was administered via gavage 6 hours prior to ischemia. Rats were randomly assigned to four groups, ischemic/reperfused (I/R) and sham-operated rats treated with the vehicle or with E.O.. Different brain areas were analysed for fatty acid changes and expression of the enzyme cyclooxygenase-2 (COX-2). Ischemia/reperfusion triggered in frontal cortex a decrease of docosahexaenoic acid (DHA), the membrane highly polyunsaturated fatty acid (HPUFA) most susceptible to oxidation. Pre-treatment with E.O. prevented this change and led further to decreased levels of COX-2, as assessed by Western Blot. In plasma of ischemic/reperfused rats, E.O. administration increased both the DHA-to-eicosapentaenoic acid (EPA) ratio and levels of the endocannabinoid congeners palmytoylethanolamide (PEA) and oleoylethanolamide (OEA). The results obtained suggest that ischemia/reperfusion triggers a cerebral insult sufficient to cause a a region specific lipid peroxidation as evidenced by the detectable, significant decrease in the tissue level of DHA, the most abundant essential fatty acid of neuronal membrane phospholipids. Acute dietary pre-treatment with E.O. triggers modifications both in the frontal cortex, where COX-2 expression decreases and the decrease of DHA is apparently prevented, and in plasma, where PEA and OEA levels increase. We suggest that the activity of PEA and OEA, as endogenous ligands of the peroxisome proliferator-activated receptor (PPAR)-alpha, by inducing the peroxisomal beta oxidation, may explain the observed increase in the DHA/EPA ratio. The latter, in fact, might account for an increased metabolism of n-3 aimed at restoring DHA within damaged brain tissue. The possibility that changes in fatty acid metabolism and plasmatic availability of PEA and OEA are correlated events represents an issue worth future investigations.

The Dose-Dependent Effects of Isoflurane on Outcome from Severe Forebrain Ischemia in the Rat

Isoflurane improves outcome against cerebral ischemia in the rat. However, the optimal neuroprotective concentration has not been defined. We examined the effects of different isoflurane concentrations on outcome from severe forebrain ischemia in the rat. Fasted rats were subjected to 0.5, 1.0, 1.5, 2.0, or 2.5 minimum alveolar concentration (MAC) isoflurane during 10 min bilateral carotid occlusion plus systemic hypotension. Each isoflurane concentration was administered only before ischemia. Arterial blood pressure was not pharmacologically manipulated. After ischemia, the anesthetic regimen was changed to fentanyl/nitrous oxide and maintained for 2 h. Pericranial temperature was maintained normothermic during the experiment. Neuromotor score, % dead hippocampal CA1 neurons, and cortical injury were measured 5 days postischemia. Preischemic arterial blood pressure decreased as MAC was increased. Animals administered >1.0 MAC frequently exhibited postischemic seizures resulting in increased mortality. There was no difference among MAC conditions for % dead CA1 neurons (93 approximately 95%). In the cortex, neuronal necrosis was less severe with 0.5 MAC and 1.0 MAC isoflurane relative to >1.0 MAC values. The neuromotor score in the 1.0 MAC isoflurane group was superior to the 2.5 MAC group. Dose-dependent effects of preischemic administration of isoflurane on histologic and behavioral outcome after severe forebrain ischemia were observed. Isoflurane MAC values <1.5 provided superior overall outcome relative to larger isoflurane concentrations.

New variability in cerebrovascular anatomy determines severity of hippocampal injury following forebrain ischemia in the Mongolian gerbil

Transient bilateral carotid artery occlusion in the Mongolian gerbil is a widely used model of forebrain ischemia due to species-specific absence of communicating arteries between the middle and posterior cerebral arteries. We have found that transient carotid occlusion induces a wide variation in histological injury of the hippocampus, suggesting that Mongolian gerbils currently available in the US have anomalous connections between the vertebral and carotid circulations. We subjected Mongolian gerbils from Harlan Sprague–Dawley and Charles River Laboratories to 5 min of bilateral carotid occlusion under continuous striatal temperature control and assessed hippocampal injury histologically 5 or 14 days later. The severity of occlusion-induced hippocampal injury depended on the presence and size of posterior cerebral communicating arteries. Injury was markedly attenuated in hippocampi having ipsilateral communicating arteries >50 μm in diameter. In contrast, severe ischemic injury occurred when either no posterior communicating artery was present, or when communicating arteries were smaller than 50 μm in diameter. Ischemic injury was independent of any communicating arteries on the contralateral side. Communicating arteries were present in 90% of gerbils from each vendor, ranging from 19 μm to 125 μm in diameter. The high prevalence of posterior communicating arteries and their profound effect on attenuating hippocampal injury indicates that an understanding of the presence and size of posterior communicating cerebral arteries in each animal is now required to interpret the extent of hippocampal injury following bilateral carotid artery occlusion in this species.

Characterizing learning deficits and hippocampal neuron loss following transient global cerebral ischemia in rats

The 2-vessel-occlusion + hypotension (2VO + H) model of transient global cerebral ischemia results in neurodegeneration within the CA1 field of the hippocampus, but previous research has failed to demonstrate robust or reliable learning/memory deficits in rats subjected to this treatment. In the present study, sensitive behavioral protocols were developed in an effort to characterize the cognitive impairments following 2VO + H more precisely. Adult rats were exposed to 10 min of bilateral carotid occlusion with simultaneous hypotension. Following recovery, 2VO + H and control rats were subjected to a series of behavioral tests (locomotor activity, sensorimotor battery, water maze [cued, place, learning set], object recognition, and radial arm maze) over an extended recovery period followed by an assessment of neuronal loss in the dorsal hippocampus. The 2VO + H treatment was associated with long-lasting spatial learning deficits in the absence of other behavioral impairments and with neurodegeneration in dorsal hippocampal CA1. Water maze protocols that placed higher memory demands upon the rats (relatively “hard” vs. “easy”) were more sensitive for detecting ischemia-induced deficits. We have shown that the use of appropriate behavioral tests (e.g., a relatively difficult place learning task) allowed for the observation of robust spatial learning deficits in a model previously shown to induce relatively subtle behavioral effects. Thus, the 2VO + H model induces both hippocampal neuronal loss and long-term learning deficits in rats, providing a potentially useful model for evaluating therapeutic efficacy.

Severe hypotension is not essential for isoflurane neuroprotection against forebrain ischemia in mice.

Volatile anesthetics provide protection in experimental models of global cerebral ischemia. To date, all models evaluated have included profound systemic arterial hypotension as a component of the ischemic insult. This study was designed to determine if isoflurane protection persists in a global insult devoid of hypotension. C57BL/6J mice having a high incidence of posterior communicating artery atresia were anesthetized with isoflurane (1.2%) or fentanyl/N2O and subjected to bilateral carotid artery occlusion for 15 min or 20 min with normotension (80-110 mmHg mean arterial pressure) or for 10 min with hypotension (35 mmHg mean arterial pressure). Three days later, neurologic function and histologic damage were assessed. Other mice underwent measurement of intraischemic cerebral blood flow (4-iodo-N-methyl-[14C]antipyrine autoradiography) or plasma norepinephrine. Isoflurane reduced the percentage of hippocampal CA1 dead neurons (e.g., 10 min bilateral carotid occlusion + hypotension: 43 +/- 18 (isoflurane) vs. 67 +/- 20 (fentanyl/N2O), P = 0.003; 20 min bilateral carotid occlusion + normotension: 49 +/- 27 (isoflurane) vs. 71 +/- 22 (fentanyl/N2O), P = 0.003). Isoflurane also reduced CA3 damage and improved neurologic function under all conditions. Intraischemic forebrain blood flow was similar during bilateral carotid occlusion plus normotension for the two anesthetic states. Plasma norepinephrine values were greater when hypotension was added to the ischemic insult. Isoflurane resulted in improved neurologic function and reduced histologic damage regardless of the presence or absence of systemic hypotension during the ischemic insult. This indicates that beneficial effects of isoflurane are most likely attributable to direct effects at the neuronal level as opposed to indirect effects resulting from interactions with profound hypotension.

Post-ischemic treatment with cannabidiol prevents electroencephalographic flattening, hyperlocomotion and neuronal injury in gerbils

The potential activity of cannabidiol, a non-psychoactive constituent of marijuana, in preventing damage caused by cerebral ischemia was studied. Cannabidiol (1.25–20 mg/kg) was given 5 min after 10 min bilateral carotid occlusion in freely-moving awake gerbils. Seven days after ischemia, it antagonized the electroencephalographic flattening of total spectral power, with a dose-dependent bell-shaped curve; the neuroprotective effect was greatest with 5 mg/kg. One day after ischemia cannabidiol completely antagonized ischemia-induced hyperlocomotion, at all doses. Rectal temperature did not change during the first hour after occlusion. Histological examination showed complete survival of CA1 neurons in cannabidiol-treated gerbils. These findings suggest a potential therapeutic role of cannabidiol in cerebral ischemia, though the clear mechanism of action remains to be elucidated.

Poly(ADP-ribose) polymerase inhibitors attenuate necrotic but not apoptotic neuronal death in experimental models of cerebral ischemia.

An excessive activation of poly(ADP-ribose) polymerase (PARP) has been proposed to play a key role in post-ischemic neuronal death. We examined the neuroprotective effects of the PARP inhibitors benzamide, 6(5H)-phenanthridinone, and 3,4-dihydro-5-[4-1(1-piperidinyl)buthoxy]-1(2H)-isoquinolinone in three rodent models of cerebral ischemia. Increasing concentrations of the three PARP inhibitors attenuated neuronal injury induced by 60 min oxygen-glucose deprivation (OGD) in mixed cortical cell cultures, but were unable to reduce CA1 pyramidal cell loss in organotypic hippocampal slices exposed to 30 min OGD or in gerbils following 5 min bilateral carotid occlusion. We then examined the necrotic and apoptotic features of OGD-induced neurodegeneration in cortical cells and hippocampal slices using biochemical and morphological approaches. Cortical cells exposed to OGD released lactate dehydrogenase into the medium and displayed ultrastructural features of necrotic cell death, whereas no caspase-3 activation nor morphological characteristics of apoptosis were observed at any time point after OGD. In contrast, a marked increase in caspase-3 activity was observed in organotypic hippocampal slices after OGD, together with fluorescence and electron microscope evidence of apoptotic neuronal death in the CA1 subregion. Moreover, the caspase inhibitor Z-VAD-FMK reduced OGD-induced CA1 pyramidal cell loss. These findings suggest that PARP overactivation may be an important mechanism leading to post-ischemic neurodegeneration of the necrotic but not of the apoptotic type.

The use-dependent sodium channel blocker mexiletine is neuroprotective against global ischemic injury

Mechanisms responsible for anoxic/ischemic cell death in mammalian CNS grey and white matter involve an increase in intracellular Ca 2+, however the routes of Ca 2+ entry appear to differ. In white matter, pathological Ca 2+ influx largely occurs as a result of reversal of Na +–Ca 2+ exchange, due to increased intracellular Na + and membrane depolarization. Na + channel blockade has therefore been logically and successfully employed to protect white matter from ischemic injury. In grey matter ischemia, it has been traditionally presumed that activation of agonist (glutamate) operated and voltage dependent Ca 2+ channels are the primary routes of Ca 2+ entry. Less attention has been directed towards Na +–Ca 2+ exchange and Na + channel blockade as a protective strategy in grey matter. This study investigates mexiletine, a use-dependent sodium channel blocker known to provide significant ischemic neuroprotection to white matter, as a grey matter protectant. Pentobarbital (65 mg/kg) anesthetized, mechanically ventilated Sprague–Dawley rats were treated with mexiletine (80 mg/kg, i.p.). Then 25 min later the animals were subjected to 10 min of bilateral carotid occlusion plus controlled hypotension to 50 Torr by temporary partial exsanguination. Animals were sacrificed with perfusion fixation after 7 days. Ischemic and normal neurons were counted in standard H&E sections of hippocampal CA1 and the ratio of ischemic to total neurons calculated. Mexiletine pre-treatment reduced hippocampal damage by approximately half when compared to control animals receiving saline alone (45 vs. 88% damage, respectively; P<0.001). These results suggest that mexiletine (and perhaps other drugs of this class) can provide protection from ischemia to grey matter as well as white matter.

Choroid plexus recovery after transient forebrain ischemia: role of growth factors and other repair mechanisms.

1. Transient forebrain ischemia in adult rats, induced by 10 min of bilateral carotid occlusion and an arterial hypotension of 40 mmHg, caused substantial damage not only to CA-1 neurons in hippocampus but also to epithelial cells in lateral ventricle choroid plexus. 2. When transient forebrain ischemia was followed by reperfusion (recovery) intervals of 0 to 12 hr, there was moderate to severe damage to many frond regions of the choroidal epithelium. In some areas, epithelial debris was sloughed into cerebrospinal fluid (CSF). Although some epithelial cells were disrupted and necrotic, their neighbors exhibited normal morphology. This patchy response to ischemia was probably due to regional differences in reperfusion or cellular metabolism. 3. Between 12 and 24 hr postischemia, there was marked restoration of the Na+, K+, water content, and ultrastructure of the choroid plexus epithelium. Since there was no microscopical evidence for mitosis, we postulate that healthy epithelial cells either were compressed together on the villus or migrated from the choroid plexus stalk to more distal regions, in order to "fill in gaps" along the basal lamina caused by necrotic epithelial cell disintegration. 4. Epithelial cells of mammalian choroid plexus synthesize and secrete many growth factors and other peptides that are of trophic benefit following injury to regions of the cerebroventricular system. For example, several growth factors are upregulated in choroid plexus after ischemic and traumatic insults to the central nervous system. 5. The presence of numerous types of growth factor receptors in choroid plexus allows growth factor mediation of recovery processes by autocrine and paracrine mechanisms. 6. The capability of choroid plexus after acute ischemia to recover its barrier and CSF formation functions is an important factor in stabilizing brain fluid balance. 7. Moreover, growth factors secreted by choroid plexus into CSF are distributed by diffusion and convection into brain tissue near the ventricular system, e.g., hippocampus. By this endocrine-like mechanism, growth factors are conveyed throughout the choroid plexus-CSF-brain nexus and can consequently promote repair of ischemia-damaged tissue in the ventricular wall and underlying brain.

Preischemic blood glucose supply to the brain modulates HSP 72 synthesis and neuronal damage in gerbils

Preischemic hyperglycemia is known to aggravate brain damage caused by transient forebrain ischemia. Because heat shock proteins (HSPs) 72 have been proposed to play a protective role against ischemic neuronal injury, we studied the HSP 72 mRNA expression and protein synthesis in gerbils subjected to 10 min bilateral carotid occlusion under normoglycemic, hyperglycemic and fasting conditions. HSP 72 mRNA expression and HSP 72 synthesis were studied using in situ hybridization and immunostaining, respectively. After 8 h of blood recirculation, HSP 72 mRNAs were expressed in all the hippocampal subfields of the three different groups, with higher expression in the hyperglycemic gerbils. After 48 h of reperfusion, HSP 72 mRNAs had almost completely disappeared in the hyper- and normoglycemic groups, and were more strongly expressed in the CA 1 neurons of the fasted group. At this time, fasted gerbils exhibited intense HSP 72 immunoreactivity in the CA 1, whereas an absence of immunoreactivity was observed in that area in the other groups. Finally, ischemia was also associated with marked astrocytic activation, as evidenced by GFAP immunostaining. Overall results indicate that preischemic differences in blood glucose supply to the brain are related to HSP 72 mRNA expression (in terms of duration) and to HSP 72 protein induction (in terms of intensity) in the vulnerable CA 1 neurons of the hippocampus. Ability of CA 1 neurons to synthesize HSP 72 proteins was associated with higher neuronal survival in the fasted group after 48 h of reflow, suggesting a protective role of HSP 72, even though evaluation of neuronal damage at 7 days indicated that neuronal death was mainly delayed in the time.

Brain concentrations of kynurenic acid after a systemic neuroprotective dose in the gerbil model of global ischemia

1. 1. Kynurenic acid (KYNA) is a kynurenine metabolite and a broad spectrum excitatory amino acid antagonist that has been shown to be neuroprotective in models of cerebral ischemia, when administered exogenously. However, the actual concentration required in the CNS to evoke significant neuroprotection has never been assessed. 2. 2. The purpose of this study was to address this question in the gerbil model of forebrain ischemia. KYNA (400–1600 mg/kg) or vehicle were administered ip 15 min before 5 min bilateral carotid occlusion. 3. 3. Seven days after reperfusion, ischemia-induced hippocampal nerve cell loss (95% in vehicle- treated) was significantly lower in KYNA-treated gerbils (65% and 52% at 1000 and 1200 mg/Kg, respectively, P < 0.01). Treatment with 1000 mg/kg produced brain KYNA concentrations that were dramatically elevated (135.9 and 42.3 μM in CSF and whole brain, vs 0.032 and 0.16 μM in controls, at 15 min after ischemia), as measured in a separate group of transcardially-perfused gerbils. Cerebral KYNA concentrations tended to return to basal values 2 hours after reperfusion. 4. 4. These results indicate that KYNA has a marked neuroprotective effect in a model of forebrain ischemia. This activity is associated with KYNA concentrations in the brain and CSF that are compatible with the in vitro affinity of the compound for ionotropic glutamate receptors.

Protection against ischemic damage by adenosine amine congener, a potent and selective adenosine A 1 receptor agonist

Although the selectivity and potency of adenosine amine congener (ADAC) at adenosine A 1 receptors are similar to other highly selective agonists at this receptor type, the chemical structure of the N 6 substituent is completely different. We now demonstrate that the characteristics of the therapeutic profile of ADAC are distinct from those observed during our previous studies of adenosine A 1 receptor agonist-mediated neuroprotection. Most significantly, chronic treatment with low microgram doses of ADAC (25–100 μg/kg) protects against both mortality and neuronal damage induced by 10 min bilateral carotid occlusion in gerbils. At higher chronic doses, the statistical significance of the protective effect is lost. Acute preischemic administration of the drug at 75–200 μg/kg also results in a statistically significant reduction of postischemic mortality and morbidity. These data indicate that, contrary to other adenosine A 1 receptor agonists whose chronic administration enhances postocclusive brain damage, ADAC may be a promising agent in treatment of both acute (e.g., cerebral ischemia) and chronic (seizures) disorders of the central nervous system in which adenosine A 1 receptors appear to be involved.

The Effects of Aprotinin on Outcome from Cerebral Ischemia in the Rat

The administration of aprotinin has been associated with a reduction in cardiac surgery-related stroke. Intrinsic neuroprotective properties of this drug have not been evaluated in laboratory outcome models of cerebral ischemia. The purpose of this study was to determine whether aprotinin exhibits neuroprotective effects against either global or focal cerebral ischemia in the rat. Fasted rats were administered aprotinin (30,000 or 60,000 KIU/kg) or vehicle (0.9% NaCl) IV before global ischemia (10 min bilateral carotid occlusion with mean arterial pressure 30 mm Hg) or focal ischemia (75 min of transient middle cerebral artery occlusion [MCAO]). Five days after global ischemia, the percentage of dead hippocampal CA1 neurons (mean +/- SD) was similar among the groups (small-dose aprotinin: 49 +/- 31, n = 15; large-dose aprotinin: 55 +/- 31, n = 13; vehicle: 47 +/- 31, n = 16; P = 0.74). After 7 days' recovery from MCAO, no difference among the groups was observed for either neurologic score (P = 0.99) or cerebral infarct volume (small-dose aprotinin: 136 +/- 80 mm (3), n = 23; large-dose aprotinin: 132 +/- 101 mm3, n = 11; vehicle: 121 +/- 81 mm3, n = 21; P = 0.87). Implications: Aprotinin offers no neuroprotection against either global or focal cerebral ischemia in the rat when administered as a single preischemic bolus. (Anesth Analg 1999;88:1-7)

Effect of postischaemic hypothermia on the mitochondrial damage induced by ischaemia and reperfusion in the gerbil

In order to test the effect of hypothermia on mitochondrial function damage following cerebral ischaemia/reperfusion, Mongolian gerbils were submitted to 30 min bilateral carotid occlusion and 2 h of reperfusion at 37°C or 30°C. After normothermic (37°C) ischaemia/reperfusion, significant decreases in mitochondrial state 3 (+ADP) oxygen consumption (−42.2%), complex II–III activity in synaptosomes (−31.7%) and complex IV were measured, in both free mitochondria and synaptosomes (−30.3% and −27.8% respectively). However, following hypothermic (30°C) reperfusion, both respiration rates and all enzyme activities remained at levels not significantly different from those in the sham operated controls.

The effects of aprotinin on outcome from cerebral ischemia in the rat.

The administration of aprotinin has been associated with a reduction in cardiac surgery-related stroke. Intrinsic neuroprotective properties of this drug have not been evaluated in laboratory outcome models of cerebral ischemia. The purpose of this study was to determine whether aprotinin exhibits neuroprotective effects against either global or focal cerebral ischemia in the rat. Fasted rats were administered aprotinin (30,000 or 60,000 KIU/kg) or vehicle (0.9% NaCl) IV before global ischemia (10 min bilateral carotid occlusion with mean arterial pressure 30 mm Hg) or focal ischemia (75 min of transient middle cerebral artery occlusion [MCAO]). Five days after global ischemia, the percentage of dead hippocampal CA1 neurons (mean +/- SD) was similar among the groups (small-dose aprotinin: 49+/-31, n = 15; large-dose aprotinin: 55+/-31, n = 13; vehicle: 47+/-31, n = 16; P = 0.74). After 7 days' recovery from MCAO, no difference among the groups was observed for either neurologic score (P = 0.99) or cerebral infarct volume (small-dose aprotinin: 136+/-80 mm3, n = 23; large-dose aprotinin: 132+/-101 mm3, n = 11; vehicle: 121+/-81 mm3, n = 21; P = 0.87). Aprotinin offers no neuroprotection against either global or focal cerebral ischemia in the rat when administered as a single preischemic bolus.

Comparative neuroprotective properties of the benzodiazepine receptor full agonist diazepam and the partial agonist PNU-101017 in the gerbil forebrain ischemia model

Recent studies have demonstrated the neuroprotective properties of the novel imidazoquinoline benzodiazepine receptor partial agonist, PNU-101017, in the gerbil forebrain ischemia model. The compound effectively reduces delayed post-ischemic (5 min bilateral carotid occlusion) hippocampal CA 1 neuronal degeneration even when its administration is witheld until 4 h after reperfusion and the effect is unrelated to hypothermia. The purpose of the present study was to determine the comparative abilities of PNU-101017 versus the full agonist diazepam to attenuate post-ischemic CA 1 damage. Male gerbils were treated either 30 min before ischemia induction or immediately after reperfusion with an initial dose of PNU-101017 (30 mg/kg i.p.) or diazepam (10 mg/kg i.p.) with a second dose being given at 2 h after reperfusion. Possible hypothermic effects of either compound were prevented by external heating. In vehicle (0.05 N HCl)-treated gerbils, the loss of hippocampal CA 1 neurons at 5 days was 85%. PNU-101017 pretreatment reduced the loss to 50% ( p<0.05 vs. vehicle) whereas pretreatment with diazepam attenuated damage to only 17% ( p<0.001 vs. vehicle). Delaying treatment with PNU-101017 until just after reperfusion still resulted in a reduction in CA 1 degeneration statistically that was indistinguishable from that seen with pretreatment. In contrast, diazepam post-treatment did not significantly decrease CA 1 neuronal loss. These results suggest that a benzodiazepine receptor partial agonist may have greater neuroprotective practicality than a full agonist for the treatment of global cerebral ischemia. The mechanistic basis for this difference may relate to the partially pro-excitatory neuronal response to endogenous GABA before and after neuronal insult.

Neuroprotective effects of the antioxidant LY231617 and NO synthase inhibitors in global cerebral ischaemia

Recent studies have shown that the novel antioxidant LY231617 protects against ischaemia-induced neuronal damage in rat models of global cerebral ischaemia. In the present studies we have examined the effects of LY231617 in the gerbil model of global cerebral ischaemia. We also examined the effects of four nitric oxide synthase inhibitors (3-bromo-7-nitroindazole, N g -nitro- l-arginine methyl ester, aminoguanidine and S-methylisothiourea sulphate) in this model. LY231617 (50 mg/kg p.o. or 30 mg/kg i.p.) was administered either 30 min prior to occlusion or immediately post-occlusion followed by three further doses at 4, 24 and 48 h after the initial dose. 3-Bromo-7-nitroindazole was administered at 40 mg/kg i.p. immediately after occlusion followed by 20 mg/kg i.p. at 3, 6, 24 and 48 h, N g -nitro- l-arginine methyl ester was administered at 10 mg/kg i.p. immediately after occlusion followed by 5 mg/kg i.p. at 3, 6, 24 and 48 h. Aminoguanidine was administered at 80 mg/kg i.p. immediately after occlusion followed by 40 mg/kg i.p. at 3, 6, 24 and 48 h and S-methylisothiourea sulphate was administered at 10 mg/kg i.p. immediately, 3, 6, 24 and 48 h after occlusion. We also examined the effects of aminoguanidine administered at 80 mg/kg i.p. immediately after occlusion followed by 40 mg/kg i.p. at 3, 6, 24, 48, 72 and 96 h and S-methylisothiourea sulphate administered at 10 mg/kg i.p. immediately, 3, 6, 24, 48, 72 and 96 h after occlusion. Control animals were either sham operated or subjected to 5 min bilateral carotid occlusion. Extensive neuronal death was observed in the CA1 layer of the hippocampus in 5-min bilateral carotid artery occluded animals 5 days after surgery. LY231617 provided significant neuroprotection against the ischaemia-induced brain damage when administration was initiated before or after occlusion ( P<0.05). The neuronal NO synthase inhibitors, 3-bromo-7-nitroindazole and a general NO synthase inhibitor, N g -nitro- l-arginine methyl ester also provided significant neuroprotection ( P<0.05). In contrast aminoguanidine and S-methylisothiourea sulphate (two inducible NO synthase inhibitors) failed to protect against the ischaemia-induced brain damage. These results indicate that free radicals and nitric oxide are involved in ischaemia-induced brain damage following global cerebral ischaemia. Antioxidants such as LY231617 or neuronal NO synthase inhibitors can prevent the ischaemia-induced neurodegeneration and may be useful as anti-ischaemic agents.