John W. Phillis

α-Phenyl-tert-butyl-nitrone reduces cortical infarct and edema in rats subjected to focal ischemia

The neuroprotective effects of the spin-trapping agent alpha-phenyl-N-tert-butyl nitrone (PBN) were evaluated in rats subjected to focal cerebral ischemia produced by permanent middle cerebral artery (MCA) and ipsilateral common carotid artery (CCA) occlusion. PBN was given i.p. at 100 mg/kg at initial times of administration of 0.5 h prior to ischemia (group 2), 0.5 (group 3), 5 (group 4) and 12 h (group 5) after ischemia. Additional doses of PBN (100 mg/kg) were administered as follows: Group 2, at 24 h; Group 3, at 5, 17, 29 and 41 h; Group 4, at 17, 29 and 41 h; Group 5, at 24 and 36 h. Animals were sacrificed 48 h after MCA occlusion and infarct volumes were calculated from triphenyetetrazolium stained 1.5 mm slices of the forebrain. PBN significantly attenuated cortical infarct volume and cerebral edema in all of the treated rats compared with those in ischemic control (group 1) rats, with no significant differences between the different PBN treated groups. The percentage of infarct volume in ischemic control rats was 22.7 +/- 1.0, while those in PBN-treated groups were: 9.6 +/- 2.0, P < 0.01 (group 2); 12.2 +/- 2.2, P < 0.01 (group 3); 11.1 +/- 2.9, P < 0.01 (group 4) and 14.4 +/- 2.5, P < 0.01 (group 5). Furthermore, neurological behavior tests showed that PBN decreased the neurological deficit scores in rats initially treated either prior to or for up to 12 h after ischemia.(ABSTRACT TRUNCATED AT 250 WORDS)

Exercise pre-conditioning reduces brain damage in ischemic rats that may be associated with regional angiogenesis and cellular overexpression of neurotrophin

There is increasing evidence that physical activity is associated with a decreased stroke risk. The purpose of this study was to determine if exercise could also reduce brain damage in rats subjected to transient middle cerebral artery (MCA) occlusion, and if the reduced brain injury is associated with angiogenesis as well as cellular expression of the nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) in regions supplied by the MCA. Adult male Sprague Dawley rats (n=36) exercised 30 min each day for 3 weeks on a treadmill on which repetitive locomotor movement was required. Then, stroke was induced by a 2-h MCA occlusion using an intraluminal filament, followed by 48 h of reperfusion. In addition to the two exercised groups of animals with or without MCA occlusion, there were two other groups of animals, with or without MCA occlusion, housed for the same duration and used as non-exercised controls. Brain damage in ischemic rats was evaluated by neurologic deficits and infarct volume. Exercise preconditioned and non-exercised brains were processed for immunocytochemistry to quantify the number of microvessels or NGF- and BDNF-labeled cells. Pre-ischemic motor activity significantly (P<0.01) reduced neurologic deficits and infarct volume in the frontoparietal cortex and dorsolateral striatum. Cellular expressions of NGF and BDNF were significantly (P<0.01) increased in cortex (neuron) and striatum (glia) of rats under the exercise condition. Significant (P<0.01) increases in microvessel density were found in striatum. Physical activity reduced stroke damage. The reduced brain damage may be attributable to angiogenesis and neurotrophin overexpression in brain regions supplied by the MCA following exercise.

Excitatory Transmitter Amino Acid Release from the Ischemic Rat Cerebral Cortex: Effects of Adenosine Receptor Agonists and Antagonists

Abstract: The effects of selective adenosine receptor agonists [N6‐cyclopentyladenosine (CPA) and N‐ethylcarboxamidoadenosine (NECA)] and antagonists {8‐cyclopentyl‐1,3‐dipropylxanthine (DPCPX) and 9‐chloro‐2‐(2‐furanyl)‐5,6‐dihydro‐1,2,4‐triazolo[1,5‐c]quinazoline‐5‐imine (CGS‐15943A)} on aspartate and glutamate release from the ischemic rat cerebral cortex were studied with the cortical cup technique. Cerebral ischemia (for 20 min) was elicited by four‐vessel occlusion. Excitatory amino acid releases were compared from control ischemic rats and drug‐treated rats. Basal levels of aspartate and glutamate release were not greatly affected by pretreatment with the adenosine receptor agonists or antagonists. However, CPA (10−10M) and NECA (10−9M) significantly inhibited the ischemia‐evoked release of aspartate and glutamate into cortical superfusates. The ability to block ischemia‐evoked release of excitatory amino acids was not evident at higher concentrations of CPA (10−6M) or NECA (10−5M). The selective A1 receptor antagonist DPCPX also had no effect on release when administered at a low dosage (0.01 mg/kg, i.p.) but blocked the ischemia‐evoked release of aspartate and glutamate at a higher dosage (0.1 mg/kg). Evoked release was inhibited by the selective A2 receptor antagonist CGS‐15943A (0.1 mg/kg, i.p.). Thus, adenosine and its analogs may suppress ischemia‐evoked release of excitatory neurotransmitter amino acids via high‐affinity A1 receptors, whereas coactivation of lower‐affinity A2 receptors may block (or reverse) the A1‐mediated response.

The effects of selective A1 and A2a adenosine receptor antagonists on cerebral ischemic injury in the gerbil.

Cerebral ischemia of 5 min duration was induced in unanesthetized Mongolian gerbils by bilateral occlusion of the carotid arteries. The extent of ischemic injury was assessed behaviorally by measuring the increases in locomotor activity following ischemia and by a histopathological assessment of the extent of CA1 hippocampal pyramidal cell injury and loss 5 days after ischemia. The A2a adenosine receptor selective antagonists 8-(3-chlorostyryl) caffeine (CSC; 0.1 mg/kg i.p.) and 4-amino-1-phenyl[1,2,4]-triazolo[4,3-a] quinoxaline (CP 66,713; 0.1 mg/kg i.p.) reduced the extent of ischemia-induced injury. An A1 selective receptor antagonist, 8-cyclopentyl-1,3-dipropylxanthine (DPCPX; 1.0 mg/kg i.p.), enhanced ischemia-evoked injury. These results suggest that adenosine A2a receptor antagonists may be useful for the prevention of cerebral injuries resulting from stroke or cardiac arrest.

Transporter reversal as a mechanism of glutamate release from the ischemic rat cerebral cortex : studies with DL-threo-β-benzyloxyaspartate

Abstract Elevated levels of the excitotoxic amino acids, glutamate and aspartate, have been implicated in the pathogenesis of neuronal injury and death induced by cerebral ischemia. This study evaluated the contribution of reversed high-affinity, Na+-dependent, glutamate transport to the ischemia-evoked release of glutamate and aspartate using dl -threo-β-benzyloxyaspartate ( dl -TBOA), a newly developed competitive, non-transported blocker of the EAAT 1–3 transporters. Changes in the extracellular levels of these and other amino acids, and of glucose and lactate in cerebral cortical superfusates during four-vessel occlusion-elicited global cerebral ischemia were examined using a cortical window technique. Basal and ischemia-evoked amino acid, glucose and lactate efflux were compared in control versus dl -TBOA (100 μM; applied topically for 35 min prior to ischemia) animals. Twenty minutes of ischemia caused large increases in aspartate, glutamate, GABA and taurine effluxes into cortical superfusates, with non-significant effects on the efflux of glycine, glutamine, alanine and serine. Application of dl -TBOA caused a 2-fold increase in basal, preischemic, extracellular glutamate levels, but did not affect those of the other compounds. In the presence of dl -TBOA, ischemia-evoked release of aspartate, glutamate, taurine and glutamine was significantly reduced; that of the other amino acids was not affected. The ischemia-evoked declines in glucose were significantly attenuated, and lactate release was enhanced above that in control animals. The amino acid data are interpreted as indicating that aspartate and glutamate releases were reduced as a consequence of dl -TBOA inhibition of reversed transport by high-affinity, Na-dependent carriers, predominantly involving the glial EAAT 2 transporter. The reduction in ischemia-evoked taurine release is interpreted as being due to a decrease in cell swelling prior to and during the initial phase of ischemia due to reduced entry of the Na+, and other ions, associated with a decreased glutamate uptake. Glucose-sparing and availability for lactate formation would also result from a reduced glutamate/Na+ uptake. These results indicate that reversed transport, primarily from glial cells by the EAAT 2 carrier, is responsible for a substantial (42 and 56%) portion of the ischemia-evoked increase in extracellular glutamate and aspartate levels, respectively. As a potent, competitive, non-transported blocker of high-affinity, Na+-dependent, glutamate transporters, dl -TBOA promises to be a valuable new compound for the study of glutamatergic mechanisms.

Increases in Cerebral Cortical Perfusate Adenosine and Inosine Concentrations during Hypoxia and Ischemia

The cerebral cortical cup technique was used to monitor changes in adenosine and inosine levels in the rat cerebral cortex during periods of hypoxia, anoxia, or hemorrhagic hypotension. Basal levels of adenosine and inosine in cortical perfusates stabilized within 10 min at concentrations of 30–50 and 75–130 nM, respectively. Comparable levels were observed in normal CSF collected from the cisterna magna. Reductions in the oxygen content of the inspired air (14, 12, 8, and 5% oxygen) resulted in increases in the adenosine and inosine levels in the cortical perfusates, the magnitude of the increase being progressively more pronounced with greater reductions in the oxygen content. Cerebral anoxia/ischemia, induced by 100% nitrogen inhalation, caused a rapid increase in the adenosine and inosine contents of the cortical perfusates. Hemorrhagic hypotension (46.1 ± 1.7 mm Hg) of 5 min duration did not result in an elevated adenosine or inosine release. The results suggest that interstitial fluid adenosine levels are likely to be in the low nM range in the normoxic animal and are capable of rapid increases during hypoxic or anoxic episodes. The findings support the adenosine hypothesis of CBF regulation.

Characterization of glutamate, aspartate, and GABA release from ischemic rat cerebral cortex

The purpose of this study was to evaluate potential mechanisms of ischemia-evoked amino acid transmitter release. Changes in extracellular levels of transmitter amino acids and lactic acid dehydrogenase (LDH) in rat cerebral cortex during and following four-vessel occlusion elicited global cerebral ischemia were examined using a cortical cup technique. Ischemia-evoked release of glutamate, aspartate and gamma-amino-butyric acid (GABA) was compared in control vs. drug-treated animals. Tetrodotoxin and antagonists of glutamate receptors (DNQX, MK-801, and AP-3) depressed the initial rate of increase in extracellular glutamate and aspartate without altering the total amount of these amino acids collected in the cortical superfusates. Cobalt, a calcium channel antagonist, failed to alter efflux. Acidic amino acid transport inhibitors (dihydrokainate, L-trans-PDC) depressed the rate of onset of glutamate and aspartate release and dihydrokainate depressed total release by 44%. PD 81723, an allosteric enhancer at the A1 adenosine receptor, depressed glutamate efflux, as did L-NAME, an inhibitor of nitric oxide synthase. Extracellular increases in GABA levels were depressed by tetrodotoxin and L-trans-PDC. The GABA transport inhibitor, nipecotic acid, increased the initial rate of onset of GABA release. Increases in LDH levels in the extracellular fluid became apparent during the period of ischemia and continued to increase during the subsequent 90 min of reperfusion. These results suggest that ischemia evokes a release of neurotransmitter amino acids that is only partially dependent upon Ca2+ influx activation or the reversal of amino acid transporters. Nonselective mechanisms, resulting from the disruption of plasma membrane integrity, may contribute significantly to the total ischemia-evoked release of excitatory amino acids.

Protection from cerebral ischemic injury in gerbils with the spin trap agent N-tert-butyl-α-phenylnitrone (PBN)

N-Tert-butyl-alpha-phenylnitrone (PBN), a spin trap agent, reduced ischemic hippocampal damage and the associated locomotor hyperactivity in Mongolian gerbils. Cerebral ischemia was induced in unanesthetized gerbils by a bilateral 5 min occlusion of the carotid arteries. PBN (100 mg/kg, i.p.) administered 30 min prior to carotid occlusion, prevented the increase in locomotor activity observed in saline-injected ischemic animals and significantly reduced damage to the hippocampal CAI pyramidal cell layer observed 5 days post-ischemia. These findings support the hypothesis of an involvement of reactive free radicals as a significant cause of ischemia-reperfusion-induced cerebral injury and suggest that PBN may be a useful agent for the prevention of cerebral ischemic damage.

The role of nitric oxide in the regulation of cerebral blood flow

The role of nitric oxide in the cerebral circulation under basal conditions and when exposed to hypoxic, hypercapnic and hypotensive stimuli, was studied in mechanically ventilated rats using a venous outflow technique, by examining the effects of inhibition of nitric oxide synthase with N-nitro-L-arginine methyl ester (L-NAME). L-NAME (10 or 30 mg/kg injected intravenously) raised mean arterial blood pressure by 14% and 24%, and increased cerebrovascular resistance (CVR) by 20% and 24%, respectively. Cerebral blood flow (CBF) was unaltered, as were blood gases and pH. The increases in MABP and CVR were attenuated by L-arginine (300 mg/kg). Following the administration of L-NAME, the increases in CBF elicited by ventilation with 8% oxygen for 25 s were unaltered, in comparison to control responses. L-NAME attenuated the increases in CBF and reduced the time for recovery to basal flow rates evoked by ventilation with 10% carbon dioxide. These effects were reversed by L-, but not by D-, arginine. Autoregulation by CBF during hypotensive episodes, as measured by comparisons of CVR values, was unaffected by L-NAME. The results suggest that endogenous nitric oxide is involved in the responses of the cerebral vasculature to elevated levels of CO2 in the arterial blood. Nitric oxide does not appear to play a major role in autoregulation to increases or decreases in MABP, or in hypoxia-evoked vasodilation.

Central effects of adenosine analogs on locomotor activity in mice and antagonism of caffeine

Mice implanted with chronic indwelling cannulas were injected in the lateral cerebral ventricle with a series of adenosine analogs and the effects on spontaneous locomotor activity were recorded. All analogs produced dose-related decreases in locomotor activity. The relative order of potency for locomotor depression was: NECA much greater than L-PIA greater than CADO greater than D-PIA. Caffeine at the lowest dose produced a significant decrease in locomotor activity. At higher doses caffeine had no effect on locomotor activity but it did antagonize the depressant effects of NECA, a finding consistent with the notion that the central stimulant action of methylxanthines is due to their antagonism of central adenosine receptors.