D. G. MacGregor

Ascorbate attenuates the systemic kainate-induced neurotoxicity in the rat hippocampus

The neuronal damage induced by systemic administration of kainic acid reproduces the cellular and regional pattern of damage produced by repeated seizures. The ability of kainic acid to induce lipid peroxidation, and the ability of free radical inhibitors to prevent ischaemically-induced cell death, has led us to examine the possible role of free radicals in kainate-induced injury. Ascorbic acid was able to reduce kainate-induced damage of the rat hippocampus, measured by means of the gliotic marker ligand [3H]PK11195. Ascorbate was significantly effective at doses of 30 mg kg-1 and above, with total protection against kainate at 50 mg kg-1. Histologically, ascorbate at 50 mg kg-1 was able to prevent kainate-induced neuronal loss in the hippocampal CA1 and CA3a cell layers. The antioxidant was also effective when administered simultaneously with, or 1 h before the kainate. Protection was also obtained by allopurinol, 175 mg kg-1 and by oxypurinol, 40 mg kg-1. Ascorbate did not modify synaptically evoked potentials or long-term potentiation in hippocampal slices, ruling out any blocking activity at glutamate receptors. It is concluded that the neuronal damage produced by systemically administered kainate involves the formation of free radicals.

Mediation of the neuroprotective action of R-phenylisopropyl-adenosine through a centrally located adenosine A1 receptor.

1 Systemic injections of kainic acid, 10 mg kg−1, into adult rats resulted in lesions in the hippocampus, as assessed by peripheral benzodiazepine ligand binding. Co‐administration of clonazepam at 1 mg kg−1 or 0.2 mg kg−1 prevented major seizures associated with kainate injections, but did not alter significantly the production of hippocampal damage. 2 The co‐administration of the adenosine A1 agonist R‐phenylisopropyladenosine (R‐PIA, 25 μg kg−1, i.p.) abolished the lesions induced by kainic acid. 3 The presence of the selective A1 antagonist, 8‐cyclopentyl‐1,3‐dipropylxanthine (250 or 50 μg kg−1, i.p.) abolished the R‐PIA neuroprotective action. 4 The A1/A2 antagonist, 8‐(p‐sulphophenyl)theophylline (20 mg kg−1, i.p.) which cannot cross the blood brain barrier, did not alter significantly the neuroprotective action of R‐PIA, indicating that the neuroprotective action of the purine may be predominantly central. 5 The time course of the neuroprotection was also examined. R‐PIA was effective when administered 2 h before or after kainate administration. 6 The results emphasise the potential utility of systemically active adenosine A1 receptor ligands in reducing CNS gliosis induced by the activation of excitatory amino acid receptors.

Inhibition by the adenosine analogue, (R-)-N6-phenylisopropyl-adenosine, of kainic acid neurotoxicity in rat hippocampus after systemic administration

1 Binding of the peripheral benzodiazepine receptor ligand, [3H]‐PK 11195, to rat hippocampal membranes has been used to quantify the reactive gliosis resulting from neuronal death induced by intraperitoneally administered kainic acid. 2 Intraperitoneal administration of kainic acid (10 mg kg−1) caused a 350–500% increase in [3H]‐PK 11195 binding measured in rat hippocampal P2 membranes 7 days later. Co‐treatment with the adenosine derivative R‐phenylisopropyladenosine (R‐PIA) (100, 25 or 10 μg kg−1, i.p.) abolished this elevation. The protective action of R‐PIA could itself be abolished by co‐treatment with 8‐phenyltheophylline (1 mg kg−1). 3 Body temperatures were recorded in the antagonist experiments and no significant changes were recorded, suggesting that the protective action of R‐PIA was not mediated by hypothermia. 4 Since systemic kainic acid‐induced neurotoxicity has been claimed as a good model of neuronal death in temporal lobe epilepsy, the results suggest that the systemic administration of purines in low doses may provide protection against certain neurodegenerative insults.

Impaired cerebral autoregulation 24 h after induction of transient unilateral focal ischaemia in the rat

Cerebral blood flow (CBF) and cerebral autoregulation have been investigated 24 h after transient focal ischaemia in the rat. Cerebral blood flow was measured autoradiographically before and during a moderate hypotensive challenge, to test autoregulatory responses, using two CBF tracers, 99mTc‐d,l‐hexamethylproyleneamine oxide and 14C‐iodoantipyrine. Prior to induced hypotension, CBF was significantly reduced within areas of infarction; cortex (28 ± 20 compared with 109 ± 23 mL/100 g/min contralateral to ischaemic focus, P = 0.001) and caudate (57 ± 31 compared with 141 ± 32 mL/100 g/min contralaterally, P = 0.005). The hypotensive challenge (mean arterial pressure reduced to 60 mmHg by increasing halothane concentration) did not compromise grey matter autoregulation in the contralateral hemisphere; CBF data were not significantly different at normotension and during hypotension. However, in the ipsilateral hemisphere, a significant volume of cortex adjacent to the infarct, which exhibited normal flow at normotension, became oligaemic during the hypotensive challenge (e.g. frontal parietal cortex 109 ± 15% to 65 ± 15% of cerebellar flow, P < 0.01). This resulted in a 2.5‐fold increase in the volume of cortex which fell below 50% cerebellar flow (39 ± 34 to 97 ± 46 mm3, P = 0.003). Moderate hypotension induced a significant reduction in CBF in both ipsilateral and contralateral subcortical white matter (P < 0.01). In peri‐infarct caudate tissue, CBF was not significantly affected by hypotension. In conclusion, a significant volume of histologically normal cortex within the middle cerebral artery territory was found to have essentially normal levels of CBF but impaired autoregulatory function at 24 h post‐ischaemia.

Release and actions of adenosine in the central nervous system

Adenosine is released from active neurons into the extracellular fluid at a concentration of about 1μmol/l. Neither the precise cellular origin nor the biochemical form of release has been firmly established, though the nucleotide is probably released partly directly, as a result of raised intracellular levels, and partly as nucleotides, which are subsequently hydrolysed. Once in the extracellular medium, adenosine markedly inhibits the release of excitatory neurotransmitters and modulatory peptides and has direct inhibitory effects on postsynaptic excitability via A1 receptors. A population of A2 receptors may mediate depolarization and enhanced transmitter release. Adenosine also modulates neuronal sensitivity to acetylcholine and catecholarnines, all these effects probably contributing to the behavioural changes observed in conscious animals. As a result of their many actions, adenosine analogues are being intensively investigated for use as anticonvulsant, anxiolytic, and neuroprotective agents.

The attenuation of kainate-induced neurotoxicity by chlormethiazole and its enhancement by dizocilpine, muscimol, and adenosine receptor agonists.

Systemically administered kainate (10 mg.kg-1) caused neuronal loss in both the hippocampus and the entorhinal regions of the rat brain. This resulted in a loss of 68.3 +/- 13.8 and 53.3 +/- 12.8% of pyramidal neurones in the hippocampal CA1 and CA3a regions, respectively. Chlormethiazole attenuated the loss of neurones in the hippocampal cell layers CA1 (cell loss 10 +/- 3.2%) and CA3a (cell loss 10 +/- 7.7%). The neuroprotective activity of chlormethiazole was apparent in the presence or absence of a low dose of clonazepam (200 micrograms.kg-1 i.p.). The kainate-induced damage could also be measured by the increase in binding of the peripheral benzodiazepine ligand ([3H]PK11195) in the hippocampus. In kainate-treated rats there was a 350-500% increase in binding indicative of reactive gliosis. Chlormethiazole prevented this elevation in a dose- and time-dependent manner, with an ED50 of 10.64 mg.kg-1 and an effective therapeutic window from 1 to 4 h posttreatment. Dizocilpine also attenuated damage significantly. The GABAA agonist muscimol was also able to attenuate the increase in [3H]PK11195 binding in a dose-dependent manner, with an ED50 of approximately 0.1 mg.kg-1. If muscimol, dizocilpine, or the adenosine A1 receptor agonist R-N6-phenylisopropyl-adenosine were administered together with chlormethiazole at their respective ED25 doses, a potentiation was apparent in the degree of neuroprotection. It is concluded that the combination of neuroprotective agents with different mechanisms of action can lead to a synergistic protection against excitotoxicity.

Prevention by a purine analogue of kainate-induced neuropathology in rat hippocampus

Systemic injection of kainic acid produces a characteristic regional and cellular pattern of neuronal loss in the central nervous system by mechanisms which may be relevant to an understanding of neurodegenerative disorders. It has previously been found, by measuring the binding of a glial marker ligand, that analogues of adenosine, such as R-N6-phenylisopropyladenosine (R-PIA), can prevent kainate-induced damage of the hippocampus at doses as low as 10 micrograms/kg, i.p. The use of gliotic markers, however, is open to misinterpretation, and the present work was designed to re-examine purine protection against kainate using histological methods. The results show that R-PIA, at a dose of 25 micrograms/kg i.p. in rats, can protect against the neuronal damage caused by kainate and that this protection could be completely prevented by the simultaneous administration of 1,3-dipropyl-8-cyclopentylxanthine, indicating the involvement of adenosine A1 receptors in the protection.

Blockade by 1,3-dipropyl-8-cyclopentylxanthine (CPX) of purine protection against kainate neurotoxicity.

The adenosine A1 receptor selective antagonist 1,3-dipropyl-8-cyclopentylxanthine (CPX) has been administered systemically to rats together with the neurotoxin kainic acid. At the lower doses of CPX tested, 10 and 50 micrograms/kg, which were sufficient to prevent the neuroprotective activity of exogenous agonists, there was no exacerbation of the neuronal damage. At 250 micrograms/kg, some enhancement of damage was found, which was also produced by 8-(p-sulphophenyl)theophylline, a non-selective xanthine which does not cross the blood-brain barrier. The results are consistent with the involvement of a central A1 receptor in the neuroprotective activity of purines, and suggest that blockade of a peripheral adenosine receptor, possibly of the A2 type, may increase neuronal damage.

Protection by an Adenosine Analogue against Kainate-Induced Extrahippocampal Neuropathology

1. The glutamate analogue kainic acid produces neuronal damage in the central nervous system. We have reported that analogues of adenosine, such as R-N6-phenylisopropyladenosine (R-PIA) can, at doses as low as 10 microg/kg IP, prevent the hippocampal damage that follows the systemic administration of kainate. The present work was designed to examine purine protection against kainate in extrahippocampal regions by using histological methods. 2. The results show that R-PIA, at a dose of 25 microg/kg IP in rats, can protect against the neuronal damage caused by kainate in the basolateral amygdaloid nuclei, the pyriform cortex and around the rhinal fissure. This protection could be prevented by the simultaneous administration of the A1 adenosine receptor antagonist 1,3-dipropyl-8-cyclopentylxanthine, confirming that the protection involved adenosine A1 receptors. No protection was observed in the posterior amygdaloid nuclei or the entorhinal cortex, suggesting the absence of relevant adenosine receptors or a different mechanism of excitotoxicity.

Group S8A serine proteases, including a novel enzyme cadeprin, induce long-lasting, metabotropic glutamate receptor-dependent synaptic depression in rat hippocampal slices

Long‐term potentiation and long‐term depression (LTD) are forms of synaptic plasticity in the central nervous system. We now report that a group of chymotrypsin‐like serine proteases, especially members of the S8A subfamily, induce LTD of evoked potentials in rat hippocampal slices. The proteolytic activity of these enzymes is required for the induction of LTD, as serine protease inhibitors prevent the effect. The depression is partly mediated by the suppression of transmitter release from glutamatergic terminals but also involves an elevation of action potential threshold with no change of post‐synaptic membrane potential or input resistance. We have also isolated a novel and more potent related enzyme, cadeprin, from Aspergillus. The LTD produced by all of these proteases is not dependent on receptors for several transmitter systems, including N‐methyl‐d‐aspartate or adenosine receptors, but is prevented by blocking group I metabotropic glutamate receptors. The activity of cadeprin, subtilisin and other S8A serine proteases may shed light on the mechanisms of LTD and a related endogenous molecule could have a physiological or pathological role as a modulator of synaptic plasticity in the mammalian hippocampus.