reperfusion injury in the gerbil

Pergamon

0306-3623(95)00042-9

Gen. Pharrnac. Vol. 26, No. 7, pp. 1545-1548,1995 Copyright © 1995ElsevierScienceInc. Printed in Great Britain.All rights reserved. 0306-3623/95 $9.50 + 0.00

Adenosine A1 Receptor Enhancer, PD 81,723, and Cerebral Ischemia/Reperfusion Injury in the Gerbil X. C A O a n d J. W. P H I L L I S * Department of Physiology, Wayne State University School of Medicine, 540 E. Canfield, Detroit, M1 48201 U.S.A. [Tel: 313-577-6745; Fax: 313-577-5494] (Received 3 January 1995)

Abstract-1. The adenosine A, receptor enhancer, PD 81,723, was tested for its neuroprotective activity in a Mongolian gerbil model of forebrain ischemia/reperfusion cerebral ischemia. 2. Gerbils were injected with PD 81,723 (1, 10 and 125 mg/kg i.p.) 20 min before a 5-min episode of forebrain ischemia. The extent of ischemic injury was assessed by monitoring the increases in locomotor activity and from the degree of damage to the CA1 hippocampal pyramidalcell layerafter 5 days of recovery. 3. By both criteria, PD 81,723, at all three dose levels,failedto protect against ischemia/reperfusionevoked cerebral injury. Key Words: Adenosine AI receptor, cerebral ischemia, enhancer, gerbil, hippocampal injury, PD 81,723

INTRODUCTION Adenosine is an important neuromodulator in the central nervous system, with inhibitory pre-synaptic and post-synaptic effects on synaptic transmission (Dunwiddie, 1985; Phillis, 1991). The effects of adenosine are mediated through two types of extracellular receptor: A~ and A2. The adenosine A1 receptor on presynaptic nerve terminals modulates transmitter release, whereas activation of an A2 receptor associated with cerebral vascular smooth muscle results in vasodilatation and an increase in cerebral blood flow. The two receptors thus appear to play complementary roles in regulating the metabolic supply-demand balance: inhibiting neuronal firing and increasing oxygen supply. In this context, adenosine has been designated a "retaliatory metabolite" (Newby, 1984). Adenosine A~ receptor activation depresses ischemiaevoked release of the excitotoxic amino acids, glutamate and aspartate, from the rat cerebral cortex (Heron et aL, 1992; Simpson et aL, 1992), and this action likely accounts for the cerebroprotective actions of A~ agonists against ischemic injury (Evans et aL, 1987; Von Lubitz et al., 1989). Conversely, prior administration of adenosine receptor antagonists enhances the degree of injury in ischemic rat brains (Rudolphi et al., 1992). Agents that augment the response of A1 receptors *To whom all correspondence should be addressed. 1545

to adenosine would be expected to be useful in conditions associated with excessive central release of glutamate and aspartate, such as stroke and cardiac arrest. Direct-acting agonists have not proven to be suitable for this role because of their widespread peripheral side effects, which include hypotension and cardiodepression (Rudolphi, 1991). Allosteric enhancers of the actions of adenosine have recently emerged as alternative agents to directly acting adenosine agonists or inhibitors of adenosine metabolism. The 2-amino-3-benzoylthiophene derivative, PD 81,723, has been shown to enhance the binding of adenosine agonists to AI receptors in brain membranes and therefore has been classified as an allosteric enhancer (Bruns and Fergus, 1990). PD 81,723 has been demonstrated to specifically potentiate A1 receptor mediated responses to both exogenous and endogenous adenosine in the heart (Kollias-Baker et aL, 1994a,b). This strategy for maximizing the effects of endogenously released adenosine offers the additional advantage of limiting the enhanced effects of endogenously released adenosine to the A1 receptors, in the absence of facilitation of its effects at A2 receptors (Kollias-Baker et aL, 1994a). Because A2 receptor activation in the brain can actually result in an increased release of the excitotoxic amino acids (O'Regan et al., 1992) and enhanced ischemic injury (Gao and Phillis, 1994), this becomes a potentially attractive feature of the use of allosteric enhancers. In the present study, the adenosine binding enhancer

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PD 81,723 is evaluated for its effects on ischemia/reperfusion evoked injury to hippocampal CA1 pyramidal cells in a gerbil forebrain ischemia model.

MATERIALS A N D METHODS

Male Mongolian gerbils (50-55 g wt.) were obtained from Tumblebrook Farms (West Brook field, MA, U.S.A.). Under pentobarbital sodium anesthesia, loops were placed around the common carotid arteries and exteriorized through the back of the neck. During the same procedure, fine silk threads, attached to the carotid loops, were allowed to exit through the ventral neck incision. After a 48-hr recovery period, the gerbils were tested for spontaneous locomotor activity in an Electronic Activity monitor (Stoelting, IL). Under manual restraint, the gerbils were then "stroked" for a 5-min period by traction on the carotid artery loops, after which the loops were withdrawn from the neck, using the fine silk threads. Successful occlusion of the carotid arteries was evident from a complete bilateral ptosis and the "hunched" posture adopted by the gerbil. Reperfusion after removal of the carotid snares was almost immediately apparent, with opening of the eyes, recovery of head and neck tone and the rapid resumption of locomotor activity. The gerbils were kept in a warmed enclosure (30-32°C) for 5 hr after the ischemic episode to prevent any fall in body temperature. Animals were retested for locomotor activity at 5 hr, 24 hr and 5 days. On the 5th day, the gerbils were re-anesthetized with pentobarbital sodium and perfused with saline and fixative via the ascending aorta. After fixation, the brains were embedded, sectioned and stained with cresyl violet. The extent of hippocampal CA1 cell loss was assessed from representative sections on slides that had their identification codes obscured. Complete details of the technical procedures and histopathological scoring indicators have been de-

scribed in previous publications (Phillis and O'Regan, 1989; Phillis and Smith-Barbour, 1993). Motor activity scores were compared using a Student's t-test and hippocampal injury scores were compared with a MannWhitney U test. The gerbils were divided into five groups. A control group (n = 12) received an intraperitoneal injection of saline 20 min before ischemia. Three groups received PD 81,723 (1 mg/kg, n = 8; 10 mg/kg, n = 22; 125 mg/kg, n = 10) dissolved in 0.01 ml dimethyl sulphoxide (DMSO) and the fifth group (n = 6) was injected with 0.01 DMSO, all 20 min before ischemia. RESULTS After bilateral carotid artery occlusion, the control ischemic gerbils displayed characteristically elevated levels of motor activity at all three testing intervals (Table 1). The percentage increases in motor activity above basal levels for animals pretreated with PD 81,723 or DMSO alone were not significantly different from those of the ischemic control gerbils at all three post-ischemic time points. Five days after the ischemic episode, widespread damage to the CA1 region of the hippocampal was evident in the brains of the control gerbils (Table 2). Many pyramidal cells either presented a shrunken appearance with condensed nuclei and minimal cytoplasms or had disappeared, leaving vacuolar-type spaces. The damage scores of the PD 81,723 and DMSO-injected animals were similar to those of the control ischemic animals (Table 2).

DISCUSSION PD 81,723, a 2-amino-3-benzoylthiophene, acts as an allosteric enhancer at the adenosine AI receptor both in binding assays and in a functional in vitro assay

Table I. Locomotor activities of Mongolian gerbils

Condition Saline + ischemia (n = 12) PD 81,723 (1 mg/kg) (n = 8) PD 81,723 (10 mg/kg) (n = 22) PD 81,723 025 mg/kg) (n = 10) DMSO (0.01 ml), (n = 6)

Pre-drug and pre-ischemia (%)

Activity as Percentage of Initial Basal Activity 5 hr

24 hr

5 days

100 ± 9 . 9 0

167.5 ± 27.8

223.6 _+ 29.8

183.5 _+ 22.7

100 ± 13.6%

135.5 ± 29.9

239.0 ± 33.8

183.1 +_ 25.9

100 ± 11.0%

159.1 + 17.4

186.6 ± 21.8

173.9 ± 14.6

100 ± 17.5070

147.0 ± 26.3

169.3 ± 24.8

207.6 ± 19.0

100 ± 12.1%

174.5 + 18.9

238.9 ± 24.4

145.6 ± 24.3

Values are means ± SEM. Initial pre-ischemic levels of activity are used as controls (100%) for subsequent measures of activity. Individual gerbils were monitored for a 30-rain period after a 10-min period for adaptation. Basal, pre-drug, pre-ischemia levels of activity were (cts/min) as follows: saline control 110.2 ± 10.9; PD 81,723 (1 mg/kg) 122.8 ± 16.7; PD 81,723 (10 mg/kg) 115.2 ± 12.7; PD 81,723 (125 mg/kg) 79.2 ± 13.9; DMSO (0.01 ml) 124.4 ± 15.0. There were no significant differences between the control, PD 81,723- or DMSO-treated gerbils.

Cerebral ischemia/reperfusion injury Table 2. Hippocampal CA1 damage scores Condition Damage scores Saline + ischemia (n = 12) 2.7 ± 0.18 PD 81,723 (1 mg/kg) (n = 8) 2.5 ± 0.19 PD 81,723 (10 mg/kg) (n = 22) 2.6 + 0.12 PD 81,723 (125 mg/kg) (n = 10) 2.2 ± 0.25 DMSO (0.01 ml) (n = 6) 2.7 + 0.58 Values are means + SEM. The histopathological scoring system was based on that used and illustrated in a previous study (Phillis and O'Regan, 1989) with 0 = no apparent necrosis and 4 = virtually complete absence of intact pyramidal cells in the CA1 region. There were no significant differencesbetween the saline control, PD 81,723- or DMSO-treated gerbils.

(Bruns and Fergus, 1990). The enhancing effect is specific for the At receptor, because no equivalent effect is evident at A2 receptors. The allosteric nature of the interaction at the At receptor is shown by a slowing of the dissociation of labeled At agonists and is specific for the agonist state o f the receptor, because At antagonist binding was not enhanced but was actually inhibited (Bruns and Fergus, 1990). P D 81,723 increased the binding o f [~H]CHA to rat brain membranes by 45% at 20 ~tM, but this effect was followed by inhibition at higher concentrations, again indicating that the enhancer possesses significant competitive antagonist activity at higher concentrations. An ability of P D 81,723 to affect neuronal activity has been demonstrated in experiments in which the enhancer potentiated the depressant action of adenosine on evoked responses in a hippocampal slice (Janusz et al., 1991) and depressed seizures in the same preparation (Janusz and Berman, 1993). At higher concentrations (> 10 ~tM), the ability of P D 81,723 to enhance adenosine's actions was reduced, presumably as a result of competitive antagonism at the adenosine receptor. P D 81,723 was tested for its ability to attenuate the release of glutamate and aspartate into cerebral cortical superfusates of rats subjected to 20 rain of four vessel occlusion cerebral ischemia. At a dose level of 10 m g / k g , it depressed the peak ischemia-evoked level of release of glutamate, without altering the release of aspartate or G A B A (Phillis et al., 1994). The total release of glutamate during the 20 min period of ischemia and subsequent 20 min o f reperfusion was not, however, significantly reduced in comparison with that in control rats. At a dose of 125 mg/kg, P D 81,723 again failed to alter ischemia reperfusion elicited releases of glutamate and aspartate. As A1 receptor activation is known to depress ischemia-evoked glutamate release, the initial reduction in glutamate release at the lower dose of P D 81,723 was not unanticipated. The disappearance of this effect when the dose was increased would be consistent with the previously mentioned ability of the c o m p o u n d to act as a competitive antagonist at the A, receptor. The gerbil model of ischemia/reperfusion evoked

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forebrain injury yields both behavioral and histopathological evidence of the extent of hippocampal C A I pyramidal neuron injury. The locomotor hyperactivity evident in the control ischemic gerbils is a characteristic phenomenon and is thought to be a consequence o f an inability to habituate to a novel environment due to the loss of CA1 pyramidal neurons (Babcock et al., 1993; Wang and Corbett, 1990). In the present experiments, P D 81,723, at a range of doses (1, 10, 125 mg/kg) failed to reduce the extent of ischemia/reperfusion evoked injury to the gerbil forebrain, as measured by alterations in locomotor activity and histopathological examination of the CA1 hippocampal pyramidal cell layer. Although the lack of a cerebroprotective effect of an adenosine At receptor enhancing c o m p o u n d on the ischemia reperfusion injury process appears to be inconsistent with the demonstrated cerebroprotective action of At receptor activation (Evans et al., 1987; Von Lubitz et al., 1989), it would be consistent with the failure of P D 81,723 to reduce the total release of excitotoxic amino acids from the cerebral cortex during ischemia reperfusion (Phillis et al., 1994). It is possible that as a"first generation" enhancer, with a "partial agonist" type of action, P D 81,723 may not be sufficiently potent in its A1 receptor enhancing abilities to exert a significant effect. This problem may be overcome with the future development of more potent enhancers, lacking a competitive antagonist action. SUMMARY P D 81,723, an allosteric enhancer of adenosine action at A1 receptors was evaluated for cerebroprotective activity in the Mongolian gerbil model of cerebral ischemia. Administered intraperitoneally at doses of 1, 10 or 125 m g / k g , P D 81,723 failed to attenuate the extent of cerebral ischemic injury, measured either as an increase in locomotor hyperactivity or as hippocampal CA1 pyramidal cell damage. Acknowledgements- Supported by USPHS grant NS 2691206 and a grant-in-aid from the American Heart Association.

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