BrainResearchBulletin,Vol.

29, pp. 659465, Printed in the USA. All rights reserved.

1992 Copyright

0361-9230/92 $5.00 + .OO 0 1992 Pergamon Press Ltd.

Effects of Hyperthermia on the Effectiveness of MK-801 Treatment in the Gerbil Hippocampus Following Transient Forebrain Ischemia HIDEAKI

HARA,’

HIROSHI

ONODERA

AND

KYUYA

KOGURE

Department of Neurology, Institute of Brain Diseases, Tohoku University School of Medicine, Sendai, Japan Received

18 July 199 1; Accepted

24 February

1992

HARA, H., H. ONODERA AND K. KCKXJRE. Eficts of hyperthermia on the effectiveness of MK-801 treatment in the gerbil hippocampus following transient forebrain ischemia. BRAIN RES BULL 29(5) 659-665, 1992.-The effects of dizocilipine maleate (MK-801), a noncompetitive N-methyl-D-aspartate (NMDA) receptor/channel antagonist, were tested on the dysfunction of neurotransmitter and signal transduction systems and morphological damage 7 days after transient forebrain ischemia in gerbils. Nemotransmitter system (adenosine Al, muscarinic choline@ receptor) and signal transduction system (inositol I ,4,5trisphosphate receptor: IPs, protein kinase C: PKC, L-type calcium channels) binding sites were mapped by in vitro quantitative receptor autoradiography. All ligands used in the present study decreased significantly in the CAI subfield 7 days after ischemia. In normothermic animals, pretreatment with MK-801 failed to protect against decreased receptor binding in the hippocampus 7 days after ischemia. Moreover, in a morphological study, pre- and posttreatment of MK-801 failed to show protective effects against ischemic neuronal damage. On the other hand, pretreatment of MK-80 I, without maintaining body temperature, prevented the neuronal death of CA1 subfield 7 days after ischemia. These results weaken the hypothesis that NMDA receptor/channel may play a pivotal role in the pathogenesis of neuronal damage after transient forebrain ischemia.

Cerebral &hernia Gerbil MK-80 I Neurotransmitter

Hippocampus Hyperthermia N-methyl-D-aspartate antagonist

Ischemic neuronal damage Hypothermia Signal transduction Receptor autoradiography

aptically released neurotransmitters, such as glutamate, are responsible for neuronal damage in the hippocampus (1). It has been reported that both noncompetitive and competitive NMDA receptor/channel antagonists prevent neuronal damage after ischemia (2,41). The most potent of these antagonists is MK80 I ( 16- 1S), a noncompetitive NMDA receptor/channel antagonist (13,44). Thus, these reports support the hypothesis that the NMDA receptor/channel plays an important role in the pathogenesis of the postischemic neuronal damage (39). However, studies in several laboratories failed to protect neurons against damage in the hippocampal CA1 subfield with antagonists for this receptor/channel after transient forebrain ischemia (3,9,22,29). Therefore, the purpose of this study was to examine differences in body temperature as a possible basis for the disparate results and to determine the effects of MK-80 1 against ischemic hippocampal neuronal damage by mean of morphologic and in vitro receptor autoradiographic technique.

THE Mongolian gerbil is an excellent model for research on stroke (23,24). Transient forebrain ischemia induces neuronal damage in specific brain areas in the gerbil (23,24) and in the rat (38,40). Neuronal degenerative processes occur especially in the hippocampus where the pyramidal cells in the CA 1 subfield seem to be the most sensitive to ischemia. Alteration of various neurotransmitter systems has been implicated in the pathogenesis of neuronal damage after ischemia (33) and the signal transduction systems may be related to the molecular mechanism of ischemic neuronal damage (20,2 1). Receptor autoradiography is a sensitive method of observing the changes of the signal transduction system (26,27). Using this autoradiography, we have reported that a potent lipid peroxidation inhibitor and pentobarbital prevented a decrease of various receptor bindings after transient forebrain ischemia in gerbils (19-21). The high sensitivity of CA1 pyramidal cells has been attributed to very high concentrations of N-methyl-D-aspat-tate (NMDA) receptors in the brain and these receptors, in turn, have been implicated in neuronal damage after ischemia (28,39). Much data has accumulated concerning the excitotoxic mechanism of hippocampal CA 1 damage after ischemia. The evidence supports the hypothesis that extracellular accumulation of syn-

METHOD

Animal

Preparation

Male adult Mongolian gerbils weighing 60-80 g were purchased from Seiwa Experimental Animals (Fukuoka, Japan).

I Requests for reprints should be addressed to Hideaki Hara Ph.D., Department of Pharmacology, New Drug Research Laboratories, Kanebo Ltd., I-5-90 Tomobuchi-cho, Miyakojima-ku, Osaka 534, Japan.

659

HARA,

TABLE

I

EFFECTS OF MK-801 ON BODY TEMPERATURE ISCHEMIA IN GERBILS

FOLLOWING

Body Temperature “C (Mean ir SE) Controlled

Uncontrolled

Time

Vehicle

MK-80 I

MK-80 I 37.8 + 0.2

Before anesthesia

38.0 + 0.1

38.0 f 0.3

Before ischemia

37.0 + 0.2

37.0 f 0.2

34.1 + 0.6

During ischemia

37.3 rt 0.5

37.1 * 0.2

33.8 * 0.7

20 min

39.6 f 0.1

39.3 k 0.2

34.2 k 0.6

I

38.8 k 0.4

38.8 k 0.2

33.5 i- 0.5

2h

38.3 f 0.2

38.3 k 0.2

32.8 k 0.7

4h

37.9 k 0.

I

37.8 t 0.3

34.0 i 0.3

After reperfusion h

II -= 5. MK-801

was administered

I

ONODERA

AND

KOGURE

mounted onto gelatincoated slides. In vitro receptor binding studies were performed as reported previously, with [3H]inositol 1,4,5_trisphosphate (IP,) (46); [‘Hlphorbol 12,13-dibutylate (PDBu) (45); [3H]PN200-l 10 (PN) (10); [3H]Ns-cyclohexyladenosine (CHA) (33); [3H]quinuclidinyl benzilate (QNB) (42). The optical density of the regions of interest was measured by a computer-assisted image analyzer (Zeiss, VIDAS image analyzer system, Germany). The relationship between optical density and radioactivity was examined with reference to tritium standards (Amersham International plc, Bucks, UK, [3H]microscales) coexposed along with the tissue sections. The optical density of the brain regions measured in the present study was in the range in which the optical density and the radioactivity of the 3H-microscales showed a near linear relationship. A possible drawback with quantitative autoradiography may be an alteration in quenching level after ischemia. As discussed in detail in our previous report, quench correction was unnecessary (33). The binding assay was performed in duplicate.

h prior to ischemia.

Stuli,stical

The animals were allowed ad lib access to food and water (fed state) and were anesthetized with a mixture of 2% halothane, 70% NZO. and 30% 02. The common carotid arteries were exposed and occluded bilaterally with Sugita No. 51 temporary aneurysm clips for 5 min. MK-801 (kindly donated by Merck and Co. Inc.. Rahway. NJ, USA) at a dose of 10 mg/kg or the vehicle (saline) was administered IP 1 h prior to or immediately after ischemia. Sham-operated animals served as controls.

The rectal temperature of most animals was maintained at 37-39°C with a heating pad and a heating lamp during the operation period and until the righting reflex reappeared. Body temperature in one group of animals was not controlled. Body temperature in all animals was monitored before anesthesia, before and during ischemia, and 20 min. 1 h, 2 h, and 4 h after reperfusion with a rectal thermometer (Nihon Koden, MGAIII. Japan).

Histological analysis was carried out according to the method of Kirino et al. (24). Briefly, on the seventh day of recirculation. the gerbils were anesthetized with pentobarbital, and fixed by transcardiac perfusion with 10% formaldehyde, and the brain was removed and placed in fresh fixative for a minimum of 48 h. Brains were dehydrated in a graded ethanol series (70- 100%). xylene baths, and embedding in paraffin. Five pm cresyl violet (pH 7.6)-stained sections of the dorsal hippocampus (1 S-2.0 mm posterior to the bregma) were examined using light microscopy. The neuronal density of the CA 1 sector was calculated by counting living neurons in the CAI sector at 400X and measuring the total length of the CA1 cell layer (3.0 + 0.5 mm) in each section with a digitizer (Wacom Co.). Cells in the CAI sector were counted on both the right and left sides, and the values (neuronal density) were averaged to calculate the cell count per millimeter for each gerbil.

Seven days after the ischemic insult, the gerbils were decapitated. The brains were dissected out of the skull and frozen in powdered dry ice, and stored at -80°C until assay. Coronal sections, 12 grn in thickness, were cut on a cryostat and thaw

.-Inul~5i.v

The values were expressed as mean + SE. Statistical comparisons were made by the one-way analysis of variance (ANOVA), Duncan’s multtple range test and the two-tailed MannWhitney c-test. A value ofp < 0.05 was considered a significant difference. RESULTS

The MK-80l-treated group exhibited the loss of righting reflex lasting more than 10 h from IO min after administration. No differences were found between control and MK-80l-treated groups with maintaining body temperature (Table I). Body temperature of control 20 min after ischemia was 15°C higher than that prior to anesthesia, and body temperature 4 h after ischemia was recovered. On the other hand, the body temperature of MK-801-treated gerbils that did not have their body temperature controlled was less than 35°C until the righting reflex reappeared.

In the sham-operated group, the CA1 pyramidal cells were well preserved. In the vehicle-treated ischemia groups with and without maintaining body temperature, most ofthe CA1 subfield were not preserved. Similarly, MK-801 (10 mg/kg, IP) administered 1 h prior to or immediately after ischemia with maintaining body temperature did not prevent neuronal damage in the CA 1 subfield. On the other hand, without maintaining body temperature, MK-801 administered 1 h prior to ischemia prevented neuronal damage (Table 2). .,t ulorudiogruphy

(Fig. 3, Tubles 3. 4)

group. [3H]1P3 binding in the CA I subfield was completely reduced 7 days after ischemia. In the CA3 subheld and dentate gyrus. binding was markedly decreased by 94% ofsham. [‘H]PDBu binding in the stratum oriens and the stratum lacunosum-moleculare of the CA1 subfield was reduced by I9 and 30%, respectively, at this period. In the CA3 subfield, however, [3H]PDBu binding sites remained unchanged in the stratum oriens. [3H]PN binding in the CA1 subfield and the stratum oriens of the CA3 subfield was significantly reduced by 44 and 24% respectively. [‘H]PN binding sites were unchanged in the stratum radiatum of the CA3 subfield and in the molecular layer of the dentate gyrus. [3H]CHA binding in the CA I subfield after l’cd~iclc-/wated

MK-80 1 AND

661

HYPERTHERMIA

FIG. I. Representative photomicrographs of the hippocampal CA I subfield 7 days after ischemia in gerbils. (A, D): sham operation. The CA I pyramidal cells are well preserved. (B, E): vehicle-treated ischemia group with maintaining body temperature. Note the marked damage to the CA 1 pyramidal cells. (C, F): MK-80 l-pretreatment group with maintaining body temperature. Most of the CAI neurons are not preserved. Scale bars: A, B, C: 250 pm; D, E, F: 25 pm.

ischemia decreased significantly 24-29 and 12%, respectively. In the CA3 subfield and the molecular layer of the dentate gyrus, [3H]CHA binding sites showed no significant changes. [3H]QNB binding in the stratum oriens and stratum radiatum of the CA 1 subfield was significantly reduced by 2 1 and 17%, respectively. In the stratum lacunosum-moleculare, the CA3 subfield and the

molecular layer of the dentate gyrus, [3H]QNB binding sites showed no significant changes. MK-801-treated group. All ligand binding decreased significantly in the CA1 subfield 7 days after ischemia. In particular, [3H]IP3 binding was completely inhibited in the CA1 subfield. In the CA3 subfield, [3H]IP3 and [3H]PN binding decreased 7

662

MK-801 AND HYPERTHERMIA

663

TABLE 2

of glutamate during and after ischemia (6,11,3 1). Clinically, hypothermia has been used for many years as prophylaxis against ischemic brain damage (8). Body temperature 20 min after ischemia was 1S”C higher than that prior to anesthesia and it was recovered 4 h after ischemia. Clifton et al. (7) have reported that without constant measurement of rectal temperature and specially warmed environments, erroneous conclusions regarding pharmacologic protection could easily be reached. Body temperature does not accurately reflect brain temperature (4), because brain t~m~mture can be greatly affected by factors such as the proximity of heating lamps, heating pads and openings in the skull for intracerebral drug injections. On the other hand, Clifton et al. (7) have reported that body temperature during ischemia had a dramatic impact on ischemia-induced cell death in gerbils, and that even a 2°C decrease in body temperature provided 100% protection from cerebral ischemia. Furthermore, they have observed that disparities between brain and body temperatures were minimized and were consistent because the gerbils were placed in a supine position with the gerbils bodies and calvariae resting on a heating table (7). Thus, the relation between brain and body temperatures is not clear at present. Differences in animal models used and experimental conditions may be a possible cause of the discrepancy. Although we did not measure brain temperature, we placed the gerbils in a supine position with the gerbils bodies and calvariae resting on a heating pad the same as a method of Clifton et al. (7). However, it is important to monitor and maintain constantly both brain and body temperatures during and after ischemia in ischemic models. NMDA receptor/channel antagonists have been reported to protect against the effects of hypoxic neuronal damage in vitro and against central nervous system damage in in vivo models of focal cerebral ischemia (14,15,25,34,36,37). However, it is less clear whether NMDA receptor/channel antagonists protect the brain against neuronal damage after transient global cerebral ischemia. initial reports of neuroprotection against global ischemia (2,13,16-l 8.28,39,41) have not been corroborated by sub-

EFFECT OF MK-801 ON NEURONAL DAMAGE OF CA1 PYRAMIDAL CELLS IN THE GERBIL HIPPOCAMPUS 7 DAYS AFTER REPERFUSION

Neuronal Density Treatment

n

(Number/mm, Mean 2 SE)

Sham (no ischemia) fschemia + vehicle fschemia + body temperature controlled and a) MK-801 pretreatment b) MK-801 posttreatment &hernia + body temperature uncontrolled and a) MK-801 pretreatment

8 13

252 -c ?.7f 23 f 4.5

13 6

3s +- 7.2 12 t 2.8

6

118+ 29*

* p