Gamma-vinyl GABA prevents hippocampal and substantia nigra reticulata damage in repetitive transient forebrain ischemia

Brab~ Research, 590 (1992) 13-17 © 1992 Elsevier Science Publishers B.V. All rights reserved 0006-8993/92/$05.{10

13

BRES 18014

Gamma-vinyl GABA prevents hippocampal and substantia nigra reticulata damage in repetitive transient forebrain ischemia A s h f a q Shuaib, Sadiq Ijaz, Sara H a s a n and Jay Kalra Cerebrorascular Research Laboratory and The Saskatchewan Stroke Research Centre, College of Medichre, Unicersityof Saskatchewan, Saskatoon, Sk. (Canada) (Accepted 31 March 1992)

Key words: Ischemia; Repetitive ischemia; y-Vinyl GABA; Alza pump

GABAergic inhibitory mechanisms may offer protection to neurons after globat ischemia. We tested the effects of y-vinyl GABA, a GABA-transaminase inhibitor, via continuous infusion in the third ventricle (Alza pumps) in a gerbil model of repetitive f~,.cbrain ischemia. We used two episodes of 3 min duration with a 'reperfusion' interval of i h between the insults. Histological analysis was done with silver staining 5 days after the insult. Our results show that there is significant protection of the hippocampus CAI region and substantia nigra reticulat;~ in treated animals compared to controls. An increase in GABA levels, decrease in glutamate, or mild hypothermia, may be potential mechanisms for this protection, GABAergic agents may prove useful agents in repetitive ischemia.

INTRODUCTION There is considerable evidence that transient forebrain ischemia results in predictable damage in 'vulnerable' regions of the brain including the striatum, hippocampus, thalamus and the substantia nigra reticulata (SNr) "a'tt'2H-3c}''~s'4t'42''~t.Damage in the hippocampus and SNr is often delayed 17'2s-'~°'43,4s. This delay in onset of damage after the insult offers hope for therapeutic intervention. Attenuation of the delayed damage in the hippocampus has been shown with several compounds including GABAergic agents 3't°'18'22'34'5°'52. The latter may exert their effects by inhibiting postischemic 'excited neurons'. There is evidence that neuronal damage after repetitive ischemia may be more severe than a single similar duration insult 1'26'27'a2'35. The onset of damage in the CA1 region of the hippocampus and substantia nigra reticulata is delayed and appears several hours to days after reperfusion 45. The time course of hippocampal damage is similar to single insult ischemia. In addition, the damage is more diffuse with frequent involvement

of the SNr and the medial geniculate nucleus 32'45. We have recently shown that the damage in the hippocampus and the SNr can be attenuated by mild hypothermia in between the ischemic episodes (Can. J. Neurosci., in press). In this communication we report the effects of a potent GABA-transaminase inhibitor, yvinyl GABA (GVG) when used continuously for 5 days after repetitive ischemic insults. MATERIALS AND METHODS For all experiments we used male adult gerbils weighing 60-80 g. Animals were housed under controlled environment with 12 h lightdark cycle and free access to food and water. After acclimatization to the vivarium for 5-11) days, the animals were prepared for intraventricular infusion of GVG. After intramuscular anesthesia with pento barbital, the gerbils were placed in a small animal stereotactic frame (Kopf Instruments, CA). A 21-gauge hypodermic needle was passed into the third ventricle and attached via a short polyethylene PE-60 tube to an AIza osmotic pump with GVG or a placebo. The pumps were subsequently buried into the subcutaneous tissue. A fixed amount of GVG (7 ng/I) was infused into the third ventricle for 5 days, until time for sacrifice. After insertion of the pumps and recovery from anesthesia, the animals were subjected to 3 episodes of transient forebrain ischemia by methods previously described4"~. Briefly, animals were anesthetized with 3% halothane and a mixture

Correspondence: A. Shuaib, Department of Medicine (Neurology), Royal University Hospital, Saskatoon, Sk., Canada S7N 0X0. Fax: (!) (306) 966-8021.

14 substantia nigra reticulata. With silver staining, dead or severely damaged neurons appear black and are very easy to identify II. if no 'black" neuron was evident on microscopy, we used a score = 0, if less than 25% neurons were damaged = !, 25-75% damaged = 2, more than 75% damaged = 3 and complete infarction = 4. There is a close relationship between cell counts and the score mentioned below (unpublished observations). All histological evaluation was done in a double blinded manner in both hemispheres and at several levels. For statistical analysis we used the KruskaI-Wallis test and individual comparisons were made using the Mann-Whitney U-test with Bonferoni correction when appropriate.

of nitrous oxide (711%) and oxygen (30%). Rectal and scalp temperatures were measured in all anim,'l,ls during the ischemic episodes and hetween the episodes. During the ischemic msults the temperatures were mainlained as close to 37°C as possible with a heat lamp. in between the insults, the anesthesia was discontinued and the temperature would remain between 36.5 and 38°C without any intervention. Kato et al. have reported that the brain temperature frequently increases in repetitive ischemia bat this increase in temperature is not associated with additional brain damage TM. After induction of anesthesia, a midline neck incision was made and the carotid arteries carefully dissected out from the surroundiug tissue This was followed by application of aneurysm clips to the carotid arteries and the occlusion was visually identified. Halothane anesthesia was decreased to !% and the nitrous oxide maintained unchanged. After 2 rain the aneurysm clips were removed and the skin lesion closed with a single suture and the animals allowed to recover. Between the ischemic insult all anesthesia was discontinued. One hour and 2 h later the procedure was carried out for another 2 rain each time. The carotid arteries were carefully visualized after each occlusion and the return of blood flow would always be evident as soon as the clips were removed. The animals were then returned to the vivarium and allowed to recover fi~r 5 days. The animals were then anesthetized with an overdose of pentobarbital and perfused initially with saline (1511 ml) and hlter with a phosphate buffer. One h later, the brains were removed and fixed in a 30% sucrose buffer until processing. The brains were then cut in 40-/.tm sections and stained with a nlodification of the Gallya's silver impregnation method 153¢'. Briefly, the sections were washed with water, pretreated with alkaline ammonium nitrate, impregnated with 0.32% silver nitrate in alkaline/ ammonium nitrate, treated with ethanolic sodium carbonate/ ammonium nitrate solution and finally developed in Nauta reducer. Representative sections were cut through the cortex, stria[urn, hippocampus, thalamus, medial geniculate nucleus and the substantia nigra rcticulata. Tissue phmes were made according to the rat atlas of Paxinos and Watstm 'm and gerbil atlas of Loskota et al. 'u. Neuronal damage was assessed by methods previously described '~, We used a scoring system simihlr to that of Pulsinalli'~t, Grotto at and Kirano -'r', We looked at the extent of damage in various regions of the brain. Whereas it is relatively easy to do cell counts of damaged (or intact) neurons in the hippt~canlpus CAI region, such COUlltS are difficult in other regions such as the basal ganglia or the

RESULTS

A total of 18 animals were used for the experiments. There were 4 deaths in the stage of pump insertion, likely related to problems with anesthesia induction. No animals died during ischemia or during the 5 days after surgery and all were available for analysis. Seven gerbils were given a placebo and 7 animals had an infusion of GVG. Rectal and scalp temperatures showed no difference in the 2 groups during the 3 h of the experiments. The temperature electrodes were subsequently removed and monitoring discontinued for the subsequent 5 days until the time of sacrifice. Results of the rectal and scalp temperatures are shown in Table I.

Behal,ior changes Animals subjected to ischemia showed a humped posture on awakening typical of severe insult, In most animals this returned to normal within half an hour after the initial 2 insults and an hour after the third

TAItLE I Scalp mul core temperature records in GVG.treated animab aml com rob This table shows the temperature measurements in the controls anti GVG-treated animals for the 3 h of the experiment. No significant changes were evident in the two groups. The numbers ! - 7 reflect the temperatures prior to the first occlusion (!), during first occlusion (2), between the first and second occlusion (3-5), during second occlusion (6) and after the second occlusion (7). i

2

3

5

4 .

GVG A AVG STD B AVG STD

.

.

.

6

7

.

37.1 [0.26]

37.1 [0.151

37.5 [0.(15]

37,5 [0,28]

37.6 t0,251

37,2 [0,1 I I

37.2 [0,261

36,1 [0.151

36.4 t0.351

36.2 [0,251

36.5 [0.36l

36.2 [(1.341

36.4 [0.101

36.3 [0.201

37,3 [0.251

37.11 t0.20l

37.2 lo,2ol

37,6 [0.17]

37,5 [0,151

37,4 [0,051

37,4 [0.101

36.2 [0.26]

36.3 [0.1 I I

36,3 10,05]

36.4 [0.101

36.5 [0.201

36.5 [0.151

36.3 tO.15]

Control (,

AVG STD D AVG STD

A. Average scalp temperature in all GVG-group animals. B. Average core temperature in all GVG-group animals. C. Average scalp temperature in all control animals. D. Average core temperature in all control animals, [ ] Standard deviation.

15

1

1

Control

IFT/Z~ GVG

ao-[

SEM

_/i Cortex

Slrialum

H I p p-C A- 1

SNR

MGN

Fig. 1. This figure shows the damage scores in the control and GVG treated animals. Details of sc~ring methods are available in the methods section. GVG-treated animals showed significant protection (P = 0.003) in the hippocampus and the substantia nigra reticulata (P = 0.03). There was no difference in the extent of damage in the cortex, striatum, thalamus (data not shown) or the medial geniculate nucleus. Hipp-CAl, hippocampus CAI region; SNR, substantia nigra reticulata; MGN, medial geniculate nucleus; GVG, gamma-vinyl GABA.

insult. No seizures were witnessed in any animals. The animals were examined for only 15-30 min twice a day for the 5 days after the surgery. During this period there appeared to be no obvious behavioral differences between the two groups.

Histological et,aluation The details of neuronal damage are shown in Fig. 1. Damage was significantly less pronounced in the hippocampus and substantia nigra reticulata in the treated animals, in control animals there was extensive damage in the cell bodies and neuropil. Whereas the cell body damage was easily evident on Nissl staining of adjacent sections, the neuropil damage was only apparent with silver staining, in several specimens (mostly in the active treatment group) neuronal damage was restricted to the neuropil only, especially in the medial geniculate n,~cleus and the substa~tia nigra reticulata. DISCUSSION The central finding in our experiments is that continuous infusion of GVG via Alza pumps into the third ventricle results in protection of the hippocampal and substantia nigra reticulata neurons. The protection is most pronounced in the hippocampus. GABA is the most abundant inhibitory amino acid in the central nervous system ~3. Glutamate decarboxylase is the major GABA synthesizing enzyme and is located mainly in the neurons. GABA-transaminase, a GABA degrading enzyme, is located in the non-synaptosomal mitochondria ~2. The effects of ischemia on GABA metabolism are variable, depending on the

region of the brain being studied. There is rapid loss of G,z:BAergic neurons in the cerebral cortex and striaturn after transient global ischemia 14'4~'. GABAergic neurons in the hippocampus however appear to be more resistant to the effects of ischemia 37. Although GABAergic neurons in the hippocampus are preserved after ischemia, recent evidence suggests that additional increase in GABAergic activity may reduce the extent of delayed neuronal cell loss in the hippocampus 24. The continuous infusion of GVG after ischemia in our experiments may offer protection in the hippocampus via similar mechanisms. The reason why damage is more severe with repetitive ischemia than a single similar duration insult is not fully understood. Severe reperfusion-related damage (when tht: second insult is produced in the hypoperfusion p e r i o d ) 47'4'~, development of severe brain edema with compression of blood vessels 4'), more prolonged release of glutamate 32 and an increase in intracellular calcium t are some of the possible factors that may contribute to the more severe damage. Except for the more severe damage, this model of ischemia appears to be quite similar to the single insult model. We have recently shown that similar to the in vivo model, the effects of repetitive ischemia appear to be cumulative in cell culture (unpublished observations, submitted for publication). Association pathway damage contributes to the severity of damage after global isehemia ~. Ablation of the excitatory input to the CAI region of the hippocampus will protect neurons in this region 2'25. This effect can be seen with transaction of the perforant pathway to the dentate gyrus or destruction of the CA3 input to the CAl region. Similarly, destruction of the inhibitory input to the CAl region from the locus coeruleus (noradrenergic) "~¢'or to the striatum from the substantia nigra compacta (dopaminergic) ~'~ may also result in an increase in the extent of neuronal damage. We have recently shown that neuronal damage in the substantia nigra reticulata is delayed and may be a result of a loss of the inhibitory GABAergic input from the striatum 45. The protection seen with GVG (which increases GABA levels) in some brain regions may be secondary to increase in inhibitory influences on the ischemic 'excited' neurons. Other mechanisms may also be contributing to the significant protection in the hippocampus and SNr with the use of GVG. It has been shown that GVG not only results in an increase in GABA levels in the CNS but also results in decrease in the production of glutamate, especially in the hippocampus 23'3'~. This decrease is modest but may be of a sufficient magnitude to offer neuronal protection. The use of GVG results in mild

16 hypothermia 4s. This decrease in the temperature (average of 2°C) remains for approximately 5 h. During the 3 h of experiments no difference in temperature was noted in our treated animals. We did not monitor the temperature after the end of experiments. It is possible that with prolonged continuous use of GVG, mild hypothermia may have occurred. Hypothermia has powerful protective effects on the CNS during ischemia4-6.,).31.44 G V G has b e e n successfully used in multiple clinical trials in m a n a g e m e n t of patients with epilepsy -'(). it has minimal side effects, is well tolerated and has proven to be a valuable addition in m a n a g e m e n t of difficult seizure patients. Histological evaluation after long-term use has shown very little neuronal c h a n g e s 7. T h e r e f o r e , unlike m a n y neuroprotective m e d i c a t i o n s with serious side effects, G V G could be used clinically to prevent damage in the h i p p o c a m p u s and SNr in unstable patients with recurrent severe cerebral insults. F u r t h e r experiments detailing its dosage, route of infusion and duration of therapy will however be necessary prior to clinical usage. In s u m m a r y we have shown that G V G when used via continuous infusion into the third ventricle offers significant protection to the h i p p o c a m p a l C A i and SNr neurons. Its m e c h a n i s m s are not well understood but may be related to an increased inhibition of neurons (by increasing local G A B A levels), or possible secondary to a decrease in glutamate concentration and hypothermia. ,.lckmm'h,dgemt,nt.~, This work was in part supported by Establishntent and Stroke Centre grants from the Saskatchewan Health Re~earch Board and a Stroke Centre grant from the Saskatchewan lteart and Stroke Foundation. Doris Kanigan helped in preparation of the manuscript. Rida Mazagri M.D. and Wendy Howlett helped in ct,!lection of data.

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