Neuroscience Research, 15 (1992) 305-309 © 1992 Elsevier Science Publishers Ireland, Ltd. All rights reserved 0168-0102/92/$05.00

305

NEURES 00595

Rapid Communication

Ischemia-induced changes in

PIP 2

levels of gerbil hippocampus

Akihiro Ishida, Kuniko Shimazaki and Nobufumi Kawai Department of Physiology, Jichi Medical School, Minamikawachi-machi, Tochigi-ken 329-04, Japan (Received 19 September 1992; accepted 28 September 1992)

Key words: Brain ischemia; Delayed neuronal death; Phosphatidylinositol 4,5-bisphosphate (PIPE): PI turnover; Hippocampus; Immunohistochemistry; CA1 pyramidal neuron

Summary We carried out an immunohistochemical study to detect changes in phosphatidylinositol 4,5-bisphosphate (PIP 2) in gerbil hippocampus at various times after transient ischemia, using an anti-PIP 2 antibody. About 24 h after transient ischemia for 5 min, an increase in the immunoreactivity was observed which was restricted to the area of CA1 pyramidal neurons. On the other hand, after less severe ischemia lasting 2 min, which did not lead to neuronal death, a decrease in PIP 2 immunoreactivity was observed at about 48 h. The results indicate that levels of PIP E following ischemia reflect dynamic changes in phosphatidylinositol (PI) turnover which may be related to neuronal degeneration.

After transient forebrain ischemia produced by clamping the carotid arteries of gerbil, selective neuronal degeneration takes place in the brain with a delay of 2-4 days. This "delayed neuronal death" (Kirino, 1982) is found in CA1 sectors of hippocampus, in dorsolateral striatum and in the cerebellar cortex (Wieloch, 1985) where glutamate receptors are abundant. Ample evidence has shown that transient ischemia induces an initial massive release of glutamate, and that the succeeding abnormal Ca 2+ accumulation inside the cell is the main triggering factor for neuronal death (Pulsinelli et al., 1982; Dienel, 1984; Desphande et al., 1987; Choi, 1988; Benveniste et al., 1988; Siej6 and Bengtsson, 1989; Schmidt-Kastner and Freund, 1991). Electrophysiological studies of hippocampal slices of gerbils at 2 - 4 days after ischemia showed an abnormal membrane properties in the CA1 pyramidal neurons (Kawai et al., 1990). Both intracellular (Kirino

Correspondence to: Dr. A. Ishida, Department of Physiology. Jichi Medical School, Minamikawachi-machi, Tochigi-kcn. 329-04, Japan. Tel.: 0285-44-2111, ext. 3124; Fax: 11285-44-8147.

et al., 1992) and whole cell recordings (Tsubokawa et al., 1992) from ischemic pyramidal neurons revealed a vulnerability of ischemic CA1 neurons to input stimulation, unusual oscillations of the membrane potential, which were enhanced by injection of D-myoinositol 1,3,5-triphosphate lIP 3) and blocked by Ca 2+ chelators, E G T A or BAPTA. These findings suggest that abnormal Ca 2+ homeostasis in ischemic neurons could be caused by disturbance of the intracellular CaE+-regulat ing system, in particular, the phosphatidylinosito[ (PI) turnover system. In order to examine the possible participation of PI turnover in the events preceding neuronal death, we carried out an immunohistochemical study using an antibody against phosphatidylinositol 4,5-bisphosphate (PIP 2) on the hippocampal neurons following transient forebrain ischemia. In total 32 adult Mongolian gerbils (8-12 weeks old, 60-90 g) were used. The gerbils were anesthetized with 3% halothane in a mixture of 30% oxygen and 70% nitrous oxide. Rectal temperature was maintained at 37-38°C throughout the operation. A midline incision was made in the ventral neck and the carotid arteries were exposed. Bilateral carotid arteries were clamped

306 with a n e u r y s m clips and the c e r e b r a l blood flow o f the f o r e b r a i n was c o m p l e t e l y d i s r u p t e d for 5 or 2 min. T h e clips were then t a k e n off so that the blood flow was r e s t o r e d . T h e gerbils were allowed to recover a n d were p e r f u s e d u n d e r p e n t o b a r b i t a l a n e s t h e s i a with 50 ml 0.9% saline followed by 50 ml 2% p a r a f o r m a l d e h y d e dissolved in 0.1 M p h o s p h a t e - b u f f e r e d saline (PBS) at the following intervals: 5 min, 12, 18 and 24 h, 2, 3 and 7 days. T h e brain was r e m o v e d and p r e s e r v e d in fixative for a b o u t 12 h a n d in 20% sucrose dissolved in 0.1 M PBS for 1 day, and t h e r e a f t e r in 30% sucrose for 2 days. T h e b r a i n s were s e c t i o n e d sagittally on a freezing m i c r o t o m e at 5 0 / x m , and some sections were m o u n t e d a n d s t a i n e d with cresyl violet to see w h e t h e r the pyram i d a l ceils in the CA1 subfield were alive. Sections w e r e s e l e c t e d at 5(t0-/zm intervals for i m m u n o h i s t o c h e m i c a l staining of PIP,. T h e y were i n e u b a t e d in 1.5% n o r m a l horse s e r u m dissolved in PBS for 1 h to block the non-specific a n t i g e n a n d also i n c u b a t e d with a n t i - P I P 2 m o u s e l g G fraction d i l u t e d 1:2000 in 0.1 M PBS o v e r n i g h t at 4°C. T h e next day the sections were r i n s e d 6 times with PBS and i n c u b a t e d with horse b i o t i n y l a t c d a n t i - m o u s e I g G a n t i b o d y solution for 1 h. A f t e r 3 rinses they were i n c u b a t e d in 0 . 3 ~ H ~ O , and

30% m e t h a n o l in PBS for 1 h to q u e n c h the e n d o g e nous p e r o x i d a s e activity, r i n s e d again and then incub a t e d with A B C r e a g e n t (avidin D H , b i o t i n y l a t e d enzyme a n d 0.5 M NaC1; V E C T A S T A I N A B C kit) for 1 h. Finally, the sections w e r e d e v e l o p e d with dia m i n o b e n z i d i n e t e t r a h y d r o c h l o r i d e in p e r o x i d a s e subs t r a t e solution and m o u n t e d on c h r o m e - g e l a t i n slide glasses. T o m e a s u r e the intensity of P I P z staining with antiP I P 2 m o n o c l o n a l a n t i b o d y ( M a t u o k a et al., 1988) of t h e C A I subfield of h i p p o c a m p u s , i m a g e analysis was carried out with SP-500 (high s p e e d analysis system; Olympus). T h e stratum p y r a m i d a l e of the CA1 subfield, and the square region of c o r t e x l o c a t e d above the h i p p o c a m p u s c o n t a i n i n g almost all layers of c o r t e x w e r e selected, and the n u m b e r s o f pixels, which was p r o p o r t i o n a l to the density of stain was c o u n t e d . T h e density was digitized with 256 levels and the total n u m b e r s of pixcls were c o u n t e d for each level to calculate the density p e r definite area. In o r d e r to exclude the non-specific stain the d e n s i t y o f the s t r a t u m p y r a re)dale of the CA1 subfield was d i v i d e d by that in t h e cortex which showed essentially no c h a n g e with the ischemic insult. In the analysis of the t e m p o r a l c h a n g e of the P1P 2 i m m u n o s t a i n i n g in CA1 p y r a m i d a l n e u r o n s

Fig. 1. Changes in ~mmunostaining against PIP 2 antibody of hippocampus following 5 rain ischcmia of gerbils. A: control gerbil wilhout ischemic insult. B-D: sections of hippocampus with reperfusion intervals of 18 h (B), 24 h (C) and 7 days (D), respectively. The upper photographs show the whole hippocampus and insets show part of the CA1 subfield of the same samples. At 7 days (D}, most of CAI pyramidal neurons were lost and glia cells were intensely stained. Bar - 1000 ,am; inset 200 ,um.

307 sections from several gerbils treated with ischemic insult for 5 or 2 rain and perfused at various recirculation intervals were stained immunohistochemically at the same time and under the same conditions. One central section of each brain was selected and measured. Gerbils in the normal control were not treated with ischemic insult. Fig. 1 shows changes in immunoreactivity of antiPIP e antibody (PIP2-Ab) following 5 min ischemia. Immediately after ischemia, PIP e immunoreactivity is essentially absent overall in the hippocampal area. However, in the preparations made 18 h following ischemia, an intense staining was observed in the CA1 subfield (Fig. 1B). The increased PIP 2 staining was also seen in the preparation at 24 h after ischemia (Fig. 1C). Thereafter, immunoreactivity in the CA1 area decreased and at 7 days following ischemia no PIP 2 reactivity was seen in the CA1 pyramidal cell layer (Fig. 1D). The gradual disappearance of immunostaining mirrors the time course of selective loss of CA1 pyramidal neurons following ischemia (Kirino, 1982;

Kirino et al., 1992). In contrast to the CA1 field, immunoreactivity in CA3 pyramidal neurons appeared to be unchanged throughout the series of the experiments. In preparations where the immunostaining was most conspicuous 18-24 h after ischemia, CA1 pyramidal neurons with their apical dendrites were extensively stained (Fig. 2A,C). It has been reported that pretreatment with a brief (2 rain) ischemia followed by a second ischemia for 5 rain did not induce severe neuronal death in the CA1 subfield (Kirino et al., 1991). We examined immunoreactivity of the hippocampus at various times following occlusion of the carotid for 2 rain. In contrast to 5 min ischemia, CA1 pyramidal neurons did not show increased immunoreactivity 1-2 days after a 2-min ischemic insult. In fact, a decrease in immunoreactivity in the CA1 subfield as compared to the control was seen in preparations 20-48 h after 2 rain ischemia (Fig. 2B,D). We carried out a quantitative comparison of immunoreactivity in the preparations treated with ischemia for 5 min and 2 min. As shown in Fig. 3, thc

Fig. 2. lmmunostaining of PIPe-reactive CAI neurons at a 18 h recirculation interval following 5 rain ischemia (A, C) and at 24 h after 2 rain ischemia (B, D). C, D are enlarged photograph of the CAI subfield in A and B, respectively. Bar = 500/xm.

308

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ischemia ischemia

•A ~ .6 a" .4. ~0

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Fig. 3. Comparison of the changes in PIP2-staining level at wlrious intervals following 5 rain (()) and 2 min (o) ischemia. Ordinate: relative density of PIP e immunoreactivity with the control value normalized to 1.(I (see text). Abscissa: time after ischemia applied at time 0. Each point indicates average of 3 experiments.

profile of the change in relative density of PIP e immunostaining greatly differed according to the duration of ischemia. In gerbils after 5 rain ischemia, immunoreactivity was higher than the control except at very short time (10 h) after ischemia, whereas in gerbils with 2 min ischemia C A I neurons exhibit decreased level of staining. The present results demonstrate that PIP 2 immunoreactivity in CA1 pyramidal neurons following ischemia is greatly affected by the duration of ischemic insult. Ischemia for 5 min, which caused complete death in CA1 pyramidal neurons, led to intense staining against PIP2-Ab in the CA1 subfield at 20-30 h after ischemia. By contrast, following 2 min ischemia which is sublethal to CA1 neurons, the PIP e immunoreactivity showed no increase, and even a decrease. Intense PIP 2 immunoreactivity produced by 5 min ischemia may reflect excessive formation of PIP e as a result of elevated Pl turnover, triggered by Ca 2+dependent protein kinases as a result of elevated Ca 2+ influx. Using the four vessel occlusion method in rats, Onodera et al. (1989) reported a transient increase in protein kinase C activity in CA1 neurons after ischemia. We have recently shown that intracellular application of IP 3 or guanosine 5'-O-3-trithiophosphate ( G T P y S ) in CA1 pyramidal neurons 24-48 h after ischemia accelerates neuronal death and conversely, injection of Ca 2+ chelators, E G T A or BAPTA prevented the cell death (Kirino et al., 1992; Tsubokawa et al., 1992). These data could be explained if following ischcmia, for a sufficiently long time (more than 5 min), the elevated intracellular Ca 2+ exceeds the critical level for triggering the activation of Ca2+-depen -

dent kinases, which promote PI turnover, and which may induce abnormal Ca 2+ homeostasis (Choi, 1988; Siej6 and Bengtsson, 1989; Schmidt-Kastner and Freund, 1991), so resulted in delayed neuronal death. The reason for the temporal decrease in PIP 2 immunoreactivity after 2 rain ischemia is currently unknown. Pretreatment with brief ischemia (2 min) renders hippocampal neurons tolerant to subsequent ischemia for 5 min (Kirino et al., 1991). In this connection, it is of interest that immunostaining against a heat shock protein (hsp 70) (Nowak, 1985) greatly increases in CA1 pyramidal cell layers after ischemia for 2 min (Kirino et al., 1991), since the role for hsp 70 in induced tolerance has been reported (Riabowol et al., 1988). This could be explained if neurons which are stressed by a sublethal stimulus synthesize proteins such as hsp 71) and become more tolerant to subsequent ischemia. Acknowledgements The authors thank Dr. T. Takenawa for providing anti-PIP 2 antibody, Drs. T. Kirino and H.P.C. Robinson for helpful discussions. This work was supported by a grant-in aid for Scientific Research from the Ministry of Education, Science and Culture of Japan (Nos. 63060006, 04267102).

References Benveniste, tt., Jorgensen, M.B., Diemer, N.ti. and Hansen, A.J. (1988) Calcium accumulation by glutamate receptor activation is involved in hippocampal cell damage after ischemia. Acta Neurol. Scand., 78: 529-536. Choi, D.W. (1988) Calcium-mediated neurotoxicity: relationship to specific channel types and role in ischemic damage. Trends Neurosci., 11: 465-469. Desphandc, ,I.K., Siej6, B.K. and Wieloch, T. (1987) Calcium accumulation and neuronal damage in the rat hippocampus following cerebral ischemia. J. Cercb. Blood Flow Mctab., 7:89 95. Dienel, G.A. (1984) Regional accumulation of calcimn in post ischemic rat brain. J. Neurochem., 43:913 025. Kawai, N., Kirino, T., Robinson, H.P.C., Miwa, A. and Tamura, A. (1990) Synaptic t r a n s m i s s i o ~ i n hippocampal pyramidal neurons of gerbil after transient ischemia. Neurosci. Res., Suppl. I1: S19. Kirino, T. (1982) Delayed neuronal death in the gerbil hippocampus following ischemia. Brain R e s , 239: 57-69. Kirino, T., Robinson, H.P.C., Miwa, A., Tamura, A. and Kawai, N. (1992) Disturbance of membrane function preceding ischemic delayed neuronal death in the gerbil hippocampus. J. Cereb. Blood Flow Metah., 12:408 417, Kirino, T., Tsujita, Y. and Tamura, A. (1991) Induced tolerance to ischemia in gerbil hippocampal neurons. J. Cereb. Blood Flow Metab.. 11:299 307. Matuoka, K., Fukami, K.. Nakanishi, O., Kawai, S. and Takenawa, T. (1988) Mitogenesis in response to P D G F and bombesin abolished by micminjection of antibody to PIP> Science, 239: 640-643.

309 Nowak, T.S., Jr. (1985) Synthesis of a stress protein following transient ischemia in the gerbil. J. Neurochem., 45: 1635-1641. Onodera, H., Araki, T. and Kogure, K. (1989) Protein Kinase C activity in the rat hippocampus after forebrain ischemia: autoradiographic analysis by [3kI]phorbol 12,13-dibutyrate. Brain Res., 481: 1-7. Pulsinelli, W.A., Brierley, J.B. and Plum, F. (1982) Temporal profile of neuronal damage in a model of transient forebrain ischemia. Ann. Neurol., 11: 491-498. Riabowol, K.T., Mizzen, L.A. and Welch, W.J. (1988) Heat shock is lethal to fibroblasts microinjected with antibodies against hsp 70. Science, 242: 433-436.

Schmidt-Kastner, R. and Freund, T.F. (1991) Selective vulnerability of the hippocampus in brain ischemia. Neuroscience, 40: 599-636. Siej6, B.K. and Bengtsson, F. (1989) Calcium fluxes, calcium antagonists, and calcium-related pathology in brain ischemia, hypoglycemia, and spreading depression. J. Cereb. Blood Flow Metab., 9: 127-140. Tsubokawa, H., Oguro, K., Robinson, H.P.C., Masuzawa, T., Kirino, T. and Kawai, N. (1992) Abnormal Ca 2+ homeostasis before cell death revealed by whole cell recording of ischemic CA1 hippocampal neurons. Neuroscience, 49: 807-817. Wieloch, T. (1985) Neurochemical correlates to selective neuronal vulnerability, Prog. Brain Res., 63: 69-85.