A;em'¢~.~cience I.ellCrs. 139 ( 19921 45 46 45

c 1992 Elsevier Scientific Publishcrs h-eland Ltd. All rights rcserved 0304-3940/92/$ 05.00

NSI. 08589

Significance of nerve growth factor content levels after transient forebrain ischemia in gerbils

Yoshihide H a s h i m o t o " , Hi rosh i K a w a t s u r a ~', Yoshio Shiga ~+ Shoei F u r u k a w a b and T a k u Shigeno ~

'ln~tilule o / Bio/oc,,ica/ Science, Mil.~ui Pha#'lHaC('ltlicolx lm'., Chiha /,l~q~an), ~',Iloh'cular Biolo,<r, Gili~ Pharmaceutical [,)H~,er.~'itv, Gilu ~ Japan) and

~l)el~arl#m'nl 0t :\k'm'o.~ur~(,ry, Sat*area :Ah'dical ('enwr. Sailama Medical Scho~J/, ,S'ai/am~/ I . lopa#7 I

( Rcccixcd 22 Novcmbcr 1991 : Revised version receivcd 10 February 1992; Accepted 12 February 1992

Ker n'ord~'. Dcluycd neuronal death; Cerebral ischcmiu: Nerve grm~th factor: Hippocampus: Enzyme immunoassay: Astroglia

hwolvemcnt of nerve growth factor (NGF) m thc pathogencsis of delayed neuronal death I DND) of CA I neurons m the hippocampus has bccn

suggested. Wc measured regional changes in the content of tissue N G F of the hippocampus in the Mongolian gerbil after 5 mm forebrain ischcmia.

The N G F content x~as found 'to dccreasc significantly in the CA3 and dentate regions by 32% two days after ischemia. By contrast in the CA I region.

the level of N G F bccamc signiticantl.~ clc\ atcd b~ 50% two weeks after ischcmia or later. The early reduction of N G F content in the afferent area

projecting to the ( 'AI sector might bc primarily linked to the pathogencsis of DND. whereas the delaycd increase ~,,ithin the CA I sector might bc a secondary local t-csl'Jonsc I11ahll} of reactive astroglia.

In understanding the pathogenesis of delayed neu- ronal death (DN D) after transient cerebral ischemia, ~e have been Ii)cusing on the role of nerve growth factor (NGF) in the hippocampus [9]. Exogenously adminis- tered N G F was effective in reducing the extent of DND. The content of N G F was significantly decreased two days after ischemia in the whole hippocampus at a time when D N D was fully developed. However, we have not yet fully investigated regional and temporal changes in the amount of tissue N G F after ischemia. We report here that therc is a reduction in N G F content in those afferent areas projecting to CA1 such as CA3 and dentate gyrus, but not in the CA I sector.

Adult Mongolian gerbils were subjected to transient forebram ischemia by occluding both carotid arteries for 5 rain under anesthesia with 2 3% halothane/O,. Rectal temperature was kept at 38°C as close as possible during and up to 1 h after ischemia. At various times after re- circulation, the brain was removed and stored l'rozen until the time of N G F determination. The hippocampus was divided into two parts, i.e., the dorsal part consisting mainly of CA1 sector and the ventral part of CA3 sector and the dentate gyrus. For the measurement of N G F content, we utilized the two-site enzyme immunoassay (EIA) system developed by Furukawa et al. [6] with a

C,,rre.V~omlenc~'. Y. Hashimoto. Institute of Biological Science. Mitsui Pharmaceuticals Inc.. Mobara. ( 'hiba 2~,~7, Japan.

slight modification [8]. Our EIA system was proven to be valid to determine N G F content in gerbil tissues, because serially diluted tissue extract of gerbil brain gave a dose- responsiveness parallel to that of rat brain extract (data not shown).

The distribution of N G F in discrete areas of the brain wits very similar to that in the rat (data not shown). The content was highest in the olfactory bulb followed by the hippocampus, the septal nucleus and the cerebral neo- cortex. In the dorsal hippocampus, the amount of N G F did not change tip to one week after ischemia but in- creased significantly by 50% 18 days after ischemia (Fig. 1). In the ventral hippocampus, there was a signifcant reduction by 32% two days after ischemia that returned to the control level by one week.

Since the first report by lto et al. [7], the idea of matu- ration death or D N D has provided a means by which cell death in the central nervous system (CNS) might be understood. Our hypothesis is that D N D is a reflection of a fiiilure of recovery processes following excitatory damage to this particular neuronal circuitry [9]. Addi- tional evidence is provided by the effect of fimbria-t\~rnix section in reducing D N D where the local N G F content in the hippocampus might have been elevated after deaf- ferentation [3]. Furthermore, the effect of protein synthe- sis inhibitor in ameliorating D N D suggests some suicide program operating due to a lack of N G F [10].

Tissue N G F content wits significantly reduced two

46

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e - v

0 2 7

Days af ter i s c h e m i a

18

14.

2:

0 2 7 18

Days af ter i s c h e m i a

Fig. 1. Changes in NGF content of (A) dorsal and (B) ventral hip- pocampus in gerbil after 5 min forebrain ischemia. The hippocampus was divided into two parts, i.e., the dorsal part mainly of CA1 sector and the ventral part mainly of CA3 sector and the dentate gyrus. Data are the means _+ S.E.M. from 4 animals, each done in quadruplicate.

*P < 0.01, versus control (Student's t-test).

days af ter i schemia in the ventra l h i p p o c a m p u s where

neurona l dea th was no t seen. This is ra ther an unex-

pected f inding and needs exp lana t ion in unde r s t and ing

the effect o f exogenous ly admin i s t e red N G F in reduc ing

dea th o f CA1 neurons . One poss ib i l i ty is that dea th o f

CA1 neurons is affected by their afferents such as CA3

and den ta te gyrus where N G F level was reduced. The

exogenous N G F might have acted via influence upon

such afferent neurons to CA1. The o ther poss ib i l i ty is

tha t react ive as t rogl ia rep laced the level o f N G F in the

CA1 region, whereas the neurona l level might have been

reduced. A l t h o u g h the g loba l level was unchanged , the

CA1 neurons were in cond i t ion o f N G F deficiency where

cer ta in suicide p r o g r a m began to opera te . I t is well

known tha t N G F is no rma l ly synthesized in the pyra -

midal cells and denta te granule cells in the h i p p o c a m p u s

[1 1]. However , as t rogl ia also have a potency to synthe-

size N G F [5]. Dur ing deve lopmen t o f D N D , it has been

shown that reactive as t rogl ia a p p e a r d o m i n a n t l y in CA 1

as G F A P posi t ive [4] or Golg i s ta ining posi t ive [9]. By

des t ruc t ion o f h i p p o c a m p a l neurons with quinol in ic

acid, Bakhi t et al. [2] found that N G F was synthesized by

G F A P posi t ive reactive as t rogl ia .

Recently, Za f r a et al. [1 1] r epor ted that N G F m R N A

was s t rongly induced in h i p p o c a m p a l neurons by non-

N M D A g lu t ama te recep tor agonist . However , we found

tha t synthesis o f N G F is reduced even though such

t ranscr ip t ion was enhanced ( H a s h i m o t o el al., unpub-

lished results). There would seem to be an uncoupl ing

between the initial neuroexc i ta t ion poss ib ly by g lu tama te

af ter ischemia and subsequent ne u ro t roph i c act ivi ty in

and a r o u n d the h ippocampus .

1 Ayer-Lelievre, C., Olson, L., Ebendal, T., Seiger, A. and Persson, H., Expression of the beta-nerve growth factor gene in hippocampal neurons, Science, 240 (1988) 1339-1341,

2 Bakhit, C., Armanini, M., Bennett, G.L., Wong, W.T.L., Hansen, S.E. and Taylor, R., Increase in glia-derived nerve growth factor following destruction of hippocampat neurons, Brain Res., 560 (1991) 76-83.

3 Bucham, A. and Pulsinelli, W., Septo-hippocampal deafferentation protects CAI neurons against ischemic injury, Brain Res., 512 (1990) 7 14.

4 DeLeo, J., Toth, L., Schubert, E, Rudolphi, K. and Kreutzberg, G.W., lschemia-induced neuronal cell death, calcium accumulation, and glial response in the hippocampus of the mongolian gerbil and protection by propentofylline (HWA 285), J. Cereb. Blood Flow Metab., 7 (1987) 745-751,

5 Furukawa, S., Furukawa, Y., Satoyoshi, E. and Hayashi, K., Syn- thesis and secretion of nerve growth factor by mouse astrogtial cells in culture, Biochem. Biophys. Res. Commun., 136 (1986) 57-63,

6 Furukawa, S., Kamo, I., Furukawa, Y., Akazawa, S., Satoyoshi, E., ltoh, K, and Hayashi, K., A highly sensitive enzyme immunoas- say for mouse fl nerve growth factor, J. Neurochem., 40 (1983) 734 744.

7 lto, U., Spatz, M., Walker, J.T.J. and Klatzo, I,, Experimental ce- rebral ischemia in Mongolian gerbils. I. Light microscopic observa- tions, Acta Neuropathol., 32 (1975) 209-223.

8 Matsui, K., Furukawa, S., Shibasaki, H. and Kikuchi, T., Reduc- tion of nerve growth factor level in the brain of genetically ataxic mice (weaver, reeler), FEBS Lett., 276 (t990) 78-80.

9 Shigeno, T., Mima, T., Takakura, K., Graham, D.I., Kato, G., Hashimoto. Y. and Furukawa, S., Amelioration of delayed neu- ronal death in the hippocampus by nerve growth factor, J. Neu- rosci., 11 (1991) 2914-2919.

10 Shigeno, T., Yamasaki, Y., Kato, G., Kusaka, K., Mima, T., Ta- kakura, K., Graham, D.I. and Furukawa, S., Reduction of delayed neuronal death by inhibition of protein synthesis, Neurosci. Lett., 120(1990) 117-119.

I 1 Zafra, F., Hengerer, B., Leibrock. J., Thoenen, H. and Lindholm, D., Activity dependent regulation of BDNF and NGF mRNAs in the rat hippocampus is mediated by non-NMDA glutamate recep- tors, EMBO J.. 9 (1990) 3545 3550.