Mild traumatic brain injury enhances muscarinic receptor-linked inositol phosphate production in rat hippocampus

Brain Research, 594 (1992) 307-310 © 1992 Elsevier Science Publishers B.V. All rights reserved 0006-8993/92/$05.00

307

BRES 25392

Mild traumatic brain injury enhances muscarinic receptor-linked inositol phosphate production in rat hippocampus T h e r e s e M. D e l a h u n t y Division of Neurosurgery, Medical Collegeof Virginia/ VirginiaCommonwealth University, Richmond, VA 23298 (USA) (Accepted 21 July 1992)

Key words: Traumatic brain injury; lschemia; Acetycholine; Muscarinic; Inositol phosphate; Hypersensitivity

Recent evidence suggests that muscarinic receptors play a role in the hippocampal hypersensitivity to imposed ischemia following mild traumatic brain injury (TBI). In rat hippocampal tissue, carbachol-stimulated inositol phosphate production was found to be enhanced by mild TBI, at 1 h post injury. This finding suggests that mild TBI may result in enhanced coupling between muscarinic receptors and phosphoinositide hydrolysis, which may contribute to post-traumatic hippocampal vulnerability to secondary ischemia.

Numerous investigations have confirmed that mild and moderate traumatic brain injury (TBI) results in sublethal neuronal pathology within the hippocampus. These changes are receptor-mediated and related to excitatory neurotransmitters (for review see ref. 5). Additionally, there is considerable evidence that acetylcholine (ACh) and muscarinic receptors are as important as excitatory amino acids in receptor-mediated TBI pathophysiology. Transient increases in CSF and hippocampal ACh levels have been observed immediately after moderate TBP '!1 and both pre- and post-injury muscarinic receptor blockade have been shown to attenuate many behavioral deficits following moderate TBI 9. Since excessive agonist surges usually down-regulate their target receptors, it is not unexpected that hippocampal muscarinic receptor binding is also diminished following moderate TBI e'l°. Mild levels of TBI which do not result in any structural damage can produce an enhanced hil:pocampal sensitivity to imposed secondary ischemia following trauma7. Additional evidence suggests that cholinergic muscarinic receptors may play a role in this enhanced hippocampal vulnerability to imposed cerebral ischemia following mild TBI 6. However, down-regulation of muscarinic receptors suggests a reduced sensitivity of cholinergic pathways and argues against the

participation of muscarinic receptor-mediated mechanisms in the increased ischemic hypersensitivity following TBI. Therefore, the effect of mild TBI on muscarinic-receptor coupled phosphoinositide hydrolysis was investigated. Mild TBI enhanced carbachol-stimulated production of inositol phosphates in hippocampus at 1 h post-injury, a time when the injured brain manifests an increased sensitivity to ischemia. The features of the central fluid percussion model utilized in this study has been extensively documented 7. Male Wistar rats (250-300 g) were surgically prepared for TBI under pentobarbital anaesthesia (54 mg/kg, i.p.). Twenty-four hours after surgery all rats were anesthetized with 4% isoflurane and 70% N20 and 30% 02 for 4 min and then subjected to a sham or mild (0.8-1 atm) epidural fluid pulse impact at the first sign of toe pinch reflex recovery. The loss and recovery of the toe pinch reflex was again documented immediately after injury. Traumatic unconsciousness was inferred by the loss of this reflex and any injured animal that did not show a transient reflex loss was placed in a separate injury group. Any rat with evidence of general or focal seizure activity was excluded from the study. One hour after injury the rats were again anesthetized with 4% isoflurane and 70% N20 and 30% 02 for 4 rain and then immediately decapitated. On each exper-

Correspondence: T.M. Delahunty, Division of Neurosurgery, P.O. Box 693, MCV Station, Richmond, VA 23298, USA.

308 imentai day, one sham and one injured rat were paired and assayed in parallel. The order of decapitation was varied with each experiment. The hippocampi from injured and sham rats were chopped into 35 mm triangular miniprisms by rotating the tissue twice by 60° and chopping with a tissue chopper. The hippocampal miniprisms were washed twice with Krebs-Ringers bicarbonate (KRB) buffer that had been bubbled with 95% 0 2 / 5 % CO 2 for at least 20 min. They were pregassed for 30 rain in 30 ml of KRB with a continuous stream of 95% 0 2 / 5 % CO2. The miniprisms were spun down and resuspended in KRB containing 10 /zCi/ml of [3H]myoinositol and incubated for 1 h at 37°C in a shaking water bath under an atmosphere of 95% 0 2 / 5 % CO 2. The tissue was then washed 3 times in KRB and resuspended in 2 ml of KRB containing 10 Mm LiCI, gassed and incubated for 10 min in the water bath. The miniprisms were spun down, resuspended into KRB containing 10 mM LiCI, and 100 ttl aliquots were added to polypropylene test tubes. Carbachol was added to each test tube in 100/zl aliquots at various concentrations. The tissue was incubated with carbachol in a continuously gassed shaking waterbath for 30 rain. The reaction was terminated by adding 20 ~tl of 5 M HCIO 4 and incubated on ice for 20 rain to extract the inositol phosphates, The extract was neutralized with 20 #l of 5 M K2CO~ and the supernatant was applied to Dowex AG 1 × 8 formate anion exchange columns and inositol phosphates separated ~''~ and counted by liquid scintillation spectrometry. The precipitates were reserved for protein determinations, Statistical comparisons were made using a two way analysis of variance (ANOVA) based on a mixed model. The technique for assaying inositol phosphate levels in miniprisms of rat hippocampal tissue was first validated. Thin layer chromatography demonstrated that the labelled inositol was incorporated only into the phosphoinositides and distribution was unchanged in the presence of carbachol. To normalize values counts per minute (cpm) of inositol phosphates were expressed per milligram of tissue protein content versus expressing the label measured in the inositoi phosphates as a fraction of the label in the phosphoinositides and inositoi phosphates added together. In the latter method, the total label incorporated into both lipids and inositol phosphates continued to increase over time despite frequent washings, Therefore the inositol phosphates generated in the presence of carbachol did not appear to increase over time when the data was expressed as a fraction of total label (Table I). Thus results were expressed as cpms per mg protein which is independent of label incorporation.

TABLE i T;me course of response to 100 I~M carbachol

Inositol phosphates generated following exposure to 100/~M carbachol for the time indicated. Inositol phosphates are expressed as fraction of total cpm in both lipids and inositoi phosphates (mean of 3 experiments) or as cpm/mg protein (mean of 2 experiments), When expressed as a fraction of total label no increase is observed over time. Ttme protein

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IP / mg

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2261 + 2310+ 2364+ 3158+ 3637+ 3793+ 4584+

30 129 54 91 25 70 186

Mild TBI significantly enhanced inositol phosphate production by carbachol in hippocampal miniprisms. The results of 7 experiments on rats with appropriate post-injury behavioral responses were pooled in Fig. 1. Each experiment consisted of a matched sham and injured animal and 4 replicates were performed for each dose. The enhancement was significant at all carbachol concentrations. For 10 -5 M carbachol the significance level was P < 0.005, F - 19.2 and d.f. -- 1,6, For 10 -4 M carbachol the significance level was P < 0.005, F = 20.2 and d,f, = 1,6. For 10 -'~ M carbachol the significance level was P < 0.001, F = 50.5 and d.f,

1,6, When the results were expressed as percent change from sham to minimize interassay variability, it was observed that the greatest enhancement occurs at 10 =s M carbachol, the most physiologically relevant dose

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CARBACHOL(M) Fig. I. Mild traumatic injury enhances carbachol-stimulated inositol phosphate production in hippocampal miniprisms. Each data point represents the mean± S.E.M. of 28 determinations, obtained in 7 independent experiments. Each experiment consisted of an injured and sham-injured animal run in parallel and all experiments were performed under identical conditions. * Indicates a significant difference from sham-injured rats. Statistical comparisons were made by separate two-way ANOVA (10 -5 M carbachol: P < 0.005, F -- 19.2, d.f. - 1,6. 10 -4 M carbachoh P < 0.005, F = 20.2, d.f. = 1,6. 10 -3 M carbachoh P < 0.001, F -- 50.5, d.f. = 1,6).

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CARBACHOL(M) Fig. 2. The percent change in response from sham values as a result of mild TBI. The dotted line represents all the data shown in Fig. 1. The dashed line represents the mean values of 4 rats that received fired percussion impact but manifested no loss of consciousness.

used (Fig. 2). Also, shown in Fig. 2 are the results obtained from 4 rats that were injured but displayed no loss of toe pinch reflex responses suggesting an injury level insufficient to produce loss of consciousness after fluid impact. The enhancement of the response to carbachoi in these rats was minimal and not significantly different from paired shams. To my knowledge this is the first demonstration of a biochemical change in a receptor-coupled phosphoinositide response following mild TBI. A 30% enhancement of carbachol stimulated inositol phosphate production in the hippocampi of mildly injured rats was observed. In contrast, the rats with behavioral responses suggesting no loss of consciousness did not show an enhanced response to carbachol. A number of important implications arise from these data. First, these data suggest that mild TBI results in a modification of musearinic receptor-stimulated phospholipase C activation. In the absence of added carbachol, no difference in levels of inositol phosphates between sham and injured rats were observed 1 h following TBI, suggesting that unstimulated phospholipase C activity was not affected at this time. Thus, at 1 h post injury, endogenous activation of phospholipase C appears unaltered by mild TBI but carbachol-stimulated phospholipase C activity is enhanced. Prior studies demonstrated decreased muscarinic receptor binding following moderate TBI 2.t°. If a similar reduction of muscarinic binding occurs after mild TBI, then the pre,~ent data suggests that receptor-effector coupling is enhanced at a site distal to the receptor, perhaps due to modification by heterologous receptor-coupled effector systems. However, until muscarinic receptor binding changes and heterologous receptor modulation

of muscarinic responses have been measured following mild TBI, such assumptions remain theoretical. Second, this finding indicates that mild TBI, which produces no overt anatomical alterations and only a brief period of traumatic unconsciousness, is sufficient to produce altered hippocampal receptor-mediated responses. Of particular significance is the potential biochemical link between post-traumatic hippocampal hypersensitivity to carbachol and post-traumatic hypersensitivity to imposed cerebral ischaemia after a comparable insult of mild TBI 7. Since cerebral ischaemia has been reported to markedly increase levels of extracellular ACh in the rat hippocampus 8, any secondary ischemic activation of muscarinic-receptor-linked phosphoinositide breakdown may be enhanced when preceded by mild TBI. Thus, in the model of mild TBI followed by imposed secondary ischaemia, a biochemical convergence involving the cholinergic system is probable, yet remains to be confirmed. In summary, these findings contribute an important advance in identifying potential biochemical mechanisms of receptor-mediated TBI pathophysiology such as altered post-traumatic sensitivity to secondary insults. Future efforts will focus on identifying the mechanisms which contribute to the TBI associated enhancement of carbachol-stimulated inositol phosphate production. I thank Drs. Larry Jenkins, Bruce Lyeth, Yi.Cheng Lu and Robert Hamm for expert assistance with animal injury and Dr. Sung Choi for advice on statistical models. This research was supported by Grants NS!9550, NS07288 and NS12587. 1 Borridge, MJ., Downes, C.P., and Hanley, M.R., Lithium amplifies agonist.dependent phosphatidylinositol rebponses in brain and salivary gland, Biochem. J., 206 (1982) 587-595 2 DeAngelis, M., Lyeth, B.G., Jenkins, L.W., Hayes, R.L., Traumatic brain injury causes a decrease in Mi and M 2 muscarinic receptor binding in rat brain, in preparation. 3 Delahunty, T.M., Cronin, M.J., and Linden, J., Regulation of G H 3 cell function via adenosine A I receptors, Biochem. J., 255 (1988) 69-77 4 Gorman, L.K., Fu, K., Hovda, D.A. Becker, D.P., and Katayama, Y., Analysis of acetylcholine release following concussive brain injury in the rat, J. Neurotraumatol., 6 (1989) 20~-210. 5 Hayes, R.L., Jenkins, L.W. and Lyeth, B.G., Neurotransmittermediated mechanisms of traumatic brain injury: acetylcholine and excitatory amino acids. In J.A. Jane, J.C. Torner, D.K. Anderson and W. Young (Eds.), Central Nervous System Trauma Status Report 1991, J. Neurotraumatol., Vol. 9 SuppL 1, Mary Ann Liebert, New York, 1992, pp. 189-200. 6 Jenkins, L.W., Lyeth, B.G., Lewelt, W., Moszynski, K. Dewitt, D.S., Balster, R.L., Miller, L.P., Clifton, G.L., Young, H.F. and Hayes, R.L., Combined pretrauma scopolamine and phencyclidine attenuate posttraumatic increased sensitivity to delayed secondary ischemia, J. NeurotraumatoL, 5 (1988) 275-282 7 Jenkins, L.W., Moszynski, K., Lyeth, B.G.: Lewelt, W., De Witt, D.S., Allen, A., Dixon, C.E., Povlishock, J.T., Majewski, T.J., Clifton, G.L., Young, H.F., Becket, D.P. and Hayes. R L., Increased vulnerability of the mildly traumatized rat h:ain to cerebral ischemia: The use of controlled secondary ischemia as a

310 research tool to identify common different mechanisms contributing to mechanical and ischemic brain injury, Brain Res., 477 (1989) 211-224. 8 Kumagae, Y. and Matsui, Y., Output, tissue levels, and synthesis of acetylcholine during and after transient forebrain ischemia In the rat, 7. Neurochem., 56 (1991) 1169-73. 9 Lyeth, B.G., Dixon, C.E., Jenkins, L.W., Harem, R.J., Alberio, A., Young, H.F., Stonnington, H.H., Hayes, R.L., Effects of scopolamine treatment, on long-term behavioral deficits following concussive brain injury to the rat, Brain Res., 452 (1988) 39-48.

10 Oleniak, L.D., Lyeth, B.G. Martin, T.J., Traumatic brain injury reduces QNB binding to muscarinic receptors in rat hippocampus, Soc. Neurosci. Abstr., 14 (1988) 1151. 11 Robinson, S.E., Martin, R.M., Davis, T.R., Gyenes,, C.A., Ryland, J.E., and Enters, E.K., The effect of acetylcholine depletion on behavior following traumatic brain injury in the rat, Brain Res., 509 (1990) 41-46.