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Increased brain content of the endogenous benzodiazepine receptor ligand, octadecaneuropeptide (ODN), following portacaval anastomosis in the rat
Peptides,Vol. 12, pp. 119-125. ©PergamonPress plc, 1991. Printedin the U.S.A.
0196-9781/91 $3.00 + .00
Increased Brain Content of the Endogenous Benzodiazepine Receptor Ligand, Octadecaneuropeptide (ODN), Following Portacaval Anastomosis in the Rat R O G E R F. B U T T E R W O R T H , * 1 M A R I E - C H R I S T I N E T O N O N , t L O U I S E DI~SY,:~ J E A N - F R A N C O I S G I G U ~ R E , * H U B E R T V A U D R Y t A N D G E O R G E S PELLETIER:~
*Laboratory of Neurochemistry, Andrd-Viallet Clinical Research Center H@ital St-Luc (University of Montreal), Montreal i'Laboratory of Molecular Endocrinology, University of Rouen, Mont-Saint-Aignan, France ~:MRC Group in Molecular Endocrinology, Laval University, Quebec Received 13 June 1990
BUTrERWORTH, R. F., M.-C. TONON, L. Dt~SY, J.-F. GIGUI~RE,H. VAUDRY AND G. PELLETIER. Increasedbrain content of the endogenous benzodiazepine receptorligand, octadecaneuropeptide(ODN),followingportacaval anastomosis in the rat. PEPTIDES 12(1) 119-125, 1991.--Evidence suggests that endogenous benzodiazepine receptor ligands such as diazepam binding inhibitor (DBI) and its metabolite octadecaneuropeptide (ODN) may be implicated in the pathogenesis of hepatic encephalopathy. Using an immunocytochemical technique and an antibody of high specific activity to synthetic ODN, we studied the effects of portacaval anastomosis (PCA) on ODN distribution in rat brain. Four weeks after PCA, ODN immunolabeling was increased in several brain regions including cerebral cortex, hippocampus, hypothalamus and thalamus. Increased ODN immunolabeling was confined to nonneuronal elements such as astrocytes and ependymal cells. Neuropathological evaluation of brain following PCA reveals asa-ocytic rather than neuronal changes. These results are consistent with a role for endogenous neuropeptide ligands for astrocytic benzodiazepine receptors in the pathogenesis of hepatic encephalopathy. Oetadecaneuropeptide Portacaval anastomosis
Endogenous benzodiazepine ligand Hepatic encephalopathy
Peripheral-type benzodiazepine receptors
THERE is evidence to suggest that alterations of benzodiazepine receptors as well as their endogenous ligands may play an important role in the pathogenesis of hepatic encephalopathy (HE) resuiting from acute and chronic liver failure (10). In particular, astrocytic or "peripheral-type" benzodiazepine receptors (PTBR's) have been found to be increased in autopsied brain tissue from patients with chronic liver disease who died in hepatic coma (17) and in the brains of rats following portacaval anastomosis (14). Furthermore, CSF concentrations of the endogenous benzodiazepine ligand diazepam binding inhibitor (DBI) have been reported in patients with HE (20). DBI, as well as its active metabolite octadecaneuropeptide (ODN), displaces benzodiazepines from PTBR's (5,15), suggesting that they may be endogenous ligands for these receptors. In the light of these observations, we have studied the effects of portacaval anastomosis on the distribution of ODN in rat brain using an immunocytochemical technique and
an antibody of high specific activity to synthetic ODN (22,23). METHOD
Surgical Techniques Adult male Sprague-Dawley rats weighing 150-175 g were anesthetized with ether and an end-to-side PCA performed according to the method of Butterworth et al. (8). Thus rats underwent a laparotomy, the inferior vena cava and portal vein were isolated, the inferior vena cava was partially clamped (anastomosis clamp, Roboz Instruments Inc.), and an elliptical piece of vein 1.5 times the portal vein diameter was removed. The portal vein was ligated and cut, and an end-to-side anastomosis performed under a dissecting microscope. Total surgery time was <15 min. Sham-operated control rats, matched for weight, were similarly anesthetized, a laparotomy was performed, and the inferior vena
1Requests for reprints should be addressed to Roger F. Butterworth, Ph.D., Laboratory of Neurochemistry, Andr6-Viallet Clinical Research Centre, H6pital Saint-Luc, 1058 St-Denis St., Montreal, Quebec H2X 3J4 Canada.
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cava and portal vein were occluded for 15 min. Following surgery, all animals were housed individually under constant conditions of temperature, humidity, and light cycles and were allowed free access to standard laboratory chow and water. Overall mortality for shunted rats was of the order of 10%.
Preparation of ODN Antiserum Synthetic ODN was covalently conjugated to bovine serum albumin (BSA, fraction V; Boehringer Mannheim) with 1-ethyl3(3-dimethylaminopropyl)-carbodiimide HC1 (ECDI, Sigma Chemical Co., St. Louis, MO) as previously described by Vaudry et al. (24). Synthetic ODN (10.6 mg) and approximately 2 ng 125I-labeled [Tyr°]ODN (106 cpm) were mixed with 30 mg of BSA and 100 mg ECDI in 2 ml of distilled water at 4°(= for 24 h in the dark. Twenty txl of homogenate were taken for Sephadex G-50 gel chromatography and the mixture was dialyzed against 1.25 ml distilled water. The coupling efficiency was 72% (i.e., yielded 8.7 moles ODN conjugated per mole of BSA). Rabbits were injected intradermally with the ODN-BSA conjugate (equivalent to 200 ixg ODN per animal) emulsified in Freund's adjuvant. Rabbits were boosted with the same amount of conjugate each month and were bled 15 days after each injection. Using radioimmunoassay, we have already observed that synthetic ODN and serial dilutions of several rat brain areas gave parallel displacement curves (22).
Immunohistochemistry Four portacaval shunted or sham-operated rats were fixed by perfusion with Bouin's fluid. Brains were embedded in paraffin and serially cut at 7 Ixm. Immunostaining was performed according to the unlabeled antibody-enzyme method of Sternberger (21). In brief, paraffin sections were deparaffinized and successively exposed to: 1) rabbit antiserum to ODN diluted to 1/500 to 1/1000, 2) goat anti-rabbit gamma globulins diluted at 1/50, and 3) the peroxidase-antiperoxidase (PAP) complex diluted at 1/100. The peroxidase was diluted in a medium containing 0.05% diaminobenzidine and 0.01% H202. Control experiments were performed on adjacent sections by substituting normal rabbit serum or ODN antiserum (diluted 1/1000) absorbed with an excess (10 -7 M) of synthetic ODN, ovine CRF, somatostatin-14, neuropeptide Y, ACTH or et-MSH. In order to quantify ODN immunoreactivity in different brain areas, paraffin sections were incubated with ODN antiserum (dilution 1/1000) during 17 h followed by a 2-h incubation with [~25I] protein A (Amersham) (0.1 ixC per section). Following immunolabeling, the sections were exposed for autoradiography with Kodak X-Omat film for 2 days. Densitometric measurements of autoradiographs of different brain areas were obtained using a digitized Amersham RAS image analysis system. Four animals were used and for each area three sections were quantified. Optical density (OD) units (after subtraction of X-ray film background) correspond to the mean OD units per pixel (1 pixel= 180 x 180 IJ.m). Statistical significance was determined according to the multiple range test of Ducan-Kramer (16). RESULTS
As shown in Figs. 1-3, ODN immunostaining was observed
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TABLE 1 Brain Regions Cerebellum Cerebral cortex Hippocampus (rostral) Hippocampus (caudal) Hypothalamus (rostral) Hypothalamus (caudal)
Portacaval Anastomosis 0.15 0.11 0.07 0.11 0.10 0.08
_ 0.02t + 0.02t __. 0.02t --- 0.01"~ _ 0.02* _.+ 0.02
Sham-Operated 0.08 0.01 0.03 0.02 0.05 0.04
--- 0.01 -+ 0.02 _ 0.01 ___0.01 ___0.01 _.+ 0.01
Data are expressed as the mean _-. S.E.M. optical density units obtained from 12 sectionscut from 4 animals. *p<0.05; tp<0.01 compared with sham-operatedanimals, F(11,24)= 29.81, p<0.001.
throughout the brain of sham-operated control rats. The magnitude and distribution of ODN immunoreactivity was comparable to that described in a previous report (22). The reaction product was found to be localized exclusively in nonneuronal cells and processes. ODN-positive cells were observed in olfactory bulb, hypothalamus, hippocampus, cerebral cortex, periaqueductal gray as well as in circumventricular organs including the pineal gland. In hypothalamus, staining was associated with astrocytes in several nuclei, including the supraoptic and arcuate nuclei. In median eminence, tanycytes were observed to be immunostained. In cerebral cortex, ODN-positive cells were located in all layers. When the antiserum was absorbed with ODN, no positive cells could be detected as shown in Figs. 1B and 2B. Significantly greater immunostainingwas observed throughout brain following portacaval anastomosis (Figs. 4, 5 and 6). However, as was the case in material from control animals, staining appeared to be restricted to nonneuronal cells and processes. In many cases, astrocytes showed intense immunostainingfollowing portacaval shunting. Optical density measurements of X-ray films also revealed stronger immunolabeling in brain areas of animals with portacaval anastomosis (Table 1). DISCUSSION Results of the present study reveal increased ODN immunostaining in several brain structures following portacaval anastomosis. In confirmation of the results of a previous study, in which electron microscopic evaluation showed that ODN immunolabeling was restricted to glial and ependymal cells (23), ODN-like immunoreactivity was found to be localized to nonneuronal cells and their processes. These findings are at variance with those of a recent report which described, using a similar immunohistochemical procedure, an exclusively neuronal localization of ODN in rat brain (2). The reason for these discrepancies is unclear but may relate to cross-reactivity of ODN antiserum with other biologically active DBI processing products such as triakontatetraneuropeptide (TTN) and eicosapentaneuropeptide (EPN) (2). Thus the increased ODN immunolabelingobserved following portacaval anatomosis in the present study may be due to increases of ODN, TTN, EPN or combinations of these peptides. Further studies are required in order to adequately resolve these issues. The distribution of ODN immunoreactivity observed in the present study closely resembles that reported from in situ hybridization studies
FACING PAGE FIG. 1. (A) Localizationof ODN in the cerebral cortex of sham-operated adult male rats. Labeled astrocytes are distributedthroughout the different layers, x 200. (B) Section consecutiveto that shown in (A). Immunoadsorptionof the antiserum with synthetic ODN (10 -7 M) has completely prevented staining. × 200.
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FACING PAGE FIG. 2. (A) Localization of ODN in the anterior part of the paraventricular nucleus of the hypothalamus of sham-operated adult male rats. Weakly stained astrocytes can be observed. V: third ventricle, x 200. (B) Section consecutive to that shown in (A). Immunoadsorptinn of the antiserum with synthetic ODN (10 -7 M) has completely abolished staining. V: third ventricle, x 200.
in which mRNA for the ODN precursor DBI was found to be localized on glia and ependymal cells, including tanycytes (3). DBI has recently been found in astrocytes where its processing and function appear to be different from that observed in neurons (15). Both DBI and ODN inhibit benzodiazepine binding to astrocytic (peripheral-type) benzodiazepine receptors (5,15). In the case of ODN, the potency of displacement of benzodiazepines from binding sites on astrocytes was found to be much greater than for the corresponding neuronal sites (5). Taken together, these observa-
/ iii
FIG. 3. Localization of ODN in the paraventricular nucleus of the thalamus in sham-operated adult male rats. Numerous weakly stained cells are present. V: lateral ventricle. × 200.
tions suggest a role for ODN or DBI in astrocytic function, mediated via the peripheral-type benzodiazepine receptor. Increased ODN immunolabeling in brain following portacaval anastomosis could have implications for the pathogenesis of hepatic encephalopathy (HE). HE resulting from chronic liver disease and portal-systemic shunting in both humans and experimental animals is characterized both neuropathologically and biochemically by astrocytic rather than neuronal changes (1, 13, 18, 19). The severity of astrocytic changes in the brains of patients who died in hepatic encephalopathy correlates well with the degree of neurological impairment (1,19). Densities of peripheral-type benzodiazepine binding sites, evaluated using the highly selective ligand PK 11195, are increased throughout brain following portacaval anastomosis (14). Increases are of a similar magnitude to the increased ODN immunoreactivity observed in the present study. A recent report described increased levels of endogenous substances with neurochemical properties characteristic of agonists for neuronal, GABA-related benzodiazepine receptors (4). However, affinities and densities of these receptors are unchanged in brain preparations from both humans and experimental animals with hepatic encephalopathy (7, 9, 17). Moreover, CSF levels of DBI are elevated in patients with HE (20) and it was initially suggested that these increases might relate to abnormalities of GABAergic function in HE. However, the finding of increased ODN immunoreactivity and increased PTBR's coupled with the finding of normal GABAergic function following portacaval anastomosis (6,7) strongly suggests that increased DBI levels in patients with HE might result from astrocytic changes in the brains of these patients. The precise mechanisms responsible for the increased ODN immunoreactivity as well as increased PTBR's in brain following portacaval anastomosis remain unclear. One possibility is that these changes result from the action of ammonia or octanoate, two neurotoxic substances with a putative role in the pathogenesis of HE. In favor of such a possibility, exposure of cultured astrocytes to pathophysiological levels of ammonia or octanoate results in increased binding of the PTBR ligand Ro5-4864 (11). Increased ODN immunoreactivity may therefore be part of the astrocytic response to exposure to these neurotoxins following portacaval anastomosis. ACKNOWLEDGEMENT These studies were supported by grants from the Medical Research Council of Canada to R.F.B. and G.P.
REFERENCES 1. Adams, R. D.; Foley, J. M. The neurological disorder associated with liver disease. Res. Publ. Assoc. Nerv. Ment. Dis. 32:199-237; 1953. 2. Alho, H.; Bovolin, P.; Jenkins, D.; Guidotti, A.; Costa, E. Cellular and subcellular localization of an octadecaneuropeptide derived from diazepam binding inhibitor: Immunohistochemical studies in the rat brain. J. Chem. Neuroanat. 2:301-318; 1989. 3. Alho, H.; Fremeau, R. T.; Tiedge, A.; Wilcox, J.; Bovolin, P.; Brosins, J.; Roberts, J. L.; Costa, E. Diazepam binding inhibitor gene expression: Location in brain and peripheral tissues of rat. Proc. Natl. Acad. Sci. USA 85:7018--7022; 1988. 4. Basile, A. S.; Gammal, S. H.; Jones, E. A.; Skolnick, P. GABAA
receptor complex in an experimental model of hepatic encephalopathy: Evidence for elevated levels of an endogenous benzodiazepine mceptur ligand. J. Neurochem. 53:1057-1063; 1989. 5. Bender, A. S.; Hertz, L. Pharmacological characteristics of diazepam receptors in neurons and astrocytes in primary cultures. J. Neurosci. Res. 18:366-372; 1987. 6. Butterworth, R. F.; Gigutre, J. F. Cerebral amino acids in portalsystemic encephalopathy: Lack of evidence for altered -/-aminobutyric acid (GABA) function. Metah. Brain Dis. 1:221-228; 1986. 7. Butterworth, R. F.; Lavoie, J.; Gigutre, J. F.; Pomier Layrargues, G.; Bergeron, M. Cerebral GABAergic and glutamatergic function in hepatic encephalopathy. Neurochem. Pathol. 6:131-144; 1987.
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FIG. 4. Localization of ODN in the cerebral cortex of animals with portacaval anastomosis. The astrocytes are numerous and strongly stained. × 200.
8. Butterworth, R. F.; Girard, G.; Gigu~re, J. F. Regional differences in the capacity for ammonia removal by brain following portacaval anastomosis. J. Neurochem. 51:486-490; 1988. 9. Butterworth, R. F.; Lavoie, J.; Gigu~re, J. F.; Pomier Layrargues, G. Affinities and densities of high affinity 3H-muscimol (GABA-A) binding sites and of central benzodiazepine receptors are unchanged in autopsied brain tissue from cirrhotic patients with hepatic encephalopathy. Hepatology 8:1084-1088; 1988. 10. Butterworth, R. F.; Pomier Layrargues, G. Benzodiazepine receptors and hepatic encephalopathy. Hepatology 11:499-501; 1990. 11. Ducis, I.; Norenberg, L. B.; Norenberg, M. D. Effect of ammonium chloride on the astrocyte benzodiazepine receptor. Brain Res. 493: 362-365; 1989. 12. Ferrarese, C.; Appollonio, I.; Frigo, M.; Gaini, S. M.; Piolti, R.; Frattola, L. Benzodizepine receptors and diazepam-binding inhibitor in human cerebral tumors. Ann. Neurol. 26:564-568; 1989. 13. Gigu~re, J. F.; Besnard, A. M.; Lavoie, J.; Pomier Layrargues, G.; Butterworth, R. F. Activities of glutamate-related enzymes in autopsied brain tissue from cirrhotic patients with hepatic encephalopathy. In: Butterworth, R. F.; Pomier Layrargues, G., eds. Hepatic encephalopathy: Pathophysiology and treatment. New Jersey: Humana Press; 1989:435--445. 14. Gigu~re, J. F.; Hamel, E.; Butterworth, R. F. Increased peripheral
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benzodiazepine binding sites following portacaval anastomosis in the rat. Soc. Neurosci. Abstr. 15:167.9; 1989. Guidotti, A.; Berkovich, A.; Ferrarese, C.; Santi, M. R.; Costa, E. Neuronal-glial differential processing of DBI to yield ligands to central or peripheral benzodiazepine recognition sites. In: Sauvenet, P.; Langer, S. Z.; Morselli, P. L., eds. Imidazopyridines in sleep disorders. New York: Raven Press; 1988:25-38. Kramer, C. Y. Extension of multiple range tests to group means with unique numbers of replication. Biometrics 12:307-310; 1956. Lavoie, J.; Pomier Layrargues, G.; Butterworth, R. F. Studies o1 central and "peripheral-type" benzodiazepine receptors in autopsied brain tissue from cirrhotic patients with hepatic encephalopathy. In: Butterworth, R. F.; Pomier Layrargues, G., eds. Hepatic encephalopathy: Pathophysiology and treatment. New Jersey: Humana Press; 1989:305-318. Lavoie, J.; Pomier Layrargues, G.; Butterworth, R. F. Increased densities of "peripheral-type" benzodiazepine receptors in autopsied brain tissue from cirrhotic patients with hepatic encephalopathy. Hepatology 11:874--878; 1990. Martin, H.; Voss, K.; Hufnagl, P.; Wack, R.; Wassilew, G. Morphometric and densitometric investigations of protoplasmic astrocytes and neurons in human hepatic encephalopathy. Exp. Neurol. 32:241250; 1987.
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FIG. 5. Localization of ODN in the paraventricular nucleus of the hypothalamus of animals with portacaval anastomosis. Numerous strongly immunostained cells can be detected. V: third ventricle, x 200.
20. Rothstein, J. D.; McKhann, G.; Guarneri, P.; Barbaccia, M. L.; Guidotti, A.; Costa, E. Cerebrospinal fluid content of diazepam binding inhibitor in chronic hepatic encephalopathy. Ann. Neurol. 26:57-62; 1989. 21. Stemberger, L. A. Immunocytochemistry. Englewood Cliffs, NJ: Prentice-Hall Inc.; 1974. 22. Tong, Y.; Tonon, M. C.; D6sy, L.; Nicolas, P.; Van&y, H.; Pelletier, G. Localization of the endogenous benzodiazepine receptor ligand octadecaneuropeptide and peripheral type benzodiazepine receptors in the rat pituitary. J. Neuroendocrinol. 2:189-192; 1990. 23. Tonon, M. C.; Desy, L.; Nicolas, P.; Vaudry, H.; Pelletier, G. Immunocytochemical localization of the endogenous benzodiazepine ligand octadecaneuropeptide (ODN) in the rat brain. Neuropeptides 15:17-24; 1990. 24. Vaudry, H.; Tonon, M. C.; Delarue, C.; Vaillant, R.; Kraicer, J. Biological and radioimmunological evidence for melanocyte-stimulating-hormones (etMSH) of extrapituitary origin in the rat brain. Neuroendocrinology 27:9-24; 1978.
FIG. 6. Localization of ODN in the paraventficular nucleus of the thalamus of animals with portacaval anastomosis. The positive cells are more strongly stained than those observed in sham-operated animals (compare with Fig. 3). V: lateral ventricles. × 200.
0196-9781/91 $3.00 + .00
Increased Brain Content of the Endogenous Benzodiazepine Receptor Ligand, Octadecaneuropeptide (ODN), Following Portacaval Anastomosis in the Rat R O G E R F. B U T T E R W O R T H , * 1 M A R I E - C H R I S T I N E T O N O N , t L O U I S E DI~SY,:~ J E A N - F R A N C O I S G I G U ~ R E , * H U B E R T V A U D R Y t A N D G E O R G E S PELLETIER:~
*Laboratory of Neurochemistry, Andrd-Viallet Clinical Research Center H@ital St-Luc (University of Montreal), Montreal i'Laboratory of Molecular Endocrinology, University of Rouen, Mont-Saint-Aignan, France ~:MRC Group in Molecular Endocrinology, Laval University, Quebec Received 13 June 1990
BUTrERWORTH, R. F., M.-C. TONON, L. Dt~SY, J.-F. GIGUI~RE,H. VAUDRY AND G. PELLETIER. Increasedbrain content of the endogenous benzodiazepine receptorligand, octadecaneuropeptide(ODN),followingportacaval anastomosis in the rat. PEPTIDES 12(1) 119-125, 1991.--Evidence suggests that endogenous benzodiazepine receptor ligands such as diazepam binding inhibitor (DBI) and its metabolite octadecaneuropeptide (ODN) may be implicated in the pathogenesis of hepatic encephalopathy. Using an immunocytochemical technique and an antibody of high specific activity to synthetic ODN, we studied the effects of portacaval anastomosis (PCA) on ODN distribution in rat brain. Four weeks after PCA, ODN immunolabeling was increased in several brain regions including cerebral cortex, hippocampus, hypothalamus and thalamus. Increased ODN immunolabeling was confined to nonneuronal elements such as astrocytes and ependymal cells. Neuropathological evaluation of brain following PCA reveals asa-ocytic rather than neuronal changes. These results are consistent with a role for endogenous neuropeptide ligands for astrocytic benzodiazepine receptors in the pathogenesis of hepatic encephalopathy. Oetadecaneuropeptide Portacaval anastomosis
Endogenous benzodiazepine ligand Hepatic encephalopathy
Peripheral-type benzodiazepine receptors
THERE is evidence to suggest that alterations of benzodiazepine receptors as well as their endogenous ligands may play an important role in the pathogenesis of hepatic encephalopathy (HE) resuiting from acute and chronic liver failure (10). In particular, astrocytic or "peripheral-type" benzodiazepine receptors (PTBR's) have been found to be increased in autopsied brain tissue from patients with chronic liver disease who died in hepatic coma (17) and in the brains of rats following portacaval anastomosis (14). Furthermore, CSF concentrations of the endogenous benzodiazepine ligand diazepam binding inhibitor (DBI) have been reported in patients with HE (20). DBI, as well as its active metabolite octadecaneuropeptide (ODN), displaces benzodiazepines from PTBR's (5,15), suggesting that they may be endogenous ligands for these receptors. In the light of these observations, we have studied the effects of portacaval anastomosis on the distribution of ODN in rat brain using an immunocytochemical technique and
an antibody of high specific activity to synthetic ODN (22,23). METHOD
Surgical Techniques Adult male Sprague-Dawley rats weighing 150-175 g were anesthetized with ether and an end-to-side PCA performed according to the method of Butterworth et al. (8). Thus rats underwent a laparotomy, the inferior vena cava and portal vein were isolated, the inferior vena cava was partially clamped (anastomosis clamp, Roboz Instruments Inc.), and an elliptical piece of vein 1.5 times the portal vein diameter was removed. The portal vein was ligated and cut, and an end-to-side anastomosis performed under a dissecting microscope. Total surgery time was <15 min. Sham-operated control rats, matched for weight, were similarly anesthetized, a laparotomy was performed, and the inferior vena
1Requests for reprints should be addressed to Roger F. Butterworth, Ph.D., Laboratory of Neurochemistry, Andr6-Viallet Clinical Research Centre, H6pital Saint-Luc, 1058 St-Denis St., Montreal, Quebec H2X 3J4 Canada.
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cava and portal vein were occluded for 15 min. Following surgery, all animals were housed individually under constant conditions of temperature, humidity, and light cycles and were allowed free access to standard laboratory chow and water. Overall mortality for shunted rats was of the order of 10%.
Preparation of ODN Antiserum Synthetic ODN was covalently conjugated to bovine serum albumin (BSA, fraction V; Boehringer Mannheim) with 1-ethyl3(3-dimethylaminopropyl)-carbodiimide HC1 (ECDI, Sigma Chemical Co., St. Louis, MO) as previously described by Vaudry et al. (24). Synthetic ODN (10.6 mg) and approximately 2 ng 125I-labeled [Tyr°]ODN (106 cpm) were mixed with 30 mg of BSA and 100 mg ECDI in 2 ml of distilled water at 4°(= for 24 h in the dark. Twenty txl of homogenate were taken for Sephadex G-50 gel chromatography and the mixture was dialyzed against 1.25 ml distilled water. The coupling efficiency was 72% (i.e., yielded 8.7 moles ODN conjugated per mole of BSA). Rabbits were injected intradermally with the ODN-BSA conjugate (equivalent to 200 ixg ODN per animal) emulsified in Freund's adjuvant. Rabbits were boosted with the same amount of conjugate each month and were bled 15 days after each injection. Using radioimmunoassay, we have already observed that synthetic ODN and serial dilutions of several rat brain areas gave parallel displacement curves (22).
Immunohistochemistry Four portacaval shunted or sham-operated rats were fixed by perfusion with Bouin's fluid. Brains were embedded in paraffin and serially cut at 7 Ixm. Immunostaining was performed according to the unlabeled antibody-enzyme method of Sternberger (21). In brief, paraffin sections were deparaffinized and successively exposed to: 1) rabbit antiserum to ODN diluted to 1/500 to 1/1000, 2) goat anti-rabbit gamma globulins diluted at 1/50, and 3) the peroxidase-antiperoxidase (PAP) complex diluted at 1/100. The peroxidase was diluted in a medium containing 0.05% diaminobenzidine and 0.01% H202. Control experiments were performed on adjacent sections by substituting normal rabbit serum or ODN antiserum (diluted 1/1000) absorbed with an excess (10 -7 M) of synthetic ODN, ovine CRF, somatostatin-14, neuropeptide Y, ACTH or et-MSH. In order to quantify ODN immunoreactivity in different brain areas, paraffin sections were incubated with ODN antiserum (dilution 1/1000) during 17 h followed by a 2-h incubation with [~25I] protein A (Amersham) (0.1 ixC per section). Following immunolabeling, the sections were exposed for autoradiography with Kodak X-Omat film for 2 days. Densitometric measurements of autoradiographs of different brain areas were obtained using a digitized Amersham RAS image analysis system. Four animals were used and for each area three sections were quantified. Optical density (OD) units (after subtraction of X-ray film background) correspond to the mean OD units per pixel (1 pixel= 180 x 180 IJ.m). Statistical significance was determined according to the multiple range test of Ducan-Kramer (16). RESULTS
As shown in Figs. 1-3, ODN immunostaining was observed
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TABLE 1 Brain Regions Cerebellum Cerebral cortex Hippocampus (rostral) Hippocampus (caudal) Hypothalamus (rostral) Hypothalamus (caudal)
Portacaval Anastomosis 0.15 0.11 0.07 0.11 0.10 0.08
_ 0.02t + 0.02t __. 0.02t --- 0.01"~ _ 0.02* _.+ 0.02
Sham-Operated 0.08 0.01 0.03 0.02 0.05 0.04
--- 0.01 -+ 0.02 _ 0.01 ___0.01 ___0.01 _.+ 0.01
Data are expressed as the mean _-. S.E.M. optical density units obtained from 12 sectionscut from 4 animals. *p<0.05; tp<0.01 compared with sham-operatedanimals, F(11,24)= 29.81, p<0.001.
throughout the brain of sham-operated control rats. The magnitude and distribution of ODN immunoreactivity was comparable to that described in a previous report (22). The reaction product was found to be localized exclusively in nonneuronal cells and processes. ODN-positive cells were observed in olfactory bulb, hypothalamus, hippocampus, cerebral cortex, periaqueductal gray as well as in circumventricular organs including the pineal gland. In hypothalamus, staining was associated with astrocytes in several nuclei, including the supraoptic and arcuate nuclei. In median eminence, tanycytes were observed to be immunostained. In cerebral cortex, ODN-positive cells were located in all layers. When the antiserum was absorbed with ODN, no positive cells could be detected as shown in Figs. 1B and 2B. Significantly greater immunostainingwas observed throughout brain following portacaval anastomosis (Figs. 4, 5 and 6). However, as was the case in material from control animals, staining appeared to be restricted to nonneuronal cells and processes. In many cases, astrocytes showed intense immunostainingfollowing portacaval shunting. Optical density measurements of X-ray films also revealed stronger immunolabeling in brain areas of animals with portacaval anastomosis (Table 1). DISCUSSION Results of the present study reveal increased ODN immunostaining in several brain structures following portacaval anastomosis. In confirmation of the results of a previous study, in which electron microscopic evaluation showed that ODN immunolabeling was restricted to glial and ependymal cells (23), ODN-like immunoreactivity was found to be localized to nonneuronal cells and their processes. These findings are at variance with those of a recent report which described, using a similar immunohistochemical procedure, an exclusively neuronal localization of ODN in rat brain (2). The reason for these discrepancies is unclear but may relate to cross-reactivity of ODN antiserum with other biologically active DBI processing products such as triakontatetraneuropeptide (TTN) and eicosapentaneuropeptide (EPN) (2). Thus the increased ODN immunolabelingobserved following portacaval anatomosis in the present study may be due to increases of ODN, TTN, EPN or combinations of these peptides. Further studies are required in order to adequately resolve these issues. The distribution of ODN immunoreactivity observed in the present study closely resembles that reported from in situ hybridization studies
FACING PAGE FIG. 1. (A) Localizationof ODN in the cerebral cortex of sham-operated adult male rats. Labeled astrocytes are distributedthroughout the different layers, x 200. (B) Section consecutiveto that shown in (A). Immunoadsorptionof the antiserum with synthetic ODN (10 -7 M) has completely prevented staining. × 200.
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OCTADECANEUROPEPTIDE IN BRAIN FOLLOWING PORTACAVAL SHUNT
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FACING PAGE FIG. 2. (A) Localization of ODN in the anterior part of the paraventricular nucleus of the hypothalamus of sham-operated adult male rats. Weakly stained astrocytes can be observed. V: third ventricle, x 200. (B) Section consecutive to that shown in (A). Immunoadsorptinn of the antiserum with synthetic ODN (10 -7 M) has completely abolished staining. V: third ventricle, x 200.
in which mRNA for the ODN precursor DBI was found to be localized on glia and ependymal cells, including tanycytes (3). DBI has recently been found in astrocytes where its processing and function appear to be different from that observed in neurons (15). Both DBI and ODN inhibit benzodiazepine binding to astrocytic (peripheral-type) benzodiazepine receptors (5,15). In the case of ODN, the potency of displacement of benzodiazepines from binding sites on astrocytes was found to be much greater than for the corresponding neuronal sites (5). Taken together, these observa-
/ iii
FIG. 3. Localization of ODN in the paraventricular nucleus of the thalamus in sham-operated adult male rats. Numerous weakly stained cells are present. V: lateral ventricle. × 200.
tions suggest a role for ODN or DBI in astrocytic function, mediated via the peripheral-type benzodiazepine receptor. Increased ODN immunolabeling in brain following portacaval anastomosis could have implications for the pathogenesis of hepatic encephalopathy (HE). HE resulting from chronic liver disease and portal-systemic shunting in both humans and experimental animals is characterized both neuropathologically and biochemically by astrocytic rather than neuronal changes (1, 13, 18, 19). The severity of astrocytic changes in the brains of patients who died in hepatic encephalopathy correlates well with the degree of neurological impairment (1,19). Densities of peripheral-type benzodiazepine binding sites, evaluated using the highly selective ligand PK 11195, are increased throughout brain following portacaval anastomosis (14). Increases are of a similar magnitude to the increased ODN immunoreactivity observed in the present study. A recent report described increased levels of endogenous substances with neurochemical properties characteristic of agonists for neuronal, GABA-related benzodiazepine receptors (4). However, affinities and densities of these receptors are unchanged in brain preparations from both humans and experimental animals with hepatic encephalopathy (7, 9, 17). Moreover, CSF levels of DBI are elevated in patients with HE (20) and it was initially suggested that these increases might relate to abnormalities of GABAergic function in HE. However, the finding of increased ODN immunoreactivity and increased PTBR's coupled with the finding of normal GABAergic function following portacaval anastomosis (6,7) strongly suggests that increased DBI levels in patients with HE might result from astrocytic changes in the brains of these patients. The precise mechanisms responsible for the increased ODN immunoreactivity as well as increased PTBR's in brain following portacaval anastomosis remain unclear. One possibility is that these changes result from the action of ammonia or octanoate, two neurotoxic substances with a putative role in the pathogenesis of HE. In favor of such a possibility, exposure of cultured astrocytes to pathophysiological levels of ammonia or octanoate results in increased binding of the PTBR ligand Ro5-4864 (11). Increased ODN immunoreactivity may therefore be part of the astrocytic response to exposure to these neurotoxins following portacaval anastomosis. ACKNOWLEDGEMENT These studies were supported by grants from the Medical Research Council of Canada to R.F.B. and G.P.
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FIG. 4. Localization of ODN in the cerebral cortex of animals with portacaval anastomosis. The astrocytes are numerous and strongly stained. × 200.
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OCTADECANEUROPEPTIDE IN BRAIN FOLLOWING PORTACAVAL SHUNT
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FIG. 5. Localization of ODN in the paraventricular nucleus of the hypothalamus of animals with portacaval anastomosis. Numerous strongly immunostained cells can be detected. V: third ventricle, x 200.
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FIG. 6. Localization of ODN in the paraventficular nucleus of the thalamus of animals with portacaval anastomosis. The positive cells are more strongly stained than those observed in sham-operated animals (compare with Fig. 3). V: lateral ventricles. × 200.