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Diurnal locomotor activity and oxidative metabolism of the suprachiasmatic nucleus in two models of hepatic insufficiency
Journal of the Neurological Sciences 212 (2003) 93 – 97 www.elsevier.com/locate/jns
Diurnal locomotor activity and oxidative metabolism of the suprachiasmatic nucleus in two models of hepatic insufficiency Laudino Lopez a,*, Jose M. Cimadevilla a, Maria A. Aller b, Jaime Arias b, M. Paz Nava c, Jorge L. Arias a a
Laboratorio de Psicobiologia, Facultad de Psicologia, Plaza Feijoo s/n, 33003 Oviedo, Spain b Catedra de Cirugia, Facultad de Medicina, UCM, Spain c Dpto. de Biologia Animal II, Facultad de Biologia, UCM, Spain
Received 30 October 2002; received in revised form 20 March 2003; accepted 21 March 2003
Abstract Subjects with hepatic cirrhosis develop alterations of several rhythmic behavioural and biochemical patterns. Since most cirrhotic patients combine portal hypertension and hepatic impairment, our work aims to assess the extent to which rhythmical changes can be due to hepatic insufficiency or portal hypertension. This was done using two experimental models in rats, portacaval shunt model (PC) and portal hypertension by a triple stenosing ligature of the portal vein (PH). We assess diurnal locomotor activity and determine the oxidative metabolism of the suprachiasmatic nucleus (SCN) by histochemical determination of cytochrome oxidase (COX). The results show that animals with PC have altered diurnal locomotor rhythm compared to control and PH rats ( p < 0.001). They also present lower COX activity in the SCN ( p < 0.05). We conclude that rhythmic alterations are due to hepatic insufficiency and not to portal hypertension. D 2003 Elsevier Science B.V. All rights reserved. Keywords: Cytochrome oxidase; Hepatic encephalopathy; Suprachiasmatic nucleus; Circadian rhythm; Hepatic cirrhosis
1. Introduction Hepatic insufficiency, mainly due to hepatic cirrhosis, is one of the main medical problems in the western world today. In addition to producing a high mortality, it also requires costly treatment and transplantation as a last resort [1,2]. Owing to this situation, several experimental models have been developed to assess and study the consequences derived from liver disease. These models include the portacaval shunt [3] and extrahepatic portal hypertension by stenosis of the vena porta [4,5]. Both procedures have confirmed value to increase our knowledge of the consequences derived from hepatic insufficiency. However, none of the models correctly reproduces the real situation of subjects with hepatic cirrhosis. In human clinical cases, the tendency is for both these models to converge: severe hepatic insufficiency (cirrhosis) that leads to portal hypertension. In experimental models, however, we have severe
* Corresponding author. Tel.: +34-985104188; fax: +34-985104144. E-mail address: [email protected] (L. Lopez).
hepatic insufficiency without portal hypertension (portacaval shunt) or portal hypertension without severe hepatic insufficiency. The portacaval anastomosis model will originate a severe hepatic and testicular atrophy [6,7], whereas the portal pressure is different to that developed by the portal hypertension model. Animals with portacaval shunt present no resistance to the emerging blood flow and, therefore, have a reduced or unchanged portal pressure compared to control rats [8,9]. In contrast, rats with portal hypertension present resistance to the emerging flow for two reasons: stenosis of the vena porta and portosystemic colateral circulation, with the ability to contract [10]. On the other hand, it is well known that subjects with cirrhosis develop a neuropsychiatric syndrome called hepatic encephalopathy [3,11,12]. Also, in experimental models of hepatic insufficiency, neurological and behavioural alterations have been observed that could be similar to those found in human hepatic encephalopathy [13,14]. One of the characteristics of this syndrome is an alteration of the different rhythmical biochemical and behavioural patterns [15 –17]. Underlying these rhythmical alterations, one can
0022-510X/03/$ - see front matter D 2003 Elsevier Science B.V. All rights reserved. doi:10.1016/S0022-510X(03)00105-9
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find the suprachiasmatic nucleus [18], the main controller of biological rhythms, which presents a diminished oxidative metabolism in rats with PC [19]. The objective of our work was to establish whether the rhythmical alterations in hepatic encephalopathy and those demonstrated in PC of the rat are also caused by portal hypertension or are merely a consequence of severe hepatic insufficiency. To do this, rats with two models of hepatic insufficiency were used and the circadian rhythm of the activity and oxidative metabolism of the suprachiasmatic nucleus (SCN) were studied by histochemical labelling of cytochrome oxidase (COX).
2. Material and methods Twenty-four Wistar rats from the vivarium of Oviedo University, Oviedo, Spain, were used. The mean weight of rats at the start of the experiments was 240 F 25 g. All the animals had ad libitum access to food and tap water and were maintained at constant room temperature (20 F 2 jC), with a relative humidity of 65 F 10% and artificial light– dark cycle of 12 h (08:00– 20:00 h/20:00 –08:00 h). The 24 animals were distributed into three groups (n = 8 each group): animals with a portacaval shunt and 35 postoperative days (PC group), animals with portal hypertension and 45 postoperative days (PH group) and a group of control animals with a pseudo-intervention and 45 days postoperative evolution (CO). The pseudo-intervention consisted in a laparotomy and clamping of the vena porta for 5 min. All the experiments were performed according to the European Communities Council Directive 86/609/ EEC. The end-to-side portacaval shunt operation was performed according to the technique described by Lee and Fischer [20]. This was modified slightly by total clamping of the inferior vena cava during PC performance [21]. The total time in which the portal vein and inferior vena cava were clamped for anastomosis was less than 15 min. The operations were performed under ethylic ether anaesthetic. A triple stenosing ligature of the portal vein was used as the surgical technique of portal hypertension [22]. Under anaesthetic by inhalation of ethyl ether, the vena porta was dissected and three stenosing ligatures were attached in the superior, medial and inferior sections. The stenoses were calibrated by a simultaneous ligature (silk 3 –0) around the vena porta and an abocath 20G. The laparotomy was closed in two layers with catgut and silk 2 – 0. A laparotomy was performed on animals in the control group under ethylic ether anaesthesia and the vena porta was clamped for 5 min. Locomotor activity was assessed using infrared sensory units, which permitted quantification of the animal’s movements (Cibertec, Madrid, Spain). All animals were assessed over a period of 15 days in light/dark conditions with 12-h illumination (lights on from 08:00 to 20:00 h). Animals had ad lib access to food and water at all times.
The animals were anaesthetised and were vasculary perfused with 0.1 M phosphate buffer (pH 7.6). All the animals were sacrificed between 11:00 and 12:00 h. The brains were sectioned in the coronal plane in a thick section that included the entire hypothalamus, and coated with a cryoprotective gel (Tissue Freezing Medium, Leica, Nussloch, Germany). Then, they were frozen by immersion in Klea 134-a and cut in a cryostat into sections 20-Am thick. The sections were histochemically processed to reveal cytochrome oxidase activity (COX) using diaminobenzidine according to a modification of the Wong-Riley method [23] by Sukekawa [24]. Quantification of COX histochemical labelling was done by optical densitometry using an image processor (Leica, QWin, Wetzlar, Germany). A total of seven sections of the suprachiasmatic nucleus per animal were quantified in each of the groups. The results are expressed as the mean F standard deviation. The one-way ANOVA and Newman – Keuls Method were used for statistical comparison of the hepatic, corporal and testicular weights between groups. Two-way ANOVA and the Newman –Keuls Method were used for the statistical comparison of the locomotor activity and COX quantification. Actograms and periodograms were done using the chronobiology package ‘‘El Temps’’n (A. Diez-Noguera, Barcelona 1999, Universitat de Barcelona).
3. Results 3.1. Testing the PC and PH At the time of sacrifice, it was confirmed that surgical intervention of the portacaval shunt was correct and that no intestinal adherences were detected around the liver. Portal hypertension was determined by measuring the pressure in the ileocolic vein and the spleen by PE-50 catheters connected to a blood pressure measuring device. The values (mm Hg) of this pressure (ileocolic vein) were 9.27 F 0.65 in the CO group and 16.02 F 0.89 in rats with PH ( p < 0.001). Pressures were not measured in the PC group since this surgical procedure is known not to alter or to reduce the portal pressure in comparison to control rats. All the animals with portal hypertension had developed portosystemic collateral circulation [25]. In Table 1, the body weights and indices of the liver/body weight and testicles/body weight at the time of sacrifice were recorded. In rats with PH, there were no significant changes in body weight or in testicular weight/body weight index compared with the CO group, although there was a significant reduction ( p < 0.022) in the liver weight/body weight index (there is a slight hepatic insufficiency that causes this to decrease by 16%). In contrast, animals with PC present a significant decline in body weight and testicular weight/body weight index compared to the CO and PH groups ( p < 0.001 in both cases). Also, the liver weight/
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Table 1 Body weight, liver weight ratio and testis ratio
CO PH PC
Body weight (g)
Liver weight/body weight (ratio)
Testis weight/body weight (ratio)
395 F 37 402 F 41 291 F 32b
3.56 F 0.38 3.09 F 0.31a 2.61 F 0.42c,d
0.902 F 0.09 0.844 F 0.07 0.468 F 0.08b
Body weight and relative weight of the liver and testis for each 100 g of body weight (mean F SD). PC = portacaval shunt (n = 8), CO = control (n = 8), PH = portal hypertension (n = 8). a p = 0.022 with CO. b p < 0.001 with CO and PH. c p < 0.001 with CO. d p = 0.015 with PH.
body weight index is lower than that of the control group CO ( p < 0.001) and of the PH group ( p < 0.015), revealing severe hepatic atrophy. 3.2. Locomotor activity Fig. 1 shows the actogram (double-plot) of four animals from each group. A difference can be observed
Fig. 2. Graph showing the locomotor activity per hour of each group (mean of 15 days). There are no differences between the groups during the light on period but differences are observed during the light off period of activity (light off) between the PC group and the CO and PH groups ( p < 0.001).
between the animals with PC and the animals with PH and CO. The rhythm amplitude is smaller and there is also less adjustment to lighting conditions in the PC group. The periodogram analysis shows that all animals from the CO and PH groups present differences in this adjustment with phase advancement (23 – 45 min) or delay (24 –25 min). Only one of the animals with PC presents a period of 24 h. Fig. 2 shows the average locomotor activity per hour over the 15 days in each group. The statistical analysis (twoway ANOVA) shows significant differences in periods of light/dark activity ( p < 0.001). There were no significant differences between the groups in the light period or inactive period ( p>0.05), but there were differences in the locomotor activity of the PC group and the PH and CO groups in the dark phase ( p < 0.001). There were no statistically significant differences between groups PH and CO. 3.3. COX activity in the suprachiasmatic nucleus Fig. 3 shows the results of COX quantification in the suprachiasmatic nucleus (SCN). Comparison of the groups
Fig. 1. Actogram (double-plot) of locomotor activity over 15 days, in light – dark conditions (lights on from 08:00 to 20:00 h). CO: control (rats 1, 3, 4, 7), PC: portacaval shunt (rats 2, 3, 5, 8), PH: portal hypertension (rats 1, 2, 6, 8). There is a difference in the activity of animals with PC compared to the CO and PH groups.
Fig. 3. Quantification of COX activity in the suprachiasmatic nucleus (SCN). The PC group has less COX activity than the CO and PH groups ( p < 0.05).
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(two-way ANOVA) reveals statistically significant differences among them ( p < 0.006). There are also significant differences in the metabolic activity of the nuclei ( p < 0.001). Finally, there is a statistically significant interaction between group and nucleus ( p = 0.013). Animals with PC have less COX activity in the suprachiasmatic nucleus than groups PH and CO ( p = 0.032 and p = 0.015, respectively).
4. Discussion Hepatic atrophy associated to portacaval anastomosis in the rat represents an experimental model of hepatic insufficiency (Table 1). In turn, the increased portal pressure in rats with portal hypertension is associated with development of collateral portosystemic circulation. Both alterations, hepatic cirrhosis and portosystemic collateral circulation, have been described in human hepatic insufficiency [26,27]. For this reason, both experimental models could be considered to be complementary to study hepatic insufficiency. As can be observed from Table 1, animals with PC present severe hepatic insufficiency caused by complete derivation of portal blood to the bloodstream. In the case of triple ligature, portal pressure values, determined by measuring the pressure in the ileocolic vein and the spleen, confirm medium-term maintenance of a portal hypertension. Animals with PC have a reduction in liver of 50% and the PH group of 16%. For the testicles, the reduction is 37.6% in the PC group and 11% in the PH group, corresponding to a non-significant reduction in the PH animals compared to the CO groups. However, analysis of the diurnal locomotor activity in the PC and PH groups reveals that the same response is not achieved in both models. Hence, PH does not appear to affect the diurnal locomotor activity in light –dark lighting conditions. At the same time, COX activity of the SCN in the PH group is not altered in comparison to the control. In contrast, the PC group not only presents alterations in locomotor behaviour but also less COX activity in the SCN confirming previously published data [19]. In conclusion, our data confirm that rats submitted to triple ligature that acquire and maintain a high portal hypertension do not present altered diurnal locomotor activity or metabolic activity of the SCN. The difference in hepatic insufficiency of these animals compared to those with portacaval shunt could be the reason for the different response of the SCN.
Acknowledgements This research was supported by grants from MCYT BSO 2001-2757 and PR-01-GE-2. We wish to thank Piedad Burgos and Begon˜a Valdes for their technical help in this
experimental work, and Caroline Coope for translating the manuscript into English.
References [1] Hurwitz ES, Holman RC, Strine TW, Chorba TL. Chronic liver disease mortality in the United States, 1979 through 1989. Am J Public Health 1995;85:1256 – 60. [2] Corrao G, Ferrari P, Zambon A, Torchio P, Arico S, Decarli A. Trends of liver cirrhosis mortality in Europe, 1970 – 1989: age-period-cohort analysis and changing alcohol consumption. Int J Epidemiol 1997; 26:100 – 9. [3] Blei AT, Omary R, Butterworth RF. Animals of hepatic encephalopathies. In: Boulton A, Baker G, Butterworth RF, editors. Animal models of neurological disease, vol. II. Clifton, NJ: Humana Press; 1992. p. 183 – 222. [4] Myking AO, Halvorsen JF. A method for graded portal vein stenosis in rats: survival related to degree of stenosis. Eur Surg Res 1973;5: 454 – 7. [5] Sikuler E, Kravetz D, Groszmann RJ. Evolution of portal hypertension and mechanisms involved in its maintenance in a rat model. Am J Physiol 1985;248:G618 – 25. [6] Zaitoun AM, Apelqvist G, Wikell C, Al-Mardini H, Bengtsson F, Record CO. Quantitative studies of testicular atrophy following portacaval shunt in rats. Hepatology 1998;28:1461 – 6. [7] Gandhi CR, Murase N, Subbotin VM, Uemura T, Nalesnik M, Demetris AJ, et al. Portacaval shunt causes apoptosis and liver atrophy in rats despite increases in endogenous levels of major hepatic growth factors. J Hepatol 2002;37:340 – 8. [8] Dasarathy S, Mullen KD, Conjeevaram HS, Kaminsky-Russ K, Wills LA, McCullough AJ. Preservation of portal pressure improves growth and metabolic profile in the male portacaval-shunted rat. Dig Dis Sci 2002;47:1936 – 42. [9] Romeo JM, Lo´pez-Farre A, Martı´n-Paradero V, Lo´pez-Novoa JM. Effect of portacaval surgical anastomosis on systemic and splanchnic hemodynamics in portal hypertensive, cirrhotic rats. Can J Physiol Pharm 1988;66:1493 – 8. [10] Chan CC, Wang SS, Lee FY, Chang FY, Lin HC, Chu CJ, et al. Endothelin-1 induces vasoconstriction on portal-systemic collaterals of portal hypertensive rats. Hepatology 2001;33:816 – 20. [11] Ferenci P, Lockwood A, Mullen K, Tarter R, Weissenborn K, Blei AT. Hepatic encephalopathy—definition, nomenclature, diagnosis, and quantification: final report of the working party at the 11th World Congresses of Gastroenterology, Vienna, 1998. Hepatology 2002;35: 716 – 21. [12] Albrecht J, Jones AE. Hepatic encephalopathy: molecular mechanisms underlying the clinical syndrome. J Neurol Sci 1999;170: 138 – 46. [13] Beaubernard C, Salomo´n F, Bismuth H. Experimental hepatic encephalopathy. Study of the organization of a diurnal sep pattern in rats with portacaval anastomosis. Biomedicine 1980;32:76 – 80. [14] Beggtsson F, Nobin A, Falck B, Gage FH, Jeppsson B. Portacaval shunt in the rat: selective alterations in behavior and brain serotonin. Pharmacol Biochem Behav 1986;24:1611 – 6. [15] Blei AT, Zee P. Abnormalities of circadian rhythmicity in liver disease. J Hepatol 1998;29:832 – 5. [16] Colantonio D, Pasqualetti P, Casale R, Desiati P, Giandomenico G, Natali G. Atrial natriuretic peptide – renin – aldosterone system in cirrhosis of the liver: circadian study. Life Sci 1989;45:631 – 5. [17] Cordoba J, Cabrera J, Lataif L, Penev P, Zee P, Blei AT. High prevalence of sleep disturbance in cirrhosis. Hepatology 1998;27:339 – 45. [18] LeSauter J, Silver R. Localization of a suprachiasmatic nucleus subregion regulating locomotor rhythmicity. J Neurosci 1999;19: 5574 – 85. [19] Lopez L, Gonzalez-Pardo H, Cimadevilla JM, Cavas M, Aller MA,
L. Lopez et al. / Journal of the Neurological Sciences 212 (2003) 93–97 Arias J, et al. Cytochrome oxidase activity of the suprachiasmatic nucleus and pineal gland in rats with portacaval shunt. Exp Neurol 2002;173:275 – 82. [20] Lee SH, Fisher B. Portacaval shunt in the rat. Surgery 1961;50: 668 – 72. [21] Arias J, Andres-Trelles F, Alsasua A. Tecnica simplificada de produccion de anastomosis portocava en la rata. Arch Farmacol Toxicol 1977;3:205 – 14. [22] Monterde G, Rodriguez-Fabian G, Vara E, Lopez L, Arias J, Aller MA, et al. Increased plasma levels of corticosterone and prolactin and decreased T3 and T4 levels in short-term prehepatic portal hypertension in rats. Dig Dis Sci 2000;45:1865 – 71.
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[23] Wong-Riley MTT. Cytochrome oxidase: an endogenous metabolic marker for neuronal activity. Trends Neurosci 1989;12:94 – 101. [24] Sukekawa K. A fresh mount method for cytochrome oxidase histochemistry. Biotech Histochem 1991;1:99 – 101. [25] Rodrı´guez-Fabian G, Monterde G, Die´guez B, Aller MA, Arias J. Hipertensio´n portal a largo plazo por triple ligadura estenosante de la vena porta. An Med Interna (Madrid) 2000;17:137 – 41. [26] de Francis R, Primignani M. Natural history of portal hipertensio´n in patients with cirrhosis. Clin Liver Dis 2001;5:645 – 63. [27] Menon KV, Kamath PS. Managing the complications of cirrhosis. Mayo Clin Proc 2000;75:501 – 9.
Diurnal locomotor activity and oxidative metabolism of the suprachiasmatic nucleus in two models of hepatic insufficiency Laudino Lopez a,*, Jose M. Cimadevilla a, Maria A. Aller b, Jaime Arias b, M. Paz Nava c, Jorge L. Arias a a
Laboratorio de Psicobiologia, Facultad de Psicologia, Plaza Feijoo s/n, 33003 Oviedo, Spain b Catedra de Cirugia, Facultad de Medicina, UCM, Spain c Dpto. de Biologia Animal II, Facultad de Biologia, UCM, Spain
Received 30 October 2002; received in revised form 20 March 2003; accepted 21 March 2003
Abstract Subjects with hepatic cirrhosis develop alterations of several rhythmic behavioural and biochemical patterns. Since most cirrhotic patients combine portal hypertension and hepatic impairment, our work aims to assess the extent to which rhythmical changes can be due to hepatic insufficiency or portal hypertension. This was done using two experimental models in rats, portacaval shunt model (PC) and portal hypertension by a triple stenosing ligature of the portal vein (PH). We assess diurnal locomotor activity and determine the oxidative metabolism of the suprachiasmatic nucleus (SCN) by histochemical determination of cytochrome oxidase (COX). The results show that animals with PC have altered diurnal locomotor rhythm compared to control and PH rats ( p < 0.001). They also present lower COX activity in the SCN ( p < 0.05). We conclude that rhythmic alterations are due to hepatic insufficiency and not to portal hypertension. D 2003 Elsevier Science B.V. All rights reserved. Keywords: Cytochrome oxidase; Hepatic encephalopathy; Suprachiasmatic nucleus; Circadian rhythm; Hepatic cirrhosis
1. Introduction Hepatic insufficiency, mainly due to hepatic cirrhosis, is one of the main medical problems in the western world today. In addition to producing a high mortality, it also requires costly treatment and transplantation as a last resort [1,2]. Owing to this situation, several experimental models have been developed to assess and study the consequences derived from liver disease. These models include the portacaval shunt [3] and extrahepatic portal hypertension by stenosis of the vena porta [4,5]. Both procedures have confirmed value to increase our knowledge of the consequences derived from hepatic insufficiency. However, none of the models correctly reproduces the real situation of subjects with hepatic cirrhosis. In human clinical cases, the tendency is for both these models to converge: severe hepatic insufficiency (cirrhosis) that leads to portal hypertension. In experimental models, however, we have severe
* Corresponding author. Tel.: +34-985104188; fax: +34-985104144. E-mail address: [email protected] (L. Lopez).
hepatic insufficiency without portal hypertension (portacaval shunt) or portal hypertension without severe hepatic insufficiency. The portacaval anastomosis model will originate a severe hepatic and testicular atrophy [6,7], whereas the portal pressure is different to that developed by the portal hypertension model. Animals with portacaval shunt present no resistance to the emerging blood flow and, therefore, have a reduced or unchanged portal pressure compared to control rats [8,9]. In contrast, rats with portal hypertension present resistance to the emerging flow for two reasons: stenosis of the vena porta and portosystemic colateral circulation, with the ability to contract [10]. On the other hand, it is well known that subjects with cirrhosis develop a neuropsychiatric syndrome called hepatic encephalopathy [3,11,12]. Also, in experimental models of hepatic insufficiency, neurological and behavioural alterations have been observed that could be similar to those found in human hepatic encephalopathy [13,14]. One of the characteristics of this syndrome is an alteration of the different rhythmical biochemical and behavioural patterns [15 –17]. Underlying these rhythmical alterations, one can
0022-510X/03/$ - see front matter D 2003 Elsevier Science B.V. All rights reserved. doi:10.1016/S0022-510X(03)00105-9
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find the suprachiasmatic nucleus [18], the main controller of biological rhythms, which presents a diminished oxidative metabolism in rats with PC [19]. The objective of our work was to establish whether the rhythmical alterations in hepatic encephalopathy and those demonstrated in PC of the rat are also caused by portal hypertension or are merely a consequence of severe hepatic insufficiency. To do this, rats with two models of hepatic insufficiency were used and the circadian rhythm of the activity and oxidative metabolism of the suprachiasmatic nucleus (SCN) were studied by histochemical labelling of cytochrome oxidase (COX).
2. Material and methods Twenty-four Wistar rats from the vivarium of Oviedo University, Oviedo, Spain, were used. The mean weight of rats at the start of the experiments was 240 F 25 g. All the animals had ad libitum access to food and tap water and were maintained at constant room temperature (20 F 2 jC), with a relative humidity of 65 F 10% and artificial light– dark cycle of 12 h (08:00– 20:00 h/20:00 –08:00 h). The 24 animals were distributed into three groups (n = 8 each group): animals with a portacaval shunt and 35 postoperative days (PC group), animals with portal hypertension and 45 postoperative days (PH group) and a group of control animals with a pseudo-intervention and 45 days postoperative evolution (CO). The pseudo-intervention consisted in a laparotomy and clamping of the vena porta for 5 min. All the experiments were performed according to the European Communities Council Directive 86/609/ EEC. The end-to-side portacaval shunt operation was performed according to the technique described by Lee and Fischer [20]. This was modified slightly by total clamping of the inferior vena cava during PC performance [21]. The total time in which the portal vein and inferior vena cava were clamped for anastomosis was less than 15 min. The operations were performed under ethylic ether anaesthetic. A triple stenosing ligature of the portal vein was used as the surgical technique of portal hypertension [22]. Under anaesthetic by inhalation of ethyl ether, the vena porta was dissected and three stenosing ligatures were attached in the superior, medial and inferior sections. The stenoses were calibrated by a simultaneous ligature (silk 3 –0) around the vena porta and an abocath 20G. The laparotomy was closed in two layers with catgut and silk 2 – 0. A laparotomy was performed on animals in the control group under ethylic ether anaesthesia and the vena porta was clamped for 5 min. Locomotor activity was assessed using infrared sensory units, which permitted quantification of the animal’s movements (Cibertec, Madrid, Spain). All animals were assessed over a period of 15 days in light/dark conditions with 12-h illumination (lights on from 08:00 to 20:00 h). Animals had ad lib access to food and water at all times.
The animals were anaesthetised and were vasculary perfused with 0.1 M phosphate buffer (pH 7.6). All the animals were sacrificed between 11:00 and 12:00 h. The brains were sectioned in the coronal plane in a thick section that included the entire hypothalamus, and coated with a cryoprotective gel (Tissue Freezing Medium, Leica, Nussloch, Germany). Then, they were frozen by immersion in Klea 134-a and cut in a cryostat into sections 20-Am thick. The sections were histochemically processed to reveal cytochrome oxidase activity (COX) using diaminobenzidine according to a modification of the Wong-Riley method [23] by Sukekawa [24]. Quantification of COX histochemical labelling was done by optical densitometry using an image processor (Leica, QWin, Wetzlar, Germany). A total of seven sections of the suprachiasmatic nucleus per animal were quantified in each of the groups. The results are expressed as the mean F standard deviation. The one-way ANOVA and Newman – Keuls Method were used for statistical comparison of the hepatic, corporal and testicular weights between groups. Two-way ANOVA and the Newman –Keuls Method were used for the statistical comparison of the locomotor activity and COX quantification. Actograms and periodograms were done using the chronobiology package ‘‘El Temps’’n (A. Diez-Noguera, Barcelona 1999, Universitat de Barcelona).
3. Results 3.1. Testing the PC and PH At the time of sacrifice, it was confirmed that surgical intervention of the portacaval shunt was correct and that no intestinal adherences were detected around the liver. Portal hypertension was determined by measuring the pressure in the ileocolic vein and the spleen by PE-50 catheters connected to a blood pressure measuring device. The values (mm Hg) of this pressure (ileocolic vein) were 9.27 F 0.65 in the CO group and 16.02 F 0.89 in rats with PH ( p < 0.001). Pressures were not measured in the PC group since this surgical procedure is known not to alter or to reduce the portal pressure in comparison to control rats. All the animals with portal hypertension had developed portosystemic collateral circulation [25]. In Table 1, the body weights and indices of the liver/body weight and testicles/body weight at the time of sacrifice were recorded. In rats with PH, there were no significant changes in body weight or in testicular weight/body weight index compared with the CO group, although there was a significant reduction ( p < 0.022) in the liver weight/body weight index (there is a slight hepatic insufficiency that causes this to decrease by 16%). In contrast, animals with PC present a significant decline in body weight and testicular weight/body weight index compared to the CO and PH groups ( p < 0.001 in both cases). Also, the liver weight/
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Table 1 Body weight, liver weight ratio and testis ratio
CO PH PC
Body weight (g)
Liver weight/body weight (ratio)
Testis weight/body weight (ratio)
395 F 37 402 F 41 291 F 32b
3.56 F 0.38 3.09 F 0.31a 2.61 F 0.42c,d
0.902 F 0.09 0.844 F 0.07 0.468 F 0.08b
Body weight and relative weight of the liver and testis for each 100 g of body weight (mean F SD). PC = portacaval shunt (n = 8), CO = control (n = 8), PH = portal hypertension (n = 8). a p = 0.022 with CO. b p < 0.001 with CO and PH. c p < 0.001 with CO. d p = 0.015 with PH.
body weight index is lower than that of the control group CO ( p < 0.001) and of the PH group ( p < 0.015), revealing severe hepatic atrophy. 3.2. Locomotor activity Fig. 1 shows the actogram (double-plot) of four animals from each group. A difference can be observed
Fig. 2. Graph showing the locomotor activity per hour of each group (mean of 15 days). There are no differences between the groups during the light on period but differences are observed during the light off period of activity (light off) between the PC group and the CO and PH groups ( p < 0.001).
between the animals with PC and the animals with PH and CO. The rhythm amplitude is smaller and there is also less adjustment to lighting conditions in the PC group. The periodogram analysis shows that all animals from the CO and PH groups present differences in this adjustment with phase advancement (23 – 45 min) or delay (24 –25 min). Only one of the animals with PC presents a period of 24 h. Fig. 2 shows the average locomotor activity per hour over the 15 days in each group. The statistical analysis (twoway ANOVA) shows significant differences in periods of light/dark activity ( p < 0.001). There were no significant differences between the groups in the light period or inactive period ( p>0.05), but there were differences in the locomotor activity of the PC group and the PH and CO groups in the dark phase ( p < 0.001). There were no statistically significant differences between groups PH and CO. 3.3. COX activity in the suprachiasmatic nucleus Fig. 3 shows the results of COX quantification in the suprachiasmatic nucleus (SCN). Comparison of the groups
Fig. 1. Actogram (double-plot) of locomotor activity over 15 days, in light – dark conditions (lights on from 08:00 to 20:00 h). CO: control (rats 1, 3, 4, 7), PC: portacaval shunt (rats 2, 3, 5, 8), PH: portal hypertension (rats 1, 2, 6, 8). There is a difference in the activity of animals with PC compared to the CO and PH groups.
Fig. 3. Quantification of COX activity in the suprachiasmatic nucleus (SCN). The PC group has less COX activity than the CO and PH groups ( p < 0.05).
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(two-way ANOVA) reveals statistically significant differences among them ( p < 0.006). There are also significant differences in the metabolic activity of the nuclei ( p < 0.001). Finally, there is a statistically significant interaction between group and nucleus ( p = 0.013). Animals with PC have less COX activity in the suprachiasmatic nucleus than groups PH and CO ( p = 0.032 and p = 0.015, respectively).
4. Discussion Hepatic atrophy associated to portacaval anastomosis in the rat represents an experimental model of hepatic insufficiency (Table 1). In turn, the increased portal pressure in rats with portal hypertension is associated with development of collateral portosystemic circulation. Both alterations, hepatic cirrhosis and portosystemic collateral circulation, have been described in human hepatic insufficiency [26,27]. For this reason, both experimental models could be considered to be complementary to study hepatic insufficiency. As can be observed from Table 1, animals with PC present severe hepatic insufficiency caused by complete derivation of portal blood to the bloodstream. In the case of triple ligature, portal pressure values, determined by measuring the pressure in the ileocolic vein and the spleen, confirm medium-term maintenance of a portal hypertension. Animals with PC have a reduction in liver of 50% and the PH group of 16%. For the testicles, the reduction is 37.6% in the PC group and 11% in the PH group, corresponding to a non-significant reduction in the PH animals compared to the CO groups. However, analysis of the diurnal locomotor activity in the PC and PH groups reveals that the same response is not achieved in both models. Hence, PH does not appear to affect the diurnal locomotor activity in light –dark lighting conditions. At the same time, COX activity of the SCN in the PH group is not altered in comparison to the control. In contrast, the PC group not only presents alterations in locomotor behaviour but also less COX activity in the SCN confirming previously published data [19]. In conclusion, our data confirm that rats submitted to triple ligature that acquire and maintain a high portal hypertension do not present altered diurnal locomotor activity or metabolic activity of the SCN. The difference in hepatic insufficiency of these animals compared to those with portacaval shunt could be the reason for the different response of the SCN.
Acknowledgements This research was supported by grants from MCYT BSO 2001-2757 and PR-01-GE-2. We wish to thank Piedad Burgos and Begon˜a Valdes for their technical help in this
experimental work, and Caroline Coope for translating the manuscript into English.
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