comparison to body weight in the sham-operated animals, this paper aims to evaluate the possible consequences of the sham operation. It concludes that ...
Portacaval shunt control animals: physiological consequences derived from the sham operation L. Lopez', P. Burgos1, L. J. Santin1, A. Begega1, J. Arias2, L. Lorente2 & J. L. Arias' lpsychobiology Laboratory, Psychology Faculty, University of Oviedo, Spain and 2Surgery Department, Medical Faculty, Complutense University of Madrid, Spain
Summary The portacaval shunt in the rat is a frequently used experimental model of porto systemic encephalopathy. Among other consequences of this surgical preparation is an important decrease in hepatic and testicular volume. Different sham-operation methods including a laparotomy were used as controls in each case. Given that the liver volume varies greatly in comparison to body weight in the sham-operated animals, this paper aims to evaluate the possible consequences of the sham operation. It concludes that control animals without manipulation, in addition to the respective controls of portacaval shunt, should be used in every case. Keywords
Portacaval shunt; hepatic encephalopathy; portosystemic encephalopathy; rat
Patients with hepatic failure and/or portosystemic shunts generally develop a series of central nervous system (eNS) disorders included in the hepatic encephalopathy syndrome (Collins & Lloyd 1992, Shafer & Jones 1990). Fulminant hepatic encephalopathy and portosystemic encephalopathy are differentiated according to the origin of the hepatic failure and disease evolution. On an experimental level, a series of models that reproduces both types of hepatic encephalopathy has been developed (Blei et al. 1992). Among the models that reproduce acute hepatic failure are those that involve an exclusion of the hepatic function (hepatectomy or devascularization of the liver) or those that use hepatotoxins, such as D-galactosamine or thioacetamide. Among the models that reproduce a portosystemic encephalopathy are experimental cirrhosis (intoxication by carbon tetrachloride) or the performance of an end-to-side portacaval shunt [PCS). Correspondence to: Dr large L. Arias, Laboratorio de Psicobiologla, Faculwd de Psicologla, cl Aniceto Sela sin, 33005 Oviedo, Spain Accepted 25 September 1996
The experimental procedure of the anastomosis between the portal vein and inferior vena cava in the rat was developed by Lee and Fisher in 1964. Since then, the portacaval shunt has been used as an animal model of portosystemic encephalopathy. The postoperative consequences derived from the shunt are minimum or null body weight increase, marked hepatic atrophy (Girard & Butterworth 1992) and testicular atrophy in male rats (Dordal et al. 19671. Together with the verification of the shunt itself, these three parameters constitute an index of the good performance of the surgery (Mullen & McCullough 1989). In this way, the liver weight/body weight ratio and the testis weight/body weight ratio represent an index of changes that occur after a PCS because it is considered that both the relative weight of the liver and the testis regarding body weight are stable in animals maintained under the same conditions, and even more so, if they have the same body weight. However, as seen in Table I, while the PCS animals have very homogeneous hepatic and Laboratory Animals (1997) 31, 225-230
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Table 1
Liver/body ratio and testes/body ratio in sham-operated and PCSrats
Reference
Rat
Conjeevaram et al. (1994a) Conjeevaram et al. (1994b) Coy et al. (1992)
S-D
Fogel et al. (1991)
W
S-D S-D
Martin & Baettig (1980) S-D Schramek et at. (1993) S-D Schroder et al. (1985)
S-D
Therrien
et al. (1994)
5-D
VanThiel et al. (1983)
W 5-D
Days Sham
Weight Sham
Liver Sham
Liver PCS
Testes Sham
cava 21
359
2.62
2.20
0.88 (a) 0.86 (a)
cava 21
329
4.61
3.15
0.97
0.74
Portal vein/inferior cava 49 (15 min) Laparotomy and 40 manipulation 120 Portal vein (12 min) 84 Portal vein/inferior 42 cava (15 min) 70 Splenocava 1/ inferior 20
418
3.51
2.16
557 436 425 293
2.7 3.00 4.73 3.1 2.94 3.3
1.9 2.2 2.47 1.35 1.59 2.3
0.93 1.05 (a) 1.07 (a)
0.52 0.55 (a) 0.58 (a)
cava 26
443
4.3
2.8
55
351 564
4.72 (a)
3.78 (a)
0.90 (a)
0.39 (a)
3.36
2.18
0.65
0.50
Sham operation Portal vein/inferior (5 min) Portal vein/inferior (5 min)
Testes PCS
cava (20 min)
VanThiel et al. (1990)
Portal vein/inferior (15 min) Portal vein (5 min)
55
(a) The testis weight and liver weight ratios are derived from the body weight and from the weights of the liver and testis. PCS=portacaval shunt, Sham=animals that are sham operated with different methods, Liver=liver/body ratio (g) ( x 100), Testes=testes/body ratio (g) (x 1001.W=Wistar, S-D=Sprague-Dawley, Days=post-operative days, Weight= body weight of the sham-operated after the post-operative period (g)
testicular atrophy, the data shown for the surgical control groups (with a sham operation that always includes a laparotomy) vary greatly in the liver compared to body weight ratio, perhaps as a consequence of the sham operation carried out. For this reason, our paper is intended to verify if the liver weight/body weight ratio can be considered as a stable and valid ratio to compare the PCS model in the same way as the testis weight/body weight ratio and also if the variations that appear in the shamoperated groups in Table 1 can be due to the effects of the sham-operation model itself. To do so, we have used animals without any type of manipulation or abdominal opening and animals with two different models of PCS sham operation (experiment 1). Furthermore, weight gain in animals with PCS surgery is an index of the correct evolution of the shunt and one of the parameters that should be considered in experimental models (Mullen & McCullough 1989). In this sense, we also wanted to evaluate the liver/body weight ratio and the testis/body weight ratio in animals that had
or had not gained weight 60 days after the PCS (experiment 2).
Material and methods Animals A total sample of 71 male Wistar rats (Vivarium of the University of Oviedo, Spain) was used. Their weights ranged from 250-350 g at the onset of the experiment. All the animals were maintained under constant conditions. Room temperature was kept at 23°C ± 1° with relative humidity of 65 ± 10% and an arti£iciallight-dark cycle of 12 h (08:00-20:00 h/20:00-08:00 h). An air conditioning system guaranteed air conditioning and air renewal in the room. The animals were placed in individual boxes for 7 days after the surgical operation (PCS or sham). After that, two or three rats were housed together in polycarbonate cages (42 x 26 x 15, Letica, Spain) with a wood base preparation (Ultrasorb, Panlab, Spain). The cages were changed approximately every 4 days during the studies. All the animals had free access to
Sham-operated and portacaval shunt
.
food (rat/mouse A04 maintenance diet, Panlab, Spain) and water.
Experimental procedures The distribution of the groups for experiment 1 was the following (group=IO): one control group without previous manipulation (group et two groups with sham operations of pes, one with 15 min of clamping of the portal vein and inferior vena cava (group SIS) and another with clamping of the portal vein for 5 min (group S5), one group with portacaval shunt (group Pq and another group with a sham-brain lesion (group SL). The postoperative period after the pes intervention or the different sham operations was 60 days. The portacaval shunt operation was performed according to the method described by Lee and Fisher (1964) with a slight modification (Arias et al. 1977) and consisted in the total clamping of the inferior vena cava during the pes performance. The time for which the portal vein and inferior vena cava were clamped for anastomosis did not exceed 15 min. The operations were done under anaesthesia by inhalation of diethyl ether (Probus, Spain). The groups with a pes sham operation were performed under anaesthesia with equithesin (pentobarbital, chloral hydrate and magnesium sulfate, 3 ml/kg i.p.). In both groups, a bilateral subcostallaparotomy with prolongation to the xiphoid apophysis followed by the dissection of the vena porta and its later clamping for 5 min or the dissection of the portal vein and inferior vena cava in its infrahepatic path with the later clamping for 15 min was performed in both groups, as was done in the pes operation. The operative field was irrigated with saline solution during the intervention. Finally, the laparotomies are closed by continuous sutura on two planes. The differences found between the control group and the sham-operation groups required a new control group of the anaesthesia used in the case of the sham-operated animals. Thus, another group was used with a sham-brain lesion caused by introducing an electrode into the medial mammillary nucleus located in the posterior hypothalamus under
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equithesin anaesthesia (Sziklas & Petrides 1993). Based on the results obtained in our laboratory (data not shown), the animals with pes at 60 days after the operation were distributed into two different groups of seven animals for experiment 2: those which increased their weight 42% (PC(+))and those in which the body weight increased only 10% (PC(-)). The animals PC( -) had a mean weight (±SD) at the time of the operation of 317±14, and their weight was 350±27 at 60 days, while those from group PC(+) had an initial weight of 303±39 and a final weight of 433±58. A control group of seven animals with no type of manipulation was also used in this experiment. The pes intervention was developed according to the method described in experiment 1. Statistics The statistical analysis was performed with Statistica for Windows 4.0 (Statsoft, Inc. 1993). The analysis of variance (one wayl with Tukey's test was used to compare the data in experiments. The minimum level of statistical significance was set at P0.9). However, groups SIS and SS had a statistically significant difference of P
