Mild hypothermia modifies ammonia-induced brain edema in rats after portacaval anastomosis

BACKGROUND & AIMS: The pathogenesis of brain edema in fulminant hepatic failure is still unresolved. Mild hypothermia (33 degrees-35 degreesC) can ameliorate brain edema after traumatic brain injury. We evaluated mild hypothermia in a model of ammonia-induced brain edema in which accumulation of brain glutamine has been proposed as a key pathogenic factor. METHODS: After portacaval anastomosis, anesthetized rats were infused with ammonium acetate at 33 degrees, 35 degrees, and 37 degreesC or vehicle at 37 degreesC. Water and glutamine levels in the brain, cardiac output, and regional and cerebral hemodynamics were measured when intracranial pressure increased 3-4-fold (ammonia infusion at 37 degrees) and matched times (other groups). RESULTS: Mild hypothermia reduced ammonia-induced brain swelling and increased intracranial pressure. Brain glutamine level was not decreased by hypothermia. Brain edema was accompanied by a specific increase in cerebral blood flow and oxygen consumption, which were normal in both hypothermic groups. When the ammonia infusion was continued in hypothermic rats, plasma ammonia levels continued to increase and brain swelling eventually developed. CONCLUSIONS: Mild hypothermia delays ammonia-induced brain edema. In this model, an increase in cerebral perfusion is required for brain edema to become manifest. Mild hypothermia could be tested for treatment of intracranial hypertension in fulminant hepatic failure.     

Cerebral blood flow and the development of ammonia-induced brain edema in rats after portacaval anastomosis.

Two mechanisms may account for brain edema in fulminant hepatic failure: the osmotic effects of brain glutamine, a product of ammonia detoxification, and a change of cerebral blood flow (CBF). We have shown brain edema, a marked increase in brain glutamine, and a selective rise in CBF in rats after portacaval anastomosis receiving an ammonia infusion. In this study, we inhibited the activity of glutamine synthetase with methionine-sulfoximine (MSO) and examined ammonia levels, brain water and CBF. Four groups received either a continuous ammonium acetate or control infusion; half of the animals had been pretreated with MSO or vehicle. The ammonia group exhibited brain edema (79.97 +/- 0.04 vs. 81.11 +/- 0. 13% water), an increase in cerebrospinal fluid (CSF) glutamine (1.29 +/- 0.21 vs. 2.84 +/- 0.39 mmol/L) and CBF (63 +/- 11 vs. 266 +/- 45 mL/min/100 g brain). When MSO was added to the ammonia infusion, ammonia levels rose further (928 +/- 51 vs. 1,293 +/- 145 mmol/L, P <.05) but CSF glutamine decreased (2.84 +/- 0.39 vs. 1.61 +/- 0.2 mmol/L, P <.01). Brain edema (80.48 +/- 0.11%) and cerebral hyperemia (140 +/- 25 mL/min/100 g brain) were significantly ameliorated in the ammonia plus MSO group. Brain output of circulating nitric oxide (NO(x)) was increased in the ammonia-infused group but normalized in the ammonia plus MSO group. In this model, the rise of CBF reflects intracranial events that occur after glutamine synthesis. Activation of nitric oxide synthase in the brain could account for these findings.     

Cerebral hyperemia and nitric oxide synthase in rats with ammonia-induced brain edema

BACKGROUND/AIM: Brain edema is a common fatal complication in acute liver failure. It is related to an acute change in brain osmolarity secondary to the glial accumulation of glutamine. Since high cerebral blood flow (CBF) precedes cerebral herniation in fulminant hepatic failure we first determined if an increase in brain water and glutamine are prerequisite to a rise in CBF in a model of ammonia-induced brain edema. Secondly, we determined if such a cerebral hyperperfusion is mediated by nitric oxide synthase (NOS). METHODS: Male rats received an end-to-side portacaval anastomosis (PCA). At 24 h, they were anesthetized with ketamine and infused with ammonium acetate (55 microM/kg per min). Studies were performed at 60, 90, 120, 150 and 180 min after starting the ammonia infusion and once the intracranial pressure had risen three-fold (mean 210'). Brain water (BW) was measured using the gravimetry method and CBF with the radioactive microsphere technique. Glutamine (GLN) in the CSF was sampled via a cisterna magna catheter. The neuronal NOS was specifically inhibited by 1-2-trifluoromethylphenyl imidazole (TRIM, 50 mg/kg intraperitoneally) and in separate studies nonspecifically by N-omega-nitro-L-arginine (L-NNA, 2 microg/kg per min intravenously) RESULTS: At 90', brain water was significantly increased (P < 0.015) as compared to the 60' group while CBF was significantly different at 150'. A significant correlation was observed between values of CBF and brain water (r = 0.88, n = 36, P < 0.001). Administration of either TRIM or L-NNA did not prevent the development of cerebral hyperperfu. sion and edema. CONCLUSION: We observed that cerebral hyperemia follows an initial rise in brain water content, rather than in the cerebrospinal fluid concentration of glutamine. The rise in CBF further correlated with brain water accumulation and was of critical importance for the development of intracranial hypertension. The unique mechanism for the rise in CBF in hyperammonemia was not prevented by NOS inhibition indicating that NO is not the mediator of high CBF and intracranial hypertension.     

Hyperplastic foci in the atrophic liver of rats after portacaval anastomosis

Hyperplastic focal areas were investigated in livers of male rats 1 and 9 months after portacaval anastomosis (PCA) by light and electron microscopy. These alterations, predominantly found in periportal areas, were characterized by light microscopy as clusters of enlarged hepatocytes along narrowed sinusoids, contrasting in the remaining liver acinus with smaller hepatocytes along widened sinusoids. No differences were observed between 1 and 9 months PCA except for glycogen content, which was homogeneously distributed in the liver at 1 month but completely lacking in foci at 9 months. The most striking ultrastructural alterations were the sinusoids delimited in these hyperplastic areas by a thickened barrier consisting of thick endothelial cells encircled by numerous subendothelial processes of the perisinusoidal fat-storing cells. Deep and widened recesses of the sinusoidal lumen separated the two-cell-thick plates of the hyperplastic cells. Hepatocytes in foci, thought to represent regenerative areas, tend to increase their exchange surface. Their progressive loss in glycogen and their two-cell-thick plates architecture should be in favour of a potential malignancy. However, the spontaneous evolution of these foci which do not necessarily give rise to nodules, as well as the lack of other features of transformation, do not support this possibility.     

Sex hormones and fertility following portacaval anastomosis in rats

Portocaval anastomosis (PCA) in the normal male rat causes profound alterations in testicular morphology and function and in plasma levels of sex steroids as well as of gonadotropins. Testicular atrophy is accompanied by a significant decrease of plasma testosterone (0.03 ng/ml versus 0.99 ng/ml) and an increase of estradiol (76.2 pg/ml versus 39.7 pg/ml) and estrone (68. pg/ml versus 45.5 pg/ml). Plasma levels of gonadotropins (LH, FSH) and prolactin are lowered too (LH: 15 ng/ml versus 28.5 ng/ml, FSH:119 versus 182 ng/ml, prolactin:53 versus 109 ng/ml). The altered sex hormone metabolism is reflected in marked changes of the morphology and nucleic acid content of the metabolizing organs i.e. liver, gonades and kidney. The results of this study are consistent with the hypothesis that portosystemic shunt per se plays an important role in the pathogenesis of the disturbed metabolism of sex hormones observed in patients with liver cirrhosis and portal hypertension.     

Phosphate-activated glutaminase activity is enhanced in brain, intestine and kidneys of rats following portacaval anastomosis

AIM: To assess whether portacaval anastomosis (PCA) in rats affects the protein expression and/or activity of glutaminase in kidneys, intestines and in three brain areas of cortex, basal ganglia and cerebellum and to explain the neurological alterations found in hepatic en-cephalopathy (HE). METHODS: Sixteen male Wistar rats weighing 250-350 g were grouped into sham-operation control (n=8) or portacaval shunt (n=8). Twenty-eight days after the procedure, the animals were sacrificed. The duodenum, kidney and brain were removed, homogenised and mitochondria were isolated. Ammonia was measured in brain and blood. Phosphate-activated glutaminase (PAG) activity was determined by measuring ammonia production following incubation for one hour at 37℃with O-phthalaldehyde (OPA) and specific activity expressed in units per gram of protein (μkat/g of protein). Protein expression was measured by immunoblotting. RESULTS: Duodenal and kidney PAG activities together with protein content were significantly higher in PCA group than in control or sham-operated rats (duodenum PAG activity was 976.95±268.87μkat/g of protein in PCA rats vs 429.19±126.92.μkat/g of protein in sham-operated rats; kidneys PAG activity was 1259.18±228.79μkat/g protein in PCA rats vs 669.67±400.8μkat/g of protein in controls, P<0.05; duodenal protein content: 173% in PCA vs sham-operated rats; in kidneys the content of protein was 152% in PCA vs sham-operated rats). PAG activity and protein expression in PCA rats were higher in cortex and basal ganglia than those in sham-operated rats (cortex: 6646.6±1870.4μkat/g of protein vs 3573.8±2037.4μkat/g of protein in control rats, P<0.01; basal ganglia, PAG activity was 3657.3±1469.6μkat/g of protein in PCA rats vs 2271.2±384μkat/g of protein in sham operated rats, P<0.05; In the cerebellum, the PAG activity was 2471.6±701.4μkat/g of protein vs 1452.9±567.8μkat/g of protein in the PCA and sham rats, respectively, P<0.05; content of protein: cerebral cortex: 162%±40% vs 100%±26%, P< 0.009; and basal ganglia: 140%±39% vs 100%±14%, P<0.05; but not in cerebellum: 100%±25% vs 100%±16%,P=ns). CONCLUSION: Increased PAG activity in kidney and duodenum could contribute significantly to the hyperam-monaemia in PCA rats, animal model of encephalopathy. PAG is increased in non-synaptic mitochondria from the cortex and basal ganglia and could be implicated in the pathogenesis of hepatic encephalopathy. Therefore, PAG could be a possible target for the treatment of HE or liver dysfunction.     

Lipoprotein catabolism in rats with portacaval anastomosis on various diets

The catabolism of lipoproteins was measured in rats with a portacaval anastomosis and in intact control rats. Radioiodinated rat high density lipoproteins or human low density lipoproteins, the major cholesterol-bearing lipoproteins in rat or man respectively, were injected intravenously into rats. More than 90% of trace amounts of these lipoproteins were removed from plasma of rats with portacaval anastomosis and controls on standard chow at closely similar rates within 24 h. Also, [125I]high density lipoproteins left the plasma at comparable rates in controls and rats with portacaval anastomosis, whether fed with a cholesterol-free chow or a carbohydrate-rich lard chow. These dietary regimens were employed to avoid artifacts through a different development of the body weight in operated and control rats. A standard laboratory chow ad libitum led to weight loss in rats with portacaval anastomosis. Pair-fed with a cholesterol-free chow both groups of rats kept the same weight, but only with a carbohydrate-rich lard chow could the natural weight gain be achieved. In all rats with portacaval anastomosis liver weights were reduced and serum cholesterol decreased by 21-31% with the major change in high density lipoproteins. The findings suggest that cholesterol concentrations are not likely to be lowered in rats with portacaval anastomosis by enhanced lipoprotein catabolism.     

Effect of portacaval or mesentericocaval anastomosis on cholesterol metabolism in rats

Portacaval anastomosis (PCA) lowered by 50% the cholesterol concentration in the plasma of rats. The free and esterified cholesterol contents in the lipoproteins were decreased with the very low density lipoproteins most affected (-85%). Cholesterol concentration as total content in the liver was reduced. The major change in the cholesterol metabolism, as studied with an isotopic equilibrium method, was the decrease in the intestinal absorption coefficient of dietary cholesterol (56.0 +/- 2.7% instead of 73.3 +/- 1.9% in controls). The rate of cholesterol transformation into bile acids was decreased (10.5 +/- 0.3 vs 14.5 +/- 0.5 mg/day/rat in controls). The rate of internal secretion of cholesterol was slightly reduced while the rate of fecal external secretion was increased, suggesting that the synthesis of cholesterol by extra-digestive tissues (including liver) was reduced after PCA. The effects of PCA on cholesterol metabolism were similar to those described for glucagon administration. Since this shunt results in hyperglucagonemia, it is suggested that this hormonal perturbation was the main factor involved in the modifications of cholesterol metabolism after PCA. Moreover, mesentericocaval anastomosis, which shunts only the intestinal blood and allows the pancreatic hormones a normal transport through the liver, did not significantly modify cholesterol metabolism. Only cholesterolemia (-28%) and the absorption coefficient of dietary cholesterol (66.0 +/- 2.3%) were slightly reduced.     

Regional blood-brain barrier transport of glucose after portacaval anastomosis

The regional influx of glucose across the blood-brain barrier was measured in rats 5 to 6 weeks after a portacaval anastomosis or sham operation. D-[¹⁴C]Glucose was infused intravenously for 15sec while arterial blood was sampled continuously for measurement of plasma radioactivity and glucose concentration. Brain tissue radioactivity was measured by quantitative autoradiography. Glucose influx and plasma clearance (permeability times surface area;PS) were calculated from the net disintegrations per minute per gram in brain, the plasma radioactivity integral, and the plasma glucose concentration. In shunted rats influx was decreased by about 22% (in the brain as a whole) compared to that in controls. This decrease was almost entirely due to the decrease in plasma glucose concentrations (27%). ThePS, normalized to take plasma concentrations into account, showed a slight decrease in most of the brain except the telencephalon. For the brain as a whole this decrease amounted to 11%. The regionalPS and glucose utilization are known to be coupled and the relationship between these was the same in sham-operated and shunted rats. The decrease inPS observed in shunted rats was commensurate with their lower rates of glucose use; thus, the transport process of glucose from plasma to brain appeared to be unaffected by portacaval shunting.     

Inhibition of aromatase improves nutritional status following portacaval anastomosis in male rats

BACKGROUND/AIMS: The portacaval anastamosis (PCA) rat is a model of the nutritional and endocrine consequences of liver cirrhosis. We hypothesized that failure to gain body weight in the PCA rat was the consequence of low testosterone levels and will be reversed by 4-hydroxyandrostenedione, a specific aromatase inhibitor. METHODS: Male Sprague-Dawley rats subjected to an end-to-side PCA or sham surgery were administered either 4-hydroxyandrostenedione or vehicle. Food intake, food efficiency, and body weight were measured, animals sacrificed 3 weeks after surgery, visceral organs harvested and plasma sex steroids measured. Hepatic RNA was extracted and dot blots done to quantify the expression of sex hormone dependent enzymes 16alpha hydroxylase and 15beta hydroxylase. RESULTS: Growth, food intake, food efficiency and plasma testosterone were lower and plasma estradiol higher in PCA than sham rats. Hepatic expression of testosterone driven 16alpha hydroxylase was lower and estradiol driven 15beta hydroxylase higher in PCA rats. These alterations were reversed by 4-hydroxyandrostenedione. CONCLUSIONS: These observations demonstrate that use of aromatase inhibitor reverses the nutritional and endocrine abnormalities in the PCA rat and suggest that this approach may be useful in cirrhotic patients.     

Region-selective reductions in activities of glutamine synthetase in rat brain following portacaval anastomosis

Portacaval anastomosis in the rat results in liver atrophy, sustained hyperammonemia and mild encephalopathy. Previous studies have demonstrated region-selective alterations of glutamine and other ammonia-related amino acids in brain following portacaval anastomosis. Ammonia removal by brain relies on glutamine synthesis and the enzyme responsible, glutamine synthetase, has an almost exclusively astrocytic localization. Glutamine synthetase activities were measured using a radioenzymatic assay in homogenates of seven brain regions of rats four weeks after end-to-side portacaval anastomosis. Enzyme activities were significantly reduced in hippocampus (by 25%, p<0.01), in cerebellum (by 29%, p<0.01) and in cerebral cortex (by 14%, p<0.05). Enzyme activities in other brain regions were within normal limits. Region-selective reductions of glutamine synthetase following portacaval anastomosis could result in disruption of neuron-glial metabolic interactions and in a deficit of glutamatergic synaptic regulation. Similar mechanisms could be implicated in the pathogenesis of hepatic encephalopathy accompanying chronic liver disease in humans.     

Liver glycogen after portacaval shunt in rats

Theories to explain the metabolic effects of portacaval shunt (PCS) have frequently failed to take into account the sequelae of PCS-induced anorexia. In this study PCS rats, followed up to 42 days postoperatively, had a lower food intake than ad libitum-fed (ALC) controls and also lost 10% body wt compared to a 20% gain of ALC rats. Liver weight was significantly reduced in PCS rats compared to ALC rats, pair-fed sham-operated (S) and pair-fed normal control (PFC) rats. However, liver glycogen was equally reduced in all animals with reduced food intake (PCS, S, and PFC rats) compared to ALC rats. Plasma glucose was significantly lower in PCS rats and PCS rats had markedly elevated plasma glucagon and lower plasma insulin concentrations than ALC rats, with the result that they had a low insulin: glucagon molar ratio of 1.3:1 compared to 10.2:1 in the ALC rats. The pair-fed control groups had intermediate I:G ratios and plasma glucose concentrations, thus an inverse relationship between mean plasma glucose and mean I:G ratio for each group was found, suggesting that the blood glucose concentration was the primary event determining the I:G ratio for each group.These controlled experiments indicate that hepatic atrophy after PCS appears to be directly related to the shunting of portal blood away from the liver, while reduced liver glycogen is related to decreased food intake. Reduction in total body weight, relative hypoglycemia, and elevated I:G ratios appear to be due to a combination of factors.     

米诺环素对大鼠脑缺血再灌注后脑水肿的影响

目的观察米诺环素对大鼠脑缺血再灌注后脑水肿的影响。方法 30只雄性SD大鼠随机分为假手术组、生理盐水组、预处理组、30 min组、4 h组,每组6只。采用线栓法制作大脑中动脉缺血再灌注模型。采用MRI弥散加权成像、T_2WI扫描、T_1WI增强扫描检测各组大鼠平均信号强度及相对扩散系数;HE染色观察各组大鼠大脑皮质组织变化。结果与生理盐水组比较,假手术组、预处理组、30 min组相对平均扩散系数明显升高,差异有统计学意义(P<0.05);假手术组、预处理组、30 min组、4 h组T_1WI增强扫描、T_2WI扫描信号强度明显降低,差异有统计学意义(P<0.05,P<0.01)。HE染色显示生理盐水组神经元、周围间质水肿明显,预处理组、30 min组、4 h组神经元及间质水肿明显减轻。结论米诺环素能缓解脑缺血后脑水肿。     

Mild hypothermia prevents brain edema and attenuates up-regulation of the astrocytic benzodiazepine receptor in experimental acute liver failure

BACKGROUND/AIMS: Mild hypothermia has proven useful in the clinical management of patients with acute liver failure. Acute liver failure in experimental animals results in alterations in the expression of genes coding for astrocytic proteins including the "peripheral-type" (astrocytic) benzodiazepine receptor (PTBR), a mitochondrial complex associated with neurosteroid synthesis. To gain further insight into the mechanisms whereby hypothermia attenuates the neurological complications of acute liver failure, we investigated PTBR expression in the brains of hepatic devascularized rats under normothermic (37 degrees C) and hypothermic (35 degrees C) conditions. METHODS: PTBR mRNA was measured using semi-quantitative RT-PCR in cerebral cortical extracts and densities of PTBR sites were measured by quantitative receptor autoradiagraphy. Brain pregnenolone content was measured by radioimmunoassay. RESULTS: At coma stages of encephalopathy, animals with acute liver failure manifested a significant increase of PTBR mRNA levels. Brain pregnenolone content and [(3)H]PK 11195 binding site densities were concomitantly increased. Mild hypothermia prevented brain edema and significantly attenuated the increased receptor expression and pregnenolone content. CONCLUSIONS: These findings suggest that an attenuation of PTBR up-regulation resulting in the prevention of increased brain neurosteroid content represents one of the mechanisms by which mild hypothermia exerts its protective effects in ALF.     

Efficient hepatic uptake of chylomicron remnant cholesterol in rats with a portacaval anastomosis

Portacaval-shunted and sham-operated male rats, fed ad libitum and of similar weight, were studied 2-3 weeks after surgery. At this time serum cholesterol levels did not differ significantly between the two groups, whereas serum triacylglycerols and phospholipids were lower in the shunted group. These animals also showed an increased serum bile acid level and an increased serum estradiol to testosterone ratio. The metabolism of native chyle labeled with [3H]cholesterol and [14C]linoleic acid or of preformed chylomicron remnants with the same labeling was studied in the groups of rats. Ten minutes after intravenous injection of chylomicron remnants 10.6 +/- 0.5% (means +/- SEM, n = 8) of the injected [3H]cholesterol and 7.6 +/- 0.4% of the [14C]linoleic acid were found per 1 g liver in the portacaval-shunted rats; the corresponding figures in the sham-operated group (n = 8) were 6.4 +/- 0.4 and 4.9 +/- 0.3, respectively (p less than 0.001 for both 3H and 14C). Thus, despite a greater than 40% reduction of liver weight induced by the shunting procedure, the total liver uptake of chylomicron remnants was not significantly decreased. The uptake of chylomicron lipids per unit liver weight was normal in the atrophic livers of portacaval-shunted rats also when very large loads of chyle were administered.