AAV vectors transduce hepatocytes in vivo as efficiently in cirrhotic as in healthy rat livers

In liver cirrhosis, abnormal liver architecture impairs efficient transduction of hepatocytes with large viral vectors such as adenoviruses. Here we evaluated the ability of adeno-associated virus (AAV) vectors, small viral vectors, to transduce normal and cirrhotic rat livers. Using AAV serotype-1 (AAV1) encoding luciferase (AAV1Luc) we analyzed luciferase expression with a CCD camera. AAV1Luc was injected through the hepatic artery (intra-arterial (IA)), the portal vein (intra-portal (IP)), directly into the liver (intra-hepatic (IH)) or infused into the biliary tree (intra-biliar). We found that AAV1Luc allows long-term and constant luciferase expression in rat livers. Interestingly, IP administration leads to higher expression levels in healthy than in cirrhotic livers, whereas the opposite occurs when using IA injection. IH administration leads to similar transgene expression in cirrhotic and healthy rats, whereas intra-biliar infusion is the least effective route. After 70% partial hepatectomy, luciferase expression decreased in the regenerating liver, suggesting lack of efficient integration of AAV1 DNA into the host genome. AAV1Luc transduced mainly the liver but also the testes and spleen. Within the liver, transgene expression was found mainly in hepatocytes. Using a liver-specific promoter, transgene expression was detected in hepatocytes but not in other organs. Our results indicate that AAVs are convenient vectors for the treatment of liver cirrhosis.     

Alteration of thymus and of immune regulatory-antifibroblast factors in acute and chronic hepatic damage

We studied in this paper the behavior of immunosuppressive and fibroblast proliferation inhibitory factors in the acute, chronic damage and cirrhotic alteration of the liver. We induced in LEW-rats acute hepatic necrosis by i.v. application of dimethylnitrosamine (DMNA: 35 mg/kg b.wt.) and by i.m. injection of CCl4 (1 ml/kg b.wt., twice a week). After 2-4 weeks we found chronic hepatic damage and after 8-10 weeks liver cirrhosis. As a control, untreated animals were used. Sera and liver factors were prepared from the animals and used for inhibition tests of fibroblast proliferation and MLC reaction. Furthermore, cell count and cell subpopulation of the thymus were determined by monoclonal antibodies (W3/25, OX-8). LF of untreated and DMNA-treated animals exhibited very strong unspecific inhibition effects of fibroblast proliferation and allogenic stimulation. However, with progression of hepatic damage (chronic hepatitis and cirrhosis) both suppressive abilities were gradually reduced. Normal sera showed very slight inhibition of allogenic stimulation but sera of animals with acute hepatic damage showed very strong inhibition. In the 2 weeks of CCl4 treatment, their inhibitory abilities were more than 40%, and with progression of hepatic damage they were gradually reduced. Normal sera or sera of animals with chronic hepatic damage could not suppress the fibroblast proliferation; however, sera of acute hepatic damage inhibited it very strongly. With chronic hepatic damage, the thymus gradually atrophied and, after 10 weeks of CCl4 treatment, it had atrophied completely. Thymocyte differentiation was found only in animals with acute hepatic damage. This suggests that factors which were liberated from the damaged hepatocytes caused differentiation of the thymocytes.     

IFN-alpha gene therapy for woodchuck hepatitis with adeno-associated virus: differences in duration of gene expression and antiviral activity using intraportal or intramuscular routes.

Gene delivery of IFN-alpha to the liver may represent an interesting strategy to maximize its antiviral efficacy and reduce side effects. We used a recombinant adeno-associated virus (AAV) encoding woodchuck IFN-alpha (AAV-IFN) to treat animals with chronic woodchuck hepatitis virus infection. The vector was given by intraportal or intramuscular route. Long-term transgene expression was detected after intraportal administration of an AAV encoding luciferase. In contrast, in the majority of the animals that received AAV-IFN through the portal vein, the expression of IFN-alpha was transient (30-40 days) and was associated with a significant but transient decrease in viral load. One animal, in which hepatic production of IFN-alpha persisted at high levels, died because of bone marrow toxicity. The disappearance of IFN-alpha expression correlated with the disappearance of AAV genomes from the liver. Intramuscular administration of AAV-IFN resulted in prolonged but fluctuating expression of the cytokine with no significant antiviral effect. In summary, this report shows that long-term expression of IFN-alpha in muscle is feasible but higher interferon levels might be needed to control viral replication. On the other hand, IFN-alpha gene delivery to the liver using an AAV vector induces a significant but transient antiviral effect in the woodchuck model.     

改良的肝硬化门脉高压伴腹水大鼠模型

目的 :建立一种制备方法简便、具有高存活率及成模率的肝硬化、门脉高压伴腹水的大鼠动物模型。方法 :80只重量为 2 0 0～ 2 5 0 g的雄性SD大鼠 ,随机选取 10只为正常对照组 ,其余 70只为肝硬化腹水造模组。造模组大鼠的处理 ,苯巴比妥溶液 (35mg % )作为饮用水诱导 1周。第 2周始每周给予 4 0 %CCl4-橄榄油溶液皮下注射 2次 ,每次剂量为 0 .2ml / 10 0g ,首剂加倍 ,第 9周始改为每 2周 3次直至第 14周。结果 :第 14周 ,肝硬化腹水造模组 70只大鼠存活 37只 ,其中 36只存在腹水。肝硬化腹水造模组的大鼠肝脾明显肿大 ;肝表面凹凸不平 ,可见大小结节 ;组织学检查显示肝硬化、假小叶的形成 ;门静脉压力较正常对照组明显升高 (17.6 7± 3.4 7vs 8.6 7± 1.0 3cmH2 O ,P <0 .0 1) ;血清总胆红素、谷丙转氨酶及谷草转氨酶较正常对照组明显升高 (P <0 .0 5 ) ,而血清白蛋白较之正常对照组明显降低 (P <0 .0 1)。结论 :本实验经对CCl4加苯巴比妥联合造模法加以改良 ,获得了一种肝硬化门脉高压伴腹水的大鼠模型 ,为进一步研究创造条件     

Gene transfer of the neuronal NO synthase isoform to cirrhotic rat liver ameliorates portal hypertension

Reduced production of nitric oxide (NO) in the cirrhotic liver results from a defect in hepatic endothelial cell nitric oxide synthase (ecNOS) and appears to contribute to the high intrahepatic resistance and portal hypertension typical of cirrhosis. Therefore, we postulated that targeting a heterologous NOS isoform to sinusoidal endothelial cells or other perisinusoidal cells, such as hepatic stellate cells, would counter the defect in NO production and reduce resistance to blood flow. Recombinant adenovirus (Ad) carrying the neuronal NOS gene (nNOS) targeted liver sinusoidal endothelial cells, stellate cells, and hepatocytes more efficiently than the corresponding cells in cirrhotic livers, but transduction rates were substantial even in cirrhotic animals. Expression of nNOS in each liver cell type, whether from normal or injured liver, caused increased NO production and inhibited endothelin-1-induced contractility of perisinusoidal stellate cells. Finally, in 2 different in vivo models of cirrhosis and portal hypertension, transduction of livers with recombinant Ad.nNOS significantly reduced intrahepatic resistance and portal pressure. The data highlight the feasibility of gene transfer to diseased liver and hepatic cells and demonstrate the potential of a novel therapy for portal hypertension caused by cirrhosis.     

Influence of hypoxia on mitochondrial function and energy status in CCl4-induced cirrhotic rat liver

The influence of hypoxia on hepatic mitochondrial function and energy status was studied in normal and carbon tetrachloride (CCl4)-induced cirrhotic rats. Under hypoxemia of 50 mm Hg-PaO2, hepatic energy status was suppressed both in normal and cirrhotic rats. After the reversal of hypoxia, it was completely restored in normal rats concomitant with a rapid elevation of hepatic mitochondrial redox state (overshoot phenomenon) and increase in the mitochondrial oxidative phosphorylative activity. By contrast, in cirrhotic rats, such an enhancement of mitochondrial function was not observed. It was clarified that cirrhotic liver mitochondrial function was not observed. It was clarified that cirrhotic liver mitochondria have little capacity to respond to the hypoxic stress. A lower resistance to hypoxic episode in cirrhotics might be attributable to the absence of mitochondrial enhancement which is a compensatory mechanism for the deranged energy metabolism of the liver.     

Effects of vitamin A administration on collagen and sulfated glycosaminoglycans contents in the livers of rats treated with carbon tetrachloride.

We have investigated the effects of nontoxic doses of vitamin A on the hepatic contents of collagen and sulfated glycosaminoglycans (SGAGs) in rats chronically treated with CCl4. When the animals were treated with this retinoid before the intoxication with CCl4, liver collagen level was significantly reduced as compared with that in rats that received only CCl4 (3.31 +/- 0.40 vs 5.00 +/- 0.61 mg/gm wet liver, mean +/- SD, respectively), although no significant differences were found for the relative proportion of type III collagen related to type I collagen. The absolute increment in the total amount of liver SGAG in the vitamin A--pretreated group was followed by a more important increase in the concentration of dermatan sulfate as compared with the CCl4 group (dermatan sulfate-to-heparan sulfate ratio: 1.15 for the CCl4 group vs 1.70 for the vitamin A--pretreated group). A significant proportion of the dermatan sulfate from this last group was of higher molecular weight when compared with the dermatan sulfate found in the liver of rats that received only CCl4. Our results indicate that the pretreatment with vitamin A modifies hepatic collagen and SGAG deposition and can inhibit or delay the development of liver cirrhosis in rats chronically treated with CCl4. We speculate that this effect could be due to the changes in the fat-storing (Ito) cells phenotype induced by vitamin A.     

Factors influencing the production of recombinant SV40 vectors.

Most gene therapy approaches employ viral vectors for gene delivery. Ideally, these vectors should be produced at high titer and purity with well-established protocols. Standardized methods to measure the quality of the vectors produced are imperative, as are techniques that allow reproducible quantitation of viral titer. We devised a series of protocols that achieve high-titer production and reproducible purification and provide for quality control and titering of recombinant simian virus 40 vectors (rSV40s). rSV40s are good candidate vehicles for gene transfer: they are easily modified to be nonreplicative and they are nonimmunogenic. Further, they infect a wide variety of cells and allow long-term transgene expression. We report here these protocols to produce rSV40 vectors in high yields, describe their purification, and characterize viral stocks using quality control techniques that monitor the presence of wild-type SV40 revertants and defective interfering particles. Several methods for reproducible titration of rSV40 viruses have been compared. We believe that these techniques can be widely applied to obtain high concentrations of high-quality rSV40 viruses reproducibly.     

13C] phenylalanine breath test and hepatic phenylalanine metabolism enzymes in cirrhotic rats

BACKGROUND: Stable isotope 13C-labelled phenylalanine breath test has been applied to enable the quantitative evaluation of hepatic functional reserve, but the mechanism underlying the changes in function has not been resolved. This study evaluated the correlation between expression of the mRNA of key enzymes mediating phenylalanine metabolism and the metabolism of L-[1-13C] phenylalanine (13C-phe) assessed by the excretion of 13C-CO2 in the breath of rats with, and without, chronic hepatic injury induced by administration of carbon tetrachloride (CCl4). MATERIALS AND METHODS: Male Sprague-Dawley (SD) rats (n = 29) were given subcutaneous injections of CCl4 to induce chronic hepatic injury. L-[1-13C] phenylalanine breath tests (PheBT) were then applied to the rats to assess hepatic function. Expression of phenylalanine hydroxylase (PHH) and tyrosine transaminase (TYT) mRNA in liver was detected by real-time fluorescence quantification RT-PCR, using TaqMan as the probe. It was then determined whether the PheBT results correlated with PHH and/or TYT mRNA expression. In addition, immunohistochemical labelling was used to visualize PHH protein expression in the control and injured liver tissue. RESULTS: There were significant decreases in PheBT and PHH mRNA expression in the cirrhotic rats relative to the uninjured controls and these two measures of liver function were correlated. However, TYT mRNA expression was not changed by CCl4-induced liver injury. The immunohistochemical analysis revealed that PHH protein was expressed predominantly in the cytoplasm of liver cells. CONCLUSIONS: The results of the PheBT were consistent with the changes in PHH gene expression following liver injury. The present findings indicate that decreased expression of the rate-limiting enzyme PHH, but not of TYT, might underlie the functional deficits detected as decreased PheBT. The 13C excretion rate constant per mass liver (PheBT-k/LW) was the most sensitive index that could be used to evaluate the PHH mRNA expression in the liver.     

Ectonucleotidase NTPDase2 is selectively down-regulated in biliary cirrhosis.

BACKGROUND: Portal fibroblasts are newly identified, potentially fibrogenic liver cells that are distinct from hepatic stellate cells. The ectonucleotidase* nucleoside triphosphate diphosphohydrolase 2 (NTPDase2) is restricted to portal fibroblasts in the normal liver. However, the fate of NTPDase2 after bile duct ligation (BDL) is unknown. AIMS: The aim of this study was to assess the effect of experimental rat and disease-mediated human biliary cirrhosis on NTPDase2 expression in the liver. METHODS: Cirrhosis was induced in experimental rats via BDL and carbon tetrachloride (CCl4) administration. Archived human liver biopsy specimens from normal liver, primary biliary cirrhosis, or hepatitis C cirrhosis were examined. Changes in expression of NTPDase2 were determined using confocal immunofluorescence, immunoblot, and real-time polymerase chain reaction. RESULTS: Confocal immunofluorescence demonstrated a decrease in NTPDase2 expression after BDL. Immunoblot and real-time polymerase chain reaction demonstrated a decrease in NTPDase2 expression by portal fibroblasts after BDL. No decrease in NTPDase2 protein was noted after CCl4 administration, and NTPDase2 messenger ribonucleic acid was markedly up-regulated after CCl4 administration. Confocal immunofluorescence demonstrated a shift of NTPDase2 expression from portal areas to central areas that colocalized with alpha-smooth muscle actin after CCl4 administration. In human biopsy specimens, NTPDase2 expression was lost in cirrhosis owing to primary biliary cirrhosis, whereas NTPDase2 expression was shifted to bridging fibrous bands in cirrhosis owing to hepatitis C. CONCLUSIONS: Loss of NTPDase2 is a common pathway in both rat and human manifestations of biliary cirrhosis. Conversely, in non-biliary cirrhosis, NTPDase2 is shifted from the portal area to bridging fibrous bands. Elucidations of the mechanisms regulating NTPDase2 expression may lead to new therapeutic approaches to fibrotic liver disease.     

Prevention of fibrosis progression in CCl4-treated rats: role of the hepatic endocannabinoid and apelin systems

Endocannabinoids behave as antifibrogenic agents by interacting with cannabinoid CB2 receptors, whereas the apelin (AP) system acts as a proangiogenic and profibrogenic mediator in the liver. This study assessed the effect of long-term stimulation of CB2 receptors or AP receptor (APJ) blockade on fibrosis progression in rats under a non-discontinued fibrosis induction program. The study was performed in control and CCl(4)-treated rats for 13 weeks. Fibrosis-induced rats received a CB2 receptor agonist (R,S)-3-(2-iodo-5-nitrobenzoyl)-1-(1-methyl-2-piperidinylmethyl)-1H-indole (AM1241) (1 mg/kg b.wt.), an APJ antagonist [Ala(13)]-apelin-13 sequence: Gln-Arg-Pro-Arg-Leu-Ser-His-Lys-Gly-Pro-Met-Pro-Ala (F13A) (75 μg/kg b.wt.), or vehicle daily during the last 5 weeks of the CCl(4) inhalation program. Mean arterial pressure (MAP), portal pressure (PP), hepatic collagen content, angiogenesis, cell infiltrate, and mRNA expression of a panel of fibrosis-related genes were measured in all animals. Fibrosis-induced rats showed increased hepatic collagen content, reduced MAP, portal hypertension, and increased expression of the assessed messengers in comparison with control rats. However, fibrotic rats treated with either AM1241 or F13A had reduced hepatic collagen content, improved MAP and PP, ameliorated cell viability, and reduced angiogenesis and cell infiltrate compared with untreated fibrotic rats. These results were associated with attenuated induction of platelet-derived growth factor receptor β, α-smooth muscle actin, matrix metalloproteinases, and tissue inhibitors of matrix metalloproteinase. CB2 receptor stimulation or APJ blockade prevents fibrosis progression in CCl(4)-treated rats. The mechanisms underlying these phenomena are coincident despite the marked dissimilarities between the CB2 and APJ signaling pathways, thus opening new avenues for preventing fibrosis progression in liver diseases.     

Determinants of hepatic function in liver cirrhosis in the rat. Multivariate analysis.

We investigated the determinants of hepatic clearance functions in a rat model of liver cirrhosis induced by phenobarbital/CCl4. Aminopyrine N-demethylation (ABT), galactose elimination (GBT), and serum bile acids (SBA) were determined in vivo. The livers were then characterized hemodynamically: intrahepatic shunting (IHS) was determined by microspheres and sinusoidal capillarization by measuring the extravascular albumin space (EVA) by a multiple indicator dilution technique. The intrinsic clearance was determined by assaying the activity of the rate-limiting enzymes in vitro. Hepatocellular volume (HCV) was measured by morphometry. ABT and SBA, but not GBT, differentiated cirrhotic from normal liver. IHS ranged from normal to 10%; all cirrhotic livers showed evidence of sinusoidal capillarization (reduced EVA). The cirrhotic livers showed a bimodal distribution of HCV, HCV being decreased in 50% of the cirrhotic livers. Multivariate analysis showed EVA and portal flow to be the main determinants of microsomal (ABT) and cytosolic (GBT) clearance function; SBA, by contrast, were determined solely by IHS. We conclude that sinusoidal capillarization is the main determinant of hepatic clearance, while serum bile acids reflect intrahepatic shunting. These findings emphasize the importance of alterations of hepatic nutritional flow to explain reduced clearance function in cirrhosis of the liver.     

Octacosanol attenuates disrupted hepatic reactive oxygen species metabolism associated with acute liver injury progression in rats intoxicated with carbon tetrachloride

We examined whether octacosanol, the main component of policosanol, attenuates disrupted hepatic reactive oxygen species metabolism associated with acute liver injury progression in rats intoxicated with carbon tetrachloride (CCl(4)). In rats intoxicated with CCl(4) (1 ml/kg, i.p.), the activities of serum transaminases increased 6 h after intoxication and further increased at 24 h. In the liver of CCl(4)-intoxicated rats, increases in lipid peroxide (LPO) concentration and myeloperoxidase activity and decreases in superoxixde dismutase activity and reduced glutathione (GSH) concentration occurred 6 h after intoxication and these changes were enhanced with an increase in xanthine oxidase activity and a decrease in catalase activity at 24 h. Octacosanol (10, 50 or 100 mg/kg) administered orally to CCl(4)-intoxicated rats at 6 h after intoxication attenuated the increased activities of serum transaminases and the increased hepatic myeloperoxidase and xanthine oxidase activities and LPO concentration and the decreased hepatic superoxide dismutase and catalase activities and GSH concentration found at 24 h after intoxication dose-dependently. Octacosanol (50 or 100 mg/kg) administered to untreated rats decreased the hepatic LPO concentration and increased the hepatic GSH concentration. These results indicate that octacosanol attenuates disrupted hepatic reactive oxygen species metabolism associated with acute liver injury progression in CCl(4)-intoxicated rats.     

What can SV40-derived vectors do for gene therapy?

The limited success of gene therapy as an approach to treating human disease largely reflects the limitations of the gene delivery vectors that have been used. Poor titers, low transduction efficiency, waning transgene expression and immunogenicity have remained obstacles in the field. As a consequence, much research in normal, immunocompetent animals has not demonstrated therapeutic levels of gene delivery, and results from most human clinical trials have been predictably discouraging. Recombinant gene transfer vectors derived from SV40 virus (rSV40) are potentially of great interest for those working in gene therapy, since these vectors are not subject to many of the problems that have limited gene delivery using other vector systems. rSV40 is made at a very high titer and infects - and so transduces - almost all nucleated cell types very efficiently, regardless of lineage or whether they are resting or dividing; they integrate and are not susceptible to transgene silencing; and they elicit no detectable immune response on the part of normal animals and so can be used to deliver multiple transgenes over time and in sequence. The recent development of 'gutless' rSV40 vectors has expanded the range of potential therapeutic transgenes that can be delivered with this system and added flexibility to the expression configurations that can be accommodated. All of these functional characteristics of SV40-derived vectors have their bases in the biology of SV40 and similar viruses, and have important implications for the potential utility of rSV40 vectors in gene therapeutics. Like all viral gene delivery systems, these vectors have their idiosyncrasies and limitations. They also allow gene delivery that bypasses many of the difficulties that have plagued the field from its inception.     

Pleomorphism of hepatic regeneration

The pleomorphism of hepatic regeneration was studied in acute liver injury and partial hepatectomy of rats. Increases in 3H-thymidine incorporation into the liver DNA fraction and in liver ornithine decarboxylase (ODC) activity were observed similarly in acute liver injury and following a two-thirds partial hepatectomy in normal rats. However, the increase in serum AFP levels was significant only in carbon tetrachloride (CCl4)-treated rats, and the portal-peripheral vein difference in plasma glucagon concentrations was great only in rats that underwent a partial hepatectomy. The weight of the remaining liver increased rapidly following a 67% hepatectomy in normal rats, but not in cirrhotic rats, of which three of five died within 6 days. Increases in liver prostaglandin E levels, 14C-orotate incorporation into the liver RNA fraction and hepatic ODC activity were observed up to 10 h following a partial hepatectomy of normal liver but not after similar removal of cirrhotic liver. Three kinds of hepatic regeneration, i.e., normal and pathologic regeneration following surgical removal of either normal or injured liver and repair of liver damage following chemically induced liver deficiency, were proposed from the observations.     

Depressed function of Kupffer cells in rats with CCl4-induced liver cirrhosis

In the present study, the Kupffer cell function of rats with CCl4-induced liver cirrhosis was tested by analyzing the changes in the host defense system. In rats without liver cirrhosis injected with CCl4 for 3 weeks concomitant with the high opsonic activity the endocytic index was significantly increased. Rats treated for 9 and 13 weeks developed cirrhosis, and their endocytic indices were not increased despite the rise in their opsonic activity. Particularly, the endocytic index of 13-week-treated rats with advanced liver cirrhosis was significantly lower than that of the other groups. The organic distribution of 51Cr-endotoxin injected intravenously exhibited characteristic changes in 9-week- and 13-week-treated rats: decreased hepatic uptake and increased splenic uptake. In contrast, pulmonary uptake was increased in all CCl4-treated rats. The superoxide production by Kupffer cells from 13-week-treated rats was greatly reduced, accompanied by the decreased superoxide dismutase activity of liver homogenate. Thus, results of this study suggest that Kupffer cell dysfunction is one of the main factors affecting host defenses in liver cirrhosis.     

Unexpected pulmonary uptake of adenovirus vectors in animals with chronic liver disease

When adenovirus vectors are injected intravenously, most of the virions are quickly taken up by the reticuloendothelial system, primarily by the liver macrophages known as Kupffer cells. However, little is known about the behavior of adenovirus vectors when there is pre-existing liver disease. To study this, we examined the biodistribution of intravenously injected vector in a rat model of cirrhosis induced by bile duct ligation. Using quantitative PCR and fluorescently tagged adenovirus vectors, we observed a significant reduction in vector uptake by the cirrhotic liver and increased accumulation in the lungs. Immunocytochemistry and electron microscopy demonstrated that this was due to changes in the reticuloendothelial system, with the vector being taken up by large numbers of pulmonary intravascular macrophages in the lungs of cirrhotic rats. Interestingly, expression of vector-encoded luciferase was significantly reduced in the livers of cirrhotic rats, but was not increased in the lungs. These data demonstrate that the biodistribution of adenovirus vectors in rats is altered by cirrhosis, which suggests the possibility that these vectors might behave unexpectedly in patients with pre-existing liver conditions, particularly since pulmonary reticuloendothelial changes are known to occur in human disease.     

Brain serotonin metabolism and behavior in rats with carbon tetrachloride-induced liver cirrhosis

Increased brain serotonin metabolism has been suggested as an etiologic factor in the development of portal-systemic encephalopathy (PSE) in connection with liver disease. We therefore investigated brain serotonin metabolism and open-field behavior (spontaneous activity and exploration) in rats with carbon tetrachloride (CCl4)-induced liver cirrhosis. Brain serotonin metabolism was evaluated in rats pretreated with an amino acid decarboxylase inhibitor. The 5-hydroxyindoles were analyzed by high-performance liquid chromatography (HPLC) with electrochemical detection. The results revealed an increased serotonin synthesis rate in all investigated brain regions in rats with histologically verified diffuse micronodular cirrhosis of the liver. Slightly impaired open-field behavior (i.e., decreased spontaneous activity) in the cirrhotic rats could not be excluded. However, the elevated brain serotonin synthesis rate could not be correlated to any abnormalities in open-field behavior.