7-day Rats Research Articles

Up‐regulation of VGLUT2 expression in hypothalamic‐neurohypophysial neurons of the rat following osmotic challenge

A second vesicular glutamate transporter (VGLUT2) has been reported to be expressed in neurosecretory neurons of the hypothalamic-neurohypophysial system. To study its role in the neurosecretory neurons, we evaluated the expression of the VGLUT2 gene in the paraventricular (PVN) and supraoptic (SON) nuclei as well as the immunoreactivity in the neurohypophysis under euhydrated and chronic hyperosmotic conditions with in situ hybridization and immunohistochemistry. The intensity of hybridization signals in the PVN, SON and thalamus of rats subjected to water deprivation for 7 days, or drinking 2% NaCl for 4 or 7 days, was compared with that of euhydrated rats (control). The overall intensity in the entire PVN or SON, but not the thalamus, was higher in osmotically stimulated rats than in controls. Within the PVN, a significantly higher intensity of signals than that of controls was found only in the dorsolateral posterior magnocellular region in 4-day salt-loaded rats and in all subregions in water-deprived or 7-day salt-loaded rats. The intensity in the SON was higher in the stimulated rats than in controls, regardless of subregions. In the neurohypophysis, VGLUT2 staining was frequently localized in vasopressin terminals of control rats and was apparently reduced in stimulated rats. These results indicate that VGLUT2 is principally expressed in magnocellular vasopressin neurons, suggesting some local effect of intrinsic glutamate on neurohypophysial hormone secretion.

White matter injury in kaolin-induced neonatal rat hydrocephalus

Hydrocephalus is a common neurological condition characterized by obstruction of cerebrospinal fluid (CSF) flow leading to enlargement of CSF-containing ventricular cavities in the brain. The primary target of injury is axons in the periventricular white matter. The stretching of axons leads to dysfunction and ultimately axotomy. Many experiments in mature animals systems indicate that white matter blood flow is decreased. A decrease in oxygen saturation should lead to production of oxygen free radicals and activation of the transcription factor hypoxia inducible factor 1 alpha (HIF). HIF further activates a cascade of cellular responses, notably, vascular endothelial growth factor (VEGF). The main effect of VEGF is to cause production of new blood vessels. Its expression can have an influence on vascular permeability and could play a role in water dysregulation in the hydrocephalic brain. Our first goal was to characterize the structural changes of a neonatal model of kaolin-induced hydrocephalus in rat. Our second goal was to investigate the molecular sequence of events resulting from ischemia. Sprague-Dawley rats underwent kaolin injection into the cisterna magna at postnatal day 1. They had enlarged ventricles by 7 days and severe dilatation by 21 days as assessed by magnetic resonance imaging (MRI) and histology. Periventricular white matter, including corpus callosum and internal capsule, was edematous at 7 days and severely atrophic at 21 days. ELISA and Western blots revealed decreased expression of myelin associated proteins including myelin basic protein (MBP). Pimonidazole hydrochloride (2-nitroimidazole) specifically binds to thiol groups of proteins, peptides and amino acids in a hypoxic environment. Following subcutaneous administration, immunohistochemistry demonstrated hypoxia-associated adducts in periventricular glial cells. Nitrotyrosine, an oxidative stress marker, could be detected by immunohistochemistry extensively in white matter and around some blood vessels. Weak labeling was also seen around large neurons in cerebral cortex. HIF and 4-Hydroxy-2-nonenal (HNE) for endogenous lipid peroxidation are currently under investigation. VEGF immunohistochemistry in normal rats revealed positive cortical neurons in 7-day old rats; this diminished at 21 days. The pattern of expression shifted to white matter glial cells in hydrocephalic rats. VEGF expression determined by ELISA supports those findings. In this model of neonatal kaolin induced hydrocephalus, hypoxia in white matter might contribute to the destructive changes.

Effects of AV3V lesion on pilocarpine-induced pressor response and salivary gland vasodilation

The cholinergic agonist pilocarpine injected intraperitoneally (ip) increases mean arterial pressure (MAP) and superior mesenteric (SM) vascular resistance and reduces submandibular/sublingual gland (SSG) vascular resistance. In the present study, we investigated the effects of electrolytic lesions of the anteroventral third ventricle (AV3V) region on the changes in MAP, SM, and SSG vascular resistances induced by ip pilocarpine. Male Holtzman rats anesthetized with urethane (1.0 g/kg) and chloralose (60 mg/kg) were submitted to sham or electrolytic AV3V lesions and had pulsed Doppler flow probes implanted around the arteries. Contrary to sham rats, in 1-h and 2-day AV3V-lesioned rats, pilocarpine (4 μmol/kg) ip decreased MAP (−41 ± 4 and −26 ± 4 mm Hg, respectively, vs. sham: 19 ± 4 mm Hg) and SM (−48 ± 11 and −45 ± 10%, respectively, vs. sham: 41 ± 10%) and hindlimb vascular resistances (−65 ± 32 and −113 ± 29%, respectively, vs. sham: 19 ± 29%). In 7-day AV3V-lesioned rats, pilocarpine produced no changes on MAP and SM and hindlimb vascular resistances. Similar to sham rats, pilocarpine reduced SSG vascular resistance 1 h after AV3V lesions (−46 ± 6%, vs. sham: −40 ± 6%), but it produced no effect 2 days after AV3V lesions and increased SSG vascular resistance (37 ± 6%) in 7-day AV3V-lesioned rats. The responses to ip pilocarpine were similar in 15-day sham and AV3V-lesioned rats. The cholinergic antagonist atropine methyl bromide (10 nmol) iv slightly increased the pressor response to ip pilocarpine in sham rats and abolished for 40 min the fall in MAP induced by ip pilocarpine in 1-h AV3V-lesioned rats. The results suggest that central mechanisms dependent on the AV3V region are involved in the pressor responses to ip pilocarpine. Although it was impaired 2 and 7 days after AV3V lesions, pilocarpine-induced salivary gland vasodilation was not altered 1 h after AV3V lesions which suggests that this vasodilation is not directly dependent on the AV3V region.

Change of Parameters of Functioning of the Cardiovascular and Respiratory Systems in Rats of Different Ages under Effects of Low Doses of the Cholinesterase Inhibitor Phosphacol

In the 4-day old rat pups and adult animals there were studied effects of subacute and acute phosphacol intoxication at the doses producing and not producing inhibition of aetylcholinesterase on ECG parameters and respiration rate. Analysis of the heart rhythm variability (HRV) was performed using an original program designed in the Labview media. The frequency diapason of cardiointervals was divided as follows: the high-frequency component (HF)-0.8-2.5 Hz, the low-frequency (LF, waves of the II order)-0.8-0.3 Hz; frequencies lower than 0.3 Hz-VLF (slow waves of the III order). Under the conditions of the subacute intoxication the heart rate frequency in the 4-day old and in adult rats increases by 36% and 13%, respectively, the respiration rate frequency increases by 73% in the newborn and does not change in adult rats. The VHR analysis indicates an increase of the tone of the parasympathetic nervous system. The level of the sympathetic activity somewhat increases in adults, but decreases in the 5-day old rat pups, which leads to an essential shift of the vagosympathetic balance towards predominance of parasympathetic influences. In adult rats, unlike the newborns, the role of the humoral-metabolic factors in regulation of the cardiac rhythm and vasomotor reactions increases significantly. Remarkably, the decrease of the nervous sympathetic effects in the rat pups leads to the positive chronotropic effect and stabilization of the cardiac rhythm. Acute phosphacol intoxication (doses of 0.25 and 1 µg/kg) is studied in the 4-day old rat pups. The low dose of the drug leads to development of a moderate bradycardia without disturbances of the cardiac rhythm. The high phosphacol dose produces pronounced bradycardia; on its background there develops a long-term transitory arrhythmia representing complexes of the heart rhythm, which alternate in the decasecond or nearminute rhythm and are separated by periodicity that is one order slower. We observed such rhythm earlier during activation of central N-cholinoreactive structures and development of bilateral pneumothorax. Results of the present study allow stating the paradoxical heart rhythm not as agonic, non-peculiar to the “living” organism, but as a special form of functioning of cardiac pacemakers due to disturbances of the heart regulatory mechanism.

Obtenção e caracterização química e nutricional de diferentes concentrados de caseína

OBJETIVO: O objetivo deste trabalho foi avaliar a composição centesimal, o perfil de aminoácidos e as características nutricionais de três concentrados de caseína, obtidos do leite bovino por diferentes processos. MÉTODOS: Os concentrados de caseína foram analisados pelos seguintes processos: uma caseína comercial, obtida por precipitação ácida seguida de neutralização; caseína obtida pela coagulação enzimática; caseína micelar obtida, respectivamente, pelos processos de microfiltração e diafiltração em membrana. A composição centesimal foi determinada por meio de procedimentos descritos no manual Official Methods of Analysis. O perfil de aminoácidos foi determinado após hidrólise ácida da proteína (HCl 6N, 105°C, 22h) em auto-analisador de aminoácidos, dotado de coluna de troca catiônica e reação pós-coluna com ninidrina. Os perfis de aminoácidos essenciais dos diferentes concentrados de caseína foram comparados e estão de acordo com o padrão Food and Agriculture Organization/World Health Organization, para crianças de 2 a 5 anos de idade. O valor nutritivo foi determinado em ratos da linhagem Wistar, recém desmamados, por meio dos índices, digestibilidade aparente da proteína, quociente de eficiência líquida da proteína e quociente de eficiência protéica operacional. RESULTADOS: A caseína comercial apresentou maior concentração de proteína (92,0%), que a caseína micelar (86,0%) e o coágulo de caseína (72,0%). Os animais nas dietas com as diferentes fontes de proteína, não apresentaram diferenças significativas quanto ao ganho de peso e ingestão de dieta. Maior digestibilidade (93,8%) foi verificada na caseína comercial, comparada à dos outros dois concentrados (91,0%). CONCLUSÃO: Os concentrados de caseína apresentaram diferenças quanto à composição centesimal, sendo a caseína comercial superior na concentração protéica. O coágulo de caseína apresentou resultados inferiores aos demais concentrados, quanto aos índices quociente de eficiência líquida da proteína e quociente de eficiência protéica operacional.

SUSCEPTIBILITY OF NEWBORN RATS TO HEPATOTOXICITY OF 1,3-DIBROMOPROPANE AND 1,1,2,2-TETRABROMOETHANE, COMPARED WITH YOUNG RATS

Newborn rat studies were conducted with oral administration of 1,3-dibromopropane (DBP) and 1,1,2,2-tetrabromoethane (TBE) from postnatal Days 4 to 21 to allow comparison of NOAELs and unequivocally toxic levels with those from 28-day young rat studies starting at 5-6 weeks of age. The unequivocally toxic level was estimated by our specified criteria, requiring simultaneous change of organ weights, histopathology, some biochemical parameters and body weights, because in this study only hypertrophy of hepatocytes was observed as a major histopathological change. DBP caused centrilobular hypertrophy of hepatocytes with alteration in biochemical parameters, as well as lowering of body weights, regardless of sex, in both newborn and young rats. NOAELs and unequivocally toxic levels were considered to be 50 and 150 mg/kg/day in newborn rats and 10 and 250 mg/kg/day in young rats, respectively. In the newborn rat study of TBE, some hepatic effects observed at the top dose of 50 mg/kg were not considered adverse because of the lack of histopathological changes. Significant lowering of body weight was noted at 200 mg/kg in the dose-finding study but histopathological data were not available. In the young rat study, there was no definite toxicity at 6 mg/kg and hypertrophic changes in liver and thyroids without body weight change occurred at 200 mg/kg. There were no clear sex differences in both the newborn and young rat studies. NOAELs were considered to be 50 and 6 mg/kg/day in newborn and young rats, respectively, but unequivocally toxic levels for both rats could not be estimated. Abnormalities of external and sexual development and reflex ontogeny in the newborn were not observed with either chemical. Based on these results, it can be concluded that the target organ of DBP and TBE is the liver in both newborn and young rats, and that while the doses at which toxic signs began to appear are higher in newborn rats, those causing clear toxicity may be paradoxically lower in the newborn case.

The Anti-thyroid Drug Methimazole Induces Neovascularization in the Neonatal Rat Analogous to ROP

To determine the effect of methimazole (MMI), an anti-thyroid drug known to reduce serum l-thyroxine (T4), and insulin-like growth factor (IGF)-1 concentrations, on retinal vascular development in neonatal rats. Sprague-Dawley rats (n=175) were raised in expanded litters of 25 in room air and were exposed to MMI from birth (given as a 0.1% solution to nursing mothers for either 4 or 10 days). Experiments ended on day 4 (n=25) or 10 (n=50) of life. A third group was exposed to MMI for the initial 4 days of life and then allowed to recover for the next 6 days (n=50). Fifty control rats were analyzed on day 4 (n=25) or 10 (n=25) of life. Left eyes were fixed, and retinas were dissected and stained with adenosine diphosphatase (ADPase). Retinas were graded for presence and severity of neovascularization (NV) in a masked manner, and retinal vascular areas were quantified. In a subsequent study, serum IGF-1 and T4 levels were measured by radioimmunoassay in an additional 200 rats exposed to treatments identical to those described. Retinal NV occurred in 31% of rats exposed to 10 days of MMI and 4% (P=0.02) of rats exposed to 4 days of MMI, followed by 6 days of recovery. None of the rats exposed to 4 days of MMI alone and none of the control animals was graded positive for NV. Retinal vascular areas were significantly reduced in rats exposed to 4 days of MMI compared with 4-day control animals (36% +/- 6% vs. 50% +/- 6%, P=0.0001). Serum IGF-1 levels were markedly reduced in 4-day MMI rats compared with age-matched control animals (42 ng/mL vs. 133 ng/mL, P=0.0001) and in 10-day MMI rats compared with 10-day control animals (133 ng/mL vs. 206.5 ng/mL, P=0.005). Serum T4 levels were similarly suppressed in the MMI-exposed litters compared with control animals at day 10 (P=0.008). In contrast, rats exposed to 4 days of MMI followed by 6 days of recovery had normal serum IGF-1 and T4 levels by day 10. The anti-thyroid drug, MMI, induces NV in neonatal rats. This may be mediated by the initial suppression of serum IGF-1. Nevertheless, the lower incidence of NV when serum IGF-1 levels are initially suppressed followed by complete recovery, is contrary to a purely permissive role for serum IGF-1, as reported previously. The relationship between the temporal course of serum IGF-1 and NV in immature retinas needs further investigation.

Age-Related Features of Ceruloplasmin Biosynthesis and Distribution in Rats

Biosynthesis of ceruloplasmin, a copper-containing glycoprotein, which plays an important role in copper transfer and bidirectional iron transport in vertebrates, was studied in 7-day old rats characterized by the embryonic type of copper metabolism. In addition to the liver, copper is synthesized in the lungs, brain, and kidneys. The appearance of labeled ceruloplasmin in the blood flow coincides with the time of liberation of de novo synthesized ceruloplasmin from the liver. [14C]-Ceruloplasmin is absorbed from the blood flow by cells of the heart, lung, and kidneys and binds to erythrocytes. The polypeptide ceruloplasmin chain does not enter the brain cells from the blood flow. Immunoreactive polypeptides of the ceruloplasmin receptor were found using immunoblotting in plasma membranes of the heart, liver, kidneys, and erythrocytes, rather than in those of the brain. It was shown by reverse transcription coupled with PCR using selective primers these cells contain molecular forms of ceruloplasmin mRNAs programming the synthesis of both secretory ceruloplasmin and ceruloplasmin connected with the plasma membrane via the glycosyl phosphatidylinositol anchor. After transition to the adult type of copper metabolism, the blood serum contents of copper and ceruloplasmin sharply increase, while the content of CP in the cerebrospinal fluid, as measured according to the oxidase and antigen activities, and copper concentration, as determined by atom-absorption spectrometry, remain low. Ontogenetic features of the system ensuring the copper homeostasis in mammals are discussed.

Down-regulation of the organic cation transporter 1 of rat liver in obstructive cholestasis.

The liver plays a major role in biotransformation and elimination of various therapeutic agents and xenobiotics, many of which are organic cations and substrates of the organic cation transporter 1 (Oct1, Slc22a1). Oct1 is expressed at the basolateral membranes of hepatocytes and proximal renal tubules. Although Oct1 is the major uptake mechanism in hepatocytes for many pharmaceutical compounds, little is known about the effects of liver injury on this process. Our aim was to investigate the effects of obstructive cholestasis on Oct1 expression and function in liver and kidney. The effects of bile duct ligation (BDL) on Oct1 protein, messenger RNA (mRNA) expression, and tissue localization were determined in rat liver and kidney with Western analysis, real-time reverse transcriptase-mediated polymerase chain reaction (RT-PCR), and immunofluorescence. To assess Oct1 function, the model substrate tetraethylammonium ([(14)C]TEA) was administered intravenously to BDL and control rats and distribution of radioactivity was determined. Oct1 protein significantly decreased in cholestatic livers to 42.1 +/- 17.7% (P <.001), 15.5 +/- 4.7% (P <.05), and 8.6 +/- 2.7% (P <.05) of controls after 3, 7, and 14 days, respectively, but not in kidneys. Hepatic Oct1 mRNA decreased to 77.2 +/- 12.7%, 40.7 +/- 8.1% (P <.05), and 50.3 +/- 7.5% (P <.05) 3, 7, and 14 days after BDL, respectively. Tissue immunofluorescence corroborated these data. Hepatic accumulation of [(14)C]TEA in 14-day BDL rats was reduced to 29.6 +/- 10.9% of controls (P <.0005). In conclusion, obstructive cholestasis down-regulates Oct1 and impairs Oct1-mediated uptake in rat liver, suggesting that hepatic uptake of small cationic drugs may be impaired in cholestatic liver injury.

Developmental plasticity of rat cerebellar cortex after cisplatin injury: Inhibitory synapses and differentiating Purkinje neurons

A single injection of cisplatin, a cytostatic agent, (5 μg/g body weight) in 10-day old rats leads later to the reorganization of the cerebellar cortex in lobules VI–VIII of the vermis. Double immunofluorescence reaction for glutamate receptor (GluR)2/3, a ionotropic glutamate receptor that labels postsynaptically Purkinje neurons, and glutamic acid decarboxylase (GAD)65, an isoform of the GABA synthesis enzyme that labels presynaptically inhibitory terminals in the molecular layer, were employed. Less-differentiated Purkinje cells were present in rats treated on postnatal day (PD)11 at the top of lobule VI and in lobules VII–VIII, in comparison with the deep zones of the same lobules and lobule III. The changes were interpreted as due to loss of trophic factors of Purkinje cell growth, e.g. signaling molecules and granule cells. However, we have shown that a remodelling of Purkinje cell dendrites occurred on PD30 (20 days after cisplatin). In fact, despite of the GluR2/3 labeling of the entire Purkinje cell dendrites, the GAD65 immunofluorescent terminals were adjacent to the proximal parts of the dendrite, while they were scarce in the distal dendritic branchlets. The findings were discussed in relation to the changed cytoarchitecture of the cerebellar cortex, which from PD17 to PD30 includes regeneration of the external germinal layer, reorientation of the main dendritic branches and of the Purkinje cell branchlets, and the presence of ectopic cells.

Oral (gavage), in utero and post-natal exposure of Sprague–Dawley rats to low doses of tributyltin chloride: Part II: effects on the immune system

The immunotoxic effects of tributyltin chloride (TBTC) were examined in the offspring of Sprague–Dawley rats exposed in utero from day 8 of gestation, through lactation and post-weaning until pups reached the age of 30 days (male and female), 60 days (female) and 90 days (male). Daily oral (gavage) doses of 0.025, 0.25 and 2.5 mg/kg body weight/day were administered in olive oil 7 days/week. Immunologic endpoints were investigated at the termination of each study. Statistically significant results ( P<0.05) included the following: At 30 days, the mean percent and absolute natural killer (NK) cell numbers were increased in male and female rats treated with the high TBTC dose. At 60 days, female rats had increased mean serum IgM levels at the low and high TBTC doses, increased mean percentage CD4 +8 + (immature) T lymphocytes at the middle and high doses, a non-linear dose–response increase in NK cell activity at the 50:1 and 100:1 effector:target cell ratios (pairwise comparisons significant at the low dose compared with control), and increased mean numbers of L. monocytogenes colony-forming bacteria on Day 2 post-infection (significant for trend) and Day 3 post infection (pairwise comparisons significant only in the middle dose). The 90-day male rats had decreased mean serum IgA levels at the middle dose group; increased IgM levels at the high dose group, increased IgG levels at the middle and high doses; decreased IgG2 a in the high dose compared to the control; a dose-related increase in the mean percentage NK cell numbers (pairwise comparisons significant at the high dose compared with the control) and increased mean NK cell activity (pairwise comparisons significant at all dose groups compared with the control). The delayed-type hypersensitivity response to oxazolone was increased in the low and middle doses and decreased in the high dose. Thymus atrophy was observed in the high TBTC dose across all ages. Thus, in utero and post-natal treatment of F1 rats with low levels of TBTC affected some aspects of humoral and cell mediated immunity as well as the number and function of cells which are involved in the host's immunosurveillance mechanisms against tumours and viral infections.

Characterization of Long-Term Survival of Syngeneic Hepatocytes in Rat Peritoneum.

Hepatocyte transplantation is a potential therapy for both acute and chronic hepatic insufficiency and also for treatment of inborn errors of metabolism affecting the liver. The peritoneum is one site for implantation and has several advantages: cells implanted there can be easily identified and observed, and it has a relatively large capacity. Long-term survival using "pure" hepatocytes in the peritoneum have been disappointing. We hypothesized that cotransplantation of hepatocytes with nonparenchymal cells would help maintain differentiated hepatocyte function. Rat liver cells transplanted intraperitoneally into August rats were sacrificed at 7 days, 1, 3, 6, 9, and 12 months and analyzed for presence, basal proliferation, and functionality of hepatocytes. To demonstrate that ectopic hepatocytes remained susceptible to exogenous growth factors affecting cell proliferation, rats 9 and 12 months after transplantation were stimulated with tri-iodothyronine and KGF. Hepatocytes were identified 7 days to >12 months, by H&E and immunohistochemically, as ectopic islands in the omental fat. Functionality was confirmed by glycogen deposition. Basal proliferation in 7-day rats was 28.0 ± 10/1000 hepatocytes in ectopic islands (cf. 5.70 ± 2.7/1000 in recipient liver). Proliferation in ectopic islands was greater than host liver. Growth factor-stimulated proliferation in ectopic islands induced a 70-fold increase in DNA synthesis. In conclusion, hepatocytes transplanted with nonparenchymal cells survive, proliferate, and function in the peritoneum of normal rats, and respond to exogenous growth stimuli. Their survival and proliferation in the presence of a normal functioning liver has implications for the potential use of the peritoneal site clinically for supplementation of liver function in metabolic disorders.

Vasodilator Reactivity to Calcitonin Gene-Related Peptide Is Increased in Mesenteric Arteries of Rats during Early Pregnancy

The objective of the present study was to determine the effect of early pregnancy on the sensitivity to, and endogenous production of calcitonin gene-related peptide (CGRP). Contractile responses of arteries of 10-day pregnant and nonpregnant rats were studied in myographs. During contractions induced by 40 mmol/l K⁺, exogenous CGRP elicited an approximately 30% stronger relaxation in mesenteric arteries in pregnancy, an effect not seen in renal and uterine arteries. Capsaicin treatment during K⁺-induced contractions caused a persistent potentiation of the contractile response in mesenteric arteries, indicating that K⁺ stimulates the endogenous release of CGRP. This potentiation was similar in the pregnant and nonpregnant state (+81 ± 23% and +82 ± 23%, respectively), suggesting no effect of pregnancy on the endogenous CGRP release. The latter was paralleled by comparable CGRP content in the arteries of both groups, indicating similar tissue CGRP availability. The results of this study support the concept that early pregnancy is associated with a rise in the vascular sensitivity to CGRP in selected areas of the vascular bed without concomitant increase in the vascular CGRP production and release.

Bovine lactoferrin decreases histopathological changes in the liver and regulates cytokine production by splenocytes of obstructive jaundiced rats.

The aim of this study was to evaluate effects of bovine lactoferrin (BLF) on histopathological changes in the liver of 14 day obstructive jaundiced (OJ) rats and production of tumor necrosis factor (TNF-alpha) and interleukin 6 (IL-6) by splenocytes from 7- and 14-day OJ rats. In the study 50 male rats of the Buffalo strain (170-270 g, mean 230 g) were used. Rats were given 10 mg BLF in 0.5 ml saline daily, using a stomach tube. BLF was applied 2 days before operation and for 13 days following operation. The specimens of liver were prepared using standard techniques. Sections, 5 mm thick were stained with hematoxylin and eosin and reviewed histologically. Microscopic estimation using semiquantitive 4-grade scale was used for evaluation of liver changes. For cytokine measurement splenocyte cultures were stimulated with 5 microg/ml lipopolysaccharide (LPS). After overnight incubation the activities of TNFalpha and IL-6 were determined using bioassays. For statistical evaluation of data the nonparametric Mann-Whitney test was applied. In rats with 14-day OJ, treated with BLF, the pathological changes in the liver were markedly reduced, in particular foci of necrosis with disseminated lymphocytes, cellular necrobiosis, bile duct proliferation and dilation. Neither proliferation of fibrous and reticular connective tissue nor activation of Kupffer cells was revealed. In the 7-day OJ rats treatment with BLF caused significant inhibition of both spontaneous (mean 253, median 275, vs mean 160, median 148 pg/ml, p=0.002) and LPS-induced TNF-alpha production (mean 4967, median 4102, vs mean 2291, median 2234 pg/ ml, p = 0.004) in the splenocyte cultures. The spontaneous (mean 120, median 81, vs mean 43, median 26 pg/ml, p=0.005) as well as LPS-induced IL-6 production (mean 422, median 378, vs mean 293, median 230 pg/ml, p = 0.025) were also lowered. On the other hand, in 14-day OJ, BLF upregulated cytokine production, in particular spontaneous (mean 148, median 158, vs mean 338, median 196, p = 0.001) and LPS-induced TNF-alpha (m ean 1331, median 1507, vs mean 2239, median 1707 pg/ml, p = 0.027). We conclude that oral administration of BLF is beneficial in alleviating deleterious effects of OJ.

Rat hepatocyte aquaporin-8 water channels are down-regulated in extrahepatic cholestasis

Hepatocytes express the water channel aquaporin-8 (AQP8), which is mainly localized in intracellular vesicles, and its adenosine 3′,5′-cyclic monophosphate (cAMP)-induced translocation to the plasma membrane facilitates osmotic water movement during canalicular bile secretion. Thus, defective expression of AQP8 may be associated with secretory dysfunction of hepatocytes caused by extrahepatic cholestasis. We studied the effect of 1, 3, and 7 days of bile duct ligation (BDL) on protein expression, subcellular localization, and messenger RNA (mRNA) levels of AQP8; this was determined in rat livers by immunoblotting in subcellular membranes, light immunohistochemistry, immunogold electron microscopy, and Northern blotting. One day of BDL did not affect expression or subcellular localization of AQP8. Three days of BDL reduced the amount of intracellular AQP8 (75%; P < .001) without affecting its plasma membrane expression. Seven days after BDL, AQP8 was markedly decreased in intracellular (67%; P < .05) and plasma (56%; P < .05) membranes. Dibutyryl cAMP failed to increase AQP8 in plasma membranes from liver slices, suggesting a defective translocation of AQP8 in 7-day BDL rats. Immunohistochemistry and immunoelectron microscopy in liver sections confirmed the BDL-induced decreased expression of hepatocyte AQP8 in intracellular vesicles and canalicular membranes. AQP8 mRNA expression was unaffected by 1-day BDL but was significantly increased by about 200% in 3- and 7-day BDL rats, indicating a posttranscriptional mechanism for protein level reduction. In conclusion, BDL-induced extrahepatic cholestasis caused posttranscriptional down-regulation of hepatocyte AQP8 protein expression. Defective expression of AQP8 water channels may contribute to bile secretory dysfunction of cholestatic hepatocytes. (H epatology 2003;37:1026-1033.)

Fat accumulation in the rat during early pregnancy is modulated by enhanced insulin responsiveness

Insulin sensitivity has been implicated in the variation of fat accumulation in early gestation by as-yet-unknown mechanisms. In the present study, we analyzed the insulin sensitivity of lipolysis and lipogenesis in lumbar adipocytes from rats at 0, 7, 14, and 20 days of gestation. In adipocytes of 7-day pregnant rats, we found a twofold decrease in both beta-agonist (isoproterenol and BRL-37344)-stimulated lipolysis and beta3-adrenoceptor protein but not in lipolysis initiated by forskolin or isobutylmethylxanthine, suggesting a modification of the lipolytic pathway at the receptor level. Whereas adipocytes from 7-day pregnant rats showed a twofold increase in fatty acid synthesis from glucose, those from 20-day pregnant animals displayed a decreased lipogenic activity. Insulin responsiveness of the lipolytic and lipogenic pathways was analyzed by dose-response experiments, giving evidence for the involvement of improved insulin responsiveness in the enhanced lipogenic and reduced lipolytic activities of adipocytes in early pregnancy. In contrast, insulin resistance is responsible for lower antilipolytic and lipogenic actions of insulin in late pregnant animals. In conclusion, the present study shows that enhanced adipose tissue insulin responsiveness during early pregnancy contributes to maternal fat accumulation, whereas decreased insulin responsiveness during late gestation modulates fat breakdown.

Changes in renal medullary transport proteins during uncontrolled diabetes mellitus in rats.

We tested whether the abundance of transport proteins involved in the urinary concentrating mechanism was altered in rats with uncontrolled diabetes mellitus (DM). Rats were injected with streptozotocin and killed 5, 10, 14, or 20 days later. Blood glucose in DM rats was 300-450 mg/dl (control: 70-130 mg/dl). Urine volume increased in DM rats from 41 +/- 7 ml/100 g body wt (BW) at 5 days to 69 +/- 3 ml/100 g BW at 20 days (control: 9 +/- 1). Urine osmolality of DM rats decreased at 5 days DM and remained low at 20 days. UT-A1 urea transporter protein in the inner medullary (IM) tip was 55% of control in 5-day DM rats but increased to 170, 220, and 280% at 10, 14, and 20 days DM, respectively, due to an increase in the 117-kDa glycoprotein form. UT-A1 in the IM base was increased to 325% of control at 5 days DM with no further increase at 20 days. Aquaporin-2 (AQP2) increased to 290% in the IM base at 5 days DM and 150% in the IM tip at 10 days; both showed no further increase at 20 days. NKCC2/BSC1 increased to 240% in outer medulla at 20 days DM, but not at 5 or 10 days. UT-B and ROMK were unchanged at any time point. The increases in UT-A1, AQP2, and NKCC2/BSC1 proteins during uncontrolled DM would tend to limit the loss of fluid and solute during uncontrolled diabetes.

The transient depression of hippocampal CA1 LTP induced by chronic intermittent ethanol exposure is associated with an inhibition of the MAP kinase pathway.

Using electrophysiological and biochemical approaches, we investigated the effects of chronic, intermittent ethanol (CIE) treatment on activation of the mitogen activated protein kinase (MAPK), also known as extracellular signal regulated protein kinase 1 and 2. In hippocampal slices taken from control rats, brief high-frequency stimulation to Schaffer collateral fibers induced a large post-tetanic potentiation (PTP) in the CA1 region that decayed to stable long-term potentiation (LTP) of field extracellular postsynaptic potentials. Western blot analyses showed that phosphorylation of MAPK was increased during PTP and returned to baseline levels during LTP. In slices from the rats removed immediately from CIE treatment, PTP and MAPK activation during the PTP was significantly less than that observed in control slices and LTP was absent. In slices from rats subjected to 1 day withdrawal from CIE treatment, both the reduction in MAPK phosphorylation during PTP and the impairment of PTP and LTP were still evident. Recovery of PTP and partial recovery of LTP was observed in slices obtained from 5-day withdrawn rats. However, MAPK activation during PTP was still attenuated significantly. Interestingly, MAPK activation was enhanced significantly during LTP in 5-day withdrawn rats as well as the sensitivity to MAPK inhibitor PD 098059. In addition to these changes in HFS-induced MAPK activation, we also observed a significant reduction in the basal phosphorylation of MAPK in slices removed from rats immediately after CIE treatment. These results implicate the MAPK signal transduction pathway as a potential cellular target of ethanol. Alterations in MAPKs could play an important role in the alcohol-induced changes in synaptic plasticity associated with the effects of alcohol abuse on learning and memory processes.

Ischemia-induced changes in monocarboxylate transporter 1 reactive cells in rat hippocampus

This study aims to demonstrate the responses of monocarboxylate transporter 1 (MCT1) immunoreactive cells to transient global ischemia in rat hippocampus using confocal and electron microscopy. The MCT1 staining in CA1 pyramidal cells of the sham-operated controls appeared evenly distributed. Most of the MCT1 immunoreactive products were associated with the cell surface; however, some intracellular reaction products are also found. This pattern of stain was not altered in the first three days after an ischemic episode. As the neuronal demise progressed, the MCT1 immunoreactive cells became patchy in the 21-day post-ischemic rats. Besides the neuronal labeling, MCT1 immunoreactivity was found in astroglia, in endothelial cells and in the adjacent ependymal lining. The latter exhibited intense labeling both in the acute and long-term surviving rats. These data suggest that MCT1 plays a role in the initial and long-term neuronal survival in the hippocampus.

Nicotinic autoreceptor function in rat brain during maturation and aging: possible differential sensitivity to organophosphorus anticholinesterases

Acetylcholine (ACh) release is modulated pre-synaptically by both muscarinic and nicotinic receptor-mediated processes. While muscarinic autoreceptors inhibit ACh release, nicotinic autoreceptors enhance ACh release and thus disruption of these processes could potentially affect cholinergic toxicity following exposure to anticholinesterases. Marked age-related differences in sensitivity to some organophosphorus (OP) anticholinesterases have been reported. We compared nicotinic autoreceptor function (NAF) during maturation and aging and evaluated its potential modulation by the common OP insecticide, chlorpyrifos (CPF). Cortical synaptosomes were pre-loaded with [ 3H]choline, superfused (0.6 ml/min) with physiological buffer and [ 3H]ACh release was evoked with potassium (KCl, 9 mM), with or without co-addition of exogenous ACh to stimulate nicotinic autoreceptors. Fractions of perfusate were subsequently collected and area under the curve (AUC) for [ 3H] was analyzed by scintillation counting. The difference in evoked release due to co-addition of exogenous ACh was defined as NAF. Under these conditions, atropine (ATR, 0.1 μM) appeared requisite for NAF; thus this muscarinic antagonist was subsequently added to all perfusion buffers. In synaptosomes from adult tissues, exogenous ACh (3–100 μM) significantly increased release in a concentration-dependent manner. The nicotinic antagonist mecamylamine (MEC, 100 μM) substantially reduced the potassium-evoked release elicited by co-addition of ACh (10 μM). Interestingly, the nicotinic agonists nicotine (NIC) and dimethylphenylpiperazinium (DMPP; 0.1–10 μM) had no effect on release. The active metabolite of CPF (i.e. chlorpyrifos oxon (CPO), 1–10 μM) inhibited NAF in vitro. Maturation-related expression of NAF was noted (AUC with co-addition of 10 μM ACh: 7-day rats, 7±6; 21-day rats, 44±6; 90-day rats, 196±37; 24-month rats, 173±52). NAF was substantially reduced (67–91%) 96 h after maximum tolerated dosages of CPF in adult and aged rats (279 mg/kg, sc) but not in juveniles (127 mg/kg, sc), even though AChE inhibition was similar among the age groups (>80%). Together these data suggest that NAF is differentially expressed during maturation and that this neuromodulatory process may be selectively altered by some OP insecticides, potentially contributing to age-related differences in response to AChE inhibitors. As NAF has been postulated to be activated under conditions of ‘impaired’ cholinergic function, selective alteration of this pre-synaptic process by OP anticholinesterases may be also important in age-related conditions associated with cholinergic hypofunction.