IGFR-I expression and structural analysis of the hard palatine mucosa in an ethanol-drinking rat strain (UChA and UChB)

The study analyzed the effects of chronic alcohol ingestion on the ultrastructure of the lining epithelium of the hard palatine mucosa of rats UChA and UChB (lines with voluntary alcohol consumption) in order to contribute to the understanding of the consequences of alcohol abuse for the morphology of the digestive system. Thirty female adult animals aged 120 days were divided into three experimental groups. (1) Ten UChA rats (genetically low ethanol consumer) with voluntary intake of 10% v/v (5.45 g/kg/day) ethanol solution and water. (2) Ten UChB (genetically high ethanol consumer) rats with voluntary intake of 10% v/v (7.16 g/kg/day) ethanol solution and water. (3) Ten Wistar rats with voluntary ad libitum water intake (control group). Both groups received Nuvital pellets ad libitum. The IGFR-I expression was intense in both experimental groups. The epithelial cells of the alcoholic rats UChA and UChB showed many alterations such as the presence of lipid droplets, altered nuclei, nuclei in corneum layer and disrupted mitochondria. It was concluded that ethanol intake induces ultrastructural lesions in the hard palatine mucosa. (C) 2011 Elsevier Ltd. All rights reserved.

Chronic Acetonemia Alters Liver Oxidative Balance and Lipid Content in Rats. A Model of Nash?

Acetone is considered to be a substance that can disturb cellular oxidative status, being also associated with the production of glucose during its metabolization. The objective of the present study was to determine the effects of chronic treatment with acetone in oxidative stress and metabolic parameters in rats. Twenty male Wistar rats were divided into two groups: control (CG) and chronic acetone group (CAG). After 28 days of acetone ingestion in a 5% aqueous solution (CAG) or water (CG) the animals were euthanized and urine, plasma and liver were collected for the determination of acetone, glucose, lipemia, hepatic fat, malondialdehyde (MDA), reduced glutathione (GSH), and vitamin E. As expected, urinary and plasma acetone levels were higher in CAG. There was no difference in hepatic MDA values between groups, whereas hepatic GSH was lower in CAG than in CG and hepatic vitamin E was higher in CAG than in CG. There was also an increase in glycemia, cholesterolemia and hepatic fat in CAG compared to CG. Chronic treatment with a 5% acetone solution produced an increase in acetonemia that was able to promote changes in hepatic oxidative metabolism and in lipid content in rats similar to those observed in nonalcoholic steatohepatitis.

Involvement of NO in the inhibitory effect of Calotropis procera latex protein fractions on leukocyte rolling, adhesion and infiltration in rat peritonitis model

Aim of the study: The latex of Calotropis procera has been used in the traditional medicinal system for the treatment of leprosy, ulcers, tumors, piles and diseases of liver, spleen, abdomen and toothache. it comprises of a non-dialyzable protein fraction (LP) that exhibits anti-inflammatory properties and a dialyzable fraction (DF) exhibiting pro-inflammatory properties. The present study was carried out to evaluate the effect of LP sub-fractions on neutrophil functions and nociception in rodent models and to elucidate the mediatory role of nitric oxide (NO). Material and methods: The LP was subjected to ion exchange chromatography and the effect of its three sub-fractions (LP(PI), LP(PII), and LP(PIII)) thus obtained was evaluated on leukocyte functions in the rat peritonitis model and on nociception in the mouse model. Results: LP sub-fractions exhibit distinct protein profile and produce a significant decrease in the carrageenan and DF induced neutrophil influx and exhibit anti-nociceptive property. The LP and its sub-fractions produced a marked reduction in the number of rolling and adherent leukocytes in the mesenteric microvasculature as revealed by intravital microscopy. The anti-inflammatory effect of LP(PI), the most potent anti-inflammatory fraction of LP, was accompanied by an increase in the serum levels of NO. Further, our study shows that NO is also involved in the inhibitory effect of LP(PI) on neutrophil influx. Conclusions: Our study shows that LP fraction of Calotropis procera comprises of three distinct sets of proteins exhibiting anti-inflammatory and anti-nociceptive properties of which LP(PI) was most potent in inhibiting neutrophil functions and its effects are mediated through NO production. (C) 2009 Elsevier Ireland Ltd. All rights reserved.

THE INFLUENCE OF VIBRISSAL SOMATOSENSORY PROCESSING IN RAT SUPERIOR COLLICULUS ON PREY CAPTURE

The lateral part of intermediate layer of superior colliculus (SCI) is a critical substrate for successful predation by rats. Hunting-evoked expression of the activity marker Fos is concentrated in SCI while prey capture in rats with NMDA lesions in SCI is impaired. Particularly affected are rapid orienting and stereotyped sequences of actions associated with predation of fast moving prey. Such deficits are consistent with the view that the deep layers of SC are important for sensory guidance of movement. Although much of the relevant evidence involves visual control of movement, less is known about movement guidance by somatosensory input from vibrissae. Indeed, our impression is that prey contact with whiskers is a likely stimulus to trigger predation. Moreover, SCI receives whisker and orofacial somatosensory information directly from trigeminal complex, and indirectly from zona incerta, parvicelular reticular formation and somatosensory barrel cortex. To better understand sensory guidance of predation by vibrissal information we investigated prey capture by rats after whisker removal and the role of superior colliculus (SC) by comparing Fos expression after hunting with and without whiskers. Rats were allowed to hunt cockroaches, after which their whiskers were removed. Two days later they were allowed to hunt cockroaches again. Without whiskers the rats were less able to retain the cockroaches after capture and less able to pursue them in the event of the cockroach escaping. The predatory behaviour of rats with re-grown whiskers returned to normal. In parallel, Fos expression in SCI induced by predation was significantly reduced in whiskerless animals. We conclude that whiskers contribute to the efficiency of rat prey capture and that the loss of vibrissal input to SCI, as reflected by reduced Fos expression, could play a critical role in predatory deficits of whiskerless rats. (C) 2011 IBRO. Published by Elsevier Ltd. All rights reserved.

Sexual differentiation of cortical spreading depression propagation after acute and kindled audiogenic seizures in the Wistar audiogenic rat (WAR)

Brain excitability diseases like epilepsy constitute one factor that influences brain electrophysiological features. Cortical spreading depression (CSD) is a phenomenon that can be altered by changes in brain excitability. CSD propagation was presently characterized in adult mate and female rats from a normal Wistar strain and from a genetically audiogenic seizure-prone strain, the Wistar audiogenic rat (WAR), both previously submitted (RAS(+)), or not (RAS(-)), to repetitive acoustic stimulation, to provoke audiogenic kindling in the WAR-strain. A gender-specific change in CSD-propagation was found. Compared to seizure-resistant animals, in the RAS- condition, mate and female WARs, respectively, presented CSD-propagation impairment and facilitation, characterized, respectively, by lower and higher propagation velocities (P<0.05). In contraposition, in the RAS(+) condition, mate and female WARs displayed, respectively, higher and tower CSD-propagation rates, as compared to the corresponding controls. In some Wistar and WAR females, we determined estrous cycle status on the day of the CSD-recording as being either estrous or diestrous; no cycle-phase-related differences in CSD-propagation velocities were detected. In contrast to other epilepsy models, such as Status Epilepticus induced by pilocarpine, despite the CSD-velocity reduction, in no case was CSD propagation blocked in WARs. The results suggest a gender-related, estrous cycle-phase-independent modification in the CSD-susceptibility of WAR rats, both in the RAS(+) and RAS(-) situation. (C) 2008 Elsevier B.V. All rights reserved.

Immunohistochemical approach reveals involvement of inducible nitric oxide synthase in rat late development

To better understand the role of nitric oxide (NO) in mammal development, specifically in the transition of the fetal stages at birth, we studied the timing of cell-specific expression of inducible NO synthase (iNOS) isoform during gestational periods of rats, mainly at the late stages of intra-uterine development. Before experimentation, the samples were collected (from 17th to 21st gestational days), fixed in 10% buffered formalin and embedded in paraffin for histological procedures. Hereafter, the sections (5 mu m thickness) obtained from different embryos were immunostained by avidin-biotin-immunoperoxidase technique, by using antibody against iNOS isoform. The most of cell immunopositive was suggestive of granulocyte-like cells and those cells were resident close to the blood vessels in different organs, such as: lung, liver or bone marrow environment. Sometimes we noted immunopositive cells in the blood flow, as reported in the thymus. In agreement, iNOS expression, obtained by western blotting analysis, showed the same profile. Together, our data shows that iNOS expression increased gradually during the late stages of rat development (from E17 to E21) and it was executed by cells close to blood vessels. Thus, we can clearly to predict that this expression was finely modulated and it contributes for time-line dependent NO production during rat late development.

Insulin-like growth factor 2 cDNA cloning and ontogeny of gene expression in the liver of the marsupial brushtail possum (Trichosurus vulpecula)

The cDNA sequence for insulin-like growth factor 2 (IGF-2) was determined from the liver of the marsupial brushtail possum (Trichosurus vulpecula) using reverse transcription followed by polymerase chain reaction (RT-PCR) with gene-specific primers. The 359 bp of possum sequence encompassed the mature peptide, 27 bp of the signal peptide, and 125 bp of the E-peptide. Alignment of the deduced amino acid sequence with those from other species indicated that the mature peptide was 71 amino acids in length, 4 amino acids longer than most other mammals. At both the nucleotide and amino acid levels there was a high degree of sequence identity with IGF-2 from other mammalian and nonmammalian species. Amino acid identity ranged from 94.4% with a variant form of human IGF-2 to 80.3% with zebrafinch IGF-2. Northern analysis revealed that radiolabeled possum IGF-2, cDNA hybridized to multiple transcripts in the liver of both adult possums and 150-day-old pouch young and that the overall level of expression was greater in pouch young. Semiquantitative RT-PCR with total RNA from liver samples of pouch young aged 12 to 150 days postpartum and adults confirmed that IGF-2 gene expression was two to three times more abundant in pouch young than in adults but there was no significant change in the level of expression during pouch life. Unlike other mammalian species, in which there is a decline in levels of liver IGF-2 gene expression around the time of birth, levels in the marsupial brushtail possum remain elevated for at least 150 days after birth. This suggests that the decline in liver IGF-2 expression in marsupials and eutherians occurs at a similar stage of development and may reflect a role for this growth factor during the postnatal growth and development of the marsupial, (C) 2001 Academic Press.

Rearrangement of diflunisal acyl glucuronide into its beta-glucuronidase-resistant isomers facilitates transport through the small intestine to the colon of the rat

Many non-steroidal anti-inflammatory drugs (NSAIDs) which form acyl glucuronide conjugates as major metabolites have shown an antiproliferative effect on colorectal tumors. This study assesses the extent to which rearrangement of an acyl glucuronide metabolite of a model NSAID into beta -glucuronidase-resistant isomers facilitates its passage through the small intestine to reach the colon. Rats were dosed orally with diflunisal (DF), its acyl glucuronide (DAG) and a mixture of rearrangement isomers (iso-DAG) at 10 mg DF equivalents/kg. The parent drug DF appeared in plasma after all doses, with maximum concentrations of 20.5 +/- 2.5, 28.8 +/- 8.3 and 11.0 +/- 1.6 mug DF/ml respectively, obtained at 3.8 +/- 0.3, 3.6 +/- 1.8 and 7.5 +/- 0.9 hr after the DF, DAG and iso-DAG doses respectively. At 48 hr, 16.2 +/- 3.3, 19.8 +/- 0.8 and 42.9 +/- 10.1% of the doses respectively were recovered in feces, with less than or equal to 1% remaining in the intestine. About half of each dose was recovered as DF and metabolites in 48 hr urine: for DF and DAG doses, the majority was in the first 24 hr urine. whereas for iso-DAG doses, recoveries in the first and second 24 hr periods were similar. The results show that hydrolysis of both DAG and iso-DAG, and absorption of liberated DF, occur during passage through the gut, but that these processes occur more slowly and to a lesser degree for iso-DAG. The intrinsic hydrolytic capacities of various intestinal segments (including contents) towards DAG and iso-DAG were obtained by incubating homogenates under saturating concentrations of DAG/iso-DAG at 37 degreesC. Upper small intestine, lower small intestine, caecum and colon released 2400, 3200, 9200 and 22800 mug DF/hr/g tissue plus contents respectively from DAG substrate, and 18, 10, 140 and 120 mug DF/hr/g tissue plus contents respectively from iso-DAG substrate. The much greater resistance of iso-DAG to hydrolysis appears attributable to its resistance to beta -glucuronidases. The data suggest that in rats dosed with DF, DAG excreted in bile would be substantially hydrolysed in the small intestine and liberated DF reabsorbed, but that portion which rearranges to iso-DAG would likely reach the colon. (C) 2001 Elsevier Science Inc. All rights reserved.

Angiotensin-converting enzyme inhibition attenuates the progression of rat hepatic fibrosis

Background & Aims: There is a significant relationship between inheritance of high transforming growth factor (TGF)-beta1 and angiotensinogen-producing genotypes and the development of progressive hepatic fibrosis in patients with chronic hepatitis C. In cardiac and renal fibrosis, TGF-beta1 production may be enhanced by angiotensin II, the principal effector molecule of the renin-angiotensin system. The aim of the present study was to determine the effects of the angiotensin converting enzyme inhibitor, captopril, on the progression of hepatic fibrosis in the rat bile duct ligation model. Methods: Rats were treated with captopril (100 mg kg(-1) day(-1)) commencing 1 or 2 weeks after bile duct ligation. Animals with bile duct ligation only and sham-operated animals sewed as controls. Four weeks after bile duct ligation, indices of fibrosis were assessed. Results: Cap topril treatment significantly reduced hepatic hydroxyproline levels, mean fibrosis score, steady state messenger RNA levels of TGF-beta1 and procollagen alpha1(I), and matrix metalloproteinase 2 and 9 activity. Conclusions: Captopril significantly attenuates the progression of hepatic fibrosis in the vat bile duct ligation model, and its effectiveness should be studied in human chronic liver diseases associated with progressive fibrosis.

Role of cytokine gene polymorphisms in acute rejection and renal impairment after liver transplantation

Although immunosuppressive regimens are effective, rejection occurs in up to 50% of patients after orthotopic liver transplantation (OLT), and there is concern about side effects from long-term therapy. Knowledge of clinical and immunogenetic variables may allow tailoring of immunosuppressive therapy to patients according to their potential risks. We studied the association between transforming growth factor-beta, interleukin-10, and tumor necrosis factor alpha (TNF-alpha) gene polymorphisms and graft rejection and renal impairment in 121 white liver transplant recipients. Clinical variables were collected retrospectively, and creatinine clearance was estimated using the formula of Cockcroft and Gault. Biallelic polymorphisms were detected using polymerase chain reaction-based methods. Thirty-seven of 121 patients (30.6%) developed at least 1 episode of rejection. Multivariate analysis showed that Child-Pugh score (P =.001), immune-mediated liver disease (P =.018), normal pre-OLT creatinine clearance (P =.037), and fewer HLA class 1 mismatches (P =.038) were independently associated with rejection, Renal impairment occurred in 80% of patients and was moderate or severe in 39%, Clinical variables independently associated with renal impairment were female sex (P =.001), pre-OLT renal dysfunction (P =.0001), and a diagnosis of viral hepatitis (P =.0008), There was a significant difference in the frequency of TNF-alpha -308 alleles among the primary liver diseases. After adjustment for potential confounders and a Bonferroni correction, the association between the TNF-alpha -308 polymorphism and graft rejection approached significance (P =.06). Recipient cytokine genotypes do not have a major independent role in graft rejection or renal impairment after OLT, Additional studies of immunogenetic factors require analysis of large numbers of patients with appropriate phenotypic information to avoid population stratification, which may lead to inappropriate conclusions.

Fate of swallowed interleukin 8 and TNF alpha and effect on the wistar rat gut

To evaluate the passage of cytokines through the gastrointestinal tract, we investigated the digestion of interleukin-8 (IL-8) and tumour necrosis factor α (TNFα), in vitro and in vivo, and their propensity to induce intestinal inflammation. We serially immuno-assayed IL-8 and TNFα solutions co-incubated with each of three pancreatin preparations at pH 4.5 and pH 8. We gavaged IL-8, TNFα and marker into 15 Wistar rats, and measured their faecal cytokine concentrations by ELISA and histologically examined their guts. IL-8 immunoreactivity was extinguished by all pancreatin preparations after 1 h of incubation at 37 °C. TNFα concentration progressively fell from 1 to 4 h with all enzyme preparations. Buffer control samples maintained their cytokine concentrations throughout incubation. No IL-8 or TNFα was detected in any rat faecal pellets. There was no significant proinflammatory effect of the gavaged cytokines on rat intestine. IL-8 and TNFα in aqueous solution could well be fully digested in the CF gut when transit time is normal and exogenous enzymes are provided, although cytokines swallowed in viscous sputum may be protected from such digestion. Copyright © 2011 Elsevier B.V. All rights reserved

Neonatal ethanol exposure reduces AMPA but not NMDA receptor levels in the rat neocortex

Fetal alcohol syndrome (FAS) is the leading cause of mental retardation in western society. We investigated possible changes in glutamate receptor levels in neonatal animals following ethanol exposure using radioligand binding and western blot analysis. We used a vapor chamber to administer ethanol to neonatal Wistar rats 3 h a day from postnatal day (PND) 4-9. A separation control group was separated from their mothers for the same time and duration as the vapor treatment, while a normal control group was left to develop normally. Daily ethanol administrations resulted in decreased brain weight and body weight, as well as microencephaly (decreased brain:body weight ratio). Neither the affinity nor maximum binding of [H-3]MK-801 (dizoclipine maleate) in the cortex of PND10 rats differed between treatment groups. Western blot analysis also failed to reveal any changes in NMDAR1, NMDAR2A, or NMDAR2B receptor levels. In contrast, the AMPA receptor subunit GluR1 was greatly reduced in vapor-treated pups compared with control pups, as revealed by western blot analysis. A similar reduction was found in westerns with an antibody recognizing the GluR2 and 4 subunits. These results indicate that ethanol reduces AMPA rather than NMDA receptors in the developing neocortex, possibly by blocking NMDA receptors during development. (C) 2002 Elsevier Science B.V. All rights reserved.

Axonal regeneration of retinal ganglion cells after optic nerve pre-lesions and attachment of normal or pre-degenerated peripheral nerve grafts

Axonal regeneration of retinal ganglion cells (RGCs) into a normal or pre-degenerated peripheral nerve graft after an optic nerve pre-lesion was investigated. A pre-lesion performed 1-2 weeks before a second lesion has been shown to enhance axonal regeneration in peripheral nerves (PN) but not in optic nerves (ON) in mammals. The lack of such a beneficial pre-lesion effect may be due to the long delay (1-6 weeks) between the two lesions since RGCs and their axons degenerate rapidly 1-2 weeks following axotomy in adult rodents. The present study examined the effects of the proximal and distal ON pre-lesions with a shortened delay (0-8 days) on axonal regeneration of RGCs through a normal or pre-degenerated PN graft. The ON of adult hamsters was transected intraorbitallv at 2 mm. (proximal lesion) or intracranially at 7 mm (distal lesion) from the optic disc. The pre-lesioned ON was re-transected at 0.5 mm from the disc after 0, 1, 2, 4, or 8 days and a normal or a pre-degenerated PN graft was attached onto the ocular stump. The number of RGCs regenerating their injured axons into the PN graft was estimated by retrograde labeling with FluoroGold 4 weeks after grafting. The number of regenerating RGCs decreased significantly when the delay-time increased in animals with both the ON pre-lesions (proximal or distal) compared to control animals without an ON pre-lesion. The proximal ON pre-lesion significantly reduced the number of regenerating RGCs after a delay of 8 days in comparison with the distal lesion. However, this adverse effect can be overcome, to some degree, by a pre-degenerated PN graft applied 2, 4, or 8 days after the distal ON pre-lesion enhanced more RGCs to regenerate than the normal PN graft. Thus, in order to obtain the highest number of regenerating RGCs, a pre-degenerated PN should be grafted immediately after an ON lesion.

Cold Storage Preservation and Warm Ischaemic Injury to Isolated Arterial Segments: Endothelial Cell Injury

Injury to endothelial calls is thought to be important to the development of the vascular lesion of chronic rejection. It was the aim of this study to develop a semiquantitative method to assess endothelial injury in arterial grafts and to document the injury produced by cold storage preservation and additional warm ischaemia. Twelve- and 24-h cold preservation of rat aortic segments, together with an additional 1 h of warm ischaemia, were assessed. Electron micrographs of representative endothelial cells were scored for cytoplasmic, nuclear and mitochondrial injury. The overall injury score was obtained by addition of the individual scores. Storage for up to 24 h in University of Wisconsin (UW) and Terasaki did not produce any injury. Twenty-four hours of storage in Euro-Collins resulted in endothelial cell death. Injury occurred after 12 h of storage in Ross, Collins and normal saline, and the injury increased following 24 h of storage. One hour of warm ischaemia did not increase the injury. Injury to endothelial cells varies with the preservation solution used and the time of cold storage, so that both the type of solution and the storage time should be taken into account in clinical studies looking at the influence of cold ischaemia time and graft outcome.