Standard Laboratory Rat Research Articles

Modulation of BDNF Expression by Bryophyllum pinnatum Extract: Implications for Oxidative Stress and Cognitive Function in Pain-induced Wistar Rats

This research work investigated modulatory effects of Bryophylum pinnatum extract on BDNF expression, and cognitive functions in repetitive pain-induced oxidative stress in Wistar rats. Animals weighing between 80–100g were acquired from the animal house of the Department of Human Physiology, Faculty of Basic Medical Sciences, University of Port Harcourt, and all animals received standard laboratory rat feeds and water ad libitum. The study was designed to assess the time dependent effects with a total of 30 rats divided into 6 groups. Group 1(Control), Group 2 (Pain Only), Group 3 (Pain + 5mg/kg Morphine), Group 4 (Pain + 10mg/kg Morphine), Group (Pain + 25mg/kg Bryophylumm Pinnatum), Group 6 (Pain + 50mg/kg Bryophylumm Pinnatum), Hydromethanolic extracts was prepared accordingly, and Gas Chromatography Mass Spectrometry (GC/MS) analysis were carried out. Neurobehavioral studies were conducted weekly to assess the effects of the interventions on cognitive and neurological parameters, using radial maze and navigational maze test. Assay of BDNF was done using the Elisa method. The Results showed that Morphine and Bryophyllum pinnatum both significantly improved BDNF expression, showed antioxidant effects, improved cognitive functions, and provided possible mechanisms of pain relief. Pharmacokinetic studies on the binding affinities and drug-likeness properties of the active compounds from these extracts revealed some favorable properties with regard to management of oxidative stress and promotion of cognitive function in states of pain. The study therefore provide indication of the therapeutic potential of Bryophyllum pinnatum in the effective management of Pain, oxidative stress and cognitive functions.

Establishment and refinement of a DEN-induced hepatocellular carcinoma model in rats

Abstract Objectives Hepatocellular carcinoma is one of the most common malignant tumors in the world with complex etiology, high tumor heterogeneity, and low efficacy of treatment. The establishment of an animal model that is close to the clinical situation of hepatocellular carcinoma and can be successfully modeled many times is of great significance to the study of the pathogenesis, diagnosis and treatment of hepatocellular carcinoma. Methods We used Diethylnitrosamine (DEN) to induce hepatocellular carcinoma in rodents and compared four models of DEN-induced hepatocellular carcinoma. Group C (Control): rats were fed a standard laboratory rat diet and freely drank normal water. Group P (Peritoneal injection): rats were administered an IP injection (50 mg/kg/week) between 5 and 23 weeks after birth. Ten microliter of DEN solution would be injected per g of rat. Group O (DEN-Fed group): rats were allowed unrestricted access to water contaminated with 0.01 % DEN between the ages of 7 and 15 weeks. 0.2 mL of DEN drinking water was consumed per gram of rat. Group P+O (Combined peritoneal injection and Oral feeding): rats were administered an IP injection (50 mg/kg) at weeks 3 and 5 post-birth, and they freely drank water contaminated with 0.012 % DEN between weeks 7 and 14 post-birth. We used an ultrasound scan, biochemical testing, haematoxylin, and eosin staining, Masson staining, Wolf scarlet staining, Ki67, CD34, a-SMA, CD8, and CD68 staining to compare between groups. Results Liver dissection and ultrasound scan showed that compared to other groups, the liver of Group P+O was darker in color, with more grey-white cancer nodules and larger localized tumors, and the structure of the tumors was slightly disorganized, with the elastography hardness of the middle lobe and the right lobe was slightly increased. The alanine aminotransferase and total bilirubin of Group P+O were higher than those of Group O but lower than those of Group P. Haematoxylin and eosin staining showed that the tumors of Group P+O were large, with large tumor cords and pseudo-glandular, the degree of differentiation was medium and surrounded by more fatty lesions. Conclusions We conclude that combined DEN treatment is more effective, stable, and has the advantage of multiple modalities, leading to faster tumor formation.

An implantable neurophysiology platform: Broadening research capabilities in free-living and non-traditional animals.

Animal-borne sensors that can record and transmit data (“biologgers”) are becoming smaller and more capable at a rapid pace. Biologgers have provided enormous insight into the covert lives of many free-ranging animals by characterizing behavioral motifs, estimating energy expenditure, and tracking movement over vast distances, thereby serving both scientific and conservational endpoints. However, given that biologgers are usually attached externally, access to the brain and neurophysiological data has been largely unexplored outside of the laboratory, limiting our understanding of how the brain adapts to, interacts with, or addresses challenges of the natural world. For example, there are only a handful of studies in free-living animals examining the role of sleep, resulting in a wake-centric view of behavior despite the fact that sleep often encompasses a large portion of an animal’s day and plays a vital role in maintaining homeostasis. The growing need to understand sleep from a mechanistic viewpoint and probe its function led us to design an implantable neurophysiology platform that can record brain activity and inertial data, while utilizing a wireless link to enable a suite of forward-looking capabilities. Here, we describe our design approach and demonstrate our device’s capability in a standard laboratory rat as well as a captive fox squirrel. We also discuss the methodological and ethical implications of deploying this new class of device “into the wild” to fill outstanding knowledge gaps.

Chebulagic acid attenuates HFD/streptozotocin induced impaired glucose metabolism and insulin resistance via up regulations of PPAR γ and GLUT 4 in type 2 diabetic rats

The aim of the present study was to evaluate the effects of chebulagic acid on enzymes involved in carbohydrate metabolism in high-fat diet/streptozotocin-induced diabetic rats. Diabetes was induced in albino male Wistar rats by feeding them with a high-fat diet comprising of 84.3% standard laboratory rat chow, 5% lard, 10% yolk powder, 0.2% cholesterol, and 5% bile salt for 2 weeks. After 2 weeks, the animals were kept in an overnight fast and injected with a low dose of streptozotocin (35 mg/kg, dissolved in 0.1 M sodium citrate buffer, pH 4.5). At the end of the experimental periods, the blood glucose, plasma insulin, hemoglobin, glycated hemoglobin levels, homeostatic model of insulin resistance, glycogen content, glycogen synthase, glycogen phosphorylase were measured in high-fat diet and streptozotocin-induced diabetic rats whereas the glucose metabolic enzymes such as hexokinase, glucose 6 phosphate dehydrogenase, glucose 6 phosphatase, fructose 1,6 bisphosphatase and protein expression of peroxisome proliferator-activated receptor–γ (PPAR-γ) and Glucose transporter 4 (GLUT4) were analyzed. Administration of chebulagic acid to HFD/STZ -induced diabetic rats for 30 days significantly decreased the levels of plasma glucose, homeostatic model assessment of insulin resistance, glycogen phosphorylase and glycosylated hemoglobin and increased the levels of insulin, glycogen content and glycogen synthase. On the other hand, the altered activity of carbohydrate metabolic enzymes, PPAR-γ and GLUT4 were brought back to near-normal levels on treatment with chebulagic acid. The effect produced by chebulagic acid on various parameters was comparable to that of metformin.

Antioxidant potential of theaflavin ameliorates the activities of key enzymes of glucose metabolism in high fat diet and streptozotocin – induced diabetic rats

ABSTRACTObjectives: The present study was to evaluate the effect of theaflavin on the activities of key enzymes of carbohydrate metabolism in high fat diet and streptozotocin – induced diabetic rats.Methods: Diabetes was induced in male albino Wistar rats by feeding them with high fat diet comprising of standard laboratory rat chow 84.3%, lard 5%, egg yolk powder 10%, cholesterol 0.2% and bile salt 0.5% for 2 weeks. After 2 weeks, the animals were kept in an overnight fast and injected with low dose of streptozotocin (40 mg/kg b.w).Results: Theaflavin (100 mg/kg b.w /day) was administered orally to diabetic rats for 30 days. At the end of the experimental period, diabetic control rats showed significant increase in plasma glucose, homeostatic model assessment of insulin resistance (HOMA-IR), glycosylated hemoglobin (HbA1c) with concomitant decrease in plasma insulin, total hemoglobin and body weight. The activities of key enzymes of carbohydrate metabolism, lipid peroxidation markers, antioxidant enzymes, glycogen content and glycogen synthase and glycogen phosphorylase were also altered in diabetic rats.Discussion: Oral administration of theaflavin to diabetic rats significantly ameliorated all the biochemical alterations to near normal levels. The results of the present study suggest that theaflavin exhibits antidiabetic effect through its antioxidant activity.

Housing environment influences stress-related hippocampal substrates and depression-like behavior

Rats are widely used animal models for biological psychiatry and neuroscience. Laboratory rats are typically housed in impoverished sensory environments. The lack of species-typical sensory environment might radically change the response of individual animals to stressful and/or threatening episodes. In this report, we demonstrate that behavioral and neural sequelae of chronic stress were modified by sensory environment of adult male rats. This includes effects of stress on the density of spines on CA3 hippocampal neurons, hippocampal neurogenesis and abundance of glucocorticoid or mineralocorticoid receptors. Enrichment also reduced depression-like behavior in a forced swim task. Stress and sensory enrichment evoked opposing effects on all the above endpoints. The sensory enrichment used in this report is of a relatively short duration provided during adulthood. This period excludes critical windows of greater plasticity during pre- and peripubertal stages. Our results suggest that standard housing practices for laboratory rats remain austere concerning sensory requirements of this species. Thus, even a moderate sensory enrichment is capable of reducing high stress-sensitivity and depressive-like behavior in standard laboratory rats.

Low dietary soy isoflavonoids increase hippocampal spine synapse density in ovariectomized rats

High dietary intake of plant estrogens (phytoestrogens) can affect brain structure and function. The effects of phytoestrogen intake within the range of normal animal and human dietary consumption, however, remain uncertain. The aim of the present study was to determine the effects of the isoflavonoids present in a standard low phytoestrogen laboratory rat chow on spine synapse density in the stratum radiatum of area CA1 of the hippocampus.Weanling rats (22days old) were fed either standard chow (Teklad 2018), a nutritionally comparable diet without soy (Teklad 2016) or a custom diet containing Teklad 2016 supplemented with the principal soy isoflavonoids, daidzein and genistein, for 40days. Rats were ovariectomized at 54days of age. Eight days later, spine synapse density on the apical dendrites of hippocampal pyramidal neurons in the stratum radiatum of area CA1 was measured by electron microscopic stereological analysis. Animals maintained on Teklad 2016 exhibited an approximately 60% lower CA1 spine synapse density than animals consuming Teklad 2018. Replacing genistein and daidzein in Teklad 2016 returned synapse density to levels indistinguishable from those in animals on Teklad 2018.These results indicate that the isoflavonoids in a standard laboratory rat diet exert significant effects on spine synapse density in the CA1 region of the hippocampus. Since changes in spine synapse density in this region of the hippocampus have been linked to cognitive performance and mood state, these data suggest that even relatively low daily consumption of soy phytoestrogens may be sufficient to influence hippocampal function.

Age-related periodontitis and alveolar bone loss in rice rats

ObjectiveTo characterize in rice rats: (a) periodontitis (PD) progress with feeding of standard laboratory rat chow (STD) during ages 4–80 weeks; and (b) PD progress with feeding of a high sucrose-casein (H-SC) diet during young adulthood. MethodsOne group (N=12) was euthanized at age 4 weeks (Baseline). Four groups (N=8-16) consumed a STD diet from baseline and were necropsied at ages 22, 30, 52, and 80 weeks. Three groups (N=10-16) consumed an H-SC diet from baseline. Two were necropsied at ages 22 and 30 weeks, respectively. The third switched to the STD diet at age 22 weeks and was necropsied at age 30 weeks. All mandibles/maxillae were assessed by histometry for degree of periodontal inflammation (PD Score), alveolar crest height (ACH, mm), and horizontal alveolar bone height (hABH, mm2). ResultsIn STD diet rats aged ≥30 weeks, all endpoints were worse (P<0.05) than at Baseline. In H-SC diet rats aged ≥22 weeks, all endpoints were worse than at Baseline (P<0.05). At age 22 weeks, all endpoints were worse in the H-SC group than in the STD group (P<0.05). By age 30 weeks, the STD and H-SC groups did not differ. Conclusions1) STD diet fed rice rats develop moderate/severe PD by age 30 weeks; 2) an H-SC diet accelerates moderate/severe PD development; and 3) switching to a STD diet does not halt/reverse PD that was accelerated by an H-SC diet. These data further clarify use of the rice rat as a PD model.

Evaluating whole transcriptome amplification for gene profiling experiments using RNA-Seq.

BackgroundRNA-Seq has enabled high-throughput gene expression profiling to provide insight into the functional link between genotype and phenotype. Low quantities of starting RNA can be a severe hindrance for studies that aim to utilize RNA-Seq. To mitigate this bottleneck, whole transcriptome amplification (WTA) technologies have been developed to generate sufficient sequencing targets from minute amounts of RNA. Successful WTA requires accurate replication of transcript abundance without the loss or distortion of specific mRNAs. Here, we test the efficacy of NuGEN’s Ovation RNA-Seq V2 system, which uses linear isothermal amplification with a unique chimeric primer for amplification, using white adipose tissue from standard laboratory rats (Rattus norvegicus). Our goal was to investigate potential biological artifacts introduced through WTA approaches by establishing comparisons between matched raw and amplified RNA libraries derived from biological replicates.ResultsWe found that 93% of expressed genes were identical between all unamplified versus matched amplified comparisons, also finding that gene density is similar across all comparisons. Our sequencing experiment and downstream bioinformatic analyses using the Tuxedo analysis pipeline resulted in the assembly of 25,543 high-quality transcripts. Libraries constructed from raw RNA and WTA samples averaged 15,298 and 15,253 expressed genes, respectively. Although significant differentially expressed genes (P < 0.05) were identified in all matched samples, each of these represents less than 0.15% of all shared genes for each comparison.ConclusionsTranscriptome amplification is efficient at maintaining relative transcript frequencies with no significant bias when using this NuGEN linear isothermal amplification kit under ideal laboratory conditions as presented in this study. This methodology has broad applications, from clinical and diagnostic, to field-based studies when sample acquisition, or sample preservation, methods prove challenging.Electronic supplementary materialThe online version of this article (doi:10.1186/s12896-015-0155-7) contains supplementary material, which is available to authorized users.

HISTOLOGICAL AND HISTOCHEMICAL CHARACTERISTICS OF RAT MYOCARDIUM IN CADMIUM TOXICOSIS

Carcinogenic effects of cadmium on lungs, testicles and prostate are well known, so as cumulative and toxic effects on kidney, liver and bones; however, there have not been many published articles about the effects of cadmium on myocardium. The aim of this study was to estimate the morphological changes in rat myocardium chronically treated by cadmium. The study was carried out on male albino Wistar rats (n=30, age=35-37 days, body mass 120g +/- 10g). The animals were raised in controlled laboratory conditions and provided with standard laboratory rat food and tap water ad libitum. The rats were divided into two groups: ten animals composed the control group and did not undergo any treatment. The 20 experimental rats were exposed to 10mg of CdCl2 /L drinking water for 90 days. After 90 days, all animals were victimized and after the macroscopic inspection of the heart, myocardial tissue was routinely processed and embedded in paraffin. Sections 5 micrometers thick were stained by HE method and histochemical PAS-AB (pH 2, 5), Masson trichrome method for demonstrating collagen fibers and Toluidine blue for mast cells identification. Cross-striated banding pattern of cardiac cells was ruined. Noticeable atrophy and hydropic degeneration of subendocardial localized cardiac cells were found, with the focal presence of myocytolysis. Endothelial cell hyperplasia and edema of the intima were present on arteriolar type blood vessels causing the focal subocclusion. Fibrocytes, histiocytes and mast cells were numerous, perivascularly localized. Mast cells were polymorphic, larger than normal, oval and mostly degranulated. Instead of scanty endomysium, there is a noticeable interstitial fibrillar fibrosis with few fields of collagen in all myocardium layers between cardiac cells, which is particularly prominent around the larger blood vessels. Cadmium has pronounced vasculotropic properties causing morphological changes of cardiomyocytes, myocardial interstitial fibrillar collagen network and on the heart small blood vessels. Acta Medica Medianae 2013;52(2):15-22.

Impaired glucose tolerance (IGT) to frank diabetes: Dietary manipulations in WNIN/GR-Ob rats

Background: Several rodent models are available to study obesity and obesity associated diabetic problems. We developed an obese mutant rat model viz., WNIN/GR-Ob from our existing WNIN (Inbred Wistar) stock of rats, which exhibit hyperglycemia on challenge with oral glucose. Since such impaired glucose tolerance (IGT) is a fore runner to frank diabetes status, we carried out a study to challenge the animals with different purified carbohydrate sources (glucose, sucrose, starch) and see the outcome. Methods: 48 obese rats of both genders and equal number of lean littermates of 35 days of age were taken for the study and were divided in to four groups, A, B, C, and D. A group received purified glucose based diet, B, received purified sucrose, C, received purified starch and the D, served as the control, receiving standard laboratory rat chow developed at our centre, containing roasted bengal gram as the source of carbohydrate. All diets were isocaloric in nature and contained 56 % carbohydrate in principle. Animals were fed for 8 weeks and parameters like food intake, body weights, and plasma glucose and insulin levels were measured in experimental and control rats at initial, 4 weeks and 8 weeks. Results: As expected, food intake, body weight and feed efficiency ratio were significantly higher in obese rats of all groups as compared to their lean littermate controls and also higher in stock diet, compared to purified diets. Both lean and obese animals showed higher values of glucose and insulin on purified diets compared to control diet. But amongst lean and obese animals, the latter showed sexual dimorphism in their response, the situation being worse in starch fed (C) group. Amongst the obese animals, the males seem to suffer more, compared to females, in starch fed group, followed by glucose and sucrose fed in that order. Conclusions: WNIN/ GR-Ob rats thus seem to be a useful animal model, vulnerable to diet manipulations, especially to carbohydrates. This has the potential to be used as a diabetic model, more akin to human systems, where diet is the major trigger for precipitating diabetes.

Switching control of sympathetic activity from forebrain to hindbrain in chronic dehydration

We investigated the mechanisms responsible for increased blood pressure and sympathetic nerve activity (SNA) caused by 2-3 days dehydration (DH) both in vivo and in situ preparations. In euhydrated (EH) rats, systemic application of the AT(1) receptor antagonist Losartan and subsequent pre-collicular transection (to remove the hypothalamus) significantly reduced thoracic (t)SNA. In contrast, in DH rats, Losartan, followed by pre-collicular and pontine transections, failed to reduce tSNA, whereas transection at the medulla-spinal cord junction massively reduced tSNA. In DH but not EH rats, selective inhibition of the commissural nucleus tractus solitarii (cNTS) significantly reduced tSNA. Comparable data were obtained in both in situ and in vivo (anaesthetized/conscious) rats and suggest that following chronic dehydration, the control of tSNA transfers from supra-brainstem structures (e.g. hypothalamus) to the medulla oblongata, particularly the cNTS. As microarray analysis revealed up-regulation of AP1 transcription factor JunD in the dehydrated cNTS, we tested the hypothesis that AP1 transcription factor activity is responsible for dehydration-induced functional plasticity. When AP1 activity was blocked in the cNTS using a viral vector expressing a dominant negative FosB, cNTS inactivation was ineffective. However, tSNA was decreased after pre-collicular transection, a response similar to that seen in EH rats. Thus, the dehydration-induced switch in control of tSNA from hypothalamus to cNTS seems to be mediated via activation of AP1 transcription factors in the cNTS. If AP1 activity is blocked in the cNTS during dehydration, sympathetic activity control reverts back to forebrain regions. This unique reciprocating neural structure-switching plasticity between brain centres emphasizes the multiple mechanisms available for the adaptive response to dehydration.

Skeletal muscle lipid metabolism changes in mice selected for high maximal metabolic rate

Exercise and fasting both increase free fatty acids, and elevated unsaturated free fatty acids are up‐regulators of lipid metabolism in skeletal muscles. We examined fatty acids in skeletal muscles in lines of Mus musculus artificially selected for high maximal metabolic rate (high‐MMR). These mice were not exercised and standard laboratory rat chow was available ad libitum. After 7 generations of directional selection for increased mass‐independent MMR, high‐MMR mice showed a 10.2% increase in mass‐independent MMR and a concomitant 2.8% increase in mass‐independent basal metabolic rate compared to control mice. Free fatty acids in the gastrocnemius muscle differed significantly between high‐MMR and control mice. For mice selected for high‐MMR, there was a significant increase in linoleic and oleic acids—consistent with elevated fatty acid oxidation in the skeletal muscle. There was no difference in muscle phospholipid fatty acid composition between high‐MMR and control mice. These findings suggest that selection for high‐MMR resulted in elevated fatty acid oxidation in the skeletal muscles. Most importantly, the results suggest that there is a genetic component to elevated unsaturated free fatty acids in skeletal muscles. Supported by NSF IOS 0344994.

Altered ATP-sensitive potassium channels may underscore obesity-triggered increase in blood pressure1

To determine whether ATP-sensitive potassium channels are altered in VSMC from arotas and mesenteric arteries of obese rat, and their association with obesity-triggered increase in blood pressure. Obesity was induced by 24 weeks of high-fat diet feeding in male Sprague-Dawley rats. Control rats were fed with standard laboratory rat chow. Blood pressure and body weight of these rats were measured every 4 weeks. At the end of 24 weeks, K(ATP) channelmediated relaxation responses in the aortas and mesenteric arteries, K(ATP) channel current, and gene expression were examined, respectively. Blood pressure and body weight were increased in rats fed with high-fat diet. K(ATP) channelmediated relaxation responses, currents, and K(ATP) expression in VSMC of both aortas and mesenteric arteries were inhibited in these rats. Altered ATP-sensitive potassium channels in obese rats may underscore obesity-triggered increase in blood pressure.

Intermittent Access to 20% Ethanol Induces High Ethanol Consumption in Long–Evans and Wistar Rats

There has been some difficulty getting standard laboratory rats to voluntarily consume large amounts of ethanol without the use of initiation procedures. It has previously been shown that standard laboratory rats will voluntarily consume high levels of ethanol if given intermittent-access to 20% ethanol in a 2-bottle-choice setting [Wise, Psychopharmacologia 29 (1973), 203]. In this study, we have further characterized this drinking model. Ethanol-naïve Long-Evans rats were given intermittent-access to 20% ethanol (three 24-hour sessions per week). No sucrose fading was needed and water was always available ad libitum. Ethanol consumption, preference, and long-term drinking behaviors were investigated. Furthermore, to pharmacologically validate the intermittent-access 20% ethanol drinking paradigm, the efficacy of acamprosate and naltrexone in decreasing ethanol consumption were compared with those of groups given continuous-access to 10 or 20% ethanol, respectively. Additionally, ethanol consumption was investigated in Wistar and out-bred alcohol preferring (P) rats following intermittent-access to 20% ethanol. The intermittent-access 20% ethanol 2-bottle-choice drinking paradigm led standard laboratory rats to escalate their ethanol intake over the first 5 to 6 drinking sessions, reaching stable baseline consumption of high amounts of ethanol (Long-Evans: 5.1 +/- 0.6; Wistar: 5.8 +/- 0.8 g/kg/24 h, respectively). Furthermore, the cycles of excessive drinking and abstinence led to an increase in ethanol preference and increased efficacy of both acamprosate and naltrexone in Long-Evans rats. P-rats initiate drinking at a higher level than both Long-Evans and Wistar rats using the intermittent-access 20% ethanol paradigm and showed a trend toward a further escalation in ethanol intake over time (mean ethanol intake: 6.3 +/- 0.8 g/kg/24 h). Standard laboratory rats will voluntarily consume ethanol using the intermittent-access 20% ethanol drinking paradigm without the use of any initiation procedures. This model promises to be a valuable tool in the alcohol research field.

Uptake and metabolism of plasma-derived erucic acid by rat brain

We examined the ability of erucic acid (22:1n-9) to cross the blood-brain barrier (BBB) by infusing [14-14C]22:1n-9 (170 microCi/kg, iv and icv) into awake, male rats. [1-14C]arachidonic acid (20:4n-6) [intravenous (i.v.)] was the positive control. After i.v. infusion, 0.011% of the plasma [14-14C]22:1n-9 was extracted by the brain, compared with 0.055% of the plasma [1-14C]20:4n-6. The [14-14C]22:1n-9 was extensively beta-oxidized (60%), compared with 30% for [1-14C]20:4n-6. Although 20:4n-6 was targeted primarily to phospholipid pools, 22:1n-9 was targeted to cholesteryl esters, triglycerides, and phospholipids. When [14-14C]22:1n-9 was infused directly into the fourth ventricle of the brain [intracerebroventricular (i.c.v.)] for 7 days, 60% of the tracer entered the phospholipid pools, similar to the distribution observed for [1-14C]20:4n-6. This demonstrates plasticity in the ability of the brain to esterify 22:1n-9 in an exposure-dependent manner. In i.v. and i.c.v. infused rats, a significant amount of tracer found in the phospholipid pools underwent sequential rounds of chain shortening and was found as [12-14C]20:1n-9 and [10-14C]oleic acid. These results demonstrate for the first time that intact 22:1n-9 crosses the BBB, is incorporated into specific lipid pools, and is chain-shortened.

Smooth endoplasmic reticulum dilation and degeneration in Purkinje neuron dendrites of aging ethanol-fed female rats

The effects of chronic ethanol consumption on the extensive Purkinje neuron (PN) dendritic arbor of male rats include dilation of the smooth endoplasmic reticulum (SER) and dendritic regression. The purpose of the present study was to examine the molecular layer of female rats for the presence of ethanol-related SER dilation and evidence of degeneration within the PN dendritic arbor. Twenty-one 12-month-old Fischer 344 female rats (n = 7/treatment group) received a liquid ethanol, liquid control, or rat chow diet for a period of 40 weeks. Ethanol-fed rats received 35% of their dietary calories as ethanol. Pair-fed rats received a liquid control diet that was isocaloric to the ethanol diet. Chow-fed rats received standard laboratory rat chow ad libitum. At the end of treatment, tissues from the anterior and posterior lobes of the cerebellar vermis were viewed and photographed with the electron microscope. The diameters of SER profiles were measured and the density of degenerating bodies within the PN dendritic arbor was quantitated. In the posterior lobe, ethanol-related SER dilation was apparent. In the anterior lobe, the density of degenerating bodies within PN dendritic shafts was significantly increased but SER dilation in PN dendritic shafts was absent. These results confirm that SER dilation and dendritic degeneration in PN dendrites may precede and contribute to ethanol-related regression in female rats. In addition, comparison of these results with data obtained in male rats from a previous study suggests that PN dendrites in females may be more sensitive to the effects of ethanol.

Inhibition of Hydrogen Sulfide Generation Contributes to Gastric Injury Caused by Anti-Inflammatory Nonsteroidal Drugs

Hydrogen sulfide (H(2)S), an endogenous gaseous mediator that causes vasodilation, is generated in mammalian tissues by cystathionine beta-synthase (CBS) and cystathionine-gamma-lyase (CSE). Here, we have investigated the role of H(2)S in a rodent model of nonsteroidal anti-inflammatory drug (NSAID) gastropathy. Rats were given acetyl salycilic acid (ASA) or an NSAID alone or in combination with NaHS, an H(2)S donor, and killed 3 hours later. Gastric blood flow was measured by laser-Doppler flowmetry, whereas intravital microscopy was used to quantify adhesion of leukocytes to mesenteric postcapillary endothelium. At a dose of 100 micromol/kg, NaHS attenuated by 60%-70% the gastric mucosal injury, and tumor necrosis factor (TNF)-alpha, intercellular adhesion molecule (ICAM)-1, and lymphocyte function-associated antigen (LFA)-1 mRNA up-regulation induced by NSAIDs (P < .05) NaHS administration prevented the associated reduction of gastric mucosal blood flow (P < .05) and reduced ASA-induced leukocyte adherence in mesenteric venules. NaHS did not affect suppression of prostaglandin E(2) (PGE(2)) synthesis by NSAIDs. Glibenclamide, a K(ATP) channel inhibitor, and DL-propargylglycine, a CSE inhibitor, exacerbated, whereas pinacidil, a K(ATP) opener, attenuated gastric injury caused by ASA. Exposure to NSAIDs reduced H(2)S formation and CSE expression (mRNA and protein) and activity by 60%-70%. By promoter deletion and mutation analysis, an Sp1 consensus site was identified in the CSE promoter. Exposure to NSAIDs inhibits Sp1 binding to its promoter and abrogates CSE expression in HEK-293 cells transfected with a vector containing the core CSE promoter. Exposure to NSAIDs inhibits Sp1 and ERK phosphorylation. These data establish a physiologic role for H(2)S in regulating the gastric microcirculation and identify CSE as a novel target for ASA/NSAIDs.

High dietary potassium chloride intake augments rat renal mineralocorticoid receptor selectivity via 11β-hydroxysteroid dehydrogenase

Glucocorticoid access to renal corticosteroid receptors is regulated by 11β-hydroxysteroid dehydrogenases (11β-HSDs), converting 11β-hydroxyglucocorticoids into inactive 11-ketones. This mechanism plays a key role in maintaining normal salt–water homeostasis and blood pressure. To study whether renal cortical proximal and distal tubular 11β-HSDs are modulated, upon shifting the electrolyte status (and may thereby contribute to adjusting the salt–water homeostasis), rats were treated for 14 days with diets with low (0.058 w/w%), normal (0.58%, which is the KCl content of standard European laboratory rat food) or high (5.8%) potassium chloride content. In proximal tubules, dietary KCl had no effect regarding corticosterone 11β-oxidation in intact cells as well as 11β-HSD1 and 11β-HSD2 protein (Western blotting) and mRNA levels (semi-quantitative RT-PCR). In distal tubules, the low KCl diet also had no effect. However, distal tubules of rats fed the high KCl diet showed increased corticosterone 11β-oxidation rates (1.6-fold, P<0.01) and 11β-HSD2 protein (4-fold, P<0.01), whereas 11β-HSD1 protein was decreased (no longer detected, P<0.05). Distal tubular 11β-HSD mRNA levels were not changed upon dietary treatment. Our results suggest that upon dietary KCl loading distal tubular mineralocorticoid receptor selectivity for aldosterone is increased because of enhanced corticosterone 11β-oxidation. This may contribute to the fine-tuning of salt–water homeostasis by the kidney.

Vitamin E suppresses cyclosporine A–induced increase in the urinary excretion of arachidonic acid metabolites including F 2-isoprostanes in the rat model

ESPITE a large body of research, the exact mechanism by which cyclosporine (CsA) causes nephrotoxicity remains unclear. A consistent finding following CsA administration is the increase in urinary excretion of arachidonic acid metabolites. 1‐3 Although experimental studies with thromboxane synthase inhibitors suggested a role for TXA2 in CsA-induced renal dysfunction, chronic administration of thromboxane synthase inhibitors in renal transplant patients treated with CsA had little effect on improving renal function. 4,5 One possible explanation would be that arachidonic acid metabolites other than TXA2 might also be involved in the mediation of renal vasoconstriction and dysfunction. This possibility deserves consideration, because recent studies suggest that free radical-catalyzed lipid peroxidation, independent of the enzymatic pathway, may produce vasoactive prostaglandins, F2-isoprostanes and E2-isoprostanes, as well as thromboxane and isothromboxane. 6‐ 9 We have reported that dosedependent renal lipid peroxidation occurs with CsA-induced renal toxicity and that vitamin E and Lazaroid compounds suppress lipid peroxidation and protect the kidney against CsA-mediated functional and structural injury. 10,11 Recent findings from others are consistent with these observations, thus providing further support for an important role for free radical-catalyzed lipid peroxidation in the pathogenesis of CsA-induced nephrotoxicity. 12‐14 Because CsA induces lipid peroxidation and this in turn can catalyze the formation of prostaglandin- and thromboxane-like molecules, 6‐ 9 we examined whether the excessive production of arachidonic acid metabolites by CsA might be linked to heightened lipid peroxidation. Compared to vehicle, 12 weeks of CsA administration was associated with a significant reduction in renal function and an increase in urinary excretion of vasoconstrictive TBX-B2 and F2-IP and vasodilatory PGF1a. Administration of vitamin E along with CsA was accompanied by marked preservation of GFR and significant suppression of urinary TBX-B2 ,F 2-IP and PGF1a. In another experiment, vitamin E administration in normal rats did not have any effect on renal function or arachidonic acid metabolite excretion. Our new findings suggest that part of the CsA-induced increase in urinary arachidonic acid metabolites may arise from free radicalcatalyzed lipid peroxidation, and that the renoprotective effect of vitamin E may arise in part from the suppression of two potent vasoconstrictors, TBX and F2-IP. METHODS Effect of Vitamin E on CsA-Induced Increase in Urinary Excretion of Arachidonic Acid Metabolites Two weeks after subcostal right nephrectomy to simulate the single kidney model of transplant, male Sprague Dawley rats, matched for body weight and serum creatinine, were assigned to 3 groups: the control (n 5 5), CsA (n 5 6), and CsA plus vitamin E (n 5 6). The initial body weights of these rats were between 250 to 275 g. The control rats received olive oil vehicle by gavage (1 mL/kg/d) and by subcutaneous injections (0.5 mL/kg/d). The CsA group received CsA 15 mg/kg/d in olive oil subcutaneously and plain olive oil by gavage. The CsA plus vitamin E group received CsA as in the CsA group, and additionally, received vitamin E 25 mg/kg/d in olive oil by gavage. Rats were pair-fed with standard laboratory rat chow and their food intake and body weights were monitored once a week. Serial hematocrit (microcapillary method) and serum protein (refractory method) were measured from tail vein blood. Using metabolic cages, 24-hour urine was collected into containers containing antioxidant (100 mL of 2% butylated hydroxytoluene in ethanol). Urine samples were spun down and aliquots were saved at 284° until the time of assay. Creatinine in the plasma and urine were measured using an auto-analyzer based on the Jaffe reaction.