Effects of ovariectomy and estrogen on ischemia-reperfusion injury in hindlimbs of female rats

The effects of estrogen and ovariectomy on indexes of muscle damage after 2 h of complete hindlimb ischemia and 2 h of reperfusion were investigated in female Sprague-Dawley rats. The rats were assigned to one of three experimental groups: ovariectomized with a 17-estradiol pellet implant (OE), ovariectomized with a placebo pellet implant (OP), or control with intact ovaries (R). It was hypothesized that following ischemia-reperfusion (I/R), muscle damage indexes [serum creatine kinase (CK) activity, calpain-like activity, inflammatory cell infiltration, and markers of lipid peroxidation (thiobarbituric-reactive substances)] would be lower in the OE and R rats compared with the OP rats due to the protective effects of estrogen. Serum CK activity following I/R was greater (P < 0.01) in the R rats vs. OP rats and similar in the OP and OE rats. Calpain-like activity was greatest in the R rats (P < 0.01) and similar in the OP and OE rats. Neutrophil infiltration was assessed using the myeloperoxidase (MPO) assay and immunohistochemical staining for CD43-positive (CD43+) cells. MPO activity was lower (P < 0.05) in the OE rats compared with any other group and similar in the OP and R rats. The number of CD43+ cells was greater (P < 0.01) in the OP rats compared with the OE and R rats and similar in the OE and R rats. The OE rats had lower (P < 0.05) thiobarbituric-reactive substance content following I/R compared with the R and OP rats. Indexes of muscle damage were consistently attenuated in the OE rats but not in the R rats. A 10-fold difference in serum estrogen content may mediate this. Surprisingly, serum CK activity and muscle calpain-like activity were lower (P < 0.05) in the OP rats compared with the R rats. Increases in serum insulin-like growth factor-1 content (P < 0.05) due to ovariectomy were hypothesized to account for this finding. Thus both ovariectomy and estrogen supplementation have differential effects on indexes of I/R muscle damage.

Green tea, black tea, and epigallocatechin modify body composition, improve glucose tolerance, and differentially alter metabolic gene expression in rats fed a high-fat diet

The mechanisms of how tea and epigallocatechin-3-gallate (EGCG) lower body fat are not completely understood. This study investigated long-term administration of green tea (GT), black tea (BT), or isolated EGCG (1 mg/kg per day) on body composition, glucose tolerance, and gene expression related to energy metabolism and lipid homeostasis; it was hypothesized that all treatments would improve the indicators of metabolic syndrome. Rats were fed a 15% fat diet for 6 months from 4 weeks of age and were supplied GT, BT, EGCG, or water. GT and BT reduced body fat, whereas GT and EGCG increased lean mass. At 16 weeks GT, BT, and EGCG improved glucose tolerance. In the liver, GT and BT increased the expression of genes involved in fatty acid synthesis (SREBP-1c, FAS, MCD, ACC) and oxidation (PPAR-α, CPT-1, ACO); however, EGCG had no effect. In perirenal fat, genes that mediate adipocyte differentiation were suppressed by GT (Pref-1, C/EBP-β, and PPAR-γ) and BT (C/EBP-β), while decreasing LPL, HSL, and UCP-2 expression; EGCG increased expression of UCP-2 and PPAR-γ genes. Liver triacylglycerol content was unchanged. The results suggest that GT and BT suppressed adipocyte differentiation and fatty acid uptake into adipose tissue, while increasing fat synthesis and oxidation by the liver, without inducing hepatic fat accumulation. In contrast, EGCG increased markers of thermogenesis and differentiation in adipose tissue, while having no effect on liver or muscle tissues at this dose. These results show novel and separate mechanisms by which tea and EGCG may improve glucose tolerance and support a role for these compounds in obesity prevention.

Age does not influence the bone response to treadmill exercise in female rats

Purpose: Because it is believed that bone may respond to exercise differently at different ages, we compared bone responses in immature and mature rats after 12 wk of treadmill running.

Methods: Twenty-two immature (5-wk-old) and 21 mature (17-wk-old) female Sprague Dawley rats were randomized into a running (trained, N = 10 immature, 9 mature) or a control group (controls, N = 12 immature, 12 mature) before sacrifice 12 wk later. Rats ran on a treadmill five times per week for 60-70 min at speeds up to 26 m[middle dot]min-1. Both at baseline and after intervention, we measured total body, lumbar spine, and proximal femoral bone mineral, as well as total body soft tissue composition using dual-energy x-ray absorptiometry (DXA) in vivo. After sacrificing the animals, we measured dynamic and static histomorphometry and three-point bending strength of the tibia.

Results: Running training was associated with greater differences in tibial subperiosteal area, cortical cross-sectional area, peak load, stiffness, and moment of inertia in immature and mature rats (P < 0.05). The trained rats had greater periosteal bone formation rates (P < 0.01) than controls, but there was no difference in tibial trabecular bone histomorphometry. Similar running-related gains were seen in DXA lumbar spine area (P = 0.04) and bone mineral content (BMC;P = 0.03) at both ages. For total body bone area and BMC, the immature trained group increased significantly compared with controls (P < 0.05), whereas the mature trained group gained less than did controls (P < 0.01).

Conclusion: In this in vivo model, where a similar physical training program was performed by immature and mature female rats, we demonstrated that both age groups were sensitive to loading and that bone strength gains appeared to result more from changes in bone geometry than from improved material properties.

Histological assessment of intermediate - and long-term creatine monohydrate supplementation in mice and rats

Creatine monohydrate (CrM) supplementation appears to be relatively safe based on data from short-term and intermediate-term human studies and results from several therapeutic trials. The purpose of the current study was to characterize pathological changes after intermediate-term and long-term CrM supplementation in mice [healthy control and SOD1 (G93A) transgenic] and rats (prednisolone and nonprednisolone treated). Histological assessment (18-20 organs/tissues) was performed on G93A mice after 159 days, and in Sprague-Dawley rats after 365 days, of CrM supplementation (2% wt/wt) compared with control feed. Liver histology was also evaluated in CD-1 mice after 300 days of low-dose CrM supplementation (0.025 and 0.05 g · kg^-1 · day^-1) and in Sprague-Dawley rats after 52 days of CrM supplementation (2% wt/wt) with and without prednisolone. Areas of hepatitis were observed in the livers of the CrM-supplemented G93A mice (P < 0.05), with no significant inflammatory lesions in any of the other 18-20 tissues/organs that were evaluated. The CD-1 mice also showed significant hepatic inflammatory lesions (P < 0.05), yet there was no negative effect of CrM on liver histology in the Sprague-Dawley rats after intermediate-term or long-term supplementation nor was inflammation seen in any other tissues/organs (P = not significant). Dietary CrM supplementation can induce inflammatory changes in the liver of mice, but not rats. The observed inflammatory changes in the murine liver must be considered in the evaluation of hepatic metabolism in CrM-supplemented mice. Species differences must be considered in the evaluation of toxicological and physiological studies.

Effect of low and high glycaemic index diets on developing the risk of metabolic syndrome in rats

Several observational studies have shown that the chronic consumption of high glycaemic index diet is associated with an increased risk of developing metabolic syndrome.  This study was performed to identify the direct influences on the lipid profile and the adipose tissue deposition and the subsequent development of the risk of metabolic syndrome in rats by feeding diets of low glycaemic index (LGI) and high glycaemic index (HGI). Fifty rat weanlings (three weeks old) were equally divided into two groups and fed on either low glycaemic index diet based on high amylose, or isocaloric high glycacmic index diet for 12 weeks. Postprandial blood and tissue samples were collected at the end of the 12 weeks of feeding. The total white adipose tissue weights of the HGl fed rats (24.74 ± 0.53 glrat) were significantly higher than the LGl fed rats (15.37 ± 0.36 gh·at). The HO! led rats had higher postprandial leptin concentrations (1.86 ± 0.17 ng/ml) than LGI fed rats (1.34 ± 0.12 ng/ml). The postprandial insulin, and postprandial insulin glucose ratio were higher in the HGI fed rats (7.06 ± 0.90 ng/ml and 0.67 ± 0.01 ng/mlxmM) compared to the LGl fed rats (3.91 ± 0.4 ng/ml and 0.44 ± 0.01 ng/mlxmM). Triglycerides of the l-IGI fed rats showed higher values (I .56 ± 0.10 mM) than the LO! fed rats (l.07 ± 0.08 mM). The results indicated that LGI feeding was beneficial in preventing the conditions enhancing the cardio vascular disease whereas long-term feeding of HGI diet may increase the risk of developing metabolic syndrome in rats.

Rapid bioassay-guided screening of toxic substances in vegetable oils that shorten the life of SHRSP rats

It has been consistently reported that vegetable oils including canola oil have a life shortening effect in Stroke-Prone Spontaneously Hypertensive Rats (SHRSP) and this toxic effect is not due to the fatty acid composition of the oil. Although it is possible that the phytosterol content or type of phytosterol present in vegetable oils may play some role in the life shortening effect observed in SHRSP rats this is still not completely resolved. Furthermore supercritical CO2 fractionation of canola oil with subsequent testing in SHRSP rats identified safe and toxic fractions however, the compounds responsible for life shortening effect were not characterised. The conventional approach to screen toxic substances in oils using rats takes more than six months and involves large number of animals. In this article we describe how rapid bioassay-guided screening could be used to identify toxic substances derived from vegetable oils and/or processed foods fortified with vegetable oils. The technique incorporates sequential fractionation of oils/processed foods and subsequent treatment of human cell lines that can be used in place of animal studies to determine cytotoxicity of the fractions with structural elucidation of compounds of interest determined via HPLC-MS and GC-MS. The rapid bioassay-guided screening proposed would require two weeks to test multiple fractions from oils, compared with six months if animal experiments were used to screen toxic effects. Fractionation of oil before bio-assay enhances the effectiveness of the detection of active compounds as fractionation increases the relative concentration of minor components.

Comparative effects of tuna oil and salmon oil on liver lipid metabolism and fatty acid concentrations in rats

The comparative effect of tuna oil (TO) and salmon oil (SO) on the plasma and liver lipid and fatty acid compositions in Sprague Dawley rats was investigated. The total triacylglycerol (TG) and total cholesterol (TC) concentrations in liver was significantly decreased in the TO group; TG level in liver was also significantly decreased in the SO group. The mRNA expression of HMG-CoA reductase in liver was significantly down-regulated in the TO and SO groups relative to the control group. The plasma TG and TC were decreased in TO, but not in SO; plasma low-density lipoprotein and very low-density lipoprotein levels in TO and SO were decreased compared with the control group. The total n-3 polyunsaturated fatty acid (PUFA) in plasma and liver phospholipids was significantly elevated in the TO and SO. Docosahexaenoic acid (22:6n-3) and eicosapentaenoic acid (20:5n-3) in tissues were significantly increased in the TO and SO, respectively. In this study, TO had a more beneficial effect on liver TC and plasma TG, TC, high-density lipoprotein in rats than SO. The likely mechanism for lowering liver and plasma cholesterol by n-3 PUFA is to suppress the mRNA expression of gene encoding HMG-CoA reductase responsible for cholesterol biosynthesis.

PRACTICAL APPLICATIONS

The beneficial effects of n-3 polyunsaturated fatty acids (PUFAs) from fish and fish oil on human health is derived from their role in modulating membrane lipid composition and affecting metabolic and signal-transduction pathways. In the present study, we demonstrated that n-3 PUFA, docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) from tuna and salmon oils can be effectively incorporated into tissue membranes. Tuna oil rich in DHA has more beneficial effect on liver total cholesterol (TC) and plasma triglyceride, TC and HDL in rats than salmon oil, which is rich in EPA. The present data could provide information for the potential application of fish oils as components of functional food, and selected for fortification with different fish oils.

The trophic effect of dietary fibre is not associated with a change in total crypt number in the distal colon of rats

Soluble fibres, such as guar gum, promote and wheat bran or methylcellulose protect from chemically induced colon carcinogenesis, relative to the effect of a fibre-free diet in rats. Mechanisms are poorly understood. Whereas all fibres are trophic to the colonic epithelium, the heterogeneity of effects on carcinogenesis may reflect different effects on the total number of crypts and, therefore, the size of the stem cell population. This study aimed to assess this hypothesis. Sprague–Dawley rats were fed one of fibre-free diets with or without 10% wheat bran, methylcellulose or guar gum for 4 weeks. The distal colons were stained with methylene blue and quantified for the number and density of crypts using an image analysis system. Epithelial proliferative kinetics was measured stathmokinetically. Methodology for quantifying crypts was valid and reproducible. Rats fed a fibre-free diet had atrophic distal colon, as shown by a decrease in crypt column height and a lower mitotic index. Fibre supplementation prevented the atrophy and was associated with crypt mouth areas that were 30–60% larger than those in the fibre-free group (P < 0.001, ANOVA), with the methylcellulose group being the largest (1.16 µm2). The crypt density of the fibre-free group was 16–19% greater than those in fibre fed groups (P + 0.006), due to the smaller size of the crypts. However, there was no difference in the total number of crypts across the four dietary groups (P > 0.1). Distal colons in all of the dietary groups contained ~105 crypts. In conclusion, although variation in the amount or type of dietary fibre exerts heterogeneous effects on the growth of the colonic epithelium and on colon carcinogenesis, the total number of crypts in the distal colon remains constant. It is, therefore, unlikely that fibres influence carcinogenic events by altering the size of the stem cell population.

The influence of specific luminal factors on the colonic epithelium : high-dose butyrate and physical changes suppress early carcinogenic events in rats

INTRODUCTION Although luminal delivery of butyrate is one putative mechanism by which biology of the colonic epithelium might be influenced by changes in luminal contents, there is a paucity of supportive cause–effect evidence. This study aimed to directly establish whether distal colonic butyrate delivery is able to alter the response of the distal colonic epithelium to a carcinogen.
METHODS Groups of male Sprague-Dawley rats with chronically intubated colons received infusions of 80 mM butyrate or 0.9 percent saline into distal colon two or five times daily. Three weeks after exposure to azoxymethane (15 mg/kg subcutaneously), the density of aberrant crypts was quantified in distal colon.
RESULTS Infusions of 0.5 ml twice daily, whether containing saline or butyrate, decreased the number of aberrant crypt foci by 45 percent compared with rats receiving no infusions (P = 0.004, analysis of variance). Similar results were obtained when infusions were restricted to the postinitiation phase. When infusions were increased to 1 ml five times daily, saline infusions similarly suppressed aberrant crypt formation (38 percent), but butyrate infusions suppressed it to a greater degree (by 64 percent; P = 0.02 compared with saline infusion, t-test).
CONCLUSIONS High levels of butyrate delivery to the distal colonic lumen alter the epithelial response to a carcinogen in otherwise healthy rats. This finding directly supports the notion that the effects of butyrate on cells in vitro do occur in vivo provided a sufficient dose is delivered. The effect of infusion of liquid per se on the epithelial response highlights the potential impact physical changes alone can have on the colonic epithelium.

Sodium appetite in adult rats following ω-3 polyunsaturated fatty acid deficiency in early development

We examined the effect of ω-3 polyunsaturated fatty acid (PUFA) deficiency during development on sodium appetite. Being raised on an ω-3 PUFA deficient diet increased the intake of 0.5 M NaCl following furosemide-induced sodium depletion by 40%. This occurred regardless of the diet they were maintained on later in life, and the increased consumption persisted for 3 days. In a second study, animals were administered furosemide and low-dose captopril. Sodium consumption of deficient raised animals was again higher than that of the control raised. Fos immunoreactivity in brain areas associated with sodium appetite and excretion were not influenced by diet. Our findings indicate that inadequate dietary ω-3 PUFA during development results in an exaggerated sodium appetite later in life.

Antiamnesic effect of stevioside in scopolamine-treated rats

The present study was undertaken to explore the potential of Stevioside in memory dysfunction of rats. Memory impairment was produced by Scopolamine (0.5 mg/kg, i.p.) in animals. Morris water-maze test was employed to assess learning and memory. Brain acetylcholinestrase enzyme (AChE) activity was measured to assess central cholinergic activity. The levels of brain thio-barbituric acid reactive species (TBARS) and reduced glutathione (GSH) were estimated to assess degree of oxidative stress. Scopolamine administration induced significant impairment of learning and memory in rats as indicated by marked decrease in Morris water-maze performance. Scopolamine administration also produced a significant enhancement of brain AChE activity and brain oxidative stress (increase in TBARS & decrease in GSH) levels. Pretreatment of Stevioside (250mg/kg dose orally) significantly reversed Scopolamine induced learning & memory deficits along with attenuation of Scopolamine induced rise in brain AChE activity and brain oxidative stress levels. It may be concluded that Stevioside exerts memory preservative effect in cognitive deficits of rats possibly through its multiple actions.