Melatonin improves inflammation processes in liver of senescence-accelerated prone male mice (SAMP8).

Aging is associated with an increase in oxidative stress and inflammation. The aim of this study was to investigate the effect of aging on various physiological parameters related to inflammation in livers obtained from two types of male mice models: Senescence-accelerated prone (SAMP8) and senescence-accelerated-resistant (SAMR1) mice, and to study the influence of the administration of melatonin (1mg/kg/day) for one month on old SAMP8 mice on these parameters. The parameters studied have been the mRNA expression of TNF-α, iNOS, IL-1ß, HO-1, HO-2, MCP1, NFkB1, NFkB2, NFkB protein or NKAP and IL-10. All have been measured by real-time reverse transcription polymerase chain reaction RT-PCR. Furthermore we analyzed the protein expression of TNF-α, iNOS, IL-1ß, HO-1, HO-2, and IL-10 by Western-blot. Aging increased oxidative stress and inflammation especially in the liver of SAMP8 mice. Treatment with melatonin decreased the mRNA expression of TNF-α, IL-1ß, HO (HO-1 and HO-2), iNOS, MCP1, NFκB1, NFκB2 and NKAP in old male mice. The protein expression of TNF-α, IL-1ß was also decreased and IL-10 increased with melatonin treatment and no significant differences were observed in the rest of parameters analyzed. The present study showed that aging was related to inflammation in livers obtained from old male senescence prone mice (SAMP8) and old male senescence resistant mice (SAMR1) being the alterations more evident in the former. Exogenous administration of melatonin was able to reduce inflammation.

Effects of growth hormone, melatonin, oestrogens and phytoestrogens on the oxidized glutathione (GSSG)/reduced glutathione (GSH) ratio and lipid peroxidation in aged ovariectomized rats.

Ovariectomy constitutes a commonly used model in rats and mice for human menopause. After ovariectomy, an imbalance between oxidant production and antioxidant levels appears in favour of the former, with increased oxidative stress and consequently an acceleration of ageing. In the present work, the levels of reduced glutathione (GSH), a relevant antioxidant, and oxidized glutathione (GSSG), an oxidant compound, as well as lipid peroxidation (through malondialdehyde (MDA) levels), were studied in liver, heart, kidney and spleen homogenates of old (24 months of age) unovariectomized and ovariectomized female Wistar rats. The results showed a significant increase of the GSSG/GSH ratio, a marker of oxidative stress, and higher MDA production in all the studied organs of ovariectomized rats as compared with unovariectomized animals. These data confirm the idea that ovariectomy accelerates the ageing process. Administration of growth hormone (GH), melatonin (MEL) and oestrogens (OE), as well as soybean phytoestrogens (PE) for 10 weeks, between 22 and 24 months of age, was able to decrease oxidative stress in the investigated organs of ovariectomized old rats, therefore slowing down the ageing process in those animals.

Effect of growth hormone treatment on lymphocyte functions in old male rats.

INTRODUCTION: Age-related changes in the communication between the neuroendocrine and the immune system have been scarcely studied. Aging in mammals is associated with an impairment of the immune response, especially regarding lymphocyte functions. Furthermore, the endocrine system is also affected by aging, one of the most significant changes being the decrease in the secretion of several hormones such as growth hormone (GH). OBJECTIVE: The aim of the present work was to study whether GH replacement therapy in old male rats could improve several lymphocyte functions. METHODS: Spleen and axillary node lymphocytes from old (24 months of age) male Wistar rats were used in the present study to investigate the effect of GH (2 mg/kg daily during 10 weeks) on chemotaxis, lymphoproliferative response to the mitogen concanavalin A, interleukin 2 release and natural killer cell activity. RESULTS: We have found that the administration of GH can reduce or even reverse the age-related changes observed in these key immune function parameters. Moreover, we have observed that the recovery of such immune functions is able to reach similar values as those exhibited by young control animals of 6 months of age. CONCLUSION: Considering that the immune system is a marker of health and a predictor of longevity, hormone replacement therapies with GH, by increasing the immune function and thus delaying or slowing down some aspects of the aging process, could facilitate successful aging.

Molecular mechanisms involved in the hormonal prevention of aging in the rat.

Previous data from our group have provided support for the role of GH, melatonin and estrogens in the prevention of aging of several physiological parameters from bone, liver metabolism, vascular activity, the central nervous system (CNS), the immune system and the skin. In the present work data on the molecular mechanisms involved are presented. A total of 140 male and female rats have been submitted to different treatments over 10 weeks, between 22 and 24 months of age. Males have been treated with GH and melatonin. Females were divided in two groups: intact and castrated at 12 months of age. The first group was treated with GH and melatonin and the second with the two latter compounds and additionally with estradiol and Phytosoya. Aging was associated with a reduction in the number of neurons of the hylus of the dentate gyrus of the hippocampus and with a reduction of neurogenesis. GH treatment increased the number of neurons but did not increase neurogenesis thus suggesting a reduction of apoptosis. This was supported by the reduction in nucleosomes and the increase in Bcl2 observed in cerebral homogenates together with an increase in sirtuin2 and a reduction of caspases 9 and 3. Melatonin, estrogen and Phytosoya treatments increased neurogenesis but did not enhance the total number of neurons. Aging induced a significant increase in mitochondrial nitric oxide in the hepatocytes, together with a reduction in the mitochondrial fraction content in cytochrome C and an increase of this compound in the cytosolic fraction. Reductions of glutathione peroxidase and glutathione S-transferase were also detected, thus indicating oxidative stress and possibly apoptosis. Treatment for 2.5 months of old rats with GH and melatonin were able to significantly and favourably affect age-induced deteriorations, thus reducing oxidative damage. Keratinocytes obtained from old rats in primary culture showed an increase in lipoperoxides, caspases 8 and 3 as well as a reduction in Bcl2 leading to enhanced number of nucleosomes that was also restored upon treatments with GH and melatonin. In conclusion, GH and melatonin treatment seem to have beneficial effects against age-induced damage in the CNS the liver and the skin through molecular mechanisms reducing oxidative stress and apoptosis.

Features of follicle-stimulating hormone-stimulated follicles in a sheep model: keys to elucidate embryo failure in assisted reproductive technique cycles.

OBJECTIVE: To evaluate the individual functionality of gonadotropin-stimulated preovulatory follicles, for understanding embryo failure in assisted reproductive technique cycles, in a sheep model. DESIGN: Observational, model study. SETTING: Public research unit. ANIMAL(S): Fifteen adult Manchega ewes. INTERVENTION(S): Synchronization of the estrous cycle with intravaginal progestagens and ovarian stimulation with FSH; evaluation of reproductive activity, plasma sampling, ovarian ultrasonography, and ovariectomies. MAIN OUTCOME MEASURE(S): Determination of estrus behavior, plasma and intrafollicular concentrations of E(2) and inhibin A, number and size of ovarian follicles, and developmental competence of oocytes. RESULT(S): These results support the usefulness of serial measurements of plasma inhibin A for assessment of follicular growth during the FSH treatment, rather than of E(2) assays commonly used. Functionality of FSH-stimulated preovulatory follicles is clearly disturbed, as confirmed by a negative correlation between follicular size and intrafollicular concentrations of inhibin A and E(2) in preovulatory follicles after individual dissection; moreover, the ability of their oocytes to resume meiosis was diminished. CONCLUSION(S): Functionality of follicles in controlled ovarian stimulation (COS), and developmental competence of their oocytes, is disturbed by the high doses of gonadotropin supplied and finally determined by follicular sizes at starting FSH treatment.

Estradiol and soy extract increase the production of new cells in the dentate gyrus of old rats.

In young rodents, estradiol increases cell proliferation in the dentate gyrus of the hippocampus. However, it is unknown if the old brain retains this response to estradiol. Here we assessed the generation of new cells in the dentate gyrus of old rats after administration of estradiol or a soy extract, since soy is used as an alternative to hormonal replacement therapy in postmenopausal women. In a first experiment, 12-month-old animals were ovariectomized and studied at 14, 18 or 22 months of age. The production of new cells, assessed by the incorporation of bromodeoxyuridine (BrdU), was similar in 14- and 18-month-old rats. However, there was a significant reduction in the number of BrdU-immunoreactive cells at 22 months of age. In a second experiment, 22-month-old ovariectomized animals were treated for 10 weeks with a weekly s.c. injection of 150 microg estradiol valerianate or with 60 mg/kg per day soy extract added to the drinking water. Both treatments increased significantly the production of new cells in the dentate gyrus. These findings indicate that the brains of old rats retain the ability to increase the production of new cells in response to estradiol and soy extracts.

Effect of growth hormone and estrogen administration on hepatocyte alterations in old ovariectomized female wistar rats.

Aging could be due to the accumulation of oxidative damage. On the other hand, growth hormone (GH) and estrogen deficiency induce deleterious effects on different tissues, and hormonal replacement could counteract these effects. We have investigated whether GH and estrogen administration modify some parameters related to oxidative stress and inflammation in hepatocytes isolated from old ovariectomized female rats. Twenty-two month-old ovariectomized animals were divided into control rats, rats treated with GH, rats treated with estradiol, and rats treated with GH+estradiol. Two-month-old intact female rats were used as young reference group. Hepatocytes were isolated, cultured, and CO and NO release, ATP, cyclic-guanosyl monophosphate (cGMP), and lipid peroxide (LPO) content of cells, as well as phosphatidylcholine (PC)synthesis, were measured. Hepatocytes isolated from old ovariectomized rats showed a decrease in ATP content and PC synthesis compared to young rats. Age also induced an increase in LPO, NO, CO, and cGMP. Treating old rats with GH significantly increased ATP and reduced CO and cGMP levels. Estradiol administration improved all the parameters that were altered. Co-administration of GH and estrogens induced a more marked effect than estrogens alone only in cGMP content. In conclusion, administration of estrogens to old ovariectomized females seemed to prevent oxidative changes in hepatocytes, whereas the effect of GH is not so evident.

Effect of melatonin administration on parameters related to oxidative damage in hepatocytes isolated from old Wistar rats.

Aging induces changes in several organs and tissues, such as the liver, and this process might be due to oxidative damage caused by free radicals and inflammatory mediators. Melatonin is a secretory product with well-known antioxidant properties. The aim of this study was to investigate the effect of melatonin administration on age-induced alterations in hepatocytes. Twenty-two-month old male Wistar rats were treated with oral melatonin for 10 wk. At the end of the treatment, hepatocytes were isolated and cultured, and different parameters were measured in both cells and medium. Aging induced a significant increase in lipid peroxidation, nitric oxide, carbon monoxide and cyclic guanosyl-monophosphate, as well as a reduction in adenosine triphosphate content and phosphatidylcholine synthesis when compared to young animals. Melatonin administration significantly ameliorated all these age-related changes in males. Melatonin administration seems to exert beneficial effects against age-induced changes in hepatocytes.

Growth hormone prevents neuronal loss in the aged rat hippocampus.

Decline of growth hormone (GH) with aging is associated to memory and cognitive alterations. In this study, the number of neurons in the hilus of the dentate gyrus has been assessed in male and female Wistar rats at 3, 6, 12, 14, 18, 22 and 24 months of age, using the optical fractionator method. Male rats had more neurons than females at all the ages studied. Significant neuronal loss was observed in both sexes between 22 and 24 months of age. In a second experiment, 22 month-old male and female rats were treated for 10 weeks with 2 mg/kg/day of GH or saline. At 24 months of age, animals treated with GH had more neurons in the hilus than animals treated with saline. These findings indicate that GH is neuroprotective in old animals and that its administration may ameliorate neuronal alterations associated to aging.

Effect of recombinant human growth hormone administration on body composition and vascular function and structure in old male Wistar rats.

The process of ageing affects negatively both cardiovascular system and body composition. On the other hand, the hormones of the somatotrophic axis, growth hormone (GH) and insulin-like growth factor-I (IGF-I), whose production is reduced by age, are involved in the regulation of the cardiovascular system. The aim of this study was to investigate the effect of GH on body composition, vascular function and structure in old male rats. Old (20 months) and adult (4 months) male Wistar rats were used. One group of old animals was treated with GH for 4 weeks. Periepididimary fat weight, Specific Gravity Index (SGI), dose responses to Acetylcholine (ACh), Isoproterenol (Iso), Phenylephrine (Phe) and ACh in the presence of NG-nitro-L: -arginine metylester (L-NAME; ACh + L-NAME), as well as vascular morphology in aortic rings, were studied. Old rats showed increased fat weight and decreased SGI as compared to adult animals. GH increased SGI and tended to reduce fat weight. Old rats showed an impairment in the vasodilator response to ACh and Iso; GH significantly improved the vasodilatation induced by Iso, whereas the response to ACh was not significantly enhanced by GH treatment. There were no significant differences between adult and old rats in the contractile response to Phe, and GH did not show any effect. Contraction induced by ACh + L-NAME was higher in old rats as compared to adults, and treatment with GH significantly reduced this response. Aortic media area was increased in old rats, and GH administration reduced this parameter. In conclusion, GH shows beneficial effects on body composition, as well as on vascular function and morphology in old male rats.

Effect of S-adenosylmethionine on age-induced hepatocyte damage in old Wistar rats.

Aging is accompanied by changes in the morphology and physiology of organs and tissues, such as the liver. This process might be due to the accumulation of oxidative damage induced by reactive oxygen (ROS) and reactive nitrogen species (RNS). Hepatocytes are very rich in mitochondria and have a high respiratory rate, so they are exposed to large amounts of ROS and permanent oxidative stress. S-Adenosylmethionine (SAMe) is an endogenous metabolite that has shown to exert protective effects on different experimental pathological models in which free radicals are involved. The aim of this study was to investigate the effect of SAMe on age-induced damage in hepatocytes. For this purpose, male and female Wistar rats of 18 and 2 months of age were used. Cells were isolated and, after incubation in the presence or in the absence of SAMe, different parameters were measured. Aging induced a significant increase in nitric oxide, carbon monoxide and cGMP, and a reduction in reduced glutathione, ATP and phosphatidylcholine synthesis, as well as in methionine- adenosyl-transferase and methyl-transferase activities. Incubation of old cells with SAMe prevented all these age-related changes, reaching values in some of the parameters similar to those found in young animals. In conclusion, SAMe seems to have beneficial effects against age-induced damage in hepatocytes.

Changes with ageing in several leukocyte functions of male and female rats.

The impairment of the immune system with aging, or 'immunosenescence', appears to contribute to the increased morbidity and mortality of aged subjects. T cell functions and Natural Killer activity seem to be the immune responses most affected by ageing. Since the immune system works more efficiently in females than in males, we have studied the changes of several immune functions with age in rats of both sexes. In addition, we have investigated if ovariectomy, a model of menopause in rats, produces a loss of this gender-related advantage. In the present work, the changes with age (2, 6, 12, 14, 18, 22 and 24 months old) in lymphocyte chemotaxis, T lymphoproliferative response to the mitogen ConA, IL-2 release and Natural Killer activity of cells from axillary nodes and spleen of male and female rats as well as of females ovariectomized at 12 months of age have been studied. An age-related decrease was found in all investigated functions, with a slightly different evolution depending on the immune organ and gender considered. In general, the data obtained show that a certain degree of immunosenescence takes place with age in rats, with males being less immunocompetent than intact age-matched females, but showing an immune response similar to that of ovariectomized animals.

Effect of recombinant human growth hormone on age-related hepatocyte changes in old male and female Wistar rats.

Aging induces changes in several organs, such as the liver, and this process might be due to damage caused by free radicals and inflammatory mediators. The growth hormone (GH)/insulin-like growth factor-1 (IGF-1) axis shows a reduction with age, and this fact could be associated with some age-related changes. The aim of this study was to investigate the effect of GH administration on age-induced alterations in hepatocytes. Two and twenty two month-old male and female Wistar rats were used. Old rats were treated with human recombinant GH for 10 wk. At the end of the treatment, hepatocytes were isolated from the liver and cultured, and different parameters were measured in cells and medium. Plasma IGF-1 was also measured. Aging significantly decreased plasma IGF-1 in males. In females, plasma IGF-1 was also reduced, but not significantly. GH treatment restored plasma IGF-1 levels to values similar to young males. Aging was associated with a significant increase in lipid peroxidation (LPO), nitric oxide (NO), carbon monoxide (CO) and cyclic guanosyl-monophosphate (cGMP), as well as a reduction in adenosyl triphosphate (ATP) and phosphatidylcholine (PC) synthesis. GH administration partially prevented all these changes in males. In females, some of the parameters were significantly improved by GH (ATP, CO, cGMP), while others showed a tendency to improvement, although differences did not reach significance. In conclusion, GH administration could exert beneficial effects against age-related changes in hepatocytes, mainly in males.

Effect of oral intake of dibutyl phthalate on reproductive parameters of Long Evans rats and pre-pubertal development of their offspring.

UNLABELLED: To investigate the influence of dibutyl phtalate (DBP) given in a soy-free rat chow on pre-pubertal development, 46 Long Evans female rats 2-month-old were divided into three experimental groups and fed three different chows: (1) control; (2) DP 0.61 g/kg chow (12 mg/kgrat/day); (3) DP 2.5 g/kg chow (50 mg/kg rat/day) for 2 months. While under this treatment, they were mated and their offspring studied. Litter size and female:male ratio were recorded. At 14 days of age 6, male pups of each group were sacrificed and testis and thymus were excised and weighed. Pups were weaned at 22 days of age and continued into three experimental groups according to diet. From day 22 onwards, vaginal opening, occurrence of first estrous, and pre-putial separation were recorded. RESULTS: The percent of pregnancies showed a marked decrease in group 3, while no difference was observed between groups 1 and 2. Sex prevalence and litter size were not affected by the different diets. Pup survival showed a decrease when mothers were fed diet 2, but it was similar in diets 1 and 3. Pup weights on day 2 showed an evident (P < 0.05) reduction in groups 2 and 3, the decrease being more marked (P < 0.001) in group 3. On day 6, pups of group 2 showed lower weights (P < 0.01) as compared with the other groups. Weight gain was significantly higher in pups of group 3. Eye opening was not affected by the different diets. Fourteen-day-old male pups' relative weight of thymus and testis showed a decrease in animals whose mothers had been fed diets 2 and 3. Vaginal opening and occurrence of first estrous showed an evident delay (P < 0.05; P < 0.01) in females fed diets 2 and 3. Significant differences (P < 0.001) in pre-putial separation were observed between treated and untreated groups. CONCLUSION: Offspring pre-pubertal development seems to be affected by oral intake of DBP by their mothers during pregnancy, the effects being more evident in the reproductive development of male pups.

Influence of maternal environment on the number of transferable embryos obtained in response to superovulatory FSH treatments in ewes.

In a first experiment, embryo viability was estimated after recovery in the uterus or the oviduct of 70 Manchega ewes following a treatment of superovulation with decreasing doses of OVAGEN. Fewer viable embryos (5.6 +/- 0.9 vs. 8.3 +/- 0.8, P < 0.05) and more degenerative embryos (31.3% vs. 6.8%, P < 0.005) were obtained from the uterus than from the oviduct respectively. In a second experiment performed on 14 ewes, embryo viability was analyzed in relation to the follicular population estimated by ultrasonography (follicles > or = 2 mm) at the first FSH administration. Progesterone (P4) and oestradiol 17beta (E2) concentrations were also determined from the beginning of the superovulation treatment to the recovery of the embryos. The number of viable embryos (4.3 +/- 1.4) was positively correlated (r = 0.824) with of 2-4 mm diameter follicles (P < 0.05), and with E2 concentrations at -12 h (r = 0.891, P < 0.01) , 0 h (r = 0.943, P < 0.0001) and +24 h (r = 0.948, P < 0.05) from estrus detection. Prolonged high levels of E2 up to 72 h with low levels of P4 on days 3 and 4 after estrus had a negative (P < 0.05) effect on embryo viability. These results indicate that ovarian response to superovulatory protocols is related to the individual variations in the number of follicles of 2-4 mm at the start of FSH treatment, and that embryo viability is conditioned by the steroid patterns during the time spent in the genital tract of the super-ovulated ewes.