Melanocortin-4 receptor agonist (RO27-3225) ameliorates soleus but not gastrocnemius atrophy in arthritic rats.

Adjuvant-induced arthritis in rats decreases body weight and muscle mass. Melanocyte stimulating hormone administration to arthritic rats decreases inflammation and skeletal muscle wasting. In this study, we investigate whether activation of melanocortin-4 receptor by RO27-3225 administration is able to prevent the effect of arthritis on the expression of muscle-specific E3 ubiquitin ligases and MyoD in two different muscles, gastrocnemius (a mainly fast type muscle) and soleus (slow type). Arthritis was induced in male Wistar rats by intradermal injection of Freund's adjuvant. Control and arthritic rats were injected with RO27-3225 (180 µg/kg i.p. twice a day) or saline, for 8 days. Body weight change, food intake and arthritis index were assessed daily. After sacrifice, serum insulin-like growth factor -1 (IGF-1) and corticosterone, as well as nuclear factor-κB(p65), cyclooxygenase-2 (COX-2), atrogene and MyoD in gastrocnemius and soleus were analysed. Administration of RO27-3225 to arthritic rats decreased arthritis scores, hind paw volume as well as nuclear factor-κB(p65) phosphorylation in gastrocnemius and soleus. However, RO27-3225 was not able to modify the effects of arthritis on serum IGF-1 and corticosterone. RO27-3225 ameliorates arthritis-induced decrease in food intake, body weight gain, epidydimal white adipose tissue and soleus weight, but not in gastrocnemius weight. Arthritis increased COX-2, atrogin-1 and MuRF1 expression in gastrocnemius and soleus, whereas RO27-3225 prevented this increase in soleus but not in gastrocnemius. Arthritis also increased MyoD expression in gastrocnemius and soleus (P < 0.01). RO27-3225 decreased MyoD expression in gastrocnemius but not in soleus of arthritic rats. In control rats RO27-3225 did not modify MyoD expression in gastrocnemius or soleus. In conclusion, our data suggest that in arthritic rats, RO27-3225 treatment decreases inflammation and muscle atrophy, preventing atrogene upregulation in slow type muscle but not in gastrocnemius. The lack of effect in the gastrocnemius can be related to the inability of RO27-3225 to prevent arthritis-induced corticosterone upregulation as well as IGF-1 downregulation.

Effects of neonatal treatment with estrogens on the development of the thymus in rats.

In order to study the effects of sex hormones administered during the neonatal period on the thymic development we have injected 5 days old female rats with a single dose (0.1mg) of estradiol benzoate. The evolution of the thymus gland after treatment was morphometrically analyzed. The thymus of the estrogen-injected animals diminished in size between the 7th and the 15th day, increasing afterwards. The cortex was the most sensitive compartment in the thymus to estrogenic treatment. Both thymic involution and enlargement were associated to changes in the frequency of large lymphoid cells in the subcapsular region and variations in the thymic cortex weights. The observed effects are tentatively attributed to alterations in the thymocyte differentiation probably due to modifications in the secretion of thymic hormones.

Decreased leptin uptake in hypothalamic nuclei with ageing in Wistar rats.

Leptin interacts with specific receptors in hypothalamic nuclei and modulates energy balance. Growing evidence has shown the association of obesity and hyperleptinaemia with non-insulin-dependent diabetes mellitus and insulin resistance. The aged Wistar rat shows peripheral insulin resistance in the absence of obesity and alterations of glucose homeostasis. However, it is not known whether, in these animals, the leptin action is altered. Here we studied the effect of ageing on plasma leptin concentration and the ability of hypothalamic nuclei to capture i.c.v.-injected digoxigenin-labelled leptin. Our data indicate that 24-month-old animals are hyperleptinaemic. However, daily food intake was greater in old animals, suggesting that they are leptin resistant. Leptin uptake in the hypothalamus was reduced in old rats. This uptake was a receptor-mediated process as demonstrated by displacement. Leptin accumulation in hypothalamic nuclei was partially colocalized with neuropeptide Y fibres. Immunohistochemical and western blot analyses showed a lower amount of the long form of leptin receptors in the hypothalamus of aged rats. Analysis by RT-PCR also demonstrated a decreased expression of leptin receptor mRNA in old animals. We conclude that the lower leptin uptake may be explained, at least in part, by a decreased amount of receptors in hypothalamic neurones of the aged rats.

Oestrogen--bromocriptine interaction in the control of luteinizing hormone and prolactin secretion in the neonatally oestrogenized female rat.

Neonatally oestrogenized female rats showed hyperprolactinaemia (prolactin, 230 micrograms/l), normal LH levels and absence of a positive feedback effect of oestrogen on secretion of LH at 5 months of age. Bromocriptine treatment for 13 days (1 mg/kg per day) caused no changes in LH levels and prolactin levels decreased to normal values (33 micrograms/l). This decrease in prolactin concentration was not followed by the recovery of phasic LH response to oestrogens. The effectiveness of oestrogens to induce prolactin secretion was greater in the neonatally oestrogenized rats than in the control group. In both cases the effect diminished after bromocriptine treatment. These results indicate that hyperprolactinaemia is not the cause of the anovulatory state in oestrogenized rats and that neonatal treatment with oestrogens alters oestrogen--prolactin relations, probably involving dopamine.

Analysis of brainstem A1 and A2 noradrenergic inputs to the preoptic area using microdialysis in the rat.

Noradrenergic inputs to the preoptic area (POA) are involved in regulating a variety of homeostatic functions. However, the accurate measurement of endogenous noradrenaline (NA) release in the POA has been difficult to achieve and consequently little has been done to characterise the different noradrenergic pathways. By combining the technique of intracranial microdialysis with tissue pre-loading of [3H]NA we have developed a sensitive index of NA release in the POA [8]. Using this method we have now examined and compared the effects of electrical stimulation of the brainstem A1 and A2 cell groups on NA release in the POA. Anaesthetised proestrus rats were implanted with microdialysis probes either unilaterally or bilaterally in the POA and stimulating electrodes positioned in either the A1 or A2 regions. Electrical stimulation (10 Hz, 10s on/off for 20 min) of the A1 region resulted in repeatable, calcium-dependent increases in radioactivity outflow from the ipsilateral POA (P < 0.01). A1-evoked release was twice as large as that observed after equivalent 10 Hz electrical stimulation of the A2 region (P < 0.05). In experiments using bilateral POA microdialysis and A1 stimulation, a significant increase in release from the contralateral POA, amounting to approximately 80% of that observed in the ipsilateral POA, was observed. Experiments involving the blockade of A1-stimulated release in the ipsilateral POA by perfusion with a calcium-free medium demonstrated that increases in radioactivity measured in the contralateral POA were not originating from the ipsilateral POA.(ABSTRACT TRUNCATED AT 250 WORDS)

Estrogen uncouples noradrenergic activation of Fos expression in the female rat preoptic area.

The preoptic area of the rat brain is a site at which gonadal steroids act to regulate sexual behaviour and gonadotrophin secretion. The expression of the immediate-early gene product, Fos, in the preoptic area was investigated in conscious ovariectomised, vehicle and estrogen-treated animals which had received an intracerebroventricular (i.c.v.) infusion of noradrenaline, and also in anaesthetised proestrous and ovariectomised rats following electrical stimulation of the brainstem A1 or A2 noradrenergic cell groups. In ovariectomised oil-treated rats, a third ventricular infusion of noradrenaline (45 micrograms) resulted in a significant (P < 0.05) increase in the numbers of Fos-immunoreactive cell nuclei throughout the preoptic area, compared to vehicle controls. In contrast, Fos expression in animals which had received estrogen replacement showed no change in response to i.c.v. noradrenaline compared with saline-treated controls. In anaesthetised, ovariectomised animals electrical stimulation of the A1 cell group resulted in a significant increase (P < 0.05) in Fos-like immunoreactivity compared with sham controls, specifically within the ventral preoptic area whilst stimulation of the A2 cell group had no significant effect. In anaesthetised, proestrous rats receiving electrical stimulation no significant changes in Fos-like immunoreactivity were detected within the preoptic area after either A1 or A2 stimulation compared with paired controls. These results show that noradrenaline-induced Fos expression in the preoptic area is dependent on estrogen status and suggest that the estrogenic regulation of reproductive functions may thus involve altered responses to noradrenaline in sub-populations of preoptic neurones.

Body weight rhythmicity in the unweaned female rat following neonatal estrogen treatment.

Male and female rats were weighted daily throughout this study to determine whether neonatal exposure to estrogens influences body weight (BW) patterns, particularly during the period before weaning when vaginal opening (VO) occurs as a consequence of this treatment. Females receiving estradiol benzoate (EB) at the age of 5 days had greater body weight than their controls soon after the treatment and until day 21. Clear 3-day periodic changes of BW, between days 9 and 20, were revealed by the spectral analysis of the results in EB-given females which did not occur in their controls. Changes of BW (at 4-day intervals) were verified in adult control females whereas age-matched EB-treated females did not show such a rhythm. Neither BW gain nor infradian rhythmicity were detected in infantile males after neonatal EB treatment. The results suggest that, as in spontaneous puberty, a relationship between the occurrence of VO and the establishment of the infradian body rhythmicity in the infantile estrogenized female may exist.

Effect of cyclooxygenase-2 inhibition by meloxicam, on atrogin-1 and myogenic regulatory factors in skeletal muscle of rats injected with endotoxin.

Cyclooxygenase-2-induction by inflammatory stimuli has been proposed as a mediator of inflammatory cachexia. We analyse whether cyclooxygenase-2 inhibition by meloxicam administration is able to modify the response of skeletal muscle to inflammation induced by lipopolysaccharide endotoxin (LPS). Male rats were injected with 1 mg kg(-1) LPS at 17:00 h and at 10:00 h the following day, and euthanized 4, 24 or 72 hours later. Atrogin-1, MuRF1, myogenic regulatory factors and cyclooxygenase-2 in the gastrocnemius were determined by real time-PCR (mRNA) and Western blot (protein). In a second experiment the effect of meloxicam administration (1 mg kg⁻¹) was analyzed. Meloxicam was administered either in a preventive manner, 1 hour before each endotoxin injection, or in a therapeutic manner, starting 2 hours after the second LPS injection and at 24 and 48 hours afterwards. There was a marked increase in MuRF1 mRNA (P<0.01) 4 hours after LPS, and in atrogin-1 mRNA 4 hours (P<0.01) and 24 hours (P<0.01) after LPS. Cyclooxygenase-2 was increased, whereas MyoD was decreased at 4, 24 and 72 h. Both types of meloxicam treatment blocked LPS-induced increase in atrogin-1. Preventive, but not therapeutic, meloxicam decreased myostatin (P<0.01) and increased Pax7 (P<0.01) and MyoD (P<0.05). Therapeutic meloxicam treatment decreased gastrocnemius myogenin. These data suggest that cyclooxygenase-2 inhibition by meloxicam administration can prevent the increase in atrogin-1 and the decrease in MyoD induced by LPS administration. However, prolonged therapeutic meloxicam treatment seems to be less effective, since it can inhibit myogenic regulatory factors.

Characterization of tritiated noradrenaline release from the rat preoptic area with microdialysis in vivo.

Present techniques are unable to provide a sensitive and accurate index of noradrenergic activity in the rat preoptic area. In this study, we have examined the brainstem A1 noradrenergic input to the preoptic area using a new technique whereby [3H]noradrenaline is preloaded into the preoptic area and release of radioactivity from this region is measured subsequently using microdialysis in vivo. Electrical stimulation of the ipsilateral A1 area for 20 min at 5, 10, and 15 Hz evoked significant increases in dialysate radioactivity that were repeatable and frequency-dependent. After removal of calcium from the perfusion medium, basal release of radioactivity was markedly reduced and the effect of A1 stimulation abolished. Changing to a 100 mM K+ medium evoked an increase in the release of radioactivity that was sixfold greater than that seen after A1 stimulation. Separation of the dialysate with HPLC showed that 33% of the increase in measured radioactivity after A1 stimulation was directly attributable to [3H]noradrenaline and the remainder to the metabolites vanillylmandelic acid, 3,4-dihydroxymandelic acid, and 3,4-dihydroxyphenylglycol. In contrast, the increase in radioactivity after K+ depolarization was due almost completely to [3H]noradrenaline. Addition of 10 microM clonidine to the perfusion medium markedly reduced basal release of radioactivity, but had no effect on evoked release following A1 stimulation. Conversely, perfusion with 10 microM yohimbine had no effect on basal release, but significantly increased evoked release after A1 stimulation. These results now provide a characterization of noradrenergic activity in the preoptic area and indicate the importance of the A1 noradrenergic input to this region. The technique of measuring radioactivity with microdialysis after preloading with [3H]noradrenaline provides a relatively simple, sensitive index of noradrenergic activity in vivo with good temporal resolution.

Controlled neonatal exposure to estrogens: a suitable tool for reproductive aging studies in the female rat.

The present study was designed to determine whether the modification of exposure time to large doses of estrogens provided a reliable model for early changes in reproductive aging. Silastic implants containing estradiol benzoate (EB) in solution were placed into 5-day-old female Wistar rats and removed 1 day (Ei1 group) or 5 days (Ei5) later. In addition, 100 micrograms [corrected] EB dissolved in 100 microliters corn oil was administered s.c. to another group (EI). Control rats received either vehicle implants or 100 microliters corn oil. Premature occurrence of vaginal opening was observed in all three estrogenized groups independently of EB exposure. However, females bearing implants for 24 h had first estrus at the same age as their controls and cycled regularly, and neither histological nor gonadal alterations could be observed at 75 days. Interestingly, they failed to cycle regularly at 5 mo whereas controls continued to cycle. On the other hand, the increase of EB exposure (Ei5, EI) resulted in a gradual and significant delay in the onset of first estrus and in a high number of estrous phases, as frequently observed during reproductive decline. At 75 days, the ovaries of these last two groups showed a reduced number of corpora lutea and an increased number of large follicles. According to this histological pattern, ovarian weight and progesterone (P) content gradually decreased whereas both groups showed higher estradiol (E2) content than controls. This resulted in a higher E2:P ratio, comparable to that observed in normal aging rats.(ABSTRACT TRUNCATED AT 250 WORDS)

Gonadal hormones affect neuronal vulnerability to excitotoxin-induced degeneration.

The role of endogenous gonadal secretions in neuroprotection has been assessed in a model of hippocampal degeneration induced by the systemic administration of kainic acid to adult male and female rats. A low dose of kainic acid (7 mg/Kg b.w.) induced a significant loss of hilar dentate neurons in castrated males and did not affect hilar neurons in intact males. The effect of kainic acid on hilar neurons in female rats was different depending on the day of the estrous cycle in which the neurotoxin was administered; while no significant effect of kainic acid was observed when it was injected in the morning of estrus, there was a significant loss of hilar neurons when it was injected in the morning of proestrus as well as when it was injected into ovariectomized rats. Estradiol or estradiol plus progesterone prevented hilar neuronal loss when injected simultaneously with kainic acid in ovariectomized rat. Progesterone by itself did not prevent neuronal loss induced by kainic acid and estogen was only effective when it was injected either 24 h before or simultaneously with kainic acid and not when it was injected 24 h after the administration of the toxin. These findings indicate that endogenous gonadal hormones protect hippocampal hilar neurons from excitotoxic degeneration. In addition, the timing of exposure to ovarian hormones and the natural fluctuation of ovarian hormones during the estrous cycle may influence the vulnerability of hilar neurons to excitotoxicity. These findings are relevant to possible modifications in neurodegenerative risk in humans as endogenous levels of gonadal hormones change during the menstrual cycle and during aging.

[Effect of constant light on the circadian rhythm of prolactin (author's transl)]. / Efecto de la iluminación constante sobre el ritmo circadiano de prolactina.

The different effects of equal periods of constant light (from days 1-65 or 70-135) upon the circadian rhythm of prolactin in male rats have been studied. Plasmatic levels of hormone have been analyzed at 5.00, 9.00, 12.00, 17.00, 21.00 and 24 h. 65 and 135 day-old controls on a 12 h light/12 h darkness schedule show PRL surges at 17.00 h. These surges disappear in both groups when submitted to constant light. A global increase of PRL levels can be observed only in 135 day-old rats, which disappears 15 days after castration. This indicates testicular involvement in the hyperprolactinemia induced by constant light.

[Plasma levels of LH and testosterone in male rats subjected peripheral de-afferentation of the vomeronasal system]. / Niveles plasmáticos de LH y testosterona en ratas macho sometidas a deaferentación periférica del sistema vomeronasal.

21 day old male rats undergoing peripheral deafferentation of the vomeronasal system (Accessory Olfactory System) show a decrease of the accessory sex organ weight as well as lower LH and testosterone plasmatic levels 30 days later when compared with intact or sham operated rats.

[Effect on the administration of LHRH on LH and prolactin plasma levels in adult female rats ovariectomized and maintained in constant light (author's transl)]. / Efecto de la administración de LHRH sobre niveles plasmáticos de LH y prolactina en ratas hembras adultas ovariectomizadas y mantenidas en iluminación constante.

The influence of prolonged periods of constant lighting on the plasma levels of LH and prolactin in adult female rats has been studied. No differences in either hormones are observed between intact animals under constant light of under a 12 hr (controls) darkness schedule. After ovariectomy LH values increase on both experimental conditions, with higher levels in the control group (p less than 0.01). After ovariectomy, a similar pattern is observed in animals under constant light, or under a 12 hr light darkness schedule, in the decrease of prolactin levels and in the increase of plasma LH levels after LHRH administration (100-1,000 ng). The stress induced by experimental manipulation, ether anesthesia and saline injection elevates plasma prolactin in both groups. LHRH administration blocks this response.

[LH and prolactin secretion under constant light in estrogen treated rats (author's transl)]. / Secreción de LH y prolactina en ratas tratadas con estrógenos y sometidas a luz constante.

Plasma LH and prolactin levels were studied in ovariectomized adult female rats submitted to light-darkness (L:D) or constant light (L:L) schedules, after the administration of estradiol benzoate (EB), 75 micrograms/day for six consecutive days. Previous to the treatment with EB LH levels were lower and prolactin levels higher in the L:L females. On days 1 and 2 after treatment, L:D females showed circadian variations of LH levels, these being higher at 17 h than at 10 h. This pattern disappeared in the L:D females. Prolactin levels increased similarly in both groups. Nine days after treatment, plasma prolactin levels remained high and the circadian pattern of LH in the L:D group disappeared.

Effects of constant light on prolactin secretion in adult female rats.

Female rats were submitted to light-darkness, to constant light on day 60 or to constant light from birth. Plasma prolactin was measured on days 80, 100 and 120 in all experimental situations before ovariectomy was performed. 10 days later, a blood sample was taken and the animals were injected subcutaneously with 75 micrograms of estradiol benzoate. Blood was again drawn on 2 consecutive days. Results showed that: (a) prolactin levels were higher in the groups submitted to prolonged periods of constant light (60--120 days); (b) after ovariectomy prolactin levels decrease but remain higher in these same groups, and (c) constant light produced a higher prolactin response to estradiol benzoate. These data indicate that the length of exposure to constant light may be a critical factor in the development of alterations in the control of prolactin secretion.

LH and prolactin in estrogenized female rats: response to LHRH.

Eighty day old female rats postnatally treated with estradiol benzoate (EB) show hyperprolactinemia and increased levels of LH with a positive correlation LH-prolactin, unlike the control animals. Fourteen days after ovariectomy this correlation disappears and the prolactin levels remain higher than control. On the contrary, the increase in LH levels is smaller in the EB group. The administration of LHRH to ovariectomized EB rats produces a decrease in prolactin levels, unobserved in the control group, as well as lower LH levels.

Luteinizing-hormone-mediated precocious puberty induced in female rats by a prepuberal pituitary graft.

The present study analyzes the mechanism of precocious puberty induced in female rats after a 'young' pituitary graft (obtained from 21-day-old animals). For this purpose, the following experiments have been performed: (1) female rats were grafted or sham-operated on day 21 with a littermate's pituitary the follicle-stimulating hormone (FSH), luteinizing hormone (LH) and estradiol plasma levels as well as the ovarian, uterine and adrenal weights were determined at different times after the graft; (2) female rats grafted or sham-operated on day 21 were treated with 0.2 ml of LH antiserum (LHAS) or the same volume of a normal horse serum (NHS); (3) female rats were injected on day 1 of life with 0.1 mg of estradiol benzoate or olive oil. Groups of these animals were decapitated daily between days 6 and 21 in order to measure gonadotropins and prolactin (PRL) pituitary content. Since on day 21 estrogenized females showed decreased gonadotropin content and normal PRL content, females in experiment 4 were grafted on day 21 with pituitaries obtained from control or neonatally estrogenized female rats. The results obtained showed that FSH, LH and estradiol plasma levels as well as ovarian and uterine weights increased after pituitary grafts. LHAS blocked the precocious puberty induced by the pituitary graft, and pituitaries obtained from neonatally estrogenized female rats were unable to modify the occurrence of puberty when grafted. In conclusion, this work evidences that precocious puberty induced by 'young' pituitary grafts was mediated by the increase in LH secretion from the graft.

Effects of discrete medial preoptic hypothalamic lesions on the androgen gonadotropin feedback system in the male rat.

The effect of discrete lesions of the Anterior Medial Preoptic Area of the Hypothalamus (MPOA) in the control of pituitary gonadotropins in the adult male Wistar rat has been studied. Electrolytic lesions were made by passing an anodal current through tungsten electrodes. Electrodes were oriented stereotaxically into the MPOA and lesion placement was histologically checked. In sham controls, electrodes were lowered into the MPOA but no current was applied. Serum LH and FSH were measured by RIA. MPOA lesioned animals showed significantly lower plasma LH (p less than 0.01) in comparison to sham lesioned group. Plasma FSH remained unaltered. To test whether these results were related to an alteration in the negative feedback system, the response to administration of Testosterone Propionate (TP) and the secretion patterns of LH and FSH after castration were analyzed. Administration of TP revealed similar LH and FSH 24 and 48 h decrements and the pattern of LH and FSH secretion after gonadectomy was not significantly different in lesioned and sham lesioned animals. Responsiveness to exogenous LHRH was not impaired by MPOA lesions. The results suggest that neural elements within the MPOA are functionally related to pituitary LH secretion in the male rat. The LH control alteration in lesioned animals is not associated with modifications in negative feedback of androgens and pituitary sensitivity to LHRH remains unaltered.

Long-term food restriction prevents ageing-associated central leptin resistance in wistar rats.

AIMS/HYPOTHESIS: Ageing is associated with insulin and leptin resistance in mammals. These alterations might be caused by the increased adiposity associated with ageing, by ageing alone or both. We studied whether leptin resistance occurs at the central level in the Wistar rat and we aimed to discriminate between the effects of ageing from those of the increased adiposity associated with ageing. METHODS: Leptin was infused intracerebroventricularly at a constant rate in young adult, old and old Wistar rats fasted for 3 months, using osmotic pumps. The effects on body weight, daily food intake, Lee index, adiposity and serum leptin values were analysed. The effect of food restriction on the expression of the long form of leptin receptor in the hypothalamus was also studied. RESULTS: Leptin decreased daily food intake and body weight in young and old Wistar rats. With a dose of 10 microg/day similar responses were obtained in young and old rats but with a dose of 0.2 microg/day, only young rats showed decreases in these parameters. Food-restriction in old rats lowered adiposity and serum leptin to values close to those of young rats, recovered responsiveness to i.c.v. administration of leptin at the dose of 0.2 microg/day and increased leptin receptor expression in the hypothalamus. CONCLUSION/INTERPRETATION: Our data show that old Wistar rats have a decreased response to leptin at the central level. Food-restriction recovers leptin responsiveness and increases leptin receptor in the hypothalamus suggesting that adiposity plays a key role in the development of leptin resistance associated with ageing.