The effect of chronic food and water restriction on open-field behaviour and serum corticosterone levels in rats

In operant conditioning experiments, two methods are commonly used to motivate laboratory rats to perform designated tasks. The first is restricting food so that rats are forced to lose 20% of body weight within one week, followed by maintenance at 80% of the baseline weight for the remainder of the experiment. The second is restricting access to water to 15 min in each 24 h period. These methods are effective in motivating the animals. There is, however, little information available on the effects on performance in tests of behaviour that are not related to operant conditioning. In addition, it is not clear if these commonly used methods of food and water restriction will lead to physiological stress as indicated by an elevation of serum corticosterone. Male rats were either food-restricted to reduce and maintain their weight at 80% of baseline weight, or were restricted to 15 min access to water every 24 h. Activity in the open field was significantly greater in food-restricted rats than in water-restricted or control rats, but freezing behaviour was similar in all experimental groups. Food-restricted rats had a higher mean serum corticosterone level than water-restricted and control rats 37 days after the start of the experimental period. These data suggested that chronically restricting food and maintenance of body weight at 80% of baseline body weight led to significant behavioural changes and physiological stress. In contrast, water restriction did not lead to changes in behaviour or corticosterone levels. A second experiment was conducted to compare the effects of food restriction to 80% of baseline body weight, as described above, with a less stringent protocol in which test rats were initially reduced to 80% of baseline weight, but were then maintained at 80% of an ad libitum fed control rat's weight. Serum corticosterone levels and adrenal gland weights were measured after the initial week of forced weight loss and after maintenance for 21 days. Forced loss of 20% of body weight in the first week led to significantly increased serum corticosterone levels and adrenal gland weights compared to ad libitum fed controls. Serum corticosterone levels and adrenal gland weights in rats maintained at 80% of their initial body weight for 21 days remained higher than ad libitum fed control rats. However, rats maintained at 80% of an ad libitum fed control rat's weight did not differ from control rats in serum corticosterone levels or adrenal gland weights at the end of the 21-day study period. Adjustment of the feeding regimen in this manner eliminated physiological evidence of chronic stress.     

Differentiation of immunopositive ACTH cells in the human fetal pituitary gland

Differentiation and localization of corticotrophic cells in the human fetus hypophysis (5-30 weeks of development) have been studied. The immune cytochemical reaction is performed in sagittal and horizontal sections 5 mcm thick. Rabbit anti adrenocorticotrophic hormone (ACTH)-, anti ACTH- and anti ACTH-sera are used. In the hypophysis anlage of a 6-week-old fetus single immune positive ACTH-cells are revealed situating at the border where the intermediate part gets into the anterior part. With age, the number of the corticotrophic cells increases and till the first third of the intrauterine development they are mainly localized along the periphery of the epithelial cords and the adenoid- or other parts of the adenohypophysis. During the second part of the intrauterine development the corticotrophic cells localize in the same places as in a mature person. The hormone-producing ability of the hypophysis coincides with the beginning of its organogenesis.     

The effect of centrally administered ghrelin on pituitary ACTH cells and circulating ACTH and corticosterone in rats

Ghrelin is a brain-gut peptide known for its growth hormone (GH)-releasing and appetite-inducing activities. This natural GH secretagogue (GHS) was originally purified from rat stomach, but it is expressed widely in different tissues where it may have endocrine and paracrine effects. The central effects of ghrelin on adrenocorticotropic hormone (ACTH) cells, ACTH release and subsequent corticosterone release from adrenal glands remains to be clarified. The aim of this study was to specifically determine the morphological features of ACTH-producing pituicytes and blood concentration of ACTH and corticosterone after central administration of ghrelin. Five doses of rat ghrelin or PBS (n=10 per group) were injected every 24 h (1 microg of ghrelin in 5 muL PBS), into the lateral cerebral ventricle of male rats. Results showed that ghrelin increased (p<0.05) absolute and relative pituitary weights compared to controls (58% and 41% respectively). Morphometric parameters, i.e. the volume of the ACTH cells, nuclear volume, and volume density were all increased (p<0.05), by 17%, 6% and 13%, respectively, 2 h after the last ghrelin treatment. Ghrelin increased circulating concentrations of ACTH and corticosterone (p<0.05) by 62% and 66%, respectively. The data provide clear documentation that intracerebroventricular ghrelin stimulates ACTH cell hypertrophy and proliferation, and promotes ACTH and corticosterone release. Determining the role of ghrelin in physiological stress responses and whether control of the peptide's activity would be useful for prevention and/or treatment of stress-induced diseases remain important research goals.     

Long-term obesity levels in female OLETF rats following time-specific post-weaning food restriction

Obesity and the metabolic syndrome represent serious health threats affecting increasing numbers of individuals, with females being more affected than males and with growing incidence among children and adolescents. In the present study, we used the OLETF rat model of early-onset obesity to examine the influence of different timing of food restriction on long-term obesity levels in females. Food restriction took place at different time windows: from weaning until postnatal day (PND) 45 (early); from weaning until PND90 (chronic); or from PND45 until PND70 (late). Follow-up continued until PND90. During and after the termination of the diet-restriction period, we focused on peripheral adiposity-related measures such as fat pad weight (brown, retroperitoneal and inguinal); inguinal adipocyte size and number; and leptin, oxytocin and glucose levels. We also examined body weight, feeding efficiency, spontaneous intake after release from diet-restriction, and plasma creatinine levels and estrous cycle characteristics as a result of the chronic diet. The results suggest that while food restriction produced significant weight and adiposity loss, OLETF females presented poor weight loss retention after the early and late short-term diets. The estrous cycle structure and time of first estrous of the OLETF rats were normalized by chronic food restriction. Females responded to early food restriction in a different manner than males did in previous studies, further emphasizing the importance of sex-appropriate approaches in the investigation and treatment of the pathologies related to obesity and the metabolic syndrome.     

Long-term obesity levels in female OLETF rats following time-specific post-weaning food restriction

Obesity and the metabolic syndrome represent serious health threats affecting increasing numbers of individuals, with females being more affected than males and with growing incidence among children and adolescents. In the present study, we used the OLETF rat model of early-onset obesity to examine the influence of different timing of food restriction on long-term obesity levels in females. Food restriction took place at different time windows: from weaning until postnatal day (PND) 45 (early); from weaning until PND90 (chronic); or from PND45 until PND70 (late). Follow-up continued until PND90. During and after the termination of the diet-restriction period, we focused on peripheral adiposity-related measures such as fat pad weight (brown, retroperitoneal and inguinal); inguinal adipocyte size and number; and leptin, oxytocin and glucose levels. We also examined body weight, feeding efficiency, spontaneous intake after release from diet-restriction, and plasma creatinine levels and estrous cycle characteristics as a result of the chronic diet. The results suggest that while food restriction produced significant weight and adiposity loss, OLETF females presented poor weight loss retention after the early and late short-term diets. The estrous cycle structure and time of first estrous of the OLETF rats were normalized by chronic food restriction. Females responded to early food restriction in a different manner than males did in previous studies, further emphasizing the importance of sex-appropriate approaches in the investigation and treatment of the pathologies related to obesity and the metabolic syndrome.     

Reproductive axis response to repeated lipopolysaccharide administration in peripubertal female rats

Immune system disorders are often accompanied by alterations in the reproductive axis. Several reports have shown that administration of bacterial lipopolysaccharide (LPS) has central inflammatory effects and activates cytokine release in the hypothalamus where the luteinizing hormone releasing hormone (Gn-RH) neurons are located. The present study was designed to investigate the effect of repeated LPS administration on the neuroendocrine mechanisms of control of the reproductive axis in peripubertal female rats (30-day-old rats). With this aim, LPS (50 μg/kg weight) was administered to the animals during 25, 27 and 29 days of age and sacrificed on 30 day of life. Gn-RH, γ?amino butyric acid (GABA) and glutamic acid (GLU), two amino acids involved in the regulation of Gn-RH secretion, hypothalamic content were measured. LH and estradiol serum levels were also determined and the day of vaginal opening examined. The results showed a significant increase in Gn-RH and GLU content (p?<?0.0001), shared by a reduction of GABA one (p?<?0.0001). LH and estradiol serum levels were decreased (p?<?0.01, p?<?0.001) and delay in the day of vaginal opening was also observed in treated animals. Present results show that repeated LPS administration impaired reproductive function, modifying the neuroendocrine mechanisms of control of the axis in peripubertal female rats.     

The effect of chronic estrogen treatment on immunoreactive beta-endorphin levels in intact female rats

Intact female rats were treated chronically with estradiol benzoate (EB) until a state of constant estrus (CE) was achieved and maintained. When compared to female rats on the day of estrus, estrogen-treated rats in constant estrus demonstrated a 33% decrease in the concentration of immunoreactive beta-endorphin (IR-BE) in the plasma, and a 45-50% decrease in the content and concentration of IR-BE in the anterior pituitary and hypothalamus. The content and concentration of IR-BE in the neurointermediate lobe of the pituitary were similar in each group. Column chromatography revealed that the reduction in IR-BE in the plasma and anterior pituitary of EB-treated CE female rats appeared to be due to a reduction in peptides coeluting with beta-endorphin and beta-lipotropin, whereas the reduction in IR-BE in the hypothalamus represented a decrease in a peptide which coeluted with beta-endorphin. These data suggest that constant estrus, induced by prolonged treatment of intact female rats with estrogen, resulted in a reduction in central and peripheral levels of IR-BE in these animals as compared to female rats on the day of estrus.     

Effects of feed restriction on fertility in female rats

BACKGROUND: Feed restriction with its resultant body weight loss impacts the rodent estrous cycle; however, the manifestation of these changes in a regulatory study design has not been documented. This study reports the effects of feed restriction in the context of an FDA regulatory submission. METHODS: Adult female rats (n = 20/group; weighing approximately 200 g each) were provided rodent chow ad lib (control) or at 20, 15, 10, or 7.5 g/rat/day (g/day) during a 2-week pre-mating phase, throughout the mating phase, and up to gestation day (GD) 7. On GD 8, all animals were provided ad lib feed until necropsy on GD 14. Estrous cyclicity, mating, and fertility parameters were evaluated. RESULTS: Ad lib rats consumed approximately 20 and 28 g/day during the pre-mating and gestation phases, respectively. All measured fertility parameters in the 20 g/day group were similar to control values. In the 15 g/day group, body weight was reduced by 16% at 2 weeks, prolonged diestrus occurred, and fertility was compromised due to reductions in corpora lutea. Within 2 weeks, mean body weight in groups receiving < or = 10 g/day was reduced by > or = 29% compared to ad lib values, and overt changes in estrous cyclicity, mating, and fertility occurred. The 7.5 g/day group was not sustainable beyond the pre-mating phase. CONCLUSIONS: For this study type, feed intake at < or = 50% ad lib values (< or = 10 g/day) was inadequate due to the magnitude and rapidity of body weight effects. Estrous parameters appeared slightly more sensitive than functional measures, as body weight changes of approximately 16% appeared near the threshold of changing routinely calculated estrous cycle parameters and were later associated with reduced fertility. In general, body weight differences of 10-15% by themselves were not adverse to normal reproduction (20 g/day).     

Effect of food restriction on cold adaptability of rats

To determine the role of the nutritional state in nonshivering thermogenesis during cold adaptation, cold adaptability was compared between cold-adapted (5 degrees C for 4-5 weeks) rats fed ad libitum and cold-adapted rats pair fed with warm controls having the same food intake. Cold-adapted pair-fed rats suffered a significant loss in body weight during cold exposure. However, brown adipose tissue (BAT) in both cold-adapted ad libitum fed and cold-adapted pair-fed rats was enlarged to the same extent as compared with that in control rats. Fat-free dry matter in BAT also increased in cold-adapted ad libitum fed and cold-adapted pair-fed rats to the same extent. Cold tolerance as assessed by the change in the colonic temperature at -5 degrees C was improved relative to control rats and was the same for cold-adapted ad libitum fed and cold-adapted pair-fed rats. Nonshivering thermogenesis as estimated by the noradrenaline-induced increase in oxygen consumption was significantly greater in the cold-exposed rats and there was no significant difference between cold-adapted ad libitum fed and cold-adapted pair-fed rats. These results suggest that an improved cold tolerance by means of nonshivering thermogenesis in brown adipose tissue is closely related to the low temperature itself but not the increased food intake which occurred in the cold.     

Atrazine-induced degranulation of thyroid mast cells in peripubertal and adult rats

Atrazine is a commonly used pesticide in the US and the non-EU countries. It is classified as an endocrine-disrupting chemical and is well-known for its reproductive toxicity in mammals and lower vertebrates. The study on atrazine effects on thyroid mast cells was performed on juvenile/peripubertal and adult male Wistar rats orally gavaged with atrazine at doses of 50 mg/kg of body weight (bw) or 200 mg/kg bw. In order to visualize the mast cell population within the thyroid gland, a histochemical staining method of toluidine blue was used. The results of the histological evaluation demonstrated a prominent increase in mast cell degranulation in both age groups and at both atrazine doses. According to the stereological analysis, a statistically significant decrease in the mast cell volume density in the young rats exposed to a higher dose of atrazine was found when compared to the corresponding control. The numerical density of mast cells significantly decreased in a higher-dose atrazine treated adults in comparison to the control. The obtained data suggest that atrazine-affected mast cells would probably have a consequent influence on thyroid follicular cells and/or thyroid microvasculature via paracrine action of released mediators, but might also be involved in already suggested thyroid cancerogenesis.     

The effect of chronic calcium treatment on thyroid C cells in ovariectomized rats

The purpose of this study was to investigate the influence of chronic calcium treatment on the structure and function of thyroid C cells in ovariectomzed adult female rats. Eighteen 3-month-old, female Wistar rats were divided into three groups. The first group was used as the sham-operated control, and the other two were surgically ovariectomized (Ovx). One month after gonadectomy, one group of Ovx rats was injected with 28.55 mg Ca-glucoheptonate (Ca)/kg b.w., while the other two groups were chronically treated with vehicle alone (Ovx and sham control). Two months after surgery, the animals were killed. In the thyroid C cells, calcitonin (CT) was localized with the peroxidase-antiperoxidase method. Stereology was used to evaluate morphometric changes in the volume of C cells, their nuclei and relative volume density. The number of C cells per unit area was calculated. Serum CT content was determined by radioimmunoassay. After chronic Ca treatment C cells were numerous with darker cytoplasm than in C cells of sham-operated control animals, but more degranulated than the corresponding cells of Ovx rats. Their volume was significantly decreased by 14% (p < 0.05), while the number was increased by 47% (p < 0.05) in comparison with corresponding controls. Serum CT concentration was decreased by 27% (n.s.) in comparison to sham-operated control. Calcium treatment of Ovx rats led to a 32% increase of serum CT concentration in relation to untreated Ovx animals. These results suggest that chronic Ca treatment of Ovx female rats positively affected CT release from thyroid C cells, without any significant changes in morphometric parameters.     

Iron restriction negatively affects bone in female rats and mineralization of hFOB osteoblast cells

We previously reported that severe iron deficiency negatively affects bone microarchitecture. Here we determined whether marginal iron restriction that reflects some human consumption patterns could have similar consequences. Thirty-two weanling female rats were randomly divided into four groups and fed the following diets for 10 weeks: (i) iron-adequate, calcium-adequate (FeA:CaA), (ii) calcium-restricted (FeA:CaR), (iii) iron-restricted (FeR:CaA), and (iv) both calcium- and iron-restricted (FeR:CaR) diets. DEXA analysis revealed that CaR decreased bone mineral density (BMD), and FeR decreased whole-body bone mineral content (BMC). Iron-restricted and calcium-restricted groups had lower BMD than did their adequate counterparts. All treatment-restricted groups had lower BMD in the fourth lumbar (L-4) vertebrae than the FeA:CaA group. Vertebrae BMD was lower in all treatment groups compared to the control group, and for BMC, the CaR groups were lower than the CaA groups and the FeR groups were lower that the FeA groups, and BMC were lower in iron- and calcium-restricted groups. The microarchitecture of the L-4 vertebrae was compromised in FeA:CaR, FeR:CaA, and FeR:CaR: (i) the connectivity density was reduced by FeR and by CaR; and (ii) trabecular number was decreased and trabecular separation was increased by FeR. Cortical thickness of the femur was reduced by both FeR and CaR. Finite element analysis revealed that L-4 vertebrae from the FeR:CaA group had greater internal stress with an applied force than the FeA:CaA group and, thus, would be more likely to break. Chelation of iron in cultured osteoblast cells impaired mineralization but had no impact upon Type I collagen deposition. Iron depletion, similar to that occurring among some human populations, reduced bone strength and microarchitecture based on the in vivo and in vitro results reported here. Impaired mineralization with iron depletion appears to be a possible mechanism for the observed bone abnormalities.     

羊骨胶原肽对青春期大鼠催乳素和促黄体生成素分泌脉冲的影响

目的研究羊骨胶原肽(sheep bone collagen peptide,SBCP)对类固醇诱导的去卵巢大鼠催乳素(pro-lactin,PRL)和促黄体生成素(luteinizing hormone,LH)分泌脉冲的影响。方法 6周龄子宫颈未开口的青春期SD大鼠实施双侧卵巢摘除手术,康复1周或3周后以类固醇替代方法诱导PRL和LH脉冲,处理组同时按1000 mg/(kg.d)的剂量以SBCP灌胃,通过颈导管采集血样,利用放射免疫技术测定各组大鼠外周血中的LH和PRL浓度。结果对于术后康复1周的大鼠,SBCP对LH脉冲振幅起到增强作用,而对PRL脉冲没有影响;对于术后康复3周的大鼠,SBCP对LH和PRL脉冲振幅均表现为降低作用,但卵巢摘除后立即给予SBCP可减弱这种降低作用。结论雌激素和孕激素替代注射的同时,补充羊骨胶原肽,不仅仍可诱导LH和PRL脉冲的产生,还可预防由于卵巢摘除带来的骨代谢紊乱,免疫力低下等不良影响,因而是对现有类固醇替代方法的改良。     

Effects of prior or concurrent food restriction on amylin-induced changes in body weight and body composition in high-fat-fed female rats

Amylin infusion reduces food intake and slows body weight gain in rodents. In obese male rats, amylin (but not pair feeding) caused a preferential reduction of fat mass with protein preservation despite equal body weight loss in amylin-treated (fed ad libitum) and pair-fed rats. In the present study, the effect of prior or concurrent food restriction on the ability of amylin to cause weight loss was evaluated. Retired female breeder rats were maintained on a high-fat diet (40% fat) for 9 wk. Prior to drug treatment, rats were either fed ad libitum or food restricted for 10 days to lose 5% of their starting body weight. They were then subdivided into treatment groups that received either vehicle or amylin (100 microgxkg(-1)xday(-1) via subcutaneous minipump) and placed under either a restricted or ad libitum feeding schedule (for a total of 8 treatment arms). Amylin 1) significantly reduced body weight compared with vehicle under all treatment conditions, except in always restricted animals, 2) significantly decreased percent body fat in all groups, and 3) preserved lean mass in all groups. These results indicate that amylin's anorexigenic and fat-specific weight loss properties can be extended to a variety of nutritive states in female rats.     

芹菜素对雌性大鼠生殖轴的影响

目的：探讨芹菜素在10-9mol/L浓度时对雌性大鼠生殖功能的影响。方法：应用侧脑室注射方法观察芹菜素对雌性大鼠生殖轴激素含量的影响。在侧脑室注射后第3天取血浆,采用放免技术测定血浆中促性腺激素释放激素（GnRH）、卵泡刺激素（FSH）、黄体生成素（LH）、雌二醇（E2）、孕酮（P）的含量。结果：在给药后第3天血浆中促性腺激素释放激素（GnRH）、卵泡刺激素（FSH）、黄体生成素（LH）的含量增加而雌二醇（E2）、孕酮（P）的含量降低,差异有显著性。结论：芹菜素抑制雌二醇（E2）合成中芳香化酶的活性实现对雌二醇（E2）、孕酮（P）分泌的抑制。芹菜素通过影响雌激素受体而使下丘脑的促性腺激素释放激素（GnRH）和腺垂体的卵泡刺激素（FSH）、黄体生成素（LH）分泌增加。     

The effect of cholecystokinin, bombesin and calcitonin on food intake in virgin, lactating and postweaning female rats

During lactation food intake increases greatly without an accompanying large increase in body weight; therefore, this physiological state is an excellent example of non-obese hyperphagia. In the present study, we found that cholecystokinin (CCK-8) decreased food intake in lactating and virgin female rats. However, female rats were more resistant to the effect of CCK on eating following weaning of the pups. Bombesin (BB) suppressed food intake in virgin female rats and in lactating rats during early and mid lactation. Rats were resistant to its satiating effect during late lactation and during the postweaning period. Calcitonin potently suppressed food intake in virgin, lactating and postweaning rats. The present findings suggest that CCK and bombesin decrease food intake more effectively in virgin female rats and during earlier phases of lactation than during late lactation or postweaning.