Mariko Shirota

Plasma ACTH levels during early, two-way avoidance acquisition in high- and low-avoidance rats (Hatano strains).

Having successfully bred for high- and low-avoidance rats (HAA and LAA, respectively) on a shuttlebox task, we performed three experiments designed to identify factors which might be related to the phenotypic differences seen in avoidance behavior. In experiment 1, shuttlebox behavior was measured to determine whether the phenotypic difference was activity related. In terms of intertrial responses, there was no difference between HAA and LAA rats in locomotor activity during the conditioning process. Experiment 2 compared adrenal weights of HAA and LAA rats at 11 weeks of age. The observation that the adrenal glands were heavier in HAA than in LAA rats suggested that these strains might differ in aspects of endocrine response. In experiment 3, plasma levels of ACTH and corticosterone were determined during early escape/avoidance acquisition in the shuttlebox. Plasma levels of ACTH after the shuttlebox testing were higher in HAA than in LAA rats. There was no difference between the two strains in plasma levels of corticosterone after testing, possibly due to a ceiling effect. These results suggest that the phenotypic differences in the acquisition of avoidance behavior of HAA and LAA rats may be related to different endocrine responses, rather than to locomotor activity.

Detection of gene expression of vascular endothelial growth factor and flk-1 in the renal glomeruli of the normal rat kidney using the laser microdissection system

Abstract. The recent development of the laser microdissection (LMD) technique enables one to target particular tissues or cells for gene or protein analyses. The purpose of this study was to detect local mRNA expression of vascular endothelial growth factor (VEGF) and its receptor, flk-1, in the glomeruli of normal rat kidneys using the LMD system. Frozen sections of the kidney of 8-week-old male Wistar rats were made. The glomeruli were dissected from the frozen sections with the LMD system, and total RNA was extracted from 200 glomeruli in each kidney. Reverse-transcription polymerase chain reaction (RT-PCR) revealed the local mRNA expression of three isoforms of VEGF, flk-1 and GAPDH in the glomeruli. Moreover, the real-time PCR was performed to evaluate the experimental condition for quantification of VEGF and flk-1 mRNA expression using this system, and the results showed that at least 10 glomeruli might be needed for quantifying local VEGF mRNA expression. However, cDNA from 200 glomeruli was not enough for quantitative evaluation of flk-1 mRNA with this system. These results demonstrate the reproducibility of the analysis of mRNA expression in the renal glomeruli using the LMD system and also suggest that the application of the LMD technique will provide information to further our understanding of the mechanisms involved in kidney diseases.

Reproductive endocrinology in Hatano high- and low-avoidance rats during the estrous cycle.

The high- and low-avoidance animals (HAA and LAA rats) were originally selected from Sprague-Dawley rats for their shuttle-box task. Reproductive endocrinology during the estrous cycle was compared between HAA and LAA rats. All HAA rats showed a regular 4-d estrous cycle, whereas most LAA rats (70.8%) showed a regular 5-d estrous cycle. The peak level of preovulatory luteinizing hormone (LH) surge level was significantly lower in LAA rats than in HAA rats on the day of proestrus. In contrast, the peak level of prolactin surge on the day of proestrus was significantly higher in LAA rats than in HAA rats. Plasma concentrations of follicle-stimulating hormone (FSH) and estradiol-17β were significantly lower in LAA rats as compared with HAA rats at 12 h on the day of estrus and from 24 h on the day of diestrus to 18 h on the day of proestrus. On the other hand, plasma concentrations of progesterone were significantly higher in LAA rats compared with HAA rats on the day of diestrus. The number of antral follicles (300–600 µm in diameter) at 12 h on the day of proestrus was significantly fewer in LAA rats than in HAA rats. The size and number of corpus luteum at 12 h on the day of estrus were significantly greater in LAA rats than in HAA rats. These results clearly demonstrated that apparent differences are observed in reproductive endocrinology between two Hatano strains. These strain differences probably originated from neural regulation of pituitary hormones.

Maternal behavior, milk ejection, and plasma hormones in Hatano high- and low-avoidance rats.

Hatano high- and low-avoidance (in a two-way active avoidance task) animals (HAA and LAA, respectively) were successfully selected from a Sprague-Dawley rat population. Pup growth of both strains was dependent on the maternal strain in a cross-fostering study. To determine whether there are strain-specific differences between HAA and LAA in maternal care, both strains of dams were subjected to a test battery as measured by nest building, home cage behavior, pup retrieval, and milk ejection tests. In addition, changes in plasma concentrations of lactotropic and corticotropic hormones such as prolactin, ACTH, and corticosterone were examined during lactation. The test battery indicated that the dams of both strains built good nests and spent an identical amount of time with their offspring. However, LAA dams showed a prolonged latency time for pup retrieval and often left pups outside the nest until the end of the test period. LAA dams also showed a decreased amount of milk ejection, whereas no strain differences were observed in milk ejection after oxytocin treatment. During lactation, a lesser increase in plasma concentrations of prolactin and a greater increase in ACTH were found in LAA dams. There were no differences between the two strains in plasma concentrations of corticosterone. These results clearly demonstrated decreases in maternal behavior and milk ejection in LAA as compared to HAA dams. The present results also suggest that maternal motivation and mechanisms responsible for maternal hormones related to suckling are involved in the degree of pup growth.

Postnatal behavior in Hatano high- and low-avoidance rats following prenatal exposure to low-dose methylazoxymethanol

The hypothesis that genetic factors influence behavioral effects was tested in rats exposed prenatally to methylazoxymethanol (MAM). We examined whether baseline behavior is an important factor influencing behavioral effects, and whether a behaviorally selected strain was useful for study of neurobehavioral teratology. Pregnant high- and low-avoidance animals (HAAs and LAAs) of the Hatano strain, selectively bred for high and low shuttlebox avoidance responses, respectively, were given an IP injection of a low dose of MAM (15 mg/kg) on day 14 of gestation. The offspring of these animals were subjected to behavioral tests for locomotor activity (running-wheel and open-field tests) and learning ability (Biel maze and shuttlebox avoidance tests). There were no significant effects of MAM on running-wheel activity or shuttlebox avoidance learning, whereas the number of errors in the Biel maze was increased in the MAM offspring of both strains. Interestingly, open-field activity of the MAM offspring was markedly decreased in LAAs but not in HAAs. Therefore, an additional experiment was performed to determine plasma levels of ACTH and corticosterone following open-field exposure. When compared to control offspring of the respective strains, plasma levels of ACTH and corticosterone were not altered by prenatal MAM treatment in LAAs. Instead, the MAM offspring in HAAs exhibited decreased ACTH levels in absence of behavioral alterations. These results demonstrated that prenatal exposure to low doses of MAM may alter postnatal behavior and endocrine response of the offspring, although to a differing degree in HAAs and LAAs. Our observations suggested that behaviorally selected strains are sensitive to neurobehavioral teratogens such as MAM.

Gastric ulceration and expression of prolactin receptor in the brain in hatano high- and low-avoidance rats

Recently, prolactin was shown to inhibit the development of stress-induced ulcers. However, the mechanism for suppression of gastric ulcers by prolactin has not been clarified. Hatano high-avoidance (HAA) and low-avoidance (LAA) strains of rats were originally selected and bred from Sprague-Dawley rats based on shuttle-boxtasks. The present study focused on the relationships among gastric ulceration and endocrine response with special reference to prolactin secretion and restraint stress in water of HAA and LAA rats. The restraint stress induced an elevation of plasma concentrations of ACTH, corticosterone, and prolactin. Peak levels of plasma ACTH during stressful condition were significantly higher in HAA rats than in LAA rats, while peak levels of prolactin were significantly lower in HAA rats than in LAA rats. The gastric erosion inde was significantly higher in HAA rats than in LAA rats 7 h after restraint stress in water. The numbers of prolactin-receptor-positive cells determined by immunohistochemistry in the paraventricular nucleus was significantly increased in LAA rats than in HAA rats 7 h after restraint stress in water. These results indicate that HAA rats were more sensitive than LAA rats to restraint stress in water. The strain differences in gastric ulceration under stress may be involved in peripheral prolactin secretion and central prolactin receptor expression. The expression of prolactin receptor in the paraventricular nucleus may be important in suppressing gastric ulceration.

Differences in adrenocortical secretory and gene expression responses to stimulation in vitro by ACTH or prolactin between high- and low-avoidance Hatano rats

Rats of the Hatano high-avoidance (HAA) and low-avoidance (LAA) strains have been genetically selected on the basis of their two-way active avoidance behavior, and have different endocrine responses to stress. The present study focused on the adrenal steroid hormone responses of the Hatano strains and identifies differences in regulation of the adrenal cortex in vitro of HAA and LAA rats. Although incubation with prolactin (PRL) and/or adrenocorticotrophic hormone (ACTH) resulted in a dose-dependent increase of corticosterone and progesterone release by adrenal cells from both HAA and LAA male rats, the responses were markedly increased for adrenal cells from LAA rats as compared with HAA rats. This finding suggested that adrenal glands of HAA rats are less sensitive to PRL and/or ACTH than adrenals from LAA rats. Several possible intra-adrenal regulators were investigated. The basal level of expression of steroidogenic acute regulatory protein (StAR) and the long form of the PRL receptor (PRLR-L) mRNAs was higher in adrenals of LAA rats. ACTH treatment of adrenal cells from HAA rats resulted in statistically significant increases in melanocortin receptor 2 (MC2R) mRNA expression, while neither ACTH nor PRL altered MC2R mRNA expression in adrenal cells of LAA rats. Conversely, the increase in PRLR-L mRNA expression induced by PRL was observed only in adrenal cells from LAA rats. Treatment of adrenal cells with PRL and/or ACTH increased the expression of StAR and CYP11A1 mRNAs for both Hatano strains. However, the induction of StAR mRNA expression was higher in LAA rats, but the CYP11A1 response was lower. These findings indicate that adrenal cells of the LAA strain have higher sensitivity to secretagogues than those of the HAA strain. These results suggest that PRL may also be important in stimulating secretion of adrenal steroid hormones.

Expression of Periostin in Normal, Atopic, and Nonatopic Chronically Inflamed Canine Skin

In humans, periostin plays a critical role in the enhancement and chronicity of allergic skin inflammation; however, whether it is involved in the pathogenesis of canine dermatitis remains unknown. The aim of this study was to examine the expression patterns of periostin in healthy, atopic, and nonatopic chronically inflamed canine skin. Biopsy specimens from 47 dogs with skin disease and normal skin tissue from 5 adult beagles were examined by light microscopy, immunohistochemistry, and in situ hybridization. In normal skin, periostin was localized just beneath the epidermis and around the hair follicles. In chronically inflamed skin, periostin expression was most intense in the dermis with inflammatory cell infiltrates. In contrast, low levels of periostin were detected in acutely inflamed and noninflamed skin. Conversely, all canine atopic dermatitis tissues characteristically showed the most intense expression of periostin in the superficial dermis, particularly at the epidermal–dermal junction. In situ hybridization showed that periostin mRNA was broadly expressed in the basal epidermal keratinocytes, outer root sheath cells, and dermal fibroblasts in normal dog skin. High expression of periostin mRNA was observed in fibroblasts in dog skin with chronically inflamed dermatitis. Moreover, in some chronically inflamed skin specimens, periostin mRNA expression was increased in basal keratinocytes. The severity score of chronic pathologic changes and CD3+ cell number in the dermis were correlated with distribution pattern of periostin in the atopic skin. These data suggest that periostin could play a role in the pathophysiology of chronic dermatitis, including atopic dermatitis, in dogs.

Dose-dependent acceleration in the delayed effects of neonatal oral exposure to low-dose 17α-ethynylestradiol on reproductive functions in female Sprague-Dawley rats.

Xenoestrogen exposure during the critical period of sexual differentiation of the brain causes delayed effects on female reproduction. We investigated the internal dose of orally administered ethynylestradiol (EE) during the critical period and its delayed effects by administering 0 (vehicle control), 0.4, or 2 μg/kg EE to female Sprague-Dawley rats for 5 days from postnatal day (PND) 1. Determination of serum EE level 24 hr after the initial dosing and 6 and 24 hr after the final dosing of 2 μg/kg indicated that the administered EE entered the circulation and cleared after every administration. Although the treatment did not affect physical development, including growth, eyelid opening, and vaginal opening, the estrous cycle was arrested from postnatal week (PNW) 12 even with 0.4 μg/kg EE, with an inverse correlation between doses and arresting ages. Although ovarian morphology at PNW 22-23 indicated that the treatment caused long-term anovulation and cystic follicle formation, the number of primordial follicles at PNW 22-23 was similar among the groups. Because this number was lower than that at PND 10 in all groups, primordial follicles may have been consumed under long-term anovulation. The treatment also caused other abnormalities, including mammary gland hyperplasia, increase in pituitary and liver weights, and decrease in the uterine weight. Because the highest circulating EE level in the 2 μg/kg-treated neonates is considered to be comparable to the physiological range of estradiol-17β, we concluded that a slight increase in the circulating estrogens during the neonatal period exerts irreversible delayed effects.

Vascular Hamartoma in the Uterus of a Female Sprague-Dawley Rat with an Episode of Vaginal Bleeding

An annular, reddened lesion with mild serosal hemorrhage and no tumorous mass formation was detected in the right uterine horn of a 37-week-old female Sprague-Dawley rat that had postpubertal vaginal bleeding. Histological examination revealed prominent proliferation of the endometrium, which occupied the uterine lumen. There were numerous aberrant vascular spaces filled with erythrocytes, proliferation of stromal cells, and inflammatory infiltrates including hemosiderin-laden macrophages in the endometrium. These vasculatures extended into the myometrium, and in a transverse section of the lesion, they were mostly distributed throughout the circumference of the uterus. They were irregular in shape and interconnected, forming a large vascular sinus and anastomosing reticular channels. In the area with serosal hemorrhage, the muscular layer covering the large irregular vascular space had undergone degeneration and necrosis. The lining cells of the vasculatures were often plump, and they protruded into the lumen and were arranged in a tombstone or hobnail manner. Immunostaining revealed that these cells were positive for von Willebrand factor and CD34. The aberrant vasculatures were not accompanied by pericytes or muscular layer, although a discontinuous muscular wall was present around some of them. From these results, the uterine lesion was diagnosed as a vascular hamartoma.