Effects of 1,5-anhydro-D-glucitol on insulin secretion both in in vitro and ex vivo pancreatic preparations.

Organ bath experiments are conventionally used to investigate the physiological actions and effects of hormones and drugs on organ responses. We developed an experimental method to reproduce insulin secretion from isolated rat pancreas preparations, to investigate substances that promote insulin secretion ex vivo. 1,5-anhydro-D-glucitol (1,5-AG) is found in foods, and exists in humans and rodents; however, whether 1,5-AG stimulates insulin secretion remains unclear. This study aimed to assess the effects of short-term 1,5-AG stimulation on insulin secretion in both ex vivo and in INS-1E (rat-derived) cells in vitro. Our results indicated that 1,5-AG had no potency to increase the proportion of insulin outflow both in ex vivo and in vitro experiments. Insulin outflow significantly increased upon stimulation with 10 µM glimepiride, a member of the sulfonylurea class of drugs, ex vivo. Glucose-stimulated insulin secretion was observed not only in INS-1E cells but also in rat pancreatic preparations. Our findings demonstrated that short-term exposure to 1,5-AG had no effect on insulin secretion in rats.

Time-dependent changes in cardiovascular function during copulatory behavior induced by the hand method in the male dog.

PURPOSE: Ejaculation in the male dog consists of three fractions. Observation of behavior and measurement of heart rate (HR), and plasma noradrenaline (NA) and adrenaline (Ad) concentrations were researched sequentially, and a fundamental examination of the features of sympathetic nerve activity during copulatory behavior induced by the hand method in the male dog was undertaken. METHODS: We investigated the breeding capability of male dogs. HR, plasma NA level and plasma Ad levels were measured during ejaculation induced by the hand method. RESULTS: HR was 125.8 ± 6.0 beats/min at rest, and peaked during mounting at 195.2 ± 8.2 beats/min. Moreover, HR at 3 min after the first fraction decreased to values similar to those at rest. Plasma NA and Ad concentrations during copulatory behavior induced by the hand method did not differ significantly from those at rest. However, although there was no significant difference, plasma NA concentration during ejaculation of the third fraction peaked at about 1.8 times the baseline value. CONCLUSIONS: In the male dog, excitation of sympathetic nerves of long duration during erection of the penis and ejaculation is questionable. However, inhibition of sympathetic nerves and activation of parasympathetic nerves is thought to occur during erection of the penis and ejaculation.

The zonula occludens protein family regulates the hepatic barrier system in the murine liver.

The hepatic barrier is indispensable for the physiological functions of the liver and is impaired under various pathological conditions. Tight junctions reportedly play a central role in hepatic barrier regulation; however, there is limited direct evidence supporting this observation, with few in vivo models or confirmations of the implicated molecular mechanisms presented to date. We inactivated the tight junction component gene, Tjp2/ZO-2, and the related molecule, Tjp1/ZO-1, in mouse livers. In humans, TJP2/ZO-2 mutations have been implicated in the development of human progressive familial intrahepatic cholestasis 4 (PFIC4). The mice deficient in either ZO-1 or ZO-2 in the liver did not exhibit major abnormalities. However, the ablation of both molecules impaired the molecular architecture as well as the structure and function of hepatocyte tight junctions, which disrupted the hepatic barrier and was lethal to the mice by 6 weeks of age. In mutant mice, bile canaliculus formation and cellular polarity were compromised; also, transporter expression and localization were deregulated. Moreover, typical hepatic zonation and bile duct formation were inhibited, and sinusoidal vessels were disorganized. These findings clarify the role of tight junctions and polarity in the hepatic barrier as well as the effect that their disruption has on liver tissue. The observations also suggest that liver-specific ZO-1-/- and ZO-2-/- mice could be used as models for PFIC4, and this will provide new insights into liver pathophysiology and clinical applications.

The Effects of Trypsin Inhibitor on Insulin Secretion Using Rat Pancreas in an Organ Bath.

BACKGROUND/AIM: We developed an experimental method to reproduce insulin secretion from isolated rat pancreas preparations using an organ bath system. However, secretion of trypsin, another pancreatic enzyme, interferes with insulin production in such systems. We aimed to ascertain the minimum trypsin inhibitor (TI), dose for obtaining a sustained, stable rate of insulin secretion. MATERIALS AND METHODS: The action of TI (1-10 µg/ml) on pancreatic preparations of male Wistar-Imamichi rats in organ bath experiments was assessed by measuring insulin, amylase, and trypsin activity. RESULTS: The level of insulin outflow remained steady in the TI-treated samples, in contrast to that in the untreated control, where insulin secretion decreased over time. The level of amylase outflow did not change significantly. Trypsin activity was significantly lower in the TI-treated samples than in the control. CONCLUSION: Even low concentrations of TI can maintain insulin secretion by inhibiting trypsin activity in organ bath experiments.

Histopathological evaluation of the effectiveness of oral Eppikajutsuto treatment for lymphatic malformation.

BACKGROUND: Lymphatic malformation (LM) is a congenital disease caused by lymphatic vessel malformation. Although standard therapies for LMs are sclerotherapy and/or surgical excision, a new therapy using Japanese herbal medicine Eppikajutsuto (TJ-28) has been recently reported as clinically effective. We aimed to experimentally confirm the therapeutic effectiveness of TJ-28 for LMs. METHODS: LM lesions were generated in the mesentery and peritoneum of mice by intraperitoneal injection of Freund's incomplete adjuvant. Mice with LMs were treated by gavage or dietary administration of TJ-28 for 2 months. Formalin-fixed paraffin-embedded tissue sections of mesentery and peritoneum tissues were histologically and immunohistochemically examined by focusing on lymph nodes and perinodal lymph vessels. RESULTS: Multiple Freund's incomplete adjuvant-associated foreign-body granulomas were formed in the mesentery and peritoneum, resulting in congestion of lymph fluid and dilatation of lymph vessels. The numbers and sizes of lymph nodes were not significantly different between TJ-28-treated and control groups. However, the luminal areas of lymphatic vessels were reduced significantly in the TJ-28 treatment group by both gavage and dietary administrations. CONCLUSION: TJ-28 conspicuously reduced congestion of lymph fluid. This is the first histopathological evaluation of LM model mice to study the effectiveness of oral TJ-28 treatment.

Plasma prolactin concentrations and copulatory behavior after salsolinol injection in male rats.

PURPOSE: The dopamine-derived endogenous compound, R-salsolinol (SAL), was recently identified as a putative endogenous prolactin (PRL)-releasing factor. However, how SAL influences copulatory behavior is unknown. In this study, we examined the relationship between SAL and copulatory behavior in male rats. METHODS: Male Sprague-Dawley rats administered SAL were exposed to female rats in estrus, the plasma PRL concentration was measured, and the behavioral frequency and time during copulatory behavior were noted. RESULTS: In the control and SAL groups, plasma PRL concentrations at 15 min before exposure to the female were 7.3 ± 2.0 and 8.0 ± 1.5 ng/ml, respectively. Moreover, plasma PRL concentrations in males immediately after exposure to the female were 7.4 ± 1.2 and 68.0 ± 5.9 ng/ml, respectively (P < 0.05). All (8/8) of the control animals ejaculated in the presence of the female, whereas only 33% (2/6) of the SAL group ejaculated. An increasing tendency for mount latency and intromission latency and a decreasing tendency for intromission frequency were observed in the SAL group. CONCLUSIONS: Copulatory behavior was inhibited in male rats after SAL injection, suggesting that SAL is a copulatory behavior inhibiting factor.

Development of an organ bath technique for isolated rat pancreas preparations to assess the effect of 1,5-AG on insulin secretion.

To investigate substances related to insulin secretion, we reported a convenient experimental method to reproduce insulin secretion from isolated rat pancreas preparations using an organ bath. While the method has experimental utility for investigating insulin secretion, optimization of the experimental design is still needed. The level of insulin outflow in the control decreased over time in our previous study. Decreasing serum 1,5-anhydroglucitol (1,5-AG) levels is also known to be shown in patients with worsening glycemic control. There is one in vitro report demonstrated that 1,5-AG induced insulin release. It appears that discussion needs to be deepened further on it. In this study, we investigated the effect of 1,5-AG on insulin secretion through to optimize the condition of endocrine function using the ex vivo organ bath technique. The level of insulin outflow in the control and 1,5-AG groups decreased over time in the organ bath experiment. To analyze the effect of trypsin on reduced insulin secretion, pancreas preparation was treated with soybean trypsin inhibitor (TI). Insulin outflow levels of the TI group were significantly higher than the control group. An enzyme indicator of tissue damage tended to be lower in the TI group. There was no significant enhancement of insulin secretion by 1,5-AG. The present study demonstrated the utility of TI application for the organ bath technique. This finding supported the development of an organ bath technique for the assessment of the effects of novel therapeutics on insulin secretion.

Time-dependent changes in cardiovascular function during copulation in male rats.

PURPOSE: Sudden cardiac death after ejaculation has been reported in humans and highlights the important relationship between sexual behavior and the heart. The rat is an extremely useful animal model for investigating reproductive function in male mammals. In this study, we examined the relationship between autonomic nervous system activity and the circulatory system during sexual behavior in male rats. METHODS: Male Wistar-Imamichi rats were exposed to female rats in estrous and heart rate (HR), systolic blood pressure (SBP), diastolic blood pressure (DBP), and plasma noradrenaline (NA) and adrenaline (Ad) concentrations were measured by telemetry during copulation. RESULTS: The resting HR was 365.5 ± 18.4 beats/min (mean ± SE), which increased to 531.2 ± 21.1 beats/min at ejaculation and decreased to 404.6 ± 30.7 beats/min 1 min after ejaculation. At rest, SBP and DBP were 123.8 ± 6.6 and 81.5 ± 4.1 mmHg, respectively, which increased to 154.5 ± 5.9 and 112.1 ± 7.3 mmHg at ejaculation. Baseline plasma Ad and NA concentrations were 151.6 ± 32.0 and 248.5 ± 22.5 pg/ml, respectively, and these increased to 393.8 ± 89.9 and 792.7 ± 154.0 pg/ml at ejaculation, after which they decreased to resting levels. The rate of increase in NA at ejaculation differed significantly from that of Ad. CONCLUSIONS: The load on the circulatory and autonomic nervous systems is controlled by a rapid decrease in HR and NA concentration immediately after ejaculation, such that the male rat is prepared for the next copulation.

Oxytocin is indispensable for conspecific-odor preference and controls the initiation of female, but not male, sexual behavior in mice.

Oxytocin (OT) has been demonstrated to be involved in various social behaviors in mammals. However, OT gene knockout (OTKO) mice can conceive and deliver successfully, though females cannot rear their pups because of lack of lactation. Here, we investigated the sociosexual behavior of both sexes in two experimental setups: olfactory preference for sexual partner's odor and direct social interaction in an enriched condition. In the preference test, mice were given a choice of two airborne odors derived from intact male and receptive female mice, or from intact or castrated male mice. Wild-type (WT) mice significantly preferred opposite-sex odors, whereas OTKO mice showed vigorous but equivalent exploration to all stimuli. In social interactions in the enriched condition, no difference in sexual behavior was found between WT and OTKO males. In contrast, WT female initiated sexual behavior at the second week test, while OTKO females required 4 weeks to receive successful mounts. Neuronal activation by odor stimulation was compared between WT and OTKO mice. The numbers of cFos-immunoreactive cells increased in the medial amygdala and the preoptic area after exposure to opposite-sex odors in WT mice, whereas the increase was suppressed in OTKO mice. We conclude that OT plays an important role in the regulation of olfactory-related social behavior in both male and female mice. The influence of OT was greater in female mice, especially during social interactions involving the acquisition of sexual experience.

Correlation between body weight (epididymal fat) and permeation rate of serum leptin through the blood-brain barrier (BBB) in male rats aged 8 months.

In this study, in order to clarify the kinetics of leptin, we focused on the ratio of leptin concentrations in cerebrospinal fluid and serum in aged male rats, and examined the weight of epididymal fat, and the passage rate of leptin through the blood-brain barrier. In the lighter animals, the epididymal fat weight was low, while leptin concentrations in the serum and cerebrospinal fluid were also low. Conversely, in the heavier animals, the weight of epididymal fat and leptin concentrations in the serum and cerebrospinal fluid were higher. With regard to the ratio of leptin in the cerebrospinal fluid and serum, the passage rate of leptin through the blood-brain barrier was lower in the heavier animals than in the lighter animals.

Reproducible insulin secretion from isolated rat pancreas preparations using an organ bath.

Diabetes mellitus is a lifestyle-related disease that is characterized by inappropriate or diminished insulin secretion. Ex vivo pharmacological studies of hypoglycemic agents are often conducted using perfused pancreatic preparations. Pancreas preparations for organ bath experiments do not require cannulation and are therefore less complex than isolated perfused pancreas preparations. However, previous research has generated almost no data on insulin secretion from pancreas preparations using organ bath preparations. The purpose of this study was to investigate the applicability of isolated rat pancreas preparations using the organ bath technique in the quantitative analysis of insulin secretion from ß-cells. We found that insulin secretion significantly declined during incubation in the organ bath, whereas it was maintained in the presence of 1 µM GLP-1. Conversely, amylase secretion exhibited a modest increase during incubation and was not altered in the presence of GLP-1. These results demonstrate that the pancreatic organ bath preparation is a sensitive and reproducible method for the ex vivo assessment of the pharmacological properties of hypoglycemic agents.

Assessing equine anxiety-related parameters using an isolation test in combination with a questionnaire survey.

The aim of the study was to determine parameters reflecting equine anxiety trait by comparing results obtained in a behavior test and an anxiety score assessed by familiar caretakers in response to a questionnaire. In the behavior test, horses were individually led into a novel room by their caretakers and loosely tethered to decrease excessive movement using the common cross-tying technique with two leads and breakable plastic cords. The horses initially remained with their caretaker for 2 min; the caretaker then left and the subject animal was left alone for 2 min. The latency to break the plastic cords, heart rate, the number of steps and pawings were recorded. When the horses were left alone, most parameters changed significantly and some showed sexual differences. A correlation analysis revealed that anxiety score assessed by caretakers showed a negative correlation with the latency to break the plastic cord and a positive correlation with heart rate only when horses were isolated. These two were suggested feasible parameters for assessing the anxiety trait of unfamiliar horses. Both the behavioral results and the anxiety scores also indicated that females were more anxious than males. Our results suggest that it would be a useful strategy for assessing other temperament traits as well to combine behavior tests with a questionnaire survey.

Incidence of anaphylactic reactions after propofol administration in dogs.

Propofol is an anesthetic agent suspended in an emulsion system that includes egg yolk lecithin and soybean oil, because of which, there is concern about the use of propofol in patients allergic to these substances. We examined the association between propofol administration and incidence of adverse events in dogs with allergy to egg yolk lecithin and soybean oil. On the basis of the findings of an allergen-specific immunoglobulin E (IgE) test, 14 dogs with high levels (high-IgE group) and 7 dogs with low levels (normal-IgE group) of IgE were selected. Following intravenous administration of propofol, the incidence of anaphylactic reactions and plasma histamine concentrations under general anesthesia maintained with isoflurane throughout surgery were compared between the two groups. The frequency of anaphylactic reactions and plasma histamine concentrations were compared by the chi-square test and Student t-test, respectively. The statistical significance for both tests was set at P<0.05. In the high- and normal-IgE groups, the average frequencies of anaphylactic reactions after propofol administration were 21.4 and 14.3%, and the mean plasma histamine concentrations were 167.9 ± 94.5 nM and 65.7 ± 40.3 nM, respectively. Animals of neither groups experienced shock-like symptoms. These results revealed that propofol might be relatively safe, although careful perioperative anesthesia monitoring and standby protocols are required when using propofol in dogs with a history of allergic diseases or high chicken- or soybean-specific IgE levels.

Vomeronasal signal deficiency enhances parental behavior in socially isolated male mice.

We previously reported that social isolation promotes parental care in sexually naïve male mice. This effect was blocked by exposure to chemosensory and auditory social signals derived from males in an adjacent compartment. In the present study, we examined whether the chemosensory signals detected in the vomeronasal organ (VNO) are involved in parental behavior by using mice deficient for a VNO-specific ion channel (Trpc2-/-) and thus impaired in VNO-input signaling. We housed virgin homozygous Trpc2-/- and heterozygous Trpc2± males for 3weeks during puberty (5-8weeks old) alone or in groups of 3-5 males. At 8weeks of age, the mice were placed with three pups in an observation cage and tested for parental behavior. The Trpc2-/- males housed under isolated conditions spent significantly longer in the vicinity of pups than did the Trpc2-/- males than had been group housed, whereas no isolation effect was observed in heterozygous Trpc2± males. Both Trpc2 knockout and isolation housing significantly increased the time males spent licking pups and crouching (arched back posture over pups to enable nursing), whereas only isolation housing increased the incidence of retrieval behavior. These results demonstrated that social signals transmitted not only through the VNO but also from other modalities, independent of each other, suppress the expression of parental behavior during puberty in sexually naïve males.

Protective Role of ICOS and ICOS Ligand in Corneal Transplantation and in Maintenance of Immune Privilege.

Purpose: The interaction between the inducible costimulatory molecule (ICOS) and ICOS ligand (ICOSL) has been implicated in the differentiation and functions of T cells. The purpose of the present study was to determine the role of ICOS-ICOSL in the immune privilege of corneal allografts. Methods: Expression of ICOS and ICOSL mRNA from mouse eyes was assessed by RT-PCR. Corneas of C57BL/6 mice were orthotopically transplanted into the eyes of ICOS-/- BALB/c recipients and BALB/c wild-type (WT) recipients treated with anti-ICOSL mAb, and graft survival was assessed. A separate set of WT and ICOS-/- BALB/c mice received an anterior chamber injection of C57BL/6 splenocytes, and induction of allospecific anterior chamber-associated immune deviation (ACAID) was assessed. In vitro, cornea was incubated with T cells from WT and ICOS-/- BALB/c mice, and destruction of corneal endothelial cells (CECs) and the population of Foxp3+ CD25+ CD4+ T cells was assessed. Results: Inducible costimulatory molecule ligand mRNA was constitutively expressed in the cornea, iris-ciliary body, and retina. Allograft survival in ICOS-/- recipients and WT recipients treated with anti-ICOSL mAb was significantly shorter than in control recipients. Anterior chamber-associated immune deviation was induced less efficiently in ICOS-/- mice. Destruction of CECs by alloreactive ICOS-/- T cells was enhanced compared with WT T cells. After coincubation with allogeneic corneal tissue, the proportion of regulatory T cells was significantly greater among WT T cells than in ICOS-/- T cells. Conclusions: The expression of ICOSL in the cornea and the ICOS-mediated induction of Foxp3+ CD4+ regulatory T cells may contribute to successful corneal allograft survival.

Changes in the heart rate and plasma epinephrine and norepinephrine concentrations of the stallion during copulation.

Aim: The physiological indexes of the copulatory behavior of the stallion have not been investigated in detail and may differ from those of other species, such as humans and rats. Methods: In order to understand the breeding capability of various stallions, their behavior during copulation was observed, and heart rate (HR) and the plasma concentrations of norepinephrine (NA) and epinephrine (Ad) were measured sequentially for a total of 13 copulations carried out during 2 days. Results: The mean HR at rest was 35.3 ± 0.9 beats per minute (b.p.m.) and it peaked during mounting (162.1 ± 5.4 b.p.m.). The HR at ejaculation was 145.7 ± 5.1 b.p.m, which was less than the peak. The plasma concentrations of NA and Ad showed similar changes to the HR; immediately after ejaculation they were, respectively, 4.7-fold and 1.9-fold higher than the resting values and there was a difference in the degree of increase of each catecholamine. Conclusions: The present results show that in the stallion the HR peaks at mounting and there is a greater change in the activity of the sympathetic nervous system because of the short-term, highly intense exercise performed during copulation. It is considered that this, combined with the particular mental stress placed on the stallion during copulation, has the potential to cause sudden cardiac death. (Reprod Med Biol 2005; 4: 143-148).

Changes of heart rate during sexual behavior in the female rat.

The authors studied the physiological and behavioral responses during sexual behavior in female rats. For this purpose, electrocardiographs were recoded from conscious and unrestrained females using a radiotelemetry system. Heart rate during sexual behavior rose sharply following the introduction of a sexually active male, displayed a peak level of 425 bpm during male ejaculation, and then rapidly decreased. This pattern of heart rate in females before and after receiving ejaculation was similar to that in males before and after ejaculation. In the rate of decrease in heart rate, however, there was a significant difference between females and males. The present study is the first to show changes of heart rate during sexual behavior in female rats.

Developmental expression of penile reflexes and copulatory behavior in male rats.

First, pubertal development of the penile reflexes, e.g., erections, cups and flips in Wistar-Imamichi male rats was investigated following sheath retraction. Second, the penile reflexes and copulatory behavior in the above males were compared between 10 and 44 weeks of age. The penile reflexes in Wistar-Imamichi rats began to appear from day 26, and all males displayed full components of the reflexes on day 47. The occurrence rates and mean numbers of erections, cups and flips in aged adults were significantly low, compared with the young adults. Also, in the observation of copulatory behavior the occurrence rate of ejaculations, and mean numbers of intromissions and ejaculations in aged adults were significantly lower than that in young adults. These results of the present study may suggest that the decrease of copulatory behavior in male rats with age results from the dysfunction of the penile reflexes with age.

Effects of i.c.v. administration of leptin on copulatory and ingestive behavior in STZ-induced diabetic male rats.

It is well known that circulating leptin concentrations correlate with adiposity in both humans and rodents and decrease after fasting, energy restriction, or weight loss. The goal of the present study was to confirm whether the decreases of copulatory behavior and the increases of ingestive behavior in STZ-induced diabetic male rats could be reversed by i.c.v. administration of leptin. Adult male Wistar-Imamichi rats aged 9 weeks were used for the studies. Males received a single injection of STZ (60 mg/kg, i.p.) and vehicle. During the experiment, individual body weight, and food and water intake were measured. The copulatory and ingestive behaviors in STZ-induced diabetic males were observed at 2 and 4 weeks after STZ. At 6 weeks after STZ, leptin (10 microg/10 microl) or aCSF (artificial cerebrospinal fluid) was injected through a lateral ventricle cannula and the above two behaviors were observed again. The i.c.v. leptin injection to STZ-induced diabetic males resulted in a significant increase of ejaculation frequencies (3.6 +/- 0.26 vs. 2.9 +/- 0.30 times) and a significant decrease in amount of food ingested (36.2 +/- 1.93 vs. 23.2 +/- 3.76 g), compared with the aCSF-injected control (p<0.01). These findings suggest that the copulatory and ingestive behaviors in i.c.v. leptin-injected STZ diabetic males were restored to levels equivalent to those in control males.

Quantitative and qualitative analysis of rat pup ultrasonic vocalization sounds induced by a hypothermic stimulus.

Ultrasonic vocalizations (USVs) are essential communicative sounds used between rodent pups and their mother. Rat pups emit USVs in stressful situations, such as when they are cold or separated from the nest. We verified the ontogenetic changes in USVs emitted by infant rats isolated from their mother during the pre-weaning period. The number of calls, and the median frequency and first peak of frequency of the calls were measured at 1, 3, 5, 7, 10, 12, and 14 days postnatal age in Wistar-Imamichi rats. Pups were placed in a cold glass beaker and USVs were recorded for 5 min. The number of calls increased to a peak on day 5 and then gradually decreased. The median frequency of calls decreased slowly during the first 12 days, and then increased slightly on day 14. Similarly, the first peak frequency of calls was the highest on day 1, and then decreased gradually by day 12. A small increase was observed on day 14. These changes in frequency were correlated with the physical development of the pups, whose body weights increased significantly with age except during postnatal days 7-10.