Hydrogen sulfide as a novel mediator for pancreatic pain in rodents.

OBJECTIVE: Hydrogen sulfide (H(2)S) is formed from l-cysteine by multiple enzymes including cystathionine-gamma-lyase (CSE) in mammals, and plays various roles in health and disease. Recently, a pronociceptive role for H(2)S in the processing of somatic pain was identified. Here, the involvement of H(2)S in pancreatic pain is examined. METHODS: Anaesthetised rats or mice received an injection of NaHS, a donor for H(2)S, or capsaicin into the pancreatic duct, and the expression of spinal Fos protein was detected by immunohistochemistry. Pancreatitis was created by 6 hourly doses of caerulein in unanaesthetised mice, and pancreatitis-related allodynia/hyperalgesia was evaluated using von Frey hairs. CSE activity and protein levels in pancreatic tissues were measured using the colorimetric method and western blotting, respectively. RESULTS: Either NaHS or capsaicin induced the expression of Fos protein in the superficial layers of the T8 and T9 spinal dorsal horn of rats or mice. The induction of Fos by NaHS but not capsaicin was abolished by mibefradil, a T-type Ca(2+) channel blocker. In conscious mice, repeated doses of caerulein produced pancreatitis accompanied by abdominal allodynia/hyperalgesia. Pretreatment with an inhibitor of CSE prevented the allodynia/hyperalgesia, but not the pancreatitis. A single dose of mibefradil reversed the established pancreatitis-related allodynia/hyperalgesia. Either the activity or protein expression of pancreatic CSE increased after the development of caerulein-induced pancreatitis in mice. CONCLUSIONS: The data suggest that pancreatic NaHS/H(2)S most probably targets T-type Ca(2+) channels, leading to nociception, and that endogenous H(2)S produced by CSE and possibly T-type Ca(2+) channels are involved in pancreatitis-related pain.

Yersinia pseudotuberculosis infection in breeding monkeys: detection and analysis of strain diversity by PCR.

In the last three decades, several monkeys reared in outdoor/indoor-outdoor breeding colonies and cages of the Primate Research Institute, Kyoto University, died of yersiniosis caused by Yersinia pseudotuberculosis, necessitating introduction of a method to detect the bacteria rapidly and thus allow preventive measures to be undertaken. A rapid nested polymerase chain reaction (PCR) method for identification of Y. pseudotuberculosis in fecal samples and a random amplified polymorphic DNA (RAPD)-PCR approach for distinguishing between bacterial strains were therefore developed. Yersinia pseudotuberculosis isolates from monkey specimens were found to be classifiable into several types. To determine the source of infection, hundreds of fecal samples of wild rats, pigeons, and sparrows were collected from around the breeding colonies and cages, and subjected to PCR analyses. Yersinia pseudotuberculosis was detected in 1.7% of the fecal samples of wild rats. The DNA fingerprints of the bacteria revealed by RAPD-PCR were the same as that of one strain isolated from macaques, suggesting the wild rat to be a possible source of infection.

The influence of rearing conditions on the physical growth of captive Japanese macaques (Macaca fuscata).

To clarify the influence of rearing conditions on the growth of various body parts of Japanese macaques (Macaca fuscata), two groups reared under different conditions, i.e., a group born and reared in open enclosures (Enclosure group) and another consisting of macaques born and reared in cages (Caged group), were somatometrically analyzed. Somatometric data on 36 measures of various body parts were collected from 77 males and 92 females. Growth in many body parts was smaller in the Caged group than in the Enclosure group. Body parts that exhibited large incremental increases were more sensitive to differences in rearing space at the infantile growth stage in both sexes. Recovery from delayed growth at the pubertal growth stage was found in many body parts. However, the size of some locomotor elements such as the wrist and hand, and ankle and foot strongly reflected limitations of space and changes due to this were irreversible. Females were more sensitive than males to such differences in rearing conditions. We conclude that open enclosures with ample rearing space are necessary for the innate growth of Japanese macaques to occur.

Neutrophil elastase inhibition reduces cerebral ischemic damage in the middle cerebral artery occlusion.

It has been reported that activated neutrophils are involved in the development of cerebral damage induced by ischemia. Activated neutrophils release a lot of mediators including toxic oxygen metabolites, elastase and cytokines which damage brain tissue. Therefore, we investigated roles of neutrophil elastase in the development of cerebral damage using an elastase inhibitor, ONO-5046. The rat middle cerebral artery (MCA) was occluded by a thrombus induced by photochemical reaction between green light and the photosensitizer dye, Rose Bengal. Photochemical reaction causes endothelial injury followed by formation of a platelet and fibrin-rich thrombus at the site of the irradiation. Photochemical reaction is routinely used in our laboratory to produce arterial occlusion in experimental animals. Twenty-four hours after the MCA occlusion, the size of cerebral damage was measured by histochemical technique. Water content in the brain was measured and neuronal deficits were examined 24 h after the MCA occlusion. ONO-5046 was administered at various doses as continuous infusion for 24 h, starting just after the MCA occlusion or from 3 h after. ONO-5046 at doses of 10 and 30 mg/kg/h significantly (p<0.05 and p<0.01, respectively) reduced the size of cerebral damage and water content (p<0.05, p<0.01, respectively) in different eight rats. Further, ONO-5046 at a dose of 30 mg/kg/h significantly (p=0.01) improved neuronal deficits. ONO-5046 which was administered starting from 3 h after the MCA occlusion, also reduced the size of cerebral damage. Neutropenia by anti-neutrophil antibody injection significantly (p<0. 01) reduced the size of cerebral damage. Elastase released from activated neutrophils may play a key role in the development of cerebral damage.

A potent inhibitor of inducible nitric oxide synthase, ONO-1714, a cyclic amidine derivative.

(1S,5S,6R,7R)-7-Chloro-3-imino-5-methyl-2-azabicyclo[4.1.0]heptane hydrochloride (ONO-1714), a novel cyclic amidine analogue, inhibits human inducible nitric oxide (iNOS) with a K(i) of 1.88 nM and rodent iNOS with similar potency in vitro. ONO-1714 was found to be 10-fold selective for human iNOS over human endothelial NOS (ecNOS). When the inhibitory activity of ONO-1714 was compared for iNOS, it was found to be 451-fold and >20,000-fold more potent than L-NMMA and aminoguanidine (AG), respectively. In terms of human iNOS selectivity, ONO-1714 was approximately 34- and 2-fold more selective for iNOS than L-NMMA and AG, respectively. ONO-1714 inhibited the LPS-induced elevation of plasma nitrate/nitrite in mice with an ID(50) value of 0.010 mg/kg, s.c. The maximum tolerated dose of ONO-1714 was 30 mg/kg, i.v. Thus, ONO-1714 represents one of the most potent iNOS inhibitors in vitro and in vivo to date and has great potentials for use as an inhibitor for clarifying the pathophysiological roles of iNOS and for use as a therapeutic agent.

The effect of an aldose reductase inhibitor (Epalrestat) on diabetic nephropathy in rats.

In order to clarify the possible contribution of the abnormal polyol pathway to the development of diabetic nephropathy, the effect of aldose reductase inhibitor on renal function and morphology was examined in streptozotocin (STZ)-induced diabetic rats. Six months after STZ injection, glomerular filtration rate and renal plasma flow showed marked decline with significant increase in nuclear-free mesangial area (MA) and relative mesangial area (RMA; MA per glomerular area) in diabetic rats. Oral administration of an aldose reductase inhibitor, Epalrestat, prevented renal hypofunction and mesangial expansion in diabetic rats without influencing the levels of blood glucose. These results suggest that the abnormal polyol pathway in diabetic rats is closely related to the development of mesangial expansion, a morphologic representative of diabetic glomerulopathy, and renal hypofunction.

Plasma catecholamine levels in SART-stressed rats and effects of drugs on stress-induced alteration in plasma and brain catecholamine levels.

1. Plasma catecholamine (CA) levels were examined in rats exposed to SART (specific alternation of rhythm in temperature) stress, a repeated cold stress. Effects of neurotropin, a sedative analgesic, and alprazolam, an anxiolytic, on the changes in plasma and brain CA levels were then studied. 2. SART stress induced remarkable increases in plasma levels of noradrenaline (NA) and dopamine (DA). The plasma adrenaline (Adr) level also increased significantly, but the extent was smaller than that of NA or DA. 3. Repeated treatments with neurotropin reduced the stress-induced increases in plasma and brain NA and DA levels significantly and dose-dependently. 4. Repeated treatments with alprazolam markedly reduced all increases in plasma and brain CA levels. 5. The above findings suggest that SART-stressed animals are in an increasing state of sympathetic neuronal activity and in a slightly increasing state of adrenal function. Neurotropin is also suggested to have modulating effects on autonomic imbalance in both catecholaminergic and cholinergic nerves.

Catecholamine levels in the brain of SART (repeated cold)-stressed rats.

1 The catecholamine levels in the brains of SART (specific alternation of rhythm in temperature)-stressed (repeated cold-stressed) rats with vagotonic-type dysautonomia, were examined by high performance liquid chromatography with electrochemical detection techniques. 2 The cerebral cortex, hypothalamus and hippocampus of the SART-stressed rats had increased levels of noradrenaline. All brain areas examined showed increased levels of dopamine. 3 These increased catecholamine levels were still maintained by day 10 of SART stress. 4 Among brain areas examined, the hypothalamus showed most rapid change. 5 Cold-stressed rats showed increased noradrenaline levels only in the basal ganglia and dopamine levels in the hippocampus. 6 Rats suffering from restraint and water immersion stress showed decreased noradrenaline levels and increased dopamine levels. 7 These results suggest that SART-stressed animals are in a disease state differing from that of other so-called stressed animals, and changes in the hypothalamus give rise to the various symptoms in SART-stressed animals.

[Three successful cases of artificial insemination in chimpanzees].

Artificial inseminations were performed on two female chimpanzees from July 1981 to April 1983 and three conceptions were obtained. Semen samples collected by rectal probe method of electroejaculation were incubated at 37 degrees C for about 20 minutes for liquefaction. Liquefied portion of the semen was sucked up into polyethylene tube about 30 cm in length attached to a syringe and was inseminated into cervix of each female of which pelvic region was raised in prone position under anesthesia. Ovulation time was speculated by swelling of sex skin in earlier two cases and by urinary LH level in last case. Three offsprings were obtained 234, 235 and 235 days after last insemination, respectively. All of three show normal developments.