Possible Involvement of the Rat Hypothalamo-Neurohypophysial/-Spinal Oxytocinergic Pathways in Acute Nociceptive Responses.

Oxytocin (OXT)-containing neurosecretory cells in the parvocellular divisions of the paraventricular nucleus (PVN), which project to the medulla and spinal cord, are involved in various physiological functions, such as sensory modulation and autonomic processes. In the present study, we examined OXT expression in the hypothalamo-spinal pathway, as well as the hypothalamo-neurohypophysial system, which includes the magnocellular neurosecretory cells in the PVN and the supraoptic nucleus (SON), after s.c. injection of saline or formalin into the hindpaws of transgenic rats that express the OXT and monomeric red fluorescent protein 1 (mRFP1) fusion gene. (i) The numbers of OXT-mRFP1 neurones that expressed Fos-like immunoreactivity (-IR) and OXT-mRFP1 intensity were increased significantly in the magnocellular/parvocellular PVN and SON after s.c. injection of formalin. (ii) OXT-mRFP1 neurones in the anterior parvocellular PVN, which may project to the dorsal horn of the spinal cord, were activated by s.c. injection of formalin, as indicated by a significant increases of Fos-IR and mRFP1 intensity intensity. (iii) Formalin injection caused a significant transient increase in plasma OXT. (iv) OXT, mRFP1 and corticotrophin-releasing hormone mRNAs in the PVN were significantly increased after s.c. injection of formalin. (v) An intrathecal injection of OXT-saporin induced hypersensitivity in conscious rats. Taken together, these results suggest that the hypothalamo-neurohypophysial/-spinal OXTergic pathways may be involved in acute nociceptive responses in rats.

Fluorescent Visualisation of Oxytocin in the Hypothalamo-neurohypophysial/-spinal Pathways After Chronic Inflammation in Oxytocin-Monomeric Red Fluorescent Protein 1 Transgenic Rats.

Oxytocin (OXT) is a well-known neurohypophysial hormone that is synthesised in the paraventricular (PVN) and supraoptic nuclei (SON) of the hypothalamus. The projection of magnocellular neurosecretory cells, which synthesise OXT and arginine vasopressin in the PVN and SON, to the posterior pituitary plays an essential role in mammalian labour and lactation through its peripheral action. However, previous studies have shown that parvocellular OXTergic cells in the PVN, which project to the medulla and spinal cord, are involved in various physiological functions (e.g. sensory modulation and autonomic). In the present study, we examined OXT expression in the PVN, SON and spinal cord after chronic inflammation from adjuvant arthritis (AA). We used transgenic rats that express OXT and the monomeric red fluorescent protein 1 (mRFP1) fusion gene to visualise both the magnocellular and parvocellular OXTergic pathways. OXT-mRFP1 fluorescence intensity was significantly increased in the PVN, SON, dorsal horn of the spinal cord and posterior pituitary in AA rats. The levels of OXT-mRFP1 mRNA were significantly increased in the PVN and SON of AA rats. These results suggested that OXT was up-regulated in both hypothalamic magnocellular neurosecretory cells and parvocellular cells by chronic inflammation, and also that OXT in the PVN-spinal pathway may be involved in sensory modulation. OXT-mRFP1 transgenic rats are a very useful model for visualising the OXTergic pathways from vesicles in a single cell to terminals in in vitro preparations.

Response of arginine vasopressin-enhanced green fluorescent protein fusion gene in the hypothalamus of adjuvant-induced arthritic rats.

Arginine vasopressin (AVP) and corticotrophin-releasing hormone (CRH) in the parvocellular neurosecretory cells of the paraventricular nucleus (PVN) play a major role in activating the hypothalamic-pituitary-adrenal axis, which is the main neuroendocrine response against the many kinds of stress. We examined the effects of chronic inflammatory/nociceptive stress on the expression of the AVP-enhanced green fluorescent protein (eGFP) fusion gene in the hypothalamus, using the adjuvant arthritis (AA) model. To induce AA, the AVP-eGFP rats were intracutaneously injected heat-killed Mycobacterium butyricum (1 mg/rat) in paraffin liquid at the base of their tails. We measured AVP, oxytocin and corticosterone levels in plasma and changes in eGFP and CRH mRNA in the hypothalamus during the time course of AA development. Then, we examined eGFP fluorescence in the PVN, the supraoptic nucleus (SON), median eminence (ME) and posterior pituitary gland (PP) when AA was established. The plasma concentrations of AVP, oxytocin and corticosterone were significantly increased on days 15 and 22 in AA rats, without affecting the plasma osmolality and sodium. Although CRH mRNA levels in the PVN were significantly decreased, eGFP mRNA levels in the PVN and the SON were significantly increased on days 15 and 22 in AA rats. The eGFP fluorescence in the SON, the PVN, internal and external layers of the ME and PP was apparently increased in AA compared to control rats. These results suggest that the increases in the concentrations of ACTH and corticosterone in AA rats are induced by hypothalamic AVP, based on data from AVP-eGFP transgenic rats.

Oestrogen-induced vigorous mounting in female rats carrying hypothalamic knife cuts.

In ovariectomized Wistar rats, 1- or 2-mm wide knife cuts were placed in a coronal plane from the surface of the cortex to the floor of the cranial cavity to interrupt posterior efferents of the ventromedial nucleus of the hypothalamus (VMN). Sham-operated females had the same knife lowered to a depth of 7 mm. Ovariectomized nonoperated females were also used. After recovery, all rats received a single injection of 2 microg oestradiol benzoate, and were tested 48 h later for male and female sexual behaviour and partner preference. When placed with highly receptive stimulus females, the rats with the 2-mm cut showed a significantly higher incidence of mounting with ejaculatory thrusts than any other groups. When placed with stud males, 1-mm cut, as well as sham-operated females, had increased lordosis quotients. Similarly, both 1-mm and 2-mm cuts and sham operation enhanced the incidence of ear wiggling. Despite the display of a transsexual behaviour (i.e. vigorous mounting), all females with the cut showed heterosexual partner preference. Thus, the cut in the present study removed the inhibitory neural effect on mounting, which presumably descends from the VMN. In the absence of this inhibition, minute amounts of oestrogen sufficed to induce vigorous mounting. Sham operation in the present study appeared to interfere with certain inhibitory neural circuitry for lordosis.

Nutritional pharmacotherapy of chronic liver disease: from support of liver failure to prevention of liver cancer.

Many patients with liver cirrhosis are in a state of protein and energy malnutrition and require careful nutritional support. Our research has revealed that approximately 30% of the patients have protein-energy malnutrition, 40% protein malnutrition, and 10% energy malnutrition; 20% are in a normal nutritional state. Supplementation with branched-chain amino acids alleviates chronic liver failure, improves the protein nutritional state, and subsequently prolongs survival. In contrast, therapeutic modalities for energy malnutrition have not yet been fully elucidated and await further studies. Improved survival of the cirrhotic patients essentially brings a higher incidence of hepatocellular carcinoma (HCC). A synthetic analogue of vitamin A (acyclic retinoid or 4,5-dehydrogeranyl geranoic acid) prevents at least the development of second primary tumors after curative treatment of preceding HCC. The mechanism of this cancer chemo-prevention is clonal deletion of premalignant and latent malignant cells by the retinoid. We describe our clinical experiences with these two nutritional pharmacotherapies of chronic liver diseases and review their basic mechanisms.

[An autopsy case of interstitial pneumonia probably induced by Sho-saiko-to].

A 66-year-old woman had been treated for 3 years by her local physician with Sho-saiko-to for chronic hepatitis C virus (HCV) infection and liver cirrhosis. She was admitted to our hospital because of cough, fever, and infiltrative shadows on chest x-ray films. Sho-saiko-to-induced pneumonitis was diagnosed and steroid therapy started. Though a temporary improvement was observed, interstitial pneumonitis relapsed and the patient died of respiratory failure and liver dysfunction. Autopsy findings showed diffuse alveolar damage and honeycombing. Furthermore, reverse-transcriptase polymerase chain reaction techniques detected HCV-RNA in specimens of fibrotic lung tissue. For comparison, HCV-RNA was not histologically detected in lung tissue specimens from 4 control subjects who were positive for HCV antibodies but who did not have interstitial lung disease. It was speculated that the progression of interstitial pneumonia in the present case may have been caused by HCV in combination with Sho-saiko-to-induced lung injury.

Oral supplementation with branched-chain amino acids improves survival rate of rats with carbon tetrachloride-induced liver cirrhosis.

We investigated whether supplementation with branched-chain amino acids (BCAA) improves survival of rats with carbon tetrachloride (CCl4) -induced cirrhosis. Liver cirrhosis was induced in 40 male Sprague-Dawley rats by administering CCl4 for 15 weeks. Twenty rats each were then assigned to the control and BCAA group and fed a casein diet or a BCAA-supplemented casein diet, respectively, for an additional 17 weeks with repeated injections of CCl4. No significant difference occurred in either mean energy or nitrogen intake or in body or liver weight between the two groups. BCAA-supplementation significantly preserved plasma albumin concentrations (P < 0.05) and inhibited significantly the occurrence of ascites and hyperammonemia (P < 0.05). The survival rate was significantly higher in the BCAA group (P=0.03), while no significant difference was found in liver histology between the groups. These results suggest that BCAA improved survival of rats with CCl4-induced cirrhosis by preventing hypoalbuminemia and hyperammonemia without directly reducing hepatic necrosis and fibrosis.

Bisphosphonate tiludronate increases bone strength by improving mass and structure in established osteopenia after ovariectomy in rats.

We examined the mechanical properties of bone in ovariectomized rats treated with tiludronate. 186 Sprague-Dawley (SD) rats, 6 months of age, were assigned to 13 groups and were maintained for 3-9 months after surgery. Ovariectomy (ovx) groups were given tiludronate orally at the respective doses of 0 (vehicle), 12.5 (low), 25 (medium), and 50 (high) mg/kg body weight daily for 3 months beginning 3 months after surgery. Rats were killed at 0 (start), 3, 6, and 9 months. Whereas bone mineral density (BMD) values of the midfemur did not increase after ovx, the values in the sham-operated groups increased age-dependently. Bending moment to failure of the femur in the sham group was larger than that of the ovx control group at 9 months. In the ovx control groups, the ultimate compressive load values of the third lumbar body were reduced compared with those in the sham groups at 3 months and thereafter. Although serum osteocalcin levels were decreased in the medium- and high-dose tiludronate groups, both serum PTH and 1,25(OH)2D levels were increased only in the high-dose group. Femoral BMD, mechanical properties, and the cortical bone area were increased by the high dose at 9 months. Lumbar ultimate compressive load and the circumscribing cortical shell area in the high-dose group were increased at 6 months and thereafter. The trabecular number values were maintained at 6 and 9 months by the high dose. These data demonstrate that tiludronate administration increased the mechanical properties of bone by preserving the age-dependent increases in the cortical bone mass and three-dimensional structure of trabecular bone. These effects seemed to be due to reduced bone turnover by the agent.

Oral supplementation with branched-chain amino acids improves transthyretin turnover in rats with carbon tetrachloride-induced liver cirrhosis.

The hypothesis that dietary branched-chain amino acid (BCAA) supplementation improves the impaired protein turnover in male Donryu rats with carbon tetrachloride-induced liver cirrhosis was tested. We supplemented cirrhotic rats orally for 2 wk with BCAA solution [26.67 mg BCAA/(100 g body weight . d)], a conventional amino acid mixture [4.25 mg BCAA/(100 g body weight . d)] or saline and fed these three groups the AIN76 basal diet to have similar intakes of total energy and total nitrogen. Normal rats without liver cirrhosis were fed the basal diet similar to the above (noncirrhotic controls). After supplementation, rats were fed intravenous transthyretin (thyroxine-binding prealbumin) doubly labeled with 125I-tyramine-cellobiose and 131I. Kinetic indices including production rate of transthyretin were analyzed from plasma transthyretin disappearance curves. Tissue sites of transthyretin degradation were assayed using a trapped ligand technique by measuring 125I-tyramine-cellobiose levels. The production rate of transthyretin was significantly lower in cirrhotic rats supplemented with saline (mean 25.46 X 10(-3) . h(-1)) compared with noncirrhotic controls (45.08 X 10(-3) . h(-1)) (P < 0.05). This was corrected by supplementing cirrhotic rats with BCAA (37.05 X 10(-3) . h(-1), P < 0.05) but not with conventional amino acid mixture (22.49 X 10(-3) . h(-1)). The accelerated degradation of transthyretin in muscles of cirrhotic rats was improved by BCAA (P < 0.05). In conclusion, dietary supplementation with BCAA improves the impaired transthyretin turnover in rats with liver cirrhosis.

Total excision of a thalamic arteriovenous malformation using an orbito-fronto-malar approach: case report.

A 37-year-old woman was admitted to Osaka Neurological Institute after the sudden onset of left hemiplegia, hemihypesthesia, and ipsilateral hemianopia on February 4, 1992. Computed tomography (CT) disclosed the presence of hemorrhage in the right thalamus extending to the ipsilateral internal capsule. Cerebral angiography after CT scanning disclosed the presence of a cerebral arteriovenous malformation (AVM) fed by copsulothalamic and lateral geniculate body arteries originating from the right anterior choroidal artery. She was operated on with removal of the AVM using a right orbito-fronto-malar approach (OFM approach), which did not require transection of the cerebral parenchyma. The anterior choroidal artery could be followed distally from its origin and small feeding branches originating from the parent artery were easily identified, and the cerebral base could be examined in greater detail than with the conventional frontotemporal approach. The nidus could be excised in its entirety without difficulty. Postoperative angiography confirmed total excision of the AVM. She was transferred to another hospital for rehabilitation on April 13, 1992. Motor strength on the left side had improved to 3/5 by that time. The OFM approach appears to be potentially useful for the resection of inferolateral thalamic AVMs, because it does not require corticotomy and feeding branches can be identified and dealt with prior to other surgical manipulations.

Tofisopam, a new 2,3-benzodiazepine. Inhibition of changes induced by stress loading and hypothalamic stimulation.

Effects of tofisopam, a new 2,3-benzodiazepine compound, were investigated on the following: gastric ulceration, induced by water-immersion stress in normal rats and by immobilization stress in olfactory-bulbectomized (OB) rats; and propulsion of the small intestine caused by water-immersion stress in rats and autonomic responses to electrical stimulation of the hypothalamus in rabbits. In the latter, the results were compared with those of diazepam and gamma-oryzanol. Tofisopam (30 and 100 mg/kg, po) significantly inhibited the gastric ulceration induced by water-immersion stress in normal rats in a dose-dependent manner. Immobilization-stress loading increased the incidence and average index of gastric ulceration in OB rats, compared with nonstressed rats. Tofisopam (100 mg/kg, po) significantly inhibited the gastric ulceration induced by stress loading in OB rats. Water-immersion stress loading induced a significant increase in intestinal propulsion in rats. This increase was reversed to control levels by tofisopam (100 mg/kg, po). Tofisopam (1.0 mg/kg, iv, or 0.1 mg/kg by intracerebrospinal injection) inhibited the constriction of ear microvessels, the decrease in earlobe temperature, and mydriasis induced by electrical stimulation of the medial hypothalamic area in rabbits. However, diazepam and gamma-oryzanol failed to inhibit the autonomic responses to medial hypothalamic stimulation. From these results, it can be concluded that tofisopam restores the autonomic abnormality induced by stress loading possibly via intervention in the central autonomic area, i.e., the hypothalamus, by an action different from that of diazepam.

[Experimental study of the effects of bromelain on the sputum consistency in rabbits].

Effects of bromelain (BR) on rabbit sputum consistency were investigated in vitro and in vivo. On the sputum showing relatively low viscosity, BR and other enzymes such as serratiopeptidase (SP), the mixed preparation of pronase and pancreatin, and lysozyme exerted lowering effects; and the effect of BR was the most potent. However, bromhexine had virtually no effect. On the sputum showing relatively high viscosity, BR exerted more potent lowering effects on the viscosity and yield value of sputum than those of SP. Furthermore, 320,000 U/head BR and 120,000 U/head SP lowered the viscosity significantly and yield value of sputum in rabbits with oral administration for 3 days. The lowering effect on the yield value of BR was more potent than that of SP. BR also increased the sputum volume in rabbits. BR and SP showed tendencies to decrease the contents of acid glycoprotein and sialic acid in sputum. It can be considered that these results support the effectiveness of BR as an expectorant in clinical use.

Anti-inflammatory properties of a newly synthesized compound, 6-chloro-4-oxyimino-1-phenyl-1,2,3,4-tetrahydroquinoline (M-7074).

Anti-inflammatory properties of a newly synthetized compound, 6-chloro-4-oximino-1-phenyl-1,2,3,4-tetrahydroquinoline (M-7074), have been investigated. Anti-edema activities of M-7074 were more potent than those of phenylbutazone in carrageenin, bradykinin and mustard edema tests in rats. M-7074 showed an inhibitory effect on adjuvant arthritis, especially on the secondary inflammatory lesions in rats. Inhibitory effect of M-7074 on cotton-pellet granuloma formation was all but equal to that of phenylbutazone in rats. M-7074 also showed inhibitory effects on ultraviolet erythema in guinea-pigs and increased vascular permeability in mice, moderate analgesic activity in rats and mice, and antipyretic activity in rats. Furthermore, inhibitory effects of M-7074 on prostaglandin biosynthesis in guinea-pig lung homogenate and arachidonic acid-induced aggregation of rabbit platelets were fairly equal to those of indomethacin. However, M-7074 showed no effect on humoral nor cellular immunity in mice. M-7074 possessed no ulcerogenic activity in rats and mice, and LD50 value of M-7074 was 8.01 g/kg, p.o. in mice. These data indicate that M-7074 is a novel anti-inflammatory agent with large margin of safety.

[Effects of tofisopam on the physiological changes induced by stress loading and hypothalamic stimulation (author's transl)].

Effects of tofisopam on the gastric ulceration induced by immobilization stress in olfactory-bulbectomized rats, propulsion of the small intestine caused by water immersion-stress in rats and autonomic responses to electrical stimulation of the hypothalamus in rabbits were investigated. Immobilization stress loading of 16.5 hours each for 10 days caused the augmentation of incidence and average index of gastric ulceration in olfactory-bulbectomized rats, compared with non-treated rats. Tofisopam 100 mg/kg, p.o. significantly inhibited the gastric ulceration in olfactory-bulbectomized rats. Water immersion-stress loading for 2 hours caused a significant increase in propulsion of the small intestine in rats. This increase was reversed to control levels after the oral administration of tofisopam in a dose of 100 mg/kg. Tofisopam at dose of 1 mg/kg i.v. inhibited the contraction of ear microvessels, the decrease in earlobe temperature and the mydriasis induced by electrical stimulation of the medial hypothalamic area in rabbits, Moreover, these inhibitions were also shown by the intra-cerebrospinal injection of tofisopam at a dose of 0.1 mg/kg. From these results, it is concluded that tofisopam could restore the autonomic abnormality induced by stress-loading and exerts such effects by acting on the hypothalamus, an area of the brain, which regulates autonomic nervous functions.

[Anti-inflammatory actions of proteases, bromelain, trypsin and their mixed preparation (author's transl)].

Anti-inflammatory actions of proteases, bromelain (BR), trypsin (TR) and their mixed preparation (KT) were studied mainly in rabbits using various experimental test methods. Inhibitory action of edema formation induced by carrageenin was observed to be dose dependent with oral administrations of KT. This inhibitory action of KT was more remarkable than actions of BR and TR, suggesting a possible synergism between the latter two. Such action was also observed with non-steroidal anti-rheumatic drugs, phenylbutazone (PB), indomethacin and acetylsalicylic acid. Oral administration of KT exerted definite inhibition or a tendency toward inhibition against paw edema induced by dextran, histamine or egg albumin or skin edema induced by anti-rabbit serum and thermal stimulation. Furthermore, inhibition of vascular permeability increase induced by histamine and bradykinin as well as a tendency toward inhibition against protein exudation in CMC-pouch method were observed. On the other hand, contrary to PB, potent inhibitory action was not manifested in the persistent proliferative inflammation models, the granuloma formation induced formalin soaked filter paper and cotton pellet and the mustard edema. Therefore, it can be deduced that the inhibitory action of KT against edema formation may be dependent mainly on the inhibitory action of vascular permeability increase and the anti-inflammatory action may be specific for acute exudative inflammation.