Ichiro Murai

Pathogenesis of idiopathic scoliosis. Experimental study in rats.

STUDY DESIGN A radiographic examination of pinealectomized rats to observe the development of scoliosis and halt the condition by administration of melatonin. OBJECTIVES To discover whether pinealectomy has the same effect in mammals as shown in the chicken, and to determine whether the bipedal condition is important for development of scoliosis. SUMMARY OF BACKGROUND DATA Pinealectomizing chickens shortly after hatching consistently resulted in scoliosis closely resembling human idiopathic scoliosis. It has not been determined whether this phenomenon is restricted solely to chickens, or if this experimental model is applicable to other animals, especially those more closely related to humans. METHODS A sham operation in five bipedal rats served as the control in this study. Pinealectomy was performed in 10 quadrupedal rats, pinealectomy in 20 bipedal rats, and pinealectomy with implantation of melatonin pellet in 10 bipedal rats. Spinal radiographs were used to measure the degree of scoliosis at 3 months after surgery. RESULTS Scoliosis developed only in pinealectomized bipedal rats and not in quadrupedal rats. It developed in none of the sham operation group and in only 1 of 10 pinealectomized bipedal rats with melatonin treatment. CONCLUSIONS Melatonin deficiency secondary to pinealectomy alone does not produce scoliosis if the quadrupedal condition is maintained. The bipedal condition, such as that in chickens or humans, plays an important role in the development of scoliosis. The findings suggest a critical influence of a postural mechanism for the development of scoliosis.

Long‐term melatonin administration reduces hyperinsulinemia and improves the altered fatty‐acid compositions in type 2 diabetic rats via the restoration of Δ‐5 desaturase activity

The objective of this study was to investigate the effect of long‐term melatonin administration on plasma levels of triglycerides, insulin and leptin, and on the fatty‐acid metabolism of plasma and hepatic lipids in type 2 diabetic rats. Otsuka Long‐Evans Tokushima Fatty (OLETF) rats, an animal model of type 2 diabetes mellitus, were divided into two groups: one untreated (n=6), and one implanted with time‐releasing melatonin pellets (1.1 mg/day for 30 wk) under the abdominal skin (n=6). Age‐matched Long‐Evans Tokushima Otsuka (LETO) rats (n=6) were used as healthy controls. The untreated diabetic rats had the increased plasma levels of triglycerides, cholesterol, insulin and leptin at 35 wk, as compared with the healthy control rats (n=6). The diabetic rats also had augmented ratios of 20:3n‐6/20:4n‐6 fatty acids, owing to diminished activity of Δ‐5 desaturase, an insulin‐permissive enzyme, in the liver. Melatonin administration to OLETF rats reduced the hypertriglyceridemia (−39%, P < 0.05), hyperinsulinemia (−33%, P < 0.01) and hyperleptinemia (–43%, P < 0.01), and restored hepatic Δ‐5 desaturase activity (148%, P < 0.005). This resulted in a return to normal ratios of 20:3n‐6/20:4n‐6 fatty acids in plasma and hepatic lipids. There was a significant correlation (r=0.64, P < 0.005) between plasma levels of insulin and the ratios of 20:3n‐6/20:4n‐6 in plasma phospholipids of all rats in the three groups. Thus, subcutaneous implantation of a melatonin‐releasing pellet thus resulted in improved lipid metabolism in diabetic rats, probably through restored insulin resistance.ALP, alkaline phosphataseGOT, glutamic oxalacetic transaminaseGPT, glutamic pyruvic transaminaseHPLC, high performance liquid chromatographyLETO rats, Long‐Evans Tokushima Otsuka ratsMUFA, monounsaturated fatty acidOLETF rats, Otsuka Long‐Evans Tokushima Fatty ratsPUFA, polyunsaturated fatty acidVLDL, very low density lipoprotein

Effect of pinealectomy on plasma levels of insulin and leptin and on hepatic lipids in type 2 diabetic rats

Abstract: We previously reported that pharmacological melatonin administration to type 2 diabetic rats reduces hyperinsulinemia and improves the altered fatty‐acid metabolism. To determine whether melatonin deficiency exacerbates diabetes‐associated conditions, we investigated the effect of pinealectomy (i.e. melatonin‐deficiency) on plasma hormone levels and lipid metabolism in type 2 diabetic Otsuka Long‐Evans Tokushima Fatty (OLETF) rats. We compared levels of insulin and leptin, and hepatic lipids in pinealectomized OLETF (PO) rats, sham‐operated OLETF (SO) rats and sham‐operated healthy Long‐Evans Tokushima Otsuka (LETO) (SL) rats 16 and 30 wk after the operation. Plasma glucose and triglycerides were increased in SO and PO rats 30 wk after operation compared with age‐matched SL rats. Pinealectomy caused an increase in free cholesterol among the plasma lipids, as compared with SO rats. Sixteen weeks after pinealectomy, typical hyperinsulinemia was observed in PO rats (3.47‐fold increase, P < 0.01) as compared with SL rats, whereas at 30 wk, the plasma levels of insulin in PO and SO rats had decreased and there was no significant difference among the three groups. Hepatic triglycerides were increased (1.54‐fold, P < 0.005) in PO rats, compared with SO rats. Hepatic acyl‐CoA synthetase (ACS) activity was significantly augmented in PO rats at 30 wk (10%, P < 0.01 versus SO group), while microsomal triglyceride transfer protein (MTP) decreased (−27% versus SO, P < 0.05); thus, the increased ACS activity and decreased MTP might have a role in the accumulation of hepatic triglycerides in PO rats. In summary, pinealectomy causes severe hyperinsulinemia and accumulation of triglycerides in the liver, probably owing to the loss of the nocturnal melatonin surge.

Role of serotonin for scoliotic deformity in pinealectomized chicken.

Study Design. The effect of intraperitoneal injection of 5-hydroxytryptophan (5-HTP) versus control in pinealectomized chickens. Objective. To find if the serotonin may have some role in the cause or treatment of idiopathic scoliosis. Summary of Background Data. One of the causes of idiopathic scoliosis is thought to be the disruption of postural reflex. Serotonin has been proposed to have a crucial role in maintaining normal postural muscle tone or postural equilibrium. Method. Forty pinealectomized chickens served as controls, and an additional 40 pinealectomized chickens received daily intraperitoneal injections of 5-hydroxy-tryptophan, a precursor of serotonin, which can pass through the blood-brain barrier. Spine radiographs were examined to measure the scoliotic deformity. Results. Scoliosis developed in all 40 pinealectomized chickens (control), whereas only 28 chickens in the 5-hydroxytryptophan-treated group (6 in severe, 22 in mild) had scoliosis developed. The remaining 12 chickens grew up with normal spines. Most chickens with mild scoliosis did not have curve progression but continued to have wedged vertebrae. Conclusion. Serotonin deficit secondary to a defect of melatonin may have disturbed postural muscle tone or postural equilibrium resulting in scoliosis in pinealectomized chicken. Prevention from the development of scoliosis or its progression in chickens treated with 5-hydroxytryptophan suggests that serotonin may have potential therapeutic value.

Pathologic mechanism of experimental scoliosis in pinealectomized chickens.

Study Design. This study was designed to investigate the pathologic mechanisms of idiopathic scoliosis using experimentally induced scoliosis in chickens. Objective. To understand the process of producing a scoliotic deformity in pinealectomized chickens. Summary of Background Data. Pinealectomy in chickens consistently produces scoliosis with anatomic characteristics similar to those of human idiopathic scoliosis. Pinealectomized chickens are an important animal model for the study of idiopathic scoliosis. Methods. In this study, 40 chickens were divided into two groups; 20 chickens treated with pinealectomy and 20 with a sham operation as control subjects on the second after hatching. The chickens in both groups then were killed at intervals ranging from 1 to 20 weeks after surgery. Their spines were examined visually and radiologically for the presence of a scoliotic curve and vertebral deformities. Results. Rotational lordoscoliosis developed in pinealectomized chickens. The chickens with severe scoliosis were characterized by apically wedge-shaped vertebrae. In contrast, no scoliosis with any vertebral deformity developed in any of the chickens that received a sham operation. Conclusions. Because there normally is evidence of lordosis in the thoracic spine of chickens, the rotational instability of the spine induced by pinealectomy may produce a scoliotic deformity as a secondary phenomenon. Pinealectomy in chickens consistently produces scoliosis with anatomic characteristics similar to those of human idiopathic scoliosis. The authors believe that disturbance of the equilibrium and the posture mechanism associated with a defect in melatonin synthesis after pinealectomy may promote the development of rotational lordoscoliosis.

Bipedal ambulation induces experimental scoliosis in C57BL/6J mice with reduced plasma and pineal melatonin levels.

Abstract:  In addition to the induction of scoliosis in chickens by pinealectomy (PINX), we previously demonstrated that removal of the pineal gland also produces scoliosis in bipedal rats, which can be inhibited by melatonin treatment. Using C57BL/6J mice with genetically low circulating melatonin levels, the main objective of the present study was to investigate whether bipedal ambulation in C57BL/6J mice has the same effects on spinal deformity as those seen in pinealectomized bipedal rats. The present study consisted of two phases. The aim of the first experiment was to determine whether the C57BL/6J mouse strain actually exhibits depressed plasma concentrations and/or pineal contents of melatonin during both the light and the dark phase of the light:dark cycle. The aims of the second experiment were to evaluate; (i) whether bipedal ambulation alone in the C57BL/6J mouse induces scoliosis, and (ii) whether PINX with bipedal ambulation in another mouse strain, i.e. C3H/HeJ, which normally exhibits diurnal fluctuations in melatonin synthesis and secretion, has effects similar to those of bipedal ambulation alone in C57BL/6J mice. C3H/HeJ mice, serving as controls, showed significant increases in both plasma concentrations and pineal contents of melatonin during the dark phase when compared with the light phase. In contrast, there were no differences in either circulating levels or pineal contents of melatonin between the light and dark phases in C57BL/6J mice. Moreover, plasma melatonin levels were below the detection limit of the assay in both phases and pineal melatonin was <10% of that in C3H/HeJ mice. Bipedal ambulation for 40 wk in C57BL/6J mice induced scoliosis at a rate of 64.3%, and two of nine scoliotic mice showed two sites of spinal deformity. This type of ambulation in C3H/HeJ mice resulted in scoliosis at a lower rate (25%), and affected animals had only a single scoliotic site. However, PINX combined with bipedal ambulation in C3H/HeJ mice produced scoliosis at a rate (70%) similar to that seen in C57BL/6J mice, and some double deformations were induced. These results confirm our previous observations in rats, and also support our hypothesis that melatonin as well as the bipedal ambulation appear to play a critical pathogenic role in scoliosis in experimental mammals.

Central effect of melatonin against stress-induced gastric ulcers in rats

WE investigated the role of melatonin in the induction of gastric lesions induced by water immersion restraint stress or centrally administered thyrotropin-releasing hormone (TRH). Melatonin (0.1–1 ng) injected intracisternally (i.c.) 30 min prior to stress dose-dependently inhibited the induction of gastric lesions by water immersion restraint stress, while 100 μg/kg, i.p. failed to protect the gastric mucosa. Preadministration of melatonin (1 ng, i.c.) significantly reduced (83%) the severity of gastric lesions induced by a TRH analogue (500 ng, i.c.). Serum melatonin concentrations 30 min after administration of 1 ng melatonin i.c. did not differ from those of rats receiving i.c. vehicle. These results suggest that melatonin plays a protective, anti-stress, role in the gastric mucosa via a mechanism involving the central nervous system.

Protective role of melatonin and the pineal gland in modulating water immersion restraint stress ulcer in rats.

We investigated the protective effect of melatonin on stress-induced gastric lesions in rats. Fasted rats were subjected to water immersion restraint stress for 4 h and the percentage of corpus mucosa containing hemorrhagic lesions was determined. Thirty minutes before restraint stress, melatonin or vehicle was administered i.p. In another experiment, pinealectomy was performed 1 week before water immersion restraint stress. Administration of melatonin at 1 and 5 mg/kg significantly decreased gastric lesions by 46 and 74%, respectively. In contrast, pinealectomy significantly enlarged the lesion area, although this effect was counteracted by melatonin at a dose of 1 mg/kg i.p. However, this protective effect of melatonin was abolished by i.p. pretreatment with indomethacin at 5 mg/kg. These results suggest that melatonin has gastroprotective properties against stress-induced gastric injury in rats and that the pineal gland contributes to gastric protection via prostaglandin-dependent mechanisms.

Roles of nocturnal melatonin and the pineal gland in modulation of water-immersion restraint stress-induced gastric mucosal lesions in rats.

The roles of melatonin and the pineal gland in the circadian variation of water‐immersion restraint stress‐induced gastric mucosal lesions in rats were investigated. Fasted rats were subjected to water‐immersion restraint stress during both the diurnal and nocturnal phases of a light:dark cycle. Pinealectomized and sham‐operated rats were also subjected to water‐immersion restraint stress at night. The lesion area after 4 hr of stress during the dark phase was significantly lower than in light‐phase controls. Pinealectomy increased the lesion area in the dark phase, compared to the sham operation, but this effect was counteracted by intracisternal melatonin preadministration at a dose of 100 ng/rat. Melatonin concentrations in control rats during the light phase were significantly increased 4 hr after water‐immersion restraint stress. In contrast, melatonin concentrations 4 hr after water‐immersion restraint stress in the dark phase were significantly depressed compared with the control levels at the corresponding time. Melatonin levels after stress exposure were markedly decreased in pinealectomized rats as compared with sham‐operated rats. These results suggest that circadian rhythm has an important role in the formation of stress‐induced gastric mucosal lesions in rats and that melatonin responses to water‐immersion restraint stress differ between day and night. The pineal gland modulates the stress response and melatonin contributes to gastric protection via a mechanism involving the central nervous system.

Circadian rhythm of melatonin and prostaglandin in modulation of stress-induced gastric mucosal lesions in rats.

We previously demonstrated the circadian variation of water‐immersion restraint stress (WRS)‐induced gastric mucosal lesions in rats.