Effects of Blindness on Sleep/Wakefulness States in Mice.

To examine the effects of blindness on sleep/wakefulness states, we compared locomotor activity and delayed recovery from isoflurane anesthesia induced by hypnotics during light and dark periods in sighted CBA/N and blind CBA/J mice. Locomotor activity around the switch from the dark to light period significantly differed in both mice. Delayed recovery induced by brotizolam was attenuated in both periods in CBA/J mice. In addition, the period specificity of delayed recovery caused by suvorexant or diphenhydramine in CBA/N mice was abolished in CBA/J mice. These results suggest that blindness impairs sleep quality.

Influence of light-dark cycle on delayed recovery from isoflurane anesthesia induced by hypnotics in mice.

We previously reported that brotizolam, but not suvorexant, delayed recovery from isoflurane anesthesia in mice. However, the effects of hypnotics may be altered by the circadian rhythm. Locomotor activity was measured using sighted (ICR and C57BL/6J) and blind (FVB/N and C3H/HeN) mice, and the effects of hypnotics on isoflurane anesthesia were compared during the light and dark periods. In sighted mice, recovery induced by brotizolam was delayed in the light period, while that by suvorexant was delayed in the dark period. In C57BL/6J mice, delayed recovery induced by brotizolam was marked, and that by suvorexant was observed in the light and dark periods. Locomotor activity was low in the last 6 h of the dark period in blind mice, and was similar to that in the light period. In blind mice, delayed recovery induced by brotizolam was identical in both periods, while suvorexant did not influence recovery from isoflurane anesthesia. These results suggest that the effects of hypnotics on isoflurane anesthesia are altered by the circadian rhythm and that daily light-dark stimuli may be required for the chronopharmacological effects of hypnotics.

A simple method using anesthetics to test effects of sleep-inducing substances in mice.

We investigated the effects of sleep-inducing agents with different mechanisms of action on the loss of the righting reflex induced by isoflurane or a mixture of medetomidine, midazolam, and butorphanol (MMB), followed by atipamezole reversal. Chlorpromazine and brotizolam delayed recovery from both types of anesthesia, whereas the melatonin receptor agonist ramelteon had no effect. The orexin receptor antagonist suvorexant delayed recovery from anesthesia only in the case of MMB, while the sleep-promoting supplement glycine only delayed recovery in the case of isoflurane. These results suggest that the simple comparison method is applicable for testing substances expected to exert sleep-inducing effects.

Three patients with Schaaf-Yang syndrome exhibiting arthrogryposis and endocrinological abnormalities.

MAGEL2 is the paternally expressed gene within Prader-Willi syndrome critical region at 15q11.2. We encountered three individuals in whom truncating mutations of MAGEL2 were identified. Patients 1 and 2, siblings born to healthy, non-consanguineous Japanese parents, showed generalized hypotonia, lethargy, severe respiratory difficulty, poor feeding, and multiple anomalies including arthrogryposis soon after birth. We carried out whole-exome sequencing, which detected a MAGEL2 mutation (c.1912C>T, p.Gln638*, heterozygous). The patients' father was heterozygous for the mutation. Patient 3 was a female infant, showed respiratory difficulty reflecting pulmonary hypoplasia, generalized hypotonia, feeding difficulty and multiple anomalies soon after birth. Targeted next-generation sequencing detected a novel heterozygous mutation in MAGEL2 (c.3131C>A, p.Ser1044*). This mutation was not found in the parents. MAGEL2 mutations, first reported to be the cause of the Prader-Willi like syndrome with autism by Schaaf et al. (2013) Nature Genetics, 45: 1405-1408 show the wide range of phenotypic spectrum from lethal arthrogryposis multiplex congenital to autism spectrum disorder (ASD) and mild intellectual disability (ID). Our results indicate that MAGEL2 mutations cause multiple congenital anomalies and intellectual disability accompanied by arthrogryposis multiplex congenita and various endocrinologic abnormalities, supporting that the view that clinical phenotypes of MAGEL2 mutations are variable.

Oral Administration of Collagen Hydrolysates Improves Glucose Tolerance in Normal Mice Through GLP-1-Dependent and GLP-1-Independent Mechanisms.

The aim of this study was to evaluate the antidiabetic properties of collagen hydrolysates (CHs). CHs exhibited dipeptidyl peptidase-IV inhibitory activity and stimulated glucagon-like-peptide-1 (GLP-1) secretion in vitro. We also determined whether CHs improve glucose tolerance in normal mice. Oral administration of CHs suppressed the glycemic response during the oral and intraperitoneal glucose tolerance tests (OGTT and IPGTT), but the effects were weaker in IPGTT than in OGTT. CHs had no effect on the gastric emptying rate. A pretreatment with the GLP-1 receptor antagonist, exendin 9-39 (Ex9), partially reversed the glucose-lowering effects of CHs, but only when coadministered with glucose. CHs administered 45 min before the glucose load potentiated the glucose-stimulated insulin secretion. This potentiating effect on insulin secretion was not reversed by the pretreatment with Ex9, it appeared to be enhanced. These results suggest that CHs improve glucose tolerance by inhibiting intestinal glucose uptake and enhancing insulin secretion, and also demonstrated that GLP-1 was partially involved in the inhibition of glucose uptake, but not essential for the enhancement of insulin secretion.

Altered gene expression profiles associated with enhanced skin inflammation induced by 12-O-tetradecanoylphorbol-13-acetate in streptozotocin-diabetic mice.

To examine the mechanisms of diabetes-enhanced inflammation, ear inflammation was induced by 12-O-tetradecanoylphorbol-13-acetate (TPA) in streptozotocin (STZ)-injected diabetic and control mice. The inflammatory response was determined from ear thickness and histology. The mRNA expression of several inflammation-related genes 8, 24 and 32 h after TPA treatment was determined by quantitative real-time RT-PCR. Ear thickness did not differ between the two groups at 8 h, but was greater in the diabetic mice than control mice at 24 and 32 h (late phase). STZ-diabetic conditions variously affected TPA-induced gene expression. The changes 8 h after TPA treatment probably reflected transcriptional regulation, and the genes were divided into three groups, up-regulated (IL-6, MCP-1, HO-1 and SOCS3), unregulated (IL-1beta, TNF-alpha and IL-10) and down-regulated (RANTES) genes. TPA-induced gene expression of cytokines, except for RANTES, peaked at 8 h and significantly declined in the late phase in control mice, while the expression of IL-1beta and TNF-alpha did not decline in the late phase in the diabetic mice. This result indicated the destabilization process for these mRNA, a type of post-transcriptional regulation, to be impaired under STZ-induced diabetic conditions; however, TPA-induced gene and protein expression of TTP, an RNA-binding protein involved in mRNA decay, were adversely enhanced in the diabetic mice. These findings suggested that STZ-induced diabetes affected the transcriptional and post-transcriptional control of TPA-induced inflammation, and greater mRNA levels of IL-1beta and TNF-alpha in the late phase were probably responsible for the diabetes-enhanced inflammation.

Nephrotic syndrome complicated by renal and cerebral infarctions in a 14-year-old girl.

Venous thrombosis is a well-known complication of nephrotic syndrome (NS), while arterial thrombosis is rare. We know of no reports of children with this complication. Here we report a case of 14-year-old girl with NS, who complicated with renal and cerebral infarctions resulting from arterial thrombosis. Urinary examination showed heavy proteinuria. She had intravascular dehydration. Serum albumin was 0.9 g/dL. Contrast-enhanced computed tomography (CT) showed a low-attenuation area in the right kidney. Decreased blood flow in the right middle cerebral artery was observed on MRA and also on multi-detector-row head CT. Urokinase and heparin were given. Cerebral infarction was treated neuroprotectively by i.v. infusion of edaravone. Comprehensive assessment of intravascular dehydration and the coagulation-fibrinolysis system is needed to guide decisions concerning prophylactic anticoagulation therapy. Better understanding of NS and its risks, as well as the necessity of drug therapy, may help teenagers to accept and cooperate with treatment.

A child with PFAPA syndrome complicated by pityriasis lichenoides et varioliformis acuta.

We encountered a boy with periodic fever, aphthous-stomatitis, pharyngitis, adenitis syndrome, complicated by a papular rash representing pityriasis lichenoides et varioliformis acuta. Proinflammatory cytokines have been implicated in both diseases and may represent the underlying common immunologic mechanism causing the two diseases.

Involvement of mast cells in the regulation of matrix metalloproteinase-9 and tissue inhibitor of metalloproteases-1 in 12-O-tetradecanoylphorbolacetate-induced inflammation in mice.

In this study, we investigated the involvement of mast cells in the regulation of matrix metalloproteinase-9 (MMP-9) in 12-O-tetradecanoylphorbolacetate (TPA)-induced inflammation, using mast cell-deficient (W/W(v)) mice and control (+/+) mice. Topical application of TPA to the ears induced acute inflammation, accompanied by mast cell degranulation in +/+ mice, which peaked at 6-12 h. There was no significant difference in ear thickness between the groups until 12 h, but the swelling was greater in W/W(v) mice than +/+ mice at 24-36 h. Western blot analysis revealed that TPA-induced marked increases in levels of proMMP-9 and tissue inhibitor of metalloproteinases-1 (TIMP-1), which existed as complexes with proMMP-9. The amount of proMMP-9-TIMP-1 complex was markedly smaller in +/+ mice than W/W(v) mice at 6 and 24 h, but had almost returned to control levels in both groups at 48 h. The free form of proMMP-9 was also slightly less abundant in +/+ mice than W/W(v) mice at 6, 24, and 48 h. Gelatin zymographic analysis revealed that levels of the active species of MMP-9 (approximately 74 and 83 kD), as well as free form of proMMP-9, increased time-dependently after the application of TPA and peaked at 24 h in +/+ mice. The 74-kD band was detected only in +/+ mice at 6 h. Our results therefore suggested that during inflammation degranulation of mast cells results in a reduction of the proMMP-9-TIMP-1 complex levels, together with a fall in the amount of free proMMP-9.

Possible involvement of mast cells in collagen remodeling in the late phase of cutaneous wound healing in mice.

We examined possible roles of mast cells in cutaneous wound healing using mast cell deficient (W/Wv) mice and their normal littermates (+/+). A round full-thickness wound was made on the back skin of these mice. The wounds closed completely within 20 days, and there was no difference in wound contraction between +/+ and W/Wv mice during the wound healing. While either chymase or tryptase activities were hardly detectable in W/Wv mice, chymase activities decreased at the impaired sites and recovered to the control level within 20 days in +/+ mice. Tryptase activities were higher than the control level on day 15 and day 20 in +/+ mice. Histological observations on day 15 and day 20 in +/+ mice revealed that mast cells were abundant at the wound edges but absent at the center. The latent and the active forms of MMP-2 and MMP-9 increased on day 10 and day 15 but recovered nearly to control levels on day 20 in both mice groups. The hydroxyproline contents in W/Wv mice were significantly higher than those in +/+ mice on day 15 and day 20. Furthermore, histological observations revealed that the collagen aggregation at the wound edges was tighter and less interwoven in W/Wv mice compared with +/+ mice. These results suggest that mast cells accumulated at the wound edge may participate in tissue remodeling in the late phase of wound healing.

The role of chemical mediators in eosinophil infiltration in allergic rhinitis in mice.

The involvement of chemical mediators other than histamine in eosinophil infiltration in the nasal mucosa was studied using histamine H(1) receptor-deficient mice. Histamine H(1) receptor-deficient mice and wild-type controls were immunized with ovalbumin and consecutive topical antigen instillation was performed. Histological alterations and eosinophil infiltration into the nasal mucosa of mice were examined. Diffuse infiltration of inflammatory cells and edema after sensitization with antigen were observed in the nasal mucosa in both wild-type and histamine H(1) receptor-deficient mice. The number of eosinophils in the nasal mucosa in mice sensitized with antigen was significantly increased as compared with controls. The number of eosinophils in the nasal mucosa was significantly decreased by cetirizine and epinastine, ramatroban and zafirlukast in wild-type mice. Not only histamine but also thromboxane A(2) and leukotrienes play important roles in allergic rhinitis, especially in the late phase participating in nasal eosinophilia.

Possible role of mucosal mast cells in the recovery process of colitis induced by dextran sulfate sodium in rats.

To clarify the role of mucosal mast cells in the lesion sites of colitis induced by dextran sulfate sodium (DSS) in rats, we investigated the histological changes and alterations relevant to mucosal mast cells in the spontaneous recovery process of colitis. Oral administration of 4% DSS solution for 11 days resulted in surface epithelial loss, crypt loss and goblet cell depletion in the rectal mucosa. A marked infiltration of inflammatory cells into the mucosa, which was consistent with a significant increase in myeloperoxidase (MPO) activity, was observed. In addition, mucosal mast cell number and rat mast cell protease (RMCP) I and II levels in the rectum increased at day 0 after DSS treatment, and most of the mucosal mast cells were degranulated. After replacing 4% DSS solution with water, re-epithelialization and restoration of goblet cells were observed at day 5 and day 10, respectively, but crypt damage was hardly recovered even at day 20. The elevated myeloperoxidase activity was significantly decreased from day 5 after DSS treatment. The increased number of mucosal mast cells was further elevated up to about 1.5-fold at day 10 and day 20 after DSS treatment and little degranulation was observed. In the spontaneous recovery process, the increased rat mast cell protease II level in the rectum was maintained for 20 days, while the increased rat mast cell protease I level was gradually decreased and recovered to control level. These results suggest that proliferated mucosal mast cells remained for 20 days, although most of infiltrated inflammatory cells disappeared in spontaneous recovery process of colitis. It may therefore be presumed that proliferated mucosal mast cells play a role in spontaneous recovery process of the colitis induced by DSS.

Inhibitory effects of propolis granular A P C on 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone-induced lung tumorigenesis in A/J mice.

We examined the effect of propolis granular A. P. C on lung tumorigenesis in female A/J mice. Lung tumors were induced by the tobacco-specific carcinogen, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) administered in drinking water for 7 weeks in mice maintained on an AIN-76A semi-synthetic diet. Propolis granular A. P. C (100 mg/kg body wt.) was administered orally daily for 6 days/week from 1 week before NNK administration and throughout the experiment. Sixteen weeks after the NNK treatment, the mice were killed and the number of surface lung tumors was measured. The number of lung tumors in mice treated with NNK alone for 7 weeks (9.4 mg/mouse) was significantly more than in that observed in control mice. Propolis granular A. P. C significantly decreased the number of lung tumors induced by NNK. These results indicate that propolis granular A. P. C is effective in suppressing NNK-induced lung tumorigenesis in mice.