上顎の形態発生におけるWnt signaling pathwayの役割

Cleft lip with or without cleft palate (CLP) usually results from a failure of the medial nasal prominences to fuse with the lateral and maxillary prominences. This failure inhibits facial morphogenesis regulated by several major morphogenetic signaling pathways. We hypothesized that CLP results from the failure of the Wnt signaling pathway. To examine whether Wnt signaling can influences upper jaw development, we applied beads soaked with Dickkopf-1 (Dkk-1), Alsterpaullone (AL) or Wnt3a to the right side of the maxillary prominence of the chick embryo. The embryo showed a defect of the maxilla on the treated side, and skeletal staining revealed hypoplasia of the premaxilla and palatine bone as a result of Dkk-1-soaked bead implantation. 5-bromo-2'-deoxyuridine (BrdU)-positive cell numbers in the treated maxillary prominence were significantly lower at both 24 and 48 hr after implantation. Down-regulation of the expression of Bmp4, Tbx22, Sox9, and Barx1 was confirmed in the maxillary prominence treated with Dkk-1, which indicated that the deformity of the maxillary bone was controlled by gene targets of the Wnt signaling pathway. Expression of N-cadherin was seen immunohistochemically in the maxillary prominences of embryos at 6 hr and increased at 24 hr after AL treatment. Wnt signaling enhanced by AL or Wnt3a up-regulated the expression levels of Msx1, Bmp4, Tbx22, Sox9, and Barx1. Our data suggest that the Wnt signaling pathway regulates maxillary morphogenesis and growth through Bmp4, Tbx22, Sox9, and Barx1. Wnt signaling might regulate N-cadherin expression via Msx1, resulting in cell aggregation for osteochondrogenesis.博士（医学）・乙第1430号・令和元年6月26日© 2019 The Japan Society of Histochemistry and CytochemistryCopyright: © 2019 The author. This is an open access article distributed under the Creative Commons Attribution License(https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, istribution, and reproduction in any medium, provided the original work is properly cited.J-STAGEへのリンク : http://dx.doi.org/10.1267/ahc.1803

Vestibular Compensation after Vestibular Dysfunction Induced by Arsanilic Acid in Mice

When vestibular function is lost, vestibular compensation works for the reacquisition of body balance. For the study of vestibular dysfunction and vestibular compensation, surgical or chemical labyrinthectomy has been performed in various animal species. In the present study, we performed chemical labyrinthectomy using arsanilic acid in mice and investigated the time course of vestibular compensation through behavioral observations and histological studies. The surgical procedures required only paracentesis and storage of 50 μL of p-arsanilic acid sodium salt solution in the tympanic cavity for 5 min. From behavioral observations, vestibular functions were worst at 2 days and recovered by 7 days after surgery. Spontaneous nystagmus appeared at 1 day after surgery with arsanilic acid and disappeared by 2 days. Histological studies revealed specific damage to the vestibular endorgans. In the ipsilateral spinal vestibular nucleus, the medial vestibular nucleus, and the contralateral prepositus hypoglossal nucleus, a substantial number of c-Fos-immunoreactive cells appeared by 1 day after surgery with arsanilic acid, with a maximum increase in number by 2 days and complete disappearance by 7 days. Taken together, these findings indicate that chemical labyrinthectomy with arsanilic acid and the subsequent observation of vestibular compensation is a useful strategy for elucidation of the molecular mechanisms underlying vestibular pathophysiologies

網膜へのレーザー凝固により惹起される脈絡膜新生血管はヘッジホッグシグナル系因子を発現する。

Choroidal neovascularization is one of the major pathological changes in age-related macular degeneration, which causes devastating blindness in the elderly population. The molecular mechanism of choroidal neovascularization has been under extensive investigation, but is still an open question. We focused on sonic hedgehog signaling, which is implicated in angiogenesis in various organs. Laser-induced injuries to the mouse retina were made to cause choroidal neovascularization. We examined gene expression of sonic hedgehog, its receptors (patched1, smoothened, cell adhesion molecule down-regulated by oncogenes (Cdon) and biregional Cdon-binding protein (Boc)) and downstream transcription factors (Gli1-3) using real-time RT-PCR. At seven days after injury, mRNAs for Patched1 and Gli1 were upregulated in response to injury, but displayed no upregulation in control retinas. Immunohistochemistry revealed that Patched1 and Gli1 proteins were localized to CD31-positive endothelial cells that cluster between the wounded retina and the pigment epithelium layer. Treatment with the hedgehog signaling inhibitor cyclopamine did not significantly decrease the size of the neovascularization areas, but the hedgehog agonist purmorphamine made the areas significantly larger than those in untreated retina. These results suggest that the hedgehog-signaling cascade may be a therapeutic target for age-related macular degeneration.博士（医学）・甲第653号・平成28年7月8日Copyright © 2016 The Japan Society of Histochemistry and Cytochemistry（日本組織細胞化学会）J-STAGEへのリンク：http://doi.org/10.1267/ahc.1503

アルサニル酸を用いたマウス内耳破壊後の前庭代償過程

When vestibular function is lost, vestibular compensation works for the reacquisition of body balance. For the study of vestibular dysfunction and vestibular compensation, surgical or chemical labyrinthectomy has been performed in various animal species. In the present study, we performed chemical labyrinthectomy using arsanilic acid in mice and investigated the time course of vestibular compensation through behavioral observations and histological studies. The surgical procedures required only paracentesis and storage of 50 µL of p-arsanilic acid sodium salt solution in the tympanic cavity for 5 min. From behavioral observations, vestibular functions were worst at 2 days and recovered by 7 days after surgery. Spontaneous nystagmus appeared at 1 day after surgery with arsanilic acid and disappeared by 2 days. Histological studies revealed specific damage to the vestibular endorgans. In the ipsilateral spinal vestibular nucleus, the medial vestibular nucleus, and the contralateral prepositus hypoglossal nucleus, a substantial number of c-Fos-immunoreactive cells appeared by 1 day after surgery with arsanilic acid, with a maximum increase in number by 2 days and complete disappearance by 7 days. Taken together, these findings indicate that chemical labyrinthectomy with arsanilic acid and the subsequent observation of vestibular compensation is a useful strategy for elucidation of the molecular mechanisms underlying vestibular pathophysiologies.博士（医学）・甲第742号・令和2年3月16日© 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/)

Cloning of a Putative Vesicle Transport-related Protein, RA410, from Cultured Rat Astrocytes and Its Expression in Ischemic Rat Brain

To elucidate the role of astrocytes in the stress response of the central nervous system to ischemia, early gene expression was evaluated in cultured rat astrocytes subjected to hypoxia/reoxygenation. Using differential display, a novel putative vesicle transport-related factor (RA410) was cloned from reoxygenated astrocytes. Analysis of the deduced amino acid sequence showed RA410 to be composed of domains common to vesicle transport-related proteins of the Sec1/Unc18 family, including Sly1p and Sec1p (yeast), Rop (Drosophila), Unc18 (Caenorhabditis elegans), and Munc18 (mammalian), suggesting its possible role in vesicular transport. Northern analysis of normal rat tissues showed the highest expression of RA410 transcripts in testis. When astrocyte cultures were subjected to a period of hypoxia followed by reoxygenation, induction of RA410 mRNA was observed within 15 min of reoxygenation, reaching a maximum by 60 min. At the start of reoxygenation, the addition of diphenyl iodonium, an NADPH oxidase inhibitor, blocked in parallel astrocyte generation of reactive oxygen intermediates and expression of RA410 message. In contrast, cycloheximide did not affect RA410 mRNA levels, indicating that RA410 is an immediate-early gene in the setting of reoxygenation. Using polyclonal antibody raised against an RA410-derived synthetic peptide, Western blotting of lysates from reoxygenated astrocytes displayed an immunoreactive band of ≈70 kDa, the expression of which followed induction of the mRNA. Fractionation of astrocyte lysates on sucrose gradients showed RA410 antigen to be predominantly in the plasma membrane. Immunoelectron microscopic analysis demonstrated RA410 in large vesicles associated with the Golgi, but not in the Golgi apparatus itself, consistent with its participation in post-Golgi transport. Consistent with thesein vitro data, RA410 expression was observed in rat brain astrocytes following transient occlusion of the middle cerebral artery. These data provide insight into a new protein (RA410) that participates in the ischemia-related stress response in astrocytes

Spared nerve injury後のマウス後根神経節におけるNGFとBDNFの発現

Neuropathic pain is initiated by a primary lesion in the peripheral nervous system and spoils quality of life. Neurotrophins play important roles in the development and transmission of neuropathic pain. There are conflicting reports that the dorsal root ganglion (DRG) in an injured nerve contribute to neuropathic pain, whereas several studies have highlighted the important contribution of the DRG in a non-injured nerve. Clarifying the role of neurotrophins in neuropathic pain is problematic because we cannot distinguish injured and intact neurons in most peripheral nerve injury models. In the present study, to elicit neuropathic pain, we used the spared nerve injury (SNI) model, in which injured DRG neurons are distinguishable from intact ones, and mechanical allodynia develops in the intact sural nerve skin territory. We examined nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) expression in the DRGs of SNI model mice. NGF and BDNF levels increased in the injured L3 DRG, while NGF decreased in the intact L5 DRG. These data offer a new point of view on the role of these neurotrophins in neuropathic pain induced by peripheral nerve injury.博士（医学）・甲第698号・平成31年3月15日© 2018 Elsevier B.V. All rights reserved