New insights into pain mechanisms through the study of genes associated with monogenic pain disorders

Pain is an intrinsic mechanism that promotes our survival by helping us to avoid injury. However, chronic pain remains a significant clinical burden and remains poorly treated. The development of new analgesic drugs may significantly improve quality of life for chronic pain patients. This thesis investigates the mechanisms of pain sensation and also suggests some novel analgesic drug targets by using molecular, genetic, and transgenic approaches. Firstly, a novel function of sodium channel Nav1.7 is explored. Microarray data showed that gene expression profiles are dramatically altered in dorsal root ganglia from Nav1.7 null mice. These changes were confirmed by real-time qRT-PCR. Altered expression of preproenkephalin (Penk) and carcinoembryonic antigen-related cell adhesion molecule 10 (Ceacam10) may contribute to the pain insensitive phenotype seen in Nav1.7 nulls. The gene expression changes were further explored using in vitro cell based assays, showing a potential role of sodium ions in controlling transcription of Penk. Secondly, we study a family with six members affected with a pain insensitive phenotype characterized by multiple painless bone fractures and frequent painless lesions caused by burning stimuli. A novel point mutation in ZFHX2, encoding a putative transcription factor expressed in small diameter sensory neurons, was identified. By analysing Zfhx2 knockout and BAC transgenic mice bearing the orthologous mutation, we confirm that ZFHX2 is crucial for normal pain perception. We study how the mutation disrupts ZFHX2 function, resulting in altered downstream expression of pain-related genes. Thirdly, a patient with small fibre neuropathy and erythromelalgia-like symptoms was genetically analysed. Using exome sequencing and detailed bioinformatics analyses, I have shortlisted three missense mutations in the genes CWC22, TMEM8B and ATL3 that are potentially pathogenic. By studying genes mutated in families with rare inherited pain disorders, this thesis broadens our understanding of pain sensation and highlights new routes to develop better analgesic drugs

A protocol for isolation and culturing of mouse primary postmitotic photoreceptors and isolation of extracellular vesicles

Here, we present a protocol for isolating and culturing mouse photoreceptors in a minimal, chemically-defined medium free from serum. We describe steps for retina dissection, enzymatic dissociation, photoreceptor enrichment, cell culture, extracellular vesicles (EVs) enrichment, and EV ultrastructural analysis. This protocol, which has been verified for cultured cells derived from multiple murine strains, allows for the study of several aspects of photoreceptor biology, including EV isolation, and cell-cell interactions such as nanotubes (NTs)

A phantom study investigating the relationship between ground-glass opacity visibility and physical detectability index in low-dose chest computed tomography

In this study, the relationship between ground-glass opacity (GGO) visibility and physical detectability index in low-dose computed tomography (LDCT) for lung cancer screening was investigated. An anthropomorphic chest phantom that included synthetic GGOs with CT numbers of -630 Hounsfield units (HU; high attenuation GGO: HGGO) and -800 HU (low attenuation GGO: LGGO), and three phantoms for physical measurements were employed. The phantoms were scanned using 12 CT systems located in 11 screening centers in Japan. The slice thicknesses and CT dose indices (CTDIvol) varied over 1.0-5.0 mm and 0.85-3.30 mGy, respectively, and several reconstruction kernels were used. Physical detectability index values were calculated from measurements of resolution, noise, and slice thickness properties for all image sets. Five radiologists and one thoracic surgeon, blind to one another\u27s observations, evaluated GGO visibility using a five-point scoring system. The physical detectability index correlated reasonably well with the GGO visibility (R2 = 0.709, p vol. Consequently, the CTDIvol also correlated reasonably well with the GGO visibility (R2 = 0.701, p vol was nearly dominant in the GGO visibility for image sets with different reconstruction kernels and slice thicknesses, used in this study

Arachidonic acid containing phosphatidylcholine increases due to microglial activation in ipsilateral spinal dorsal horn following spared sciatic nerve injury

Peripheral nerve injury induces substantial molecular changes in the somatosensory system that leads to maladaptive plasticity and cause neuropathic pain. Understanding the molecular pathways responsible for the development of neuropathic pain is essential to the development of novel rationally designed therapeutics. Although lipids make up to half of the dry weight of the spinal cord, their relation with the development of neuropathic pain is poorly understood. We aimed to elucidate the regulation of spinal lipids in response to neuropathic peripheral nerve injury in mice by utilizing matrix-assisted laser desorption/ionization imaging mass spectrometry, which allows visualization of lipid distribution within the cord. We found that arachidonic acid (AA) containing [PC(diacyl-16:0/20:4)+K]+ was increased temporarily at superficial ipsilateral dorsal horn seven days after spared nerve injury (SNI). The spatiotemporal changes in lipid concentration resembled microglia activation as defined by ionized calcium binding adaptor molecule 1 (Iba1) immunohistochemistry. Suppression of microglial function through minocycline administration resulted in attenuation of hypersensitivity and reduces [PC(diacyl-16:0/20:4)+K]+ elevation in the spinal dorsal horn. These data suggested that AA containing [PC(diacyl-16:0/20:4)+K]+ is related to hypersensitivity evoked by SNI and implicate microglial cell activation in this lipid production

A Clinical Evaluation of a Plastic Temporary Filling Material (Dura Seal®)

Recently Dura Seal, a kind of plastic, has been marketed as a temporary filling material. This clinical study was done to determine whether or not Dura Seal has the properties advertised by the manufacturer. A total of 135 teeth, including 110 vital teeth and 25 pulpless teeth, were prepared for metal inlay cavities. The prepared cavities were sealed with Dura Seal for an average of 12.0 days. Dura Seal was found to be easily removable, to show no pulp damage, and to have considerable mechanical strength

Effect of aspirin treatment on serum levels of lipoprotein (a) : analysis from the apolipoprotein (a) isoforms

We have found that aspirin lowers elevated serum lipoprotein(a) [Lp(a)] levels via reduction of the transcriptional activity of apolipoprotein(a) [apo(a)] gene with suppression of apo(a) mRNA expression. In the present study, we evaluated the effect of aspirin treatment on serum Lp(a) level and analyzed its relation to type of apo(a) isoform. Serum levels of Lp(a) were measured by turbidimetric immunoassay before and after the oral administration of aspirin therapy (81 mg/day) in 57 patients with coronary artery disease or cerebral infarction. Apo(a) isoforms were determined by immunoblotting method. In patients with high serum Lp(a) levels (more than 30 mg/dl), aspirin reduced serum Lp(a) levels to approximately 80 % of the baseline after one month. Their levels sustained significantly low even after six months. The effect of aspirin in reducing elevated serum Lp(a) levels were stronger in patients with smaller-sized type or double-band type of apo(a) isoforms. The transcriptional efficiency of apo(a) gene is thought to be increased in patients with these apo(a) isoforms. Therefore, these findings suggest that aspirin reduces apo(a) gene transcription preferentialy in patients with high transcriptional efficiency of this gene

Study of risk factors of the abutment screw fracture of titanium dental implant

【目的】チタン製インプラントは今日では欠損補綴治療の１つとして，信頼され普及し取り入れられてきている．インプラント治療は，プラークコントロールや咬合調整等の定期的なメインテナンスの継続が長期安定につながると考えられているが，長期間の使用に伴い偶発症が報告されている．申請者らは，上部構造装着後５年経過したインプラント補綴装置におけるアバットメントスクリューの頭部と首部の間での破折を経験した．アバットメントスクリュー破折の原因として，上部構造の適合不良，スクリューの緩みによる可動性の発現，強い咬合力などが考えられるとの報告はあるが詳細な原因について客観的に確証されていない．またインプラントが存在する口腔内環境は，常に唾液に囲まれており飲食物によって温度やpH が変動することによる腐食の可能性も考えられるが，その関連性も明らかではない．そこで申請者らは，チタン合金製アバットメントスクリュー破折の原因および詳細な物理的因子や環境因子について，破折したアバットメントスクリュー破断面の観察，三次元非線形有限要素法による応力解析，および口腔内環境に類似させた腐食反応実験を行った．【材料と方法】１．破断面の観察および元素分析：走査型電子顕微鏡を用いてアバットメントスクリューの破断面のSEM 画像の観察とエネルギー分散型X 線分析（EDS）による元素分析を行った．２．三次元非線形有限要素法による応力解析：破折インプラントと同一のインプラントを樹脂包埋し正中線にて切断し寸法を測定して解析モデルを作製した．解析にはANSYSVer.13 を使用して非線形解析を行った．３．口腔内類似環境下における腐食反応：アバットメントスクリューと同じ組成である，Ti-６Al-４V のチタン合金板に耐力相当の曲げ応力を負荷し，Na2SO4 水溶液，生理的食塩水に浸漬し37℃恒温槽内において腐食試験を行った．標準試料として無負荷板のNa2SO4 水溶液の浸漬も行った．1 週間，2 週間，3 週間，4 週間浸漬後の試験片のSEM 観察とEDS による元素分析を行った．【結果】１．破断面の観察および元素分析：破断面には腐食孔と疲労破壊の様相が認められ，表面にはS などの付着物が検出された．２．三次元非線形有限要素法による応力解析：インプラントの上部構造物辺縁隆線部に垂直方向へ応力を負荷させた場合に，アバットメントスクリュー破折部位と同一部位に応力集中が認められた．３．口腔内類似環境下における腐食反応：すべての試験片で孔食などの腐食所見はみられなかった．浸漬４週間後の応力を負荷した試験片中央部と端部には，膜状の付着物が観察されたがS は検出されなかった．【考察】本症例は天然歯である下顎左側第１小臼歯が頬側に転位しており，下顎左側第２小臼歯の咬合面近心頬側にファセッテが認められる．これらのことから，左側側方運動時に下顎左側第２小臼歯に過度に咬合力の負担が繰り返し負荷され破折に至ったと考えられる．破折したアバットメントスクリューの破断面に孔食が見られ，またEDS 分析でSが存在した．三次元非線形有限要素解析の結果は，上部構造物の辺縁隆線部に垂直方向へ応力を負荷させた場合，アバットメントスクリュー頭部への応力が集中することが明らかになり、これが破折の原因と考えられた．また本症例で使用しているアバットメントスクリューの座面部の形状は，頭部の下丸みと呼ばれる形状が付与されておらず，応力が集中しやすい形となっており，その形態も破折原因の一つであると考えられた．本研究の腐食実験では，明らかな孔食および応力腐食割れ所見はなく，S も検出されなかったことより、破折面の孔食原因は硫化物によるものと予想されたが，その原因を特定することはできなかった．応力腐食割れが起こるための条件は「材料」・「環境」・「応力」の三要素が重複する特定の条件で発生することより，本腐食実験ではこの三要素の特定の条件が揃わなかったのではないかと考え、腐食については今後さらなる検討が必要であるとしている．2014博士（歯学）松本歯科大