Diabetes'negative impact on radiculopathy induced by application of nucleus pulposus onto DRG in spontaneous diabetic rats

博士（医学）福島県立医科大

Environmental Occurrence of Polycyclic Aromatic Hydrocarbons and Nitro-Polycyclic Aromatic Hydrocarbons in Particulate Matters in Ho Chi Minh City, Viet Nam and Osaka, Japan

大阪府立大学Viet Nam National UniversityPromoting Environmental Pesearch in Pan-Japan Sea Area : Young Researchers\u27 Network, Schedule: March 8-10,2006,Kanazawa Excel Hotel Tokyu, Japan, Organized by: Kanazawa University 21st-Century COE Program, Environmental Monitoring and Prediction of Long- & Short- Term Dynamics of Pan-Japan Sea Area ; IICRC(Ishikawa International Cooperation Research Centre), Sponsors : Japan Sea Research ; UNU-IAS(United Nations University Institute of Advanced Studies)+Ishikawa Prefecture Government ; City of Kanazaw

p38 MAPK Facilitates Crosstalk Between Endoplasmic Reticulum Stress and IL-6 Release in the Intervertebral Disc

Degenerative disc disease is associated with increased expression of pro-inflammatory cytokines in the intervertebral disc (IVD). However, it is not completely clear how inflammation arises in the IVD and which cellular compartments are involved in this process. Recently, the endoplasmic reticulum (ER) has emerged as a possible modulator of inflammation in age-related disorders. In addition, ER stress has been associated with the microenvironment of degenerated IVDs. Therefore, the aim of this study was to analyze the effects of ER stress on inflammatory responses in degenerated human IVDs and associated molecular mechanisms. Gene expression of ER stress marker GRP78 and pro-inflammatory cytokines IL-6, IL-8, IL-1β, and TNF-α was analyzed in human surgical IVD samples (n = 51, Pfirrmann grade 2–5). The expression of GRP78 positively correlated with the degeneration grade in lumbar IVDs and IL-6, but not with IL-1β and TNF-α. Another set of human surgical IVD samples (n = 25) was used to prepare primary cell cultures. ER stress inducer thapsigargin (Tg, 100 and 500 nM) activated gene and protein expression of IL-6 and induced phosphorylation of p38 MAPK. Both inhibition of p38 MAPK by SB203580 (10 µM) and knockdown of ER stress effector CCAAT-enhancer-binding protein homologous protein (CHOP) reduced gene and protein expression of IL-6 in Tg-treated cells. Furthermore, the effects of an inflammatory microenvironment on ER stress were tested. TNF-α (5 and 10 ng/mL) did not activate ER stress, while IL-1β (5 and 10 ng/mL) activated gene and protein expression of GRP78, but did not influence [Ca2+]i flux and expression of CHOP, indicating that pro-inflammatory cytokines alone may not induce ER stress in vivo. This study showed that IL-6 release in the IVD can be initiated following ER stress and that ER stress mediates IL-6 release through p38 MAPK and CHOP. Therapeutic targeting of ER stress response may reduce the consequences of the harsh microenvironment in degenerated IVD

Expression and Activity of TRPA1 and TRPV1 in the Intervertebral Disc: Association with Inflammation and Matrix Remodeling

Transient receptor potential (TRP) channels have emerged as potential sensors and transducers of inflammatory pain. The aims of this study were to investigate (1) the expression of TRP channels in intervertebral disc (IVD) cells in normal and inflammatory conditions and (2) the function of Transient receptor potential ankyrin 1 (TRPA1) and Transient receptor potential vanilloid 1 (TRPV1) in IVD inflammation and matrix homeostasis. RT-qPCR was used to analyze human fetal, healthy, and degenerated IVD tissues for the gene expression of TRPA1 and TRPV1. The primary IVD cell cultures were stimulated with either interleukin-1 beta (IL-1β) or tumor necrosis factor alpha (TNF-α) alone or in combination with TRPA1/V1 agonist allyl isothiocyanate (AITC, 3 and 10 µM), followed by analysis of calcium flux and the expression of inflammation mediators (RT-qPCR/ELISA) and matrix constituents (RT-qPCR). The matrix structure and composition in caudal motion segments from TRPA1 and TRPV1 wild-type (WT) and knock-out (KO) mice was visualized by FAST staining. Gene expression of other TRP channels (A1, C1, C3, C6, V1, V2, V4, V6, M2, M7, M8) was also tested in cytokine-treated cells. TRPA1 was expressed in fetal IVD cells, 20% of degenerated IVDs, but not in healthy mature IVDs. TRPA1 expression was not detectable in untreated cells and it increased upon cytokine treatment, while TRPV1 was expressed and concomitantly reduced. In inflamed IVD cells, 10 µM AITC activated calcium flux, induced gene expression of IL-8, and reduced disintegrin and metalloproteinase with thrombospondin motifs 5 (ADAMTS5) and collagen 1A1, possibly via upregulated TRPA1. TRPA1 KO in mice was associated with signs of degeneration in the nucleus pulposus and the vertebral growth plate, whereas TRPV1 KO did not show profound changes. Cytokine treatment also affected the gene expression of TRPV2 (increase), TRPV4 (increase), and TRPC6 (decrease). TRPA1 might be expressed in developing IVD, downregulated during its maturation, and upregulated again in degenerative disc disease, participating in matrix homeostasis. However, follow-up studies with larger sample sizes are needed to fully elucidate the role of TRPA1 and other TRP channels in degenerative disc disease

Correlation study of observation data of polycyclic aromatic hydrocarbons and chemical transport model calculation data of other pollutants at Wajima

金沢大学大学院自然科学研究科場所：金沢大学自然科学研究科図書館棟1階，講演会場：図書館棟1階　大会議室，ポスター会場：図書館棟1階12会議室,主催・共催：文部科学省２１世紀COE「環日本海域の環境計測と長期・短期変動予測」， 大気環境学会， 金沢大学工学

Correlation study of observation data of polycyclic aromatic hydrocarbons and chemical transport model calculation data of other pollutants at Wajima

金沢大学大学院自然科学研究科場所：東京大学弥生講堂，共催：文部科学省２１世紀COE「環日本海域の環境計測と長期・短期変動予測」，大気環境学

Onycholyda sichuanica Shinohara, Naito and Huang 1988

Onycholyda sichuanica Shinohara, Naito and Huang, 1988 (Fig. 3) Onycholyda sichuanica Shinohara, Naito and Huang, 1988: 92; Xin and Wu, 2010: 10; Shinohara and Wei, 2012: 56. Onycholyda fanjingshanica Jiang, Wei and Zhu, 2004: 44; Shinohara and Wei, 2012: 56. N. syn. Distribution. China (Sichuan, Gansu, Guizhou, Guangxi, Hubei, Hunan, Anhui, Fujian). Host plants. Rubus spp. Field observations and rearing records. On June 8, 2013, Shinohara found seven leaf-rolls (four of them in Fig. 3 A–D), containing ten larvae, on Rubus spp. at Huatai, ca. 900 m, Mt. Hupingshan, Hunan Province. Of the seven leaf-rolls, one contained three larvae (three egg shells), one contained two larvae (one of them sample no. 486, two egg shells, Fig. 3D, E), while the other five contained one larva, respectively (sample nos. 480–482, 488, 489; for four of them, no egg shells were found, whereas five egg shells were found on the remaining one leaf, Fig. 3B, F). Of the ten larvae, one larva (sample no. 489) matured and was fixed in ethanol on June 9, two (sample nos. 481, 482) on June 10, two (sample nos. 480, 488) on June 11, one (sample no. 486) on June 12, while the remaining four were fixed in ethanol on June 14 before maturation. Six of the ten larvae were used for molecular analysis (Table 1). All the leaf-rolls and remains of the egg shells were found on the under surface of the leaves and all the egg shells were located on the lateral veins near the base of the leaf (Fig. 3 B–F). The larval abodes were irregular in shape (Fig. 3 A–D). Larva. Late instar (Fig. 3G, H): Head black; antenna creamy white, each antennomere more or less dark brownish; trunk pale greenish white, terminal segment largely creamy white; thoracic legs and subanal appendage white. Mature larva (Fig. 3I): Similar to the preceding, but antenna pale brown and trunk, including thoracic legs and subanal appendage, vivid pale green. Remarks. Shinohara and Wei (2012) suspected that O. sichuanica (only males known) and O. fanjingshanica (only females known) are opposite sexes of the same species, because the two “species” had much in common in morphology and were obtained together in the same area on Mt. Yunshan, Hunan Province. Our analysis based on mitochondrial COI+tRNA(Leu)+COII gene sequences perfectly supported the hypothesis that the two taxa are conspecific, and we hereby propose to treat them as synonyms. The adult specimens from Mt. Yunshan (sample nos. 763, 764, 779, 780) and larval material from Mt. Hupingshan (sample nos. 480–482, 486, 488, 489), both localities in Hunan Province, agreed in mitochondrial COI+tRNA(Leu)+COII gene sequences and we determine the larvae as O. sichuanica. This is the first record of the larvae and host plant of this sawfly. The previously unknown larva of this species is well characterized by the entirely black head and entirely pale green trunk in late-instar and prepupal stages (Fig. 3 G–I). It is quite similar to that of O. flavicostalis except for the entirely pale cervical sclerite (compare Fig. 3 G–I with Fig. 2D). The larva of the Japanese O. esakii also has a similar color pattern, but the cervical sclerite is black (Shinohara and Kojima 2009). The observed larvae of O. sichuanica were solitary or living together in a small group.Published as part of Shinohara, Akihiko, Kiyoshi, Takuya, Wei, Meicai & Kameda, Yuichi, 2018, Identification of Larvae and Recognition of New Synonymy for Two Chinese Onycholyda Sawflies (Insecta: Hymenoptera: Pamphiliidae) based on Molecular Data, pp. 75-81 in Species Diversity 23 (1) on page 80, DOI: 10.12782/specdiv.23.75, http://zenodo.org/record/458514

Onycholyda flavicostalis Shinohara, in Shinohara and Wei 2012

Onycholyda flavicostalis Shinohara, 2012 (Fig. 2) Onycholyda flavicostalis Shinohara, in Shinohara and Wei, 2012: 57. Distribution. China (Shaanxi, Hubei, Hunan, Sichuan). Host plants. Rubus adenophorus Rolfe., Rubus sp. Field observations and rearing records. Case A. One leaf-roll on Rubus adenophorus containing seven gregarious middle-instar larvae was found in Mt. Yunshan, Hunan Province on May 2, 2009. The leaf-roll was on the underside of the leaf and remains of the egg shell were not found. The larvae were fixed on May 8 (including sample nos. 783–785, Table 1). Case B. Nine leaf-rolls, one each containing one larva, were found on R. adenophorus at Yaolangou, Muyu, Shennongjia, Hubei Province, on May 18, 2010. All the nine larvae were fixed in ethanol on May 19 and 21 (one of them, sample no. 466). Another leaf-roll containing three larvae was also found on R. adenophorus at the same locality on the same day. The three larvae were fixed in ethanol on May 22 (sample nos. 470–472). For all the ten leaf-rolls examined, remains of the egg shells were not found. Case C. Two leaf-rolls were found on R. adenophorus in Qianjiaping, Shennongjia, Hubei Province on May 18, 2012. One of them (Fig. 2A) contained one larva and the other (Fig. 2B) contained two larvae. Each of the leaf-rolls was on the underside of the leaf and remains of the egg shell were not found. The larvae matured (one of them in Fig. 2C) and were fixed on May 22 (including sample nos. 775, 777). Case D. Two groups of eggs, consisting of five eggs and three eggs, respectively, were found on the same leaf of R. adenophorus in Guanmenshan, Shennongjia, Hubei Province on May 23, 2012. The groups of eggs were deposited on the lateral veins in a line in the basal part of the underside of a leaf (Fig. 2E, F). The eggs hatched on May 27 and the larvae were fixed on June 6 (one of them sample no. 781). Case E. One leaf-roll containing one larva was found on Rubus sp. along a narrow unpaved car road at about 1600 m in altitude about 32 km from Baoguosi on Mt. Emeishan, Sichuan Province, on May 14, 2015. The larva was fixed in ethanol on May 18 (sample no. 587). Egg. Normal for pamphiliids, yellowish white, deposited in a row along lateral veins in basal part of the leaf (Fig. 2E, F). Larva. Mature larva (Fig. 2C, D). Head black, with epicranial suture and part of clypeus greenish; antenna creamy white, each antennomere more or less dark brownish; labrum and mandible dark brown, palpi pale brown, and other mouthparts greenish, partly marked with blackish brown; trunk pale greenish, terminal segment largely creamy white; cervical sclerite partly blackish; thoracic legs and subanal appendage greenish white; suranal hook dark brown. Remarks. Our molecular analysis has clearly shown that the larvae feeding on Rubus adenophorus from Shennongjia, Hubei Province, and Mt. Yunshan, Hunan Province, and those feeding on Rubus sp. from Mt. Emeishan, Sichuan Province, belong to O. flavicostalis, which was described from Mt. Yunshan, Hunan Province. This is the first record of the host plant and larva of O. flavicostalis and the first distribution record of this sawfly from Hubei and Sichuan Provinces. Onycholyda flavicostalis and O. odaesana Shinohara and Byun, 1993, occur together on Mt. Yunshan in Hunan Province (Shinohara and Wei 2012) and the larvae of the two species feed on the same host plant, R. adenophorus (Shinohara and Wei 2010; present study). The larva of O. flavicostalis is a gregarious or solitary leaf-roller with a black head, whereas the larva of O. odaesana is solitary and has a pale brown head with three black spots. Among the non-Chinese species, the larva of O. esakii (Takeuchi, 1938) from Japan is a gregarious leaf-roller and has a color pattern very similar to that of O. flavicostalis (Shinohara and Kojima 2009). We are not able to distinguish the larvae of the two species based on a current knowledge besides the differences in distribution and the host plant species. The mature larva of O. sichuanica also has an entirely black head and a pale green trunk but the cervical sclerite is entirely pale in this species, unlike those of O. flavicostalis and O. esakii. As noted in the section of field observations above, the female of O. flavicostalis deposits eggs in a row (up to seven eggs together) and the middle-instar larvae live gregariously in one leaf-roll, whereas the late-instar larvae may live in a small group or even solitarily. The range of variation in the larval gregariousness of this species should be confirmed by obtaining further information.Published as part of Shinohara, Akihiko, Kiyoshi, Takuya, Wei, Meicai & Kameda, Yuichi, 2018, Identification of Larvae and Recognition of New Synonymy for Two Chinese Onycholyda Sawflies (Insecta: Hymenoptera: Pamphiliidae) based on Molecular Data, pp. 75-81 in Species Diversity 23 (1) on pages 77-80, DOI: 10.12782/specdiv.23.75, http://zenodo.org/record/458514

Measurement of mechanical withdrawal thresholds and gait analysis using the CatWalk method in a nucleus pulposus-applied rodent model

Abstract Background There are some previous reports of gait analysis using a rodent pain model. Applying the CatWalk method, objective measurements of pain-related behavior could be evaluated, but this method has not been investigated using the nucleus pulposus (NP) applied model, which was developed as a model of lumber disc herniation. We aimed to measure mechanical withdrawal thresholds and analyze gait patterns using the CatWalk method for the evaluation of the pain-related behavior caused by NP application. Methods Twenty-four nine-week-old female Sprague-Dawley rats were randomly divided into two experimental groups, the NP group (n = 12), in which autologous NP from the tail was applied to the left L5 dorsal root ganglion, and the sham-operated group (n = 12). Measurements of mechanical withdrawal thresholds were performed using von Frey filaments touching the left footpads, and gait analysis was performed using the CatWalk method. These experiments were conducted 1 day before surgery and 7, 14, 21, and 28 days after surgery. Data were statistically analyzed using the Wilcoxon rank-sum test. Results The NP group showed significantly lower withdrawal thresholds than the sham group at days 14 and 21. Stand (duration of contact of a paw with the glass plate) was significantly higher in the NP group at days 7 and 14, whereas step cycle (duration between two consecutive initial contacts of the same paw) and duty cycle (stand as a percentage of step cycle) were the same at day 7. Long initial dual stance (duration of ground contact for both hind paws simultaneously, but the first one in a step cycle of a target hind paw) of the right hind paw was measured at days 7 and 14. The left hind paw per right hind paw ratio of the stand index (speed at which the paw loses contact with the glass plate) and mean intensity (mean intensity of the complete paw) changed at day 7 or 14. Phase dispersion (parameter describing the temporal relationship between placement of two paws) of the hind paws decreased at day 7. Conclusions Rats with applied NP showed a decreased withdrawal threshold and abnormal gait. The differences in gait parameters between the NP and sham groups were observed at an earlier time point than the withdrawal thresholds. Gait analysis could be an effective method for understanding pain caused by applied NP