- 著者
- Carol MWALE Takafumi SUNAGA Yanlin WANG Eugene C BWALYA H M Suranji WIJEKOON Sangho KIM Masahiro OKUMURA
- 出版者
- JAPANESE SOCIETY OF VETERINARY SCIENCE
- 雑誌
- Journal of Veterinary Medical Science (ISSN:09167250)
- 巻号頁・発行日
- vol.85, no.4, pp.515-522, 2023 (Released:2023-04-22)
- 参考文献数
- 42
- 被引用文献数
- 1
Bupivacaine, levobupivacaine and ropivacaine are potent, long acting, amide-type local anesthetics that have several clinical applications including intra-articular administration. The objectives of this study were to evaluate their in vitro effects on cell viability and caspase activity to elucidate whether they activate the extrinsic or intrinsic pathways of apoptosis in canine articular chondrocytes. Chondrocytes in monolayer culture were treated with culture medium as the control, or with 0.062% (0.62 mg/mL) bupivacaine, 0.062% levobupivacaine, and 0.062% ropivacaine for 24 hr. Cell viability was evaluated using the live/dead, 3-(4,5-dimehylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT), and Cell Counting Kit-8 (CCK-8) assays. Evaluation of caspase-3, caspase-8, and caspase-9 activity was performed using colorimetric assays. The MTT and CCK-8 assays were used to evaluate the effect of caspase inhibitors on local anesthetic chondrotoxicity. All three local anesthetics decreased chondrocyte viability after 24 hr (P<0.001). Apoptosis was induced through both the extrinsic and intrinsic pathways. Bupivacaine increased caspase-3, caspase-8, and caspase-9 activity (P<0.001). Levobupivacaine increased caspase-3 (P=0.03) while ropivacaine did not significantly upregulate activity for all three caspases. Caspase inhibition did not suppress bupivacaine chondrotoxicity whereas inhibition of caspase-8 and caspase-9 decreased ropivacaine chondrotoxicity and mildly attenuated levobupivacaine chondrotoxicity. In summary, the level of chondrotoxicity, the type of caspase activated, the level of caspase activation, and the response to caspase inhibitors was dependent on the type of local anesthetic. Therefore, ropivacaine may be a safer choice for intra-articular administration compared to levobupivacaine and bupivacaine.
- 著者
- Ekkapol AKARAPHUTIPORN Eugene C. BWALYA Sangho KIM Takafumi SUNAGA Ryosuke ECHIGO Masahiro OKUMURA
- 出版者
- JAPANESE SOCIETY OF VETERINARY SCIENCE
- 雑誌
- Journal of Veterinary Medical Science (ISSN:09167250)
- 巻号頁・発行日
- pp.20-0091, (Released:2020-07-07)
- 被引用文献数
- 3
Pentosan polysulfate (PPS) is a semi-synthetic sulfated polysaccharide compound which has been shown the benefits on therapeutic treatment for osteoarthritis (OA) and has been proposed as a disease modifying osteoarthritis drugs (DMOADs). This study investigated the effects of PPS on cell proliferation, particularly in cell cycle modulation and phenotype promotion of canine articular chondrocytes (AC). Canine AC were treated with PPS (0–80 µg/ml) for 24, 48 and 72 hr. The effect of PPS on cell viability, cell proliferation and cell cycle distribution were analyzed by MTT assay, DNA quantification and flow cytometry. Chondrocyte phenotype was analyzed by quantitative real-time PCR (qPCR) and glycosaminoglycan (GAG) quantification. PPS significantly reduced AC proliferation through cell cycle modulation particularly by maintaining a significantly higher proportion of chondrocytes in the G1 phase and a significantly lower proportion in the S phase of the cell cycle in a concentration- and time-dependent manner. While the proportion of chondrocytes in G1 phase corresponded with the significant downregulation of cyclin-dependent kinase (CDK) 1 and 4. Furthermore, the study confirms that PPS promotes a chondrogenic phenotype of AC through significant upregulation of collagen type II (Col2A1) mRNA and GAG synthesis. The effect of PPS on the inhibition of chondrocyte proliferation while promoting a chondrocyte phenotype could be beneficial in the early stages of OA treatment, which transient increase in proliferative activity of chondrocytes with subsequent phenotypic shift and less productive in an essential component of extracellular matrix (ECM) is observed.
- 著者
- Ekkapol AKARAPHUTIPORN Takafumi SUNAGA Eugene C. BWALYA Ryosuke ECHIGO Masahiro OKUMURA
- 出版者
- JAPANESE SOCIETY OF VETERINARY SCIENCE
- 雑誌
- Journal of Veterinary Medical Science (ISSN:09167250)
- 巻号頁・発行日
- pp.20-0118, (Released:2020-04-30)
- 被引用文献数
- 3
This study investigated the effects of culture time on phenotype stability of canine articular chondrocytes (CACs) in non-passaged long-term monolayer culture. Third passage (P3) CACs isolated from four cartilage samples were seeded at three different initial seeding densities (0.2 х 104, 1.0 х 104 and 5.0 х 104 cells/cm2) and maintained in monolayer condition up to 8 weeks without undergoing subculture after confluence. The characteristic changes of chondrocytes during the culture period were evaluated based on the cell morphology, cell proliferation, glycosaminoglycans (GAGs) content, DNA quantification, mRNA expression and ultrastructure of chondrocytes. Chondrocytes maintained under post-confluence condition exhibited a capability to grow and proliferate up to 4 weeks. Alcian blue staining and Dimethylmethylene blue (DMMB) assay revealed that the extracellular matrix (ECM) synthesis was increased in a time-dependent manner from 2 to 8 weeks. The chondrocyte mRNA expression profile was dramatically affected by prolonged culture time, with a significant downregulation of collagen type I, whereas the expression of collagen type II, aggrecan, Sox9 and matrix metalloproteinase 13 (MMP-13) were significantly upregulated. In addition, transmission electron microscopy (TEM) result indicated dilation of rough endoplasmic reticulum (RER) in these long-term monolayer cultured chondrocytes. These findings demonstrate that the chondrocytes phenotype could be partially redifferentiated through the spontaneous redifferentiation process in long-term cultures using standard culture medium without the addition of chondrogenic supplements or tissue-culture scaffolds.
- 著者
- Takafumi SUNAGA Namgil OH Kenji HOSOYA Satoshi TAKAGI Masahiro OKUMURA
- 出版者
- JAPANESE SOCIETY OF VETERINARY SCIENCE
- 雑誌
- Journal of Veterinary Medical Science (ISSN:09167250)
- 巻号頁・発行日
- vol.74, no.6, pp.745-750, 2012 (Released:2012-07-04)
- 参考文献数
- 31
- 被引用文献数
- 1 3
Canine osteoarthritis occurs frequently and causes secondary synovitis. Administration of nonsteroidal anti-inflammatory drugs (NSAIDs) is one of the major therapeutic options for pain management of joint diseases. Tepoxalin has an unique property as an NSAIDs that suppresses both cyclooxygenase and lipoxygenase. The purpose of this study was to evaluate antiproliferative effects of tepoxalin on cultured canine synovial cells. Cytotoxic effects of tepoxalin, carprofen, meloxicam and AA-861 on cultured canine synoviocytes were evaluated by MTT colorimetric assay. Apoptosis was detected by morphological observations with Giemsa or annexin V/Hoechst 33342 staining and by the inhibition of caspase-3 activity with N-Ac-Asp-Glu-Val-Asp-CHO (Ac-DEVD-CHO). Cytotoxic effects of tepoxalin were evident in comparison with the effects of carprofen or meloxicam. The same tendency of cytotoxicity was observed when 5-lipoxygenase was inhibited by AA-861. The morphological findings and contradictory effects of Ac-DEVD-CHO with regard to the cytotoxicity proved the proapoptotic effects of tepoxalin. In conclusion, tepoxalin might control osteoarthritic synovitis by inducing apoptosis in proliferating synoviocytes, while most NSAIDs that selectively inhibit cyclooxygenase-2 most likely would not suppress synovial proliferation.
- 著者
- Takafumi SUNAGA Namgil OH Kenji HOSOYA Satoshi TAKAGI Masahiro OKUMURA
- 出版者
- JAPANESE SOCIETY OF VETERINARY SCIENCE
- 雑誌
- Journal of Veterinary Medical Science (ISSN:09167250)
- 巻号頁・発行日
- pp.1201080756, (Released:2012-01-12)
- 被引用文献数
- 1 3
Canine osteoarthritis occurs frequently and causes secondary synovitis. Administration of non-steroidal anti-inflammatory drugs (NSAIDs) is one of major therapeutic options for pain management of joint diseases. Tepoxalin has an unique property as NSAIDs, which suppress both cyclooxygenase and lipoxygenase. The purpose of this study was to evaluate anti-proliferative effects of tepoxalin on cultured canine synovial cells. Cytotoxic effects of tepoxalin, carprofen, meloxicam and AA-861 on cultured canine synoviocytes were evaluated by MTT colorimetric assay. Apoptosis was detected by morphological observations with giemsa or annexin-V/hoechst 33342 staining, and by the inhibition of caspase-3 activity with N-Ac-Asp-Glu-Val-Asp-CHO (Ac-DEVD-CHO). Cytotoxic effects of tepoxalin were evident in comparison with those of carprofen or meloxicam. Same tendency of the cytotoxicity was observed when 5-lipoxigenase was inhibited by AA-861. Morphological findings and contradictory effects of Ac-DEVD-CHO to the cytotoxicity proved pro-apoptotic effects of tepoxalin. In conclusion, tepoxalin might control osteoarthritic synovitis by inducing apoptosis on proliferated synoviocytes, while most NSAIDs with selective inhibition of cyclooxygenase-2 most likely would not suppress synovial proliferation.