Viability of Equine Chondrocytes After Exposure to Mepivacaine and Ropivacaine In Vitro.

Chondrocyte health is altered when exposed to local anesthetics, raising concerns as to the long-term effects of local anesthetics intra-articularly for diagnosis and analgesia. To investigate the drug with the lowest toxic potential, the effect of ropivacaine and mepivacaine on chondrocytes was evaluated. Articular cartilage from normal metacarpophalangeal joints of five equine cadaver specimens was used to establish chondrocyte cultures. Following seven days, chondrocytes were exposed to standard culture medium (DMEM), ropivacaine 7.5 mg/ml (ROP7.5), ropivacaine 10 mg/ml (ROP10), mepivacaine 20 mg/ml (MEP20), mepivacaine 30 mg/ml (MEP 30), and 0.9% saline solution (SAL). Chondrocyte viability was evaluated by trypan blue exclusion, MTT, and flow cytometry via cellular staining with propidium iodide. No differences were observed between treatments following trypan blue exclusion assay. A difference was observed between DMEM and all other treatment groups (P < .0001) with a significant viability drop using the MTT assay. Mepivacaine 20 mg/ml and MEP30 exposure between showed greatest decrease in cellular viability compared to SAL, ROP7.5, and ROP10 (P < .0001). Cellular viability decreased as measured by flow cytometry in all groups compared to DMEM and ROP7.5 (P < .02). Interestingly, the trypan blue, MTT, and flow cytometry assays yielded different results. Although there was no difference using trypan blue, MTT demonstrated that ropivacaine-treated cells had lower viability than DMEM, and cytometry found that ROP7.5 did not differ from DMEM. Results in vitro suggest that short-term exposure to ropivacaine may result in less chondrotoxicity than mepivacaine. In vivo studies are warranted investigating long-term effects of local anesthetics on equine articular cartilage.

Ultrasonographic, macroscopic and histological characterization of the proximal insertion of the suspensory ligament in Crioulo horses / Caracterização ultrassonográfica, macroscópica e histológica da inserção proximal do ligamento suspensório em cavalos Crioulos

Although ultrasound (US) is a routine diagnostic modality, it still presents limitations for the diagnosis of lesions such as those in the proximal insertion of the suspensory ligament (PISL) because of its composition, which includes muscle fibers and adipose tissue interspersed with the ligament fibers. The objective of the present study was to describe the ultrasonographic, macroscopic and histological aspects of the PISL of thoracic limbs (TL) and pelvic limbs (PL) in Crioulo horses (CH). We selected 34 specimens of TL (right and left) and 10 specimens of PL of horses with a mean age of 5.7 years, from a private clinic or sent to the Department of Veterinary Pathology of UFSM, which died from different causes. The animals had no previous history of lameness in selected limbs associated with PISL injuries. The 34 specimens of PISL of TL were divided into CH (n=25) and Thoroughbred horses (TBH) (n=9), which composed the control group, and 11 specimens of PISL of PL were divided into CH (n=8) and TBH (n=3), which also served as control. The US examination was performed in the PISL using a Sonosite Edge device, 5-10 MHz linear transducer, with cross-sectional and longitudinal palmaromedial and palmarolateral images of the proximal surface of metacarpus III, II and IV (MCIII/MCII/MCIV). In PL, the evaluation was performed four centimeters below the chestnut in the plantaromedial aspect of metatarsus III and II (MTIII/MTII). PISL lobulated shape and size were compared with those of the contralateral limb, as well as the regularity of the palmar bone surface of MC III, II and IV. Subsequently, dissection of the PISL lobes was performed, as well as its macroscopic evaluation, which preceded the histological processing of the samples. In specimens of the CH breed, PISL showed echogenicity varying from peripheral dorsal hyperechogenic zones that merge into echogenic and hypoechogenic zones, where lobulation occurs. In the samples from the TBH group, PISL was also lobulated, but with differences in the echogenicity pattern such as diffuse hypoechogenicity and echogenicity. Macroscopically, CH samples presented a large amount of adipose tissue that corresponds to the dorsal peripheral zone of PISL, which ends in the connective tissue that delimits the ligamentous lobes. On a macroscopic cross-section of PISL, muscle fibers in red are mixed with white ligament fibers in the center of the ligament. This macroscopic finding was not observed in TBH samples, in which muscle fibers overlap ligament fibers throughout the ligament extension and a small amount of fat is present in the dorsal periphery of the ligament. PISL of PL had a triangular shape with echogenicity characteristics very similar to those observed in TL. In ultrasonographic, macroscopic and histological evaluation, PISL samples of TL and PL in CH showed a larger amount of peripheral dorsal adipose tissue, as well as a larger number of merged ligament and muscle fibers compared with those in TBH.(AU)

Embora uma modalidade diagnóstica rotineira, a ultrassonografia ainda possui algumas limitações para o diagnóstico de lesões como as que afetam a inserção proximal do ligamento suspensório (IPLS). Uma dessas limitações é relacionada à composição desse ligamento que inclui fibras musculares e tecido adiposo intercalados entre as fibras ligamentares. O objetivo do presente trabalho foi descrever os aspectos ultrassonográficos (US), macroscópicos e histológicos da IPLS de membros torácicos (MT) e membros pélvicos (MP) de equinos da raça crioulo (CC). Foram selecionados 34 espécimes de MT (direito e esquerdo) e 10 espécimes de MP de equinos com idade média de 5,7 anos, que vieram a óbito por diferentes causas, oriundos de uma clínica privada ou destinados ao Laboratório de Patologia Veterinária da UFSM. Não havia histórico prévio de claudicações nos membros selecionados que pudessem estar relacionadas a lesões da IPLS. Os 34 espécimes da IPLS MT foram divididos pela raça CC (n=25) e Puro Sangue Inglês (PSI) (n=9), o qual serviu como grupo controle e, 11 espécimes da IPLS do MP divididos em raça CC (n=8) e PSI (n=3) também como grupo controle. O exame US foi realizado na IPLS com um aparelho Sonosite Edge, transdutor linear de 5-10 MHz, com imagens transversais e longitudinais palmaromedial e palmarolateral da face proximal do metacarpiano (MC) III, II e IV. No MP a avaliação foi realizada quatro centímetros abaixo da castanha no aspecto plantaromedial do metatarsiano III e II (MTIII / MTII). Foram também observadas à forma lobulada da IPLS e o tamanho em comparação ao membro contralateral, bem como a regularidade da superfície óssea palmar do MC III, II, IV. Posteriormente foi realizada a dissecação dos lobos IPLS, bem como a avaliação macroscópica dos mesmos que antecedeu o processamento das amostras para histologia. Em espécimes CC, a IPLS possui uma ecogenicidade que varia de zonas periféricas dorsais hiperecogênicas que se mesclam a zonas ecogênicas e hipoecogênicas onde ocorre a sua lobulação. Nas amostras do grupo PSI, a IPLS também é lobulada, mas com diferenças no padrão de ecogenicidade como, hipoecogenicidade e ecogenicidade difusas. Macroscopicamente, as amostras CC apresentaram uma grande quantidade de tecido adiposo que corresponde à zona periférica dorsal da IPLS, a qual termina no tecido conjuntivo que delimita os lobos ligamentares. Em uma secção transversal macroscópica da IPLS as fibras musculares em vermelho se mesclam as fibras ligamentares brancas no centro do ligamento. Este achado macroscópico não foi observado na raça PSI, onde as fibras musculares intercalam as fibras ligamentares em toda a extensão do ligamento e pequenas quantidades de gordura estão presentes na periferia dorsal do mesmo. A IPLS no MP possui um formato triangular com características de ecogenicidade muito similares as citadas no MT. Na avaliação US, macroscópica e histológica as amostras da IPLS em MT e MP de equinos na raça CC demonstraram uma maior quantidade de tecido adiposo dorsal periférico bem como, uma maior quantidade de fibras musculares e ligamentares mescladas em comparação às amostras PSI.(AU)

Evaluation of Three Methods of Platelet-Rich Plasma for Treatment of Equine Distal Limb Skin Wounds.

Wounds localized on the distal limbs in horses are difficulty healing. No studies have been conducted to evaluate homologous platelet-rich plasma (PRP) in horses. The aim of this study was to evaluate the effect upon applying autologous PRP, homologous PRP, and autologous PRP gel on the wound healing process of wounds on the distal limb of horses. To study a possible correlation between the platelet count and wound healing time, four skin wounds of 4 cm2 were surgically created on the dorsolateral aspect of the third metacarpus in eight healthy adult horses. Each skin wound was randomly treated according to the established treatment groups (G): GI, treated with autologous PRP injection on the wound edges; GII treated topically with autologous PRP gel; and GIII homologous PRP injection on the wound edges and control group treated with saline solution injected on the wound edges. The animals were monitored every fifteen days throughout the wound healing process. The wound size was determined using a tape measure. Skin biopsies were obtained on the 15th and 30th day after the first PRP treatment. Autologous PRP gel treatment reduced the healing time in 15 days compared to the wounds of the control group. Histologically, GII presented a higher frequency of mild inflammation and mild to moderate neovascularization of biopsies 1 and 2. Platelet-rich plasma in gel form showed the best result on wound healing of the distal limb of horses in both clinical and histopathological evaluations. The homologous PRP is recommended for the recovery of animals with compromised hemodynamic conditions.

Cooling and Cryopreservation of Equine Platelet-Rich Plasma With Dimethyl Sulfoxide and Trehalose.

Equine platelet-rich plasma (PRP) has been used in horses to repair bone, articular and tendinous lesions, laminitis, and even endometritis. However, platelets have a very limited lifespan, which makes it difficult to prepare and use PRP, except in loco. With the aim to produce PRP with higher platelet viability for clinical purposes, the effects of the cryoprotectants dimethyl sulfoxide (DMSO) and trehalose were evaluated on cooled (4°C) and cryopreserved (-196°C) equine PRP. The protocols of cooling and cryopreservation were performed independently, comparing the following treatments: fresh PRP, PRP + 6% DMSO, PRP + 300 mM of trehalose, and PRP only. The PRP samples were prepared by double centrifugation of the blood of six ponies, further divided into four aliquots. The cooled or cryopreserved aliquots were stored for 14 days. All samples were evaluated for the platelet count, the mean platelet volume, and the release of transforming growth factor beta 1 (TGF-ß1). The number of platelets in the fresh PRP and cooled samples was similar; however, platelet count was higher in the fresh PRP than in cryopreserved samples. The release of TGF-ß1 was higher in the fresh PRP (105891 ± 52398 pg/mL), but the stored samples still released significant amounts of this growth factor (27291 ± 9625 pg/mL).

Comparison of the effects of triamcinolone acetonide or platelet-rich plasma on expression of extracellular matrix-related genes in equine healthy chondrocytes in vitro / Comparação dos efeitos do acetonido de triancinolona ou plasma rico em plaquetas na expressão de genes relacionados à matriz extracelular de condrócitos in vitro de equinos adultos

ABSTRACT: In healthy cartilage, chondrocytes maintain an expression of collagens and proteoglycans and are sensitive to growth factors and cytokines that either enhance or reduce type II collagen synthesis. In osteoarthritis, pro-inflammatory cytokines, such as IL-6, induce overexpression of metalloproteinases (MMP) and decreasing synthesis of aggrecan. Use of chondroprotectors agents, such as Platelet-Rich Plasma (PRP) and triamcinolone (TA) are alternatives to reduce the progression of joint damage. In this study, we used chondrocytes extracted from metacarpophalangeal joints of healthy horses as the experimental model. Cells were treated in vitro with PRP or TA. No differences were observed between these treatments in comparison to the control group when the expressions of MMP9, MMP13, IL-6 and ACAN genes were evaluated (P<0.05). With these results, we can suggest that the treatments were not deleterious to equine cultured chondrocyte, once they did not stimulate MMPs and IL-6 synthesis or caused changes in ACAN.

RESUMO: Na cartilagem saudável, os condrócitos mantêm a expressão de colágenos e proteoglicanos, sendo sensíveis a fatores de crescimento e citocinas que aumentam ou reduzem a síntese de colágeno tipo II. Na osteoartrite, citocinas pró-inflamatórias, como a IL-6, estimulam a expressão de metaloproteinases (MMP) e reduzem a síntese de agrecano. O uso de condroprotetores, como o Plasma Rico em Plaquetas (PRP) e triancinolona (TA) é uma alternativa para se reduzir a progressão do dano articular. Neste estudo foram usados condrócitos extraídos das articulações metacarpofalangeanas de equinos saudáveis. As células foram tratadas in vitro com TA ou PRP. Não foram observadas diferenças entre os tratamentos comparando-se com o grupo controle quanto à expressão genética de MMP-9, MMP-13, IL-6 e ACAN (p<0,05). Assim, pode-se sugerir que os tratamentos não foram deletérios ao cultivo de condrócitos, uma vez que não estimularam a síntese de MMP e IL-6 e nem causaram alterações no ACAN.

Can Triamcinolone acetonide, platelet-rich plasma, and pentosan polysulfate sodium induce oxidative stress in cultured equine chondrocytes? / Acetonido de triancinolona, plasma rico em plaquetas e pentosano polissulfato sódico podem ocasionar estresse oxidativo em cultivo de condrócitos de equinos?

ABSTRACT: Progressive deterioration and loss of articular cartilage are the final degenerative events common to osteoarthritis (OA). Reactive oxygen species (ROS) play an important role in this chondrocyte catabolic activity, leading to cell death and matrix components breakdown. Intra-articular corticosteroid injections such as triamcinolone acetonide have been used to control pain and inflammation associated with OA. New treatments for OA, platelet-rich plasma and pentosan polysulphate sodium have also been used and further investigations are necessary to determine their safety in joint cells. In this in vitro study, the use of these three substances (triamcinolone acetonide, platelet-rich plasma, and pentosan polysulphate sodium) in healthy chondrocytes did not alter the antioxidant status when compared to control groups, indicating that they could be considered safe in healthy conditions.

RESUMO: A deterioração progressiva e perda da cartilagem articular são os eventos finais da osteoartrite (OA). Espécies reativas de oxigênio (ROS) têm papel importante na atividade catabólica de condrócitos, levando a morte celular e quebra dos componentes da matriz. Injeções intra-articulares de corticosteroides, como com o acetonido de triancinolona, são usadas para controle da dor e inflamação associadas à OA. Novos tratamentos para a OA, como o plasma rico em plaquetas e o pentosano polissulfato sódico, também tem sido utilizados e necessitam de maiores investigações para determinar sua segurança para as células articulares de equinos. Neste estudo in vitro, o uso destas três substâncias (acetonido de triancinolona, pentosan polissulfato de sódio de plasma rico em plaquetas) em condrócitos saudáveis de equinos não alterou o status antioxidante quando comparado aos grupos controle, indicando que puderam ser considerados seguros em condições saudáveis.

Aspectos ultrassonográficos, anatômicos e histológicos normais da articulação metacarpofalangeana equina / Normal ultrasonographic, anatomical and histological aspects of the equine metacarpophalangeal joint

O objetivo deste estudo foi descrever e caracterizar as estruturas que compõem a articulação metacarpofalangena equina por meio de análise ultrassonográfica, anatômica e histológica. Membros torácicos equinos (=70), obtidos em instalações frigoríficas, foram submetidos a exame ultrassonográfico post mortem. Destes, 30 membros apresentaram imagens ultrassonográficas consideradas sem alterações. Posteriormente foi realizada a dissecação dos mesmos e o estudo anátomo-histológico. Critérios como tamanho, forma, arquitetura e ecogenicidade foram observados a fim de caracterizar as imagens ultrassonográficas, anatômicas e histológicas normais das estruturas ligamentares, tendíneas, capsulares e cartilaginosas da articulação metacarpofalangeana equina.(AU)

The purpose of this study was to describe and characterize the equine metacarpophalangeal joint structures through ultrasonographic, anatomical and histological analysis. Seventy forelimb specimens were obtained from a slaughterhouse and submitted to ultrasonographic evaluation. Thirty specimens without ultrasonographic detectable lesions were selected for dissection and subsequent anatomical and histological evaluation. Criteria such as size, shape, architecture and echogenicity were observed in order to characterize normal ligaments, tendons, joint capsule and articular cartilage of the metacarpophalangeal joint.(AU)

Avaliação de sete protocolos para obtenção de plasma rico em plaquetas na espécie equina / Evaluation of seven platelet-rich plasma processing protocols in the equine species

O presente estudo teve por objetivo avaliar a capacidade de concentração plaquetária e sua correlação com os níveis do fator de crescimento TGF-B1, a presença de leucócitos e de hemácias nos diferentes protocolos utilizados na obtenção do plasma rico em plaquetas (PRP) de equinos, através do método manual. Dez equinos, sadios, com idade média de 7 anos (±2,39), pesando em média 500kg (±67,1) foram utilizados neste estudo. Os protocolos testados variaram na velocidade e no tempo nas duas centrifugações. As variáveis analisadas nas amostras de PRP foram: concentração de plaquetas, presença de leucócitos e hemácias, e níveis de TGF-β1 quantificados pelo teste ELISA. Os protocolos testados não diferiram na capacidade de concentração de plaquetas e nos níveis de TGF-β1. Entretanto, houve diferença significava entre o protocolo I e os demais por este apresentar maior número de hemácias e leucócitos nas amostras de PRP, sendo por esse motivo considerado um protocolo inadequado para processamento do volume de sangue utilizado. Os demais protocolos podem ser utilizados para obtenção de PRP terapêutico em equinos.

PRP is plasma that contains a high numbers of platelets and growth factors in a small volume. The aim of this study was to evaluate seven different protocols to obtain PRP by the manual method according to their capacity to concentrate platelets, leukocyte and erythrocyte contamination and correlation between platelet count and TGF-β1 growth factor levels in PRP samples. Ten healthy horses with a mean age of 7 years (±2.39), weighing on average 500 kg (±67.1) were used in this study. The protocols tested varied according to the speed and time used at the two centrifugations. PRP samples were analyzed regarding platelet concentration, leukocyte and erythrocyte contamination and TGF-β1 levels quantified by ELISA. No significant differences among protocols were observed regarding the ability to concentrate platelets and TGF-β1. However, protocol I showed significantly higher erythrocyte and leukocyte counts in PRP samples than the other protocols, reason why it was considered inadequate for the volume of blood processed in this experiment. The remaining protocols are suitable for extracting PRP.