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.

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.

Prednisona e meloxicam no tratamento de ratos submetidos ao trauma agudo da medula espinhal / Prednisone and meloxicam in the treatment of rats underwent to acute spinal cord injury

O objetivo deste estudo foi investigar o efeito da prednisona e do meloxicam na terapia de ratos submetidos ao modelo experimental de trauma agudo da medula espinhal, induzida pelo cateter de Fogarty 2Fr, mediante a avaliação dos parâmetros de estresse oxidativo, dos testes neurológicos e do exame histopatológico da medula espinhal. Foram utilizados 90 ratos Wistar, distribuídos em seis grupos, denominados controle salina ou GCS (n=15), controle prednisona ou GCP (n=15), controle meloxicam ou GCM (n=15), trauma mais salina ou GTS (n=15), trauma mais prednisona ou GTP (n=15) e trauma mais meloxicam GTM (n=15). Cada grupo foi redistribuído em três subgrupos de igual número, de acordo com o tempo de tratamento no pós-operatório de 24h, 72h e sete dias. Todos os grupos foram submetidos à laminectomia e, nos grupos GTS, GTM e GTP, após a exposição da medula espinhal, foi realizado o trauma medular compressivo, utilizando o cateter de Fogarty 2Fr. Os grupos GCS e GTS foram tratados com solução salina, os GSM e GTM receberam meloxicam e os GSP e GTP prednisona, sendo administrados pela via intraperitoneal. Em todos os ratos, foram avaliados os parâmetros de estresse oxidativo, testes neurológicos e exame histopatológico da medula espinhal. Os animais dos grupos GTS, GTM e GTP, nos diferentes tempos (24h, 72h e sete dias), tiveram pontuação zero na escala de Basso, Beattie e Bresnahan (BBB); no plano inclinado, permaneceram com pontuação três e perderam a percepção da dor profunda. Os grupos GTM e GTP apresentaram menor atividade da catalase e de níveis de TBARS, quando comparado ao grupo GTS. Foi constatada degeneração Walleriana e necrose da substância cinzenta de intensidades variáveis, não apresentando diferença entre os grupos submetidos ao trauma. O meloxicam e a prednisona apresentam possível efeito antioxidante, mas não impedem a necrose e a degeneração Walleriana da medula espinhal de ratos.

The aim of the study was investigate the use of the prednisone and meloxicam in treatment of rats underwent to the experimental model of acute spinal cord injury with 2Fr Fogarty catheter, with evaluation of the oxidative stress, neurological test and histopathological analysis of the spinal cord. Ninety rats were separated into six equal groups denominated saline control or SCG, prednisone control or PCG, meloxicam control or MCG, saline and injury or STG, prednisone and injury PTG and meloxicam and injury MTG. Each group was divide into three subgroups according to treatment time in the postoperative period of 24h, 72h and seven days. All the rats underwent laminectomy and in the groups STG, MTG and PTG, after exposure of the spinal cord it was performed a compressive spinal cord injury with a 2Fr Fogarty catheter. The SCG and STG were treated with saline, MSG and MTG, with meloxicam and PSG and PTG with prednisone. All rats were evaluated for oxidative stress, neurological tests and histopathology of the spinal cord. Neurological tests were performed with Basso, Beattie e Bresnahan score (BBB), inclined plane and deep pain 24 hours before and after surgery and repeated every 48 hours until the day of euthanasia. The groups STG, MTG and PTG in the different times were zero point in the BBB scale and three points in the inclined plane and absence of deep pain. MTG and PTG had lower catalase activity and TBARS levels when compared to the STG. In the histopathological analysis it was found Wallerian degeneration and necrosis of gray matter of intensity variation. Meloxicam and prednisone can exhibit antioxidant effect, but the necrosis and Wallerian degeneration were not stop in rats underwent to acute spinal cord injury.

Oxidative stress parameters in blood, liver, and kidney of diabetic rats treated with curcumin and/or insulin.

This study evaluated the effects of curcumin and/or insulin on antioxidant enzyme activity in blood, liver, and kidney, as well as on lipid peroxidation and delta aminolevulinic dehydratase (δ-ALA-D) activity, and a histopathological analysis of streptozotocin-induced diabetic rats. The animals were divided into six groups (n = 6): control/saline (C); control/curcumin (CCur); diabetic/saline (D); diabetic/insulin (DIns); diabetic/curcumin (DCur); and diabetic/insulin/curcumin (DInsCur). After 30 days of treatment with curcumin and/or insulin, the animals were sacrificed and the liver, kidney, and serum were used for experimental determinations. Results of histopathological analysis showed that the treatment with insulin ameliorate renal and hepatic lesions from both DIns and DInsCur groups. TBARS levels were significantly increased in serum, liver, and kidney in D group and the administration of curcumin and insulin prevented this increase in DIns and DCur groups. The activities of catalase (CAT), superoxide dismutase, and δ-ALA-D presented a significant decrease in the liver and kidney D group when compared to C group (P < 0.05). The animals treated with curcumin and insulin presented an increase of CAT activity, revealing a positive interaction between both substances. The treatments with curcumin or insulin prevented oxidative stress in blood, through modulation of enzymatic antioxidant defenses. These findings contributed to the comprehension that antioxidants from medicinal plants could be used as adjuvant in the treatment of this endocrinopathy and not as single therapy.

Caffeic acid treatment alters the extracellular adenine nucleotide hydrolysis in platelets and lymphocytes of adult rats.

This study evaluated the effects of caffeic acid on ectonucleotidase activities such as NTPDase (nucleoside triphosphate diphosphohydrolase), Ecto-NPP (nucleotide pyrophosphatase/phosphodiesterase), 5'-nucleotidase and adenosine deaminase (ADA) in platelets and lymphocytes of rats, as well as in the profile of platelet aggregation. Animals were divided into five groups: I (control); II (oil); III (caffeic acid 10 mg/kg); IV (caffeic acid 50 mg/kg); and V (caffeic acid 100 mg/kg). Animals were treated with caffeic acid diluted in oil for 30 days. In platelets, caffeic acid decreased the ATP hydrolysis and increased ADP hydrolysis in groups III, IV and V when compared to control (P<0.05). The 5'-nucleotidase activity was decreased, while E-NPP and ADA activities were increased in platelets of rats of groups III, IV and V (P<0.05). Caffeic acid reduced significantly the platelet aggregation in the animals of groups III, IV and V in relation to group I (P<0.05). In lymphocytes, the NTPDase and ADA activities were increased in all groups treated with caffeic acid when compared to control (P<0.05). These findings demonstrated that the enzymes were altered in tissues by caffeic acid and this compound decreased the platelet aggregation suggesting that caffeic acid should be considered a potentially therapeutic agent in disorders related to the purinergic system.

Role of acute phase proteins in the immune response of rabbits infected with Trypanosoma evansi.

The aim of this study was to characterize the response of acute phase proteins (APP) in rabbits experimentally infected with Trypanosoma evansi (T. evansi), and to relate the findings with serum immunoglobulins levels, in order to verify the relation between APP and the immune response of rabbits. A total of 12 animals were used in this experiment and divided into 2 groups, control and infected, of six rabbits each. The experimental period was 118 days, and blood was collected on days 0, 5, 20, 35, 65, 95 and 118 post-infection (PI). The infection with T. evansi stimulated APP and immunoglobulins production, once the infected animals showed an increase in C-reactive protein, haptoglobin, alpha 2-macroglobulin and IgM levels. The elevation in IgM levels observed in this study, when related to the increase in C-reactive protein and haptoglobin levels, suggests the involvement of these proteins in host defense against flagellated protozoa, with possible participation in the control of the parasitemia in rabbits infected with T. evansi.

Parasitismo por Calodium hepaticum em dois cães / Calodium hepaticum parasitism in two dogs

Calodium hepaticum já foi relatado parasitando o parênquima hepático de diversas espécies de mamíferos, porém é infrequente em cães. O presente artigo tem o objetivo de descrever dois casos de capilariose hepática em cães, diagnosticados em um período de um mês na região de Santa Maria, Rio Grande do Sul, Brasil. O primeiro cão apresentava sintomatologia clínica de insuficiência cardíaca, e ovos de Calodium hepaticum foram encontrados ocasionalmente no exame histopatológico do fígado. O segundo animal apresentava mucosas ictéricas e ascite. No hemograma, ficou evidente a presença de anemia arregenerativa e, na avaliação de bioquímica sérica, percebeu-se aumento de fosfatase alcalina, alanina aminotransferase e hipoalbuminemia. A análise do liquido cavitário foi compatível com transudato modificado. No exame histopatológico, foram observados numerosos ovos bioperculados de Calodium hepaticum, dispostos aleatoriamente pelo parênquima hepático, com reação granulomatosa e fibrosa adjacente aos ovos. A existência de maior número de cães infectados demonstra a necessidade de controle da população de roedores.

Calodium hepaticum has been reported as a parasite of the hepatic parenchyma of many mammal species, but it is not frequent in dogs. The present article aimed to describe two cases of hepatic capillariasis in dogs, diagnosed in a period of one month, in e Santa Maria city, Rio Grande do Sul state, Brazil . The first dog presented clinical sintomathology of cardiac insufficiency and Calodium hepaticum eggs were occasionally found by histopathological examination of the liver. The second animal presented icteric mucous membranes and ascites. The hemogram revealed non-regenerative anaemia and in the biochemistry serum were observed elevation of alkaline phosphatase, alanina aminotransferase and hypoalbuminemia. The cavitary liquid analysis was compatible with modified transudate. In the histopathological examination, numerous bioperculated eggs of Calodium hepaticum were observed aleatorily distributed in the hepatic parenchyma, with granulomatous and fibrous reaction adjacent to the eggs. The occurrence of two cases in a short period of time suggests that other dogs may be infected and requires a rodent population control.