UTILIZAÇÃO DE CÉLULA-TRONCO MESENQUIMAL ASSOCIADA AO EXERCÍCIO COMO ABORDAGEM TERAPÊUTICA PARA OTIMIZAR A REGENERAÇÃO DE NERVO ISQUIÁTICO EM CAMUNDONGOS

Lesões nervosas periféricas (PNI) podem causar dor neuropática, perda de massa muscular e um longo tempo para reabilitação. A capacidade regenerativa e a recuperação funcional da dependem do grau da lesão, da idade e da distância entre o dano do nervo e o músculo-alvo. Principalmente, essa regeneração é pobre e limitada. O exercício é usado em ensaios clínicos para reduzir a atrofia muscular e estimular a plasticidade. A terapia com células-tronco mesenquimais (MSC) promove a produção de neurotrofinas, substratos e um microambiente ideal para o reparo de tecidos. Nós analisamos os efeitos regenerativos da terapia com MSC e exercícios de natação (SW) no modelo de compressão do nervo isquiático de camundongo. As MSC foram injetadas (300ul, 1x106, ip.) 7 dias após a lesão e o SW foi iniciado 14 dias após a lesão, realizado 3 vezes por semana durante 20 minutos. Foram estudados 4 grupos: DMEM (inoculação de meio de cultura), DMEM + SW (meio de cultura e exercício), MSC (apenas inoculação de células-tronco mesenquimais), MSC + SW (associação de exercício e terapia celular). A análise funcional (LSS e SFI) foi feita antes da cirurgia, um dia após e semanalmente durante 8 semanas, quando os animais foram sacrificados para avaliação morfológica (microscopia de luz e eletrônica). Nossos resultados mostraram que a terapia combinada com MSC e SW apresentou um maior número de fibras mielinizadas e não mielinizadas e um grande número de fibras dentro da razão G ideal. Os resultados mostraram maior sobrevida dos neurônios motores e sensoriais e melhor recuperação das funções motoras. Concluímos que o uso de células-tronco mesenquimais e a natação aceleram a regeneração axonal promovendo a recuperação precoce das funções motoras e sensoriais.

Peripheral nervous (PN) injuries can cause neuropathic pain, loss of muscle mass, and a long time to rehabilitation. The PN regenerative capacity and functional recovery are dependent on the degree of PN injuries, age, and the distance from the nerve damage to the target muscle. Mostly, this regeneration is poor and limited. Exercise is used in clinical trials to reduce muscle atrophy and stimulate plasticity. The mesenchymal stem cells (MSC) therapy combines regenerative and tissue replacement to promote neurotrophins production and substrates for regeneration. We analyzed the regenerative effects of MSC therapy and swimming exercise (SE) in the mouse sciatic nerve compression model. MSC was injected (300ul, 1x106,ip.) 7 days after injury. SE was started 14 days after injury, performed 3 times a week for 20 minutes. We studied 4 groups: DMEM (cell culture medium injected), DMEM+SW (DMEM and SE), MSC (mesenchymal stem cells injected), MSC+SW (combined SE and cell therapy). Functional analysis (LSS and SFI) was made before surgery, one day after, and weekly for 8 weeks when animals were euthanized for morphological assessment (light microscopy and electronica). Our results showed that combined MSC therapy and SE presented a higher number of myelinated and unmyelinated fibers and a large number of fibers within the ideal G-ratio. The results showed motor and sensory neurons survival and better recovery of motor and sensory functions. We conclude that the use of mesenchymal stem cells and swimming accelerates axonal regeneration promoting early recovery of motor and sensory functions.

Recuperação funcional de cães paraplégicos com doença do disco intervertebral toracolombar sem percepção à dor profunda submetidos ao tratamento cirúrgico: 15 casos (2006-2010) / Functional recovery in paraplegic dogs with thoracolumbar intervertebral disc disease without deep pain perception submitted to surgical treatment: 15 cases (2006-2010)

O objetivo deste estudo retrospectivo foi avaliar a recuperação funcional de cães paraplégicos sem percepção à dor profunda (PDP) com doença do disco intervertebral (DDIV) toracolombar submetidos à hemilaminectomia dorsolateral. Foram incluídos somente cães com DDIV entre os segmentos da medula espinhal T3 e L3, que estavam paraplégicos sem PDP submetidos à cirurgia descompressiva. Foi observada recuperação funcional satisfatória em 73,3% dos cães (n=11), sendo um, aos cinco dias, sete entre 15 e 30 dias e três acima de 30 dias do procedimento cirúrgico. A duração da perda da PDP antes da cirurgia em cinco cães recuperados foi entre 12 e 48 horas e, em seis cães, acima de 48 horas. Cães paraplégicos sem PDP em decorrência da DDIV toracolombar podem apresentar recuperação funcional satisfatória quando submetidos ao tratamento cirúrgico mesmo sem percepção a dor profunda com tempo superior a 48 horas. Futuras pesquisas serão necessárias para avaliar a eficiência do tratamento cirúrgico, principalmente para aqueles que perderam a PDP acima de 48 horas.(AU)

This retrospective study was to evaluate the functional recovery of paraplegic dogs without deep pain perception (DPP) with intervertebral disc disease (IVDD) submitted to dorsolateral hemilaminectomy. Only dogs with IVDD between spinal cord segments T3 and L3, which were paraplegic without DPP and were submitted to decompressive surgery were included in the study. Satisfactory functional recovery was observed in 73.3% of the dogs (n=11). Recovery time after surgery was one day (one dog), between 15 and 30 days (seven dogs) and over 30 days 30 days 3 dogs). The duration of the lack of DPP before surgery was 12-48 hours, in five recovered dogs and over 48 hours in six recovered dogs It can be concluded that paraplegic dogs with thoracolumbar IVD and lack of DPP may present satisfactory functional recovery when submitted to surgical treatment even when the absence of deep pain perception has settled for more than 48 hours. Further research is needed to better evaluate the effectiveness of surgical treatment, mainly for dogs with lack of DPP over 48 hours.(AU)

Recuperação funcional em cães com doença do disco intervertebral toracolombar sem percepção à dor profunda: 37 casos (2002-2010) / Functional recovery in dogs with thoracolumbar intervertebral disk disease without deep pain perception: 37 cases (2002-2010)

O objetivo deste trabalho foi avaliar a recuperação funcional de 37 cães com diagnóstico de doença do disco intervertebral (DDIV) toracolombar, sem percepção da dor profunda superior a 48 horas e não submetidos ao tratamento cirúrgico. Os dados identificados foram: raça, idade, sexo, localização da lesão, perda da percepção da dor profunda, duração dos sinais clínicos, recuperação funcional, retorno da percepção da dor profunda, recidivas, eutanásias ou morte. Foi observada recuperação funcional em 11 cães (55 por cento), sendo seis deles entre 30 e 60 dias após o início dos sinais clínicos. Dos onze cães que tiveram recuperação funcional satisfatória, dois (18 por cento) não tiveram retorno da percepção à dor profunda. Pode-se concluir que cães com diagnóstico de DDIV sem percepção à dor profunda superior a 48 horas e não submetidos ao tratamento cirúrgico podem apresentar recuperação funcional satisfatória e são necessários, no mínimo, 30 dias do início dos sinais clínicos para estabelecer um prognóstico quanto ao retorno dos movimentos voluntários.(AU)

The aim of this study was to evaluate functional recovery in 37 cases with diagnostic of thoracolumbar intervertebral disk disease in dogs without deep pain perception (more than 48 hours) and did not underwent surgical treatment from 2002 to 2010. The following data were obtained: Breed, age, sex, neuroanatomic localization, loss of deep pain perception, duration of clinical signs, functional recovery, deep pain recovery, recurrence and euthanasia or death. A satisfactory functional recovery was observed in 11 dogs (55 percent), mostly between 30-60 days after the beginning of the clinics signal (six dogs). Two of 11 dogs with satisfactory functional recovery did not recovered deep pain perception. The results showed that dogs with presumptive diagnoses of thoracolumbar intervertebral disk disease with more than 48 hours and that did not underwent surgical treatment are capable of a functional satisfactory recovery and should be waited 30 days after clinical signs begin to establish a prognosis on the recovery of voluntaries movements.(AU)

UTILIZAÇÃO DE CÉLULAS MONONUCLEARES DE MEDULA ÓSSEA PARA O TRATAMENTO DE SEQUELAS NEUROLÓGICAS DE CINOMOSE CANINA CURITIBA

A cinomose é uma doença infecciosa causada por um Morbillivirus pertencente a família Paramyxoviridae. Até 30% dos cães infectados com o vírus da cinomose apresentam complicações neurológicas e aproximadamente 10% morrem por leucoencefalite aguda. Os cães que recuperam podem apresentar sequelas permanentes. Até o momento, não existe nenhum tratamento específico para animais com sinais neurológicos da cinomose, assim, são necessárias novas intervenções terapêuticas. O transplante de células-tronco tem emergido como um novo e promissor modelo de terapia. Células mononucleares derivadas da medula óssea (CMNMO) podem ser isoladas e aplicadas como estratégia terapêutica em estudos clínicos e pré-clínicos. Porém, existem alguns desafios associados a essa abordagem terapêutica. Um deles é a falta de estudos sobre a caracterização das CMNMO dos cães. Heparina e citrato-fosfato-dextrose-adenina-1 (CPDA-1) são comumente utilizados para coletar medula óssea. Porém, o efeito destes anticoagulantes em termos de isolamento dessas células também não é conhecido. Outro ponto que precisa ser mais estudado é a via de administração para ação eficiente das células transplantadas. A via intravenosa (IV) tem sido utilizada porque é o método mais fácil e menos invasivo de transplante de células. A marc

Canine distemper is an infectious disease caused by a Morbillivirus belonging to Paramyxoviridae family. Up to 30% of dogs infected with canine distemper virus showed neurological complications and approximately 10% die from acute leukoencephalitis. In addition, dogs that recover may have permanent sequels. There is no specific therapy for animals showing distemper neurological signs, and new therapeutic interventions are necessary, and cell transplantation has emerged as a promising new therapy model. Bone marrow mononuclear cells (BMMNC) can be easily isolated and broadly applied as a therapeutic strategy in a large number of preclinical and clinical studies. However, there are some challenges associated with this therapeutic approach. One of them is the lack of studies on the characterization of canine BMMNC. Heparin and citrate-phosphate-dextrose-adenine-1 (CPDA-1) are commonly used to harvest bone marrow. However, the comparison of effect of anticoagulants at harvest on terms of stem cell yield has not been studied. Another point that needs to be further studied is the route of administration for efficient cell delivery. Intravenous (IV) delivery has been used because it is easier and less invasive method of cell transplantation. The labelling and tracking of cells allow to evaluate if IV transplanted cells are attracted to the site of injury. The PKH26 is a fluorescent labeling molecule, noncytotoxic and is stable for long time period. Because of these characteristics, this dye seems to be ideal for cells tracking. The characterization of subsets of BMMNC and a better understanding of the functions of these cells may contribute to the knowledge of their potential and thus increase the possibilities to their use for clinical trials in veterinary medicine. The BMMMC administered intravenously have a lung passage 30 times larger than the mesenchymal stem cells (MSC), facilitating the migration of these cells to the injured tissues. However, the migration of these cells to the central nervous system (CNS), after intravenous administration, in animals with canine distemper sequels has not been studied. The PKH is a fluorescent labelling molecule, which is incorporated in the lipid bilayer of the cytoplasmic membrane, which has been used for the identification of cell migration. After incorporated into the cells in vitro, the PKH is not transferred to the medium or to unmarked cells, and has no toxic or immunogenic effect. BMMNC can be labeled with PKH and used for transplant intravenously, so this dye appears to be ideal for monitoring the migration of these cells. The aims of this study were the comparison of the yield of bone marrow-derived mononuclear cells harvested from dogs with two different anticoagulants, evaluate cell migration after allogeneic BMMNC transplantation in 10 animals with neurological complications of canine distemper, characterize phenotypically canine BMMNC and, evaluate the safety and efficacy of allogeneic BMMNC transplantation for treatment of neurological sequels of canine distemper in others 23 dogs. For comparison of the anticoagulants, the bone marrow from five dogs was harvest in heparin or CPDA-1, isolated in a density gradient, and stained for CD9 and CD44 for characterization by flow cytometry. The means were compared using Students paired t-test. Samples harvested with CPDA-1 yielded an average of 5.16 x 106 (±1.76 x 106) to 20.20 x 106 (±1.55 x 106) mononuclear cells/mL, whereas the yield of samples harvested with heparin varied between 4.56 x 106 (±0.69 x 106) and 24.30 x 106 (±2.12 x 106) mononuclear cells/mL. By flow cytometry, the mean percentage of double-stained cells varied from 1.96% (±0.64%) to 5.01 % (±0.73%) for CPDA-1 and from 2.23% (±0.70%) to 7.27 % (±0.97%) for heparin. No significant statistical differences were observed on yield or CD9 and CD44 expression. Bone marrow was harvested from eight donors and BMMNC were isolated, labeled with PKH26 dye and IV transplanted into the patients, to assess the labelling and tracking of cells. The cell migration was assessed in the cerebrospinal fluid (CSF) of patients at different periods of time and the percentage of labeled cells with PKH 26 dye was evaluated quantitatively by flow cytometry and qualitatively by fluorescence microscope. In the quantitative analysis by flow cytometer, it was observed a wide variation in the percentage of labeled cells in different CSF collection times. Also, there was an increase on the percentage of PKH26-labeled cells in the CSF, with highest percentage between 4 and 5 ½ hours after infusion with subsequent decrease. In the qualitative analysis, it was showed the presence of labeled BMMNC recruited by central nervous system on CSF. In order to evaluate the safety and efficacy of allogeneic BMMNC transplantation for treatment of neurological sequels of canine distemper, it was used a single blind randomized controlled trial in 46 dogs divided into treatment group and control group with weekly follow-up for 35 days. Bone marrow was harvested from 23 healthy donors and BMMNC were isolated by density gradient centrifugation. BMMNC from four donors were labeled with anti-CD8a, CD9, CD14, CD29, CD34, CD44, CD45, and CD90 for phenotypic characterization. Dogs from the treatment group received 1 x 108 BMMNC intravenously. Regarding the safety of cell transplantation, no serious adverse events related to the procedure were recorded during the 35 days of the study. Functional recovery was evaluated according to the Olby scoring system with some modifications. For the treatment group, the median and interquartile range of the functional score, with 0, 7, 14, 21, 28 and 35 days after injection were 7 (4-13), 9 (5-14), 12 (6-15), 14 (6-15), 14 (6-16), and 14 (6-16) respectively, whereas for the placebo group they were 6 (4-12), 7 (4- 13), 7 (4-12), 7 (4-12), 6 (3-12), and 6 (3-12). The differences observed were statistically significant (p < 0.05), showing the effectiveness of BMMNC transplantation in dogs with distemper leukoencephalitis. In conclusion, this study demonstrates that BMMNC can be efficiently labeled with PKH26 dye and these cells can be monitored after IV transplantation in animals with neurological complications of canine distemper.

DEAMBULAÇÃO APÓS O USO DE APARELHOS DE FIXAÇÃO EXTERNA OU PINOS INTRAMEDULARES NA TÍBIA DE CÃES SADIOS

In this study we evaluate the contribution of external skeletal fixation devices and intramedullary pins in the functional recovery of healthy dogs. Sixteen healthy dogs have been divided into two experimental groups of 8 dogs each. The first group was submitted to external skeletal fixation devices and the second group was submitted to intramedullary pins in the left Tibia. The external skeletal fixation was taken out by the thirtieth day of the clinical evaluation. The dogs that were submitted to external fixation devices had functional recovery of the studied limb of 17.3 days (in average), while those submitted to intramedullary pins had the functional recovery of the limb in 4.2 days. We concluded that the intramedullary pins have less interference in the functional recovery of the affected limb when compared to the external skeletal fixation devices.

Avalia-se, clínica e comparativamente, o aparelho de fixação externa e pinos intramedulares, quanto ao tempo de recuperação das funções normais e deambulação de cães sadios. Foram utilizados 16 cães sadios separados em dois grupos experimentais de oito animais. No primeiro grupo, os cães foram submetidos à aplicação de um aparelho de fixação externa e, no segundo grupo, submetidos à inserção de dois pinos intramedulares na tíbia esquerda. Os aparelhos de fixação externa foram retirados após 30 dias de avaliação clínica. Observou-se que os cães com fixadores externos tiveram retorno funcional em média de 17,3 dias, enquanto os submetidos à inserção intramedular tiveram retorno aos 4,2 dias. Com esses resultados, conclui-se que o uso de pinos intramedulares tem menor interferência na deambulação em relação ao aparelho de fixação externa.

Deambulação após o uso de aparelhos de fixação externa ou pinos intramedulares na tíbia de cães sadios

In this study we evaluate the contribution of external skeletal fixation devices and intramedullary pins in the functional recovery of healthy dogs. Sixteen healthy dogs have been divided into two experimental groups of 8 dogs each. The first group was submitted to external skeletal fixation devices and the second group was submitted to intramedullary pins in the left Tibia. The external skeletal fixation was taken out by the thirtieth day of the clinical evaluation. The dogs that were submitted to external fixation devices had functional recovery of the studied limb of 17.3 days (in average), while those submitted to intramedullary pins had the functional recovery of the limb in 4.2 days. We concluded that the intramedullary pins have less interference in the functional recovery of the affected limb when compared to the external skeletal fixation devices.

Avalia-se, clínica e comparativamente, o aparelho de fixação externa e pinos intramedulares, quanto ao tempo de recuperação das funções normais e deambulação de cães sadios. Foram utilizados 16 cães sadios separados em dois grupos experimentais de oito animais. No primeiro grupo, os cães foram submetidos à aplicação de um aparelho de fixação externa e, no segundo grupo, submetidos à inserção de dois pinos intramedulares na tíbia esquerda. Os aparelhos de fixação externa foram retirados após 30 dias de avaliação clínica. Observou-se que os cães com fixadores externos tiveram retorno funcional em média de 17,3 dias, enquanto os submetidos à inserção intramedular tiveram retorno aos 4,2 dias. Com esses resultados, conclui-se que o uso de pinos intramedulares tem menor interferência na deambulação em relação ao aparelho de fixação externa.