Bilateral patellar agenesis in dog: A case report.

A bilateral patellar agenesis is an extremely rare congenital condition in which the patient does not develop both patellas and can present secondary alterations as bone, muscle and postural changes. There are some hypotheses that it has a genetic background presenting dominant characteristic. It is not yet standardized a gold treatment for this affection, but according to rare reports, clinical and surgical treatments are possible. This is a case report based on imagining exams of a mix breed male puppy that was born with bilateral patellar agenesis, an affection not yet reported in canine species.

Excess maternal and postnatal thyroxine alters chondrocyte numbers and the composition of the extracellular matrix of growth cartilage in rats.

Purpose/Aim: The aim of this study was to evaluate the effects of excess maternal and postnatal thyroxine on chondrocytes and the extracellular matrix (ECM) of growth cartilage. MATERIALS AND METHODS: We used 16 adult female Wistar rats divided into two groups: thyroxine treatment and control. From weaning to 40 days of age, offspring of the treated group (n = 8) received L-thyroxine. Plasma free T4 was measured. Histomorphometric analysis was performed on thyroids and femurs of all offspring. Alcian blue histochemical staining and real-time reverse transcription polymerase chain reaction measurements of gene expression levels of Sox9, Runx2, Aggrecan, Col I, Col II, Alkaline phosphatase, Mmp2, Mmp9, and Bmp2 were performed. Data were analyzed for statistical significance by student's t-test. RESULTS: Excess maternal and postnatal thyroxine reduced the intensity of Alcian blue staining, altered the number of chondrocytes in proliferative and hypertrophic zones in growth cartilage, and reduced the gene expression of Sox9, Mmp2, Mmp9, Col II, and Bmp2 in the growth cartilage of all offspring. Additionally, excess thyroxine altered the gene expression of Runx2, Aggrecan and Col I, and this effect was dependent on age. CONCLUSIONS: Excess thyroxine in neonates suppresses chondrocyte proliferation, stimulates chondrocyte hypertrophy and changes the ECM composition by reducing the amount of proteoglycans and glycosaminoglycans (GAGs). Prolonged exposure to excess thyroxine suppresses chondrocyte activity in general, with a severe reduction in the proteoglycan content of cartilage and the expression of gene transcripts essential for endochondral growth and characteristics of the chondrocyte phenotype.

Excess Maternal Thyroxine Alters the Proliferative Activity and Angiogenic Profile of Growth Cartilage of Rats at Birth and Weaning.

Objective The aim of this study was to unravel the mechanisms by which thyroxine affects skeletal growth by evaluating proliferative activity and angiogenic profile of growth cartilage of neonatal and weanling rats. Methods Sixteen adult Wistar rats were equally divided into 2 groups: control and treated with thyroxine during pregnancy and lactation. The weight, measurement of plasma free T4 and thyroids, femurs' histomorphometric analysis, and proliferative activity and angiogenic profile by immunohistochemical or real-time reverse transcriptase-polymerase chain reaction in growth cartilage was performed. Data were analyzed using Student's t test. Results The free T4 was significantly higher in the treated rats. However, the height of the follicular epithelium of the thyroid in newborns was significantly lower in the treated group. The excess maternal thyroxine significantly reduced the body weight and length of the femur in the offspring but significantly increased the thickness of trabecular bone and changed the height of the zones of the growth plate. Furthermore, excess maternal thyroxine reduced cell proliferation and vascular endothelial growth factor (VEGF) expression in the growth cartilage of newborn and 20-day-old rats ( P < 0.05). There was also a reduction in the immunohistochemical expression of Tie2 in the cartilaginous epiphysis of the newborns and FLK-1 in the articular cartilage of 20-day-old rats. No significant difference was observed in Ang2 expression. Conclusions The excess maternal thyroxine during pregnancy and lactation reduced endochondral bone growth in the progeny and reduced the proliferation rate and VEGF, Flk-1, and Tie2 expression in the cartilage of growing rats without altering the mRNA expression of Ang1 and Ang2.

Association of riluzole and dantrolene improves significant recovery after acute spinal cord injury in rats.

BACKGROUND CONTEXT: Damage to the spinal cord can result in irreversible impairment or complete loss of motor, sensory, and autonomic functions. Riluzole and dantrolene have been shown to provide neuroprotection by reducing neuronal apoptosis after brain and spinal cord injury (SCI) in several animal models of neurologic disorders. As these drugs protect the injured spinal cord through different mechanisms, we investigated the cumulative effects of riluzole and dantrolene. PURPOSE: This study aimed to investigate the neuroprotective efficacy of the combined administration of riluzole and dantrolene in experimental thoracic SCI. STUDY DESIGN: Twenty-nine Wistar rats were laminectomized at T12 and divided in five groups. Rats in GI (n=6) underwent laminectomy alone and were treated with placebo. Rats in GII (n=6) underwent laminectomy followed by SCI and were treated with placebo. Rats in GIII (n=5) underwent laminectomy followed by SCI and were treated with riluzole and placebo 15 minutes and 1 hour after laminectomy, respectively. Rats in GIV (n=6) underwent laminectomy followed by SCI and were treated with placebo and dantrolene 15 minutes and 1 hour after laminectomy, respectively. Rats in GV (n=6) underwent laminectomy followed by SCI and were treated with riluzole and dantrolene 15 minutes and 1 hour after laminectomy, respectively. A compressive trauma was performed to induce SCI. METHODS: Behavioral testing of hind limb function was performed using the Basso Beattie Bresnahan locomotor rating scale, which revealed significant recovery in the group treated with the association of riluzole and dantrolene compared with other groups. After euthanasia, the spinal cord was evaluated using light microscopy and immunochemistry with anti-NeuN and transferase dUTP nick-end-labeling (TUNEL) staining. RESULTS: Animals treated with the association of riluzole and dantrolene showed a larger number of NeuN-positive neurons adjacent to the epicenter of injury (p≤.05). Furthermore, the TUNEL staining was similar between animals treated with riluzole and dantrolene and those that did not receive spinal cord trauma (p>.05). CONCLUSIONS: These results showed that riluzole and dantrolene have a synergistic effect in neuroprotection after traumatic SCI by decreasing apoptotic cell death.

ω-conotoxin MVIIA intralesional injection in spinal cord injury in rats / Aplicação intralesional da ω-conotoxina MVIIA no trauma medular em ratos

This study aimed to investigate the neuroprotective effect of ω-conotoxin MVIIA (MVIIA) intralesional application in rats submitted to spinal cord injury. Male Wistar rats, weighing 300g±23.4, were distributed in five groups: negative control (SHAM), placebo (PLA), 5μM MVIIA, 10μM MVIIA and 20μM MVIIA MVIIA. After laminectomy of the 12th thoracic vertebra (SHAM), the PLA, 5μM MVIIA, 10μM MVIIA and 20μM MVIIA groups were subjected to acute compressive spinal cord trauma for five minutes, and then five minutes later, the animals received specific treatment in a standard total volume of 2µL, by intralesional route, using sterile PBS as placebo. Locomotor activity was assayed using Basso Beattie Bresnahan (BBB) scale to show the patterning of SCI. With 48 hours of injury, the animals were euthanized, the liquor sample was collected in atlantooccipital space, and also the spinal segment, including the epicenter and caudal region to injury. Assays were performed for mitochondrial viability, serum glutamate, production of reactive oxygen species (ROS) and lipid peroxidation (LP) were performed. The study design was randomized and the data submitted to ANOVA and comparison of means by SNK test, and data from BBB scale were evaluated using Kruskal-Wallis test (P<0.05). There was no significant difference between groups in BBB scores. The MVIIA did not promote decrease in the levels of glutamate, ROS, LP, and did not preserve the mitochondria in the intralesional application five minutes after spinal cord injury in rats.

Objetivou-se investigar o efeito neuroprotetor da aplicação intralesional da MVIIA em ratos submetidos ao trauma medular. Foram utilizados ratos Wistar, machos, com peso entre 300g±23.4, distribuídos em cinco grupos: controle negativo (SHAM), placebo (PLA), 5µM MVIIA, 10µM MVIIA e 20µM MVIIA. Após a laminectomia da vértebra torácica 12 (SHAM), os grupos PLA, 5µM MVIIA, 10µM MVIIA e 20µM MVIIA foram submetidos ao trauma medular agudo compressivo por cinco minutos e, cinco minutos após o trauma, receberam o tratamento específico em volume total padrão de 2µL, pela via intralesional, sendo utilizado como placebo o PBS estéril. A atividade locomotora foi avaliada pela escala proposta por Basso Beattie Bresnahan (BBB), com intuito de mostrar a padronização do trauma medular. Com 48 horas do trauma, os animais foram submetidos à eutanásia, coletou-se amostra do líquor no espaço atlantooccipital e um segmento medular, incluindo o epicentro e região caudal à lesão. Foram realizados ensaios de viabilidade mitocondrial, dosagem de glutamato, produção de espécies reativas de oxigênio (ERO) e peroxidação lipídica (PL). O delineamento do estudo foi inteiramente casualizado e os dados submetidos ao ANOVA, com comparação de médias pelo teste de SNK e os dados do teste BBB foram comparados utilizando o teste Kruskal-Wallis (P<0.05). Em relação aos escores do BBB, não houve diferença entre os grupos. A MVIIA não promoveu a diminuição dos níveis do glutamato, ERO, PL e não preservou a mitocôndria na aplicação intralesional, cinco minutos após o trauma medular em ratos.

Ischemia-reperfusion model in rat spinal cord: cell viability and apoptosis signaling study.

This work aimed at determining the ideal ischemia time in an in vitro ischemia-reperfusion model of spinal cord injury. Rat spinal cord slices were prepared and then exposed or not to oxygen deprivation and low glucose (ODLG) for 30, 45, 60, 75 and 90 minutes. Cell viability was assessed by triphenyltetrazolium (TTC), lactate dehydrogenase (LDH) release, and fluorochrome dyes specific for cell dead (ethidium homodimer) using the apotome system. Glutamate release was enzymatically measured by a fluorescent method. Gene expression of apoptotic factors was assessed by real time RT-PCR. Whereas spinal cord slices exposed to ODLG exhibited mild increase in fluorescence for 30 minutes after the insult, the 45, 60, 75 and 90 minutes caused a 2-fold increase. ODLG exposure for 45, 60, 75 or 90 minutes, glutamate and LDH release were significantly elevated. nNOS mRNA expression was overexpressed for 45 minutes and moderately increased for 60 minutes in ODLG groups. Bax/bcl-xl ratio, caspase 9 and caspase 3 mRNA expressions were significantly increased for 45 minutes of ODLG, but not for 30, 60, 75 and 90 minutes. Results showed that cell viability reduction in the spinal cord was dependent on ischemic time, resulting in glutamate and LDH release. ODLG for 45 minutes was adequate for gene expression evaluation of proteins and proteases involved in apoptosis pathways.

Perspectivas da aplicação das conotoxinas bloqueadoras de canais para cálcio dependentes de voltagem no trauma medular / Perspectives for the application of voltage-dependent calcium channels blockers conotoxin in spinal cord injury

O bloqueio dos canais para cálcio dependentes de voltagem é uma estratégia importante no tratamento do trauma medular, pois previne o influxo exacerbado do cálcio que participa ativamente em processos neurodegenerativos agudos, resultando em neuroproteção com melhora das funções neurológica. Dentre esses bloqueadores, as toxinas de caramujos marinhos são peptídeos com adequada estabilidade estrutural, estudadas pelas ações específicas em canais iônicos e receptores que interferem diretamente na liberação de neurotransmissores e na neuromodulação dos neurônios motores e sensitivos da medula espinal. Elas já são utilizadas no tratamento de desordens neurológicas e mostram-se promissoras no desenvolvimento de novas terapias para o trauma medular. Portanto, objetivou-se discorrer sobre a fisiopatologia do trauma medular e a possível utilização terapêutica das toxinas de caramujo marinho, atuantes nos principais canais para cálcio dependentes de voltagem.

Blocking voltage dependent calcium channels is an important strategy in acute spinal trauma treatment, because it prevents the exacerbated calcium influx which participates actively in acute neurodegenerative processes, resulting in neuroprotection with improvement of neurological and electrophysiological functions. The cone snail toxins are peptides with adequate structural stability, which have been studied by specific actions on ion channels and receptors that directly interfering in the release of neurotransmitters and neuromodulation of sensory and motor neurons of the spinal cord. They are already used in the treatment of neurological disorders and appear to be promising in the development of new therapies for spinal trauma. Therefore, it was aimed to discuss the pathophysiology of spinal cord trauma, and possible therapeutic use of marine snail toxins that acts in voltage-dependent calcium channels.

Effects of dantrolene on apoptosis and immunohistochemical expression of NeuN in the spinal cord after traumatic injury in rats.

Dantrolene has been shown to be neuroprotective by reducing neuronal apoptosis after brain injury in several animal models of neurological disorders. In this study, we investigated the effects of dantrolene on experimental spinal cord injury (SCI). Forty-six male Wistar rats were laminectomized at T13 and divided in six groups: GI (n = 7) underwent SCI with placebo and was euthanized after 32 h; GII (n = 7) underwent laminectomy alone with placebo and was euthanized after 32 h; GIII (n = 8) underwent SCI with dantrolene and was euthanized after 32 h; GIV (n = 8) underwent SCI with placebo and was euthanized after 8 days; GV (n = 8) underwent laminectomy alone with placebo and was euthanized after 8 days; and GVI (n = 8) underwent SCI with dantrolene and was euthanized after 8 days. A compressive trauma was performed to induce SCI. After euthanasia, the spinal cord was evaluated using light microscopy, TUNEL staining and immunochemistry with anti-Caspase-3 and anti-NeuN. Animals treated with dantrolene showed a smaller number of TUNEL-positive and caspase-3-positive cells and a larger number of NeuN-positive neurons, both at 32 h and 8 days (P ≤ 0.05). These results showed that dantrolene protects spinal cord tissue after traumatic SCI by decreasing apoptotic cell death.

Cortisol sérico e glicemia em cadelas tratadas com tramadol e submetidas à ovário-histerectomia / Serum cortisol and blood glucose in bitches treated with tramadol and submitted to ovariohysterectomy

O tramadol é um analgésico opióide usado em medicina veterinária, embora existam poucos estudos sobre este fármaco. O objetivo deste trabalho foi avaliar o efeito analgésico promovido pela administracão do tramadol, mensurando o cortisol sérico e a glicemia de cadelas. Para isso, foram utilizadas 15 fêmeas, submetidas a ovário-histerectomia sob anestesia geral com isofluorano. Os animais foram divididos em três grupos. Grupo 1 (Tep) receberam tramadol pela via epidural (1,0mg kg-1 diluído em água bidestilada ao volume final de 3,0mL) e, após 15 minutos, 3,0mL de água bidestilada pela via intravenosa. No grupo 2 (Tiv), foi administrado 3,0mL de água bidestilada pela via epidural e, após 15 minutos, tramadol pela via intravenosa (1,0mg kg-1 diluído em água bidestilada ao volume final de 3,0mL). No grupo 3 (CT), os animais receberam 3,0mL de água bidestilada pela via epidural e, após 15 minutos, 3,0mL de água bidestilada pela via intravenosa. A eficácia de cada regime analgésico foi avaliada durante 12 horas após a administracão da injecão epidural. Não houve diferenca significativa entre os grupos experimentais para as variáveis estudadas (P>0,05). Foram observadas diferencas significativas dentro de cada tratamento. No grupo Tep houve aumento do cortisol sérico aos 25 minutos do trans-operatório (M3), em relacão ao valor obtido imediatamente após a inducão anestésica (M2) (P>0,05). Nos tratamentos Tiv e CT, verificou-se elevacão das variáveis, duas (M4) e quatro (M5) horas da injecão epidural (P<0,05), quando comparadas ao pré-tratamento (M1). Nesses períodos, as variáveis estudadas no tratamento Tep foram estatisticamente semelhantes ao pré-tratamento (M1). Com base nos resultados, conclui-se que: 1) a metodologia empregada foi sensível para determinar os momentos de maior estresse cirúrgico, dentre os estudados e 2) o tramadol por via epidural produz analgesia mais duradoura quando comparado à administracão intravenosa.

Técnica histoquímica aplicada ao tecido ósseo desmineralizado e parafinado para o estudo do osteócito e suas conexões / Histochemical tecnique for osteocyte and its connections study in descalcified and paraffined bone tissue

O osteócito vem sendo alvo de pesquisas recentes. A avaliação in situ de sua morfologia, da atividade e das características de suas conexões é difícil, portanto é realizada apenas com técnicas avançadas. Devido à importância desse tipo celular na manutenção da matriz óssea, este estudo propõe uma técnica de coloração pela prata como alternativa para o estudo do osteócito e suas conexões em tecido ósseo desmineralizado e parafinado. Cortes de 4æm do fêmur de ratas foram desmineralizados, desparafinados em xilol e hidratados em concentrações decrescentes de álcool etílico (ETOH) e água miliQ. Para a impregnação foram utilizadas soluções de nitrato de prata a 50 por cento e de ácido fórmico a 1 por cento com 2 por cento de gelatina microbiológica em estufa a 40ºC. Essa técnica permite visualizar facilmente as bordas lacunares dos osteócitos e suas conexões, proporcionando uma alternativa simples e eficaz para o estudo da morfologia desse tipo celular até então ainda não proposta com essa finalidade.