The Presence of the Neuronal Nitric Oxide Synthase Isoform in the Intervertebral Disk.

Intervertebral disk degeneration is a progressive and debilitating disease with multifactorial causes. Nitric oxide (NO) might contribute to the cell death pathway. We evaluated the presence of the constitutive form of the neuronal NOS (nNOS) in both health and degenerated intervertebral disk through qPCR and immunohistochemistry. We also analyzed the potential role of nNOS modulation in the tail needle puncture model of intervertebral disk degeneration. Male Wistar rats were submitted to percutaneous disk puncture with a 21-gauge needle of coccygeal vertebras. The selective nNOS pharmacological inhibitor N (ω)-propyl-L-arginine (NPLA) or a nNOS-target siRNA (siRNAnNOShum_4400) was injected immediately after the intervertebral disk puncture with a 30-gauge needle. Signs of disk degeneration were analyzed by in vivo magnetic resonance imaging and histological score. We found that intact intervertebral disks express low levels of nNOS mRNA. Disk injury caused a 4 fold increase in nNOS mRNA content at 5 h post disk lesion. However, NPLA or nNOS-target siRNA slight mitigate the intervertebral disk degenerative progress. Our data show evidence of the nNOS presence in the intervertebral disk and its upregulation during degeneration. Further studies would disclose the nNOS role and its potential therapeutical value in the intervertebral disk degeneration.

Does a small size needle puncture cause intervertebral disc changes?

PURPOSE: Small size needles have been regularly used for intradiscal injection of innocuous/potential therapeutic compounds in experimental conditions, but also in clinic procedures, such as discography. Our aim was to investigate if a 30-gauge needle could trigger observable changes on intact intervertebral discs. We compared these effects to those induced by a large size needle (21-gauge), a well-known intervertebral disc degenerative model based on needle puncture. METHODS: Coccygeal intervertebral discs (Co8-9) of adult male Wistar rats were punctured with a 21-gauge needle, while the coccygeal levels Co7-8 and Co9-10 remained intact. The 30-gauge needle was used to inject a safe volume of saline (2 µl) on both intact (Co9-10) and punctured (Co8-9) discs. MRI and histological score were performed at 2, 15 and 42 days after procedure. RESULTS: MRI analyses revealed significant reduction on signal intensity of 21-gauge punctured discs. Intact discs which received a saline injection through a 30-gauge needle also revealed significant alterations in the MRI signal when compared with control discs. No histological changes were observed in the intact saline injected discs at any time analyzed. CONCLUSION: Since significant intervertebral image changes were observed with a 30-gauge needle, cautious interpretation of the pharmacological inoculation findings is required.

Does a small size needle puncture cause intervertebral disc changes?

PURPOSE:Small size needles have been regularly used for intradiscal injection of innocuous/potential therapeutic compounds in experimental conditions, but also in clinic procedures, such as discography. Our aim was to investigate if a 30-gauge needle could trigger observable changes on intact intervertebral discs. We compared these effects to those induced by a large size needle (21-gauge), a well-known intervertebral disc degenerative model based on needle puncture.METHODS:Coccygeal intervertebral discs (Co8-9) of adult male Wistar rats were punctured with a 21-gauge needle, while the coccygeal levels Co7-8 and Co9-10 remained intact. The 30-gauge needle was used to inject a safe volume of saline (2 µl) on both intact (Co9-10) and punctured (Co8-9) discs. MRI and histological score were performed at 2, 15 and 42 days after procedure.RESULTS: MRI analyses revealed significant reduction on signal intensity of 21-gauge punctured discs. Intact discs which received a saline injection through a 30-gauge needle also revealed significant alterations in the MRI signal when compared with control discs. No histological changes were observed in the intact saline injected discs at any time analyzed.CONCLUSION: Since significant intervertebral image changes were observed with a 30-gauge needle, cautious interpretation of the pharmacological inoculation findings is required.

Advantages of a combined method of decalcification compared to EDTA.

Decalcification of mineralized tissues is an essential step during tissue processing in the routine histopathology. The time required for complete decalcification, and the effect of decalcifier on cellular and tissue morphology are important parameters which influence the selection of decalcifying agents. In this study, we compared a decalcifying solution (ETDA) composed of both acid and chelating agents to a classical and well-known decalcifying agent (EDTA). To this purpose, the optic density of bone radiographs, residual calcium analysis, bone sample weight, and histological and immunohistochemical analysis were performed. Our data suggest that, similarly to EDTA, the ETDA solution completely removes the calcium ions from the samples enabling easy sectioning. However, unlike the EDTA, this agent takes much less time. Furthermore, both agents showed comparable decalcification efficacy, and similarly, they did not produce cellular, tissue or antigenicity impairments. Therefore, ETDA may be a suitable option when it is necessary an association between a rapid and complete removal of calcium minerals, and a suitable preservation of structure and antigenicity of tissues.

Protective effects of cannabidiol on lesion-induced intervertebral disc degeneration.

Disc degeneration is a multifactorial process that involves hypoxia, inflammation, neoinnervation, accelerated catabolism, and reduction in water and glycosaminoglycan content. Cannabidiol is the main non-psychotropic component of the Cannabis sativa with protective and anti-inflammatory properties. However, possible therapeutic effects of cannabidiol on intervertebral disc degeneration have not been investigated yet. The present study investigated the effects of cannabidiol intradiscal injection in the coccygeal intervertebral disc degeneration induced by the needle puncture model using magnetic resonance imaging (MRI) and histological analyses. Disc injury was induced in the tail of male Wistar rats via a single needle puncture. The discs selected for injury were punctured percutaneously using a 21-gauge needle. MRI and histological evaluation were employed to assess the results. The effects of intradiscal injection of cannabidiol (30, 60 or 120 nmol) injected immediately after lesion were analyzed acutely (2 days) by MRI. The experimental group that received cannabidiol 120 nmol was resubmitted to MRI examination and then to histological analyses 15 days after lesion/cannabidiol injection. The needle puncture produced a significant disc injury detected both by MRI and histological analyses. Cannabidiol significantly attenuated the effects of disc injury induced by the needle puncture. Considering that cannabidiol presents an extremely safe profile and is currently being used clinically, these results suggest that this compound could be useful in the treatment of intervertebral disc degeneration.

Modelo de degeneração do disco intervertebral por punção da cauda de ratos Wistar: avaliação histológica e radiográfica / Modelo de degeneración del disco intervertebral por punción de la cola de ratas Wistar: evaluación histológica y radiográfica / Injury model by needle puncture of the Wistar rats' tail: histological and radiographic assessments

OBJETIVO: descrever a caracterização histológica e radiográfica do método de indução da degeneração do disco intervertebral da cauda de ratos Wistar induzida por meio de punção. MÉTODOS: ratos Wistar machos adultos foram anestesiados, radiografados e submetidos à punção dos discos intervertebrais localizados entre a sexta e a sétima e a oitava e nona vértebras coccígeas. Para a punção foi utilizada agulha de 20G, que foi introduzida até o ânulo fibroso, e foi realizada dupla rotação de 360º, mantendo-se a mesma posição durante 30 segundos antes da retirada. O disco intermediário aos segmentos lesados (7-8) não foi puncionado e foi utilizado como controle. Foi selecionado o período pós-lesão de 30 dias (n=9) para sacrifício e análise dos discos intervertebrais. Os animais foram radiografados 30 dias após a lesão para análise da altura do disco intervertebral. Os segmentos da cauda foram removidos, fixados e desmineralizados, processados e corados com Hematoxilina-Eosina para avaliação histológica. RESULTADOS: a análise radiográfica revelou a redução significativa da altura dos discos lesados em relação ao controle. A avaliação histológica revelou alterações no núcleo pulposo e ânulo fibroso dos discos lesados em relação ao controle. Não foram observadas diferenças na intensidade de lesão entre os discos proximal e distal. CONCLUSÃO: a degeneração do disco intervertebral da cauda de ratos Wistar induzida por meio de punção mostrou ser método reprodutível para estudo da degeneração do disco intervertebral. Esse modelo mostrou validade para avaliação experimental de novas intervenções terapêuticas nos processos de degeneração do disco intervertebral.

OBJECTIVE: to report the induction of intervertebral disc degeneration of the rat caudal spine by needle puncture and its radiographic and histologic characterization. METHODS: adult male Wistar rats were anesthetized, submitted to the X-Ray and then to the needle puncture (20G) of intervertebral disc between the sixth and seventh (proximal segment) and the eighth and ninth (distal segment) coccygeal vertebrae. Radiographies were taken 30 days after lesion for analysis of intervertebral disc height. The intermediate disc (7-8) to injured segments was not punctured and was considered as control. All segments were removed, fixed and demineralized, processed and stained with Hematoxylin-Eosin for histological evaluation. RESULTS: radiographic analysis revealed significant reduction in disc height of lesioned discs compared to control. Similarly, histological analysis revealed significant changes in the nucleus pulposus and annulus fibrosus of the lesioned discs (proximal and distal) relative to the control. There was no difference in the intensity of injury between the proximal and distal discs. CONCLUSION: the experimental model of tail intervertebral disc degeneration by needle puncture reproduced the steps of the intervertebral disc degeneration, assessed by different instruments, and it can be used for experimental evaluation of new therapeutic interventions for intervertebral disc degeneration process.

OBJETIVO: describir la caracterización histológica y radiográfica del método de inducción de la degeneración del disco intervertebral de la cola de ratas Wistar, inducida por medio de la punción. MÉTODOS: ratas Wistar machos adultos fueron anestesiados, radiografiados y sometidos a la punción de los discos intervertebrales localizados, entre la sexta y la séptima; y la octava y novena vértebras coccígeas. Para la punción, fue utilizada una aguja de 20G, que fue introducida hasta el ánulo fibroso, y fue realizada una dupla rotación de 360º, manteniendo esta posición durante 30 segundos, previamente a la retirada. El disco intermediario a los segmentos lesionados (7-8) no fue puncionado y fue considerado como control. Fue seleccionado el periodo post-lesión de 30 días (n=9) para sacrificio y análisis de los discos intervertebrales. Los animales fueron radiografiados 30 días después de la lesión para análisis de la altura del disco intervertebral. Los segmentos de la cola fueron removidos, fijados y desmineralizados, procesados y coloreados con hematoxilina-eosina para evaluación histológica. RESULTADOS: el análisis radiográfico mostró una reducción significativa de la altura de los discos lesionados en relación al control. La evaluación histológica mostró alteraciones en el núcleo pulposo y el ánulo fibroso de los discos lesionados en relación al control. No fueron observadas diferencias en la intensidad de la lesión entre los discos proximal y distal. CONCLUSIÓNES: la degeneración del disco intervertebral de la cola de ratas Wistar inducida por medio de punción mostró ser un método reproducible para el estudio de la degeneración del disco intervertebral. Ese modelo mostró validez para la evaluación experimental de nuevas intervenciones terapéuticas en los procesos de la degeneración del disco intervertebral.

Integração do enxerto ósseo corticoesponjoso homólogo, tratado quimicamente e esterilizado em óxido de etileno: estudo em cães / Integration of cortical cancellous bone homograft chemically treated and sterilized with ethylene oxide: a study in dogs

Objetivo: Aprofundar a investigação sobre o processo de integração do enxerto ósseo homólogo tratado quimicamente e esterilizado em óxido de etileno, em cães. Métodos: De cães doadores foram retirados cilindros ósseos de 1,0cm de diâmetro e 1,0cm de altura da epífise distal dos fêmures, no sentido transversal do osso, que sofreram preparação em passagens sucessivas em água oxigenada, éter, álcool e esterilizados em óxido de etileno. Para o implante desses enxertos foram usados cães receptores alocados em dois grupos. No grupo experimental, 10 animais foram operados e, por meio de acesso lateral, foi realizada uma cavidade cilíndrica transversal de 1,0cm de diâmetro na epífise distal do fêmur direito, com uso de trefina. Foi aspirada medula óssea do osso ilíaco, que foi usada para embeber o bloco de osso já preparado e a ser utilizado, que, em seguida, foi encaixado na cavidade previamente criada. Três semanas após, os animais foram reoperados, agora como enxertia realizada no outro fêmur (esquerdo), com a mesma técnica. Todos os cães receptores foram sacrificados após seis semanas da primeira cirurgia, de modo a fornecer enxertos com três e seis semanas após implante. Os animais do grupo controle receberam enxerto autólogo retirado do fêmur oposto em uma única sessão operatória e constituíram dois subgrupos: um sacrificado com três semanas (10 animais), e o outro, sacrificado com seis semanas (10 animais).