Genotoxic parameters of human degenerated intervertebral discs are linked to the pathogenesis of disc degeneration.

BACKGROUND: Degenerative disc disease (DDD) is a prevalent disorder that brings great incapacity and morbidity to the world's population. Its pathophysiology is not fully understood. DNA damage can influence this process, but so far, there have been few studies to evaluate this topic and its true importance in DDD, as well as whether there is a relation between degeneration grade and DNA damage. The objective of this study is to evaluate the degree of damage to the DNA and the relation to the severity of DDD and measure its response to this insult compared to live/dead cell parameters and reactive oxygen species activity in human discs. METHODS: An experimental study was performed with 15 patients with grade IV or V Pfirrmann classification who underwent spinal surgery. Five patients were operated on two levels, resulting in 20 samples that were submitted to the comet assay to measure DNA damage. Of these, six samples were submitted to flow cytometry, and apoptosis, necrosis, cell membrane integrity, intracellular esterase activity, reactive oxygen species (ROS), caspase 3 and mitochondrial membrane potential were evaluated. RESULTS: All samples had DNA damage, and the average of index damage (ID) was 78.1 (SD ± 65.11) and frequency damage (FD) was 49.3% (SD ± 26,05%). There was no statistical difference between the Pfirrmann grades and genotoxic damage. Likewise, all samples that underwent flow cytometry showed apoptosis and ROS to many different degrees. CONCLUSIONS: DNA damage occurs in high-grade degeneration of human discs and contributes to activation of the apoptosis pathway and ROS production that can accelerate disc degeneration.

Hyaluronic Acid In Vitro Response: Viability and Proliferation Profile of Human Chondrocytes in 3D-Based Culture.

OBJECTIVES: This study aimed to evaluate the efficacy of hyaluronic acid in the viability and proliferation profile of human femoral-tibial joint cartilage affected by osteoarthritis using in vitro models of chondrocytes in a 2-dimensional (2D)- and 3-dimensional (3D)-based culture model by spheroids. DESIGN: In vitro study of knee cartilage affected by osteoarthritis that required surgical treatment. Samples were cultured and exposed to hyaluronic acid (100 and 500 µM; intervention group) or vehicle solution. In monolayer or 2D culture, proliferation and cell viability were measured, and nuclear morphometry was analyzed by 4',6'-diamino-2-fenil-indol (DAPI) staining. The 3D-based culture established from the culture of articular cartilage of patients submitted to total knee arthroplasty evaluated the diameter, viability, and fusion ability of the chondrospheres created. RESULTS: Samples from 3 patients resulted in viable cultures, with chondrocyte cells exhibiting a potential for cell proliferation and viability to establish a culture. Hyaluronic acid (100 and 500 µM) improved chondrocyte viability and proliferation up to 72 hours in contact when compared with the control group, and no nuclear irregularities in morphology cell characteristics were observed by DAPI. In the 3D evaluation, hyaluronic acid (500 µM) improved the cellular feedback mechanisms, increasing the survival and maintenance of the chondrospheres after 7 days of analysis, showing the intrinsic capacity of chondrospheres grouped in the attempt to rearrange and reestablish new articular tissue. CONCLUSIONS: The 2D- and 3D-based culture models with hyaluronic acid improved chondrocyte viability and proliferation and demonstrated the ability of freshly formed chondrospheres to undergo fusion when placed together in the presence of hyaluronic acid.

Hyperbaric Oxygen Therapy After Acute Thoracic Spinal Cord Injury: Improvement of Locomotor Recovery in Rats.

STUDY DESIGN: A controlled laboratory study. OBJECTIVE: The aim of this study was to analyze the effectiveness of hyperbaric therapy (HT) using mild and moderate models of spinal cord injury (SCI). SUMMARY OF BACKGROUND DATA: SCI can cause permanent impairment with socioeconomic consequences. The motor deficit occurs by two mechanisms: destruction of neuronal cells and local inflammatory response, resulting in hypoxia. HT acts by increasing oxygen in the injured area. METHODS: Thoracic laminectomy was performed in 72 female Wistar rats. The MASCIS impactor was used at 12.5 mm (n = 35) and 25 mm (n = 35) of height to perform, respectively, mild and moderate SCI. Muscle strength was assessed through the Basso, Beattie, and Bresnahan scale (BBB) on days 1, 7, 14, 21, and 28 after SCI. The animals were randomized into five subgroups with seven animals each: (1) control group had SCI without HT; (2) HT 30 minutes after SCI; (3) HT 30 minutes after SCI and daily for 7 days; (4) HT 12 hours after SCI; and (5) HT 12 hours after SCI and daily for 7 days. HT was performed at 2.5 atm for 1 hour. RESULTS: There was a linear relationship between injury severity and motor deficit until day 21, with similar BBB scores on day 28. A pattern of uniform lesions was observed in the mild SCI, with lower variation of BBB when compared with moderate SCI. All animals that underwent HT had significant improvement in motor function and histology when compared with control group. Regardless of the injury model, animals submitted to 7-day protocols had an early improvement in motor function and a smaller area of histological injury. CONCLUSION: The present study reported that the sooner HT is begun after mild and moderate SCI and the larger the number of sessions, the greater and earlier is the motor recovery and smaller is the tissue injury. LEVEL OF EVIDENCE: N/A.

Histological markers of degeneration and regeneration of the human intervertebral disk / Marcadores histológicos de degeneração e regeneração do disco intervertebral humano / Marcadores histológicos de la degeneración y regeneración del disco intervertebral humano

ABSTRACT Objective: To define histological scores for intervertebral disc degeneration that would enable the definition of morphological characteristics of disease, besides improving knowledge of the lumbar degenerative disc disease by means of immunohistochemical markers. Methods: Hematoxylin and Eosin, Alcian/PAS, Masson Trichrome and Safranin O/FCF staining was used on the intervertebral disc degeneration sections of patients with lumbar degenerative disc disease. The protein markers defined in immunohistochemistry were cell proliferation (Ki-67) and apoptosis (p53). Results: The study data enabled the determination of Safranin O/FCF stain as the most effective one for evaluating parameters such as area, diameter, and number of chondrocyte clusters. The importance of using stains in association, such as Safranin O/FCF, Masson Trichrome, Alcian/PAS and Hematoxylin and Eosin, was also determined, as they are complementary for the histopathological verification of intervertebral disc degeneration. By expressing proteins using the immunohistochemistry technique, it was possible to consider two stages of disc degeneration: cell proliferation with chondrocyte cluster formation, and induction of apoptosis. Conclusion: This study enabled the histological and immunohistochemical characterization to be determined for lumbar degenerative disc disease, and its degrees of evolution, by determining new disc degeneration scores.

RESUMO Objetivo: Definir escores histológicos de degeneração do disco intervertebral que permitam a identificação de características morfológicas da doença, além de melhorar o conhecimento sobre a discopatia degenerativa lombar por meio de marcadores imuno-histoquímicos. Métodos: As colorações histológicas de hematoxilina e eosina, azul de alcian/PAS, tricrômica de Masson e safranina O/FCF foram utilizadas em cortes de disco intervertebral degenerado de pacientes com discopatia degenerativa lombar. Os marcadores proteicos definidos na imuno-histoquímica permitiram a avaliação da proliferação celular (Ki-67) e da apoptose (p53). Resultados: Os dados do estudo permitiram a determinação da coloração de safranina O/FCF como a mais eficaz para avaliar os parâmetros tais como a área, o diâmetro e o número de agrupamentos de condrócitos. Também se determinou a importância do uso das colorações histológicas de forma associada, como safranina O/FCF, tricrômica de Masson, azul de alcian/PAS e hematoxilina e eosina, uma vez que elas são complementares para a verificação histopatológica da degeneração do disco intervertebral. Pela técnica da expressão de proteínas com técnica imuno-histoquímica, foi possível considerar dois estágios de degeneração do disco: proliferação de células com a formação de agrupamentos de condrócitos, seguida pela indução de apoptose. Conclusão: Este estudo permitiu definir a caracterização histológica e imuno-histoquímica em discopatia degenerativa lombar e seus graus de evolução, determinando novos escores de degeneração discal.

RESUMEN Objetivo: Definir valores histológicos de degeneración del disco intervertebral que permitan la definición de las características morfológicas de la enfermedad y mejorar el conocimiento de la enfermedad degenerativa del disco lumbar mediante marcadores inmunohistoquímicos. Métodos: Las coloraciones histológicas con hematoxilina y eosina, azul alcián/PAS, tricrómico de Masson y safranina O/FCF se utilizaron en secciones de los discos intervertebrales degenerados de pacientes con enfermedad degenerativa del disco lumbar. Los marcadores de proteínas definidos por inmunohistoquímica permitieron la evaluación de la proliferación celular (Ki-67), y la apoptosis (p53) . Resultados: Los datos de la determinación permitieron establecer la tinción con safranina O/FCF como la más eficaz para evaluar parámetros tales como el área, diámetro y número de agrupaciones de condrocitos. Se determinó también la importancia del uso asociado de diversas tinciones, como safranina O/FCF, tricrómico de Masson, azul de alcián/PAS y hematoxilina y eosina, ya que son complementarias para la verificación histopatológica de la degeneración del disco intervertebral. Por la técnica de la expresión de proteína con análisis inmunohistoquímica, fue posible establecer dos etapas de la degeneración del disco: la proliferación de células con la formación de agrupaciones de condrocitos y la inducción de la apoptosis. Conclusión: Este estudio permitió definir la caracterización histológica e inmunohistoquímica en la enfermedad degenerativa del disco lumbar y sus grados de evolución, mediante la determinación de nuevas puntuaciones de degeneración del disco.

Motor and histological findings in a model of sci: comparison between posterior and lateral clips / Achados motores e histológicos em um modelo de trm: comparação entre colocação posterior e lateral de clipes / Hallazgos motores e histológicos en un modelo de trm: comparación de la colocación lateral y posterior de clips

Objective: To evaluate the locomotor and histological impact on the spinal cord comparing lateral and posterior clip placement. Method: Randomized experimental trial. Twenty female Wistar rats, weighing between 250 and 300 grams and aged 12-14 weeks were randomized in two groups according to the placement of the clip: lateral group (N=10) and posterior group (N=10). After exposing the thoracic segment of the spine (T8-T10), a laminectomy was performed at the T9 level under microscopic view. The spinal cord injury was made using a 5 mm long aneurysm clip with a closing pressure of 50 grams. Locomotor behavior was evaluated by the Basso, Beattie and Bresnahan scale in days 1, 7, 14, 21, and 28 after surgery. The area of injury was assessed by histological analysis and measured by a software. Results: The histological evaluation showed a larger mean area of 4.8±1mm² of lesion (P=0.03) in the lateral group when compared with the posterior group mean area of 2.3±2mm². There was no significant difference between lateral and posterior groups with respect to locomotor scores from day 1 to 28 (P=0.361). Conclusion: The lesion area observed in the spinal cord histology after lateral placement of a clip was significantly bigger than in the posterior placement. The motor evaluation showed similar BBB scores regardless of the type of clamping method. .

Objetivo: Avaliar o impacto locomotor e histológico da colocação lateral ou posterior do clipe na medula espinal. Método: Estudo experimental randomizado. Vinte ratas Wistar, que pesavam entre 250 e 300 gramas, com 12 a 14 semanas de vida, foram randomizadas em dois grupos com relação à colocação do clipe: grupo lateral (N=10) e grupo posterior (N=10). Após expor o segmento torácico da medula (T8-T10), uma laminectomia foi realizada no nível de T9 sob visão de microscópio. A lesão medular foi realizada com um clipe de aneurisma de 5 mm de comprimento com pressão de fechamento de 50 gramas. O comportamento locomotor foi avaliado pela escala de Basso, Beattie e Bresnahan nos dias 1, 7, 14, 21 e 28 após a cirurgia. A área da lesão foi avaliada por análise histológica e foi medida por um software. Resultados: A avaliação histológica mostrou média maior da área de lesão medular (P=0.03) na utilização da clipagem lateral 4,8±1 em comparação com a posterior (2,3±2). Não houve diferença significativa entre os grupos lateral e posterior com relação ao escore locomotor do dia 1 ao 28 (P=0.361). Conclusão: A área de lesão medular observada na histologia da medula espinal depois de colocação de clipe lateral foi significativamente maior em comparação com a clipagem posterior. A avaliação motora mostrou resultados similares independente do ...

Objetivo: Evaluar el impacto locomotor e histológico de la colocación lateral y posterior del clip en la médula espinal. Métodos: Ensayo aleatorio experimental. Veinte ratas Wistar, que pesaban entre 250 y 300 gramas, con 12 a 14 semanas de vida fueron asignadas al azar en dos grupos de acuerdo a la colocación del clip: grupo lateral (N=10) y grupo posterior (N=10). Después de exponer el segmento torácico de la médula (T8-T10), se realizó una laminectomía en el nivel de T9 bajo visión microscópica. La lesión medular se realizó mediante un clip de aneurisma con 5 mm de largo y presión de cierre de 50 gramos. El comportamiento locomotor se evaluó mediante la escala de Basso, Beattie y Bresnahan los días 1, 7, 14, 21 y 28 después de la cirugía. El área de la lesión se evaluó mediante el análisis histológico y su medida por un software. Resultados: El estudio histológico mostró un área media mayor de lesión de 4,8±1mm² (P=0,03) en el grupo lateral en comparación con el grupo posterior (2,3±2). No hubo diferencia significativa entre los grupos lateral y posterior con respecto a las puntuaciones del aparato locomotor desde el día 1 al 28 (P=0,361). Conclusión: El área de la lesión observada en la histología de la médula espinal después de la colocación lateral de un clip fue significativamente mayor que en la colocación posterior. La evaluación locomotora mostró ...

Stem-cell treatment in disc degeneration: What is the evidence? / Células tronco no tratamento de doença degenerativa discal: Qual é a evidência? / Células madre en el tratamiento de degeneración de disco: ¿Cual es la evidencia?

To review the potential role of stem cells in treating degenerative disc disease of the intervertebral disc (IVD). A review was performed of articles from the Medline database concerning stem cells and degenerative disc disease (DDD). To discuss the data, the papers were classified as: review, in vitro, experimental, and clinical. The currently available treatments were basically for symptom reduction, not to revert the IVD degenerative process. The use of mesenchymal stem cells (MSC) is being proposed as an option of treatment for DDD. In vitro studies have shown that the MSC are able to differentiate into NP cells and that the MSC also reduce the inflammatory levels of the degenerated IVD. Besides, experimental studies demonstrated that the MSC remained viable when injected into the IVD, and that they were able to regenerate partially from the degenerated IVD and its structure. The few clinical studies found in the literature presented diverging results. The use of MSC is being widely studied and shows promising results for the treatment of DDD. Although many advances are being achieved in studies in vitro and experimental, there is a lack of clinical studies to prove the role of MSC in DDD management.

Revisar o potencial papel das células-tronco para o tratamento de doença degenerativa do disco intervertebral (IVD). Foi realizada uma revisão dos trabalhos da base de dados Medline em relação a células-tronco e doença degenerativa discal (DDD). Para fins de discussão dos dados, os trabalhos foram divididos em: revisão, in vitro, experimentais e clínicos. Os tratamentos disponíveis atualmente focam basicamente na redução dos sintomas, e não na reversão do processo degenerativo do DIV. O uso de células-tronco mesenquimais (CTM) vem sendo proposto como uma opção de tratamento para DDD. Estudos in vitro demonstraram que as CTM são capazes de se diferenciarem em células do NP e que as CTM também diminuem os níveis inflamatórios do DIV degenerado. Além disso, estudos experimentais demonstraram que as CTM permaneciam viáveis quando injetadas no DIV e que eram capazes de regenerar parcialmente do DIV degenerado e sua estrutura. Os poucos estudos clínicos presentes na literatura apresentam resultados divergentes. O uso de CTM esta sendo amplamente estudado e mostra resultados promissores para o tratamento da DDD. Embora muitos avanços venham sendo alcançados em estudos in vitro e experimentais, ainda faltam estudos clínicos para comprovar o papel das CTM no manejo da DDD.

Revisar el papel potencial de las células madre en el tratamiento de la enfermedad degenerativa del disco intervertebral (IVD). Se realizó una revisión de los artículos de la base de datos Medline sobre células madre y la enfermedad degenerativa del disco (DDD). Para fines de discusión de los datos, los trabajos se dividieron en: revisión, in vitro, experimentales y clínicos. Los tratamientos disponibles actualmente enfocan básicamente la reducción de los síntomas, y no la reversión del proceso degenerativo del IVD. El uso de células madre mesenquimales (MSC) se propone como una opción de tratamiento para DDD. Estudios in vitro demostraron que las MSC son capaces de diferenciarse en células NP y que las MSC también reducen los niveles inflamatorios de la IVD degenerado. Además, estudios experimentales demostraron que las MSC se mantuvieron viables cuando inyectadas en el IVD y que eran capaces de regenerarse parcialmente del IVD degenerado y su estructura. Los pocos estudios clínicos presentes en la literatura presentan resultados divergentes. El uso de MSC se está estudiando ampliamente y muestra resultados prometedores en el tratamiento de la DDD. Aunque se hayan logrado muchos avances en estudios in vitro y experimentales, aún faltan estudios clínicos para comprobar el papel de las MSC en el manejo de la DDD.

Aeromonas associated diarrhoeal disease in south Brazil: prevalence, virulence factors and antimicrobial resistance

Aeromonas were isolated from 27 (6.6 percent) of 408 patients admitted with acute gastroenteritis in two hospitals at Rio Grande do Sul, Brazil. Isolates were classified as A. hydrophila (51.8 percent), A. caviae (40.8 percent), and A. veronii biotype sobria (7.4 percent). The highest prevalence of Aeromonas associated infections occurred in lactants and children. Virulence genes (aerA -aerolysin/hemolysin, ahpA -serine-protease, satA - glycerophospholipid-cholesterol acyltransferase, lipA -lipase, and ahyB -elastase) and virulence factors (hemolytic, proteolitic, lipolitic activities, and biofilm formation) were identified in most A. hydrophila and A. veronii biotype sobria isolates, with lower frequencies on A. caviae. All Aeromonas isolates were resistant to ampicillin, ticarcillin/clavulanic acid, cephalotin, and cephazolin, and most of them (>70 percent) exhibited resistance to imipenem, carbenicillin, amoxillin/sulbactan, and piperacillin. Multiple-resistance, more than four antibiotics, was evidenced in 29.6 percent of the isolates. The most efficient antibiotics were the quinolones (ciprofloxacin and norfloxacin), and the aminoglycosides (amikacin and netilmicin).

Aeromonas foram isoladas de 27 (6.6 por cento) dos 408 pacientes admitidos com gastroenterite aguda em dois hospitais do Rio Grande do Sul, Brasil. Os isolados foram classificados com A. hydrophila (51.8 por cento), A. caviae (40.8 por cento), e A. veronii biotype sobria (7.4 por cento). A maior prevalência de Aeromonas ocorreu em lactantes e crianças. Genes (aerA -aerolisina/hemolisina, ahpA -serina-protease, satA - glicerofosfolipidio-colesterol aciltransferase, lipA -lipase, e ahyB -elastase) e factores (atividade hemolítica, proteolítica, lipolítica, e formação de biofilme) de virulência foram identificados na maioria dos isolados de A. hydrophila e A. veronii biotype sobria, com freqüências menores em A. caviae. Todos os isolados de Aeromonas apresentaram resistência a ampicilina, ticarcilina/ácido clavulânico, cefalotina e cefazolina, e a maior parte (>70 por cento) exibiram resistência a imipenem, carbenicilina, amoxacilina/sulbactam e piperacilina. Resistência múltipla foi evidenciada em 29,6 por cento dos isolados. Os antibióticos mais eficientes foram as quinolonas (ciprofloxacina e norfloxacina) e os aminoglicosídicos (amicacina e netilmicina).