Ultrastructural study of the short-term effects of chymopapain on the intervertebral disc

The initial effects of chymopapain, a chemonucleolytic agent, on the intervertebral disc of dogs were studied by light and electron microscopic techniques. Fragments of nucleus pulposus and annulus fibrosis were incubated with chymopapain up to 24 h in vitro. Proteoglycans and matrix proteins were rapidly removed, while collagen fibers remained intact up to 24 h. For several hours, most cells remained normal in appearance with only slight swelling and an increased number of vacuoles. After exposure to the protease for 24 h cells in both the annulus and nucleus showed extensive membrane damage and some were necrotic, but many survived relatively intact. These results suggest that, similar to the results of the digestion of cartilage with other proteases, the cells of the disc can survive brief chymopapain exposure during chemonucleolysis procedures and could serve as a source for regenerating tissue. The nature of the regeneration may depend on the extracellular scaffold that remains and the nutrition available to tissue as well as the age and biomechanical state of the disc. As for clinical significance, chemonucleolysis is an important nonsurgical alternative for treating prolapsed disc. The cells of nucleus and annulus can survive short-term exposure to treatment, and thus be responsible for partial regeneration of the tissue. This regeneration may be important in preventing long-term degenerative disease in the facet joints caused by increased pressure due to decreased disc height.     

Effects of collagenase and chymopapain on spinal nerves and intervertebral discs of cynomolgus monkeys

In order to test the safety and efficacy of Nucleolysin, a collagenase for intradiscal chemotherapy, laminectomies were performed on the L2-3 intervertebral discs of four groups of three young adult Cynomolgus monkeys. One primate from each group was injected with half the recommended human dose of Nucleolysin, chymopapain, or the same volume of sterile water. The remaining half of the human dose of each drug or equal volume of sterile water was equally divided and placed upon the right L-3 and L-4 nerve roots at their vertebral foramina. The right L-4 nerve root was first compressed for 10 seconds with an aneurysm clip. These procedures were done to simulate inadvertent contact of enzyme with spinal nerves in patients undergoing chemonucleolysis. After 4 weeks of observation, the 12 primates were humanely killed and examined post mortem. The effects of both enzymes were limited to those tissues with which they came in direct contact. Complete digestion of the nucleus pulposus of all enzyme-injected intervertebral discs was observed. Variable portions of the anulus fibrosus (from 2.3% to 57.4%) were also dissolved. Direct contact of Nucleolysin with lumbar nerve roots caused minor perineural reaction and no more intraneural changes than seen in sterile water controls. Chymopapain induced mild to severe perineural skeletal muscle necrosis and fibrosis with perineural arterial lesions as well as a degenerative neuropathy which was more marked in the traumatized nerve. The results of this study suggest that Nucleolysin and chymopapain are approximately equally effective on intervertebral discs, and that Nucleolysin is less injurious to spinal nerve roots and perineural tissue at the doses used.     

Residual chymopapain activity after chemonucleolysis in normal intervertebral discs in dogs.

Studies were carried out to demonstrate residual chymopapain activity in intervertebral discs after chemonucleolysis; protease assay, enzyme-linked immunosorbent assay, and immunohistochemical localization of the chymopapain in the disc tissue were done. Chymopapain, one milligram per level, was injected into the normal lumbar intervertebral discs of adult mongrel dogs and the discs were excised after two weeks. Proteolytically active chymopapain was still present in the extract of intervertebral disc at this time. The proteolytic activity was decreased by sulfhydryl inhibitors but not by inhibitors of metalloproteases or serine proteases. Protease and enzyme-linked immunosorbent assays showed that 0.60 +/- 0.48 per cent and 0.49 +/- 0.38 per cent of the original dose was present two weeks after the injection. Chymopapain was shown by immunohistochemical staining to be diffusely located throughout the extracellular matrix of the anulus fibrosus and the nucleus pulposus. Some cells, located mainly in the inner portion of the anulus, contained vacuoles filled with immunoreactive product.     

Biomechanical analysis of canine intervertebral discs after chymopapain injection. A preliminary report

With the renewed interest in using chymopapain (CP) as a chemonucleolytic agent for treatment of sciatica and low-back pain, the present study was undertaken to investigate the biomechanical property changes in canine lumbar discs after CP injections. The short-term (30- to 120-minute) in vitro effects of such an enzymatic agent appear to be the same as those of saline solution, causing increased disc heights, stiffness values, and creep rates. In the in vivo study, after three weeks, CP-injected discs had significant reductions in disc height and compressive stiffness, but the creep rate was increased substantially. However, at three months after injection, these biomechanical properties began to reverse and approached those of the uninjected controls. Buffer solution (cysteine and EDTA) was tried, but the sample size was too small to provide conclusive information. The results suggest that CP causes a disc to change its material property, but such effects appear to be time-related.     

Levels of keratan sulfate-bearing fragments rise predictably following chemonucleolysis of dog intervertebral discs with chymopapain

Chymopapain (1 mg) was injected into each of four-lumbar intervertebral discs of adult mongrel dogs. As expected, at 2 weeks, all injected discs exhibited marked loss of height (mean: 50% of original height) indicative of severe proteoglycan depletion. The appearance of keratan sulfate-bearing fragments in plasma was monitored by an ELISA-inhibition assay which uses a monoclonal antibody (1/20/5-D-4) specific for an epitope present only in the longest keratan sulfate chains. Levels of plasma keratan sulfate rose within 30 minutes and reached a maximum between 24 and 72 hours later. Levels then declined progressively but were still elevated at 2 weeks postinjection. Keratan sulfate-bearing fragments in plasma were purified by ion exchange chromatography on DEAE-Sephacryl and fractionated by sieve chromatography on Sepharose CL-6B. These plasma keratan sulfate-bearing fragments were found to be similar in size to keratan sulfate-bearing fragments generated by chymopapain digestion of dog nucleus pulposus proteoglycans, but slightly larger than single keratan sulfate chains obtained by alkaline borohydride treatment of dog nucleus pulposus proteoglycans. The results of this study show that measurements of blood levels of keratan sulfate could prove useful in monitoring effective degradation of disc proteoglycans in chemonucleolysis in man and help discriminate between ineffective enzyme placement, and alternative mechanisms of treatment failure.     

The effects of chymopapain on prolapsed human intervertebral disc. A clinical and correlative histochemical study

Light and electron microscopy were used to demonstrate extensive matrix degradation in a failed chymopapain-treated disc as compared with an untreated degenerative control disc. Both specimens contained viable cells with patches of degenerative cells. There was a loss of disc height in the chymopapain-treated disc, but no improvement of symptoms. These results illustrated that even in well-circumscribed cases with documented disc protrusion into the spinal canal, the disc space narrowing following injection may aggravate the symptoms of nerve root entrapment, especially if a low-grade lateral recess stenosis is associated with the disc protrusion. The persistence or aggravation of symptoms probably is related to these anatomic considerations. The low level of enzyme activity in the injected area may or may not be correlated with the clinical response to treatment.     

The innervation of the cervical intervertebral discs

Microdissection and histologic studies were undertaken to determine the innervation of the cervical intervertebral discs. The cervical sinuvertebral nerves were found to have an upward course in the vertebral canal, supplying the disc at their level of entry and the disc above. Branches of the vertebral nerve supplied the lateral aspects of the cervical discs. Histologic studies of discs obtained at operation showed the presence of nerve fibers as deeply as the outer third of the anulus fibrosus. These anatomic findings provide the hitherto missing substrate for primary disc pain and the pain of provocation discography.     

Surface strain on human intervertebral discs

The biomechanical functions of the internal components of the intervertebral disc are not well understood. The surface deformation of 17 human cadaveric lumbar intervertebral discs was studied by photogrammetry by adhering small optical targets to the disc surface and thereby recording the length, bulge, and vertical height of lines on the disc surface representing annular fibers. Discs were studied in pure compression, flexion and extension, axial rotation, and shear. Two definitions of a fiber were investigated: first with the end-points of the fiber on the vertebra ("bone-to-bone" definition), second, where the end points of the fiber were just before the disc vertebra junction (the "disc-only" definition). Measurements were compared with a "constant-volume" physical model and with a mathematical model of the intervertebral disc. Fiber strains were 6% or less under physiological conditions. Comparison of results from the two definitions of fiber length showed greater strains for the disc-only definition in compressive loading. Fiber strains were less than in the constant-volume model of comparable dimensions in compressive loading by a factor of about two, thus suggesting fluid loss or end-plate deformations in the physiologic conditions. The mathematical model indicated that the surface strain for intervertebral discs is very sensitive to the disc-height: diameter ratio and to fluid loss from the disc but is less sensitive to the helix angle of the fibers.     

Protrusion of thoracic intervertebral discs

Summary A critical analysis of 23 cases of thoracal disc prolapse observed by the authors as well as of 168 cases gathered from the literature is made.The different etiological factors are considered and the signification of the different kind of traumas is stressed.It is shown that in cases with a long clinical evolution local pain may be absent and the absence of radicular pain in some cases may be due to the relative great distance between a median disc prolapse and the posterior root.In search for a more adequate technique for the removal of the prolapsed disc an analysis of the different surgical approaches is made.The results obtained in 19 cases aut of the total number of cases are presented because in 2 cases a coexistent vertebral tumour has been found and in 2 cases the time follow-up was too short. There was one post-operative death, 1 late death, 3 patients were not benefited by the operation, in 6 the result was fair, good in 4 cases and excellent in 4 cases.

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Zusammenfassung Die Autoren geben eine kritische Analyse von 23 eigenen und 168 Fällen des Schrifttums mit thorakalem Bandscheibenvorfall. Die einzelnen ätiologischen Faktoren werden dargelegt und die verschiedenen Arten von Traumen hervorgehoben. Es wurde gezeigt, daß bei Fällen mit langer klinischer Entwicklung örtliche Schmerzen fehlen können und daß das Fehlen von radikulären Schmerzen bei manchen Fällen auf dem relativ großen Abstand zwischen einem medialen Diskusprolaps und der hinteren Wurzel beruht. Auf der Suche nach einer zweckmäßigeren Technik für die Entfernung des Bandscheibenvorfalles werden die verschiedenen operativen Zugänge analysiert.Aus dem eigenen Krankengut werden die Behandlungsergebnisse nur von 19 Fällen wiedergegeben, weil bei 2 Fällen gleichzeitig Wirbeltumoren bestanden und bei 2 anderen Fällen die Beobachtungszeit noch zu kurz war. In der Serie findet sich ein postoperativer Todesfall und ein späterer Todesfall; 3 Fälle wurden durch die Operation nicht gebessert; bei 6 Fällen war das Ergebnis befriedigend, bei 4 gut und bei 4 ausgezeichnet.

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Resumen Se hace un análisis crítico de 23 casos de hernias y protusiones de los discos torácicos observados por los autores, así como de 168 casos recogidos de la literatura.Se consideran los diferentes factores etiolicos y la significación de las distintas clases de traumas.Puede ocurrir que en casos con una larga evolución clínica no exista dolor local y la ausencia de dolor radicular en algunos casos puede ser debida a la distancia relativamente grande entre el disco mediano prolapsado y la raiz posterior.En busca de una técnica más adecuada para la extirpación de los discos torácicos prolapsados se analizan las diferentes vias quirúrgicas de acceso.Se presentan los resultados obtenidos en 19 casos del total, ya que en 2 casos se encontró un tumor vertebral coexistente y en los otros 2 el tiernpo de observación era muy corto. Hubo una muerte postoperatoria, otra tardía, 3 enfermos que no se beneficiaron con la operación, otros 6 en los cuales los resultados fueron medianos, buenos en 4 y excelentes en los otros 4 casos.

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Résumé Analyse de 23 cas de hernies discales thoraciques vus par les auteurs, et de 168 cas rassemblés dans la littérature.Etude des différents facteurs étiologiques, et valeur des différentes sortes de traumatismes. On montre que dans les cas ayant une longue évolution clinique, la douleur locale peut manquer, et l'absence de douleur radiculaire dans quelques cas peut être dûe à la distance relativement grande, qui sépare une hernie discale médiale et la racine postérieure.Cherchant la technique d'exérèse du disque la meilleure, on fait une analyse des différentes voies d'abord chirurgicales.Les résultats obtenus dans 19 cas sont présentés (dans 2 cas, coexistait une tumeur vertébrale, et dans 2 autres cas, le recul était insuffisant). Il y a eu un décès post-opératoire, un décès tardif, 3 malades ne furent pas améliorés par l'opération, chez 6 le résultat fut moyen, bon dans 4 cas, excellent dans 4 cas.

     

番茄红素对D-半乳糖诱导衰老大鼠椎间盘的影响

目的 观察D-半乳糖诱导衰老大鼠模型椎间盘等的变化及番茄红素对其影响.方法 将30只SD大鼠随机分成对照组、模型组和干预组.模型组和干预组每天颈部皮下注射D-半乳糖,对照组用等量生理盐水替代,干预组每天用番茄红素灌胃.各组每周称体重,12周后处死并取椎间盘及血清观测.结果 与对照组相比,模型组椎间盘明显退变,干预组椎间盘轻微退变;模型组体重减轻(P<0.05)、抗氧化水平下降明显(P<0.05.P<0.01).而干预组较模型组体重及抗氧化水平有所升高(P<0.05·P<0.01).结论 D-半乳糖在诱导大鼠衰老的过程中可诱发椎间盘退变.而番茄红素可延缓椎间盘退变的进展.