Chemonucleolysis--an experimental histopathological study

To investigate the histopathological changes of the intervertebral disc after chemonucleolysis, experiments were performed on 21 monkeys. After the injection of chymopapain, animals were sacrificed at definite intervals up to 1 year. Histopathological studies on these specimens revealed disappearance of proteoglycan from the nucleus pulposus, inner annulus fibrosus and cartilaginous endplates as early as 1 day after injection. At 4 weeks, regeneration of proteoglycan was indicated by the recovery of positive Safranin-O (S-O) staining in the area of the nucleus pulposus. After 24 weeks, the entire intervertebral disc showed uniform S-O staining indicating further regeneration of proteoglycan. The matrix of the reformed nucleus pulposus contained increased amount of fibrous elements compared to the controls. These results indicate proteoglycan regeneration by chondrocytes after chemonucleolysis. The reformed nucleus was histopathologically different from the control.     

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.     

Changes with age in proteoglycan synthesis in cells cultured in vitro from the inner and outer rabbit annulus fibrosus. Responses to interleukin-1 and interleukin-1 receptor antagonist protein

STUDY DESIGN: Proteoglycan synthesis was examined in cells isolated from the inner and outer annulus fibrosus of young and old rabbits. Their responses to interleukin-1 alpha and interleukin-1 receptor antagonist protein were investigated. OBJECTIVES: To evaluate the age-related changes and the anatomically related differences in the function of intervertebral disc cells. SUMMARY OF BACKGROUND DATA: Proteoglycan content in the human intervertebral disc decreases with age. Age-related changes in intervertebral disc cell function, however, have not been fully investigated. METHODS: Japanese white rabbits aged 2 months (young group) and 3 years (old group) were used. The inner and outer layer of the annulus fibrosus were separated. The proteoglycan synthesis and release were measured in cells cultured with or without human recombinant interleukin-1 alpha and interleukin-1 receptor antagonist protein. RESULTS: The proteoglycan synthesis significantly decreased and the release rate significantly increased in the old rabbits, compared with the young ones. In the inner annulus, the inhibition of proteoglycan synthesis due to interleukin-1 alpha was greater in the old rabbits than in the young ones. In the old rabbits, interleukin-1-induced inhibition was more pronounced in the inner annulus than in the outer annulus. Interleukin-1 receptor antagonist protein suppressed inhibition of proteoglycan synthesis by interleukin-1 alpha in the two layers in both age groups. CONCLUSIONS: Both the decline in proteoglycan synthesis and the increased cell sensitivity to interleukin-1 alpha with age may contribute to the degradation of discs. The increase in cell response to interleukin-1 alpha in the inner annulus of rabbits may explain why the inner annulus and nucleus pulposus degrade earlier than the outer annulus in human discs. Interleukin-1 receptor antagonist protein could be useful in inhibiting the degradation of the disc.     

Chymopapain, chemonucleolysis, and nucleus pulposus regeneration

In the adult mongrel dog, in vivo injection of chymopapain into the intervertebral disc resulted in disc-space narrowing at two weeks, with a complete loss of proteoglycan (as indicated by safranin-O staining) from the nucleus pulposus, the cartilaginous end-plates, and the annulus fibrosus. As demonstrated by [35S]sulphate-labeling and proteoglycan isolation, the nucleus pulposus retained the ability to synthesize proteoglycans, but these were degraded by endogenous proteolytic activity. Three months after chymopapain treatment the intervertebral disc showed an increase in height. There was a return of intense safranin-O staining in the annulus and the cartilaginous end-plates, and very prominently in the nucleus. The proteoglycans that were present were recovered as aggregates, with the proteoglycan monomer being slightly larger than in the controls. Six months after chymopapain treatment the intervertebral disc had increased further in height, and normal histology had been restored. The chemical composition and physical properties of the proteoglycans that were isolated from the nucleus pulposus were essentially the same as those from the controls. These observations suggest that the nucleus can regenerate following the injection of chymopapain. Clinical Relevance: Our observations demonstrate that chymopapain has a profound but reversible effect on the intervertebral disc. The radiographic narrowing of the intervertebral disc following chymopapain injection correlates with the loss of proteoglycan content and structure. The restoration of normal disc height following chymopapain injection is explained by reconstitution of the intervertebral disc with normal proteoglycans. In experimental animals, chemonucleolysis with chymopapain appears to be less likely than surgical excision to permanently alter the biochemistry of the nucleus pulposus.     

Histological development of intervertebral disc herniation

Sagittal and horizontal sections of 257 intervertebral discs obtained at autopsy and material obtained from 441 operations for herniation of a disc were examined histologically. In the material that was taken at autopsy, myxomatous degeneration of the annulus fibrosus increased in proportion to the age of the subject. The bundles in the internal layer of the annulus fibrosus reversed their usual direction and showed myxomatous degeneration, sometimes resulting in posterior and anterior convex bulging in the internal layer of the anterior and posterior parts of the annulus fibrosus, respectively. When material from a disc was surgically removed as a single free fragment (as in a complete extrusion or a sequestration type of herniation), annulus fibrosus with myxomatous degeneration was found in most material, while the nucleus pulposus rarely was. These results suggest that, from the standpoint of pathomechanism, a protrusion type of herniation of the annulus fibrosus exists in which only the annulus fibrosus is protruded due to reversal of the bundles of the annulus fibrosus, without involvement of the nucleus pulposus. This type of herniation would be a separate entity from the protrusion type of herniation of the nucleus pulposus that occurs when the nucleus pulposus is protruded through a fissure in the annulus fibrosus.     

Histological changes in intervertebral discs after smoking and cessation: experimental study using a rat passive smoking model

Background Passive smoking has been reported to induce intervertebral disc degeneration in rats, and the objective of the present study was to histologically investigate changes in smoking-induced intervertebral disc degeneration after cessation of smoking. Methods Four-week-old rats were subjected to passive smoking for 8 weeks in a smoking box [20 cigarettes a day: one cigarette an hour (inhaled over 3 minutes and followed by ventilation with room air for 5 minutes)] to induce intervertebral disc degeneration. Smoke-free periods of different lengths were then established, and intervertebral discs were histologically analyzed. Results Immediately after 8 weeks of passive smoking, intervertebral discs exhibited cracks, tears, and misalignment of the annulus fibrosus, and increased fibrous tissue was seen in the nucleus pulposus. In addition, the level of interleukin-1β in intervertebral discs was higher in the smoking group than in the non-smoking group. After cessation, progression of degeneration ceased, and the matrix of the nucleus pulposus and annulus fibrosus exhibited increased fibrous connective tissue and proteoglycan. However, there were no changes in annulus fibrosus misalignment. Interleukin-1β levels also remained significantly elevated after 8 weeks of cessation. Conclusions While the annulus fibrosus degeneration caused by smoking was partially irreversible after cessation of smoking, the amount of mucin (proteoglycan) in the nucleus pulposus and annulus fibrosus tended to increase after cessation, thus suggesting the possibility that smoking-induced intervertebral disc degeneration can be repaired to some degree by cessation of smoking.     

Experimental model of disc herniations in rats for study of nucleolytic drugs

An experimental model of disc herniation in tail discs of rats is described. Constant result on nucleus hernia and intervertebral narrowing were obtained by an easy manipulation on numerous rats. Intradiscal injection of aprotinin produced a widening of the disc height. Trypsin, collagenase, chymopapain, and hyaluronidase induced a narrowing of disc height; trypsin induced macroscopic necrosis of the soft surrounding tissues; and collagenase had a destructive effect on nucleus pulposus, annulus fibrosus, and even on end-plates. Chymopapain and hyaluronidase acted mainly on nucleus pulposus. Hyaluronidase could be of interest as a nucleolytic drug and needs further studies on optimal dosage and lack of side effects in the surrounding tissues before injecting it into human discs.     

实验性脊柱内固定后相应区域椎间盘超微结构观察

目的　观察脊柱内固定后相应区域椎间盘的超微结构变化。　方法　日本大耳白兔2 4只,随机分成实验组和对照组,每组12只。实验组骨膜下游离T1 0 ～L3棘突和关节突,克氏针制成“L”形,将钢丝横行穿过T1 1、1 2 ,L1、2 的关节突关节,并与置于T1 1 ～L3棘突两旁的克氏针系紧,对相应区域的脊柱行内固定术。对照组未行手术,仅喂养至实验完成。术后6个月,对两组动物摄X线片观察1次,随后处死动物。取两组动物的L1 椎间盘组织(髓核、纤维环内侧及纤维环外侧)行透射电镜观察,对两组T1 2 、L2 椎间盘组织分别行水平面和矢状面透射电镜及扫描电镜观察。　结果　X线片显示,实验组与对照组椎体及椎间隙差别不明显;透射电镜与扫描电镜观察,实验组椎间盘的髓核、纤维环内层细胞的结构改变较纤维环外层早;对照组的髓核、纤维环内层细胞的结构改变与纤维环外层差别不明显。在退变的椎间盘基质中,蛋白多糖颗粒和特殊结构明显减少。髓核与纤维环基质内有蛋白多糖颗粒和一种特殊结构,而特殊结构在髓核与纤维环内层的形态不一致。　结论　脊柱内固定术后6个月,实验组在异常应力环境下发生椎间盘退变。髓核、纤维环内层基质内的特殊结构分布有特殊规律,与蛋白多糖颗粒在椎间盘退变中的生物学行为密切相关。     

Effects of axial traction stress on solute transport and proteoglycan synthesis in the porcine intervertebral disc in vitro

Summary The effects of axial traction stress on intradiscal hydration, solute transport and proteoglycan synthesis were examined in 658 porcine coccygeal intervertebral discs in vitro. Measurements were performed in three tissue fractions: nucleus pulposus, inner and outer annulus fibrosus. At 0.80 MPa traction stress, the equilibrium hydration did not change in the nucleus pulposus. However, in the inner and outer annulus, the equilibrium hydration was reduced, and the change led to an increase of the effective fixed charge density. Diffusion of solute to the nucleus pulposus was significantly suppressed at 0.80 MPa traction stress. The fluid flow of the intervertebral disc tended to be suppressed during the creep recovery process after compression. The proteoglycan synthesis rate in the outer annulus was markedly suppressed by traction stress of 0.80 MPa for 4 h, but not that in the nucleus pulposus. These results suggest that a prolonged excessive axial traction stress induces a decrease in tissue hydration in the annulus fibrosus, and this may lead to an increase in the fractional volume of solid in the matrix and tissue osmotic pressure, resulting in diffusion inhibition of solute and suppression of proteoglycan synthesis. Thus, prolonged and excessive spinal traction may accelerate disc degeneration.Presented at the annual meeting of the International Society for the Study of the Lumbar Spine, May 12–16, 1991, Heidelberg, Germany     

Postmortem changes in ultrastructures of the mouse intervertebral disc

To elucidate the effects of nutrition and oxygen deficiencies on the intervertebral disc, cell components of mouse intervertebral discs and their postmortem changes were observed by electron microscopy. The annulus fibrosus could be divided into an inner and outer region. The main cell components of the annulus fibrosus were fibroblast-like cells in the outer region and chondrocytes in the inner region. The nucleus pulposus consisted of massively packed notochordal cells. The cartilage plates could also be divided into two zones: articular cartilage and growth cartilage containing chondrocytes. Postmortem degenerative changes proceeded from the peripheral to the central parts of the intervertebral disc, ie, showing degeneration of first the fibroblast-like cells, next the chondrocytes, and finally, the notochordal cells. The findings suggest that cells situated at the periphery predominantly depend on aerobic metabolism, whereas the cells situated more centrally depend on anaerobic metabolism. Furthermore, postmortem changes of the nucleus pulposus were similar to age-related changes. The age-related changes or degeneration in the intervertebral disc appear to be related to deficiencies of nutrition or oxygen caused by changes in structures of the disc and the surrounding tissues.     

Histochemical and ultrastructural observations on brown degeneration of human intervertebral disc

Thirty-eight fresh human intervertebral discs collected during anterior interbody fusion surgery were histochemically and ultrastructurally analyzed for pigments. Macroscopically, five stages of degeneration were classified according to the color, fibrosis, and fragility of the nucleus pulposus of the discs. In order to demonstrate lipofuscin granules, specimens were subjected to special staining procedures, including carbol fuchsin lipofuscin stain, the Schmorl's reaction, and autofluorescence. Lipofuscin granules were distributed from the inner layer of the annulus fibrosus to the nucleus pulposus. Such granules were numerous in cases of slight or severe degeneration, whereas fewer granules were found in cases of moderate degeneration. However, the stage of macroscopic degeneration of the intervertebral disc did not necessarily correlate with the incidence of lipofuscin granules. By ultrastructural observation, the morphological features of the components of the intervertebral disc and the ultrastructure of the lipofuscin granule were clarified. The ultrastructure of the "brown degeneration" disc exhibited markedly increased amorphous electron-dense bodies located among collagen fibrils in the matrix.     

Elastic fibers in human intervertebral discs.

Although previous studies failed to demonstrate elastic fibers in intervertebral discs, electron microscopy of twenty human intervertebral discs obtained at autopsy and operation revealed characteristic elastic fibers in both the annulus fibrosus and the nucleus pulposus. Their contribution to the mechanical properties of the intervertebral disc remains to be determined.     

腰椎间盘MRI高信号区的组织病理学特点和临床意义

目的研究椎间盘源性下腰痛患者腰椎间盘纤维环后方MRI高信号区的组织病理学特征及其临床意义。方法对52例经保守治疗无效、CT片显示无腰椎间盘突出的下腰痛患者行腰椎MR检查及腰椎间盘造影术。男39例,女13例;平均年龄38.8岁。选择纤维环后方出现高信号区的部分病例行腰椎后路椎间盘切除、椎体间融合、椎弓根螺钉内固定术,术中收集包括高信号区部位的椎间盘。对标本行矢状面连续组织学切片,光镜下观察高信号区椎间盘组织的组织病理学结构,并分析其临床意义。结果在行腰椎间盘造影的52例142个椎间盘中,17例17个椎间盘显示高信号区,且在椎间盘造影过程中全部呈现2或3级的纤维环破裂和疼痛复制反应。敏感性和特异性均为100%。高信号区与纤维环破裂程度分级呈正相关,说明纤维环破裂程度分级越高,越易出现高信号区(R=0.462,P<0.01)。共收集11例患者11个椎间盘,组织学研究发现对应高信号区的椎间盘组织表现为沿纤维环裂隙形成的不同程度的血管化肉芽组织,有成熟的瘢痕化胶原组织。结论症状性下腰痛患者的腰椎MRI上有椎间盘高信号区,可以作为椎间盘源性下腰痛诊断的重要征象。     

Water, fixed charge density, protein contents, and lysine incorporation into protein in chymopapain-digested intervertebral disc of rabbit

Chymopapain (Discase) was injected at a dose of 0.125 nanokatal unit into the intervertebral discs of rabbits, and sequential changes in the metabolism of water, proteoglycan, collagen, and noncollagenous protein were investigated separately in the nucleus pulposus, anterior, and posterior anulus fibrosus. One week after chymopapain injection, the water and proteoglycan content was lower in all of the fractionated tissues of the anterior and posterior anulus and nucleus pulposus of the discs than in the control discs. In the anterior and posterior anulus, the proteoglycan content recovered after 12 weeks, but there was no recovery in the nucleus pulposus. The collagen content continued to increase up to the 12th week in the nucleus pulposus, while the noncollagenous protein content decreased in all tissue fractions after 1 week. In the anterior and posterior anulus, the content of noncollagenous protein recovered after 3 to 6 weeks, but there was no recovery in the nucleus pulposus. The lysine incorporation in collagen and noncollagenous protein was inhibited in all tissue fractions after 12 weeks, suggesting a decrease in synthetic activity. The intradiscal pressure calculated from proteoglycan hydration at 1 to 6 weeks after chymopapain injection showed a marked decrease to 0.8 to 0.9 atm, but it recovered to 1.6 atm after 12 weeks.     

Matrix replenishment by intervertebral disc cells after chemonucleolysis in vitro with chondroitinase ABC and chymopapain

BACKGROUND CONTEXT: One of the advantages of chemonucleolysis for the treatment of a herniated intervertebral disc is the potential for the disc to self-repair. It has been suggested that the enzymes used for chemonucleolysis differentially affect the potential of the disc cells to promote repair. PURPOSE: To test the ability of nucleus pulposus and anulus fibrosus cells to repair the extracellular matrix degraded in vitro by either chondroitinase ABC or chymopapain. STUDY DESIGN: An alginate cell culture system was used to monitor the progress of matrix repair after chemonucleolysis in vitro. METHODS: Rabbit nucleus pulposus or anulus fibrosus cells precultured for 10 days in alginate gel were briefly exposed to low concentrations of chondroitinase ABC or chymopapain and then returned to normal culture conditions for up to 4 weeks. At each time point, the contents of DNA and matrix macromolecules and proteoglycan synthesis were measured. RESULTS: The DNA content of enzyme-treated alginate beads during the following 4 weeks of culture was higher in the chondroitinase ABC group than in the chymopapain group (NP, p<.01, and AF, p<.05). The content of proteoglycan in beads containing nucleus pulposus and anulus fibrosus cells in the chondroitinase ABC group was higher than that in the chymopapain group (NP and AF, p<.001). The rate of proteoglycan synthesis and the content of collagen did not, however, differ between those two groups. CONCLUSIONS: Intervertebral disc cells exposed to chondroitinase ABC reestablish a matrix richer in proteoglycan than cells exposed to chymopapain. This may be because of differences in the substrate spectrum of each enzyme. Although these results cannot be translated directly to the in vivo situation, they suggest the possibility that cells in discs subjected to chondroitinase ABC-induced chemonucleolysis retain a greater ability to replenish their extracellular matrix with proteoglycans than cells in discs exposed to chymopapain.     

Three-dimensional observation of collagen framework of lumbar intervertebral discs.

Lumbar intervertebral discs obtained from rats, dogs and humans were examined by scanning electron microscopy. The nucleus pulposus was constructed of a loose network of fine fibrils and formed lamellated membranes in the peripheral areas. The annulus fibrosus was composed of concentric lamellae of fibrous bundles that ran uniformly in each lamella and crossed over to the bundles of adjoining lamellae. The lamellae were made of fine fibrils measuring 0.1-0.2 mu in diameter, corresponding to matured collagen fibrils. The cartilage plate consisted of a close meshwork of collagen fibrils which interconnected with the annular fibrils. From these results, it was concluded that the intervertebral disc was well developed for shock absorption at the light microscopic and ultrastructural levels. In specimens treated with chymotrypsin, the extra-fibrillar substances were easily digested in the nucleus, as well as in the annulus. The intervertebral disc may thus be easily affected by chemical agents.     

Enhanced prolylhydroxylase activity in the posterior annulus fibrosus of canine intervertebral discs following long-term running exercise

Summary The effect of long-term exercise on the intervertebral disc collagen concentration (hydroxyproline), collagen-synthesizing enzymes (prolyl-4-hydroxylase, PH, and galactosyl-hydroxylysyl glucosyltransferase, GGT) and hydroxypyridinium crosslinks was studied in ten female beagle dogs. The dogs were run on a treadmill for 1 year starting at the age of 15 weeks. The daily running distance was gradually increased to 40 km, which distance the dogs ran for the final 15 weeks. Ten untrained dogs from the same breeding colony served as controls. The nucleus pulposus and anterior and posterior halves of the annulus fibrosus of C2–3, T10–12, L4–5 disc segments were analysed. Crosslinks were measured from the anterior annulus fibrosus of the T10–11 disc. Hydroxyproline and hydroxypyridinium concentrations remained similar in both groups. PH and GGT were significantly elevated by running in the posterior annulus fibrosus of the thoracic and lumbar discs and in the lumbar nucleus pulposus. In the thoracic nucleus pulposus GGT was reduced significantly. The results suggest activated collagen metabolism in the posterior annulus fibrosus of the thoracic and lumbar discs as a result of locally increased strains on the spine.The research was carried out at the Department of Anatomy, University of Kuopio     

椎间盘源性下腰痛的发病机制

目的探讨椎间盘源性下腰痛的发病机制。方法收集腰椎后路切除的17例椎间盘源性下腰痛患者的19个经腰椎间盘造影术证实的疼痛腰椎间盘;同时收集12个在MRI T2加权像上信号强度明显减弱、无腰痛症状的生理老化椎间盘和10个正常对照椎间盘,行组织学检查和P物质、神经丝蛋白和血管活性肠肽的免疫组织化学染色检查。结果椎间盘源性下腰痛患者的疼痛椎间盘在组织学上的显著特征表现为,一条从髓核至纤维环外层的血管化肉芽组织条带区,其间伴有1个或多个裂隙;肉芽组织条带区与椎间盘造影术后CT上显示的纤维环裂隙一致,肉芽组织之外的纤维环结构基本正常。生理老化椎间盘和正常对照椎间盘表现为与年龄相关的改变。免疫组织化学染色显示,疼痛椎间盘中P物质、神经丝蛋白和血管活性肠肽3种神经肽阳性神经纤维分布数量和比例,较正常对照椎间盘和生理老化椎间盘明显增多;神经纤维主要沿伴有裂隙的肉芽组织条带区分布;疼痛椎间盘髓核中可见P物质和神经丝蛋白的阳性神经纤维分布。结论椎间盘后方神经分布广泛的肉芽组织条带区是椎间盘造影术疼痛和椎间盘源性下腰痛的起源部位。肉芽组织条带可能起源于椎间盘的创伤修复过程。生理老化椎间盘和疼痛椎间盘的差异是后者形成组织学上的肉芽组织条带区。     

退变颈椎间盘中IL-17表达与分布的研究

目的 观察退变颈椎间盘中自细胞介素-17(IL-17)的表达与分布,并探讨其与颈椎间盘退变发生发展的关系.方法 实时荧光相对定量PCR(RQ-PCR)检测30例退变颈椎间盘及10例正常对照椎间盘中IL-1β、IL-17、肿瘤坏死因子-α(TNF-α)和孤核受体(retinoid-related orphan re-ce...     

Analysis of rabbit intervertebral disc physiology based on water metabolism. I. Factors influencing metabolism of the normal intervertebral discs

Basic factors influencing the metabolism of intervertebral discs of rabbits were quantitatively analyzed based on the water metabolism. The blood flow surrounding the intervertebral disc was calculated using pharmacokinetic concepts from the data obtained by time-related tritiated water distribution analyses. The blood flow was estimated as 0.056 (mg/min/mg tissue) in the anterior annulus, 0.106 in the posterior annulus, 0.120 in the lateral annulus, and 0.084 in the nucleus pulposus, respectively (Experiment 1). Water content and fixed charge density in the intervertebral disc fractions also were measured (Experiment 2). The cations and uncharged small solutes transported into the disc tissue ranged in descending order from nucleus pulposus, lateral annulus, posterior annulus, to anterior annulus. The authors also calculated theoretically the swelling pressure of the proteoglycan in the intervertebral disc fractions from the results of Experiment 2. It was concluded that swelling pressure was highest in the nucleus pulposus, and lowest in the anterior annulus. The water in the posterior annulus is less exchangeable than in the other disc tissue fractions.