The role of mast cells in disc herniation inflammation.

STUDY DESIGN: A study of herniated lumbar disc tissue samples and control disc material to determine the presence of mast cells in disc herniations. OBJECTIVES: To analyze whether mast cells have any involvement in disc herniation pathophysiology and lumbar pain, because mast cells may have an important role in acute and chronic inflammatory responses. SUMMARY OF BACKGROUND DATA: Studies of inflammatory cells, biochemical mediators of inflammation, and tissue degrading enzymes have suggested that these factors may be involved--and perhaps play an important role--in the pathophysiology of lumbar pain and radiculopathy. Mast cells are known to play an important role in acute and chronic inflammatory responses. It was therefore of interest to clarify their possible role in intervertebral disc herniation inflammation. METHODS: Fifty herniated lumbar discs from 50 patients who had undergone disc surgery and three normal control discs were obtained. Sections from every disc then were examined histologically and immunocytochemically for mast cells by using monoclonal antibodies to either of two types of specific proteases of mast cells, tryptase and chymase. RESULTS: By none of the methods could any mast cells be observed in any of the control disc samples. With toluidine blue staining, mast cells were observed in 9 of 50 (18%) of discs. Mast cells immunoreactive to either tryptase or chymase were observed in 10 of 50 disc samples (20%) and immunoreactive for tryptase and chymase simultaneously in 4 of 50 disc samples (8%). However, the majority of the samples studied (80%) demonstrated immunoreactivity to neither tryptase nor chymase. Among the samples studied were five disc protrusions that totally lacked mast cells. CONCLUSIONS: A minority of disc herniations exhibited mast cells, as verified by toluidine blue staining and immunocytochemistry. The results may suggest a role of mast cells in intervertebral disc herniation inflammation, but only in a subset of these cases. Massive infiltration by mast cells never was observed.

A comparative immunohistochemical study of inflammatory cells in acute-stage and chronic-stage disc herniations.

STUDY DESIGN: Herniated lumbar disc specimens were obtained from patients undergoing surgical discectomy for persistent radicular pain (radiculopathy) and stained for inflammatory cells to determine their occurrence in relation to the duration of radicular pain and to analyze the role of the time factor in the inflammatory response. OBJECTIVES: To analyze the presence of inflammatory cells and their involvement in the pathophysiology of radicular pain and to determine whether there is a clear difference in the occurrence of inflammatory cells between the earlier phase of radicular pain (after herniation) and the later chronic stage. SUMMARY OF BACKGROUND DATA: Previously, inflammatory cells were reported in herniated disc tissues, and macrophages were most prevalent. Biologically active inflammatory mediators have also been repeatedly observed. However, there have been no observations regarding possible differences in the occurrence of inflammatory cells in radicular pain of different durations. METHODS: Forty-four herniated lumbar discs were obtained from 44 patients undergoing disc surgery. Two groups of 22 age- and gender-matched patients with comparable affected disc levels were studied. In the first group (acute group) pain duration ranged from 3 days to 21 days. In the second group (chronic group) pain duration was 6 months or longer. All disc herniation specimens were subjected to indirect immunocytochemistry to study and compare the presence of inflammatory cells. RESULTS: Inflammatory cells, predominantly macrophages, were observed in both groups. Macrophages were abundantly present in eight (36%) disc samples in the acute group; in three (14%) samples only few scattered macrophages were observed. In the chronic group, in nine (41%) disc samples, abundant macrophages were observed; in six (27%) there were a few scattered macrophages. In the acute group, in three (14%) disc samples abundant activated T lymphocytes were observed; in two (9%) there were only a few activated T lymphocytes, whereas in the chronic group abundant activated T lymphocytes were not seen; only a few scattered activated T lymphocytes were observed in five (23%) disc tissue samples. In two (9%) samples in the acute group, B cells were abundantly present, and in two (9%) only a few B cells were observed. In the chronic group, abundant B cells were seen in no samples, and only a few B cells were noted in one (5%) sample. Only in the acute group and only in lateral disc herniations were abundant lymphocytes observed. In disc samples from intraspinal herniations, acute and chronic, there were only abundant macrophages, not lymphocytes. CONCLUSIONS: Because of the small size of the study groups and the low prevalence particularly of lymphocytes in both groups, no major group differences were noted. The prevalence of macrophages was highest, similar in both groups, and was similar to the results in prior studies. The results indicate no major differences in the occurrence of inflammatory cells in acute and chronic disc herniations. They also indicate that only macrophages may have a clinical relevance in disc tissue inflammation.

An immunohistochemical study of nerve structures in the anulus fibrosus of human normal lumbar intervertebral discs.

STUDY DESIGN: The innervation of the anulus fibrosus of human macroscopically normal intervertebral discs from five patients was investigated immunohistochemically. OBJECTIVES: Immunoreactivity to general nerve markers (synaptophysin and protein gene product 9.5) and to neuropeptides (substance P and C-flanking peptide of neuropeptide Y) was studied. SUMMARY OF BACKGROUND DATA: In the lumbar disc of a newborn, free nerve endings have been demonstrated in the outer layers of anulus fibrosus. In degenerated and herniated discs, nerve structures have been shown to penetrate deeper into the anulus fibrosus. There are only a few studies on the innervation of normal adult intervertebral disc tissue. METHODS: Thin frozen sections of human normal lumbar intervertebral disc tissue were immunostained for general nerve markers and neuropeptides. RESULTS: Synaptophysin and protein gene product 9.5 immunoreactive nerve structures were observed penetrating 3.5 mm and 1.1 mm into the anulus, respectively. Immunoreactivity to C-flanking peptide of neuropeptide Y and substance P were observed at a maximum depth of 0.9 and 0.5 mm in the anulus, respectively. Antibodies to the former have been used to study sympathetic nerves, whereas substance P is a transmitter present in sensory nerves. CONCLUSIONS: In anulus fibrosus samples from macroscopically normal discs, a general marker for nerve endings can be found at a depth of a few millimeters, whereas neuropeptide markers show nerves only in the outermost layers of the anulus fibrosus. This absence of demonstrable nerves in deeper anulus fibrosus in normal discs is probably not a methodologic artifact, because blood vessels have also been demonstrated only at the disc surface. It is, however, possible that neuropeptide nerves also penetrate to a depth of a few millimeters, but that methodologic limitations permit the visualization of only the neuropeptide nerves closest to the disc surface. The results of the present study lend support to previous suggestions that, except at the surface, a normal intervertebral disc is almost without innervation.

Inflammatory cells, motor weakness, and straight leg raising in transligamentous disc herniations.

STUDY DESIGN: Possible statistically significant relationships between inflammatory cells and either motor weakness or straight leg raising were determined. OBJECTIVES: To look for any clinically relevant links between inflammatory cells in disc herniations and signs of radiculopathy. SUMMARY OF BACKGROUND DATA: Many studies have during recent years shown a presence of various types of inflammatory cells in disc herniations, but their clinical relevance has been questioned. To be clinically relevant, a presence of inflammatory cells should show a clear relationship to clinical evidence of nerve root involvement. Macrophages repeatedly demonstrated in a high proportion of disc herniations studied are of particular interest. Their major role may be in disc herniations tissue resorption and not in sciatica. METHODS: A total of 96 disc herniations, all transligamentous, were analyzed by immunohistochemistry for presence of macrophages, T or B lymphocytes, and activated T lymphocytes separately. From recorded patient data, motor weakness and straight leg raising data were compared with a presence or absence of abundant (+ = at least 20 cells in a group) inflammatory cells. When not abundant, inflammatory cells were classified as "only few cells" (+) and grouped together with "no cells" (-). Patients with or without motor weakness were compared. Straight leg raising was compared for a positive (at <70 degrees ) or a negative test, and separately using the median as cut-off value. Groups were compared by chi-square analysis with the level of statistical significance set at P<0.05. RESULTS: None of the four inflammatory cell types showed any significant association with motor weakness. Nor was any association observed when comparing positive and negative straight leg raising. With the median (straight leg raising = 47.5 degrees ) as cut-off, only activated T cells showed a weak (chi2 = 4.40, P<0.05) relationship with tighter straight leg raising, but none of the other cell types did. Even when straight leg raising was < 47.5 degrees, three times more disc herniations lacked (n = 34) inflammatory cells than showed (n = 13) inflammation. In a subgroup of only sequestrated discs, the findings were similar. However, in the patients with a bilaterally positive straight leg raising (n = 25), the prevalence of at least one inflammatory cell type was much higher in sequestrated discs (80%) than in extrusions (33%). This may suggest more subtle interrelationships between type of disc herniation, straight leg raising, and inflammatory cells. CONCLUSIONS: The results of this study do not support a clinically relevant role for disc herniation inflammatory cells in sciatica. For the cells to be clinically relevant, a strong relationship between a presence of inflammatory cells and either or both of motor weakness and a tight straight leg raising should have been observed. The authors conclude that macrophages, which have been demonstrated in a high proportion of disc herniations in previous studies, are probably more important for disc tissue resorption processes than for producing sciatica. Other types of inflammatory cells are more rarely observed and may have no clinical meaning at all. However, more subtle interrelationships, considering the various types of disc herniations, should be further explored.

Basic fibroblast growth factor immunoreactivity in blood vessels and cells of disc herniations.

STUDY DESIGN: Basic fibroblast growth factor immunoreactivity was studied in disc herniation tissue. OBJECTIVES: The first objective was to analyze in which tissue components, if any, fibroblast growth factor is expressed in the disc herniation. The second objective was to compare such expression with that in fresh cadaver disc tissue. SUMMARY OF BACKGROUND DATA: Disc herniation tissue contains vascular ingrowth, which promotes the formation of granulation tissue. Fibroblast growth factor is a potent inducer of angiogenesis and also regulates extracellular proteolysis. METHODS: Twenty-seven disc herniation tissue and five macroscopically normal fresh cadaver discs were treated with an identical immunohistochemical protocol. Serial frozen sections were stained with a polyclonal basic fibroblast growth factor antibody and a polyclonal antibody to von Willebrand factor, which localizes endothelial cells. The immunostaining data were compared with relevant clinical data. RESULTS: Histologically, 74% of the samples contained anulus fibrosus and 59% nucleus pulposus. Basic fibroblast growth factor immunoreactivity was detected in 81% of the samples. There were immunopositive small blood vessels and scattered immunopositive disc cells (67%). Not all observed blood vessels were basic fibroblast growth factor immunopositive. In control discs, no immunoreactivity was observed. CONCLUSIONS: The observed presence of fibroblast growth factor in small blood vessels suggests an active angiogenesis as a result of disc injury. Cellular expression of fibroblast growth factor may be linked to proteolytic activity in disc extracellular matrix.

Inflammatory cells in experimental intervertebral disc injury.

STUDY DESIGN: Inflammatory cells were located by immunocytochemistry in areas of experimental intervertebral disc injury in pigs. OBJECTIVES: To study the occurrence of T lymphocytes and macrophages 1 week, 1 month, and 3 months after partial-thickness transverse scalpel injuries in pig lumbar discs. SUMMARY OF BACKGROUND DATA: Inflammatory cells and mediators recently have been observed in disc herniation tissue that was removed at disc prolapse surgery. The prevalence of inflammatory cell infiltrates in such clinical disc tissue material also has been studied. There are no studies, however, that have analyzed, using immunocytochemical methodology, the occurrence of, types of, and time dependence of inflammatory cells in an experimental disc injury model. The role of inflammation in intervertebral disc injury and repair has not been determined. METHODS: Transverse scalpel injuries 5-mm long and 4-mm deep were cut in the anterolateral anulus of L5-L6 and L4-L5 discs in 16 pigs. The cuts in the center of the anulus did not reach the nucleus pulposus and never produced a disc prolapse. In every pig, two non-adjacent lumbar discs (L1-L2 and L2-L3) were used as controls. Four discs per animal were studied in parallel by two different complementary immunohistochemical staining protocols. T lymphocytes and macrophages were located immunohistochemically using CD3 and CD68 antibodies, respectively. Discs were removed for analysis from four pigs at 1 week, from six pigs at 1 month, and from six pigs at 3 months. Inflammatory cells were categorized by two independent observers as being entirely absent (-), only few scattered cells (+), and at least one larger cellular infiltrate (+2). RESULTS: In none of the discs could extensive inflammatory cell infiltration be observed. T lymphocytes were present in significantly more sections cut from injured discs than in sections cut from control discs. The difference was highly significant particularly at 1 week and 1 month after disc removal. Only the 1-month-after-injury sections from injured discs exhibited significantly more macrophages than those from control discs. CONCLUSIONS: The results suggest the presence of only modest inflammatory cell infiltration in experimental intervertebral disc injury at all follow-up times. The inflammatory response in partial-thickness anterior experimental intervertebral disc injury, in the absence of disc prolapse, seems to be dominated by a T lymphocyte response. The macrophage response is apparently strongest at 1 month after such injury. These findings differ from what has been observed in herniated disc tissue.

Comparison of the prevalence of inflammatory cells in subtypes of disc herniations and associations with straight leg raising.

STUDY DESIGN: The prevalence of inflammatory cells in 205 disc herniations (DHs) and nine macroscopically normal discs for comparison was studied immunohistochemically. Inflammatory cells were separately analyzed in subtypes of DH. Immunohistochemical data were related to clinical parameters, the straight leg raising test (SLR) in particular. OBJECTIVES: The objectives of the study were to compare the occurrence of inflammatory cells in various subtypes of DH and to determine the association between clinical data and inflammatory cell occurrence in a more extensive sample of DH, with separate analysis of DH subtypes. SUMMARY OF BACKGROUND DATA: Previous studies have suggested a common occurrence of inflammation and inflammatory cells, particularly macrophages, in DHs. No studies on any larger material comprising different subtypes of DH have been done. METHODS: For immunohistochemistry the alkaline phosphatase antialkaline phosphatase method was used. Monoclonal antibodies to T cells in general (CD2), activated T cells (CD25), B cells (CD22), and macrophages (CD68) were used. Obtained immunostaining results were then compared with clinical data, e.g., duration of pain, SLR, and type of DH (sequesters 86, extrusions 103, protrusions 16). Associations were studied by the chi2 test or Fisher's exact test, as applicable (level of significance P < 0.05). RESULTS: Abundant T cells were seen in 17% of the 205 DHs, activated T cells in 17%, B cells in 16%, and macrophages in 37%. All cell types were 2-3 times more prevalent in sequestrated discs than in extrusions. In protrusions macrophages were abundantly seen in 25% (4 of 16) and no other inflammatory cells. In patients with positive SLR and a sequestrated disc abundant lymphocytes were seen three times more often than in extrusions. When patients with bilaterally negative SLR were compared with those with tight SLR (< or =30 degrees ) with respect to inflammatory cell occurrence, some significant differences were noted (CD68, P < 0.025; CD25, P = 0.04). A comparison between SLR bilaterally positive and bilaterally negative also showed associations for all four inflammatory cell types (P = 0.016 to P = 0.029). There was no correlation between inflammatory cells and duration of pain. Abundant inflammatory cells were never seen in control discs. CONCLUSIONS: When SLR was positive and the DH type was sequestered, inflammatory cells were most commonly seen. Our results showed some statistically significant associations between inflammatory cells and SLR, most clearly when comparing bilaterally positive and negative SLR. Interestingly, a bilaterally positive SLR showed an association with all four inflammatory cell types analyzed. Tight SLR also showed an association, particularly with macrophages. In addition to tissue resorption, they may participate in sciatic pain. Even though lymphocytes were less prevalent, they may have some role in sequestered discs and bilaterally positive SLR.

Inflammatory cells in full-thickness anulus injury in pigs. An experimental disc herniation animal model.

STUDY DESIGN: Inflammatory cells were studied by indirect immunocytochemistry in experimental full-thickness anulus fibrosus lesions in pigs. OBJECTIVES: First, to determine the occurrence, by immunocytochemistry, of T lymphocytes and macrophages in experimentally produced, anterolateral full-thickness disc lesions in pigs, and second, to compare the presence of inflammatory cells in 1) the injury area, 2) the adjacent noninjured part of the disc, and 3) control discs. SUMMARY OF BACKGROUND DATA: Previous studies on disc herniation material obtained from human disc surgeries have demonstrated inflammatory cells in a subgroup of herniations. Macrophages were most prevalent, being more numerous than lymphocytes. Macrophages have furthermore been suggested to be important in the resorption process of extruded disc tissue. No similar studies on an animal model of disc herniation, however, have so far been presented. METHODS: A full thickness anular incision, 10 mm long, was made with a scalpel in the L3-L4 or L4-L5 intervertebral discs of 12 adult pigs. The incision was made in the anterolateral part of the disc. Nucleus material was observed outside the injury site when tissue samples were taken, suggesting a disc herniation. Tissue then was analyzed from the area of injury, from the area adjacent to the injury, and from separate control discs from three additional pigs of the same age. Thin frozen sections were studied by indirect immunocytochemistry (alkaline phosphatase anti-alkaline phosphatase method) using monoclonal anti-human antibodies applicable to porcine tissues, T lymphocytes (CD3), and macrophages (CD68). Cells were graded as: -, absent; (+), only a few scattered cells; and +, abundant cells. Disc tissue samples were taken 1 month (three discs), 2 months (four discs), and 3 months (five discs) after the operation. RESULTS: Macrophages were present more commonly than T cells, and were abundant in seven of 12 discs (58%), with T cells abundant in four of 12 discs (33%). Only a few macrophages were present in the injured tissue from one additional disc, and scattered T cells were seen in four additional discs. Abundant macrophages were also observed in one of two discs in the adjacent noninjured area, whereas only a few T lymphocytes at the most were present in such noninjured disc tissue. In four (33%) and three (25%) injured discs, respectively, no macrophages or T lymphocytes could be found. No inflammatory cells were observed in three of 12 discs (25%). The three control discs showed no inflammatory cells. CONCLUSIONS: Inflammatory cells, predominantly macrophages, were present in a subsample of experimental discs with full-thickness anulus defects, as has previously been observed for human disc herniations. In this animal model, macrophages may have spread to adjacent noninjured parts of the disc. The induced herniation in this animal model is, however, anterolateral and may not fully correspond to clinical disc herniations, most of which are posterolateral. However, the results from this model support a role for inflammation in disc herniation.

Prevalence, morphology, and topography of blood vessels in herniated disc tissue. A comparative immunocytochemical study.

STUDY DESIGN: Ninety disc herniations removed during surgery were studied by immunocytochemistry, using two different endothelial cell markers, to study the prevalence, morphology, and topography of blood vessels in disc herniations. OBJECTIVES: To increase the specific localization of even very small blood vessels present in disc herniations by using specific antibodies to endothelial cells; to study blood vessels comparatively with two different endothelial cell antibodies, comparing their prevalence; and to study blood vessel morphology and topographic relationships of blood vessels to other tissue elements, particularly disc cells. SUMMARY OF BACKGROUND DATA: In many previous macroscopic studies and in studies using conventional histologic methodology, blood vessels have been observed in degenerated and injured intervertebral discs. In a smaller patient sample, the authors previously observed blood vessels in approximately 80% of disc herniations by immunocytochemistry, the blood vessels co-localizing with macrophage cells. Many of these blood vessels are the product of very active neovascularization after disc tissue injury. The presence of such blood vessels has not, however, been studied in greater detail or in larger patient samples. Immunocytochemistry offers superior visualization and more specific localization and was thus used in the present study. METHODS: Thin frozen sections from 90 disc herniations were immunostained in parallel with von Willebrand factor and Ulex europaeus antibodies, both of which localize endothelial cells specifically. Indirect immunocytochemistry by avidin-biotin-peroxidase complex or alkaline phosphatase-antialkaline phosphatase were used for immunolocalization. Blood vessels were classified as being: +, abundant: (+), very few; or +, totally absent. RESULTS: The prevalence of blood vessels in disc herniations was found in 82 of 90 (91%) disc herniations with von Willebrand factor antibody and in 75 of 90 (83%) disc herniations with Ulex europaeus antibody. In 59 disc herniations (66%), blood vessels were observed with both antibodies in parallel, whereas they were observed with neither antibody in only six of 90 disc herniations. Furthermore, the ratio of abundant to very few blood vessels was 73:9 with von Willebrand factor antibody and 63:12 with Ulex europaeus antibody, further supporting the abundance of blood vessels in disc herniations. Blood vessels were most prevalent in sequestrated discs, but they were also observed in six of eight protrusions. Dense blood vessel networks were observed to penetrate the disc tissue, and blood vessels were also present in areas of inflammatory cell infiltration. Topographically, blood vessels were, on several occasions and with both antibodies, seen to pass close by or to surround disc cells. CONCLUSIONS: By immunocytochemistry with endothelial cell markers, blood vessels can be observed to be numerous, and their prevalence in herniated discs is very high, presumably as a result of a very intense neovascularization process after the disc injury. A close apposition to disc cells may suggest attempts to increase the nutrition of these cells and will influence the metabolism of the cells.

Immunocytochemical localization of immunoglobulins in disc herniations.

STUDY DESIGN: Disc herniation and control discs were studied for the presence of immunoglobulins immunocytochemically. OBJECTIVES: To study a possible presence of immunoglobulin complexes in herniated disc tissue and to locate them at the tissue level by immunocytochemistry; to compare immunohistologic findings with those obtained in control disc tissue; and to compare the prevalences of immunoglobulin M and immunoglobulin G. SUMMARY OF BACKGROUND DATA: In herniated disc tissue, high activity of inflammatory phospholipase A2 was previously demonstrated, and inflammatory cells were noted immunohistochemically. Immunoglobulins G and M were observed biochemically but have not been located at the tissue level. METHODS: Fifty-two disc herniations and three macroscopically normal fresh cadaver discs were managed by an identical immunocytochemical protocol, using monoclonal antihuman antibodies to immunoglobulins M and G. RESULTS: In 29 of 52 disc herniations (56%), immunoglobulin M deposits were observed, and in 18 of 52 disc herniations (35%) immunoglobulin G could be demonstrated. Almost all the disc herniations where immunoglobulin G was present also contained immunoglobulin M deposits (except for two). In the control discs studied, neither immunoglobulin could be observed immunohistochemically. The immunoglobulin deposits were noted in areas where blood vessels were also present. Morphologically, immunoglobulin immunoreactivity resembling immune complexes was observed. CONCLUSIONS: The results lend support to previous suggestions of inflammation and immune reaction in disc herniations, including previous biochemical studies suggesting immunoglobulin deposition. The exact role of the demonstrated immunoglobulins in disc tissue pathophysiology will have to be clarified further.

Immunohistochemical demonstration of sensory and autonomic nerve terminals in herniated lumbar disc tissue.

STUDY DESIGN: Thirty-five lumbar disc herniations removed at surgery were studied by indirect immunocytochemistry. OBJECTIVES: To localize immunohistochemically both sensory and autonomic nerve terminals in disc herniations. SUMMARY OF BACKGROUND DATA: Using various more or less specific histologic and histochemical methods, investigators have reported the presence of free nerve terminals in disc tissue. However, very few studies have, to date, convincingly demonstrated nerve terminals in disc tissue that morphologically resemble the tiny nerve terminals of sensory and autonomic nerve fibers. METHODS: Amplification of the peroxidase reaction product in avidin-biotin-peroxidase complex immunostaining by the glucose oxidase-diaminobenzidine-nickel sulfate method was used to visualize small punctate nerve terminals at high magnification. Thin frozen sections from disc herniation tissue prefixed in Zamboni fixative were incubated with antibodies to synaptophysin to visualize nerve terminals in general, and with antibodies to substance P and C-flanking peptide of neuropeptide Y to further characterize nerve terminals as either sensory or sympathetic. RESULTS: Nerve terminals could be demonstrated in 29 (83%) of the 35 disc herniations. They were observed with the synaptophysin antibody in 17 of 35 (49%) disc herniations, with substance P in 16 of 35 (46%) disc herniations, and with C-flanking peptide of neuropeptide Y in 13 of 35 (37%) disc herniations. Morphologically, the nerve terminals were seen as tiny immunoreactive dots. Some of the nerve terminals were observed close to disc cells, possibly suggesting direct interaction. CONCLUSIONS: Small nerve terminals in disc herniations, both sensory substance P endings and sympathetic C-flanking peptide of neuropeptide Y endings, could be involved in mechanisms of discogenic pain, disc tissue neurogenic inflammation, tissue repair processes after injury, and control of local blood circulation in the newly formed blood vessels. Disc cells may be directly affected by the neuropeptides released from nearby nerve terminals.

A controlled immunohistochemical study of inflammatory cells in disc herniation tissue.

STUDY DESIGN: The presence and abundance of inflammatory cells was studied immunocytochemically in lumbar disc herniations (DH) and macroscopically normal discs for comparison. OBJECTIVES: The objective of the study was to characterize inflammatory cells that appear in herniated disc tissue and to study the relative abundance of various types of inflammatory cells. SUMMARY OF BACKGROUND DATA: Only few macrophages were observed in control discs, whereas abundant macrophages were present in half of the DH. Other types of inflammatory cells were less often abundant in the present material. In about a third of the DH interleukin-1 beta-expressing cells were also observed. METHODS: Twenty-four DH and control tissue from five discs were studied immunocytochemically, using specific monoclonal antibodies to various types of inflammatory cells and interleukin-1 beta. The results were compared with corresponding clinical data. Macrophages were studied with an antibody to CD68 antigen and Ber-MAC3 antibody separately. RESULTS: The obtained results suggest a variable inflammatory cell response in DH, which seems to be often dominated by macrophages at the time of operation. Thus previous suggestions of sometimes very active inflammation in DH tissue are supported. CONCLUSIONS: Inflammation may be important in disc tissue pathophysiology, possibly also in discogenic pain mechanisms.

A controlled biochemical and immunohistochemical study of human synovial-type (group II) phospholipase A2 and inflammatory cells in macroscopically normal, degenerated, and herniated human lumbar disc tissues.

STUDY DESIGN: Group II phospholipase A2 enzyme activity was studied biochemically and immunohistochemically in tissue samples from disc prolapses, degenerated discs, and macroscopically normal discs. In parallel, phospholipase A2 and inflammatory cells were studied by indirect immunocytochemistry. OBJECTIVES: To compare phospholipase A2 activity in normal discs and abnormal discs by an identical assay for phospholipases A2, and to compare the occurrence of inflammatory cells with phospholipase A2 activity and immunoreactivity. SUMMARY OF BACKGROUND DATA: It has been suggested that a high phospholipase A2 enzyme activity in herniated disc tissue could be significant in abnormal states such as sciatica and discogenic pain. No comparison between healthy disc tissue and samples of abnormal discs (degenerated or herniated) has been carried out. In particular, an identical assay for phospholipase A2 for such tissue samples, supported by immunohistochemical staining data, has never been applied in parallel to normal and abnormal disc tissue, and neither have such results been compared with the demonstration of inflammatory cells. METHODS: Group II phospholipase A2 enzyme activity was determined, in parallel, using an identical assay for tissue samples from 11 macroscopically normal discs, 33 disc herniations, and six discs showing degeneration by discography. For determination of phospholipase A2 enzyme activity, a radioassay using 1-palmitoyl-2-(1-14C)linoleoyl-L-3-phosphatidylethanolamine as the phospholipid substrate was used. Total tissue DNA as an estimate of total tissue cell number was measured in parallel with phospholipase A2 activity. All tissue samples also were studied by indirect immunocytochemistry, locating phospholipase A2 and T and B lymphocytes. RESULTS: Neither degenerated nor herniated disc tissue samples demonstrated a higher phospholipase A2 activity than control disc tissue samples. Average phospholipase A2 activity was actually higher in the control samples than in herniated disc samples (Mann-Whitney test, P < 0.001), possibly a result of a higher total DNA (P < 0.005). The observed level of phospholipase A2 activity was lower than that of inflammatory human synovial fluid. Neither was there marked immunoreactivity for phospholipase A2, which was observed in chondrocytes in areas of cartilage and occasional disc cells, supporting the biochemical results. Lymphocytes were more numerous only in herniated disc samples (15%), and their presence showed little overlap with phospholipase A2 immunoreactivity. CONCLUSIONS: Synovial-type (Group II) phospholipase A2 enzyme activity is not particularly high in disc tissue and does not appear to be higher in herniated or degenerated discs than control disc tissue. Immunoreactivity to phospholipase A2 is seen only occasionally and is strong only when cartilage tissue is present. Neither are inflammatory lymphocytes commonly observed.

Transforming growth factor beta receptor induction in herniated intervertebral disc tissue: an immunohistochemical study.

Transforming growth factor beta (TGF-beta) is a potent inducer of angiogenesis and fibrogenesis. There is presently little information about the pathophysiological function of TGF-beta in herniated disc tissue. In order to analyze the cellular role and activation of TGF-beta after disc herniation we immunostained frozen material from 38 disc herniation operations and from eight macroscopically normal discs from organ donors. Polyclonal TGF-beta-I, TGF-beta-II and TGF-beta receptor type II antibodies were used with the avidin biotin complex (ABC-) immunoperoxidase method. All the herniated discs were TGF-beta immunopositive. Such immunoreactivity was mainly associated with disc cells. In a few samples, capillaries were also TGF-beta immunopositive. Immunopositivity was similarly observed in the control discs. To analyze possible differences between the two groups, we calculated the ratio of immunopositive disc cells. For all three antibodies, a statistically significantly (Mann-Whitney test, P = 0.0001) higher number of disc cells showed immunopositivity in the herniated discs. The increase in TGF-beta receptor immunopositivity suggested induction of TGF-beta receptors in herniated discs. Our results support an active regulatory role for TGF-beta in disc cell metabolism.

Platelet-derived growth factor and vascular endothelial growth factor expression in disc herniation tissue: and immunohistochemical study.

Angiogenesis is essential in tissue growth and regeneration. There are several factors that are able to stimulate vascular endothelial cell growth, including platelet-derived growth factor (PDGF) and vascular endothelial growth factor (VEGF). Disc herniation tissue (DHT) contains vascular ingrowth, which promotes granulation tissue formation. In this study we observed 50 disc herniations for PDGF and VEGF immunoreactivity. PDGF immunopositivity was detected in 38 samples (78%). In 28 samples (56%) there were PDGF immunopositive capillaries, PDGF immunopositive disc cells were detected in 19 samples (38%) and PDGF immunopositive fibroblasts in 6 DHT samples (12%). VEGF immunopositive capillaries were identified in 44 DHT samples (88%). For neither growth factor was immunopositivity dependent on preoperative radicular pain duration. In extrusions (n = 25) VEGF immunopositive capillaries were detected in 23 samples (92%) and PDGF immunopositivity in 21 samples (84%). PDGF immunopositivity was more commonly associated with capillaries than with nuclei of disc cells. In sequesters (n = 20) VEGF immunopositive capillaries were identified in all samples and PDGF immunopositivity in 16 (80%). As in extrusions, PDGF immunoreaction was more prevalent in capillaries than in disc cells. Patient age did not relate to VEGF expression. In all age groups it was higher than 80%. Thus capillaries in disc herniation tissue are evidently newly formed and our results demonstrate that PDGF and VEGF participate in the neovascularization process. The presence of PDGF in fibroblasts and in disc cells suggests that this growth factor regulates the function of these cells, possibly the proliferation of the cells and the production of extracellular matrix components.

Concomitant immunocytochemical study of macrophage cells and blood vessels in disc herniation tissue.

Twenty disc herniations (DH) were studied immunocytochemically for macrophages and blood vessels. Serial thin frozen sections were immunostained with an antibody specific for tissue macrophages (monoclonal antibody to CD68 antigen) and the endothelium of blood vessels (polyclonal antibody to von Willebrand factor). With this method, blood vessels, often abundant, were observed in as many as 16/20 (80%) of the DH studied, 12 disc extrusions and 8 sequestrated discs, whereas abundant macrophages were noted in 11/20 (55%) of the DH. Macrophages were present only in areas with blood vessels and had presumably infiltrated the tissue from them. As has been noted previously, some blood vessels are apparently newly formed as a result of tissue injury, whereas others were present in the disc prior to herniation. This is suggested by the lack of a clear correlation between the presence or absence of blood vessels and the preoperative duration of radicular pain. In areas of the DH where cartilage fragments occurred, both macrophages and blood vessels were particularly abundant.

Comparative immunohistochemical study of group II (synovial-type) and group IV (cytosolic) phospholipases A2 in disc prolapse tissue.

Phospholipase A2 (PLA2) has been suggested to be present in herniated disc tissue and it could possibly be involved in sciatica/ discogenic back pain mechanisms. In the present study the occurrence of two different phospholipase A2 enzymes, (1) low molecular weight (14 kDa) group II synovial-type (sPLA2) and (2) high molecular weight (85 kDa) group IV cytosolic (cPLA2), were compared. Fifty-three disc prolapses obtained at disc operations were analyzed by immunohistochemistry, using anti-human monoclonal antibodies to sPLA2 and cPLA2, respectively. Only cell-associated (disc cells, hyaline cartilage chondrocytes) sPLA2 and cPLA2 immunoreactivity could be observed. The results showed that sPLA2 was more common (25/53, 47%) than cPLA2 (13/53, 25%). sPLA2 and cPLA2 were simultaneously present in 13 of 53 samples (25%). However, both PLA2 enzymes were predominantly present in hyaline cartilage cells (sPLA2: 16/53, cPLA2: 5/53), being less commonly observed in disc cells (sPLA2: 6/53, cPLA2: 3/53). In addition, three samples for sPLA2 and two samples for cPLA2 exhibited immunoreactivity in cartilage and disc cells simultaneously. sPLA2 was observed in no other locations, but in 3 of 53 samples cPLA2 was observed more diffusely in areas of granulation tissue, possibly in macrophages. No gender- or age-related dependence for either type of PLA2 enzyme immunoreactivity could be observed. Neither did their occurrence relate to clinical data such as straight leg raising or neurological deficit. The results do not support a major role for either of the two disc-cell-associated PLA2s in disc pathophysiology. For both enzymes, the major pool appears to reside in cartilage tissue cells, presumably in dislodged end-plate fragments. Disc cells are apparently unlikely candidates for major PLA2 storage.