In vivo detection of intervertebral disk injury using a radiolabeled monoclonal antibody against keratan sulfate.

UNLABELLED: In the intervertebral disk, proteoglycans form the major part of the extracellular matrix, surrounding chondrocytelike disk cells. Keratan sulfate is a major constituent of proteoglycans. METHODS: We have radioiodinated a monoclonal antibody raised against keratan sulfate. This antibody was injected into rats (n = 6), and the biodistribution was studied. A model of intervertebral disk injury was developed, and two tail disks in each animal with both acute (2 wk old) and subacute (7 wk old) injuries were studied for in vivo antibody uptake. RESULTS: The biodistribution at 72 h was as follows: blood, 0.0018 percentage injected dose per gram of tissue (%ID/g); lung, 0.0106 %ID/g; esophagus, 0.0078 %ID/g; kidney, 0.0063 %ID/g; liver, 0.0047 %ID/g; spleen, 0.0046 %ID/g; heart, 0.0036 %ID/g; thyroid, 0.0034 %ID/g; muscle, 0.0017 %ID/g; and bone, 0.0016 %ID/g. In the subacute stage, a significant difference (P < 0.006) was found in antibody uptake between injured disks (n = 12) and adjacent healthy disks (n = 12). In vivo gamma imaging showed increased uptake in other animals having lumbar disk injuries (2, 7, and 17 d after injury). Cartilage tissue, such as the trachea, was studied separately and showed extremely high antibody uptake, 0.10 %ID/g. Rat trachea was also visualized on gamma images. CONCLUSION: Our data suggest that antibodies against nucleus pulposus components, such as proteoglycans, can be used for in vivo detection of intervertebral disk injury. This finding is in spite of the minimal circulation present in intervertebral disks.

Tissue reactions around two alloplastic ligament substitute materials: experimental study on rats with carbon fibres and polypropylene.

The early connective tissue reactions around two alloplastic and prosthetic materials, carbon fibre and polypropylene were examined in 30 rats. Each of the materials was implanted into three sites: under the skin, intraperitoneally and into a femoral bone tunnel. Microscopic and scanning electron microscopic findings at 1, 3, 6, 9 and 12 wk post-operatively are described. The amount of granulation tissue around the carbon fibres was more prominent in all the environments, but the connective tissue remained less mature and less organized during the whole follow-up compared to that of the polypropylene. There was no ingrowth of bone between the bundles of the artificial materials in the bony tunnels. The differences in the early cellular reactions and scar tissue maturation in the interstices of these two materials are probably due to the structural differences rather than to the surface properties of the materials.

Fibroblasts in synovitis in rheumatoid arthritis but not in noninflammatory synovial tissue in meniscus lesions express the carboxyterminal domain of procollagen type I.

Synovial tissue (ST) sections from patients with rheumatoid arthritis (RA) and meniscus lesions were stained using monoclonal antibodies against the carboxyterminal domain of human type I procollagen (alpha-pC) in avidin-biotin-peroxidase complex (ABC) staining. This gave a good signal-noise ratio and identified some synovial B-type lining cells and stromal fibroblasts in inflammatory RA ST but not in noninflammatory ST from patients with meniscus lesions. The authors' findings provide immunohistochemical evidence that the local fibroblasts in inflammatory ST in RA are activated, probably as a result of various humoral mediators produced in situ (by inflammatory mononuclear cells) in RA but not in normal noninflammatory ST.

Serotonin-like immunoreactivity in rat carotid body.

Serotonin-like immunofluorescence was demonstrated in the glomus cells of the rat carotid body. Similar immunoreactivity was noted in mast cells in the organ, while no immunoreactive nerve fibers were seen. It is suggested that glomus cell serotonin could participate in the modulation of chemoreceptor activity.

Intracellular free [Ca2+] and [Na+] in response to capsaicin in cultured dorsal root ganglion cells.

Intracellular free Ca2+ and Na+ concentrations in cultured rat dorsal root ganglion cells were studied using the intracellular fluorescent probes fura-2 and SBFI, respectively. Capsaicin increased both intracellular [Ca2+] and [Na+]. A rise in cytosolic free Ca2+ could still be seen if Na+ was removed from the outer medium. The results suggest that capsaicin opens up ion channels which are permeable both to Ca2+ and Na+.

Relationship of the Pain Disability Index (PDI) and the Oswestry Disability Questionnaire (ODQ) with three dynamic physical tests in a group of patients with chronic low-back and leg pain.

OBJECTIVE: To determine the relationship between overall disability in daily activities, assessed with the Pain Disability Index (PDI) and the Oswestry Disability Questionnaire (ODQ), and impaired performance on three physical tests in patients with chronic low-back pain. DESIGN AND SUBJECTS: The PDI and ODQ were administered in a cross-sectional study, before beginning a back rehabilitation program, to 45 patients with low-back pain of > or = 3 months' duration, with or without radiation to the legs. All patients also performed repetitive sit-up, arch-up, and squatting tests. SETTING: Tertiary care center. RESULTS: Modestly significant (p < 0.05) or significant (p < 0.01) inverse correlations (Pearson's r = 0.30-0.41) were noted between the PDI and the ODQ and all three physical performance tests. When normative data were used, the correlation (Spearman's rs = -0.45) between PDI and the squatting test remained significant (p < 0.01), whereas it was modestly significant (rs = -0.33, p < 0.05) between the ODQ and squatting test and between the PDI and arch-up test (rs = -0.35, p < 0.05). Compared with patients presently working, those on sick leave had significantly higher scores on the PDI and ODQ (Wilcoxon's two-sample test: p < 0.001) and also significantly worse performance on all physical tests (p < 0.001). CONCLUSIONS: The PDI and ODQ, as measures of self-perceived disability, and impaired performance on repetitive squatting, arch-up, and sit-up tests, as measures of physical capability, show some overlap in low-back-pain patients. Both types of disability measures are clearly influenced by the patient's work status.

Intercorrelation and test-retest reliability of the Pain Disability Index (PDI) and the Oswestry Disability Questionnaire (ODQ) and their correlation with pain intensity in low back pain patients.

OBJECTIVE: To determine the intercorrelation between subjective disability, as assessed with the Pain Disability Index (PDI) and the Oswestry Disability Questionnaire (ODQ) and their correlation with visual analogue scale (VAS) pain intensity ratings. DESIGN AND SUBJECTS: Questionnaires were administered to 94 patients with chronic low back pain with or without radiation into the legs of at least 3 months' duration. SETTING: Tertiary care center. RESULTS: High correlations were noted between the ODQ and PDI (r = 0.83) and PDI factor 1 (r = 0.84), a subscale of the PDI. Lower correlations were noted between pain intensity (VAS) scores and the ODQ (r = 0.62) and the PDI (r = 0.69). A weaker correlation (r = 0.41) was noted between the ODQ and PDI factor 2. Intraclass correlation coefficients (ICC) for test-retest reliability in 20 patients (time interval 1 week) were for the ODQ ICC = 0.83, PDI ICC = 0.91, PDI percentage score ICC = 0.91, PDI factor 1 ICC = 0.87, and PDI factor 2 ICC = 0.73, respectively. CONCLUSIONS: The present results suggest that either the PDI or the percentage score PDI and also the even shorter-to-administer PDI factor 1 may be useful and reliable tests for the assessment of subjective disability in low back pain patients. As noted by the moderate intercorrelations with pain intensity scores, both the PDI and the ODQ address a broader concept of disability than that directly related to pain intensity.

Relationship of subjective disability with pain intensity, pain duration, pain location, and work-related factors in nonoperated patients with chronic low back pain.

OBJECTIVE: A cross-sectional study on patients with chronic low back pain to compare relationships between subjective disability and pain intensity, pain duration, pain location, and work-related factors. DESIGN AND SUBJECTS: One hundred and seven outpatients with low back pain, with or without radiation to the legs, of at least 3 months' duration, were administered a comprehensive back patient questionnaire. The questionnaire included the Pain Disability Index (PDI) for the assessment of overall perceived disability and assessments of pain intensity, duration, and location and two pretested short-form work questionnaires. None of the patients had undergone a back operation. SETTING: Tertiary care center. RESULTS: There was evidence of significant interrelationships between the PDI and pain intensity (low back pain, r = 0.53, p < 0.001; leg pain, r = 0.32, p < 0.01; and buttock pain, r = 0.36, p < 0.01), pain location (significantly higher scores with distal pain radiation), and work-related factors (a work load sum score of r = 0.31, p < 0.01; significantly higher scores in patients on sick leave). CONCLUSIONS: The results suggest that subjective disability in patients with chronic low back pain overlaps with both pain and work-related factors. The observations support the multidimensionality of low back disability.

Fibroblast markers in labial salivary gland biopsies in progressive systemic sclerosis.

Labial salivary gland (LSG) biopsies from 13 patients were studied. Three were normal glands, five showed fibrosis induced by progressive systemic sclerosis (PSS) and five more had PSS-induced fibrosis combined with and focal sialadenitis compatible with Sjögren's syndrome (SS). Monoclonal antibodies to proline-4-hydroxylase (alpha PH or 5B5-A) and the carboxyterminal domain of human type I procollagen (alpha pC or M-38) were used as fibroblast markers. Immunostaining was done with avidin-biotin-peroxidase complex (ABC). Using various sample controls (including cultured fibroblasts and specimens enriched for lymphocytes, plasma cells, granulocytes, monocytes and dendritic cells) as well as analysis of various LSG resident cells, the specificity of the alpha PH and alpha pC markers for fibroblasts was established. Cross reactions were only seen with plasma cells and acinar cells containing the beta subunit of PH or disulfide isomerase involved in SS-SH interchange reactions in these secretory cells. All fibroblasts in fibroblast monolayer cultures at their logarithmic phase of growth stained with the fibroblast markers studied, but false negative staining was seen with resting, mature fibroblasts in dense connective tissue in LSG sections. Therefore, it can be concluded that proline 4-hydroxylase and the COOH-terminal domain of type-I procollagen both indicate fibroblast involvement in collagen (type l) synthesis and thus identify active but not resting fibroblasts. PH+ fibroblast-like cells and pC+ fibroblasts were both more frequent in PSS LSGs than in normal glands, suggesting active local fibroblast involvement in PSS.(ABSTRACT TRUNCATED AT 250 WORDS)

Immunohistochemical demonstration of nociceptors in the ligamentous structures of the lumbar spine.

Substance P, a physiologically potent neuropeptide is known to participate in the sensory, and especially nociceptive, transmission of neural impulses. On histologic grounds, the nerve terminals of the sinuvertebral nerve formerly have been suggested to be sensory in character and to mediate the low-back pain syndrome. Samples of paramedullary ligamentous structures were collected on disc operations. A positive immunoreaction as an indicator of substance P was confirmed in some nerve terminals of the posterior longitudinal ligament. Neither the yellow ligament nor the intervertebral disc showed such nociceptive-type nerves.

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.

Silver impregnation and immunohistochemical study of nerves in lumbar facet joint plical tissue.

Impingement of plical synovial tissue in a facet joint could cause pain. Plical tissue was removed during surgery for recurrent disc herniation or spinal stenosis. The presence of nerves was studied with silver impregnation, immunofluorescence, and avidin-biotin-peroxidase complex (ABC) immunostaining. Heterologous antisera to protein gene product (PGP) 9.5, substance P, calcitonin gene-related peptide (CGRP), and galanin were used to stain nerves. After silver impregnation, nerve-like structures were observed perivascularly. Such nerves located close to blood vessels were also immunoreactive for PGP 9.5, a more general cytoplasmic neural marker, whereas only few perivascular small varicosities were seen with antisera to substance P and galanin and none with antiserum to CGRP. In addition, PGP-9.5-, substance-P-, and galanin-immunoreactive nerves were occasionally seen very near to fat globules. Very few peptide-immunoreactive nerve varicosities were seen with immunofluorescence, and none of the PGP-9.5-immunoreactive nerves that were observed with ABC immunostaining were immunoreactive for neuropeptides as well. One mechanism for pain production could be mechanical compression of fatty tissue, but it is considered more likely that nerves in this particular tissue are mainly involved in local vasoregulation and that they are not sensory nociceptive nerves.

Straight leg raising test and lumbar cerebrospinal fluid levels of vasoactive intestinal polypeptide and somatostatin in patients with low back pain.

STUDY DESIGN: Straight leg raising was recorded before myelography in 77 patients. At myelography, samples of cerebrospinal fluid were drawn and later analyzed for neuropeptides vasoactive intestinal polypeptide and somatostatin. OBJECTIVES: The study sought to examine correlations, if any, between a positive straight leg raising test and cerebrospinal fluid neuropeptide levels. METHODS: The straight leg raising test was recorded for all patients before a myelography examination was performed because of intractable leg pain symptoms. Forty-seven of the patients were men and 30 were women. Cerebrospinal fluid samples were obtained from all patients upon myelography. Levels of the neuropeptides vasoactive intestinal polypeptide and somatostatin were analyzed in a blind manner by radioimmunoassay, using commercially available radioimmunoassay kits. RESULTS: The results are compatible with previous observations that suggest cerebrospinal neuropeptide levels are altered in conjunction with neural injury or pain syndromes. In the present mixed back pain patient population, which included radicular pain symptoms due to disc herniation and lumbar stenosis, alterations in vasoactive intestinal peptide levels in particular were observed with a positive straight leg raising test. CONCLUSIONS: Nerve root injury, as suggested by a positive straight leg raising test, appears to be neurochemically linked to altered cerebrospinal fluid vasoactive intestinal peptide levels.

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.