Erkki Karaharju

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.

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.

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 llgamentous 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.

Effect of rigid plate fixation on structure and mineral content of cortical bone.

The effect of rigid plate fixation on the structure and mineral content of cortical bone were studied by light and fluorescence microscopy and by chemical analysis of calcium and hydroxyproline content in 27 rabbits from one day to 17 weeks after the attachment of a 5 hole ASIF-plate onto intact femur. Between the second and seventeenth postoperative weeks the plated cortical bone became progressively thinner and porotically transformed. Subendosteal resorption was partly offset by the formation of new subperiosteal bone that resulted in a slightly wider than normal, thin-walled tubular bone. At 17 weeks the mean calcium content of the tubular bone under the plate was 15% less than the initial postoperative content (p less than 0.05). The corresponding decrease in calcium content in the metaphyseal and epiphyseal areas of both the operated and the control bone was highly significant. The mean values for hydroxyproline in plated and control bones remained largely unchanged.

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 vasular 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 immuunohistochemical 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 fib roblast, growth factor immunopositive. I 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 proteolytia activity in disc extracellular matrix.

Ring chromosomes in parosteal osteosarcoma contain sequences from 12q13–15: A combined cytogenetic and comparative genomic hybridization study

Seven parosteal osteosarcoma (POS) samples, six of which were cytogenetically characterized, were studied by using comparative genomic hybridization (CGH). All samples showed DNA sequence copy number changes (mean, six aberrations/tumor; range, 1–13); gains were more frequent than losses. Gain of 12q13–15 sequences was found in every tumor and correlated with the presence of ring chromosomes. High‐level amplification, which was detected in four tumors, was seen only in chromosome 12, with 12q13–14 as the minimal common region. By using chromosome painting, one of the rings of one case was shown to be composed entirely of chromosome 12 material. Together with previous data, our findings show that gain of 12q13–15 sequences is a characteristic feature of POS and that these sequences are contained within the ring chromosomes. Genes Chromosom Cancer 16:31–34 (1996).

External fixation of the pelvic girdle with a trapezoid compression frame

The compression obtained with external anchorage in the anterior and posterior part of the pelvic girdle has been studied with a measuring device in the sacroiliac joint and and the symphysis of a specimen pelvis. Two models of external frames were tested, in both of which anchorage to the pelvic bone was brought about with 3 pins inserted in the iliac crests. The results were best with a trapezoid frame and bar exerting a compressive force on the pelvic girdle. When mounted with an inclination of 70 degrees to the long axis of the body, the frame exerted considerable compression on the posterior part of the pelvic girdle. This mounting afforded good stability of the detached hemipelvis. The trapezoid compression frame is recommended as an alternative to conventional methods of treating unstable fractures and dislocations of the pelvic girdle.

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 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.

Innervation of synovial membrane and meniscus

Substance P-immunofluorescent nerves, which are closely connected to pain transmission, were shown in human knee synovial membrane and menisci. Both tissues also contained enkephalin-immunofluorescent nerves, which are probably involved in the modulation of pain transmission. Previous suggestions on the presence of nociceptive receptors in these non-cartilaginous joint structures, made on a histological basis, are thus confirmed by a specific immunohistochemical method.