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Innervation of Spinal Ligaments of Patients with Disc Herniation
Path. Res. Pract. 187, 936-938 (1991)
Innervation of Spinal Ligaments of Patients with Disc Herniation 1 An Immunohistochemical Study L. H. Yahia Institute of Biomedical Engineering Ecole Polytechnique de Montreal, Canada
N. Newman Hotel-Dieu de Montreal, Canada
SUMMARY
The purpose of this work was to demonstrate whether neural elements are present in the spinal ligaments removed from patients with disc herniation. The tissue samples were stained by an immunohistochemical technique using antibodies to neurofilament protein (NFP) subunits as specific markers. Numerous NFP-immunoreactive nerve fibers and free nerve endings were demonstrated within the ligamentous structures. These findings were discussed relative to the low-back pain of disc herniated patients.
Introduction The nerve supply of the posterior ligaments of the lumbar spine is at present debatable. Hirsch et al.2 using the methylene blue immersion technique, found fine free fibers and complex unencapsulated endings in the supraspinous (SSL) and interspinous (ISL) ligaments. Jackson et al.3, on the other hand, were unable to identify such nerve endings in the same structures. Pion chon et a1. 5 have applied immunohistochemistry for S-100 protein to the staining of neural elements, and found only free nerve endings in these ligaments. Recently, we have used a modified gold chloride staining technique, and revealed the presence of Ruffini receptors and Pacini an corpuscles, in addition to the free nerve endings 6 • This investigation was then extended to a study of neural elements found in spinal ligaments obtained from degenerated lumbar spine where metaplasia of the ligamentous tissue into fibrocartilage is known to occur7 . The staining results were not satisfactory due probably to lack of stain penetration into 1 This work was supported by the "Fondation de Recherche et d'Education en Orthopedie de Montreal (FREOM)".
© 1991 by Gustav Fischer Verlag, Stuttgart
the fibrocartilaginous tissue as suggested by Zimny et al.2. In view of these poor results, it was decided to use the antibodies to neurofilament protein subunits as specific markers to locate the neural tissue of the spinal ligaments immunohistochemically. Material and Methods (i) Patients. Surgical specimens of the ISL and SSL ligaments were obtained from 7 patients, who underwent surgery for discectomy. The tissue samples were removed from the L 1/L 4 spinal levels. No specimens were removed from patients except those necessary to accomplish the required operation. The age ranged from 30 to 60 years, at the average of 45.8 years. As a control group for immunohistochemical staining, five specimens of the lumbosacral plexus were obtained during autopsy. (ii) Immunohistochemical procedure. The samples were fixed in 10% formalin, routinely embedded in paraffin and cut at 5-20 !tm thickness. The slices were allowed to adhere to glass slides and rinsed with phosphate buffer solution (PBS) at pH 7.6. The sections were treated with 3 % peroxide for 20 min and, again, rinsed in PBS. Before being digested with 0.1 % trypsin in 0.5% CaCh solution (15 min), they were immersed in distilled 0344-0338/91/0187-0936$3.50/0
Innervation of Spinal Ligaments in Disk Herniated Patients · 937
Fig. 1. NFP-immunoreactive nerve bundle showing perineurium (P) and endoneurium (END) in the interspinous ligament (x 630). water for 10 min at 37.5 °C. The floating sections were then incubated with monoclonal antibodies (raised in mice against human brain) to neurofilament polypeptides for 18-20 h. Antibodies against the 70 and 200 kdalton subunits (Dimension Lab., Missisauga, Ontario) were used at a dilution of 1 : 50 with PBS. After being rinsed in PBS the sections were incubated in goat anti-mouse antibody solution (dilution 1 :20 with PBS) for 1 h . Finally, the sections were stained with diamino benzidine (DAB) solution (Sigma Chemical Co., St-Louis, Mo) for 10 min. The solution was made by dissolving 12 mg ofDAB in 5 ml of PBS and one drop of hydrogen peroxide (H 20 2). The slides were then rinsed in PBS, dehydrated and mounted.
Results With the neurofilament protein (NFP) antiserum, it was possible to demonstrate nerve structures in all the ligamentous samples studied. The SSL and ISL ligaments showed various profiles of immunoreactive axons when stained with the monoclonal antibodies against 70 and 200 kdalton NFP subunits. Both individual axons and bundles ofaxons were seen in these ligaments (Fig. 1). Observation of horizontal sections showed that NFP-positive nerve fibers were located freely in-between the collagen fiber bundles (Fig. 2). Most of the NFP-positive nerves terminated within the ligaments as free endings; no specialized nerve terminals including Ruffini corpuscles were found in these ligaments. In the lumbosacral plexus (control), a major population of the axons was reactive to the NFP antiserum, although the intensity of the immunoreaction varied among axons (Fig. 3).
Fig. 2. NFP-immunoreactive nerve fibers (arrows) in the supraspinous ligament. The nerve fibers are closely related to the collagen fibers (C) and end freely within the ligament (x 630) .
Discussion Previously, using a gold chloride staining method, we have found free nerve endings in spinal ligaments and, in addition, specialized nerve receptors resembling Ruffini and Pacinian corpuscles 6 • In studies of spinal ligaments in young scoliotic patients, nerve endings were more numerous than in similar tissue taken from adults with disc degeneration and herniation. The present study demonstrates numerous neurofilament protein-immunoreactive nerve fibers in the supraspinous and interspinous ligaments of disc herniated patients. Most of the nerve fibers terminate as simple, free nerve endings resembling those described by Korkala et a1. 4 in the human posterior longitudinal ligament. These nerves were suggested to be responsible for pain production. Their presence in the pathological ligaments examined here has potential clinical significance in relation to low-back pain. In a parallel study7, we have seen degenerative changes in similar ligaments. The earliest ultrastructural evidence of degeneration appears as necrosis of cells, increase of proteoglycan concentration, and a transformation of fibroblasts into chondrocyte-like cells?, 8. One consequence of the chondroid metaplasia is that the deposition of calcium pyrophosphate crystals, which normally involves only cartilage itself, now also includes the chondroid-transformed ligament9 . Therefore, the free nerve endings revealed may be locally excited by these breakdown products of proteoglycans, crystals or cell debris as suggested by Brown 1 and Zimmermann 1o •
938· L. H. Yahia and N. Newman
Hopefully, therapy could be focused if the site and mechanism of pain were better known. Reviews which implicate the nerve structure and pattern usually differentiate these sites: the spinal ligaments, the apophyseal joint capsule and ligament, the posterior vertebral ligament, and flavum tissue. The immunohistological methods may expand our knowledge of differences and lead to more selective and more specific therapy. Acknowledgements The authors wish to thank Louise Clement for her assistance. This research was funded by the "Fondation de Recherche et d'Education en Orthopedie de Montreal (FREOM).
References
Fig. 3. A cross-section of a lumbosacral plexus (control) stained with antibody against NFP. Almost all axons are immunoreactive to NFP (x 250).
Finally, studies evolving from this report such as a comparison of distribution and number of nociceptive receptors in normal and pathological tissues should be an area for active research and observation. These studies might also illustrate mechanisms of nerve excitation whether it is accomplished by compression of nerves, ischemia or fibrosis, associated inflammatory tissue, or other differences. The clinician, faced with multiple back pain syndromes, is challenged by the enigma of the origin of pain.
1 Brown MD (1989) The source of low-back pain and sciatica. Sem in Arthritis and Rheumatism 18(4): 67-72 2 Hirsch C, Ingelmark BE, Miller M (1963) The anatomical basis for low-back pain. Acta Orthop Scand 33: 1-17 3 Jackson HC, Winkelmann RK, Bickel WH (1966) Nerve endings in the human lumbar spinal column and related structures. J Bone Joint Surg (Am) 48(7): 1272-1281 4 Korkala 0, Gronblad M, Liesi P, Karaharju E (1985) Immunohistochemical demonstration of nociceptors in the ligamentous structures of the lumbar spine. Spine 10: 156-157 5 Pionchon H, Tommasi M, Pia let J, Bancel B, Chazal J, Escande G, Scheye T, Vanneuville G (1986) Etude de l'innervation des ligaments rachidiens al'etage lombaire. Bull Ass Anat 70: 63-67 6 Yahia LH, Newman N, Rivard CH (1988) Neurohistology of lumbar spine ligaments. Acta Orthop Scand 59(5): 508-512 7 Yahia LH, Drouin G, Maurais G, Garzon S, Rivard CH (1989) Degeneration of the human lumbar spine ligaments. An ultrastructural study. Path Res Pract 184: 369-375 8 Yahia LH, Drouin G, Maurais G, Rivard CH (1989) Etude de la structure microscopique des ligaments posterieurs du rachis lomb a ire. Int Orthop 13: 207-216 9 Yahia LH, Garzon S, Strykowski H, Rivard CH (1990) Ultrastructure of the human interspinous ligament and ligamentum f1avum. Spine 15: 262-268 10 Zimmermann M (1989) Pain mechanisms and mediators in osteoarthritis. Sem in Arthritis and Rheumatism 18(4): 22-29 11 Zimny ML, St-Onge MF, Albright DJ (1987) The use of agents which improve staining of fresh human fibrocartilage. Stain Technol 62(5): 341-348
Received January 22, 1990 . Accepted in revised form November 28, 1990
Key words: Spinal ligaments - Innervation - Neurofilaments - Disc herniation Dr. L. H. Yahia, Institut de genie biomedical, Ecole Poly technique, C. P. 6079, Succ. "A", Montreal, Quebec, H3 C 3 A 7,Canada
Innervation of Spinal Ligaments of Patients with Disc Herniation 1 An Immunohistochemical Study L. H. Yahia Institute of Biomedical Engineering Ecole Polytechnique de Montreal, Canada
N. Newman Hotel-Dieu de Montreal, Canada
SUMMARY
The purpose of this work was to demonstrate whether neural elements are present in the spinal ligaments removed from patients with disc herniation. The tissue samples were stained by an immunohistochemical technique using antibodies to neurofilament protein (NFP) subunits as specific markers. Numerous NFP-immunoreactive nerve fibers and free nerve endings were demonstrated within the ligamentous structures. These findings were discussed relative to the low-back pain of disc herniated patients.
Introduction The nerve supply of the posterior ligaments of the lumbar spine is at present debatable. Hirsch et al.2 using the methylene blue immersion technique, found fine free fibers and complex unencapsulated endings in the supraspinous (SSL) and interspinous (ISL) ligaments. Jackson et al.3, on the other hand, were unable to identify such nerve endings in the same structures. Pion chon et a1. 5 have applied immunohistochemistry for S-100 protein to the staining of neural elements, and found only free nerve endings in these ligaments. Recently, we have used a modified gold chloride staining technique, and revealed the presence of Ruffini receptors and Pacini an corpuscles, in addition to the free nerve endings 6 • This investigation was then extended to a study of neural elements found in spinal ligaments obtained from degenerated lumbar spine where metaplasia of the ligamentous tissue into fibrocartilage is known to occur7 . The staining results were not satisfactory due probably to lack of stain penetration into 1 This work was supported by the "Fondation de Recherche et d'Education en Orthopedie de Montreal (FREOM)".
© 1991 by Gustav Fischer Verlag, Stuttgart
the fibrocartilaginous tissue as suggested by Zimny et al.2. In view of these poor results, it was decided to use the antibodies to neurofilament protein subunits as specific markers to locate the neural tissue of the spinal ligaments immunohistochemically. Material and Methods (i) Patients. Surgical specimens of the ISL and SSL ligaments were obtained from 7 patients, who underwent surgery for discectomy. The tissue samples were removed from the L 1/L 4 spinal levels. No specimens were removed from patients except those necessary to accomplish the required operation. The age ranged from 30 to 60 years, at the average of 45.8 years. As a control group for immunohistochemical staining, five specimens of the lumbosacral plexus were obtained during autopsy. (ii) Immunohistochemical procedure. The samples were fixed in 10% formalin, routinely embedded in paraffin and cut at 5-20 !tm thickness. The slices were allowed to adhere to glass slides and rinsed with phosphate buffer solution (PBS) at pH 7.6. The sections were treated with 3 % peroxide for 20 min and, again, rinsed in PBS. Before being digested with 0.1 % trypsin in 0.5% CaCh solution (15 min), they were immersed in distilled 0344-0338/91/0187-0936$3.50/0
Innervation of Spinal Ligaments in Disk Herniated Patients · 937
Fig. 1. NFP-immunoreactive nerve bundle showing perineurium (P) and endoneurium (END) in the interspinous ligament (x 630). water for 10 min at 37.5 °C. The floating sections were then incubated with monoclonal antibodies (raised in mice against human brain) to neurofilament polypeptides for 18-20 h. Antibodies against the 70 and 200 kdalton subunits (Dimension Lab., Missisauga, Ontario) were used at a dilution of 1 : 50 with PBS. After being rinsed in PBS the sections were incubated in goat anti-mouse antibody solution (dilution 1 :20 with PBS) for 1 h . Finally, the sections were stained with diamino benzidine (DAB) solution (Sigma Chemical Co., St-Louis, Mo) for 10 min. The solution was made by dissolving 12 mg ofDAB in 5 ml of PBS and one drop of hydrogen peroxide (H 20 2). The slides were then rinsed in PBS, dehydrated and mounted.
Results With the neurofilament protein (NFP) antiserum, it was possible to demonstrate nerve structures in all the ligamentous samples studied. The SSL and ISL ligaments showed various profiles of immunoreactive axons when stained with the monoclonal antibodies against 70 and 200 kdalton NFP subunits. Both individual axons and bundles ofaxons were seen in these ligaments (Fig. 1). Observation of horizontal sections showed that NFP-positive nerve fibers were located freely in-between the collagen fiber bundles (Fig. 2). Most of the NFP-positive nerves terminated within the ligaments as free endings; no specialized nerve terminals including Ruffini corpuscles were found in these ligaments. In the lumbosacral plexus (control), a major population of the axons was reactive to the NFP antiserum, although the intensity of the immunoreaction varied among axons (Fig. 3).
Fig. 2. NFP-immunoreactive nerve fibers (arrows) in the supraspinous ligament. The nerve fibers are closely related to the collagen fibers (C) and end freely within the ligament (x 630) .
Discussion Previously, using a gold chloride staining method, we have found free nerve endings in spinal ligaments and, in addition, specialized nerve receptors resembling Ruffini and Pacinian corpuscles 6 • In studies of spinal ligaments in young scoliotic patients, nerve endings were more numerous than in similar tissue taken from adults with disc degeneration and herniation. The present study demonstrates numerous neurofilament protein-immunoreactive nerve fibers in the supraspinous and interspinous ligaments of disc herniated patients. Most of the nerve fibers terminate as simple, free nerve endings resembling those described by Korkala et a1. 4 in the human posterior longitudinal ligament. These nerves were suggested to be responsible for pain production. Their presence in the pathological ligaments examined here has potential clinical significance in relation to low-back pain. In a parallel study7, we have seen degenerative changes in similar ligaments. The earliest ultrastructural evidence of degeneration appears as necrosis of cells, increase of proteoglycan concentration, and a transformation of fibroblasts into chondrocyte-like cells?, 8. One consequence of the chondroid metaplasia is that the deposition of calcium pyrophosphate crystals, which normally involves only cartilage itself, now also includes the chondroid-transformed ligament9 . Therefore, the free nerve endings revealed may be locally excited by these breakdown products of proteoglycans, crystals or cell debris as suggested by Brown 1 and Zimmermann 1o •
938· L. H. Yahia and N. Newman
Hopefully, therapy could be focused if the site and mechanism of pain were better known. Reviews which implicate the nerve structure and pattern usually differentiate these sites: the spinal ligaments, the apophyseal joint capsule and ligament, the posterior vertebral ligament, and flavum tissue. The immunohistological methods may expand our knowledge of differences and lead to more selective and more specific therapy. Acknowledgements The authors wish to thank Louise Clement for her assistance. This research was funded by the "Fondation de Recherche et d'Education en Orthopedie de Montreal (FREOM).
References
Fig. 3. A cross-section of a lumbosacral plexus (control) stained with antibody against NFP. Almost all axons are immunoreactive to NFP (x 250).
Finally, studies evolving from this report such as a comparison of distribution and number of nociceptive receptors in normal and pathological tissues should be an area for active research and observation. These studies might also illustrate mechanisms of nerve excitation whether it is accomplished by compression of nerves, ischemia or fibrosis, associated inflammatory tissue, or other differences. The clinician, faced with multiple back pain syndromes, is challenged by the enigma of the origin of pain.
1 Brown MD (1989) The source of low-back pain and sciatica. Sem in Arthritis and Rheumatism 18(4): 67-72 2 Hirsch C, Ingelmark BE, Miller M (1963) The anatomical basis for low-back pain. Acta Orthop Scand 33: 1-17 3 Jackson HC, Winkelmann RK, Bickel WH (1966) Nerve endings in the human lumbar spinal column and related structures. J Bone Joint Surg (Am) 48(7): 1272-1281 4 Korkala 0, Gronblad M, Liesi P, Karaharju E (1985) Immunohistochemical demonstration of nociceptors in the ligamentous structures of the lumbar spine. Spine 10: 156-157 5 Pionchon H, Tommasi M, Pia let J, Bancel B, Chazal J, Escande G, Scheye T, Vanneuville G (1986) Etude de l'innervation des ligaments rachidiens al'etage lombaire. Bull Ass Anat 70: 63-67 6 Yahia LH, Newman N, Rivard CH (1988) Neurohistology of lumbar spine ligaments. Acta Orthop Scand 59(5): 508-512 7 Yahia LH, Drouin G, Maurais G, Garzon S, Rivard CH (1989) Degeneration of the human lumbar spine ligaments. An ultrastructural study. Path Res Pract 184: 369-375 8 Yahia LH, Drouin G, Maurais G, Rivard CH (1989) Etude de la structure microscopique des ligaments posterieurs du rachis lomb a ire. Int Orthop 13: 207-216 9 Yahia LH, Garzon S, Strykowski H, Rivard CH (1990) Ultrastructure of the human interspinous ligament and ligamentum f1avum. Spine 15: 262-268 10 Zimmermann M (1989) Pain mechanisms and mediators in osteoarthritis. Sem in Arthritis and Rheumatism 18(4): 22-29 11 Zimny ML, St-Onge MF, Albright DJ (1987) The use of agents which improve staining of fresh human fibrocartilage. Stain Technol 62(5): 341-348
Received January 22, 1990 . Accepted in revised form November 28, 1990
Key words: Spinal ligaments - Innervation - Neurofilaments - Disc herniation Dr. L. H. Yahia, Institut de genie biomedical, Ecole Poly technique, C. P. 6079, Succ. "A", Montreal, Quebec, H3 C 3 A 7,Canada