Histological Changes of Ligamenta Flava in Lumbar Disc Herniation and Spinal Canal Stenosis

Samples of ligamenta flava were obtained after surgical operations from 50 patients with a lumbar disc herniation, another 50 patients with a lumbar canal stenosis, and 25 patients with spinal fractures who were used as control group. Ligamenta flava from control patients aged below 46 years consisted of large elastic fibers, thin bundles of collagen fibers, and few spindle-shaped fibroblast cells. In close proximity to the laminal insertion, the ligamentum flavum had fibrocartilagineous features. in the control patients who were aged 46 or older, the areas that had fewer and thinner elastic fibers and a more abundant collagen component were visible occasionally. The spindleshaped fibroblast cells were fewer compared with control patients aged below 46 years. Also remnants of necrotic cells and few, short, thin, interwoven, fragmented, non-branching elastic fibers, as well as small calcified areas, were occasionally visible. In close proximity to the laminal insertion, the ligamentum flavum had larger fibrocartilaginous features with more collagen fibers compared with younger patients. In patients with disc herniation, the ligamenta flava had nearly similar morphologic features to those of the control patients of similar ages. The ligamenta flava from patients with lumbar spinal stenosis aged below 46 years showed areas of fibrosis in which the cells were often represented by fibroblast cells and in stenotic patients older than 46 years, central portion of ligamentum flavum showed areas of fibrosis, in which the elastic fibers appear normal in some areas, showed little changes in others and in most of these areas showed great changes. Fibrous septa, degenerating elastic fibers as well as small calcified areas were observed often. In conclusion, Lumbar ligamentum flavum as any tissue in human body undergo degenerative changes during aging. In lumbar canal stenosis, the degenerative changes were more obvious compared with normal spine or lumbar disc herniation. In stenotic patients, ligamenta flava show a significant decrease in the elastic component as a result of fibrosis and chondroid metaplasia of the tissue, as well as degeneration of the elastic fibers. These changes, and the presence of calcified areas within the tissue, decrease the elasticity of the ligaments. An elastic tissue can be deformed under traction and gradually return to its normal size, proportional to the decrease of the elastic tension. Ligamenta flava do not normally bulge into the spinal canal when spine is in the neutral position. Introduction n the last decades, several studies have analyzed the microscopic morphology of ligamenta flava. However, some studies were carried out in animals 1,2 , whereas others have Correspondence to: Thamer A Hamdan, Dean, College of medicine, University of Basrah, IRAQ. concentrated on normal human ligaments or the calcification processes in ligamenta flava of the cervical spine 3,4 . Only three investigations have analyzed the microscopic morphology of ligamenta flava in patients with I Histological changes of ligamenta flava T. A Hamdan Bas J Surg, September, 11, 2005 lumbar disc herniation or spinal stenosis. One of the two 5 , studied the ultrastructural features of ligaments from patients with a herniated disc, using as controls, ligamenta flava obtained from young adults only. The second investigation 6 analyzed, at histological and histochemical levels, ligamenta flava from patients with lumbar canal stenosis or degenerative spondylolisthesis, using as a control, the ligamentum flavum of a teenager patients. The scarcity of information on the microscopic features of human ligamenta flava in degenerative spinal diseases is surprising, because these ligaments have been implicated often in the pathogenesis of degenerative condition of the spine, particularly lumbar spinal stenosis 7-9 . In this condition, ligamenta flava compress the nerve structures, although controversy still exists on whether these ligaments increase in thickness as a result of fibrosis or degenerative changes 10-12 . The third investigation analyzed at histological, histochemical, and transmission electron microscopic study, the morphologic features of ligamenta flava in patients with lumbar disc herniation and spinal stenosis. Because connective tissues, including the ligamentum flavum, undergo morphologic changes with aging 12-15 , used as controls, ligamenta flava from patients in the second to the seventh decade of life to differentiate between age-related and pathologic changes. In the present histological microscopic study, we analyzed the morphologic features of ligamenta flava in patients with lumbar disc herniation and lumbar spinal stenosis. We used as controls, ligamenta flava from patients in the second to the eighth decade of life. Materials and Methods This descriptive study was conducted in Basrah province from Iraq in 2003. Preparation and examination was conducted in the department of Anatomy, Histology and Embryology in the College of Medicine-University of Basrah. Samples of ligamenta flava were obtained after surgical operations carried at Ebn-Al-Beetar private hospital from 50 patients with a lumbar disc herniation, another 50 patients with a lumbar canal stenosis, and 25 patients with spinal fractures who were used as control group. In the group with herniated discs, there were 33 men and 17 women, ranging in age from (17-57) years old (mean age 42). Disc herniation was located at level L3-L4 in 18 patients, at level L4-L5 in 27 patients, at level L5-S1 in 5 patients. With lumbar stenosis included 26 men and 24 women, aged 35-84 years (mean age 57). The control group includes 22 men and 3 women, aged (16-65) years (mean age 49). Samples of fractures were located from lower thoracic area of 3 patients and in thoracolumbar area of 5 patients and in lumbar area of 14 patients and in lumbosacral area of 3 patients. From each patient, specimens of ligamentum flavum were taken either from the central portion of the ligament or from the area which is in close proximity to the attachment to the distal lamina. In each case, specimens were obtained from the most superficial layer, and from the middle or deep layers of the ligamentum flavum. All specimens were collected directly after surgical operations and tissue samples were placed in 10% formalin used as fixative solution. Preparation of tissue for microscopic examination 16 . 1Fixation: The pieces of the tissue were immersed in 10% formalin for about 12 hours or over night. 2Dehydration: Tissues were directly transferred from the fixative solution to successively increasing gradients of alcohol concentration Histological changes of ligamenta flava T. A Hamdan Bas J Surg, September, 11, 2005 (70% then 90%) two changes one hour for each. 3Clearing: Xylene used as clearing agents to replace the alcohol in the tissue. This process completed by two changes through xylene one hour each. 4Embedding: Tissue transfer to melted paraffin at 58-60 C o to keep the wax in liquid state during the procedure. The original wax was replaced by fresh wax two times for about (1-6 hours). The clearing agent replaced by paraffin wax and paraffin penetrates all intercellular and intracellular spaces making the tissues more resistant to sectioning. 5Paraffin: Is allowed to be hardened in the form of a block contains the piece of the tissue. 6Sectioning: A paraffin block containing piece of tissue fixed on a rotary microtome used for paraffin section to cut the tissue into sections in about (3-5 μm thick) by using a sharp knife. Paraffin ribbon consisting of tissue slices adhering to one another come from sectioning process, carefully separated and floated on a surface of the hot bath at (3040C o ), then mounted over an albuminized glass slide. Preparation for staining: Paraffin material that infiltrates within the slices of tissue was removed before staining processes can be started by using the following procedures 4 : 1We immerse a glass slide bearing a paraffin section in clearing agent for (15-30 minutes). 2We wash with absolute alcohol for 2 minutes. 3Then transfer to successively weaker solutions of alcohol 90%, 70% and 50%, 2 minutes for each. 4We wash with running tap water for 2 minutes. Staining of tissue section: By using Verhoeff's haematoxylin method which is the most commonly used, it can be prepared quickly and requires only a short staining time 16 . Procedure: a) We bring paraffin wax sections to water. It is not necessary to treat the section with iodine if it has been in a mercuric chloride fixative before staining, as the mercuric chloride deposits are removed by the staining solution. b) Then we stain with haematoxylin solution for (15-30 minutes). c) After that we differentiate with 2% aqueous solution ferric chloride for (10-30 seconds). d) Then we wash off the ferric chloride solution with tap water. e) We examine the section under the microscope for correct differentiation. If differentiation is carried too far the section can be restained, provided that it has not been washed with alcohol. f) Then wash with water. g) We place in 95% alcohol for (5minutes) to remove the iodine. h) Then wash in water for at least (5minutes). i) After that we counter stain with van Gieson stain which has been diluted with an equal amount of distilled water for (30 seconds). j) Then we rinse with 95% alcohol. k) We dehydrate with absolute alcohol. l) Then clear with xylene. m) At last we mount in D.P.X (Diastase, plastosin, xylene). By using verhoeffs' method, elastic fibers are selectively stained deep blue colour. Using Van Giesons as a Counterstain, acid fuchsin gives a red color to collagenous fibers. Cellular detail of fibroblasts is not revealed, but the nucli stain deep blue. Degenerated elastic fibers are also stained but can Histological changes of ligamenta flava T. A Hamdan Bas J Surg, September, 11, 2005 be distinguished from the normal by their less intense staining and presenting less distinct outlines. Then the slides were examined under light microscope and selected images were taken by using a special light microscope provided with photographic camera.