Journal of Orthopaedic Research 978-90 Raven Press, Ltd., New York 0 1991 Orthopaedic Research Society
Histochemical and Ultrastructural Observations on Brown Degeneration of Human Intervertebral Disc Tsutomu Ishii, Haruo Tsuji, Akimi Sano, Yoshiharu Katoh, Hisao Matsui, and Nobuo Terahata Department of Orthopaedic Surgery, Faculty of Medicine, Toyama Medical and Pharmaceutical University, Toyama, Japan
Summary: Thirty-eight fresh human intervertebral discs collected during antenor interbody fusion surgery were histochemically and ultrastructurally analyzed for pigments. Macroscopically, five stages of degeneration were classified according to the color, fibrosis, and fragility of the nucleus pulposus of the discs. In order to demonstrate lipofuscin granules, specimens were subjected to special staining procedures, including carbol fuchsin lipofuscin stain, the Schmorl’s reaction, and autofluorescence. Lipofuscin granules were distributed from the inner layer of the annulus fibrosus to the nucleus pulposus. Such granules were numerous in cases of slight or severe degeneration, whereas fewer granules were found in cases of moderate degeneration. However, the stage of macroscopic degeneration of the intervertebral disc did not necessarily correlate with the incidence of lipofuscin granules. By ultrastructural observation, the morphological features of the components of the intervertebral disc and the ultrastructure of the lipofuscin granule were clarified. The ultrastructure of the “brown degeneration” disc exhibited markedly increased amorphous electron-dense bodies located among collagen fibrils in the degeneration-Degenmatrix. Key Words: Intervertebral disc-Brown eration-Lipofuscin-Ultrastructure.
was first reported by Saunders (29) and Coventry (9). Thereafter, few studies have been carried out concerning this issue. Focusing attention on this phenomenon, we attempted to elucidate the mechanism of intervertebral disc degeneration by histochemical and ultrastructural observations of the pigments, which are indications of “brown degeneration.” Lipofuscin granules, which have thus far been considered to be the primary characteristic of “brown degeneration,” are yellowish-brown pigments scattered throughout the cytoplasm. These pigments have also been called age pigments. Lipofuscin granules in the nerve cells and myocardial cells have been widely studied, and their tendency to increase with aging has been reported (21,22,31). However, many points remain unclear as to the re-
Dysfunction of the lumbar intervertebral disc, which is the main component of the functional spinal unit, results in a high incidence of low back pain. The pathology of this condition shows a degenerative process. The intervertebral disc has been actively studied from the morphological (8,10, 11,13), biochemical (13,18,35), and biomechanical (14) aspects. “Brown degeneration” in the intervertebral disc is not uncommon among the degenerative processes. Brown degeneration of the intervertebral disc Received August 7 , 1989; accepted July 20, 1990. Address correspondence and reprint requests to H. Tsuji at Department of Orthopaedic Surgery,Toyama Medical and Pharmaceutical University, 930-01 Toyama, Japan.
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lationship of lipofuscin granules and the aging of cells. No reports have discussed the existence of these granules in the chondrocytes of the intervertebral disc. The present study was done to clarify the relationship between discoloration and intervertebral disc degeneration. MATERIALS AND METHODS Thirty-eight intervertebral discs from 34 patients with varying intervertebral disc diseases, obtained during anterior en-bloc discectomy and interbody fusion operation, were investigated. There were 25 patients with herniated nucleus pulposus, five with symptomatic disc degeneration, and eight with degenerative spondylolisthesis. The patients were 25 men and 9 women, with a mean age of 43.4 4 10.2 years (range 20-65). The discs examined included four L3/4, 26 L4/5, and eight L5/S1. No patients with diabetes mellitus were included. Macroscopic Classification of the Discs The intervertebral discs were macroscopically classified into the following five stages of degeneration, referring to completely normal discs in our autopsy observations and Galante’s classification of intervertebral discs (14).
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FIG. 1. Relationship between patient’s age and stage of disc degeneration.
Preparation of Specimens and Observations The intervertebral disc tissue removed, composed of the anterior annulus fibrosus and the nucleus pulposus and approximately 30 X 30 x 10 mm in size, was divided into two slices. One was used for light microscopic observation, and the other one was used for ultrastructural observation. For light microscopy, each specimen was fixed in modified Millonig 10% phosphate-buffered formalin solution at pH 7.4, embedded in paraffin, and subjected to hematoxylin-eosin stain, carbol fuchsin lipofuscin
Stage Z:
Sticky nucleus pulposus with semitransparent gelatinous areas and no discoloration. Negative or extremely slight degeneration as a whole. 0.
/
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The nucleus pulposus is straw color and slightly fibrous yet is still soft and lustrous, corresponding to slight degeneration.
Stage ZZ:
AF
Stage IZI: The nucleus pulposus is yellow or partly brownish, fibrous, and relatively hardcorresponding to moderate degeneration. Stage ZV: The
nucleus pulposus is completely fibrous, contains less water, and is fragile and yellowish-brown or brownish in color-corresponding to moderate to severe degeneration. Stage V: The nucleus pulposus is coarse, extremely fragile, and markedly desiccated, and is deep yellow to brown in color-corresponding to extreme degeneration.
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FIG. 2. Total cell count per unit area of 3.3 rnm2 in relation to the stage of degeneration. The total cell count in the inner layer of the annulus fibrosis (0)tended to increase with the progression of degeneration. In the nucleus pulposus (0)the pattern was similar with the exception of stage 111. and A indicate statistical difference (P < 0.5) for the nucleus pulposus and for the inner layer of the annulus fibrosus, respectively. and AA, P < 0.01.
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stain (AFIP lipofuscin stain) (20), Schmorl's reaction (20), periodate-Schiff s procedure (PAS) stain, toluidine blue stain, or Safranin-0 stain. The lipofuscin granules were carefully observed in all sheets of the specimens, and the number of granules was recorded as the number of lipofuscin per mm2. Lipofuscin granules in the unstained sections were also confirmed by fluorescent microscopy at excitation spectra between 334 nm and 365 nm. The chondrocyte count, chondrocyte pair count, and cluster count were determined by observing the entire area of the tissue specimen using a 3.3 mm2 eyepiece scale at 40-fold magnification. For electron microscopy, 20 blocks, 1 X 1 X 1 mm in size each, were obtained from the nucleus pulposus (10 blocks) and the inner layer of the annulus fibrosus (10 blocks) of each intervertebral disc immediately after removal. The blocks were fixed in 2% glutaraldehyde solution (pH 7.4, 0.1 M cacodylate buffer) containing ruthenium red (RR) 1,000 ppm for 3 h in a cool, dark place. The specimens were subsequently washed with 0.1 M cacodylate buffer containing 7% saccharose and fixed in
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2% OsO, solution containing RR 2,000 ppm (pH 7.4, 0.1 M cacodylate buffer) for 1 h in a cool, dark place. After dehydration, the specimens were embedded in Epon 812. For microscopy, 1-p,m-thick toluidine blue-stained sections and uranyl acetatestained and lead citrate-stained ultrathin sections of each specimen were observed under a JEOL 200CX electron microscope (JEOL, Tokyo).
RESULTS Macroscopic Findings Based on the criteria of disc degeneration, four discs were identified as stage I, eight discs stage 11, 12 discs stage 111, eight discs stage IV, and six discs stage V. The mean age of the patients with stage I was 28.8 6.0 years; stage 11, 36.8 2 5.5; stage 111, 45.6 2 9.5; stage IV, 50.1 f 10.2; and stage V, 52.0 6.4. The parallel correlation of the increase in stage and increase in age was obvious (Fig. 1).
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5 Stage
1 2 3 4 5 stage 1 2 3 4 5 Stage FIG. 3. Chondrocyte pair count according to cluster size. Small cluster count, medium cluster count, and large cluster count per unit area of 3.3 mm2 in relation to the stage of disc degeneration. In the inner layer of the annulus fibrosus (0),the chondrocyte pair count was high for slight or severe degeneration and rather low for moderate degeneration; there was no correlation between the cluster count and the stage of degeneration. In the nucleus pulposus (0). the chondrocyte pair count was similar to those in the annulus fibrosus. However, the cluster count tended to increase with the progression of degeneration. and A indicate statistical difference (p < 0.5) for the nucleus pulposus and for the inner layer of the annulus fibrosus, respectively.
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Light Microscopic Findings In the inner layer of the anterior annulus fibrosus, the chondrocytes occupying lacunae were generally scattered throughout the matrix of the collagen bundle framework. In the middle layer of the anterior annulus fibrosus, so-called chondrocyte pairs (two semicircular cells in a single lacuna) and cloning or clusters of several chondrocytes were found. In the outer layer of the annulus fibrosus, fibroblast-like cells were noted. Blood vessels were occasionally found in the outermost layer of the annulus fibrosus. In the transitional area between the inner layer of the annulus fibrosus and the nucleus pulposus, the annular architecture was no longer present, and chondrocytes, chondrocyte pairs, and clusters of various sizes were scattered throughout irregular fibrous matrix. For each layer of the annulus fibrosus and the nucleus pulposus, the relationship of the total chondrocyte count, chondrocyte pair count, and cluster count per unit area to the stage of disc degeneration was determined. Since the clusters varied in size,
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they were classified into three categories for counting: small clusters were composed of 3-10 cells, medium clusters were composed of 11-20 cells, and large clusters were composed of 21 or more cells. The relationship of the cluster totals in the nucleus pulposus and in the inner layer of the annulus fibrosus and the macroscopic stage of degeneration are shown in Figs. 2 and 3. In the inner layer of the annulus fibrosus, the total chondrocyte count tended to increase with the progression of degeneration (Fig. 2), whereas the chondrocyte pair count was high in slight and severe degeneration stages and was rather low in the moderate degeneration stage (Fig. 3, upper left). There was little correlation between the cluster count in the annulus fibrosus and the stage of degeneration, with an extremely low incidence of medium and large clusters (Fig. 3, upper right and lower trace). The total cell count (Fig. 2) and the chondrocyte pair count in the nucleus pulposus and the inner layer of the annulus fibrosus were similar, with the exception of Stage 111. However, the cluster count in the nucleus pulposus tended to increase
FIG. 4. A: AFlP lipofuscin stain of the nucleus pulposus of a 51-year-old man in stage IV degeneration revealed red-stained . Schmorl's reaction of the inner layer granules in the cytoplasm, suggestive Of lipofuscin granules. AFlP lipofuscin stain ~ 1 9 0B: of the annulus fibrosus of a 62-year-old man in stage IV degeneration showing granules stained blue in both the intracellular and extracellular area, suggestive of lipofuscin granules. Schmorl's reaction ~ 3 8 5 .C: Fluorescent micrograph of the nucleus pulposus of a 51-year-old man in stage IV degeneration showing granules emitting orange autofluorescence at excitation spectra between 334 nm and 365 nm, which is strongly suggestive of lipofuscin ~ 1 9 0 .
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with the progression of degeneration (p < 0.05). In particular, the medium and large clusters were characteristically increased in the degeneration nucleus pulposus (p < 0.05). The hematoxylin-eosin-stained sections revealed yellowish-brown granules in both intracellular and extracellular regions of chondrocytes. These granules were stained red by the AFIP lipofuscin stain (Fig. 4A) and blue by the Schmorl’s reaction (Fig. 4B) and emitted an orange autofluorescence at excitation spectra between 334 nm and 365 nm (Fig. 4C), suggesting the presence of lipofuscin granules. Fluorescence micrography also disclosed that blue to green materials of large, irregular shape and granules were distributed within the chondrocytes and in the territorial and interterritorial matrix. These granules were densely distributed in the areas from the inner layer of the annulus fibrosus to the nucleus pulposus and were seen in various cells. The cells containing lipofuscin granules consisted of chondrocytes (61.2%), chondrocyte pairs (23.8%), small clusters (13.9%),medium clusters (0.7%), and large clusters (0.6%). Regarding the relationship of the incidence of lipofuscin granules to the macroscopic stage of degeneration, the incidence of these granules tended to be high in Stages I and V discs. Thus, there appeared to be no particular correlation between the degenerative stage and the number of lipofuscin granules (Fig. 5). The relationship between lipofuscin granule count and patient age shown in Fig. 6 suggested that lipofuscin granules tend to appear in adulthood.
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FIG. 6. Relationship between lipofuscin granule count and age. 0 ,nucleus pulposus; 0, annulus fibrosus (inner layer).
Electron Microscopic Findings Chondrocytes
In general, chondrocyte cytoplasm was relatively abundant in rER, and cisternal enlargement of rER was found in approximately 16% of all chondrocytes. Golgi apparatus and mitochondria were also observed. Lysosomes were also found, but the incidence seemed to be relatively low. It was possible to observe the central body in the Golgi apparatus near the nucleus in only two of the 121 chondrocytes observed. In addition, lipid droplets were found in the cytoplasm in 36% of all chondrocytes. These droplets were often enlarged, and in some cases occupied almost the entire area of the cytoplasm (Fig. 7). Approximately 10% of chondrocytes contained a few lipofuscin granules, as described in detail later. Almost all chondrocytes were surrounded by areas rich in proteoglycan granules, forming the so-called territorial matrix. Territorial Matrix
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FIG. 5. Population of lipofuscin granules per mm2according to the degeneration stage. There was no particular correlation, but the incidence of the granule tended to be high in the Stages I and V discs. 0 ,nucleus pulposus; 0, annulus fibrosus (inner layer).
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Many proteoglycan granules were found in the areas surrounding the chondrocyte, within distances of 3 4 pm from the cell membrane. This ruthenium red-positive material typically shows islet formation in the territorial matrix. However, in the present study such a formation was relatively rare in the specimens observed. There were many electron-dense bodies, referred to as lipidic debris, in the marginal areas of the territorial matrix. A few lipofuscin-like granules were also found in the lipidic debris layer (Fig. 8).
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FIG. 7. Electron micrograph of a chondrocyte containing a lipid droplet in the nucleus pulposus of Stage IV. Lipofuscin granules are also shown in the cytoplasm. Ruthenium red stain ~ 1 , 7 0 0
Chondrocyte Pairs A chondrocyte pair is composed of two independent cells having a common temtorial matrix. The shape is semicircular or elliptic. Some chondrocyte pairs contained evident lipid droplets, vacuoles, or lipofuscin granules (Fig. 9). Although chondrocyte pairs appear to be the result of cell division, the protoplasms of the two chondrocytes were found to be in clear contact with each other in some of the chondrocyte pairs. It is noteworthy that in some cases, one chondrocyte was intruding into the territorial matrix of the adjacent chondrocyte; this suggests a developmental process of chondrocyte pairs. Cluster A chondrocyte cluster is an aggregation of independent cells, although it looks like a multinuclear cell by light microscopy. Clusters composed of 3-7 cells were observed by electron microscopy. It was rare for all cells in the cluster to be viable; some cells were necrotic or contained markedly increased
vacuoles, enlarged rER, and lipofuscin granules (Fig. 10). Distinct lipidic debris was found in the marginal areas of the cluster. In addition, banded structures were relatively frequently noted in the vicinity of the cluster. There was no contact between the cells like that seen in the chondrocyte pairs. Lipofuscin Granules
Lipofuscin granules were found both inside and outside the chondrocyte. These granules were generally composed of nonhomogenous electron-dense bodies of various degrees. Some granules accompanying the vacuoles and/or membrane contained fine granules that had a very high density (Fig. 11). Most of the intracellular lipofuscin granules were located in the marginal areas of the cell. About 76% of all lipofuscin granules contained vacuoles of varying sizes; some vacuoles were surrounded by electrondense bodies, and others were larger than electrondense bodies. The membrane of the lipofuscin granule was visible in about half of the lipofuscin granules observed. Lipid droplets were frequently found
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FIG. 8. Electron micrograph of a chondrocyte in the nucleus pulposus in Stage 111 showing proteoglycans and various shapes of electron-dense bodies, the lipidic debris in the pericellular matrix. A lipofuscin-like granule is also found (arrowhead).
Ruthenium red stain ~5,100.
in the vicinity of these lipofuscin granules. In addition, large vacuoles or lysosomes were occasionally found near the lipofuscin granules. Lipofuscin-like granules were also found in the cell debris, their basic structure presumably being the same as that of intracellular lipofuscin granules (Fig. 12). However, a few lipofuscin-like granules in the lipidic debris layer and in the territorial matrix did not have any vacuoles or membrane (Fig. 8). Matrix
Collagen fibrils measuring 40-110 nm in diameter were found in the matrix. Each fibril exhibited cyclic striations with intervals of approximately 50 nm. There were no obvious striations in the very thin fibrils, 15 nm in diameter, whereas the 20nm-diameter fibrils showed striations. Granules measuring 10-50 nm in diameter, suggestive of proteoglycan, were attached to the surface of the collagen fibril. Some fibrils showed no relationship of the proteoglycan granules to the striations. Collagen sheath structures of highly electron-dense sub-
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stances enveloping the collagen fibrils were also observed (3). In the matrix an amorphous electrondense material (27,28,33) that filled the interfibrillar spaces was found in areas 4-5 pm or farther from the chondrocyte. When two parts macroscopically different in color and obtained from the same intervertebral disc were compared, amorphous electrondense materials showing the different shapes of lipofuscin granules were markedly increased in the brown part (Fig. 13). Round, electron-dense, matrix vesicle-like bodies with a membrane, which are said to play an important role in tissue calcification, were seldom found between collagen fibrils (2). The diameter of the matrix vesicle-like body was approximately 0.1 km. Elastic fibers about 1 pm in diameter were only rarely observed. DISCUSSION
In the present study, fresh human intervertebral discs were graded according to macroscopic classification by the color and elasticity of the nucleus pulposus and the inner and middle layers of the an-
FIG. 9. A mate of one chondrocyte pair in the inner layer of the annulus fibrosus of Stage 111. Lipofuscin granules containing vacuoles can be seen. Ruthenium red stain x5.100.
FIG. 10. A small cluster in the inner layer of the nucleus pulposus in Stage 111. A necrotic cell within the cluster revealed lipofuscin granules, vacuoles, and enlarged rER in the cytoplasm (right side). Lipidic debris is shown in the pericellular matrix. Ruthenium red stain xl.700.
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FIG. 11. Lipofuscin granule nonhomogenous electron-dense body is demonstrated in the cytoplasm of a chondrocyte. Various vacuoles and thin membranes can be found in the lysosome. Ruthenium red stain x12.700.
FIG. 12. Lipofuscin-like granules seen at cell necrosis in the nucleus pulposus of Stage 111. Ruthenium red stain ~2,500. J Orthop Res, Vol. 9, N o . 1 , 1991
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FIG. 13. Amorphous electron-dense materials in the matrix of different areas of a disc with (A) and without (B) brown discoloration. The amorphous electron-dense bodies of different shapes from lipofuscin granules demonstrated between collagen fibrils are markedly increased in the upper photograph as compared with the lower photograph. Ruthenium red stain ~3,400.
nulus fibrosus. This classification is relatively well correlated with age. Only a few reports of “brown degeneration” in the intervertebral disc are available in the literature. In 1970, Peereboom reported that “age pigments” in a region showing “brown degeneration” in the annulus fibrosus were PASpositive and emitted autofluorescence (26). Since lipofuscin emits fluorescence of about 470 nm with an excitation spectrum of 360 nm, it is highly probable that these pigments were not truly lipofuscin. The pigments observed in the present study, however, emitted orange fluorescence by excitation at 330 nm and 365 nm and were positive for the lipofuscin stain and the Schmorl’s reaction. Therefore, these pigments were considered to be lipofuscin. In the intervertebral disc, brown degeneration was macroscopically more conspicuous in the area from the nucleus pulposus to the inner
layer of the annulus fibrosus; the incidence of lipofuscin granules was particularly high in these areas. Histological degeneration of the intervertebral disc was characterized by a marked increase in chondrocyte pairs and clusters in the nucleus pulposus and in the inner layer of the annulus fibrosus. In particular, in severely degenerated discs, large clusters alone were seen sparsely distributed, with almost no isolated chondrocytes. Regarding the nature of the pigments in question, it is thought that numerous pigments appear in postmitotic nerve and myocardial cells and also that these pigments increase in number with age (21, 22,31). Attention is now being drawn toward the possibility that pigments are an important morphological feature of aging. However, these pigments are present in general tissues, including liver cells and periosteal cells, and are not necessarily corre-
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FIG. 14. An isolated lipofuscin-llike granule is demonstrated in the territorial matrix close to the chondrocyte, suggesting a process of granule release of the cell. Ruthenium red stain ~5,100.
lated with age (17,34). We found no obvious correlation between the stage of disc degeneration and the incidence of pigments. This lack of correlation was also suggested from the high incidence of pigments in chondrocytes and the fact that the incidence of such pigments was low in chondrocyte pairs and clusters that characterize regressive change in tissues. Although there was no decisive evidence from the ultrastructural observations, it can be speculated that intracellular lipofuscin is released outside the chondrocyte during the cell division process, since chondrocytes seem to have the ability to release lipofuscin (Fig. 14). The outflow of intracellular lipofuscin might also be caused by cell collapse from an increase in necrotic cells. Calculation of the extracellular lipofuscin granules could not be achieved in this study, because the histochemical nature of this pigment might be altered in the extracellular matrix. Further observation is required. The above-mentioned speculation is derived from the fact that lipofuscin granules are more likely to appear in a so-called active zone, or the transitional area between the nucleus pulposus and the inner layer of the annulus fibrosus. However, since ceroid granules, which are very similar to lipofuscin
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granules, appear in the cerebrum (24) and villi of the intestinal tissue (19) in vitamin E-deficient animals, the appearance of lipofuscin granules should be regarded as a phenomenon closely related to the regressive process in tissue. Recent studies have demonstrated that these granules are positive for reactions of unsaturated fatty acid, aldehyde, phospholipid, and various amino acids (6). These granules are known to be derived from the reaction of a substance having an amino base and malondealdehyde, a cleavage product of lipid peroxide converted from a multivalent unsaturated fatty acid (5,6). Regarding the mechanism of disc degeneration, it seems that lipofuscin production and cell degeneration are closely linked in the process of the appearance of superoxide and the production of lipid peroxide (30). In the present study, fresh and large specimens of human intervertebral discs were removed without trauma from the anterior approach, proving very useful for electron microscopic observation. The following morphological features of the chondrocytes observed in this study were similar to those reported by previous studies: rER, Golgi apparatus, lipid droplets, proteoglycan in the territorial matrix, lipidic debris (16), and banded structure (4,7). How-
BROWN DEGENERATION OF HUMAN DISC ever, there was no obvious morphological difference between chondrocytes with and without lipofuscin granules. As for the morphological origin, lipofuscin granules are generally considered to originate from cell organelles and particularly from lysosomes (12). However, there are some reports suggesting that the mitochondria (25), Golgi apparatus (l), and rER (32), and other cell organelles are the origin. Other reports propose that these granules are produced without any connection whatsoever to cell organelles (15). The next question is that of the fate of lipofuscin produced in the cell. Since a similar structure has been found in the extracellular lipidic debris, it is hypothesized that lipofuscin is released outside the cell or flows into the matrix along with cell necrosis. Lipofuscin is usually metabolized by wandering leukocytes or histiocytes. However, in the intervertebral disc, which is avascular tissue, lipofuscin seems to remain in the matrix for some time and becomes diffused after some metabolic process. As the disc degenerates, isolated chondrocytes form into chondrocyte pairs and, subsequently, into clusters. Details of this process and its significance require further studies. It is possible that a chondrocyte pair is produced by the immigration of one chondrocyte into another. It is also possible that the cluster formation is an associated defense reaction of chondrocytes to cope with the deterioration of the external environment. Regarding discoloration in the intervertebral disc, occasionally no lipofuscin granules are seen microscopically in the brown discs. The amorphous electron-dense material, which is not lipofuscin, is markedly increased in the matrix of the brown degeneration disc. Thus the material that may play a role in the discoloration of the disc is considered morphologically to be cell debris, but some possible chemical alteration in the cell debris, such as a nonenzymatic browning process, cannot be ruled out (23). Further investigation is needed. Acknowledgment: This work was supported by the Grant-in-Aid for scientific Research of the Japanese Ministry of Education, Science, and Culture.
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3. 1Buckwalter JA, Maynard JA, Cooper RR: Sheathing of colI agen fibrils in human intervertebral discs. J Anat 125:615t518, 1978 4. 1Buckwalter JA, Maynard JA, Cooper RR: Banded structures 1n human nucleus pulposus. Clin Orthop 139:259-266, 1979 5. (Zhio KS, Tappel AL: Synthesis and characterization of the 1Suorescent products derived from malonaldehyde and mino acids. Biochemistry 8:2821-2827, 1969 6. (Zhio KS, Fletcher URB, Tappel AL: Peroxidation of subI:ellular organelles: formation of lipofuscinlike fluorescent 1pigments. Science 166:1535-1536, 1969 7. Zornah MS, Meachim G, Parry EW: Banded structures in 1the matrix of human and rabbit nucleus pulposus. J Anat 107:351-362, 1970 8. ICloventry MB, Ghormley RK, Kernohan JW: The intervertebral disc: its microscopic anatomy and pathology. I. Anatomy, development, and physiology. J Bone Joint Surg 27:105-112, 1945 9. ICoventry MB, Ghormley RK, Kernohan JW: The intervertebral disc: its microscopic anatomy and pathology. 11. Changes in the intervertebral disc concomitant with age. J Bone Joint Surg 27:233-247, 1945 10. ICoventry MB, Ghormley RK, Kernohan JW: The intervertebral disc: its microscopic anatomy and pathology. 111. Pathological changes in the intervertebral disc. J Bone Joint Surg 27:460-473, 1945 11. Coventry MB: Anatomy of the intervertebral disc. Clin Orthop 67:%15, 1969 12. Essner E, Novikoff AB: Localization of acid phosphatase activity in hepatic lysosome by means of electron microscopy. J Biophys Biochem Cytol9:773-784, 1961 13. Flanklin L, Hull EW: Lipid content of the intervertebral disc. Clin Chem 12:253-257, 1966 14. Galante J: Tensile properties of the human lumbar annulus fibrosus. Acta Orthop Scand lOO(supp1): 1-91, 1967 15. Gedigk P, Wessel W: Elektronenmikroskopische Untersuchung des Vitamin-E-Mangel-Pigmentes in Myometrium der Ratte. Virchows Arch [A] 337:367-382, 1964 16. Ghardially FN: Fine structure of synovial joints. London, Buttenvorths Publishers, 1983, pp 236-260 17. Goldfisher S,Bernstein J: Lipofuscin (aging) pigment granules of the newborn human liver. J Cell Biol 42:253-261, 1969 18. Happey F: Studies of the structure of the human intervertebral disc in relation to its functional and aging process. The Joints and Synovial Fluid, Vol. 2, ed. by L. Sokoloff, New York, Academic Press, 1980, pp 95-137 19. Katz ML, Groome AB, Robinson WG: Localization of lipofuscin in the duodenums of vitamin Edeficient rats. J Nutr 115:1355-1365, 1985 20. Luna LG: Manual of Histologic Staining Methods of the Armed Forces Institute of Pathology, 3rd ed, New York, McGraw Hill, 1968, pp 185-186 21. Malkoff DB, Strehler BL: The ultrastructure of isolated and in situ human cardiac age pigment. J Cell Biol 16:611416, 1963 22. Mann DMA, Yates PO: Lipofuscin pigments: their relationship to aging in the human nervous system. I. The lipofuscin content of nerve cells. Brain 97:481488, 1974 23. Monnier VM, Cerami A: Nonenzymatic browning in vivo: Possible process for aging of long-lived protein. Science 211:491493, 1981 24, Nishioka N, Takahata N, Iizuka R: Histochemical studies on the life pigments in the nerve cells. A comparison with lipofuscin and ceroid pigment. Acta Neuropathol 11: 174-181, 1968 25. Payne F: Changes in the endocrine glands of the fowl with age. J Gerontol4:193-199, 1949
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T. ISHII ET AL. 26. Peereboom JWC: Age-dependent changes in the human intervertebral disc fluorescent substance and amino acids in the annulus fibrosus. Gerontologia 16352-367, 1970 27. Postacchini F, Bellocci M, Ricciardi-Pollini PT, Modesti A: An ultrastructural study of recurrent disc herniation: a preliminary report. Spine 7:492-497, 1982 28. Postacchini F , Bellocci M, Massobrio M: Morphologic changes in annulus fibrosus during aging: an ultrastructural study in rats. Spine 9596403, 1984 29. Saunders JBCM, Inman VT: Pathology of the intervertebral disc. Arch Surg 60:389, 1940 30. Sheldahl JA, Tappel AL: Fluorescent products from aging dorsophila melanogaster: an indicator of free radical lipid peroxidation damage. Exp Gerontol9:3341, 1974
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31. Strehler BL, Mark DD, Mildvan AS, Gee MV: Rate and magnitude of age pigment accumulation in the human myocardium. J Gerontol 14:43&439, 1959 32. Strehler BL: On the histochemistry and ultrastructure of age pigment. Adv Gerontol Res 1:343-384, 1964 33. Sylvest J, Hentzer B, Kobayasi T: Ultrastructure of prolapsed disc. Acta Orthop Scand 48:3240, 1977 34. Tauchi H, Hananouchi M: Accumulation of lipofuscin pigment in human hepatic cells from different races and in different environmental conditions. Mech Ageing Dev 12:183195, 1980 35. Taylor TKF, Akeson WH: Intervertebral disc prolapse: a review of morphology and biochemical knowledge concerning the nature of prolapse. Clin Orthop 7654-79, 1971
Histochemical and Ultrastructural Observations on Brown Degeneration of Human Intervertebral Disc Tsutomu Ishii, Haruo Tsuji, Akimi Sano, Yoshiharu Katoh, Hisao Matsui, and Nobuo Terahata Department of Orthopaedic Surgery, Faculty of Medicine, Toyama Medical and Pharmaceutical University, Toyama, Japan
Summary: Thirty-eight fresh human intervertebral discs collected during antenor interbody fusion surgery were histochemically and ultrastructurally analyzed for pigments. Macroscopically, five stages of degeneration were classified according to the color, fibrosis, and fragility of the nucleus pulposus of the discs. In order to demonstrate lipofuscin granules, specimens were subjected to special staining procedures, including carbol fuchsin lipofuscin stain, the Schmorl’s reaction, and autofluorescence. Lipofuscin granules were distributed from the inner layer of the annulus fibrosus to the nucleus pulposus. Such granules were numerous in cases of slight or severe degeneration, whereas fewer granules were found in cases of moderate degeneration. However, the stage of macroscopic degeneration of the intervertebral disc did not necessarily correlate with the incidence of lipofuscin granules. By ultrastructural observation, the morphological features of the components of the intervertebral disc and the ultrastructure of the lipofuscin granule were clarified. The ultrastructure of the “brown degeneration” disc exhibited markedly increased amorphous electron-dense bodies located among collagen fibrils in the degeneration-Degenmatrix. Key Words: Intervertebral disc-Brown eration-Lipofuscin-Ultrastructure.
was first reported by Saunders (29) and Coventry (9). Thereafter, few studies have been carried out concerning this issue. Focusing attention on this phenomenon, we attempted to elucidate the mechanism of intervertebral disc degeneration by histochemical and ultrastructural observations of the pigments, which are indications of “brown degeneration.” Lipofuscin granules, which have thus far been considered to be the primary characteristic of “brown degeneration,” are yellowish-brown pigments scattered throughout the cytoplasm. These pigments have also been called age pigments. Lipofuscin granules in the nerve cells and myocardial cells have been widely studied, and their tendency to increase with aging has been reported (21,22,31). However, many points remain unclear as to the re-
Dysfunction of the lumbar intervertebral disc, which is the main component of the functional spinal unit, results in a high incidence of low back pain. The pathology of this condition shows a degenerative process. The intervertebral disc has been actively studied from the morphological (8,10, 11,13), biochemical (13,18,35), and biomechanical (14) aspects. “Brown degeneration” in the intervertebral disc is not uncommon among the degenerative processes. Brown degeneration of the intervertebral disc Received August 7 , 1989; accepted July 20, 1990. Address correspondence and reprint requests to H. Tsuji at Department of Orthopaedic Surgery,Toyama Medical and Pharmaceutical University, 930-01 Toyama, Japan.
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lationship of lipofuscin granules and the aging of cells. No reports have discussed the existence of these granules in the chondrocytes of the intervertebral disc. The present study was done to clarify the relationship between discoloration and intervertebral disc degeneration. MATERIALS AND METHODS Thirty-eight intervertebral discs from 34 patients with varying intervertebral disc diseases, obtained during anterior en-bloc discectomy and interbody fusion operation, were investigated. There were 25 patients with herniated nucleus pulposus, five with symptomatic disc degeneration, and eight with degenerative spondylolisthesis. The patients were 25 men and 9 women, with a mean age of 43.4 4 10.2 years (range 20-65). The discs examined included four L3/4, 26 L4/5, and eight L5/S1. No patients with diabetes mellitus were included. Macroscopic Classification of the Discs The intervertebral discs were macroscopically classified into the following five stages of degeneration, referring to completely normal discs in our autopsy observations and Galante’s classification of intervertebral discs (14).
1 L
1
I
1
2
I
3
5
4
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FIG. 1. Relationship between patient’s age and stage of disc degeneration.
Preparation of Specimens and Observations The intervertebral disc tissue removed, composed of the anterior annulus fibrosus and the nucleus pulposus and approximately 30 X 30 x 10 mm in size, was divided into two slices. One was used for light microscopic observation, and the other one was used for ultrastructural observation. For light microscopy, each specimen was fixed in modified Millonig 10% phosphate-buffered formalin solution at pH 7.4, embedded in paraffin, and subjected to hematoxylin-eosin stain, carbol fuchsin lipofuscin
Stage Z:
Sticky nucleus pulposus with semitransparent gelatinous areas and no discoloration. Negative or extremely slight degeneration as a whole. 0.
/
/
/-h
The nucleus pulposus is straw color and slightly fibrous yet is still soft and lustrous, corresponding to slight degeneration.
Stage ZZ:
AF
Stage IZI: The nucleus pulposus is yellow or partly brownish, fibrous, and relatively hardcorresponding to moderate degeneration. Stage ZV: The
nucleus pulposus is completely fibrous, contains less water, and is fragile and yellowish-brown or brownish in color-corresponding to moderate to severe degeneration. Stage V: The nucleus pulposus is coarse, extremely fragile, and markedly desiccated, and is deep yellow to brown in color-corresponding to extreme degeneration.
1
2
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FIG. 2. Total cell count per unit area of 3.3 rnm2 in relation to the stage of degeneration. The total cell count in the inner layer of the annulus fibrosis (0)tended to increase with the progression of degeneration. In the nucleus pulposus (0)the pattern was similar with the exception of stage 111. and A indicate statistical difference (P < 0.5) for the nucleus pulposus and for the inner layer of the annulus fibrosus, respectively. and AA, P < 0.01.
*
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stain (AFIP lipofuscin stain) (20), Schmorl's reaction (20), periodate-Schiff s procedure (PAS) stain, toluidine blue stain, or Safranin-0 stain. The lipofuscin granules were carefully observed in all sheets of the specimens, and the number of granules was recorded as the number of lipofuscin per mm2. Lipofuscin granules in the unstained sections were also confirmed by fluorescent microscopy at excitation spectra between 334 nm and 365 nm. The chondrocyte count, chondrocyte pair count, and cluster count were determined by observing the entire area of the tissue specimen using a 3.3 mm2 eyepiece scale at 40-fold magnification. For electron microscopy, 20 blocks, 1 X 1 X 1 mm in size each, were obtained from the nucleus pulposus (10 blocks) and the inner layer of the annulus fibrosus (10 blocks) of each intervertebral disc immediately after removal. The blocks were fixed in 2% glutaraldehyde solution (pH 7.4, 0.1 M cacodylate buffer) containing ruthenium red (RR) 1,000 ppm for 3 h in a cool, dark place. The specimens were subsequently washed with 0.1 M cacodylate buffer containing 7% saccharose and fixed in
c
*
I
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2
,
*
3
4
-!
5 Stage
2% OsO, solution containing RR 2,000 ppm (pH 7.4, 0.1 M cacodylate buffer) for 1 h in a cool, dark place. After dehydration, the specimens were embedded in Epon 812. For microscopy, 1-p,m-thick toluidine blue-stained sections and uranyl acetatestained and lead citrate-stained ultrathin sections of each specimen were observed under a JEOL 200CX electron microscope (JEOL, Tokyo).
RESULTS Macroscopic Findings Based on the criteria of disc degeneration, four discs were identified as stage I, eight discs stage 11, 12 discs stage 111, eight discs stage IV, and six discs stage V. The mean age of the patients with stage I was 28.8 6.0 years; stage 11, 36.8 2 5.5; stage 111, 45.6 2 9.5; stage IV, 50.1 f 10.2; and stage V, 52.0 6.4. The parallel correlation of the increase in stage and increase in age was obvious (Fig. 1).
*
*
1
2
3
4
5 Stage
1 2 3 4 5 stage 1 2 3 4 5 Stage FIG. 3. Chondrocyte pair count according to cluster size. Small cluster count, medium cluster count, and large cluster count per unit area of 3.3 mm2 in relation to the stage of disc degeneration. In the inner layer of the annulus fibrosus (0),the chondrocyte pair count was high for slight or severe degeneration and rather low for moderate degeneration; there was no correlation between the cluster count and the stage of degeneration. In the nucleus pulposus (0). the chondrocyte pair count was similar to those in the annulus fibrosus. However, the cluster count tended to increase with the progression of degeneration. and A indicate statistical difference (p < 0.5) for the nucleus pulposus and for the inner layer of the annulus fibrosus, respectively.
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Light Microscopic Findings In the inner layer of the anterior annulus fibrosus, the chondrocytes occupying lacunae were generally scattered throughout the matrix of the collagen bundle framework. In the middle layer of the anterior annulus fibrosus, so-called chondrocyte pairs (two semicircular cells in a single lacuna) and cloning or clusters of several chondrocytes were found. In the outer layer of the annulus fibrosus, fibroblast-like cells were noted. Blood vessels were occasionally found in the outermost layer of the annulus fibrosus. In the transitional area between the inner layer of the annulus fibrosus and the nucleus pulposus, the annular architecture was no longer present, and chondrocytes, chondrocyte pairs, and clusters of various sizes were scattered throughout irregular fibrous matrix. For each layer of the annulus fibrosus and the nucleus pulposus, the relationship of the total chondrocyte count, chondrocyte pair count, and cluster count per unit area to the stage of disc degeneration was determined. Since the clusters varied in size,
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they were classified into three categories for counting: small clusters were composed of 3-10 cells, medium clusters were composed of 11-20 cells, and large clusters were composed of 21 or more cells. The relationship of the cluster totals in the nucleus pulposus and in the inner layer of the annulus fibrosus and the macroscopic stage of degeneration are shown in Figs. 2 and 3. In the inner layer of the annulus fibrosus, the total chondrocyte count tended to increase with the progression of degeneration (Fig. 2), whereas the chondrocyte pair count was high in slight and severe degeneration stages and was rather low in the moderate degeneration stage (Fig. 3, upper left). There was little correlation between the cluster count in the annulus fibrosus and the stage of degeneration, with an extremely low incidence of medium and large clusters (Fig. 3, upper right and lower trace). The total cell count (Fig. 2) and the chondrocyte pair count in the nucleus pulposus and the inner layer of the annulus fibrosus were similar, with the exception of Stage 111. However, the cluster count in the nucleus pulposus tended to increase
FIG. 4. A: AFlP lipofuscin stain of the nucleus pulposus of a 51-year-old man in stage IV degeneration revealed red-stained . Schmorl's reaction of the inner layer granules in the cytoplasm, suggestive Of lipofuscin granules. AFlP lipofuscin stain ~ 1 9 0B: of the annulus fibrosus of a 62-year-old man in stage IV degeneration showing granules stained blue in both the intracellular and extracellular area, suggestive of lipofuscin granules. Schmorl's reaction ~ 3 8 5 .C: Fluorescent micrograph of the nucleus pulposus of a 51-year-old man in stage IV degeneration showing granules emitting orange autofluorescence at excitation spectra between 334 nm and 365 nm, which is strongly suggestive of lipofuscin ~ 1 9 0 .
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with the progression of degeneration (p < 0.05). In particular, the medium and large clusters were characteristically increased in the degeneration nucleus pulposus (p < 0.05). The hematoxylin-eosin-stained sections revealed yellowish-brown granules in both intracellular and extracellular regions of chondrocytes. These granules were stained red by the AFIP lipofuscin stain (Fig. 4A) and blue by the Schmorl’s reaction (Fig. 4B) and emitted an orange autofluorescence at excitation spectra between 334 nm and 365 nm (Fig. 4C), suggesting the presence of lipofuscin granules. Fluorescence micrography also disclosed that blue to green materials of large, irregular shape and granules were distributed within the chondrocytes and in the territorial and interterritorial matrix. These granules were densely distributed in the areas from the inner layer of the annulus fibrosus to the nucleus pulposus and were seen in various cells. The cells containing lipofuscin granules consisted of chondrocytes (61.2%), chondrocyte pairs (23.8%), small clusters (13.9%),medium clusters (0.7%), and large clusters (0.6%). Regarding the relationship of the incidence of lipofuscin granules to the macroscopic stage of degeneration, the incidence of these granules tended to be high in Stages I and V discs. Thus, there appeared to be no particular correlation between the degenerative stage and the number of lipofuscin granules (Fig. 5). The relationship between lipofuscin granule count and patient age shown in Fig. 6 suggested that lipofuscin granules tend to appear in adulthood.
0
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-
li5 4 ]
.
0
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10
20
30
40
50
60
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FIG. 6. Relationship between lipofuscin granule count and age. 0 ,nucleus pulposus; 0, annulus fibrosus (inner layer).
Electron Microscopic Findings Chondrocytes
In general, chondrocyte cytoplasm was relatively abundant in rER, and cisternal enlargement of rER was found in approximately 16% of all chondrocytes. Golgi apparatus and mitochondria were also observed. Lysosomes were also found, but the incidence seemed to be relatively low. It was possible to observe the central body in the Golgi apparatus near the nucleus in only two of the 121 chondrocytes observed. In addition, lipid droplets were found in the cytoplasm in 36% of all chondrocytes. These droplets were often enlarged, and in some cases occupied almost the entire area of the cytoplasm (Fig. 7). Approximately 10% of chondrocytes contained a few lipofuscin granules, as described in detail later. Almost all chondrocytes were surrounded by areas rich in proteoglycan granules, forming the so-called territorial matrix. Territorial Matrix
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. I -
Q
7
1
2
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Stage
FIG. 5. Population of lipofuscin granules per mm2according to the degeneration stage. There was no particular correlation, but the incidence of the granule tended to be high in the Stages I and V discs. 0 ,nucleus pulposus; 0, annulus fibrosus (inner layer).
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Many proteoglycan granules were found in the areas surrounding the chondrocyte, within distances of 3 4 pm from the cell membrane. This ruthenium red-positive material typically shows islet formation in the territorial matrix. However, in the present study such a formation was relatively rare in the specimens observed. There were many electron-dense bodies, referred to as lipidic debris, in the marginal areas of the territorial matrix. A few lipofuscin-like granules were also found in the lipidic debris layer (Fig. 8).
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FIG. 7. Electron micrograph of a chondrocyte containing a lipid droplet in the nucleus pulposus of Stage IV. Lipofuscin granules are also shown in the cytoplasm. Ruthenium red stain ~ 1 , 7 0 0
Chondrocyte Pairs A chondrocyte pair is composed of two independent cells having a common temtorial matrix. The shape is semicircular or elliptic. Some chondrocyte pairs contained evident lipid droplets, vacuoles, or lipofuscin granules (Fig. 9). Although chondrocyte pairs appear to be the result of cell division, the protoplasms of the two chondrocytes were found to be in clear contact with each other in some of the chondrocyte pairs. It is noteworthy that in some cases, one chondrocyte was intruding into the territorial matrix of the adjacent chondrocyte; this suggests a developmental process of chondrocyte pairs. Cluster A chondrocyte cluster is an aggregation of independent cells, although it looks like a multinuclear cell by light microscopy. Clusters composed of 3-7 cells were observed by electron microscopy. It was rare for all cells in the cluster to be viable; some cells were necrotic or contained markedly increased
vacuoles, enlarged rER, and lipofuscin granules (Fig. 10). Distinct lipidic debris was found in the marginal areas of the cluster. In addition, banded structures were relatively frequently noted in the vicinity of the cluster. There was no contact between the cells like that seen in the chondrocyte pairs. Lipofuscin Granules
Lipofuscin granules were found both inside and outside the chondrocyte. These granules were generally composed of nonhomogenous electron-dense bodies of various degrees. Some granules accompanying the vacuoles and/or membrane contained fine granules that had a very high density (Fig. 11). Most of the intracellular lipofuscin granules were located in the marginal areas of the cell. About 76% of all lipofuscin granules contained vacuoles of varying sizes; some vacuoles were surrounded by electrondense bodies, and others were larger than electrondense bodies. The membrane of the lipofuscin granule was visible in about half of the lipofuscin granules observed. Lipid droplets were frequently found
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FIG. 8. Electron micrograph of a chondrocyte in the nucleus pulposus in Stage 111 showing proteoglycans and various shapes of electron-dense bodies, the lipidic debris in the pericellular matrix. A lipofuscin-like granule is also found (arrowhead).
Ruthenium red stain ~5,100.
in the vicinity of these lipofuscin granules. In addition, large vacuoles or lysosomes were occasionally found near the lipofuscin granules. Lipofuscin-like granules were also found in the cell debris, their basic structure presumably being the same as that of intracellular lipofuscin granules (Fig. 12). However, a few lipofuscin-like granules in the lipidic debris layer and in the territorial matrix did not have any vacuoles or membrane (Fig. 8). Matrix
Collagen fibrils measuring 40-110 nm in diameter were found in the matrix. Each fibril exhibited cyclic striations with intervals of approximately 50 nm. There were no obvious striations in the very thin fibrils, 15 nm in diameter, whereas the 20nm-diameter fibrils showed striations. Granules measuring 10-50 nm in diameter, suggestive of proteoglycan, were attached to the surface of the collagen fibril. Some fibrils showed no relationship of the proteoglycan granules to the striations. Collagen sheath structures of highly electron-dense sub-
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stances enveloping the collagen fibrils were also observed (3). In the matrix an amorphous electrondense material (27,28,33) that filled the interfibrillar spaces was found in areas 4-5 pm or farther from the chondrocyte. When two parts macroscopically different in color and obtained from the same intervertebral disc were compared, amorphous electrondense materials showing the different shapes of lipofuscin granules were markedly increased in the brown part (Fig. 13). Round, electron-dense, matrix vesicle-like bodies with a membrane, which are said to play an important role in tissue calcification, were seldom found between collagen fibrils (2). The diameter of the matrix vesicle-like body was approximately 0.1 km. Elastic fibers about 1 pm in diameter were only rarely observed. DISCUSSION
In the present study, fresh human intervertebral discs were graded according to macroscopic classification by the color and elasticity of the nucleus pulposus and the inner and middle layers of the an-
FIG. 9. A mate of one chondrocyte pair in the inner layer of the annulus fibrosus of Stage 111. Lipofuscin granules containing vacuoles can be seen. Ruthenium red stain x5.100.
FIG. 10. A small cluster in the inner layer of the nucleus pulposus in Stage 111. A necrotic cell within the cluster revealed lipofuscin granules, vacuoles, and enlarged rER in the cytoplasm (right side). Lipidic debris is shown in the pericellular matrix. Ruthenium red stain xl.700.
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FIG. 11. Lipofuscin granule nonhomogenous electron-dense body is demonstrated in the cytoplasm of a chondrocyte. Various vacuoles and thin membranes can be found in the lysosome. Ruthenium red stain x12.700.
FIG. 12. Lipofuscin-like granules seen at cell necrosis in the nucleus pulposus of Stage 111. Ruthenium red stain ~2,500. J Orthop Res, Vol. 9, N o . 1 , 1991
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FIG. 13. Amorphous electron-dense materials in the matrix of different areas of a disc with (A) and without (B) brown discoloration. The amorphous electron-dense bodies of different shapes from lipofuscin granules demonstrated between collagen fibrils are markedly increased in the upper photograph as compared with the lower photograph. Ruthenium red stain ~3,400.
nulus fibrosus. This classification is relatively well correlated with age. Only a few reports of “brown degeneration” in the intervertebral disc are available in the literature. In 1970, Peereboom reported that “age pigments” in a region showing “brown degeneration” in the annulus fibrosus were PASpositive and emitted autofluorescence (26). Since lipofuscin emits fluorescence of about 470 nm with an excitation spectrum of 360 nm, it is highly probable that these pigments were not truly lipofuscin. The pigments observed in the present study, however, emitted orange fluorescence by excitation at 330 nm and 365 nm and were positive for the lipofuscin stain and the Schmorl’s reaction. Therefore, these pigments were considered to be lipofuscin. In the intervertebral disc, brown degeneration was macroscopically more conspicuous in the area from the nucleus pulposus to the inner
layer of the annulus fibrosus; the incidence of lipofuscin granules was particularly high in these areas. Histological degeneration of the intervertebral disc was characterized by a marked increase in chondrocyte pairs and clusters in the nucleus pulposus and in the inner layer of the annulus fibrosus. In particular, in severely degenerated discs, large clusters alone were seen sparsely distributed, with almost no isolated chondrocytes. Regarding the nature of the pigments in question, it is thought that numerous pigments appear in postmitotic nerve and myocardial cells and also that these pigments increase in number with age (21, 22,31). Attention is now being drawn toward the possibility that pigments are an important morphological feature of aging. However, these pigments are present in general tissues, including liver cells and periosteal cells, and are not necessarily corre-
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FIG. 14. An isolated lipofuscin-llike granule is demonstrated in the territorial matrix close to the chondrocyte, suggesting a process of granule release of the cell. Ruthenium red stain ~5,100.
lated with age (17,34). We found no obvious correlation between the stage of disc degeneration and the incidence of pigments. This lack of correlation was also suggested from the high incidence of pigments in chondrocytes and the fact that the incidence of such pigments was low in chondrocyte pairs and clusters that characterize regressive change in tissues. Although there was no decisive evidence from the ultrastructural observations, it can be speculated that intracellular lipofuscin is released outside the chondrocyte during the cell division process, since chondrocytes seem to have the ability to release lipofuscin (Fig. 14). The outflow of intracellular lipofuscin might also be caused by cell collapse from an increase in necrotic cells. Calculation of the extracellular lipofuscin granules could not be achieved in this study, because the histochemical nature of this pigment might be altered in the extracellular matrix. Further observation is required. The above-mentioned speculation is derived from the fact that lipofuscin granules are more likely to appear in a so-called active zone, or the transitional area between the nucleus pulposus and the inner layer of the annulus fibrosus. However, since ceroid granules, which are very similar to lipofuscin
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granules, appear in the cerebrum (24) and villi of the intestinal tissue (19) in vitamin E-deficient animals, the appearance of lipofuscin granules should be regarded as a phenomenon closely related to the regressive process in tissue. Recent studies have demonstrated that these granules are positive for reactions of unsaturated fatty acid, aldehyde, phospholipid, and various amino acids (6). These granules are known to be derived from the reaction of a substance having an amino base and malondealdehyde, a cleavage product of lipid peroxide converted from a multivalent unsaturated fatty acid (5,6). Regarding the mechanism of disc degeneration, it seems that lipofuscin production and cell degeneration are closely linked in the process of the appearance of superoxide and the production of lipid peroxide (30). In the present study, fresh and large specimens of human intervertebral discs were removed without trauma from the anterior approach, proving very useful for electron microscopic observation. The following morphological features of the chondrocytes observed in this study were similar to those reported by previous studies: rER, Golgi apparatus, lipid droplets, proteoglycan in the territorial matrix, lipidic debris (16), and banded structure (4,7). How-
BROWN DEGENERATION OF HUMAN DISC ever, there was no obvious morphological difference between chondrocytes with and without lipofuscin granules. As for the morphological origin, lipofuscin granules are generally considered to originate from cell organelles and particularly from lysosomes (12). However, there are some reports suggesting that the mitochondria (25), Golgi apparatus (l), and rER (32), and other cell organelles are the origin. Other reports propose that these granules are produced without any connection whatsoever to cell organelles (15). The next question is that of the fate of lipofuscin produced in the cell. Since a similar structure has been found in the extracellular lipidic debris, it is hypothesized that lipofuscin is released outside the cell or flows into the matrix along with cell necrosis. Lipofuscin is usually metabolized by wandering leukocytes or histiocytes. However, in the intervertebral disc, which is avascular tissue, lipofuscin seems to remain in the matrix for some time and becomes diffused after some metabolic process. As the disc degenerates, isolated chondrocytes form into chondrocyte pairs and, subsequently, into clusters. Details of this process and its significance require further studies. It is possible that a chondrocyte pair is produced by the immigration of one chondrocyte into another. It is also possible that the cluster formation is an associated defense reaction of chondrocytes to cope with the deterioration of the external environment. Regarding discoloration in the intervertebral disc, occasionally no lipofuscin granules are seen microscopically in the brown discs. The amorphous electron-dense material, which is not lipofuscin, is markedly increased in the matrix of the brown degeneration disc. Thus the material that may play a role in the discoloration of the disc is considered morphologically to be cell debris, but some possible chemical alteration in the cell debris, such as a nonenzymatic browning process, cannot be ruled out (23). Further investigation is needed. Acknowledgment: This work was supported by the Grant-in-Aid for scientific Research of the Japanese Ministry of Education, Science, and Culture.
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ganglia of senile rats. An electron microscope study. J Geronto1 12:364369, 1957 2. Brighton CT: The growth plate. Orthop Clin North Am 15:57 1-594, 1984
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3. 1Buckwalter JA, Maynard JA, Cooper RR: Sheathing of colI agen fibrils in human intervertebral discs. J Anat 125:615t518, 1978 4. 1Buckwalter JA, Maynard JA, Cooper RR: Banded structures 1n human nucleus pulposus. Clin Orthop 139:259-266, 1979 5. (Zhio KS, Tappel AL: Synthesis and characterization of the 1Suorescent products derived from malonaldehyde and mino acids. Biochemistry 8:2821-2827, 1969 6. (Zhio KS, Fletcher URB, Tappel AL: Peroxidation of subI:ellular organelles: formation of lipofuscinlike fluorescent 1pigments. Science 166:1535-1536, 1969 7. Zornah MS, Meachim G, Parry EW: Banded structures in 1the matrix of human and rabbit nucleus pulposus. J Anat 107:351-362, 1970 8. ICloventry MB, Ghormley RK, Kernohan JW: The intervertebral disc: its microscopic anatomy and pathology. I. Anatomy, development, and physiology. J Bone Joint Surg 27:105-112, 1945 9. ICoventry MB, Ghormley RK, Kernohan JW: The intervertebral disc: its microscopic anatomy and pathology. 11. Changes in the intervertebral disc concomitant with age. J Bone Joint Surg 27:233-247, 1945 10. ICoventry MB, Ghormley RK, Kernohan JW: The intervertebral disc: its microscopic anatomy and pathology. 111. Pathological changes in the intervertebral disc. J Bone Joint Surg 27:460-473, 1945 11. Coventry MB: Anatomy of the intervertebral disc. Clin Orthop 67:%15, 1969 12. Essner E, Novikoff AB: Localization of acid phosphatase activity in hepatic lysosome by means of electron microscopy. J Biophys Biochem Cytol9:773-784, 1961 13. Flanklin L, Hull EW: Lipid content of the intervertebral disc. Clin Chem 12:253-257, 1966 14. Galante J: Tensile properties of the human lumbar annulus fibrosus. Acta Orthop Scand lOO(supp1): 1-91, 1967 15. Gedigk P, Wessel W: Elektronenmikroskopische Untersuchung des Vitamin-E-Mangel-Pigmentes in Myometrium der Ratte. Virchows Arch [A] 337:367-382, 1964 16. Ghardially FN: Fine structure of synovial joints. London, Buttenvorths Publishers, 1983, pp 236-260 17. Goldfisher S,Bernstein J: Lipofuscin (aging) pigment granules of the newborn human liver. J Cell Biol 42:253-261, 1969 18. Happey F: Studies of the structure of the human intervertebral disc in relation to its functional and aging process. The Joints and Synovial Fluid, Vol. 2, ed. by L. Sokoloff, New York, Academic Press, 1980, pp 95-137 19. Katz ML, Groome AB, Robinson WG: Localization of lipofuscin in the duodenums of vitamin Edeficient rats. J Nutr 115:1355-1365, 1985 20. Luna LG: Manual of Histologic Staining Methods of the Armed Forces Institute of Pathology, 3rd ed, New York, McGraw Hill, 1968, pp 185-186 21. Malkoff DB, Strehler BL: The ultrastructure of isolated and in situ human cardiac age pigment. J Cell Biol 16:611416, 1963 22. Mann DMA, Yates PO: Lipofuscin pigments: their relationship to aging in the human nervous system. I. The lipofuscin content of nerve cells. Brain 97:481488, 1974 23. Monnier VM, Cerami A: Nonenzymatic browning in vivo: Possible process for aging of long-lived protein. Science 211:491493, 1981 24, Nishioka N, Takahata N, Iizuka R: Histochemical studies on the life pigments in the nerve cells. A comparison with lipofuscin and ceroid pigment. Acta Neuropathol 11: 174-181, 1968 25. Payne F: Changes in the endocrine glands of the fowl with age. J Gerontol4:193-199, 1949
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