The Aging-related Oxidative Stress Status Of Intervertebral Disc In Normal Aging Rats

BackgroundLow back pain (LBP) continues to be a major cause of morbidity in china and the world. Low back pain is a common disease in orthopedically clinic which puzzles such a large amount of People and effect their lives and works. It is reported that over 80% of the population will suffer from LBP. As a report received from National Center for Health Statistics, LBP is the most common factor that causes older patient limit of activity. Although the exact cause has yet to be defined, the intervertebral disk and its age-related changes have been most frequently implicated. Human intervertebral disc undergoes multifactorial biochemical and morphologic degenerative changes during the process of aging. The frequency of degeneration, especially lumbar degeneration increases sharply with age and is regarded as a major cause of discogenic low back pain. It is generally accepted that degeneration of discs begins during the second decade of life, and it is well known that other structures of spine do not change as fast as the intervertebral discs. The most extensive degenerative changes occur in the nucleus pulposus, including degradation of proteoglycans aggregates with its fragmentation, dehydration, and loss of viable cells. Data available in literature, suggests that the most critical factor contributing to the degeneration, is increasing volume of the disc due to its growth, resulting in longer nutritional pathways and insufficient nutrition. Basic science research has demonstrated that the intervertebral disc is an avascular tissue. While the annulus fibrosus is predominantly collagenous, the matrix of the central nucleus pulposus is rich in proteoglycans. With aging, the substance of proteoglycans significantly decreases which is believed to be a critical factor in intervertebral disc degeneration.For organisms living in an aerobic environment, exposure to reactive oxygen species (ROS) is continuous and unavoidable. ROS encompass a variety of diverse chemical species including superoxide anions, hydroxyl radicals and hydrogen peroxide. Some of these species, such as superoxide or hydroxyl radicals, are extremely unstable, whereas others, like hydrogen peroxide, are freely diffusible and relatively long-lived. These various radical species can either be generated exogenously or produced intracellularly from several different sources. Oxidative stress occurs as a consequence of excessive production of reactive oxygen species or impaired antioxidant defense systems. Antioxidant enzymes include two types of superoxide dismutase (SOD), which specifically scavenges superoxide radicals: copper-zinc SOD, which is located in the cytosol and Mn-SOD, which is located in the mitochondria. SOD is the first enzymatic step in the defense system against oxidative stress. One of the major presentations of oxidative stress is lipid peroxidation, Lipid peroxidation is initiated by free radicals attack of membrane lipids, generating large amounts of reactive products. And Malondialdehyde (MDA) is an important marker of oxidative stress. The oxidation of protein plays an essential role in the pathogenesis of a large number of degenerative diseases, which is now widely recognized. Biomarkers of protein oxidation are often applied when a battery of markers of oxidative stress status is being studied. A new marker of protein oxidation, advanced oxidation protein products (AOPP), has begun to call the attention of some investigators. AOPP were described by Witko-Sarsat et al for the first time. It is believed that oxidative stress is caused by an imbalance between the production of reactive oxygen and a biological system's ability to neutralize the reactive intermediates. Oxidative damage occurs because of both intrinsic and extrinsic mechanisms. Intrinsic and extrinsic damage are the primary causes of cell aging. The cell uses a series of intrinsic antioxidants to protect itself from free radical damage. Naturally occurring extrinsic antioxidants have also been widely shown to offset and alleviate these changes. Although ageing is likely to be a multifactorial process, there is now significant evidence implicating the aging and oxidative stress. Ageing is a factor that causes intervertebral disc degeneration. And to date, there are few preliminary reports studying the role of oxidative Stress in the age-related changes of intervertebral disc. So we speculate that oxidative stress might play a role in the age-related changes of intervertebral disc.ObjectiveTo assess the aging-related oxidative stress status of intervertebral disc in normal aging rats, and to study the relationship between oxidative stress and the age-related changes of intervertebral disc, and to learn the role of oxidative stress in the age-related changes of intervertebral disc in rats.Method1. A total of 60 wista male rats were divide into 3 groups, youth group (3 months),adult group (9 months) and senile group (18 months).Artery blood and intervertebral disc were collected from every rats, the neat starch of the pulpous nucleus tissues was adopted.2. Histology:Five samples select randomly from every group. Fixed samples were decalcified en bloc with a rapid decalcifier solution. After decalcification, the vertebrae in each sample were cut off on the center with a scalpel. The discs from L5/S1or L6/S1 with their 2 adjacent half vertebrae were sectioned midsagittally parallel to the direction of puncture with a scalpel, dehydrated and embedded. Paraffin-embedded sectioning was carried out on each disc. Two 6μm midsagittal sections were prepared for mounting and staining with safranin o-fast green, and hematoxylin and eosin respectively.3. Histological grading scale:The scale is based on 5 categories of degenerative changes with scores ranging from 5 points (1 in each category) for a normal disc to 15 points (3 in each category) for every disc. Including cellularity of the anulus fibrosus,morphology of the anulus fibrosus,border between the anulus fibrosus and nucleus pulposus,cellularity of the nucleus pulposus and morphology of the nucleus pulposus.4. Biochemistry:The concent of NO in serum and in homogenate of intervertebral disc were detected by the method of nitric acid reductase. The activity of SOD was measured by xanthine oxidase method. The levels of MDA were determined by thiobarbituric acid colorimetry. The concent of AOPP in serum and in homogenate of intervertebral disc was determined by spectrophotometry.5. Statistical Analysis:Appropriate data management and statistical analysis was performed using Excel 2003and SPSS 13.0. Data were summarized with routine descriptive statistics and expressed as the mean±tandard error.3group data were tested using one-way analyses of variance (ANOVA) with a LSD test. Differences at P< 0.05 were regarded as significant.Result1. The histologic appearances of intervertebral discs:in the youth group,we found that the nucleus pulposus were round nearly, comprising at least half of the disc area in midsagittal sections. Normal cellularity with stellar shaped nuclear cells evenly distributed throughout the nucleus. Well-organized collagen lamellae without ruptured or serpentine fibers.in the adult group, we found that there were degenerate, such as the nucleus pulposus were rounded or irregularly shaped, comprising one quarter to half of the disc area in midsagittal sections, Slight decrease in the nunber of cells with some clustering, Cellularity of the anulus fibrosus were neither fibroblasts nor chondrocytes comprise more than 75% of the cells. in the senile group, the degenerate of intervertebral were obviously, we can saw that the nucleus pulposus were Irregularly shaped, comprising less than one quarter of the disc area in midsagittal sections, the anulus fibrosus Inward bulging, ruptured or serpentine fibers in more than one third of the annulus. Border between the anulus fibrosus and nucleus pulposus were moderate or severe interruption. Histological Grading Scale show that there were significant difference among 3 proups (F=42.818, P=0.000). The scale in youth group were obviously less than that in adult group (P=0.000;P=0.000), the scale in adult group were obviously less than that in senile group (P=0.002)2. Biochemistry(1) The concent of NO:The concent of NO in serum and intervertebral were increase gradually with aging. The concent of NO in serum in 3 groups respective were 15.1403±3.9712μmol/L,15.8841±4.3315μmol/L and 16.0915±4.8031μmol/L. The concent of NO in intervertebral disc in 3 groups respective were 18.6357±16.3681μmol/gprot,37.7185±20.6398μmol/gprot and 49.1593±28.7779μmol/gprot. and we found that there were no significant difference in The concent of NO of serum among 3 groups (F=0.261, P=0.771);But the concent of NO in intervertebral disc of the youth, adult and senile group were significant different (F=9.374, P=0.000). The concent of NO in intervertebral disc of adult and senile were significant higher than that of youth group (P=0.000;P=0.010). there are no significant different between adult group and senile group (P=0.114).(2) The activity of SOD was measured by xanthine oxidase method.we found that The activity of SOD in serum and intervertebral disc were decrease gradually with aging (F=7.777, P=0.000;F=6.448, P=0.003), in youth group respective were 187.6387±6.4206U/ml and 98.5516±24.8518U/mgprot, in adult group respective were182.2228±5.5937U/ml and 86.5944±26.8894U/mgprot.in senile group respective were179.9398±6.9553U/mland 69.2931±25.9436U/mgprot. The activity of SOD in serum of youth group were significant higher than that of adult and senile group (P=0.009;P=0.000). There are no significant different between adult group and senile group (P=0.114). The activity of SOD in intervertebral disc of senile group were significant lower than that of youth and adult group (P=0.001;P=0.039). there are no significant different between youth group and adult group (P=0.150)(3) The levels of MDA were determined by thiobarbituric acid colorimetry. Data show that the levels of MDA in serum in 3 groups respective were 6.7615±2.2892 nmol/ml,8.3463±2.7170 nmol/ml and 12.1078±2.4669 nmol/ml. The levels of MDA in intervertebral disc in 3 groups respective were 5.4613±3.7687nmol/mgprot, 6.2193±4.1821 nmol/mgprot and 8.9931±4.8440 nmol/mgprot. The concent of MDA in serum and intervertebral disc were increase gradually with aging(F=24.184, P=0.000; F=3.760, P=0.029). The levels of MDA in serum of youth group were significant lower than that of adult and senile group (P=0.050;P=0.000), and that of adult lower than senile group(P=0.000). The levels of MDA in intervertebral disc of senile group were significant higher than that of youth and adult group (P=0.012; P=0.045). There are no significant different between youth group and adult group (P=0.578).(4) The concent of AOPP in serum and in homogenate of intervertebral was determined by spectrophotometry. The concent of AOPP in serum and intervertebral disc were decrease gradually with aging(F=42.040, P=0.000; F=19.645, P=0.000), in youth group respective were 37.2310±8.9431μmol/L and 55.7073±21.2910μmol/L, in adult group respective were69.2388±17.2419μmol/L and 93.3990±25.3464μmol/L, in senile group respective were82.9855±20.2328μmol/L and 107.4738±33.0502μmol/L. The concent of AOPP in intervertebral disc in adult group and senile group were obviously higher than that in younth group (P=0.000;P=0.000). And aslo in serum (P=0.000;P=0.000). The concent of AOPP in serum in senile group were obviously higher than that in adult group (P=0.009)Conclusion1. The intervertebral disc in normal aging rats suffer degenerate with aging.2. There is severe Oxidative Stress in the intervertebral disc; Oxidative Stress may play an important intermediary role in the age-related changes of intervertebral disc.