Imbalanced Protein Expression Patterns of Anabolic,Catabolic, Anti-Catabolic and Inflammatory Cytokines in Degenerative Cervical Disc Cells: New Indications for Gene Therapeutic Treatments of Cervical Disc Diseases

Degenerative disc disease (DDD) of the cervical spine is common after middle age and can cause loss of disc height with painful nerve impingement, bone and joint inflammation. Despite the clinical importance of these problems, in current publications the pathology of cervical disc degeneration has been studied merely from a morphologic view point using magnetic resonance imaging (MRI), without addressing the issue of biological treatment approaches. So far a wide range of endogenously expressed bioactive factors in degenerative cervical disc cells has not yet been investigated, despite its importance for gene therapeutic approaches. Although degenerative lumbar disc cells have been targeted by different biological treatment approaches, the quantities of disc cells and the concentrations of gene therapeutic factors used in animal models differ extremely. These indicate lack of experimentally acquired data regarding disc cell proliferation and levels of target proteins. Therefore, we analysed proliferation and endogenous expression levels of anabolic, catabolic, ant-catabolic, inflammatory cytokines and matrix proteins of degenerative cervical disc cells in three-dimensional cultures. Preoperative MRI grading of cervical discs was used, then grade III and IV nucleus pulposus (NP) tissues were isolated from 15 patients, operated due to cervical disc herniation. NP cells were cultured for four weeks with low-glucose in collagen I scaffold. Their proliferation rates were analysed using 3-(4, 5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide. Their protein expression levels of 28 therapeutic targets were analysed using enzyme-linked immunosorbent assay. During progressive grades of degeneration NP cell proliferation rates were similar. Significantly decreased aggrecan and collagen II expressions (P<0.0001) were accompanied by accumulations of selective catabolic and inflammatory cytokines (disintegrin and metalloproteinase with thrombospondin motifs 4 and 5, matrix metalloproteinase 3, interleukin-1β, interleukin-1 receptor) combined with low expression of anti-catabolic factor (metalloproteinase inhibitor 3) (P<0.0001). This study might contribute to inhibit inflammatory catabolism of cervical discs.     

Structure,function, aging and turnover of aggrecan in the intervertebral disc

Background

Aggrecan is the major non-collagenous component of the intervertebral disc. It is a large proteoglycan possessing numerous glycosaminoglycan chains and the ability to form aggregates in association with hyaluronan. Its abundance and unique molecular features provide the disc with its osmotic properties and ability to withstand compressive loads. Degradation and loss of aggrecan result in impairment of disc function and the onset of degeneration.

Scope of review

This review summarizes current knowledge concerning the structure and function of aggrecan in the normal intervertebral disc and how and why these change in aging and degenerative disc disease. It also outlines how supplementation with aggrecan or a biomimetic may be of therapeutic value in treating the degenerate disc.

Major conclusions

Aggrecan abundance reaches a plateau in the early twenties, declining thereafter due to proteolysis, mainly by matrix metalloproteinases and aggrecanases, though degradation of hyaluronan and non-enzymic glycation may also participate. Aggrecan loss is an early event in disc degeneration, although it is a lengthy process as degradation products may accumulate in the disc for decades. The low turnover rate of the remaining aggrecan is an additional contributing factor, preventing protein renewal. It may be possible to retard the degenerative process by restoring the aggrecan content of the disc, or by supplementing with a bioimimetic possessing similar osmotic properties.

General significance

This review provides a basis for scientists and clinicians to understand and appreciate the central role of aggrecan in the function, degeneration and repair of the intervertebral disc.     

Increase of nerve growth factor levels in the human herniated intervertebral disc: can annular rupture trigger discogenic back pain?

Introduction

Nerve growth factor (NGF) has an important role in the generation of discogenic pain. We hypothesized that annular rupture is a trigger for discogenic pain through the action of NGF. In this study, the protein levels of NGF in discs from patients with disc herniation were examined and compared with those from discs of patients with other lumbar degenerative disc diseases.

Methods

Patients (n = 55) with lumbar degenerative disc disease treated by surgery were included. Nucleus pulposus tissue (or herniated disc tissue) was surgically removed and homogenized; protein levels were quantified using an enzyme-linked immunosorbent assay (ELISA) for NGF. Levels of NGF in the discs were compared between 1) patients with herniated discs (herniated group) and those with other lumbar degenerative disc diseases (non-herniated group), and 2) low-grade and high-grade degenerated discs. Patient’s symptoms were assessed using a visual analog scale (VAS) and the Oswestry disability index (ODI); the influence of NGF levels on pre- and post-operative symptoms was examined.

Results

Mean levels of NGF in discs of patients were significantly higher in herniated discs (83.4 pg/mg total protein) than those in non-herniated discs (68.4 pg/mg).No significant differences in levels of NGF were found between low-grade and high-grade degenerated discs. Multivariate analysis, adjusted for age and sex, also showed significant correlation between the presence of disc herniation and NGF levels, though no significant correlation was found between disc degeneration and NGF levels. In both herniated and non-herniated groups, pre-operative symptoms were not related to NGF levels. In the herniated group, post-operative lower extremity pain and low back pain (LBP) in motion were greater in patients with low levels of NGF; no significant differences were found in the non-herniated group.

Conclusions

This study reports that NGF increased in herniated discs, and may play an important role in the generation of discogenic pain. Analysis of patient symptoms revealed that pre-operative NGF levels were related to post-operative residual lower extremity pain and LBP in motion. The results suggest that NGF in the disc is related to pain generation, however, the impact of NGF on generation of LBP varies in individual patients.

Electronic supplementary material

The online version of this article (doi:10.1186/ar4674) contains supplementary material, which is available to authorized users.     

A Combinatorial Relative Mass Value Evaluation of Endogenous Bioactive Proteins in Three-Dimensional Cultured Nucleus Pulposus Cells of Herniated Intervertebral Discs: Identification of Potential Target Proteins for Gene Therapeutic Approaches

Painful degenerative disc diseases have been targeted by different biological treatment approaches. Nucleus pulposus (NP) cells play a central role in intervertebral disc (IVD) maintenance by orchestrating catabolic, anabolic and inflammatory factors that affect the extracellular matrix. IVD degeneration is associated with imbalances of these factors, resulting in a catabolic inflammatory metabolism. Therefore, accurate knowledge about their quantity and quality with regard to matrix synthesis is vital for a rational gene therapeutic approach. NP cells were isolated from 63 patients operated due to lumbar disc herniation (mean age 56 / range 29 - 84 years). Then, three-dimensional culture with low-glucose was completed in a collagen type I scaffold for four weeks. Subsequently cell proliferation evaluation was performed using 3-(4, 5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide and intracellular concentration of 28 endogenously expressed anabolic, catabolic, inflammatory factors and relevant matrix proteins was determined by enzyme-linked immunosorbent assay. Specimen-related grades of degeneration were confirmed by preoperative magnetic resonance imaging. Independent from gender, age and grade of degeneration proliferation rates remained similar in all groups of NP cells. Progressive grades of degeneration, however, showed a significant influence on accumulation of selective groups of factors such as disintegrin and metalloproteinase with thrombospondin motifs 4 and 5, matrix metalloproteinase 3, metalloproteinase inhibitor 1 and 2, interleukin-1β and interleukin-1 receptor. Along with these changes, the key NP matrix proteins aggrecan and collagen II decreased significantly. The concentration of anabolic factors bone morphogenetic proteins 2, 4, 6 and 7, insulin-like growth factor 1, transforming growth factor beta 1 and 3, however, remained below the minimal detectable quantities. These findings indicate that progressive degenerative changes in NP may be problematic with regard to biologic treatment strategies. Hence, gene therapeutic interventions regulating relevant bioactive factors identified in this work might contribute to the development of regenerative treatment approaches for degenerative disc diseases.     

MRI Assessment of Lumbar Intervertebral Disc Degeneration with Lumbar Degenerative Disease Using the Pfirrmann Grading Systems

Background

To evaluate by MRI intervertebral disc degeneration in patients with lumbar degenerative disease using the Pfirrmann grading system and to determine whether Modic changes correlated with the Pfirrmann grades and modified Pfirrmann grades of disc degeneration.

Methods

The clinical data of 108 surgical patients with lumbar degenerative disease were reviewed and their preoperative MR images were analyzed. Disc degeneration was evaluated using the Pfirrmann grading system. Patients were followed up and low back pain was evaluated using the visual analog scale (VAS) and the effect of back pain on the daily quality of life was assessed using Oswestry disability index (ODI).

Results

Forty-four cases had normal anatomical appearance (Modic type 0) and their Pfirrmann grades were 3.77±0.480 and their modified Pfirrmann grades were of 5.81±1.006. Twenty-seven cases had Modic type I changes and their Pfirrmann grades were 4.79±0.557 and their modified Pfirrmann grades were 7.00±0.832. Thirty-six cases exhibited Modic type II changes and their Pfirrmann grades and modified Pfirrmann grades were 4.11±0.398 and 6.64±0.867, respectively. One case had Modic type III changes. Kruskal-Wallis test revealed significant difference in modified Pfirrmann grade among Modic type 0, I and II changes (P<0.01) but no significant difference between Modic type I and II changes (P>0.05). Binary regression analysis showed that Modic changes correlated most strongly with disc degeneration. Follow up studies indicated that the VAS and ODI scores were markedly improved postoperatively. However, no difference was noted in VAS and ODI scores among patients with different Modic types.

Conclusion

Modic changes correlate with the Pfirrmann and modified Pfirrmann grades of disc degeneration in lumbar degenerative disease. There is no significant correlation between Modic types and surgical outcomes.     

Osteoarthritis of the hands, hips and knees in an Australian twin sample--evidence of association with the aggrecan VNTR polymorphism.

Age-related changes in the composition of the cartilage matrix may be associated with the development of osteoarthritis, a relatively late-onset disease characterised by the destruction of joint cartilage. In order to investigate whether differences in the VNTR polymorphic region of aggrecan affect cartilage functionality and therefore the development of osteoarthritis, we examined the aggrecan polymorphic genotypes of a sample of 134 Australian twins aged over 50 (including 34 monozygotic and 27 dizygotic twin pairs). Clinical measures of hand, hip and knee osteoarthritis, as well as self-reported bone and joint pain, were tested for association with the aggrecan polymorphism. The results were consistent with either a deleterious effect of allele 27, or a protective effect of alleles 25 and 28, providing some additional evidence for an association between the aggrecan VNTR polymorphism and osteoarthritis of the hands, hips and knees.     

Percutaneous radiofrequency lumbar rhizolysis (rhizotomy).

Low back pain may arise from degenerative changes in the posterior joints of the lumbar spine. These joints are innervated by a branch of the posterior primary ramus, which follows an anatomically constant course. Pain impulses from these joints can be interrupted by coagulating this nerve with a radiofrequency wave, the probe having been placed in the area of the nerve percutaneously. Percutaneous lumbar rhizolysis was carried out under local anesthesia on an outpatient basis in 82 patients, most of whom had multiple level rhizolysis. Rhizolysis was successful in 67% of patients with mechanical low back pain without evidence of disc herniation and nerve-root compression or psychogenic pain, who had not previously undergone an operation for relief of the pain.     

Clinical experience in cell-based therapeutics: disc chondrocyte transplantation A treatment for degenerated or damaged intervertebral disc

Disc herniation treated by discectomy results in a significant loss of nucleus material and disc height. Biological restoration through the use of autologous disc chondrocyte transplantation offers a potential to achieve functional integration of disc metabolism and mechanics. Chondrocytes that have been removed from damaged cartilaginous tissues maintain a capacity to proliferate, produce and secrete matrix components and respond to physical stimuli such as dynamic loading. Nucleus regeneration using autologous cultured disc-derived chondrocytes (ADCT) has been demonstrated in a canine model and in clinical pilot studies. In 2002 a prospective, controlled, randomised, multi-center study, EuroDISC, comparing safety and efficacy of autologous disc chondrocyte transplant, chondrotransplant DISC, plus discectomy (ADCT), with discectomy alone was initiated. A dog model was used to investigate the hypothesis that autologous disc chondrocytes can be used to repair damaged intervertebral disc. Disc chondrocytes were harvested and expanded in culture under controlled and defined conditions, returned to the same animals from which they had been sampled (autologous transplantation) via percutaneous delivery. The animals were analyzed at specific times after transplantation by several methods to examine whether disc chondrocytes integrated with the surrounding tissue, produced the appropriate intervertebral disc extracellular matrix, and might provide a formative solution to disc repair. The clinical goals of the EuroDISC study, were to provide long-term pain relief, maintain disc height and prevent adjacent segment disease. Interim analysis was performed after 2 years; Oswestry (low back pain/disability), Quebec Back-Pain Disability Scale, as well as Prolo and VAS score were used for the evaluation. Disc height was assessed by MRI. In the context of degenerative changes in an injury model: () autologous disc chondrocytes were expended in culture and returned to the disc by a minimally invasive procedure after 12 weeks; () disc chondrocytes remained viable after transplantation as shown by bromodeoxyuridine incorporation and maintained a capacity for proliferation after transplantation as depicted by histology; () transplanted disc chondrocytes produced an extracellular matrix that displayed composition similar to normal intervertebral disc tissue. Positive evidence of Proteoglycan content was supported by accepted histochemical staining techniques such as Safranin O-Fast Green; () both Type II and Type I collagens were demonstrated in the regenerated intervertebral disc matrix by immunohistochemistry after chondrocyte transplantation; and () when the disc heights were analyzed for variance according to treatment a statistically significant-correlation between transplanting cells and retention of disc height was achieved. A clinically significant reduction of low back pain in the ADCT-treated group was shown by all three pain score systems. The median total Oswestry score was 2 in the ADCT-treated group compared with 6 in the control group. Decreases in the disability index and VAS score in ADCT-treated patients correlated strongly with the reduction of low back pain. Decreases in disc height over time were only found in the control group, and of potential significance, intervertebral discs in adjacent segments appeared to retain hydration when compared to those adjacent to levels that had undergone discectomy without cell intervention. Autologous chondrocyte transplantation is technically feasible and biologically relevant to repairing disc damage and retarding disc degeneration.     

Degenerative Pathways of Lumbar Motion Segments - A Comparison in Two Samples of Patients with Persistent Low Back Pain

Background

Magnetic resonance imaging (MRI) is used to identify spinal pathoanatomy in people with persistent low back pain. However, the clinical relevance of spinal degenerative MRI findings remains uncertain. Although multiple MRI findings are almost always present at the same time, research into the association with clinical outcomes (such as pain) has predominantly focused on individual MRI findings. This study aimed to: (i) investigate how multiple MRI lumbar spine findings cluster together within two different samples of patients with low back pain, (ii) classify these clusters into hypothetical pathways of degeneration based on scientific knowledge of disco-vertebral degeneration, and (iii) compare these clusters and degenerative pathways between samples.

Methods

We performed a secondary cross-sectional analysis on two dissimilar MRI samples collected in a hospital department: (1) data from the spinal MRI reports of 4,162 low back pain patients and (2) data from an MRI research protocol of 631 low back pain patients. Latent Class Analysis was used in both samples to cluster MRI findings from lumbar motion segments. Using content analysis, each cluster was then categorised into hypothetical pathways of degeneration.

Results

Six clusters of MRI findings were identified in each of the two samples. The content of the clusters in the two samples displayed some differences but had the same overall pattern of MRI findings. Although the hypothetical degenerative pathways identified in the two samples were not identical, the overall pattern of increasing degeneration within the pathways was the same.

Conclusions

It was expected that different clusters could emerge from different samples, however, when organised into hypothetical pathways of degeneration, the overall pattern of increasing degeneration was similar and biologically plausible. This evidence of reproducibility suggests that Latent Class Analysis may provide a new approach to investigating the relationship between MRI findings and clinically important characteristics such as pain and activity limitation.     

Genome-wide association study in Dachshund: identification of a major locus affecting intervertebral disc calcification

Intervertebral disc calcification and herniation commonly affects Dachshund where the predisposition is caused by an early onset degenerative process resulting in disc calcification. A continuous spectrum of disc degeneration is seen within and among dog breeds, suggesting a multifactorial etiology. The number of calcified discs at 2 years of age determined by a radiographic evaluation is a good indicator of the severity of disc degeneration and thus serves as a measure for the risk of developing intervertebral disc herniation. The aim of the study was to identify genetic variants associated with intervertebral disc calcification in Dachshund through a genome-wide association (GWA) study. Based on thorough radiographic examinations, 48 cases with ≥ 6 disc calcifications or surgically treated for disc herniation and 46 controls with 0-1 disc calcifications were identified. GWA using the Illumina CanineHD BeadChip identified a locus on chromosome 12 from 36.8 to 38.6 Mb with 36 markers reaching genome-wide significance (P(genome) = 0.00001-0.026). This study suggests that a major locus on chromosome 12 harbors genetic variations affecting the development of intervertebral disc calcification in Dachshund.     

Disc degeneration implies low back pain

Background

Low back pain exerts a tremendous burden on individual patients and society due to its prevalence and ability to cause long-term disability. Contemporary treatment and prevention efforts are stymied by the absence of a confirmed cause for the majority of low back pain patients.

Methods

A system dynamics approach is used to build a physiologically-based model investigating the relationship between disc degeneration and low back pain. The model’s predictions are evaluated under two different types of study designs and compared with established observations on low back pain.

Results

A three-compartment model (no disc degeneration, disc degeneration with pain remission, disc degeneration with pain recurrence) accurately predicts the age-specific prevalence observed in one of the largest population-based surveys (R ²?=?0.998). The estimated transition age at which intervertebral discs lose the growth potential and begin degenerating is 13.3 years. The estimated disc degeneration rate is 0.0344/year. Without any additional change being made to parameter’s values, the model also fully accounts for the age-specific prevalence of disc degeneration detected with a lumbar MRI among asymptomatic individuals (R ²?=?0.978).

Conclusions

Dual testing of the proposed mechanistic model with two independent data sources (one with lumbar MRI and the other without) confirm that disc degeneration is the driving force behind and cause of age dependence in low back pain. Observed complexity of low back pain epidemiology arises from the slow dynamics of disc degeneration coupled with the fast dynamics of disease recurrence.

     

Analysis of Polymorphism of the Number of Tandem Repeats in the Aggrecan Gene Exon G3 in the Families with Idiopathic Scoliosis

In our previous study we showed that the inheritance of pronounced forms of idiopathic scoliosis was described by an autosomal-dominant major gene model assuming incomplete sex- and age-dependent penetrance. In the present study a search for the major gene was carried out by means of testing candidate genes. The aggrecan gene with known polymorphism of the number of tandem repeats in exon G3 was considered to be one of these candidate genes. Various alleles of this gene provide attachment of different number of chondroitin sulfate chains to a proteoglycan core protein, thereby changing functional properties of cartilage. Using the TDT analysis of 33 unrelated families consisting of a proband and his/her parents, we examined the existence of associations between the aggrecan alleles and the disease. Among nine alleles identified, three alleles with tandem repeats numbers of 25, 26, and 27 prevailed. We did not reveal preferable transmission of any of these alleles to the proband (TDT-statistics for different alleles varied from 0 to 0.71). There was also no correlation between the number of tandem repeats and the disease severity. Thus, either the polymorphism of the number of tandem repeats is not the direct reason for development of idiopathic scoliosis in the families tested, or its effect is too low to be detected using the samples examined.     

Alterations in biochemical components of extracellular matrix in intervertebral disc herniation: role of MMP-2 and TIMP-2 in type II collagen loss

Alterations in the composition of intervertebral disc extracellular matrix, mainly collagen and proteoglycans, may cause changes in mechanical properties of the disc, leading to dysfunction, nerve root compression, and herniation with severe clinical manifestations. Matrix metalloproteinases may be involved in degradation by hydrolysing extracellular matrix components. Inhibitors of matrix metalloproteinases, in contrast, function in the maintenance of degradation control. In this study, we investigated: (i) whether the level of matrix degradation correlated with the duration of the symptomatic disease, (ii) roles of matrix metalloproteinase-2 (MMP-2) and tissue inhibitor of matrix metalloproteinases-2 (TIMP-2) in intervertebral disc degeneration. Nucleus pulposus of intervertebral discs were obtained from 22 patients and analysed for collagen and proteoglycan contents, and pro-MMP-2, TIMP-2 levels. Collagen content was determined as hydroxyproline and proteoglycan content was measured as glycosaminoglycans. The loss in matrix components did not correlate with the duration of the degenerative disc disease. Pro-MMP-2 levels were higher at early stages of the degenerative disc disease (r = -0.495, P < 0.05). TIMP-2 levels were similar in all samples. Pro-MMP-2 and TIMP-2 levels negatively correlated in herniated discs samples (r = -0.855, P < 0.01). Pro- MMP-2 levels negatively correlated with the collagen content in herniated disc material. Our findings may suggest a silent period of active disease prior to symptomatic outcome during which irreversible matrix loss occurs. Involvement of other proteolytic enzymes at different stages of the disease should also be investigated to help to control the degradation cascade at relatively early stages of disc degeneration before the clinical onset of disease.     

Analysis of polymorphism of the number of tandem repeats in the aggrecan gene exon G3 in the families with idiopathic scoliosis

In our previous study we showed that the inheritance of pronounced forms of idiopathic scoliosis was described by an autosomal-dominant major gene model assuming incomplete sex- and age-dependent penetrance. In the present study a search for the major gene was carried out by means of testing candidate genes. The aggrecan gene with known polymorphism of the number of tandem repeats in exon G3 was considered to be one of these candidate genes. Various alleles of this gene provide attachment of different number of chondroitin sulfate chains to a proteoglycan core protein, thereby changing functional properties of cartilage. Using the TDT analysis of 33 unrelated families consisting of a proband and his parents, we examined the existence of associations between the aggrecan alleles and the disease. Among nine alleles identified, three alleles with tandem repeats numbers of 25, 26, and 27 prevailed. We did not reveal preferable transmission of any of these alleles to the proband (TDT-statistics for different alleles varied from 0 to 0.71). There was also no correlation between the number of tandem repeats and the disease severity. Thus, either the polymorphism of the number of tandem repeats is not the direct reason for development of idiopathic scoliosis in the families tested, or its effect is too low to be detected using the samples examined.     

TGF-β synergizes with ML264 to block IL-1β-induced matrix degradation mediated by Krüppel-like factor 5 in the nucleus pulposus

Intervertebral disc degeneration causes low back pain.Interleukin-1β (IL-1β) is a well-known inflammatory mediator that is involved in disc degeneration but its molecular mechanisms on catabolic and anabolic events in nucleus pulposus (NP) cells remain unclear. Krüppel-like factor 5 (KLF5) is associated with inflammation and was previously shown to cause cartilage degradation. In this study, we revealed that KLF5 is involved in IL-1β activated NF-kB cascade by enhancing both p65 phosphorylation and p65 acetylation. Moreover, the catabolic effect of KLF5 can be abolished by transforming growth factor-β (TGF-β) via promoting the proteasomal degradation of KLF5. Therefore, a KLF5 inhibitor ML264 was further proved to synergize with TGF-β to attenuate IL-1β-induced intervertebral disc degeneration. These results indicate the critical role of KLF5 in regulating intervertebral disc metabolism and suggest KLF5 inhibitor such as ML264 as potential compound for treatment of degenerative disc disease.     

中药熏蒸联合推拿治疗腰椎间盘突出症的临床效果

目的：探讨中药熏蒸联合推拿治疗腰椎间盘突出症的临床效果。方法：选择2013 年12 月到2015 年1 月我院收治的112 例 腰椎间盘突出症患者为研究对象,根据治疗方法的不同分为研究组和对照组。对照组给予常规推拿治疗措施,研究组给予常规推 拿措施联合中药熏蒸治疗。比较两组患者的住院时间,下肢放射痛、麻木、腰痛的发生率及临床疗效。结果：研究组患者的住院时 间及下肢放射痛、麻木、腰痛的发生率均较对照组明显缩短或降低,其治疗总有效率为92.9%,显著高于对照组,均有统计学差异 (P＜0.05)。结论：中药熏蒸联合推拿方法治疗腰椎间盘突出症的临床效果优于单一推拿治疗措施,可有效改善患者的临床症状,缩 短疗程,提高治疗有效率,值得临床推广使用。     

Glu298Asp polymorphism of the endothelial nitric oxide synthase gene in Turkish patients with ischemic stroke

The low plasma nitric oxide concentrations and reduced vascular reactivity are considered major proatherogenic mechanisms in cardiovascular diseases. The present study aimed to assess the allelic frequency and the genotypic distribution of the Glu298Asp gene polymorphism at exon 7 of endothelial nitric oxide synthase (eNOS) gene in Turkish ischemic stroke patients compared to appropriate healthy controls, and to correlate the genetic findings with stroke subtypes. The study population included 146 (75 males, 71 females) patients with ischemic stroke which were categorized according to the Trial of ORG 10172 in Acute Stroke Treatment (TOAST) and 133 (34 males, 99 females) healthy subjects. The eNOS polymorphism was identified with a PCR followed by RFLP with the restriction enzyme BanII. Genotypes were defined as GG, GT, and TT according to the presence of the G and T alleles. In this case-control study, we did not find any significant difference in either the genotypic distribution or allelic frequency of Glu298Asp gene polymorphism between the patients and the controls. In addition, there was also no significant difference for the genotype distribution and the allelic frequency among the stroke subtypes. The results suggested the lack of the association between the Glu298Asp gene polymorphism and ischemic stroke or subtypes of ischemic stroke in the Turkish population.