Effect of hyperglycemia on apoptosis of notochordal cells and intervertebral disc degeneration in diabetic rats

Laboratory investigation

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Object

Diabetes mellitus is thought to be an important etiologic factor in intervertebral disc degeneration. It is known that notochordal cells gradually disappear from the nucleus pulposus (NP) of the intervertebral disc with age by undergoing apoptosis. What is not known is whether diabetes has an effect on apoptotic rates of notochordal cells. The purpose of this study was to investigate the effect of hyperglycemia on apoptosis of notochordal cells and intervertebral disc degeneration in age-matched OLETF (diabetic) and LETO (control) rats.

Methods

Lumbar disc tissue (L1–2 through L5–6), including cranial and caudal cartilaginous endplates, was obtained from 6- and 12-month-old OLETF and LETO rats (40 rats, 10 in each of the 4 groups). The authors examined the NP using TUNEL, histological analysis, and Western blot for expression of matrix metalloproteinase (MMP)–1, -2, -3, and -13, tissue inhibitor of metalloproteinase (TIMP)–1 and -2, and Fas (apoptosis-related protein). The apoptosis index of notochordal cells was calculated. The degree of transition of notochordal NP to fibrocartilaginous NP was classified on a scale ranging from Grade 0 (no transition) to Grade 4 (transition > 75%). The degree of expression of MMP-1, -2, -3, and -13, TIMP-1 and -2, and Fas was evaluated by densitometry.

Results

At 6 and 12 months of age, OLETF rats showed increased body weight and abnormal 2-hour glucose tolerance tests compared with LETO rats. The apoptosis index of notochordal cells was significantly higher in the OLETF rats than in the LETO rats at both 6 and 12 months of age. The degree of transition of notochordal NP to fibrocartilaginous NP was significantly higher in the OLETF rats than in the LETO rats at 6 and 12 months of age. The expression of MMP-1, -2, -3, and -13, TIMP-1, and Fas was higher in the OLETF rats at 6 and 12 months of age. The expression of TIMP-2 was significantly higher in the OLETF rats than in the LETO rats at 6 months of age, but not at 12.

Conclusions

The findings suggest that diabetes is associated with premature, excessive apoptosis of NP notochordal cells. This results in an accelerated transition of a notochordal NP to a fibrocartilaginous NP, which leads to early intervertebral disc degeneration. It remains to be determined if these premature changes are due to hyperglycemia or some other factors associated with diabetes. Understanding the mechanism by which diabetes affects disc degeneration is the first step in designing therapeutic modalities to delay or prevent disc degeneration caused by diabetes mellitus.

Abbreviations used in this paper: LETO = Long-Evans Tokushima Otsuka; MMP = matrix metalloproteinase; NP = nucleus pulposus; OLETF = Otsuka-Long-Evans-Tokushima fatty; TIMP = tissue inhibitor of metalloproteinase; TUNEL = terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end-labeling.

Article Information

Address correspondence to: Jong-Beom Park, M.D., Ph.D., Department of Orthopaedic Surgery, Uijeongbu St. Mary's Hospital, The Catholic University of Korea, 61–1 Kumho-dong, Uijeongbu-si, Kyunggi-do, Korea 480–717. email: spinepjb@catholic.ac.kr.

© AANS, except where prohibited by US copyright law.

Headings

Figures

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    Photomicrographs demonstrating the transition grading of notochordal NP to fibrocartilaginous NP. The degree of transition is graded with reference to its severity (range 0–4) in a midsagittal section (× 100) from each sample. Grade 0 indicates no fibrocartilaginous NP (all notochordal); Grade 1, < 25% fibrocartilaginous NP; Grade 2, 25–50%; Grade 3, 50–75%; and Grade 4, > 75%. H & E and Masson trichrome. Original magnification × 40 (left column) and × 100 (right column).

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    A and B: Line graphs showing results of 2-hour glucose tolerance test in 6-month-old (A) and 12-month-old (B) rats. C: Bar graph showing comparison of 2-hour glucose levels in LETO and OLETF rats at the 2 age points (119 ± 16 mg/dl vs 225 ± 34 mg/dl, p = 0.001; 112 ± 14 mg/dl vs 142 ± 21 mg/dl, p < 0.05, respectively).

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    Photomicrographs (A–D) and bar graph (E) demonstrating results of TUNEL assay for apoptosis of notochordal cells. Apoptosis of notochordal cells in NP of OLETF and LETO rats was evident in specimens from 6- and 12-month old animals. At both time points, the apoptosis index of notochordal cells was significantly increased in OLETF rats compared with the index in LETO rats (24.8 ± 4.2% vs 18.6 ± 3.1%, p < 0.05; 34.6 ± 3.8% vs 24.4 ± 2.1%, p < 0.01, respectively).

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    Photomicrographs (A–D) and bar graph (E) demonstrating transition grading of notochordal NP to fibrocartilaginous NP. Masson trichrome stain demonstrated higher transition grades in OLETF rats compared with LETO rats at 6 and 12 months of age. The degree of transition of notochordal NP to fibrocartilaginous NP was significantly increased in OLETF rats at 6 and 12 months of age compared with LETO rats (2.3 ± 0.4 vs 1.4 ± 0.3, p < 0.01; 3.6 ± 0.3 vs 2.6 ± 0.5, p < 0.01, respectively).

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    Western blots (A) and bar graphs showing the results of Western blotting (B and C). Western blotting demonstrated the expression of MMP-1, -2, -3, and -13, TIMP-1 and -2, and Fas in the NP of OLETF and LETO rats at 6 and 12 months of age. Expression of MMP-1, -2, -3, and -13, TIMP-1 and -2, and Fas was significantly increased in OLETF rats at 6 months of age compared with LETO rats (all, p < 0.01). Expression of MMP-1, -2, -3, and -13, TIMP-1, and Fas was significantly increased in OLETF rats at 12 months of age compared with LETO rats (all, p < 0.05). While the expression of TIMP-2 was slightly higher in OLETF rats at 12 months of age compared with LETO rats, the difference was not significant (p = 0.251).

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