Anat Cell Biol.  2020 Sep;53(3):313-324. 10.5115/acb.20.047.

Grape seed extract inhibits nucleus pulposus cell apoptosis and attenuates annular puncture induced intervertebral disc degeneration in rabbit model

Affiliations
  • 1Department of Human Anatomy, Federal University of Technology, Akure, Ondo State, Nigeria
  • 2Department of Biomedical Technology, Federal University of Technology, Akure, Ondo State, Nigeria

Abstract

Low back pain is a musculoskeletal disorders implicated to disc degeneration. Grape seed extracts (GSEs) is a natural flavonoids rich compound with antioxidants and anti-inflammatory properties. This study is aimed at investigating the inhibitory and anabolic response of GSE on annular punctured induced disc degeneration in rabbit model. TwentyEight New Zealand white rabbits (weighing about 2.0–3.5 kg) were used with institutional animal care committee’s approval. The animals were divided into four groups (n=7 per group). Group A (non-punctured group) received distilled water orally for 4 weeks. Group B (punctured group) received distilled water for 4 weeks. Group C (punctured treated group) received distilled water for 4 weeks and thereafter received 500 mg/kg of GSE for another 4 weeks. Group D received 500 mg/kg of GSE immediately after puncture for 4 weeks. At the end of the experiment, the animals were sacrificed with intramuscular injection of ketamine followed by intravenous injection of sodium pentobarbital. The percentage disc height index of the punctured group showed significant decrease compared to the control and treated groups. Histological and immunohistochemical studies showed distortion in the disc morphology, decrease in chondrocyte like cells, disorganization of collagen and elastic fibers, increase Bax expression levels in the punctured group compared to control and treated groups which was attenuated after GSE administration. GSE has preventive and restorative effects on punctured induced disc preventing the degradation of collagen fibrils within the disc tissues.

Keyword

Disc degeneration; Grape seed extract; Apoptosis; Gene expression; Histology

Figure

  • Fig. 1 Effect of GSE on DHI in annular punctured induced disc degeneration in rabbit (n=7). Dist., distilled; GSE, grape seed extract; % DHI, percentage disc height index. *P<0.05 as compared to group A; **P<0.05 as compared to group C; ***P<0.05 as compared to groups A and D.

  • Fig. 2 Photomicrograph of IVD histology in rabbit administered with Grape seed extract in the control and punctured treated groups (H&E, ×100). (A) Group A: showing normal IVD cytoarchitecture with numerous CLCs in the NP. (B) Group B: showing absence of CLCs in the NP and disorganization of collagen fibers in the AF. (C) Group C: showing nearly similar features with group 1. (D) Group D: showing restoration of NP contents. CLCs, chondrocytes like cells; IAF, inner annulus fibrosus; IVD, intervertebral disc; NP, nucleus pulposus; OAF, outer annulus fibrosus; VEP, vertebral end plate. Scale Bars=250 μm (A–D).

  • Fig. 3 Effect of GSE on histological grading score in annular punctured induced disc degeneration in rabbit (n=7). Dist., distilled; GSE, grape seed extract. *P<0.05 as compared to group A; **P<0.05 as compared to group C; ***P<0.05 as compared to groups A and D.

  • Fig. 4 (A–C) Effect of GSE on morphometric analysis of CLCs in annular punctured induced disc degeneration in rabbit (n=7). Group A, non-punctured group; Group B, punctured non-treated group; Group C, treated group 4 weeks after punctured; Group D, treated group immediately after punctured. CLCs, chondrocytes like cells; Dist., distilled; GSE, grape seed extract; IAF, inner annulus fibrosus; NP, nucleus pulposus; OAF, outer annulus fibrosus. *P<0.05 as compared to group A; **P<0.05 as compared to group C; ***P<0.05 as compared to groups A and D.

  • Fig. 5 (A) Photomicrograph of IVD collagen fibers organization in control rabbit showing parallel arrangements of collagen fibres in AF. (B) Photomicrograph of IVD collagen fibers organization in punctured non-treated rabbit showing disorganization and loss of continuity a of the arrangement of collagen fibres within the outer and IAF. (C) Photomicrograph of IVD collagen fibers organization in treated rabbit after 4 weeks of punctured showing nearly normal re-organization in the distorted arrangements of collagen fibers in both outer and IAF. (D) Photomicrograph of IVD collagen fibers organization in treated rabbit immediately after punctured showing parallel arrangements of collagen fibres in AF (Haematoxylin van Gieson, ×100). AF, annulus fibrosus; IAF, inner annulus fibrosus; IVD, intervertebral disc; OAF, outer annulus fibrosus; VEP, vertebral end plate. Scale Bars=100 μm (A, B, D), 250 μm (C).

  • Fig. 6 (A) Photomicrograph of IVD elastic fibers organization in control rabbit showing parallel arrangements of elastic fibres in AF. (B) Photomicrograph of IVD elastic fibers organization in punctured non-treated rabbit showing disorganization in the arrangement of elastic fibres within the outer and IAF. (C) Photomicrograph of IVD elastic fibers organization in treated rabbit after 4 weeks of punctured showing nearly normal arrangements of elastic fibers in both outer and IAF. (D) Photomicrograph of IVD elastic fibers organization in treated rabbit immediately after punctured showing parallel arrangements of elastic fibres in AF (Verhoeff van Gieson, ×100). AF, annulus fibrosus; IAF, inner annulus fibrosus; IVD, intervertebral disc; NP, nucleus pulposus; OAF, outer annulus fibrosus; VEP, vertebral end plate. Scale Bars=100 μm (A-D).

  • Fig. 7 (A–D) Effect of GSE on levels of gene expression in annular punctured induced disc degeneration in rabbit (n=7). Group A, non-punctured group; Group B, punctured non-treated group; Group C, treated group 4 weeks after punctured; Group D, treated group immediately after punctured. CoL 1, Collage type I; CoL 2, Collagen type II; Dist., distilled; GSE, grape seed extract; MMP-13, Matrix Metaloproteinase-13. *P<0.05 as compared to group A; **P<0.05 as compared to group C; ***P<0.05 as compared to groups A and D.

  • Fig. 8 Effect of GSE on expression levels of Bax in annular punctured induced disc degeneration in rabbit (n=7). Dist., distilled; GSE, grape seed extract. *P<0.05 as compared to group A; **P<0.05 as compared to group C; ***P<0.05 as compared to groups A and D.


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