Effect of ultrasound-guided needle-knife release of the ligamentum flavum on the expression of integrin alpha5 and beta1 in degenerative rabbit lumbar intervertebral discs

doi:10.12307/2025.204

Abstract

Abstract: BACKGROUND: Needle-knife release of the ligamentum flavum can effectively improve symptoms in patients with lumbar degeneration, and ultrasound guidance can increase the precision of needle-knife release; however, the specific effects of needle-knife release of the ligamentum flavum on the degenerated intervertebral discs and the possible mechanisms remain to be clarified.
OBJECTIVE: To investigate the effect of ultrasound-guided needle-knife release of the ligamentum flavum.
METHODS: Twenty-four New Zealand rabbits were randomized into control (n=6) and model (n=18) groups. A rabbit model of lumbar disc degeneration model was established in the model group by cutting the supraspinous and interspinous ligaments of the L5/6 and L6/7 segments to maintain a standing posture and apply axial load to the lumbar spine. After successful modeling, the model rabbits were subdivided into a control group, a model group, an ultrasonic needle-knife group, and a sham needle-knife group according to a random number table method, with six animals in each group. The ultrasonic needle-knife group underwent ultrasound-guided needle-knife release of the right yellow ligament of L7/S1, once every week, for a total of four times. The needle-knife approach in the sham needle-knife group was the same as that in the ultrasound needle-knife group, but the ligamentum flavum was not released. At 30 days after the intervention, MRI was used to observe the changes in the signal intensity of the nucleus pulposus within the L7/S1 segment. Hematoxylin-eosin staining was used to observe the morphological changes of the L7/S1 segment. Immunohistochemical staining was used to detect the expression of type I and II collagen in the nucleus pulposus of the L7/S1 segment. RT-PCR and western blot were used to detect the expression of integrin α5 and β1, p38, and nuclear factor κB in the L7/S1 segment.
RESULTS AND CONCLUSION: MRI findings indicated that the nucleus pulposus of the intervertebral disc of rabbits in the model group was gray-black in color, and the gray value of the nucleus pulposus was significantly lower than that of the control group (P < 0.01). The brightness of the nucleus pulposus of the intervertebral disc of the rabbits in the ultrasonic needle-knife group was elevated compared with that of the model group, and the gray value of the nucleus pulposus was higher than that of the model group (P < 0.01). Results from hematoxylin-eosin staining showed that in the model group, the shape of the nucleus pulposus was irregular, the number of nucleus pulposus cells was reduced, the extracellular matrix was compressed, the fibrous ring was ruptured, the structure and boundary of the end plate were unclear, and the chondrocytes were arranged disorderly. Compared with the model group, the ultrasonic needle-knife group showed an increase in the number of the nucleus pulposus, an improvement in the rupture of the fibrous ring, and more regular arrangement of cartilage endplate cells. Results from immunohistochemical staining showed an increase in positive expression of type I collagen (P < 0.01) and a decrease in positive expression of type II collagen in the nucleus pulposus of the model group compared with the control group as well as a decrease in positive expression of type I collagen and an increase in positive expression of type II collagen in the nucleus pulposus of the ultrasonic needle-knife group compared with the model group (P < 0.01). RT-PCR and western blot assays showed that the mRNA and protein expression of integrin α5, integrin β1, p38, and nuclear factor κB in the intervertebral discs of rabbits in the model group were increased compared with that in the control group (P < 0.01); the mRNA and protein expression of integrin α5, integrin β1, p38, and nuclear factor κB in the intervertebral discs of rabbits in the ultrasonic needle-knife group was decreased compared with that in the model group (P < 0.01). To conclude, ultrasound-guided needle-knife release of the ligamentum flavum can improve the degree of lumbar disc degeneration in rabbits, which may be related to the inhibition of p38 and nuclear factor-κB expression by modulating integrin α5 and β1 expression.

Key words: lumbar disc degeneration, needle-knife, ligamentum flavum, ultrasound guidance, integrin

CLC Number:

Chen Can, Zhao Yu, Hu Binhan, Du Mengfan, Liu Junning, Niu Susheng, Zhang Yan. Effect of ultrasound-guided needle-knife release of the ligamentum flavum on the expression of integrin alpha5 and beta1 in degenerative rabbit lumbar intervertebral discs[J]. Chinese Journal of Tissue Engineering Research, 2025, 29(2): 331-338.

Figures/Tables 7

2.1 实验动物数量分析 24只兔全部进入结果分析。 2.2 兔腰椎间盘退变模型鉴定造模12周后MRI成像可见对照组L7/S1椎间盘结构基本完整，髓核呈高亮信号，髓核与纤维环的分界清晰；造模组髓核信号呈灰-黑色，与对照组相比椎间盘高度轻度下降，髓核与纤维环的分界模糊(图3A)，按Pfirrmann等级可评估为Ⅲ级退变。造模组L7/S1节段腰椎间盘髓核灰度值低于对照组(P < 0.01)(图3B)。 2.3 针刀干预后各组兔椎间盘MRI检查结果及灰度值比较针刀干预后30 d，MRI成像可见对照组椎间盘结构基本完整，髓核呈高亮信号，髓核与纤维环的分界清晰；模型组椎间盘髓核和纤维环的界限不清晰，髓核呈灰-黑色，椎间盘高度略有下降；与模型组相比，超声针刀组椎间盘纤维环与髓核界限较清晰，髓核亮度升高；假针刀组形态与模型组相近，见图4A。模型组、假针刀组髓核灰度值低于对照组(P < 0.05，P < 0.01)，模型组与假针刀组、超声针刀组与对照组髓核灰度值比较差异均无显著性意义 (P > 0.05)；超声针刀组髓核灰度值高于模型组、假针刀组(P < 0.05，P < 0.01)，见图4B。 2.4 针刀干预后各组兔椎间盘苏木精-伊红染色及Masuda评分比较针刀干预后30 d苏木精-伊红染色显示，对照组椎间盘结构完整，髓核形状为椭圆形，有大量圆形的软骨细胞，纤维环显示规则的层状结构，软骨终板结构完整，软骨细胞丰富；模型组和假针刀组观察到了不规则形状的髓核，髓核细胞数量减少，细胞外基质被压缩，纤维环破裂或呈“蛇形”，终板结构与边界不清，软骨细胞排列紊乱；与模型组相比，超声针刀组髓核细胞数量增多，纤维环破裂情况改善，软骨终板细胞排列较为规则，见图5A。模型组、假针刀组Masuda组织学评分明显高于对照组(P < 0.01)，模型组与假针刀组、超声针刀组与对照组Masuda组织学评分比较差异均无显著性意义(P > 0.05)；超声针刀组Masuda组织学评分低于模型组、假针刀组 (P < 0.01，P < 0.05)，见图5B。 2.5 针刀干预后各组兔椎间盘中髓核Ⅰ型胶原、Ⅱ型胶原表达针刀干预后30 d免疫组化染色显示，对照组髓核中Ⅱ型胶原呈高表达，Ⅰ型胶原基本无染色，反映了健康的髓核组织；模型组与假针刀组髓核中Ⅱ型胶原表达显著减少，并且出现了Ⅰ型胶原阳性表达，表明椎间盘发生了退变；相较于模型组，超声针刀组髓核中Ⅱ型胶原表达增加，Ⅰ型胶原表达降低，表明针刀干预对椎间盘退变有一定的抑制作用，见图6A。免疫组化积分吸光度值定量分析结果显示，模型组和假针刀组Ⅰ型胶原阳性表达高于对照组(P < 0.01)，Ⅱ型胶原阳性表达低于对照组(P < 0.01)；模型组和假针刀组Ⅰ型胶原、Ⅱ型胶原阳性表达比较差异均无显著性意义(P > 0.05)；超声针刀组Ⅰ型胶原阳性表达高于对照组(P < 0.01)、低于模型组和假针刀组(P < 0.01)，Ⅱ型胶原阳性表达低于对照组(P < 0.01)、高于模型组和假针刀组(P < 0.01)，见图6B。 2.6 各组兔椎间盘中整合素α5、整合素β1、p38、核因子κB mRNA基因表达情况 RT-PCR检测显示，模型组和假针刀组整合素α5、整合素β1、p38、核因子κB mRNA相对表达均高于对照组(P < 0.01)，模型组和假针刀组整合素α5、整合素β1、p38、核因子κB mRNA相对表达比较差异均无显著性意义(P > 0.05)；超声针刀组整合素β1 mRNA相对与对照组比较差异无显著性意义(P > 0.05)，整合素α5、p38、核因子κB mRNA相对表达均高于对照组(P < 0.05)；超声针刀组整合素α5、整合素β1、p38、核因子κB mRNA相对表达均低于模型组(P < 0.01)，见图7。 2.7 各组兔椎间盘中整合素α5、整合素β1、p38、核因子κB蛋白表达情况 Western Blot检测显示，模型组和假针刀组整合素α5、整合素β1、p38、核因子κB 蛋白相对表达均高于对照组(P < 0.01)，模型组和假针刀组整合素α5、整合素β1、p38、核因子κB 蛋白相对表达比较差异均无显著性意义(P > 0.05)；超声针刀组整合素α5、p38蛋白相对表达量表达与对照组比较差异无显著性意义(P > 0.05)，超声针刀组整合素β1、核因子κB 蛋白相对表达高于对照组(P < 0.05，P < 0.01)；超声针刀组椎间盘中整合素α5、整合素β1、p38、核因子κB 蛋白相对表达均低于模型组(P < 0.01)，见图8。"

References

[1] MERTIMO T, KARPPINEN J, NIINIMAKI J, et al. Association of lumbar disc degeneration with low back pain in middle age in the Northern Finland Birth Cohort 1966. BMC Musculoskelet Disord. 2022;23(1):359.
[2] BUCHBINDER R, VAN TULDER M, OBERG B, et al. Low back pain: a call for action. Lancet. 2018;391(10137):2384-2388.
[3] GBD 2019 Diseases and Injuries Collaborators. Global burden of 369 diseases and injuries in 204 countries and territories, 1990-2019: a systematic analysis for the Global Burden of Disease Study 2019. Lancet. 2020;396(10258):1204-1222.
[4] 林秀华,刘存斌,耿凯,等.超声引导下针刀松解黄韧带治疗腰椎间盘突出症的临床效果[J].中国医药导报,2022,19(9):157-160.
[5] 石翀,王海龙,邱祖云,等.超声引导下针刀经皮松解黄韧带治疗腰椎间盘突出症的初步疗效观察[J]. 中日友好医院学报,2022, 36(3):178-179,181.
[6] 戴敏,李开平,何宁宁.超声可视化针刀技术治疗腕管综合征的安全性及临床疗效观察[J].中华中医药学刊,2020,38(6):193-196,273.
[7] 王超.过度压缩应力通过p38/NF-κB调控髓核细胞退变的机制及椎间盘蠕变特性研究[D].上海:中国人民解放军海军军医大学,2021.
[8] KANDA Y, YURUBE T, MORITA Y, et al. Delayed notochordal cell disappearance through integrin α5β1 mechanotransduction during ex-vivo dynamic loading-induced intervertebral disc degeneration J Orthop Res. 2021;39(9):1933-1944.
[9] 白雪东,王德利,侯黎升,等.直立体位下无创轴向加载建立兔椎间盘退变动物模型[J].中国脊柱脊髓杂志,2017,27(6):545-552.
[10] 杨雄.基于韧带损伤腰椎力学失稳模型的建立与应用[D].重庆:重庆大学,2019.
[11] RUAN D, HE Q, DING Y, et al. Intervertebral disc transplantation in the treatment of degenerative spine disease: a preliminary study. Lancet. 2007;369(9566):993-999.
[12] 赵宇,郭双,陈灿,等.超声引导下针刀松解兔腰椎黄韧带的解剖学研究[J].中国医药导报,2023,20(13):4-8.
[13] MASUDA K, AOTA Y, MUEHLEMAN C, et al. A novel rabbit model of mild, reproducible disc degeneration by an anulus needle puncture: correlation between the degree of disc injury and radiological and histological appearances of disc degeneration. Spine (Phila Pa 1976). 2005;30(1):5-14.
[14] 路翀,姚明鹤,崔海舰,等.从筋骨理论探讨腰椎间盘退变动物模型的构建及其在中医药研究中的应用[J].中华中医药学刊,2021, 39(7):131-134.
[15] 林星星,董宝强,马铁明.对经筋理论中若干“点”概念的辨析与整合[J].中国中医基础医学杂志,2018,24(5):584-585,626.
[16] POSTACCHINI F, GUMINA S, CINOTTI G, et al. Ligamenta flava in lumbar disc herniation and spinal stenosis. Light and electron microscopic morphology. Spine (Phila Pa 1976). 1994;19(8):917-922.
[17] 任昊天,富昱,董宝强.基于“经筋理论”探讨针刀“解结”法治疗冈上肌肌腱炎的作用机制[J].实用中医内科杂志.[2023-06-14].
[18] 刘福水,方婷,周凡媛.针刀“调筋治骨”法治疗颈椎病力学机制探讨[J].中华中医药学刊,2018,36(12):2862-2865+3094.
[19] SUN C, ZHANG H, WANG X, et al. Ligamentum flavum fibrosis and hypertrophy: Molecular pathways, cellular mechanisms, and future directions FASEB J. 2020;34(8):9854-9868.
[20] 李志伟,高峰,孟强,等.高频肌骨超声诊断踝关节外侧副韧带损伤及其评估预后的价值[J].中国骨与关节损伤杂志,2022,37(8): 820-823.
[21] 刘星,袁锋,刘柃.超声可视化针刀治疗技术的临床价值和学术价值探讨[J].辽宁中医药大学学报,2023,25(11):20-23.
[22] 王善建,陈秀平.超声引导下针刀松解夹脊穴配合深刺次穴治疗腰椎间盘突出症的疗效观察[J].颈腰痛杂志,2022,43(5):700-702.
[23] GUO J, YAN H, XIE Y, et al. A lumbar disc degeneration model established through an external compressive device for the study of microcirculation changes in bony endplates. Heliyon. 2023;9(5):e15633.
[24] LIU S, SUN Y, DONG J, et al. A Mouse Model of Lumbar Spine Instability. J Vis Exp. 2021;(170). doi: 10.3791/61722.
[25] HEI L, GE Z, YUAN W, et al. Evaluation of a rabbit model of adjacent intervertebral disc degeneration after fixation and fusion and maintenance in an upright feeding cage. Neurol Res. 2021;43(6):447-457.
[26] AO X, WANG L, SHAO Y, et al. Development and Characterization of a Novel Bipedal Standing Mouse Model of Intervertebral Disc and Facet Joint Degeneration. Clin Orthop Relat Res. 2019;477(6):1492-1504.
[27] FU F, BAO R, YAO S, et al. Aberrant spinal mechanical loading stress triggers intervertebral disc degeneration by inducing pyroptosis and nerve ingrowth. Sci Rep. 2021;11(1):772.
[28] LAO Y, XU T, JIN H, et al. Accumulated Spinal Axial Biomechanical Loading Induces Degeneration in Intervertebral Disc of Mice Lumbar Spine. Orthop Surg. 2018;10(1):56-63.
[29] MERTER A, KARACA MO, YAZAR T. Biomechanical effects of sequential resection of the posterior ligamentous complex on intradiscal pressure and resistance to compression forces. Acta Orthop Traumatol Turc. 2019;53(6): 502-506.
[30] 谢瑞.基于筋伤理论探讨退行性腰椎失稳的力学、病理学机制及手法干预研究[D].北京:中国中医科学院,2021.
[31] HEUER F, SCHMIDT H, KLEZL Z, et al. Stepwise reduction of functional spinal structures increase range of motion and change lordosis angle. J Biomech. 2007;40(2):271-270.
[32] YING Y, CAI K, CAI X, et al. Recent advances in the repair of degenerative intervertebral disc for preclinical applications. Front Bioeng Biotechnol. 2023;11:1259731.
[33] 陈斌,刘洪,张良志,等.可视化针刀抑制髓核细胞凋亡从而改善颈椎病兔的椎间盘退变[J].针刺研究,2022,47(11):1005-1011.
[34] GUO J, YAN H, XIE Y, et al. A lumbar disc degeneration model established through an external compressive device for the study of microcirculation changes in bony endplates. Heliyon. 2023;9(5):e15633.
[35] WU ZL, CHEN YJ, ZHANG GZ, et al. SKI knockdown suppresses apoptosis and extracellular matrix degradation of nucleus pulposus cells via inhibition of the Wnt/β-catenin pathway and ameliorates disc degeneration. Apoptosis. 2022;27(1-2):133-148.
[36] WANG N, BUTLER JP, INGBER DE. Mechanotransduction across the cell surface and through the cytoskeleton. Science. 1993;260(5111):1124-1127.
[37] NETTLES DL, RICHARDSON WJ, SETTON LA. Integrin expression in cells of the intervertebral disc. J Anat. 2004;204(6):515-520.
[38] KURAKAWA T KAKUTANI K, MORITA Y, et al. Functional impact of integrin α5β1 on the homeostasis of intervertebral discs: a study of mechanotransduction pathways using a novel dynamic loading organ culture system. Spine J. 2015;15(3):417-426.