The Effect Of Ultrasound-guided Needle Knife Release Of Lumbar Ligamenta Flavum Hypertrophy In Rabbits And Its Effect On The TGF-β1/Smad2 Pathway

Part I:Study on the approach of needle knife release of ligamentum flavum in rabbits under ultrasound guidanceObjectiveAccording to the anatomy of the lumbar spine of normal male New Zealand rabbits,the possibility of needle knife approach was analyzed,and the ultrasound-guided needle knife release of the rabbit ligament was designed according to the anatomical results.MethodsTwelve 3-month-old male New Zealand rabbits were randomly divided into anatomical group（6 rabbits）and approach group（6 rabbits）.The anatomical group measured the spinous process height and mastoid process height of L₅,L₆and L₇vertebrae,length and width of L_4/5,L_5/6,L_6/7and L₇/S₁vertebrae,and observed the morphology of ligamenta xanthoides.The Angle between spinous process and mastoid process was measured in the approach group after anesthesia.The ultrasonic probe was placed beside the spinous process on the longitudinal axis to enter the needle in a plane way.The needle knife reached the yellow ligament along the longitudinal axis approach and approached the yellow ligament from the medial edge of the inferior articular process of the upper vertebral body in the target segment.The depth of the yellow ligament was determined according to the thickness of the yellow ligament measured by anatomy and combined with the ultrasonic image,and the yellow ligament on both sides of the L_4/5,L_5/6,L_6/7and L₇/S₁segments were released.The insertion depth（U-h）,insertion Angle（U-Ag）,vertical distance（R）from the posterior midline and vertical distance（S）from the Angle of the sacrum were measured,and the needle path was dissected and observed.Results1.Characteristics of the bony structure of the rabbit lumbar vertebraeNew Zealand rabbits have 7 lumbar vertebrae,and the highest line of iliac spines on both sides is between L₆-L₇.The spinous process of L_1-L₆protrudes headside and presents an acute Angle with the horizontal line,and the Angle between the spinous process of L₇and the horizontal line is about 90°.The mastoid process protrudes dorsolateral on both sides of the spinous process.There was no significant difference in the spinous height of L₅,L₆and L₇vertebrae（P>0.05）,the average height was（9.15±0.96）mm,and the height of the mastoid process was similar to that of the spinous process（P>0.05）.2.Rabbit laminar space and ligament flavum morphologyThe ligament flavum at the posterior wall of the spinal canal is divided into left and right parts,which are longer longitudinally and shorter laterally,and have a"V"shape.At the facet joint level,ligament flavum is relatively thick in the middle and relatively thin medially and laterally.Compared with L_4/5,the laminar gap length of L_5/6,L_6/7and L₇/S₁segments was longer（P<0.001,P<0.01）.Compared with L_5/6,the length of L₇/S₁laminar space is slightly shorter（P<0.05）.Compared with L_6/7,the length of L₇/S₁laminar gap is slightly shorter（P<0.01）.There was no significant difference in length of L_5/6and L_6/7（P>0.05）,and the length of laminar interstitial space of L_4/5was shorter than that of L_5/6and L_6/7segments（P<0.01）.There was no significant difference in laminar gap width in L_4/5,L_5/6,L_6/7and L₇/S₁segments（P>0.05）.3.The ultrasonic transverse axis approach measures the angle between the spinous process and the mastoidThere was no significant difference between the angle between mastoids and spinous processes on both sides of the same vertebral body（P>0.05）when the probe was placed on the vertical transverse axis of the posterior median line,and there was no significant difference in the angle between spinous processes and mastoids in L₅,L₆and L₇vertebral bodies（P>0.05）.The mean angle between the spinous process and the mastoid is（37.84±7.26）°.4.Ultrasonic longitudinal axis approach related data measurementThe long axis of the probe was placed along the rabbit posterior median line,and the scanning plane was between the spinous process and the facet joint.After the root of the vertebral arch plate was clearly displayed,the acupuncture plane was determined,and the probe was fixed.The middle point of the probe tail was used as the needle-knife entry point.The ligamentum luteum was released by needle knife along the longitudinal axis at the medial margin of the inferior articular process of the upper vertebra in the target segment.There were no significant differences in R,and U-h on the left and right sides of L_4/5,L_5/6,L_6/7and L₇/S₁on ultrasonic images（P>0.05）.There were no significant differences in R and U-h between L_4/5,L_5/6,L_6/7and L₇/S₁（P>0.05）.The U-Ag value of L₇/S₁segment was larger than L_4/5segment（P<0.01）,and that of L_5/6segment was larger than L_4/5segment（P<0.05）.There was no significant difference in U-Ag value among other segments.S decreased gradually from L_4/5to L₇/S₁（P<0.001,P<0.05）.5.Anatomical longitudinal axis approach to observe safetyAfter leaving the needle,autopsy showed that the needle knife approach and the puncture path did not injure blood vessels and nerves.The needle tip is located on the medial ligament flavum of the facet joint,and there is no obvious bleeding locally,and the dura is intact.Conclusions1.In the process of releasing the ligamenta flavum of rabbit lumbar spine with ultrasound-guided needle knife,compared with the transverse approach,the parallel longitudinal approach is safer and more effective because the Angle between the mastoid process and the spinous process is acute.2.There were anatomical differences between L_4/5,L_5/6,L_6/7and L₇/S₁segments,and the insertion Angle of the needle knife should be adjusted according to the intervention segment.PratⅡ:The effect of ultrasound-guided needle knife on the release of hypertrophy of yellow ligament in rabbits and its effect on TGF-β1/Smad2 pathwayObjectiveTo investigate the effect of ultrasound-guided acupotomy on the treatment of yellow ligament hypertrophy and its effect on TGF-β1/Smad2 pathway activated by Angptl2.MethodsThere were 30 3-month-old male New Zealand rabbits,9 were divided into normal groups according to the numerical random method,and the remaining 21 were modeled.The model was performed by cutting off the L_5/6and L_6/7supraspinous ligaments and the interspinous ligament to maintain a standing posture.After 3m molding,3 rabbits in the normal group and 3 rabbits after molding were taken according to the random number method for identification.After successful identification,the modeled rabbits were divided into model group,needle knife group and dummy needle knife group,each group of 6,the needle knife group was needle knife under ultrasound guidance to release the right ligament flavum of L₇/S₁,and the pseudo needle knife group reached the right ligament flavum of L₇/S₁under ultrasound guidance without release,and intervened once every 7d,a total of 4 interventions.The motor function of the hindlimbs of rabbits was scored 4w after the intervention,the oblique plate test evaluated the muscle strength of the hindlimbs of rabbits,the range of motion of the lumbar spine was measured by X-ray,the ligament flavum and intervertebral discs were observed by MRI,the fiber morphology of ligament flavum was observed by HE staining,the collagen fiber morphology of ligament flavum was observed by Masson staining,the expression of Angptl2,TGF-β1 and Smad2 m RNA was detected by RT-PCR,and the expression of Angptl2,TGF-β1 and Smad2 proteins was detected by Western Blot.Results1.Model identificationMRI T2WI axial bitmap showed that the thickness of the L₇/S₁ligament flavum in rabbits after modeling was significantly greater than that of the normal group.The thickness of the L₇/S₁ligament freus was（1.51±0.16）mm in the normal group and（1.90±0.13）mm in the model group,which was statistically significant compared with the normal group（P<0.05）.HE staining showed that the cross-sectional area of L₇/S₁ligament flavum in rabbits after molding was significantly larger than that of normal group.Compared with the normal group,the cross-sectional area of rabbits after molding was significantly increased（P<0.05）.Masson staining showed that the fibers of the yellow ligament of rabbit were loose and disordered after modeling,wavy,and the content of collagen fibers was higher than that of the normal group.Compared with the normal group,the collagen volume integral number of the model group was significantly increased（P<0.05）.2.Rabbit motor function score after interventionCompared with the normal group,the BBB score of the model group and the sham knife group was lower（P<0.001,P<0.05）,and there was no significant difference in the BBB score of the needle knife group（P>0.05）.The BBB score of the needle knife group was significantly higher than that of the model group（P<0.001）,and there was no significant difference between the pseudo knife group and the model group（P>0.05）.3.Oblique plate test after interventionCompared with the normal group,the angle of the model group and the dummy knife group were significantly reduced（P<0.001）,and the angle of the needle knife group was slightly smaller than that of the normal group（P<0.05）.The angle of the needle knife group was significantly higher than that of the model group（P<0.01）,and there was no significant difference between the rabbits in the pseudoneedle knife group and the model group（P>0.05）.4.X-ray examination after interventionX-ray examination showed that compared with the normal group,the model group,the acupotomology group and the fake acupotomology group had no significant increase in the range of motion at the hyperextension position,and there was no statistical difference in the FR value among the groups（P>0.05）.In the hyperflexion position,the activity of model group,acupotomology group and sham acupotomology group was increased compared with the normal group,and ER value was significantly increased（P<0.01）,but there was no significant difference in ER value between model group,acupotomology group and sham acupotomology group（P>0.05）.L_5/6,L_6/7and L₇/S₁segments in each group had no obvious vertebral spondylolisthesis or rotation5.MRI examination after interventionIn axial T2WI images,high signals were observed in the right ligamentum flavum of L₇/S₁in the acupotomy group,and the boundary was fuzzy,while the left ligamentum flavum of L₇/S₁was relatively thin.L₇/S₁ligamentum flavum was thicker in both model group and sham needle-knife group.The L₇/S₁intervertebral disc nucleus pulposus was full with uniform gray in normal sagittal position on T2WI and acupotomy group.Signal intensity of L₇/S₁intervertebral disc nucleus pulposus decreased in model group and sham needle knife group.6.HE staining of ligament flavum after interventionThe cross section morphology of the ligamenta flava was observed under 50 times light HE staining,and the ligamenta flava in the model group and the pseudoacupotomy group was more hypertrophic than that in the normal group.The thickness of ligamenta flavum in acupotomy group was smaller than that in model group and fake acupotomy group,but still larger than that in normal group.Compared with normal group,the cross-sectional area of L₇/S₁ligaments in model group,acupotomology group and sham acupotomology group was increased（P<0.01,P<0.05,P<0.001）.Compared with the model group,the needle-knife group had a smaller cross-sectional area（P<0.05）.Compared with the acupotomy group,the cross-sectional area of the sham acupotomy group was significantly increased（P<0.01）.There was no significant difference between model group and sham needle knife group（P>0.05）.The fibers in the unreleased region of ligamentum flavum were arranged regularly in the normal group and the acupotomy group under the 200-fold light microscope.In the acupotomy group,there was obvious scar tissue in the ligamenta flavum in the area of acupotomy release.The number of cells in the ligamenta flava was significantly higher in the model group and the sham needle knife group,and the fibers of the ligamenta flava were more dense,and some of them were twisted.7.Masson staining of ligament flavum after interventionMasson staining results showed that the fibers of the ligamenta flava in the normal group were arranged in a regular way,with red elastic fibers as the main fibers and blue collagen fibers less.The fibers of ligamenta flavum in the model group and the fake needle knife group were disordered,with more blue collagen fibers,and some fibers were twisted.Compared with the model group and the fake acupotomology group,the fibers of the yellow ligament in the unreleased area of the acupotomology group were arranged more regularly,and the blue collagen fibers were less,but the collagen fibers were more than the normal group.In the acupotomy group,there was more collagen fiber hyperplasia in the ligamenta flavum in the area of acupotomy release.Compared with normal group,collagen fiber volume fraction in model group,acupotomy group and fake acupotomy group was significantly increased（P<0.001,P<0.05）.Compared with model group,the collagen fiber volume fraction in acupotomy group was decreased（P<0.05）.Compared with acupotomy group,collagen fiber volume fraction in the sham acupotomy group was significantly increased（P<0.01）.8.Effects of ultrasound-guided acupotomy on m RNA expression levels of Angptl2,TGF-β1 and Smad2 in rabbit L₇/S₁yellow ligamentCompared with the normal group,the protein expressions of Angptl2,TGF-β1 and Smad2 in the model group and the sham acutometry group were increased（P<0.001,P<0.01,P<0.05）,but there was no statistical significance in the protein expressions of Angptl2,TGF-β1 and Smad2 between the normal group and the acutometry group（P>0.05）.Compared with the model group,the protein expression levels of Angptl2,TGF-β1and Smad2 in the acupotomy group were decreased（P<0.01,P<0.05）,but there were no significant differences in the protein expression levels of Angptl2,TGF-β1 and Smad2between the model group and the sham acupotomy group（P>0.05）.Compared with the acupotomy group,the protein expression levels of Angptl2,TGF-β1 and Smad2 in the sham acupotomy group were increased（P<0.01,P<0.05）.9.Effects of ultrasound-guided acupotomy on protein expression levels of Angptl2,TGF-β1 and Smad2 in rabbit L₇/S₁yellow ligamentCompared with the normal group,the protein expressions of Angptl2,TGF-β1 and Smad2 in the model group and the sham acutometry group were increased（P<0.001,P<0.05）,but there was no statistical significance in the protein expressions of Angptl2,TGF-β1 and Smad2 between the normal group and the acutometry group（P>0.05）.Compared with the model group,the protein expression levels of Angptl2,TGF-β1 and Smad2 in the acupotomy group were decreased（P<0.001,P<0.05）,but there were no significant differences in the protein expression levels of Angptl2,TGF-β1 and Smad2between the model group and the sham acupotomy group（P>0.05）.Compared with the acupotomy group,the protein expression levels of Angptl2,TGF-β1 and Smad2 in the sham acupotomy group were increased（P<0.001,P<0.05）.Conclusions1.Ultrasound-guided needle-knife release of the ligamentum flavum can partially improve the motor function of the hind limb.After needle-knife release,the instability degree of the corresponding lumbar vertebra was not aggravated,and the degeneration of the lumbar intervertebral disc in the corresponding segment was partially improved.2.Ultrasound-guided needle-knife release of ligamentum flavum can significantly improve the fiber arrangement disorder.Angptl2-mediated TGF-β1/Smad2 pathway was also down-regulated.