17β-Estradiol Protects Intervetebral Disc From Degeneration In Rat And Curative Effects Of Dynesys Dynamic Stabilization System For Lumbar Intervertebral Disc Degeneration Diseases

Part one 17β-estradiol inhibits intervertebral disc degeneration by down-regulating MMP-3,MMP-13 and Caspase-3 in a rat modelObjective:17β-Estradiol（E₂）has been previously reported for its function of protecting rat NP cell by inhibiting apoptosis and inflammatory factors in in-vitro studies.However,whether E₂ has protective effect in vivo remains unclear.This study was aimed to further explore whether E₂ had protective effects on intervertebral disc degeneration（IVDD）in a rat model.Methods:Forty 3-month-old female Sprague-Dawley（SD）rats were randomly divided into four groups:Sham,OVX,E₂ and ICI.All the rats except Sham group received the tail acupuncture technique to establish the animal models of the intervertebral disc degeneration.Then Sham group only underwent the resection of a bit fat and was treated with the solvent of E₂;OVX group underwent bilateral ovariectomy and was treated with the solvent of E₂;E₂ group was treated with E₂ based on OVX;ICI group was treated with E₂ and pretreated with ICI182780（inhibitor of the estrogen receptor）based on OVX.After 28 days of intervention,the degeneration of intervertebral disc was evaluated by X-ray,Hematoxylin and eosin（HE）staining,Immunohistochemistry（IHC）,Western blot and RT-qPCR.Radiography was used to evaluate degree of IVDD by measuring Disc Height Index（DHI）.Hematoxylin and eosin（HE）staining for intervertebral disc was used to accomplish the morphological observation for degeneration by optical microscope,According to the previously published grading scales,the modified histological grading of HE staining was used to assess morphological changes in both annulus fibrosus（AF）and nucleus pulposus（NP）.Immunohistochemistry（IHC）,Western blot were applied to qualitatively and quantitatively detect the expression of matrix metallop-roteinase（MMP）-3,MMP-13 and Caspase-3,Western blot was also applied to detect the expression of collagen Type II.The RT-qPCR is used to detect expression level of mRNA,including MMP-3,MMP-13,Caspase-3,and collagen Type II.Results:According to X-ray results,compared with the sham,radiographs showed marked intervertebral disc narrowing in the OVX group（P<0.05）,which was reversed by E₂（P<0.05）.However,the protective effect of E₂ on disc height was inhibited by ICI182780（inhibitor of the estrogen receptor）（P<0.05）.HE staining showed typical apoptosis characteristics of intervertebral disc cells in OVX,which were reversed by E₂.A loss of well-distributed pattern,as well as a gradual reduction in the number of NP cells with some areas of clustering were clearly observed in the OVX group,testifying to the tissue’s degradation.Significant differences in histological scores were found between the sham group and OVX group,OVX group and E₂ group,E₂ group and ICI group（P<0.05）.IHC showed that,the expression of Caspase-3,MMP-3 and MMP-13 was strongly positive in the pericellular area of NP and at the border of NP and AF.The positive staining of Caspase-3,MMP-3 and MMP-13 became significantly weakened when treated with E₂.However,the anti-apoptosis effect of E₂ was entirely reversed by ICI182780.The levels of expression of Caspase-3,MMP-3,MMP-13 and collagen Type II were quantitatively determined by western blot experiments.Compared with the sham group,the OVX group showed a significant increase on the expression of Caspase-3,MMP-3 and MMP-13（P<0.05）,but a decrease in the level of expression of collagen Type II.On the other hand,E₂downregulated Caspase-3,MMP-3 and MMP-13 proteins level but upregulated collagen Type II.Importantly,treatment with ICI182780 showed comparable results with the OVX group,characterized by an increase in Caspase-3,MMP-3 and MMP-13 but a decrease in collagen（P<0.05）.To further investigate these processes at the molecular level,the gene expression of these proteins was ascertained by RT-q PCR experiments.Compared with the sham,the mRNA levels of OVX group presented a significant increase in the expression of Caspase-3,MMP-3,MMP-13,but decrease in collagen Type II（P<0.05）.The addition of E₂ had the opposite effect on mRNA expression levels compared to the OVX group,which was further reversed by the addition of ICI182780（P<0.05）.Conclusion:17β-Estradiol inhibits IVDD by down-regulating MMP-3,MMP-13 and Caspase-3 in a rat model.This research thus provides us a potentially promising avenue for IVDD therapy and prevention.Part two Curative effects of Dynesys dynamic stabilization system for lumbar intervertebral disc herniationObjective:At present,lumbar interbody fusion and internal fixation is still the common method for the treatment of lumbar disc herniation with instability or possible to be instable post-operatively.Strong fixation between segments is helpful for the lumbar vertebrae to sustain the weight of the body and to restore the reduced intervertebral height.However,after rigid fixation,the motion of the fixed segment is almost completely lost and that results in a substantial increase of stress in the adjacent segment.As a result,the adjacent segments are inclined to degenerate and be suffered from the adjacent segment disease.At the same time,the fusion surgery usually makes patients suffered from bigger wound and long hospitalization.Besides,the lumbar fusion of this operation is not proportional to its long-term clinical satisfaction and many patients may suffer from the failed back surgery syndrome.These phenomena have drawn an increasing attention of orthopedic clinicians.The clinical application of the instrument is not long in China,and and there is still a lot of controversy about its effectiveness and indications.This study reported the curative effects and the associated problems of Dynesys dynamic stabilization system for these kinds of lumbar intervertebral disc herniation during a two-year follow-up,in an attempt to provide experimental basis for the clinical application of this system.Methods:Fifty patients with lumbar intervertebral disc herniation treated with Dynesys dynamic stabilization system from February 2015 to February2017 were selected.Operating time,length of surgical wound and the amount of blood loss of all patients were recorded.They were followed up for two years via telephone reservations and outpatient reviews.The time points for follow-up was at 6 months,1 year and 2 years after operation.Oswestry dysfunction index（ODI）and visual analogue scale（VAS）scores of patients were recorded before and at each time of follow-up after surgery.Lumbar MRI was performed before operation and in the last follow-up.Woodend classification and apparent diffusion coefficient（ADC）were used to evaluate the occurrence of lumbar disc degeneration.The intervertebral disc space height and intervertebral range of motion（ROM）of lumbar fixed segments were measured by imaging.The intervertebral disc space height=（ventral height+dorsal height+central height）/3.Superior margin of upper vertebral body and inferior margin of lower vertebral body on the operative segment were connected into a line.And intervertebral ROM referred to the angle between the line in the status of extension and flexion.Results:Fifty patients were selected,including 27 males and 23 females at the age of 29-60 years old and their mean age was（44.7±5.2）years old.The fixation was performed at L2-L3 in two patients,L3-L4 in three patients,L4-L5 in twenty patients and L5-S1 in twenty-five patients.Before operation,there was no degeneration in adjacent segments.All the 50 patients completed the operation successfully,their symptoms were significantly improved,and all were followed up.Operative incision length was（7.47±2.8）cm,operating time was（105.43±25.4）min and the amount of blood loss was（137.18±61.2）ml.Postoperative wounds healed up well.During the follow-up period,there were no complications such as spinal cord nerve injury,deep infection and pedicle fracture.After operation,no pedicle screw loss,loosening or breakage appeared.ODI and VAS scores of patients before surgery were（68.92±10.12）and（6.14±1.13）respectively.Compared with preoperative ODI and VAS scores,postoperative scores were significantly decreased.There were statistical differences in the scores at each time point between pre-and post-operation（all P<0.05）.What’s more,there were no statistical differences among scores at post-operative 6-month,1-and 2-year（P>0.05）.That suggested that the clinical symptoms of patients were significantly relieved afte surgey and the effects were persistent during the 2 years.After 2 years’ follow-up,there was one case of lumbar disc deterioration changed from grade II to grade III,one from grade I to grade II,and one exceptional case reversed rom grade II to grade I,while the others had no significant changes.The esults of MRI image of intervertebral disc showed that pre-operative ADC alue of patients was（1.03±0.22）*10^-3mm2/S.At the end of two-year follow p after operation,the ADC was（0.99±0.26）*10^-3mm2/S,which was a little ecreased and the difference was not statistically significant（P>0.05）.The ntervertebral disc space height of fixed segments of patients before surgery as 9.76±1.38 mm and the intervertebral ROM was（6.17±1.74）°.After urgery,the intervertebral disc space height changed to 11.24±1.58 mm and ntervertebral ROM changed to（2.49±1.18）°,and the differences were tatistical significant（both P<0.05）.Conclusion:As one of the outstanding representatives of spinal non-fusion instrument,Dynesys dynamic stabilization system is an effective device for the treatment of lumbar disc herniation with instability or possible to be instable post-operatively.after decompression,the additional applicaton of Dynesys system can significantly relieve the symptoms of back and leg pain,improve self-care ability and the quality of life of patients.Imaging follow-up results showed that Dynesys dynamic fixation system could prevent lumbar intervertebral disc degeneration and remain intervertebral ROM of fixed segments.Part three Comparison of Nucleotomy alone,Nucleotomy with Fusion and ucleotomy With Dynesys Dynamic Stabilization System for Treating Lumbar Intervertebral Disc HerniationObjective:Nucleotomy is the main surgical treatment for lumbar disc herniation in the early time,but spinal instability and the related symptoms are often found in the long term.Therefore,Nucleotomy with variety additional lumbar fusion and internal fixation has gradually become the common method for treatment of lumbar disc herniation.However,due to its inherent defects of biomechanics,this operative method has lead to the known adjacent segment disease,which has drawn an increasing attention of orthopedic clinicians.To settle the matter,Dynesys dynamic stabilization system as one the non-fusion instrument,has came into being since 1994.This study was carried out to compare the Effectiveness of nucleotomy（N）,nucleotomy with fusion and internal fixation（F）,and n ucleotomy with additional Dynesys dynamic stabilization system（D）for treating lumbar disc herniation.It was intended to provide theoretical support when choosing the strategy of surgery by the data of follow-up for 2 years.Methods:From September 2015 to September 2017,one hundred and two patients with lumbar intervertebral disc herniation who had been treated with one of the three oprations were selected.All of them were divided into 3groups according the method of operation.Operating time,length of surgical wound and the amount of blood loss of all patients were recorded.They were followed up for two years via telephone reservations and outpatient reviews.The time points for follow-up was at 3 months,6 months,1 year and 2 years after operation.Oswestry dysfunction index（ODI）and visual analogue scale（VAS）scores of patients were recorded before and at each at each time of follow-up after surgery.Lumbar MRI and X-ray was performed before operation and in the last follow-up.Woodend classification and apparent diffusion coefficient（ADC）were used to evaluate the degeneration of the treated and superior lumbar disc.The intervertebral disc space height and intervertebral range of motion（ROM）of lumbar fixed segments and the superior segments were measured by imaging.The intervertebral disc space height=（ventral height+dorsal height+central height）/3.Superior margin of upper vertebral body and inferior margin of lower vertebral body on the operative segment were connected into a line.And intervertebral ROM referred to the angle between the line in the status of extension and flexion.In this study,the comparison of the fixed segments according to the the degree of degeneration and intervertebral ROM was only done between the nucleotomy group and the Dynesys group.We chose the superior segments as the subjects to investigate the ASD associated problems.Results:One hundred and two patients were included in the study and they were divided into Nucleotomy group（N,n=27）,Fusion group（F,n=45）and Dynesys group（D,n=30）.There was no statistical difference among the three groups about the general data of patients in perioperative period（P>0.05）.All the 102 patients completed the operation successfully,their symptoms were significantly improved,and all were followed up.Operative incision length of the 3 groups（N,F and D）was respectively5.15±1.3 cm,8.17±2.1 cm and 7.52±2.4 cm.The difference between the N group and the other two groups was statistically significant（P<0.05）.operating time of the 3 groups（N,F and D）were 67.47±21.2min,129.42±29.6min,110.12±23.1min respectively,and the differences between each two of the three groups were statistically significant（P<0.05）.The amount of blood loss of the 3 groups was 97.18±41.2 ml,218.54±87.3 ml,139.43±75.6 ml respectively,and the differences between each two of the three groups were statistically significant（P<0.05）.ODI and VAS scores of the 3 groups were 61.1±10.5/6.2±1.5,65.6±13.1/6.8±1.7,59.7±9.5/6.3±1.1respectively.Compared with these preoperative ODI and VAS scores,postoperative scores of the 3 groups were all significantly decreased（all P<0.05）.While both of the scores of D group were steady in subsequent follow-up（P>0.05）and only 1 patient was recognized as FBSS（3.3%）,the scores of N group showed an upward trend and there was statistical differences between the scores of the 2-year and the 3-month follow-up（P<0.05）.Eight patients of N group（29.6%）were diagnosed as FBSS in the last follow-up.For F group,the trend of changes of the 2 scores was similar to that of N group,but the changes was less than the latter and there was no statistical differences between the scores of the 2-year and the 3-month follow-up（P>0.05）.There were 4 patients diagnosed as FBSS at last in F group（8.9%）.Degenerative progress was found in 5 and 9 patients in D and N groups respectively.The results of MRI image of the fixed segments showed that pre-operative ADC value of patients in N group was（1.05±0.25）*10^-3mm²/S,but it decreased to（0.83±0.34）*10^-3mm²/S at the end of two-year follow up after operation and the difference was statistically significant（P<0.05）.However,there was no statistical differences between the values of D group with the value was respectively（1.07±0.21）*10^-3mm²/S and（0.99±0.19）*10^-3mm²/S pre-and post-operatively.The ROM of the fixed segments was respectively（6.36±2.77）°and（6.53±1.97）°pre-and post-operatively in F group and the difference was not significant statistically.The pre-operative ROM of fixed segment in D group was（7.11±2.43）°,but it inclined to（2.47±1.16）°at the end of two-year follow up and the difference was statistically significant（P<0.05）.With regard to the superior segments of the treated ones,there were 3,2 and 10 cases in group N,D and F which were identified with egenerative deteriorations respectively and Compared with the other two groups,there were statistically significant differences between group F and the other groups（P<0.05）.The results of MRI image of the superior segments showed that,for patients of group N,the pre-operative ADC value was（1.13±0.15）*10^-3mm²/S and the post-operative value was（1.07±0.27）*10^-3mm²/S,without statistically significant differences（P>0.05）.The same results appeared in the group D with the pre-and post-operative was（1.17±0.31）*10^-3mm²/S and（1.12±0.23）*10^-3mm²/S respectively,and the differences was also not statistically significant（P>0.05）.Different from the two above groups,as to group F,the pre-operative ADC value was（1.09±0.35）*10^-3mm²/S,but it dropped to（0.91±0.37）*10^-3mm²/S in the2-year follow-up and the differences was statistically significant（P<0.05）.As to the ROM of the superior segments of the treated ones,in group N,it was pre-operative（8.18±2.3）°and post-operative（9.41±1.19）°with a little increase,but the difference was not significant statistically（P>0.05）.In group D,it was pre-operative（8.25±2.56）°and post-operative（10.01±3.48）°,but the little more rise show significant difference statistically（P<0.05）.With respect to group F,the value increased sharply from pre-operative（8.43±1.27）°to post-operative（11.31±2.37）°in the 2-year follow-up and the change showed significant difference statistically（P<0.05）.Conclusion:All of the three methods relieved the symptoms of lumbar disc herniation at the early postoperative stage,but the curative effect was not Persisting by nucleotomy alone,and it may cause accelerated degeneration at the treated segments and thus the recurrence of symptoms.Adding fusion and internal fixation after nucleotomy Could make the curative effect lasting more time,but it was easy to lead to ASD and caused long term complications.Nucleotomy with additional Dynesys dynamic stabilization system could relieved the symptoms of LDH for a long time,and what is more,no obvious ASD was found during the period the the 2-year follow-up.