大鼠失神经骨骼肌7．0T MR磷谱表现

Study on denervated rat skeletal muscle by 7.0 T 31p-MR phosphorus spectroscopy

目的 通过7.0 T MR磷谱（31P-MRS）探讨大鼠失神经支配骨骼肌的变化规律.方法 实验动物为体重200～250 g雄性SD大鼠.实验组18只,选取右后肢作为手术侧,于腹股沟水平向下切除10 mm长度股神经,以建立失神经营养支配骨骼肌萎缩模型.对照组6只为假手术组（只进行右后肢皮肤切口及神经探测）.于造模前和造模后不同时间点（造模后3 d及1、2、4、6、8、10周）,在7.0 T动物MR成像系统上采集股直肌31P-MRS信号.用外标法（浓度为10 mmol/L磷酸肌酸钠水模溶液）和jMRUI软件实时定量β-ATP、磷酸肌酸（PCr）和无机磷酸盐（Pi）的绝对浓度.同时计算其相对浓度PCr/Pi、Pi/β-ATP、PCr/β-ATP、PCr/（PCr＋Pi）和Pi/（PCr＋Pi）.细胞内pH值（pHi）由Pi信号相对于PCr信号的化学位移计算.实验组或对照组纵向各时间点之间各代谢物的比较采用单因素方差分析（ANOVA）中的Bonferroni检验.实验组与对照组各个代谢物在横向各时间点之间的比较分析采用独立样本t检验.P〈0.05表示有统计学意义.结果 对照组大鼠股直肌β-ATP、PCr和Pi平均浓度分别为（6.654±0.178）、（25.656±0.695）、（1.594±0.096）μmol/g.实验组大鼠骨骼肌失神经后各实验时间点,β-ATP、PCr、Pi、PCr/Pi、PCr/PCr＋Pi、Pi/PCr＋Pi与对照组间比较差异均有统计学意义（P〈0.05）.除第3天外,实验组Pi/β-ATP、PCr/β-ATP、pHi与对照组间的差异均有统计学意义（P〈0.05）.实验组大鼠失神经损伤第3天,股直肌β-ATP和PCr与失神经前相比分别下降了约11.5％和19.7％;第4周时,分别下降了约63.1％和68.8％;此后下降速度减慢,至第10周,下降74.0％和82.3％.Pi、Pi/PCr＋Pi、Pi/ATP水平随失神经支配时间延长进行性升高.正常大鼠pHi为7.033±0.017.失神经营养支配后pHi逐渐下降.结论 7.0 T 31P MRS可以定量显示正常和失神经骨骼肌不同时间点能量代谢和pHi值的变化趋势.骨骼肌失神经后早期即发生功能障碍并呈进行性加重,且β-ATP、PCr、PCr/Pi变化主要发生在失神经后4周内,其有可能成为评价失神经骨骼肌能量代谢变化的可靠标志物.

Objective To investigate the changes of 31P-MRS in denervated skeletal muscle at 7.0 T MR system. Methods In the experiment group, a total of 18 male Sprague-Dawley rats aged 6-8 weeks old and weighing 200-250 g were obtained. The right posterior femoral nerve were transected, and the proximal stumps were ligated by using 5-0 nylon stitches to preclude spontaneous repair. A sham surgery （incision and exploration of the nerve） was performed at the same time （n=6）. Before rat model established and at varying times after initial surgery （3 d, and 1, 2, 4, 6, and 8 weeks）. 31p-MR spectra of rat quadriceps femoris were acquired on 7.0 T Agilent small animal MR imaging system. We quantified the phosphocreatine （PCr）, adenosine triphosphate（β-ATP）, and inorganic phosphate（Pi） using Creatine phosphate disodium salt （10 mmol/L） as an external standard. The ratios, include PCr/Pi, Pi/13-ATP, PCr/13-ATP, PCr/（PCr＋Pi） and Pi/（PCr＋Pi） were quantified in jMRUI. All data were analyzed by one way analysis of variance （ANOVA） with posttest inter-group comparisons using Bonferroni test. Comparative analysis methods between the experimental and control group were performed via independent samples t test. P〈0.05 was considered as statistically significance. Results The average measured concentrations of J3-ATP, PCr, and Pi in control groups were （6.654±0.178） μmol/g, （25.656±0.738） trmol/g, and （1.594±0.096） txmol/g, respectively. There were significant statistically differences in 13-ATP, PCr, Pi, PCr/Pi, PCr/PCr＋Pi,Pi/PCr＋Pi between the study and control group（P〈0.05） at any measurement time point after denervation. Pi/13-ATP, PCr/15-ATP and pHi in the experimental group significantly statistically differed from those of the control group （all P〈0.05）at each time point except on day 3 after operation. The total concentrations of 13-ATP and PCr were reduced by 11.5% and 19.7% respectively on day 3. Thereafter, 13-ATP and PCr declined rapidly by 63.1% and 68.8% at week 4 respectively, then decreased slowely by 74.0% and 82.3% till week 10. The change of PCr/Pi is similar to β-ATP and PCr, but more remarkable. Pi, Pi/PCr＋Pi, Pi/ATP showed a progressively increase till week 10. The intra-cellular pH （pHi） of normal rat muscles was 7.033 ± 0.017, While the pHi gently in experiment rat muscles decreased during the entire experiment. Conclusion nP-MRS with 7.0 T can quantify the temporal changes of energy metabolism and pHi in normal and denervated rat muscles. It shows that the dysfunction of energy metabolism are progressive with time and that they begin within a short period following the nerve section, the change of β-ATP, PCr, PCr/Pi take place primarily within 4 weeks after denervation. β-ATP, PCr, and PCr/Pi may be potential biomarkers of energy metabolism in the evaluation of denervated muscle atrophy.