显微拉曼光谱技术联合Karnovsky-Roots染色法快速鉴别周围神经纤维的功能性质

Rapid differentiation between the functional properties of peripheral nerve fascicles via raman micro-spectroscopy and Karnovsky-Roots staining

目的研究周围神经内部运动神经纤维和感觉神经纤维经Karnovsky-Roots染色后的显微拉曼光谱特征及该法对两种不同功能性质的神经纤维进行快速识别。方法取新西兰大白兔20只，分别取其双侧骶1脊神经前根和后根各20例标本作30μm冰冻切片，切片在Karnovsky-Roots染色30rain前后分别在显微拉曼光谱仪下用波长633nm的激光束聚焦于神经纤维断面进行拉曼光谱采集。取其中各1例脊神经前根和后根标本染色8h后作同样处理。对2100cm。和1440cm。拉曼强度的比值进行统计分析，获得可以区分运动神经纤维和感觉神经纤维的分类算法；将其余各20例脊神经前根和后根标本经染色30min后的拉曼光谱与解剖学“金标准”比较，行诊断性试验。结果（1）染色30min后，运动神经纤维的拉曼光谱在2100～2160cm。出现明显的拉曼强度，在感觉神经纤维的拉曼光谱中则没有；（2）染色30rain后运动神经纤维I2100／I1440比值与感觉神经纤维相比差异具有统计学意义；I2100／I1440〉0．2的神经纤维可以认为是染色结果阳性，即为运动神经纤维。结论经纤维经Karnovsky-Roots染色30min后，运动神经纤维与感觉神的拉曼光谱可根据I2100／I1440比值加以区分。显微拉曼光谱可以显著加快Karnovsky-Roots染色鉴别运动神经纤维和感觉神经纤维功能性质的速度。

Objective To acquire the Raman spectra of peripheral nerve fascicles after 30 min of Karnovsky-Roots staining,and to differentiate between motor and sensory fascicles by Raman micro- spectroscopy. Methods Twenty New Zealand rabbits were sacrificed to bilaterally harvest their S1 anterior and posterior spinal nerve roots. Twenty anterior and posterior root samples were randomly chosen from each group and made into 30 μm frozen sections. Sections of every sample were scanned with Raman micro-spectroscopy （633 nm laser） before and after 30 rain of Karnovsky-Rootsstaining. An additional slide of both an anterior root and a posterior root was selected, stained for 8 h and scanned. The value of I2100/I1440 was statistically analyzed and a classification algorithm was formulated. Diagnostic test was performed for the remaining twenty anterior and twenty posterior spinal nerve roots using with the aforementioned classification algorithm, comparing our method against anatomical proof which was considered as ＂the golden standard＂. Results （1） Raman scatterings were detected near 2 100 - 2 160 cm-1 in motor fascicles after 30 rain of Karnvosky-Roots staining. No such modification would manifest in sensory fascicles. （2） I2100/I1440 was statistically different in motor fascicles than in sensory fascicles after staining. Fascicles could be classified as positively stained （motor fascicles） when I2100/I1440 〉 0.2. Conclusions The two types of nerve fascicles can be differentiated from their counterparts by the value of I2100/I1440 in their Raman spectra. Raman micro-spectroscopy can be used to rapidly distinguish motor and sensory nerve fascicles after 30 min of Karnovsky-Roots staining.