The Experimental Study Of Identifying Sensory Nerve Fascicles In Rats By Nanofluorescent Immunohistochemical Method

Object There are sensory and motor nerve fibers in peripheral nerve, topographic and end-organ specificity of nerve regeneration have been shown to exist.If we failed to match sensory and motor nerve fascicles of injured nerve,the functional recovery can not be achieved. Though many scholars have made plenty of study on related problems, there is not a ideal method to differentiate nerve fascicles.We study the nerve identification using Annexin V antibody labled by CdTe nanocrystals, try to explore a quick, easy and special method, in order to provide a new avialable and realistic method of nerve identification for clinic.Methods 1. 25 Wistar rats were divided into 5 groups randomly,each 5.Before surgery they were anesthetized by intra-abdominal injection of 1% sodium thiopentone solution(25mg/kg). Under sterile condition,the sural nerves of left side were transected in the middle of calf and a short segment(4-5mm) of each nerve was excised to avoid the severed nerve realigning,then the skin incision were sutured and sterile dressed.The rats were killed respectively at 1,2, 3, 4 and 8 weeks after operation.The scars of the nerve were excised under surgical microscope, 1cm segment of proximal and distal stump was taken respectively for histochemical staining, observing the expression of Annexin V at defferent time after operation. 2. 10 Wistar rats were divided into two groups randomly,each 5. One group served as control, another as experimental group.1cm nerve was taken from the sciatic nerve trunk, its motor branches and sural nerve on the left side, then the specimens were quickly frozed and transverse sectioned (thick 5um) with a cryostat microtome. The experimental group use Annexin V antibody labeled by CdTe nanocrystals as incubating liquid,the controlled use normal goat serum,then the specimens were incubated in 37℃incubator.Taken out to observe under fluorescent microscop at 5min,10min,15min,20min and 30min respectively.Result In the first part,we observe the expression of Annexin V in severed sural nerve of rat at defferent time after operation.The results showed that the expression of Annexin V in the proximal stumps of severed sural nerve all presented strongly green fluorescent at defferent postinjury time,the expression of Annexin V in the distal stumps is muted at four weeks and eight weeks after the sural nerve severed,but the nerve fascicles still enough to be seen.In the other part of the experiment,we apply AnnexinV antibody labeled by CdTe nanocrystals to identicate sensory nerve fascicles in rats. The results indicated that the red fluorescence was not seen in controlled group.While in experimental group, different from the sensory branches of sciatic nerve and sural nerve,the motor branches of sciatic nerve could not see the fluorescence,the fluorescence intensity at 10min was not enough to show the nerve fascicles clearly, we could see the nerve fascicles with red fluorescence clearly at 15min, the fluorescence intensity at 20min and 30min had little difference with that of at 15min.Conclusion AnnexinV mainly express at myelin and axons of rat sural nerves, using AnnexinV antibody labeled by CdTe nanocrystals can identify sensory nerve fascicles of rat rappidly, it is possible to provide a new method for clinical differentiation of peripheral nerve nerve fascicles, and it is available for old nerve injuries also.