Establishment Of The Observation Model Of Dorsal Root Ganglion And Its Preliminary Application In Pain Research By In Vivo Imaging In Awake Mice

Objective:To establish the observation model of dorsal root ganglion and to preliminaryly apply it in pain rcasearch and observe the discharge change of the dorsal root ganglion neurons in the model of pain by in vivo imaging in awake mice. To provide a feasible method of the observation of dorsal root ganglion in awake mice.Method:This research is divided into three parts:1. Establishment of the observation model of dorsal root ganglion by in vivo imaging in awake mice:The objects of the study were adult YFP and GFP fluorescent transgenic mice (20-25g). We fixed spinal in mice by a homemade device, and then we imaged the side of spinal cord, the back of spinal cord and dorsal root ganglion with the two-photon fluorescence microscope by an observation window, and checked the movement of the dorsal root ganglion and the behavior of the model.Verify indicators including:the imaging time; the average velocity, the foot distance, stride length, rest time, grooming time and rearing time of 1-3 days,4-6 days,8-10 days and 14-15 days; Postoperative instant speeds and the top speeds of 1-3 days,4-6 days,8-10 days and 14-15days. And through the dorsal root ganglion inherent inflammation related microglia concentration analysis of the 1st and 7th days after surgery, postoperative inflammatory response in mice were detected. In addition,fixed effect was checked by two-photon fluorescence microscope imaging and spinal gray region difference records.2.The injection of AAV-CAG-GcaMP6 verus to the dorsal root ganglion and the observation of the dorsal root ganglion in awake mice:The objects of the study were adult C57BL/6j (C57) (20-25g). Through a self-made spinal fixation device, we fixed on the spine of mice, and exposed the dorsal root ganglion. And then we injected AAV-CAG-GcaMP6 virus.14 to 18 days later, we verified the initial fixation, by two-photon microscope imaging and AAV-CAG-GcaMP6 virus transfection effect after injection.3. Observation of the dorsal root ganglion in pain model:The objects of the study were adult YFP and GFP fluorescent transgenic mice (20-25g). We fixed Spinal in mice by a homemade device, and then we imaged the side of spinal cord, the back of spinal cord and dorsal root ganglion with the two-photon fluorescence microscope by a observation window, and checked the movement of the dorsal root ganglion and the behavior of the model. We constructed the pain model of mice by plantar injection of formalin. Via two-photon fluorescence microscope we verified the initial fixation, by two-photon microscope imaging and AAV-CAG-GcaMP6 virus transfection effect after injection.Result:1.We got a good fixed effect and could observe the dorsal root ganglion for a long time.The locomotor function was preserved after implantation and long-term imaging of dosal root ganglion. Within 15 days after the operation, imaging, can stablely display dorsal root ganglion neurons, neurons in nucleus, axons and dendrites. Microglia increased obviously at the 7th day. We tested mice for gait abnormalities during normal movement. Mice with inked paws ran the length of a narrow enclosure, after which we analyzed the patterns of their footprints to measure base of support (lateral distance between hindlimb placement), stride length and running speed for mice with spinal chamber implants as well as sham controls (shaved and anesthetized but not receiving surgery; three mice per group. We found no remarkable differences in gait attributable to the implant at any time point. In the second assay, we assessed spontaneous activity, rearing behaviors and movement speed by video-monitored open-field testing. We determined the cumulative time spent immobile, grooming, rearing and speed distributions from post hoc video analysis. Grooming time in mice with implants was significantly higher on days 1-6 after surgery than in sham controls, but this difference did not persist over time. We observed no differences in immobility or rearing times. Although we observed a slight reduction in top ambulatory speed in mice with implants, as compared to sham controls, it was not statistically significant. Finally, mice with implants did not exhibit any difficulty in grooming hindquarters or climbing. With this device, we can realize the dorsal root ganglion injection and electrical signals record. Homemade device can satisfy the AAV-CAG-GcaMP6 virus injection and two-photon fluorescence microscopy imaging requirements.Virus injection of 14 to 18 days later can be clearly observed in the dorsal root ganglion neurons, and we found that the dorsal root ganglion neurons showed corresponding discharge with the electrical stimulation.3. The dorsal root ganglion observation model of mice can combined with pain research well. Through homemade device in the establishment of the dorsal root ganglion observation window, the virus injection dorsal root ganglion and the observation of the dorsal root ganglion neurons, we found that dorsal root ganglion neurons discharge faster than the control, so we speculated these neurons may be pain related neurons in the dorsal root ganglia.Conclusion:We successfully established the observation model of dorsal root ganglion and preliminaryly applyed it in pain reasearch and observed the discharge change of the dorsal root ganglion neurons in the model of pain by in vivo imaging in awake mice. This research might provide a feasible method of the observation of dorsal root ganglion in awake mice.