A facet joint landmark-guided approach for multi-segmental DRG injection with limited bone removal in mice

Targeted gene delivery to the dorsal root ganglion (DRG) has been developed as a valuable approach for pain research. Nevertheless, conventional DRG microinjection approaches suffer from notable drawbacks: vertebral bone resection and facet joint destruction induce spinal instability, extensive tissue adhesion impedes repeated surgery, and multi-segment targeting remains challenging.

To establish a multi-segmental DRG injection workflow, we developed a vertebral counting scheme based on mouse anatomical dissection. To guide our selection of target segments for viral delivery, Thy1-YFP-H transgenic mice was used to trace segmental DRG innervation patterns with good resolution. Anatomical dissection was then performed to examine the precise spatial relationship between each target DRG and its neighboring facet joints. With this anatomical roadmap, together with a custom designed temporary spinal stabilizer, we established a standardized injection procedure applicable to user-specified levels. Post-operatively, animal behavior was serially assessed, and finally in vivo GCaMP calcium imaging was conducted to verify functional viral transduction.

Vertebral counting based on the V-shaped white lines on the spinous processes enabled reliable identification of lumbar segmentation. YFP tracing clarified distinct segmental innervation patterns: L1-L2 fibers do not contribute to the sciatic nerve; L3-L5 collectively form the sciatic nerve; L6 fibers, plus minor L5 branches, constitute the pudendal nerve. Additional anatomical dissection confirmed that drilling at the base of the inferior articular process of the cranial vertebra provided a direct access route to the L3-L6 DRGs. Serial behavioral monitoring post-injection revealed transient functional disturbances, most of which resolved by postoperative day 21. Finally, concurrent jGCaMP8s imaging confirmed successful functional transduction: air-puff stimulation elicited robust calcium responses in transduced DRG neurons, whereas no such signals were detected in wild-type controls.

We have established a reproducible viral delivery method that minimizes bone resection and avoids gross facet joint destruction through laminar drilling, while enabling multi-segmental injection into mouse L3-L6 DRGs. The combination of longitudinal behavioral monitoring and stimulus-evoked calcium imaging supported the surgical feasibility and functional transduction of this approach. This method thus provides an experimental toolkit that should be useful for dissecting somatic-visceral pain crosstalk through multi-segmental DRG genetic manipulation.