Traumatic Peripheral Nerve Injury in Mice

Traumatic peripheral nerve injury (TPNI) is a common cause of morbidity after orthopedic trauma^1,2,3. Yearly, approximately 3% of trauma patients suffer nerve injury^1,4, at an incidence of 3,50,000 cases⁵, resulting in 50,000 surgical repairs⁶. TPNIs occur in a wide range of severity, and the functional recovery directly depends on the type and severity of these injuries^7,8,9. Less severe trauma (e.g., mild crush, incomplete laceration, etc.) will injure the myelin sheath and axons first, while more severe forces (e.g., severe crush, complete lacerations, etc.) will disrupt the connective nerve tissues; for example, the endoneurium, perineurium, and epineurium in addition to the myelin and axons^1,10. Patients with TPNI hope that nerve function will eventually return, and muscle atrophy will be reversed. Decades of research have provided no precise treatments to enhance or ensure complete recovery despite advances in the treatment procedures^11,12.

Nerve transections will not heal without surgical repair, which is often performed under the microscope. Repairs are typically performed end-to-end, making an effort to ensure that the repair site is under no tension. Nerve grafting is used to ensure that repairs are tensionless^13,14. Despite the seemingly advanced methods used in these repairs, functional recovery is generally unimpressive^11,12. Rehabilitation is often incomplete and unsatisfying. The optimal functional recovery requires regenerating axons to cross the injury site (nerve bridge) and innervate the target organ. These processes are complicated by axonal misdirection or growth stunting, resulting in muscle atrophy and eventual failure to recover^15,16,17,18. It has been shown that functional outcomes following nerve repair (e.g., end-to-end suturing, isografting, etc.) depend on the accuracy of fascicular apposition^19,20. Proper directionality of the transected nerve stumps and their fascicles is thus critical in nerve repair, without which poor functional recovery can be expected even with optimal axonal regeneration. Microsurgical suture repair itself is a traumatic process, and little has occurred in terms of novel methods to improve outcomes drastically. The field lacks reproducible nerve transection animal models, which result in predictable gaps that allow reliable recovery measurements on a functional and tissue level. Such methods, if available, would allow characterization of nerve regeneration without the problems of variable changes in neural vascularization and post denervation atrophy^21,22. Many groups endeavor to use better models that limit this kind of variability. One way is to ensure that nerve repairs are minimally manipulated, and nerve stumps are perfectly opposed.

This is best accomplished by using a standardized peripheral nerve transection technique called stepwise cut and fibrin glue (STG). Repairs in this STG model are secured with fibrin glue, and gap distances are standardized and minimized^21,22. Fibrin glue itself is employed in humans for these repairs, likely for the same reasons, along with its beneficial effects on post-repair scar formation^23,24. The key to the present method is that the nerve repair begins before the laceration is completed, ensuring a fixed injury pattern. This current method exhibited a close commonality to the characteristic pathophysiology of nerve transection with the gold-standard epineural suturing, and the negative impact of fibrin glue was not observed on nerve regeneration. Repairing of the sciatic nerve transection with fibrin glue in mice ameliorates the elongation of axon compared to early nerve regeneration via suturing, and these findings are consistent with STG. STG also benefits from the minimal manipulation principle, where the nerve is never touched for suture positioning²¹. This effectively standardizes the nerve trauma associated with repair in the model. Similar principles were used to investigate misalignment by flipping the nerve before gluing²². This allowed direct comparison of nerve injuries where almost the same amount of manipulation contributed to differences in alignment without increased gap or trauma. This facilitated the direct examination of the effect of alignment on nerve-injury-induced neurovascular changes^21,22, muscle atrophy^21,22, and functional recovery^21,22. The present investigation is all that allows the study of purposefully and precisely misaligned nerve stumps.

Most of the nerves in TPNI are not severed, have no gap or defect, and seem to be capable of recovery, and yet in many of these cases, limbs remain permanently dysfunctional from nerve injuries and confound interventions. Experimental TPNIs are customarily performed on rodent sciatic nerve crush injuries (SNCIs) using locking needle drivers (NDs), forceps, or similar devices, and an experienced surgeon to create a precise and reproducible crush injury^{25,26,27,28,29,30}. SNCI animal models depend on innate operator precision to limit pressure variation, but this is never measured explicitly. This results in variability between animals and studies, with no clear guidance on the standardized pressure. It is thus anticipated that the capability to precisely deliver and report a coherent, accurate series of injuries with various known intensities may benefit the TPNI field. A perfect model can provide an SNCI of a known nerve injury severity to each animal by any lab or researcher for authentic inter-study and device replicability. To address this deficiency, a unique calibrated digital device was constructed containing a Force Sensitive Resistor (FSR), proficient in reporting the pressure (real-time) applied to a nerve. This device was then tested for the replicability of various crush injury pressures deployed by diverse types of forceps and NDs³¹.

Finally, a specific method was developed to address gaps in nerve³². The nerve gaps in the literature are induced by removing a nerve section and then repairing it back into the defect^13,33,34. The manipulation required for this surgical procedure is often compounded with suturing, and the stumps of the nerve retract variably^21,32,34. It was based on the reasoning that using isogenic oversized nerve grafts, the nerve stump retraction will never an issue³². The method required the simultaneous operation on two or three animals at once, taking a 7 mm graft to place into a 5 mm defect induced in another animal. The defect size of the second animal was then used to graft a still smaller defect in another animal if needed. This resulted in a comprehensive method for simultaneous surgery to graft defects with donor nerves that are always larger than the nerve defect to ensure tensionless repair. On coupling with the requirement of minimal manipulation, this offers an avenue to study graft length directly in syngeneic animals without asymmetric graft gaps that are ubiquitous in the literature^20,32,34.