Repair and regeneration of peripheral nerve injuries that ablate branch points

Abstract

The peripheral nervous system has an extensive branching organization, and peripheral nerve injuries that ablate branch points present a complex challenge for clinical repair. Ablations of linear segments of the PNS have been extensively studied and routinely treated with autografts, acellular nerve allografts, conduits, wraps, and nerve transfers. In contrast, segmental-loss peripheral nerve injuries, in which one or more branch points are ablated so that there are three or more nerve endings, present additional complications that have not been rigorously studied or documented. This review discusses: (1) the branched anatomy of the peripheral nervous system, (2) case reports describing how peripheral nerve injuries with branched ablations have been surgically managed, (3) factors known to influence regeneration through branched nerve structures, (4) techniques and models of branched peripheral nerve injuries in animal models, and (5) conclusions regarding outcome measures and studies needed to improve understanding of regeneration through ablated branched structures of the peripheral nervous system.

Introduction

There are many kilometers of peripheral nerves (PNs) in the adult human body (Catala and Kubis, 2013). Large diameter PNs extending out of the brain and spinal cord progressively branch into smaller PNs before reaching their target innervation tissues. PNs containing many thousands of axons can extend long distances, a meter or more in some cases, and innervate many different motor and sensory target tissues as they extend and branch distally.

PNs are frequently injured, and PN injuries (PNIs) that ablate nerve segments are the most common and costly cause of temporary and permanent nervous system dysfunction (Secer et al., 2008; Taylor et al., 2008; Birch et al., 2012a, b; Ring, 2013; Brattain, 2014; Adiguzel et al., 2016; Battiston et al., 2017). In the short term following ablation PNI, there is an immediate and complete loss of sensory and motor function in the tissues previously innervated by the damaged nerve. Atrophy of the denervated muscle begins within days of the PNI and, unless reinnervated, is irreversible after 18–24 months (Grinsell and Keating, 2014). Fibrosis of denervated tissue increases over time and prevents neuromuscular junction reestablishment within months, even if regenerating axons eventually reach the muscle (Wang et al., 2019).

The peripheral nervous system (PNS) is capable of unaided regeneration after injury, with the extent of regeneration dependent on the ability of surviving axons to traverse the injury, regenerate through the distal nerve structure, and reinnervate an appropriate sensory or motor target tissue. When the injury results in a segmental PNI where direct neurorrhaphy of the ends is not possible due to excessive tension, a bridging material is often inserted into the gap or nerve transfer is considered. Currently available bridging materials include autografts, acellular nerve allografts (ANAs), viable allografts with immunosuppression, or degradable conduits/wraps (Mackinnon et al., 2001; Campbell, 2008; Griffin et al., 2013; Safa and Buncke, 2016; Pan et al., 2020). These materials are designed to guide regenerating axons to the proximal and distal stumps, minimize infiltration of non-nerve fibrous tissue into the PNI, and physically stabilize the regenerating nerve.

Bridging materials are developed and marketed for linear ablations, although the extensive branching anatomy of the PNS means that many ablation PNIs are likely to damage one or more branch points. Branched PNIs add complexity to surgical management and increase the likelihood of denervated tissue atrophy compared to linear ablations. The clinical and experimental management of branched ablations is neither well documented nor rigorously studied. This review aims to address some of these knowledge gaps.

Search Strategy

Publications and case reports for this review were compiled from the referenced sources following a search of the literature between August and December of 2022. Clinical case reports and animal research models had to include a clear reference to an injury or repair involving ablation of a branch point to be included.

Anatomy of Branch Points

The human femoral nerve exemplifies the branching anatomy of the PNS, as shown in Figure 1 (reproduced with permission from Gustafson et al. (2009)). Approximately 4–6 cm below the inguinal ligament, the femoral nerve branches into anterior and posterior divisions (Figure 1A). The anterior division has several major branches to the sartorius and pectineus muscles and the anterior cutaneous sensory branch(es) to the skin of the anteromedial thigh. The posterior division first branches into the saphenous cutaneous sensory nerve, which extends down the leg to innervate the medial aspects of the leg and foot. The more distal posterior nerve, now entirely motor, successively branches to innervate the vastus medialis, vastus intermedialis, vastus lateralis, and rectus femoris of the quadriceps.

Figure 1.

Anatomical and fascicular organization of the main trunk of the human femoral nerve and its major branches.

(A) Harvested femoral nerve with labeled branches. (B) Fascicular organization of the femoral nerve traced distally from all branches. a: Proximal nerve stump, b: branch to sartorius muscle, c: nerve trunk distal to sartorius muscle branch and proximal to pectineus branch, d: branch to pectineus muscle, e: nerve trunk distal to pectineus branch and proximal to medial/cutaneous branch, f: main nerve that branches into the medial/cutaneous and saphenous branches, g: branch to the vastus medialis muscle, h: branch to the vastus intermedius muscle, i: main nerve branch that further branches to innervate the vastus lateralis and rectus femoris muscles, j: branch to the vastus lateralis muscle, k: branch to the rectus femoris muscle. Reprinted from Gustafson et al. (2009).

Detailed fascicle tracing shows how fascicles are organized within nerves before branching points. Fascicles within the femoral nerve are grouped within the main nerve trunk prior to the physical bifurcation (Figure 1B from Gustafson et al., 2009). Fascicular organization within the nerve trunk begins several centimeters proximal to a branch point. For example, axons innervating the sartorius muscle are organized into groups of fascicles that are mostly anterolateral within the nerve sheath of the main trunk of the femoral nerve proximal to the physical bifurcation (Figure 1B). Similar patterns are seen in Figure 1B for the fascicles that branch to innervate the pectineus muscle and other branches of the femoral nerve.

These studies have reported variability in fascicular organization (Aizawa, 1992; Gustafson et al., 2009). For example, fascicles comprising the sartorius branch before its bifurcation are sometimes found medially rather than anterolaterally within the main trunk of the femoral nerve. In addition, the most proximal nerve segment branching from the main trunk of the femoral nerve distal to the inguinal ligament may be the medial cutaneous, sartorius, or pectineus nerves. That is, the location of the axon and fascicles of a given branch within the unbranched trunk can be predicted with some confidence based on the location within the nerve, but absolute certainty is not possible. Additional fascicle tracing in many more anatomical specimens may more clearly define which organizations are most common in branching structures to facilitate the repair of ablation-type PNIs involving branch points.

There are at least three characteristics of branch points that should be considered. One property is how fascicles/fibers destined for a particular branch segregate within the main nerve trunk (Figure 1B). A second property is where, proximal to distal, the branching nerve establishes an epineurium distinct from that of the main nerve trunk. The third characteristic is where the branch separates from the connective tissue enveloping the main trunk. Figure 2 shows the posterior cutaneous, tibial, peroneal, and sural branches of a rat sciatic nerve. The arrows labeled B point to the posterior cutaneous branch and indicate where it can be observed distinct from the main sciatic epineurium and after it has separated from the connective tissue surrounding the main nerve trunk.

Figure 2.

Anatomy of the sciatic nerve.

Proximal sciatic nerve extending from the (A) external obturator tendon and (B) branching away from the trunk at the posterior cutaneous nerve before extending approximately 2 mm more and branching again into the (C) sural nerve, (D) tibial nerve, and the (E) peroneal nerve. Unpublished data.

Current Practices in Surgical Management of Ablation Peripheral Nerve Injuries that Include Branch Points

The field of PN regeneration is overwhelmingly focused on linear segmental defects, although the highly branched organization of the PNS means that long segments of unbranched nerve are rare. There is very little information in the literature on the clinical repair of branched PNIs. Individual case reports provide the only information available on the clinical management of branched PNIs. Figure 3 shows the strategies for management of branched PNIs identified from published case reports.

Figure 3.

Illustration of the clinical treatment strategies used to treat peripheral nerve injuries that ablate branch points.

Created with BioRender.com.

Bypassing branches

When segmental injuries occur to nerve segments with branches of lesser functional importance, these minor branch points can be bypassed to prioritize regeneration to more functionally important areas. Bypassing minor branches prioritizes regeneration of the main nerve at the expense of permanent loss of sensation or motor control of tissues innervated by the bypassed branches. This is a common surgical consideration. Branch bypass is not limited to human studies. The rat sciatic nerve is a very commonly used model for ablation of PNIs (Navarro, 2016). Ostensibly linear ablations using this model typically remove a segment between the external obturator tendon at the proximal end and the peroneal-tibial bifurcation at the distal end. Such ablations may bypass the posterior cutaneous nerve (Figure 2).

Cabling branches at the distal end of a linear bridge

In this strategy, branch reconstruction is performed using standard linear bridging materials. The distal branch stumps are sutured to the distal end of the linear bridge. This strategy is possible in cases where the distal branch stumps are close enough to be sutured to the same distal end of the bridging material. McKee et al. (2020) described a case in which an allograft with immunosuppression was used to repair the ablation of a ~2.2 cm branch segment of the digital nerve. The allograft was chosen by the patient over an autograft to avoid secondary surgery and donor site morbidity.

Cabling of bridging materials from the proximal stump

Cabling of autografts is performed for linear defects when the diameter of the bridging material, typically sensory autografts, is significantly smaller than the injured nerve (Isaacs, 2013). Cabling can be adapted to repair ablated branch points by attaching multiple cables to the proximal stump, which are then sutured distally to the different stumps of each branch. Leechavenchongs et al. (2001) describe two cases of proximally cabling autografts to repair ~2 cm digital nerve branch ablations. This strategy of cabling multiple bridging materials at the proximal end and distally to separate branches has also been documented with vein grafts (Terzis and Kostas, 2007).

Branched bridging materials

Anatomical matching between the injury and the bridging material is an important consideration for linear defects and can also be considered for branching defects. Anatomically matched branched bridging materials replicate the bifurcation point within the bridge, simplifying surgical repair. Several cases using branching autografts for facial nerve reconstruction have shown positive results, with branching autografts harvested from the thoracodorsal, sural, greater auricular, and medial antebrachial cutaneous nerves (Haller and Shelton, 1997; White et al., 2006; Lu et al., 2010; Biglioli et al., 2013, 2014). Hu et al. (2016) investigated the use of branching ANAs from the sural nerve for facial nerve reconstruction and reported improved outcomes when there were fewer branches in the ANA as well as in cases with shorter graft lengths.

Nerve transfer

Nerve transfer is a commonly used strategy in which all or part of a healthy (and ideally redundant) nerve is rerouted and connected to a damaged nerve (Domeshek et al., 2019). A primary advantage of nerve transfer is that the terminus of the healthy nerve is often closer to the target innervation tissue, so that axons extending from the transferred healthy nerve have shorter regenerative distances. In the context of PNI at branch points, nerve transfer has been applied in two ways: single branch and multiple branch nerve transfer. Dougherty et al. (2021) reviewed a technique for single facial nerve transfer in which one of the branches of an ablated facial nerve is transferred to a nerve of the masseter muscle to simplify the remainder of the reconstruction. Functional recovery from the transferred nerve preceded that of the branches treated with bridging materials without nerve transfer (Owusu et al., 2016). A similar strategy was used in a case report by Motomiya et al. (2021), where a single flexor carpi ulnaris funiculus was transferred to the biceps brachii after injury to the musculocutaneous nerve branch and biceps brachii to prioritize elbow flexion. Multiple nerve transfer avoids any reconnection to the initial proximal stump and instead transfers all distal stumps to different nerves. Tung and Mackinnon (2001) documented two cases in which multiple distal branches of the pronator teres were transferred to the flexor digitorum superficialis rather than attempting a direct bridge to the nerve proximal to the injury.

Factors that Influence Regeneration Through Peripheral Nerve Injuries that Ablate Branching Nerve Structures

Regenerative distance and misrouting are contributing factors to the regenerative success or failure after PNIs. PNIs that are longer distances from their innervation tissues have poorer outcomes than PNIs relatively close to their innervation tissues (Hu et al., 2016). Misrouting occurs when axons regenerate through different pathways at branches of the PNS to reinnervate different tissues than they originally innervated. Animal studies show that up to 33% of motor axons may misroute when there is even a single branch point distal to the PNI (Masand et al., 2012). Misrouting becomes a more significant factor with longer regenerative distances and with a greater number of distal branch points.

When misrouting does occur, it has several consequences. Sensorimotor misrouting, such as when a motor axon regenerates into an exclusively sensory branch, can lead to the permanent loss (pruning) of the misrouted axons, discussed in more detail below. In cases when motor axons reinnervate different musculature than they originally innervated, functional restoration depends on the degree of central and peripheral plasticity to compensate for the new innervation. For example, consider a motor neuron whose axon in the common peroneal nerve originally innervated the tibialis anterior but after injury regenerated to innervate either the extensor digitorum longus or the peroneus brevis. With the original innervation this neuron functioned during dorsiflexion and extension of toes. This functionality would be partially conserved if the axon regenerated into the extensor digitorum longus after injury. In contrast, regeneration into the peroneus brevis would have this axon contributing to foot eversion and plantarflex of the ankle. The degree that central and peripheral plasticity can compensate for such changes is considerable, but not infinite (Navarro, 2009).

How regenerating axons are influenced by the branching structures in the nerve has been primarily investigated in the context of PNIs that occur in a linear/non-branched segment proximal to a branch point. These studies have often been conducted from the perspective of assessing axonal pathfinding designed to determine what occurs when the axons regenerate through the bisection or linear defect, reenter the distal nerve structure, and encounter the branch point that is distal to the PNI/bridging material.

The femoral nerve model has been particularly useful for studying regenerative processes at branch points because it contains a well-defined branch where the saphenous nerve, containing only sensory axons, branches away from the rest of the nerve that is predominantly motor with some spindle and tendon afferents (Irintchev, 2011). Misrouting can be quantified in this model by retrograde labeling where dyes travel retrograde in regenerated axons to trace the source of the regenerated axon into the spinal column. Localization of the dyes into the dorsal or ventral aspects of the spinal column showes the proportion of neurons that are motor or sensory. Some of the key observations of this model indicate that regenerating motor axons that regenerate down the saphenous sensory branch and cannot reestablish neuromuscular junctions may be eliminated through a process called pruning (Brushart, 1988; Madison et al., 1996). The extent of pruning that occurs for misrouted motor axons is not definitive because studies that have purposely misrouted motor axons into the saphenous branch of the femoral nerve show prolonged survival of such misrouted axons (Midha et al., 1997). Collateralization to proper target tissues may occur to maintain the viability of such misrouted axons (Redett et al., 2005).

The femoral model has also been useful for assessing preferential motor reinnervation that occurs when a “pathfinder or pioneer” motor axon regenerates and establishes neuromuscular junctions with a muscle. Subsequent regenerating motor axons are more likely to follow the regenerative path of the established motor axon (Brushart, 1988; Madison et al., 1996; Brushart et al., 1998). Preferential motor reinnervation has been attributed to cues provided by the pioneer motor axon as well as differences in the biological cues within motor branches that may influence pathway selection prior to any end-organ reinnervation of leading axons (Martini et al., 1994; Madison et al., 2007). The result of preferential motor reinnervation is fewer misrouted motor axons at branch points such as the posterior femoral nerve where there is a distinct separation of motor and sensory branches. More recent data using in vitro organotypic models showed that the presence of sensory axons inhibited the extension of motor axons (Brushart et al., 2020), raising the possibility that the regeneration of motor neurons is influenced by both positive and negative cues. Preferential motor reinnervation may have undesirable effects in cases when both branches are mixed or motor. The branch with the most proximally located musculature may attract a disproportionate number of regenerating motor axons.

For the regeneration of sensory axons at branch points, early studies looking into sensory axon reinnervation show that sensory axon reinnervation in unrepaired transection PNI is not preferential toward distal sensory nerves (Politis, 1985; Brushart and Seiler, 1987). Sensory axons instead regenerate randomly toward the nearest injured distal nerve stump due to diffusible factors guiding regenerating axons across the gap toward distal nerve stumps (Politis et al., 1982; Mackinnon et al., 1986). Alternatively, when non-fasciculated nerves have been directly sutured, there is no evidence of these diffusible factors guiding the reinnervation of sensory axons, which appears to occur randomly despite dedicated nerve morphology in the distal stump (Weiss and Edds, 1945). Current information suggests sensory reinnervation may be random and that preferential motor reinnervation may be the driving force that reduces the risk of motor misrouting at branch points.

Techniques and Models of Branched Peripheral Nerve Injuries in Animal Models

Repair of ablation PNIs in animal models is predominantly focused on linear ablations, exemplified by the challenge to identify the longest unbranched PN segment for which linear bridging materials can be tested (Navarro, 2016). This focus on linear ablations is especially evident for smaller animal models with few long nerves, where even one of the most common linear ablation models, the rat sciatic nerve, is actually branched (Figure 2). A few animal studies address branched ablations that often use the same materials used for linear defects, as discussed below.

Branched conduits

Biodegradable nerve conduits/wraps were one of the first commercially available devices to address linear ablations. Silicon Y-shaped conduits have been injected with human umbilical cord stem cells to repair a 5 cm segmental defect in the rat posterior femoral nerve (Muheremu et al., 2016). The main comparison of the study was made between Y-shaped conduits that had a 3 mm trunk (linear section prior to the branch point) and 4 mm long branches versus Y conduits with a 4 mm trunk and 3 mm branches and sham controls. Results indicated that even short differences in the length of the trunk segment of Y conduits prior to the branch point may be consequential. Other studies have shown that it was possible to generate highly specific branched conduits and nerve scaffolds capable of branching, or branched nerve sheets, by advanced fabrication methods, but these have yet to be validated in branched PNIs in vivo (Hadlock et al., 1998; Suri et al., 2011; Zhu et al., 2018; Shahriari et al., 2019; Narita et al., 2021).

Branched vascular grafts

The branching anatomy of vasculature is somewhat similar to that of the PNS and therefore is a rich source of branching graft materials of varying sizes. Venous grafts are autologous and have shown significant success for linear defects in many models (Sabongi et al., 2015). Hizay et al. (2012) transplanted 5 mm aortic vascular grafts from inbred rats to guide the regeneration of the ablations of the zygomatic and buccal branches of the facial nerve. This graft design bypassed the smaller marginal mandibular and ramus colli branches of the facial nerve. The common iliac branches were used to guide the regenerating zygomatic and buccal nerves while the visceral and parietal aortic branches were removed, and the holes cauterized. Controls included uninjured animals and direct coaptation where the 5 mm segment was sutured back into place after the removal of the lesser branches of the facial nerve (autograft). Outcomes showed axons regenerated down both branched in the aortic vascular grafts, but it was functionally inferior to autografts.

Branched ANAs

ANAs are a potentially facile approach because branching nerve structures can be obtained from donors, decellularized, and stored. Hu et al. (2010) assessed ANAs for use in the rabbit facial nerve containing five branch points with a total length of 2 cm. Injuries were repaired in four protocols 1) matching branching facial ANA, 2) viable branching facial allograft from inbred donors, 3) linear peroneal ANA, or 4) linear, viable peroneal nerve inbred allograft. Peroneal grafts either had the branches sutured to the distal end of the linear graft or cabled with the ends directly sutured into the muscle targets of the main facial branches. Their data showed that regeneration with anatomically matching grafts, facial ANAs, was superior to that of anatomically mismatched peroneal ANAs.

Branched viable autografts and allografts

Viable (not decellularized) allografts can be anatomically matched to the defect and contain viable donor cells that aid in the regenerative process. While nerve allografts are not as immunogenic as other donor tissues, they still evoke a host response and immunosuppression is required for best results (Roballo and Bushman, 2019; Roballo et al., 2022). Roballo and Bushman (2019) explored branched autografts and localized immunosuppression of branched allografts as an alternative to drug-based systemic (whole body) immunosuppression in Lewis rats with Sprague-Dawley donors. A 2 cm length of sciatic nerve was removed that included the peroneal and tibial branch point, which was bridged with the autograft (same segment sutured back into place), or 2 cm branched allografts obtained from the same section of the sciatic nerve from donors. The sural and posterior cutaneous nerves were bypassed. Branched allografts were tested with and without allogeneic regulatory T cells locally placed around the allografts with a degradable hydrogel. Outcomes showed robust regeneration for the branched autografts, as well as the branched allografts with regulatory T cells. In contrast, regeneration through branched allografts without Tregs was impaired.

Allgood et al. (2022) compared outcomes for 2.5 cm branched defects of the sciatic nerve in Lewis rats bridged with 2.5 cm autografts and inbred allografts sutured into the defect in different orientations. For sciatic autografts in the original orientation, the peroneal and tibial nerves in the branched grafts were sutured to their respective distal nerve structures while for switched grafts the peroneal branch within the graft was sutured to the tibial (and graft tibial to host peroneal) (Figure 4). These branches were of disparate sizes, with areas of 0.13 mm² for the peroneal branch and 0.29 mm² for the tibial branch. A similar scheme was used for branched sciatic inbred allografts. Branched inbred femoral allografts were likewise tested in two configurations, with the motor branch of the graft sutured to the tibial branch and the saphenous branch of the graft sutured to the peroneal branch in the first orientation and to opposite distal nerve ends in the switched orientation (i.e., motor to peroneal and saphenous to tibial). Outcome measures for this 36-week study included compound muscle action potentials (CMAPs) of the peroneal and tibial nerves, CatWalk gait analysis, toluidine blue cross sections of each branch and retrograde labeling at endpoint.

Figure 4.

Illustration of the most notable outcomes of the branched nerve grafting experiment done by Allgood et al. (2022).

After injury, degenerated and unmyelinated axons are present in and distal to the graft. As regeneration proceeds, myelinated axons can be seen at week 16 with new axons forming until week 36. It is hypothesized that collateralization occurs to increase the number of axons in the distal stumps by 36 weeks which contributes to the larger axon counts at this time point. Average CMAP recordings of each branch can be found in green boxes. (*) indicate the notable CMAP values discussed in the text. Additionally, there is increased nerve area in the (A) sciatic and (B) femoral grafts indicated by arrows. CMAP: Compound muscle action potential. Created with BioRender.com.

Figure 4 shows some of the outcomes from this comparative study. For example, the graft source and orientation played a role earlier in the regenerative process, but all groups achieved relatively equal levels of functional reinnervation by the 36-week endpoint. CMAP amplitudes of the extensor digitorum brevis and flexor digitorum brevis muscles in the foot, respectively innervated by distal element of the peroneal and tibial nerves, indicated that regeneration at the earlier time point of 16 weeks in this study favored the larger diameter branch within the grafts (tibial branch for sciatic grafts, motor branch for femoral grafts) rather than which distal nerve was sutured to the graft branch. Higher CMAP amplitudes were observed for plantar musculature innervated via the tibial branches within sciatic grafts compared to plantar musculature innervated via peroneal branches within grafts (Figure 4A). In femoral grafts, greater CMAP amplitudes were observed at 16 weeks for plantar musculature innervated via the motor branches within the femoral grafts (Figure 4B). CMAP amplitudes had, to some extent, equalized between branches at 36 weeks but some differences remained. For example, CMAP amplitudes for plantar musculature innervated through the tibial nerve were greater in the femoral graft group when innervated through the motor branch within the graft compared to the saphenous.

Nerve morphometry showed that the cross-sectional area of smaller branches within grafts increased in size by the 36-week endpoint. For example, the peroneal branch of the sciatic nerve had a cross sectional area of 0.13 mm² in uninjured nerves but had increased in size to 0.24 mm² after being sutured to the tibial stump for 36 weeks. Axonal counts showed that the number of regenerated axons at the 36-week endpoint was in some cases greater than the number of axons present within uninjured nerves. For example, 1291 axons were observed on average in the peroneal branch of uninjured animals. Within the graft segments connected to the distal peroneal branch, there were 1743 within the peroneal branch of the graft connected to the distal peroneal nerve but 2398 average axons within the tibial branch of grafts connected to the distal peroneal branch. Despite the higher axon numbers, CMAP amplitudes at endpoint were smaller than those of uninjured peroneal nerves. This may be due to differences in the diameter of axons in the two conditions (9.2 μm for uninjured, 3.8 μm for regenerated) and the potential for collateralization to have occurred during the regenerative process (Stein and Pearson, 1971; Redett et al., 2005). Lastly, retrograde labeling showed no significant differences in any group, despite the femoral graft having a dedicated sensory and motor branch.

Conclusions

The branching organization of the PNS is such that it would be uncommon to encounter an ablation of 1 cm or longer that did not include at least one branch point. It is therefore likely that the concept of the simple (non-branching) linear ablation PNI is over-represented. The focus on development of bridging devices for linear segmental PNIs is understandable given the poor, non-restorative outcomes that persist for these defects, but additional consideration should be to branching PNIs given the actual anatomy of the PNS.

Several changes to study design and description are needed to better understand regeneration and recovery after branching PNIs. The first being that more precise descriptions of the branching pattern of the injuries and the specific sites of anastomoses of bridging materials are needed. Many case reports seemed likely to have addressed branching injuries due to the location and length of the injuries and bridging materials used, but these details were not specified and therefore such reports were excluded from consideration in this review. The inability to conclusively determine the branching anatomy of PNIs and treatment strategies based on the descriptions provided in clinical reports is a potential limitation of this review.

To improve repair of ablation PNIs that include branch points, it is also necessary to consistently quantify the extent of regeneration down each of the branches distal to the ablated branch point. House-Brackmann scoring for facial nerve function and other types of behavioral scoring are relevant to patient outcomes, but functional redundancy in reinnervated sensorimotor tissues may obscure the extent that regeneration occurred for different branches after ablation of a branched segment of nerve. Conducting branch specific assessments, such as monofilament assays of sensory perception or electrophysiology, would be informative additions to gross behavioral function. Non-invasive imaging techniques such as magnetic resonance imaging are often helpful for the identification of PNIs but are rarely used to assess recovery in patients (Md Swanger et al., 2010). Some methods of magnetic resonance imaging can resolve nerve regeneration of even very small (100 µm) bundles that correspond to histological and functional indications of regeneration in animal models (Bendszus et al., 2004; Wanner et al., 2019). Such imaging would also resolve the volume of individual muscle groups.

Assessing regeneration down all distal branches is more easily accomplished in animal models. Nerve morphology obtained from cross sections generate a wealth of informative data such as axon number, % myelination, G-ratio, axon density and overall nerve morphology. Morphology should be obtained and quantified from all branches affected by the ablation. Retrograde labeling can be conducted in a manner that preserves nerve morphology and gives useful information on axonal pathfinding (Shehab and Hughes, 2011; Zhou et al., 2015). Electrophysiological measurements, such as action potentials conducted across lesion sites and CMAPs for all branches, are informative and complementary additions to behavioral outcomes.

Control groups that enable comparison across multiple animal studies are a necessary addition for branched ablations. The lack of consistent and validated strategies (i.e., clinical predicates) for the regeneration of branched PNIs means that autografting is the only and best current option. Autografts may be cabled sensory grafts when feasible or could be the same branched segment of nerve removed to create the branched defect. While the latter is not clinically realistic, it would provide a reproducible basis of comparison to what may be considered the best possible regeneration given the injury. Studies with branched autografts of up to 2.5 cm of the sciatic nerve at the peroneal/tibial branch point in rats show robust and reliable regeneration down both branches (Santos Roballo et al., 2019; Allgood et al., 2022).

Lastly, more comprehensive anatomical characterization of fascicular mapping within main nerve trunks and into distal branches is needed. Precise fascicular alignment across sites of anastomosis in non-branching injuries improves outcomes by presumably maximizing the reinnervation of target tissues by fibers that originally innervated them (Brushart et al., 1983). Fascicular tracing within the main nerve trunks and through the physical branch points will determine how reproducible this organization is within diverse patient populations. Such data may facilitate strategies to maximize fascicular continuity across ablations of branched segments, such as the cabling of autografts from the proximal stump (Figure 3). For example, cabling autografts from the proximal stump of an ablated femoral nerve branch point may lead to better outcomes if the autograft cable connected to the distal stump of the sartorius nerve was proximally sutured to the anterolateral aspect of proximal stump – and likewise for other distal branches. Significant variability in pre-branch point fascicular organization might reduce the efficacy of this strategy.

Additional file: Open peer review report 1 ^{(79.2KB, pdf)} .