Sensory regeneration in dorsal root avulsion.

Brachial as well as lumbosacral plexus avulsion injuries are usually caused by high kinetic traumas, such as car-pedestrian, car and motorcycle accidents or falls from great heights. Traction forces affecting the head and shoulders or extremities pull the spinal nerve sleeves away from the spinal cord and rupture the postganglionic spinal root from the cord. In so called central avulsion injuries, the spinal root is avulsed at the interface between the central and peripheral nervous system (CNS and PNS). This results not only in the disconnection of the root from the cord but also in a longitudinal spinal cord injury. The complexity of the injury leads to degeneration of the spinal root and a marked inflammatory response of the spinal cord followed by the formation of a glial scar (Kachramanoglou et al., 2011). Over the years, a multitude of studies used disruptions of the dorsal root to study regeneration of sensory axons across the CNS-PNS interface. Typically, sensory axons regenerate readily through the dorsal root and dorsal rootlets but are arrested at the zone of transition between PNS and CNS. This area is usually referred to as the dorsal root transitional zone (DRTZ) or dorsal root entry zone (DREZ). The DRTZ is characterized by CNS astrocytic tissue extending into the central part of the rootlet while its periphery is formed by Schwann cell sheets of the PNS. After injury to the dorsal root, the DRTZ undergoes gliosis resulting in the extension of astrocytic tissue further into the rootlet and the formation of a glial scar (Carlstedt, 2008). The two commonly used models to analyze sensory axon regeneration across the DRTZ are the dorsal root crush and dorsal root rhizotomy (DRR) model. In dorsal root crush, the root is forcefully squeezed causing the disruption of nerve fibers without interrupting the endoneurial tube. In DRR, the root is completely transected using micro-scissors. In both models, the CNS-PNS interface is left untouched during the procedure. Using the dorsal root crush model, several attempts to overcome the axonal growth inhibiting environment present at the DRTZ succeed to regenerate sensory fibers. Enzymatic digestion of growth inhibiting chrondroitin sulfate proteoglycans (CSPGs) using bacterial chondroitinase ABC supports sensory axon ingrowth, but only when it is combined with growth promoting treatments (Steinmetz et al., 2005). Also infusion with blocking agents aiming at the downstream targets of myelin associated inhibitory protein Nogo, myelin-associated glycoprotein (MAG) and oligodendrocyte myelin glycoprotein (OMgp) lead to the regeneration of myelinated axons (Harvey et al., 2009). Other even more successful approaches use neurotrophic factors to stimulate axonal outgrowth pathways. Intrathecal delivery of nerve growth factor (NGF), neurotrophin-3 or glial derived neurotrophic factor (GDNF), systemic administration of the GDNF family member artemin or viral expression of NGF or fibroblast growth factor-2 induce extension of peptidergic and/or non-peptidergic sensory axons across the site of injury and allow regrowth of sensory axons into the dorsal horn. So far, only systemic delivery of artemin achieves topographically correct projections of both peptidergic and non-peptidergic sensory fibers into the dorsal horn (Smith et al., 2012). Taken together, the dorsal root crush model provides an excellent platform to identify molecules that promote or inhibit sensory regeneration through the reactive DRTZ. DRR provides an even greater challenge for sensory regeneration due to the complete transection of the root, leaving not even the nerve sheets intact. Using the DRR model, ingrowth of sensory axons into the dorsal horn was first achieved after injection of olfactory ensheathing glia into the DRTZ and dorsal horn and successive micro-suturing of the dorsal root to the cord (Ramón-Cueto and Nieto-Sampedro, 1994). Further developing this approach, olfactory ensheathing cells (OEC) applied to the cut surfaces of dorsal root and spinal cord followed by the application of the tissue adhesive fibrin glue result in the entry of sensory axons into the spinal cord. Interestingly, OECs interact with both CNS astrocytes and Schwann cells to form a growth permissive tissue bridge at the PNS-CNS interface (Li et al., 2004). Partial recovery of sensory and motor functions after DRR was shown after the application of a fibrin sealant alone and was further improved when sealant was applied together with mononuclear cells (Benitez et al., 2014). In conclusion, sensory regeneration after DRR was most successful when cell transplantation was combined with reattachment of the root. Recently, a new model to study dorsal root injury was introduced. The dorsal root avulsion (DRA) model is characterized by the surgical pulling of individual dorsal roots away from the spinal cord until the complete rupture of the root from the cord. This procedure causes the disruption of the dorsal root and contributing rootlets, the complete disruption of the DRTZ and injury to the dorsal column and horn along the spinal cord segment connected to the avulsed root (Figure 1A). DRA results in a rapid invasion of neutrophils into the dorsal horn followed by a macrophage and microglial response and extensive astrogliosis. All of these events are markedly elevated and prolonged compared to DRR and are not confined to the ipsilateral side. Recovery of vascularization occurs over the first month following DRR but is absent after DRA. Both DRR and DRA result in a loss of spinal cord neurons, but only in DRA a second wave of neurodegeneration occurs two weeks after the injury. Taken together, DRA leads to extensive spinal cord trauma and follows a chronic progression over the first month (Chew et al., 2011). Its close resemblance to the events occurring after central avulsion injuries in patients renders it the ideal model to study sensory regeneration in a clinically relevant perspective. We adapted the DRA model to study sensory regeneration in this unique setting. These attempts proved to be especially challenging due to the varying degree of damage to the traumatized dorsal horn, rendering it of great importance to develop ways to create root avulsions in a reproducible manner. In all cases, DRA led to a complete disruption of the DRTZ and extensive glial scarring at the site of injury. DRA was performed along the L3–5 segment of the lumbar spinal cord disrupting the dorsal roots that contain the main contribution to the sciatic nerve in mice.