Spinal root avulsion: an excellent model for studying motoneuron degeneration and regeneration after severe axonal injury

Carolin Ruven, Tak-Kwong Chan, Wutian Wu,

1 Department of Anatomy, LKS Faculty of Medicine, The University of Hong Kong, Hong Kong Special Administrative Region, China; 2 GHM Institute of CNS regeneration, Jinan University and The University of Hong Kong, Guangzhou, Guangdong Province, China

Spinal root avulsion: an excellent model for studying motoneuron degeneration and regeneration after severe axonal injury

Carolin Ruven1, Tak-Kwong Chan1, Wutian Wu1,2

1 Department of Anatomy, LKS Faculty of Medicine, The University of Hong Kong, Hong Kong Special Administrative Region, China; 2 GHM Institute of CNS regeneration, Jinan University and The University of Hong Kong, Guangzhou, Guangdong Province, China

Spinal root avulsion is an excellent model for studying the response of motoneurons to severe injury to their axons (Koliatsos et al., 1994). In this model (‘Avulsion Model’), spinal roots are torn off from spinal cord without removing the vertebra at different levels of spinal segments, usually at cervical and lumbar segments. Step-by-step procedures are described in detail elsewhere (Chu and Wu, 2009). The Avulsion Model resembles very well brachial plexus injuries in human beings. Around 70% of severe brachial plexus injuries in human involved avulsion of one or more roots (Narakas, 1985) and the main causes of traumatic brachial plexus injuries were motor vehicle accidents, sport injuries and difficult deliveries (Terzis et al., 2001). The Avulsion Model involves injury to both central nervous system (CNS) and peripheral nervous system (PNS) while nerve axotomy, transection and crush injuries only involve PNS.

The avulsion model provides an opportunity to study the phenomena of neuronal death and survival after spinal cord injuries. After avulsion injuies in rat, massive death of motoneurons occurs (Wu, 1993; Wu and Li, 1993). Motoneurons in the injured spinal cord segment begin to die one week after injury and 70% of the motoneurons degenerate by 3 weeks. By 6 weeks, almost all motoneurons degenerate (Li et al., 1995). In contrast, distal axotomy of spinal nerves does not cause any motoneuron death due to the presence of the remaining part of peripheral nerves. Studies have shown that at least 4 mm of remaining peripheral nerve is needed to avoid degeneration of a motoneuron (Gu et al., 1997). In the Avulsion Model, spinal roots are pulled away from spinal cord so that nerve roots are totally removed and cell bodies in the spinal cord do not get any external support from peripheral nerve (Wu, 1993; Koliatsos et al., 1994). It has been shown that glial cell derived neurotrophic factor (GDNF) and brain derived neurotrophic factor (BDNF) from Schwann cells in peripheral nerves are the factors that keep the motoneurons in the spinal cord from dying (Li et al., 1995; Novikov et al., 1995; Chai et al., 1999; Wu et al., 2003). It is known that the changes within a cell have similar characteristics in both necrotic and apoptotic deaths (Li et al., 1998), but the exact mechanisms of how the motoneurons die are still unknown. Avulsion injury also triggers the in fl ammation response that brings macrophages and microglia to the lesion site (Koliatsos et al., 1994; Yuan et al., 2004). During the process of degeneration, motoneurons undergo many biochemical and structural changes (Li et al., 1998; Yang et al., 2006). Expression of nitricoxide synthase (NOS) is highly upregulated after an injury suggesting the important role of NOS in the degeneration of motoneurons (Wu, 1993). Expression of NOS can be inhibited by neurotrophic factors GDNF and BDNF that are produced by Schwann cells in peripheral nerve and that have shown the ability to prevent the degeneration of motoneurons (Novikov et al., 1995; Wu et al., 1995; Wu et al., 2003). These fi ndings help us to understand and learn the mechanisms of cell deaths in CNS.

The rapid loss of motoneurons in the Avulsion Model also gives us an opportunity to test different methods of enhancing the survival and regeneration of motoneurons. As spinal roots are pulled off only from one side of spinal cord leaving the other side intact, the number of motoneurons in the contralateral side of spinal cord can serve as a control (Wu, 1993). Re-implantation of the avulsed ventral root was shown to be most effective in preventing motoneuronal deaths but the delay of surgery causes makes implantation technically very dif fi cult due to retraction of the nerve stum (Carlstedt et al., 1993; Hallin et al., 1999; Carlstedt et al., 2000; Blits et al., 2004; Eggers et al., 2010; Carlstedt and Hayton, 2012; Su et al., 2013). An alternative method is to implant a peripheral nerve graft that could enhance the survival of motoneurons, axonal regeneration and functional recovery (Wu et al., 1994; Wu, 1996; Su et al., 2013). Survival and regeneration of motoneurons in the injury site is promoted by GDNF and BDNF produced by Schwann cells in the nerve graft (Frostick et al., 1998). These neurotrophic factors can be used alone as therapeutic agents because they can be easily applied on the surface of spinal cord close to the ventral root of the avulsion site. GDNF and BDNF are suggested to prevent motoneuronal death by inhibiting the expression of NOS in injured motoneurons (Novikov et al., 1995; Wu et al., 1995; Wu et al., 2003).

Besides studying neuronal death and survival, the Avulsion model is also good for studying axonal regeneration. Since spinal roots are torn off from spinal cord, the cell bodies of motoneurons are no longer connected with their axons. Target muscles lose innervation from motoneurons when injury is left unattended. To avoid this, we have to fi nd possible ways to promote axonal regeneration. For functional recovery, regenerated axons have to myelinate and reach the target muscles. Axonal regeneration can be promoted by re-implantating the avulsed ventral root, or transplanting peripheral nerve graft or conduit (Carlstedt et al., 1993; Wu et al., 1994; Wu, 1996; Gu et al., 2004; Gu et al., 2005; Eggers et al., 2010; Su et al., 2013; Zhan et al., 2013). Brachial plexus avulsion model in rats is convenient for functional recovery studies because of the short distance that axons have to regrow. Therefore, axonal regeneration under different conditions can be tested with relatively short observation time.

In rats, brachial plexus root avulsion results in progressive atrophy of forelimb muscles due to the loss of innervation from motoneurons. In the Avulsion Model, both forelimb and hind limb muscles can be observed for atrophy when avulsion is made in the cervical spinal segment, while atrophy only occurs in hind limb muscles when avulsion is made in the lumbar segment. Similarly, traumatic injuries to human spinal cord often results in the paralysis of muscles innervated by the nerve roots at or below the lesion site. So, this model gives us an insight into processes occurring in traumatic muscle atrophy and diseases like spinal muscular atrophy (SMA). Briefly, muscle atrophy involves the reduction of muscle fi ber size and muscle weight, increased amount of connective and fat tissue and overall weakness of muscles (Grimby et al., 1976; Scelsi et al., 1982; Castroet al., 1999; Round et al., 2003). So far, transplantation of neural progenitor cells or other pluripotent cells into peripheral nerve seems to be a promising strategy to reduce muscle atrophy (Erb et al., 1993; Thomas et al., 2000; Su et al., 2012; Su et al., 2013).

In summary, the Avulsion Model will allow us to discover the mechanisms of motoneuronal death and survival that may in turn enhance our understanding about the pathological processes occurring in neurodegenerative diseases such as Alzheimer, Parkinson, and Huntington’s disease.

Blits B, Carlstedt TP, Ruitenberg MJ, de Winter F, Hermens WT, Dijkhuizen PA, Claasens JW, Eggers R, van der Sluis R, Tenenbaum L, Boer GJ, Verhaagen J (2004) Rescue and sprouting of motoneurons following ventral root avulsion and reimplantation combined with intraspinal adeno-associated viral vectormediated expression of glial cell line-derived neurotrophic factor or brain-derived neurotrophic factor. Exp Neurol 189:303-316.

Carlstedt T, Aldskogius H, Hallin RG, Nilsson-Remahl I (1993) Novel surgical strategies to correct neural de fi cits following experimental spinal nerve root lesions. Brain Res Bull 30:447-451.

Carlstedt T, Anand P, Hallin R, Misra PV, Norén G, Seferlis T (2000) Spinal nerve root repair and reimplantation of avulsed ventral roots into the spinal cord after brachial plexus injury. J Neurosurg Spine 93:237-247.

Carlstedt T, Havton L (2012) The longitudinal spinal cord injury: Lessons from intraspinal plexus, cauda equina and medullary conus lesions. Handb Clin Neurol 109:337-354.

Castro MJ, Apple DF Jr, Staron RS, Campos GE, Dudley GA (1999) In fl uence of complete spinal cord injury on skeletal muscle within 6 mo of injury. J Appl Physiol 86:350-358.

Chai H, Wu W, So KF, Prevette DM, Oppenheim RW (1999) Longterm effects of a single dose of brain-derived neurotrophic factor on motoneuron survival following spinal root avulsion in the adult rat. Neurosci Lett 274:147-150.

Chu TH, Wu W (2009) Spinal root avulsion and repair model. In: Animal models of acute neurological injuries (Chen J, Xu XM, Xu ZC, Zhang J, Ed), pp 487-496. New York: Humana Press.

Eggers R, Tannemaat MR, Ehlert EM, Verhaagen J (2010) A spatio-temporal analysis of motoneuron survival, axonal regeneration and neurotrophic factor expression after lumbar ventral root avulsion and implantation. Exp Neurol 223:207-220.

Erb DE, Mora RJ, Bunge RP (1993) Reinnervation of adult rat gastrocnemius muscle by embryonic motoneurons transplanted into the axotomized tibial nerve. Exp Neurol 124:372-376.

Frostick SP, Yin Q, Kemp GJ (1998) Schwann cells, neurotrophic factors, and peripheral nerve regeneration. Microsurgery 18:397-405.

Grimby G, Broberg C, Krotkiewska I, Krotkiewski M (1976) Muscle fi ber composition in patients with traumatic cord lesion. Scand J Rehabil Med 8:37-42.

Gu HY, Chai H, Zhang JY, Yao ZB, Zhou LH, Wong WM, Bruce I, Wu W (2004) Survival, regeneration and functional recovery of motoneurons in adult rats by reimplantation of ventral root following spinal root avulsion. Eur J Neurosci 19:2123-2131.

Gu HY, Chai H, Zhang JY, Yao ZB, Zhou LH, Wong WM, Bruce IC, Wu WT (2005) Survival, regeneration and functional recovery of motoneurons after delayed reimplantation of avulsed spinal root in adult rat. Exp Neurol 192:89-99.

Gu Y, Spasic Z, Wu W (1997) The effects of remaining axons on motoneuron survival and NOS expression following axotomy in the adult rat. Dev Neurosci 19:255-259.

Hallin RG, Carlstedt T, Nilsson-Remahl I, Risling M (1999) Spinal cord implantation of avulsed ventral roots in primates; correlation between restored motor function and morphology. Exp Brain Res 124:304-310.

Koliatsos VE, Price WL, Pardo CA, Price DL (1994) Ventral root avulsion: an experimental model of death of adult motor neurons. J Comp Neurol 342:35-44.

Li L, Houenou LJ, Wu W, Lei M, Prevette DM, Oppenheim RW (1998) Characterization of spinal motoneuron degeneration following different types of peripheral nerve injury in neonatal and adult mice. J Comp Neurol 396:158-168.

Li L, Wu W, Lin LF, Lei M, Oppenheim RW, Houenou LJ (1995) Rescue of adult mouse motoneurons from injury-induced cell death by a glial cell line-derived neurotrophic factor. Proc Natl Acad Sci U S A 92:9771-9775.

Narakas AO (1985) The treatment of brachial plexus injuries. Int Orthop 9:29-36.

Novikov L, Novikova L, Kellerth JO (1995) Brain-derived neurotrophic factor promotes survival and blocks nitric oxide synthase expression in adult rat spinal motoneurons after ventral root avulsion. Neurosci Lett. 200:45-48.

Round JM, Barr FM, Moffat B, Jones DA (1993) Fibre areas and histochemical fi bre types in the quadriceps muscle of paraplegic subjects. J Neurol Sci 116:207-211.

Scelsi R, Marchetti C, Poggi P, Lotta S, Lommi G (1982) Muscle fi ber type morphology and distribution in paraplegic patients with traumatic cord lesion. Histochemical and ultrastructural aspects of rectus femoris muscle. Acta Neuropathol 57:243-248.

Su H, Yuan Q, Qin D, Yang X, Wong WM, So KF, Wu W (2013) Ventral root re-implantation is better than peripheral nerve transplantation for motoneuron survival and regeneration after spinal root avulsion injury. BMC Surg 13:21.

Su H, Zhang W, Yang X, Qin D, Sang Y, Wu C, Wong WM, Yuan Q, So KF, Wu W (2012) Neural progenitor cells generate motoneuron-like cells to form functional connections with target muscles after transplantation into the musculocutaneous nerve. Cell Transplant 21:2651-2663.

Terzis JK, Vekris MD, Soucacos PN. (2001) Brachial plexus root avulsions. World J Surg 25:1049-1061.

Thomas CK, Erb DE, Grumbles RM, Bunge RP (2000) Embryonic cord transplants in peripheral nerve restore skeletal muscle function. J Neurophysiol 84:591-595.

Wu W (1993) Expression of nitric oxide synthase (NOS) in injured CNS neurons as shown by NADPH-diaphorase histochemistry. Exp Neurol 120:153-159.

Wu W (1996) Potential roles of gene expression changes in adult rat spinal motoneurons following axonal injury: a comparison among c-jun, low af fi nity nerve growth factor receptor (LNGFR), and nitric oxide synthase (NOS). Exp Neurol 141:190-200.

Wu W, Han K, Li L, Schinco FP (1994) Implantation of PNS graft inhibits the induction of neuronal nitric oxide synthase and enhances the survival of spinal motoneurons following root avulsion. Exp Neurol 129:335-339.

Wu W, Li L (1993) Inhibition of nitric oxide synthase reduces motoneuron death due to spinal root avulsion. Neurosci Lett 153:121-124.

Wu W, Li L, Penix JO, Gu Y, Liu H, Prevette DM, Oppenheim RW (1995) GDNF and BDNF inhibit the induction of NOS and prevent the death of adult rat spinal motoneurons following root avulsion. Soc Neurosci Abstr. 21:279.

Wu W, Li L, Yick LW, Chai H, Xie Y, Yang Y, Prevette DM, Oppenheim RW (2003) GDNF and BDNF alter the expression of neuronal NOS, c-Jun, and p75 and prevent motoneuron death following spinal root avulsion in adult rats. J Neurotrauma 20:603-612.

Yang Y, Xie Y, Chai H, Fan M, Liu S, Liu H, Bruce I, Wu W (2006) Microarray analysis of gene expression patterns in adult spinal motoneurons after different types of axonal injuries. Brain Res 1075:1-12.

Yuan Q, Xie Y, So KF, Wu W (2003) In fl ammatory response associated with axonal injury to spinal motoneurons in newborn rats. Dev Neurosci 25:72-78.

Zhan X, Gao M, Jiang Y, Zhang W, Wong WM, Yuan Q, Su H, Kang X, Dai X, Zhang W, Guo J, Wu W (2013) Nano fi ber scaffolds facilitate functional regeneration of peripheral nerve injury. Nanomedicine 9:305-315.

Copyedited by Zheng BH, Snow DM, Li CH, Song LP, Liu WJ, Zhao M

Wutian Wu, M.D., Professor, Department of Anatomy, LKS Faculty of Medicine, The University of Hong Kong, 21 Sassoon Road, Hong Kong Special Administrative Region, China, wtwu@hku.hk.

10.4103/1673-5374.125338 http://www.nrronline.org/

Accepted: 2014-01-02