Root Avulsion In Adult Rats Research Articles

Human dental pulp stem cell monolayer and spheroid therapy after spinal motor root avulsion in adult rats

Spinal cord injuries result in severe neurological deficits and neuronal loss, with poor functional recovery. Mesenchymal stem cells have shown promising results; therefore the present objective of this work was to compare motor recovery after treatment with human dental pulp stem cells (hDPSC) cultivated in monolayer (2D) or as spheroids (3D), following avulsion and reimplantation of spinal motor roots in adult rats. Thus, 72 adult female Lewis rats were divided into 4 groups: avulsion (AV); avulsion followed by reimplantation (AR); avulsion associated with reimplant and 2D cell therapy (AR + 2D), and avulsion associated with reimplant and 3D cell therapy (AR + 3D). The application of the cells in 2D and 3D was performed by microsurgery, with subsequent functional assessment using a walking track test (Catwalk system), immunohistochemistry, neuronal survival, and qRT-PCR in 1-, 4-, and 12-weeks post-injury. The animals in the AR + 2D and AR + 3D groups showed the highest neuronal survival rates, and immunofluorescence revealed downregulation of GFAP, and Iba-1, with preservation of synaptophysin, indicating a reduction in glial reactivity, combined with the maintenance of pre-synaptic inputs. There was an increase in anti-inflammatory (IL-4, TGFβ) and a reduction of pro-inflammatory factors (IL-6, TNFα) in animals treated with reimplantation and hDPSC. As for the functional recovery, in all analyzed parameters, the AR + 2D group performed better and was superior to the avulsion alone. Overall, our results indicate that the 2D and 3D cell therapy approaches provide successful immunomodulation and motor recovery, consistent with advanced therapies after spinal cord injury.

PLGA-PEG-PLGA hydrogel with NEP1-40 promotes the functional recovery of brachial plexus root avulsion in adult rats.

Adult brachial plexus root avulsion can cause serious damage to nerve tissue and impair axonal regeneration, making the recovery of nerve function difficult. Nogo-A extracellular peptide residues 1-40 (NEP1-40) promote axonal regeneration by inhibiting the Nogo-66 receptor (NgR1), and poly (D, L-lactide-co-glycolide)-poly (ethylene glycol)-poly (D, L-lactide-co-glycolide) (PLGA-PEG-PLGA) hydrogel can be used to fill in tissue defects and concurrently function to sustain the release of NEP1-40. In this study, we established an adult rat model of brachial plexus nerve root avulsion injury and conducted nerve root replantation. PLGA-PEG-PLGA hydrogel combined with NEP1-40 was used to promote nerve regeneration and functional recovery in this rat model. Our results demonstrated that functional recovery was enhanced, and the survival rate of spinal anterior horn motoneurons was higher in rats that received a combination of PLGA-PEG-PLGA hydrogel and NEP1-40 than in those receiving other treatments. The combined therapy also significantly increased the number of fluorescent retrogradely labeled neurons, muscle fiber diameter, and motor endplate area of the biceps brachii. In conclusion, this study demonstrates that the effects of PLGA-PEG-PLGA hydrogel combined with NEP1-40 are superior to those of other therapies used to treat brachial plexus nerve root avulsion injury. Therefore, future studies should investigate the potential of PLGA-PEG-PLGA hydrogel as a primary treatment for brachial plexus root avulsion.

Down‐regulation of apurinic/apyrimidinic endonuclease 1 (APE1) in spinal motor neurones under oxidative stress

Apurinic/apyrimidinic endonuclease 1 (APE1) is an intermediate enzyme in base excision repair which is important for removing damaged nucleotides under normal and pathological conditions. Accumulation of damaged bases causes genome instability and jeopardizes cell survival. Our study is to examine APE1 regulation under oxidative stress in spinal motor neurones which are vulnerable to oxidative insult. We challenged the motor neurone-like cell line NSC-34 with hydrogen peroxide and delineated APE1 function by applying various inhibitors. We also examined the expression of APE1 in spinal motor neurones after spinal root avulsion in adult rats. We showed that hydrogen peroxide induced APE1 down-regulation and cell death in a differentiated motor neurone-like cell line. Inhibiting the two functional domains of APE1, namely, DNA repair and redox domains potentiated hydrogen peroxide induced cell death. We further showed that p53 phosphorylation early after hydrogen peroxide treatment might contribute to the down-regulation of APE1. Our in vivo results similarly showed that APE1 was down-regulated after root avulsion injury in spinal motor neurones. Delay of motor neurone death suggested that APE1 might not cause immediate cell death but render motor neurones vulnerable to further oxidative insults. We conclude that spinal motor neurones down-regulate APE1 upon oxidative stress. This property renders motor neurones susceptible to continuous challenge of oxidative stress in pathological conditions.

Intrathecal application of short interfering RNA knocks down c-jun expression and augments spinal motoneuron death after root avulsion in adult rats

The immediate-early gene, c-jun, is expressed in spinal motoneurons after spinal root avulsion. The expression of c-jun was suggested to be necessary for motoneuron survival and regeneration after avulsion. In the present study, a small interfering RNA (siRNA) was delivered intrathecally to the injured spinal segments immediately after root avulsion in rats to knock down expression of the c-jun gene in injured spinal motoneurons so as to explore the role of c-jun in the motoneurons in vivo. Our results showed that the siRNA not only inhibited the expression of both c-jun mRNA and protein but also augmented the death of injured motoneurons at day 14 post-injury. These findings indicated that induction of c-jun gene expression plays a pivotal role in the survival of injured motoneurons. Meanwhile, these results suggest that siRNAs applied intrathecally can effectively mediate the expression of the c-jun gene in injured motoneurons.

Induction of c-Jun phosphorylation in spinal motoneurons in neonatal and adult rats following axonal injury

This study aims to address if phosphorylation of the transcription factor c-Jun is associated with lesion-induced death of spinal motoneurons, and if this cellular response is modulated by glial-cell-line-derived neurotrophic factor (GDNF). We found that after both distal axotomy and root avulsion, spinal motoneurons in neonatal rats expressed phosphorylated c-Jun (p-c-Jun) and almost all injured motoneurons in these animals died. Similarly, root avulsion in adult rats also induced p-c-Jun expression that preceded the loss of motoneurons. In contrast, neither motoneuron death nor p-c-Jun induction was found after distal axotomy of spinal nerves in adult rats. Application of GDNF after distal axotomy in the neonatal model prevented motoneuron death but did not alter the expression of p-c-Jun in the surviving motoneurons. We conclude that c-Jun phosphorylation correlates with the cellular events leading to motoneuron death and that its expression cannot be modulated by GDNF. We further showed that expression of p-c-Jun was not correlated with the expression of growth-associated protein-43 (GAP-43), whose expression was closely correlated both temporally and spatially with periods of axonal outgrowth, suggesting that p-c-Jun may not be related with axonal regeneration of injured motoneurons.

Implantation of Neurotrophic Factor-Treated Sensory Nerve Graft Enhances Survival and Axonal Regeneration of Motoneurons After Spinal Root Avulsion

We previously showed that motor nerves are superior to sensory nerves in promoting axon regeneration after spinal root avulsion. It is, however, impractical to use motor nerves as grafts. One potential approach to enhancing axonal regeneration using sensory nerves is to deliver trophic factors to the graft. Here, we examined the regulation of receptors for brain-derived neurotrophic factor, glial cell line-derived neurotrophic factor, ciliary neurotrophic factor, and pleiotrophin after root avulsion in adult rats. We then tested their survival-promoting and neuroregenerative effects on spinal motoneurons. The results showed that receptors for brain-derived neurotrophic factor and glial cell line-derived neurotrophic factor were upregulated and that these trophic factors promoted survival and axonal regeneration of motoneurons when they were injected into the sensory nerve graft before implantation. In contrast, receptors for ciliary neurotrophic factor and pleiotrophin were downregulated after avulsion. Ciliary neurotrophic factor did not promote survival and axonal regeneration, whereas pleiotrophin promoted axonal regeneration but not survival of injured spinal motoneurons. Our results suggest that infusion of trophic factors into sensory nerve grafts promote motoneuron survival and axonal regeneration. The technique is technically easy and is, therefore, potentially clinically applicable.

Motor nerve graft is better than sensory nerve graft for survival and regeneration of motoneurons after spinal root avulsion in adult rats

In the present study, we compared the effects of implanting peripheral sensory nerve and motor nerve on motoneuron survival and regeneration after spinal root avulsion in adult rats. Our results showed that 116% more motoneurons regenerated axons into the motor than the sensory nerve graft and 59% of motoneurons survived in the motor nerve-implanted group compared to 48% in the sensory nerve-implanted group. We demonstrated by real time PCR that levels of BDNF and GDNF mRNA were significantly higher in the motor than the sensory nerve five days after implantation into the spinal cord. This may account for the superiority of motor over sensory nerve in promoting motor axon regeneration and motoneuron survival. Lastly, we also showed that implanting two sensory nerves enhances motoneuron regeneration over implanting a single nerve.

Delayed implantation of a peripheral nerve graft reduces motoneuron survival but does not affect regeneration following spinal root avulsion in adult rats.

Adult spinal motoneurons can regenerate their axons into a peripheral nerve (PN) graft following root avulsion injury if the graft is implanted immediately after the lesion is induced. The present study was designed to determine how avulsed motoneurons respond to a PN graft if implantation takes place a few days to a few weeks later. Survival, regeneration, and gene expression changes of injured motoneurons after delayed PN graft implantation were studied. The survival rates of spinal motoneurons were 78%, 65%, 57%, or 53% if a PN graft was implanted immediately, 1, 2, or 3 weeks after root avulsion, respectively. Interestingly, most of the surviving motoneurons were able to regenerate their axons into the graft regardless of the delay. All regenerating motoneurons expressed p75, but not nNOS, while all motoneurons that failed to regenerate expressed nNOS, but not p75. p75 and nNOS may, therefore, be used as markers for success or failure to regenerate axons. In the group with immediate graft implantation, 85% of the surviving motoneurons extended axons into the PN graft, while in the groups in which implantation was delayed 1, 2, or 3 weeks, 84%, 82%, and 83% of the surviving motoneurons, respectively, were found to have regenerated into the grafts. These findings indicate that avulsed spinal motoneurons retain the ability to regenerate for at least 3 weeks, and perhaps for as long as they survive. Therefore, the delayed implantation of a PN graft after root avulsion may provide a continued conducive environment to support regeneration.

GDNF and BDNF alter the expression of neuronal NOS, c-Jun, and p75 and prevent motoneuron death following spinal root avulsion in adult rats.

In the present study, we examined the effects of glial cell line-derived neurotrophic factor (GDNF), brain-derived neurotrophic factor (BDNF), ciliary neurotrophic factor (CNTF), and insulin growth factor (IGF-1) on adult motoneuron survival following spinal root avulsion. The expression of neuronal nitric oxide synthase (nNOS), c-Jun, and the low-affinity neurotrophin receptor (P75) following treatment with these neurotrophic factors was also examined. In control animals, approximately 80% of spinal motoneurons were nNOS positive at 3 weeks following the lesion, whereas in GDNF or BDNF treated animals no nNOS positive motoneurons were found at the same time point. Following injury and treatment with GDNF and BDNF increased numbers of motoneurons were c-Jun and P75 positive. By 6 weeks following the lesion, only approximately 28% of motoneurons persisted in control animals whereas about 90% of motoneurons survived injury following treatment with either GDNF or BDNF. In contrast, CNTF and IGF-1 were ineffective in either inhibiting nNOS expression or preventing motoneuron death. Our results provide in vivo evidence that the survival of injured adult mammalian motoneurons can be promoted by specific neurotrophic factors, and that this effect is associated with inhibition of nNOS expression and up-regulation of c-Jun and P75 expression.

GM-1 ganglioside treatment reduces motoneuron death after ventral root avulsion in adult rats

After ventral root avulsion, a large percentage of the wounded spinal motoneurons die within 2 weeks. Neuronal death is preceded by a series of morphological and physiological changes probably as a result of both apoptotic and necrotic mechanisms. Associated with neuronal death, inflammation develops in the wounded area, resulting in additional neuronal loss as well as the degeneration of fibers from different tracts of the ventral and lateral funiculi. In the present study we tested the neuroprotective and local anti-inflammatory effects of monosialoganglioside (GM-1) after an initial 150 mg/kg body weight dose, followed by the daily administration of 100 mg/kg body weight for 2 weeks. The results showed a statistically significant enhancement of surviving motoneurons which showed good morphological preservation. Also, GM-1 treatment reduced by less then a half, the number of degenerating fibers into the ventral and lateral funiculi. Taken together, our results indicate that the administration of GM-1 in high doses during the critical period of motoneuron death after avulsion is neuroprotective, and diminishes local inflammation.