Co-expression of GAP-43 and nNOS in avulsed motoneurons and their potential role for motoneuron regeneration

doi:10.1016/j.niox.2010.07.006

Nitric Oxide

Volume 23, Issue 4, 15 December 2010, Pages 258-263

https://doi.org/10.1016/j.niox.2010.07.006 Get rights and content

Abstract

Neuronal nitric oxide synthase (nNOS) is induced after axonal injury. The role of induced nNOS in injured neurons is not well established. In the present study, we investigated the co-expression of nNOS with GAP-43 in spinal motoneurons following axonal injury. The role of induced nNOS was discussed and evaluated. In normal rats, spinal motoneurons do not express nNOS or GAP-43. Following spinal root avulsion, expression of nNOS and GAP-43 were induced and colocalized in avulsed motoneurons. Reimplantation of avulsed roots resulted in a remarkable decrease of GAP-43- and nNOS-IR in the soma of the injured motoneurons. A number of GAP-43-IR regenerating motor axons were found in the reimplanted nerve. In contrast, the nNOS-IR was absent in reimplanted nerve. These results suggest that expression of GAP-43 in avulsed motoneurons is related to axonal regeneration whereas nNOS is not.

Introduction

Nitric oxide (NO) is a free radical gas synthesized by nitric oxide synthase (NOS). Three main classes of NOS have been identified and visualized by the use of different antibodies: neuronal NOS (nNOS) in neurons, inducible NOS (iNOS) in macrophages, and endothelial NOS (eNOS) in endothelial cells. Previous studies have shown that NO is involved in neuronal degeneration in some studies [1], [2], [3], [4] but beneficial for regeneration in others [5], [6], [7]. The possible role of NO/NOS in growth and regeneration of injured neurons has been explored in various lesion experiments [8]. For example, axotomy produces an increase of nNOS-positive cells in the dorsal root ganglia (DRG) demonstrated by NADPH-diaphorase histochemistry [9], NOS immunocytochemistry [10], [11], and in situ hybridization [12]. Subsequently, the increased nNOS after peripheral nerve injury is transported toward the periphery, which indicates the involvement of NO/NOS in regeneration [13]. The involvement of NO/NOS in axonal regeneration is further supported by the evidence that nNOS colocalize with growth-associated phosphoprotein 43 (GAP-43) [8], a protein closely related with axon regeneration, in growth cone [14], [15], [16], [17], [18], [19].

In motoneurons, however, the induction of nNOS may differ depending on the nucleus studied [20], [21], the age of the experimental animals [22] and severity of axonal damage [23]. The role of the induced nNOS in lesioned motoneurons is also very controversial. On the one hand, it has been suggested that nNOS is toxic to the injured motoneurons. For example, nNOS is induced in facial motoneurons in adult or neonatal rats after facial nerve avulsion, which lead to motoneuron loss and furthermore administration of NOS inhibitors in avulsed rats reduces motoneuron death [24]; on the other hand, nNOS has been suggested to be beneficial to the axonal regeneration of the injured spinal motoneurons [5], [25]. The latter role for NO in injured motoneurons is supported by studies in lizard. In lizards, tail loss transects spinal nerves and the cut axons elongate in the regrowing tail [25]. This natural paradigm of robust regenerative response of injured spinal motoneurons involve nNOS induction in the axotomized motoneurons [25]. This induced nNOS is further found to be co-expressed with GAP-43 in motoneurons that innervate the regenerated tail [5].

Our previous studies have found that nNOS is induced in spinal motoneurons [20], [26], [27]. Recently, we have also found that GAP-43 is induced in avulsed motoneurons and its expression is related closely with regenerative capacity of avulsed motoneurons [28]. Co-expression of nNOS and GAP-43 in DRG and in axotomized lizard motoneurons encourages us to investigate whether nNOS and GAP-43 are co-expressed in adult rat motoneurons after root avulsion. Since motoneurons are able to regenerate their axons into the peripheral nerve if the avulsed ventral root is reimplanted [29], [30], [31], [32], in the present study we also examine whether the induced nNOS is co-localized with GAP-43 in regenerating axons.

Section snippets

Animals

Female adult Sprague–Dawley rats were used. All surgical interventions and subsequent care and treatment were approved by the Committee on the Use of Live Animals for Teaching and Research of the University of Hong Kong.

Spinal root avulsion

Anesthetized animals were placed on the surgical table and a dorsal laminectomy was carried out. The dura was opened and both the ventral root and dorsal root with the ganglion of the seven cervical spinal cord (C7) were selectively avulsed from the spinal cord by traction under

Expression of GAP-43 and nNOS in spinal motoneurons following root avulsion

In control normal animals, GAP-43-IR and nNOS-IR were absent in ventral horn of spinal cord (Fig. 1A and B, respectively), which were consistent with previous studies [26], [27], [28], [35]. Following spinal root avulsion, NOS-IR motoneurons began to be seen at 1 week and reached peak at 2 weeks post-lesion in the ventral horn (Table 1 and Fig. 1D). Similarly, GAP-43-IR could also be observed in the avulsed motoneurons (Fig. 1C). GAP-43 induction exhibited a time-course similar to that described

Discussion

The major findings of the present study are: (1) nNOS-IR induced in avulsed motoneurons is co-localized with GAP-43-IR, (2) GAP-43-IR was transported centrifugally to peripheral part of regenerating motor axons but not remained in the cell bodies of motoneurons following reimplantation of avulsed roots, (3) nNOS production was inhibited following reimplantation of avulsed roots.

Acknowledgments

This study was supported by HKU Spinal Cord Injury Foundation and grants from the University of Hong Kong and Hong Kong Research Grants Council (RGC).

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NADPH-diaphorase histochemistry in the rat revealed a maximum of 24% to 27% nitrergic motoneurons of the total pool after crushing, while in more severe injuries the proportion increased: 48–60% after transection and 80–98% after avulsion. This model has been further supported by more recent reports after XIIth nerve crushing and spinal root avulsion (Cheng et al., 2010; Yuan et al., 2010a; Wei et al., 2011). Upregulation of nNOS after motoneuron axotomy seems to be part of the program recapitulating a dedifferentiated phenotype of motoneurons, as transient expression of nNOS occurs in rat hypoglossal and spinal motoneurons during early postnatal development (Vazquez et al., 1999; Gao et al., 2008).
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Co-expression of GAP-43 and nNOS in avulsed motoneurons and their potential role for motoneuron regeneration

Abstract

Introduction

Section snippets

Animals

Spinal root avulsion

Expression of GAP-43 and nNOS in spinal motoneurons following root avulsion

Discussion

Acknowledgments

Neuroscience

Neurosci. Lett.

Brain Res.

Prog. Brain Res.

Prog. Brain Res.

Neurosci. Lett.

Neurosci. Lett.

Exp. Neurol.

Neuroscience

Exp. Neurol.

Exp. Neurol.

Prog. Neurobiol.

Trends Neurosci.

Exp. Neurol.

Exp. Neurol.

Neurosci. Lett.

Neuroscience

Brain Res.

Nitric oxide: role in neurotoxicity

Clin. Exp. Pharmacol. Physiol.

A novel neuroprotective agent with antioxidant and nitric oxide synthase inhibitory action

Free Radic. Res.

Neuroprotective effect of sigma(1)-receptor ligand 4-phenyl-1-(4-phenylbutyl) piperidine (PPBP) is linked to reduced neuronal nitric oxide production

Stroke

Neuroprotection via inhibition of nitric oxide synthase by bis(7)-tacrine

Neuroreport

NO-cGMP mediated galanin expression in NGF-deprived or axotomized sensory neurons

J. Neurochem.

Nitric oxide synthase inhibitor attenuates number of regenerating spinal motoneurons in adult rats

Neuroreport

Nitric oxide synthase and growth-associated protein are coexpressed in primary sensory neurons after peripheral injury

J. Comp. Neurol.