Artemin induced functional recovery and reinnervation after partial nerve injury

Abstract

Systemic artemin promotes regeneration of dorsal roots to the spinal cord after crush injury. However, it is unclear whether systemic artemin can also promote peripheral nerve regeneration, and functional recovery after partial lesions distal to the dorsal root ganglion (DRG) remains unknown. In the present investigation, male Sprague Dawley rats received axotomy, ligation, or crush of the L5 spinal nerve or sham surgery. Starting the day of injury, animals received intermittent subcutaneous artemin or vehicle across 2weeks. Sensory thresholds to tactile or thermal stimuli were monitored for 6weeks after injury. Immunohistochemical analyses of the DRG and nerve regeneration were performed at the 6-week time point. Artemin transiently reversed tactile and thermal hypersensitivity after axotomy, ligation, or crush injury. Thermal and tactile hypersensitivity reemerged within 1week of treatment termination. However, artemin-treated rats with nerve crush, but not axotomy or ligation, subsequently showed gradual return of sensory thresholds to preinjury baseline levels by 6weeks after injury. Artemin normalized labeling for NF200, IB4, and CGRP in nerve fibers distal to the crush injury, suggesting persistent normalization of nerve crush-induced neurochemical changes. Sciatic and intradermal administration of dextran or cholera toxin B distal to the crush injury site resulted in labeling of neuronal profiles in the L5 DRG, suggesting regeneration functional restoration of nonmyelinated and myelinated fibers across the injury site into cutaneous tissue. Artemin also diminished ATF3 and caspase 3 expression in the L5 DRG, suggesting persistent neuroprotective actions. A limited period of artemin treatment elicits disease modification by promoting sensory reinnervation of distal territories and restoring preinjury sensory thresholds.

Keywords: Artemin; Hyperalgesia; Peripheral nerve injury; Regeneration; Sensory neurons.

Figure 1

Systemic artemin promotes recovery of tactile and thermal hypersensitivity following peripheral nerve crush, axotomy or ligation. The rats received subcutaneous injections of 1mg/kg artemin on a schedule of MWF (arrows) or vehicle for two weeks. Artemin treated rats showed partial reversal of L₅ nerve-crush induced tactile (A) and thermal hypersensitivity (B) by the end of the first week of treatment. Tactile hypersensitivity returned by the first week following termination of artemin treatment, followed by a gradual recovery of sensory thresholds to pre-injury values across the remaining test period (A). Thermal hypersensitivity returned by the first week following termination of artemin treatment, followed by a gradual return of sensory thresholds towards pre-injury values in both vehicle and artemin treated rats (B). Artemin treated rats showed partial reversal of L₅ axotomy-induced tactile (C) and thermal (D) hypersensitivity by the end of the first week of treatment. Tactile and thermal hypersensitivity returned following termination of artemin treatment and persisted for the remainder of the testing period. Artemin treated rats showed partial reversal of L₅ ligation-induced tactile (E) and thermal (F) hypersensitivity by the end of the first week of treatment. Tactile and thermal hypersensitivity returned following termination of artemin treatment and persisted for the remainder of the testing period. sham-operated rats did not demonstrate any significant changes in tactile (G) or thermal (H) sensory thresholds either with artemin or vehicle treatment. Error bars indicate mean ± SEM. n = 8 per group. Asterisks indicate significance (P < 0.05) between artemin and vehicle treated groups.

Figure 2

Systemic artemin across 2 weeks immediately following injury restores neurochemical alterations in sensory fibers 6 weeks post- L₅ spinal nerve crush. Artemin treatment after nerve crush increased NF200, CGRP and IB4 labeled fibers observed in L₅ spinal nerve distal to crush region. Axotomy or nerve ligation of the L₅ spinal nerve greatly diminished NF200, CGRP and IB4 labeling in the spinal nerve distal to nerve injury. Labeling after L₅ ligation was sparse after artemin treatment, indicated by arrows, and labeling after axotomy was not affected by artemin treatment. Scale bar indicates 20 µm for all images.

Figure 3

Artemin blocks neurochemical signs in the DRG of nerve crush-induced neuropathy 6 weeks following injury. A. Nerve crush resulted in increased NPY labeling and decreased CGRP as well as IB4 labeling within the ipsilateral L₅ DRG. Artemin attenuated the nerve-crush induced increase in NPY and the decreased CGRP and IB4 labeling. B. The graph shows quantification of NPY, CGRP and IB4 labeled neuronal profiles in the ipsilateral L₅ DRG. The counts confirm that artemin ameliorated the nerve-crush induced increase in NPY and the decreased CGRP and IB4 labeling. Asterisks indicate significant differences (P < 0.05) compared to vehicle treatment after nerve crush. C. Nerve crush induced ATF3 labeled neuronal profiles were markedly reduced with artemin treatment. A similar decrease in nerve-crush induced caspase-3 labeled neuronal profiles was observed in artemin treated rats. Scale bar indicates 50 µm for all images. D. The graph shows quantification of ATF3 and caspase-3 labeled neuronal nuclei from counted sections confirming that artemin decreased the percentage of ATF3 and caspase-3 labeled neurons following nerve crush. Asterisks indicates significant differences (P < 0.05) compared to vehicle treatment after nerve crush.

Figure 3

Artemin blocks neurochemical signs in the DRG of nerve crush-induced neuropathy 6 weeks following injury. A. Nerve crush resulted in increased NPY labeling and decreased CGRP as well as IB4 labeling within the ipsilateral L₅ DRG. Artemin attenuated the nerve-crush induced increase in NPY and the decreased CGRP and IB4 labeling. B. The graph shows quantification of NPY, CGRP and IB4 labeled neuronal profiles in the ipsilateral L₅ DRG. The counts confirm that artemin ameliorated the nerve-crush induced increase in NPY and the decreased CGRP and IB4 labeling. Asterisks indicate significant differences (P < 0.05) compared to vehicle treatment after nerve crush. C. Nerve crush induced ATF3 labeled neuronal profiles were markedly reduced with artemin treatment. A similar decrease in nerve-crush induced caspase-3 labeled neuronal profiles was observed in artemin treated rats. Scale bar indicates 50 µm for all images. D. The graph shows quantification of ATF3 and caspase-3 labeled neuronal nuclei from counted sections confirming that artemin decreased the percentage of ATF3 and caspase-3 labeled neurons following nerve crush. Asterisks indicates significant differences (P < 0.05) compared to vehicle treatment after nerve crush.

Figure 4

Artemin promotes regeneration of non-myelinated and myelinated axons through nerve crush injury into sciatic nerve 6 weeks post L₅ spinal nerve crush. A. Artemin or vehicle treatment did not markedly change the number of cell bodies within the L₅ DRG showing retrograde labeling of dextran from sciatic nerve in sham-operated rats, corresponding to non-myelinated fibers. Artemin treatment increased dextran labeling in L₅ DRG of rats treated with nerve crush, but not in rats that had undergone axotomy or nerve ligation. Scale bar indicates 50 µm for all images. B. The graph shows quantification of dextran-labeled neurons from counted sections in percentages. Asterisks indicates significant differences (P < 0.05) compared to vehicle treatment with the same surgery. C. Artemin or vehicle treatment did not markedly change the number of cell bodies within the L₅ DRG showing retrograde labeling of CTB from sciatic nerve in sham-operated rats, corresponding to myelinated fibers. Artemin treatment increased CTB labeling in L₅ DRG of rats treated with nerve crush, but not in rats that had undergone axotomy or nerve ligation. Scale bar indicates 50 µm for all images. D. The graph shows quantification of CTB-labeled neurons from counted sections in percentages. Asterisks indicate significant differences (P < 0.05) compared to vehicle treatment with the same surgery.

Figure 4

Artemin promotes regeneration of non-myelinated and myelinated axons through nerve crush injury into sciatic nerve 6 weeks post L₅ spinal nerve crush. A. Artemin or vehicle treatment did not markedly change the number of cell bodies within the L₅ DRG showing retrograde labeling of dextran from sciatic nerve in sham-operated rats, corresponding to non-myelinated fibers. Artemin treatment increased dextran labeling in L₅ DRG of rats treated with nerve crush, but not in rats that had undergone axotomy or nerve ligation. Scale bar indicates 50 µm for all images. B. The graph shows quantification of dextran-labeled neurons from counted sections in percentages. Asterisks indicates significant differences (P < 0.05) compared to vehicle treatment with the same surgery. C. Artemin or vehicle treatment did not markedly change the number of cell bodies within the L₅ DRG showing retrograde labeling of CTB from sciatic nerve in sham-operated rats, corresponding to myelinated fibers. Artemin treatment increased CTB labeling in L₅ DRG of rats treated with nerve crush, but not in rats that had undergone axotomy or nerve ligation. Scale bar indicates 50 µm for all images. D. The graph shows quantification of CTB-labeled neurons from counted sections in percentages. Asterisks indicate significant differences (P < 0.05) compared to vehicle treatment with the same surgery.

Figure 5

Artemin promotes reinnervation by non-myelinated and myelinated fibers of peripheral cutaneous tissues. A. Rhodamine-dextran or CTB labeling in L₅ DRG 1 week following intradermal administration into the ipsilateral hindpaw, 6 weeks following sham or nerve crush. Artemin treatment did not alter rhodamine-dextran or CTB labeling in sham-operated rats. Artemin treatment increased rhodamine-dextran as well as CTB labeling in L₅ DRG of nerve crush treated rats compared to vehicle treated rats. Scale bar indicates 50 µm for all images. B. Quantification of rhodamine-dextran and CTB labeled neurons from L₅ DRG sections confirm an increase in the percentage of cells labeled for rhodamine-dextran or CTB with artemin treatment. Asterisks indicate significant differences (P < 0.05) compared to vehicle treatment after nerve crush.

Figure 5

Artemin promotes reinnervation by non-myelinated and myelinated fibers of peripheral cutaneous tissues. A. Rhodamine-dextran or CTB labeling in L₅ DRG 1 week following intradermal administration into the ipsilateral hindpaw, 6 weeks following sham or nerve crush. Artemin treatment did not alter rhodamine-dextran or CTB labeling in sham-operated rats. Artemin treatment increased rhodamine-dextran as well as CTB labeling in L₅ DRG of nerve crush treated rats compared to vehicle treated rats. Scale bar indicates 50 µm for all images. B. Quantification of rhodamine-dextran and CTB labeled neurons from L₅ DRG sections confirm an increase in the percentage of cells labeled for rhodamine-dextran or CTB with artemin treatment. Asterisks indicate significant differences (P < 0.05) compared to vehicle treatment after nerve crush.