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Analysis of axonal regeneration in the central and peripheral nervous systems of the NG2-deficient mouse.

Hossain-Ibrahim MK, Rezajooi K, Stallcup WB, Lieberman AR, Anderson PN - BMC Neurosci (2007)

Bottom Line: Although a substantial body of evidence underpins this hypothesis, it is challenged by recent findings including strong expression of NG2 in regenerating peripheral nerve.In the CNS, we used anterograde tracing with BDA to study corticospinal tract (CST) axons after spinal cord injury and transganglionic labelling with CT-HRP to trace ascending sensory dorsal column (DC) axons after DC lesions and a conditioning lesion of the sciatic nerve.Anatomical correlates of regeneration were assessed by: retrograde labelling of regenerating dorsal root ganglion (DRG) cells with DiAsp; immunostaining with PGP 9.5 to visualise sensory reinnervation of plantar hindpaws; electron microscopic analysis of regenerating axons in tibial and digital nerves; and by silver-cholinesterase histochemical study of motor end plate reinnervation.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Anatomy and Developmental Biology, University College London, Gower Street, London WC1E 6BT, UK. mkhi@hotmail.com

ABSTRACT

Background: The chondroitin sulphate proteoglycan NG2 blocks neurite outgrowth in vitro and has been proposed as a major inhibitor of axonal regeneration in the CNS. Although a substantial body of evidence underpins this hypothesis, it is challenged by recent findings including strong expression of NG2 in regenerating peripheral nerve.

Results: We studied axonal regeneration in the PNS and CNS of genetically engineered mice that do not express NG2, and in sex and age matched wild-type controls. In the CNS, we used anterograde tracing with BDA to study corticospinal tract (CST) axons after spinal cord injury and transganglionic labelling with CT-HRP to trace ascending sensory dorsal column (DC) axons after DC lesions and a conditioning lesion of the sciatic nerve. Injury to these fibre tracts resulted in no difference between knockout and wild-type mice in the ability of CST axons or DC axons to enter or cross the lesion site. Similarly, after dorsal root injury (with conditioning lesion), most regenerating dorsal root axons failed to grow across the dorsal root entry zone in both transgenic and wild-type mice. Following sciatic nerve injuries, functional recovery was assessed by analysis of the toe-spreading reflex and cutaneous sensitivity to Von Frey hairs. Anatomical correlates of regeneration were assessed by: retrograde labelling of regenerating dorsal root ganglion (DRG) cells with DiAsp; immunostaining with PGP 9.5 to visualise sensory reinnervation of plantar hindpaws; electron microscopic analysis of regenerating axons in tibial and digital nerves; and by silver-cholinesterase histochemical study of motor end plate reinnervation. We also examined functional and anatomical correlates of regeneration after injury of the facial nerve by assessing the time taken for whisker movements and corneal reflexes to recover and by retrograde labelling of regenerated axons with Fluorogold and DiAsp. None of the anatomical or functional analyses revealed significant differences between wild-type and knockout mice.

Conclusion: These findings show that NG2 is unlikely to be a major inhibitor of axonal regeneration after injury to the CNS, and, further, that NG2 is unlikely to be necessary for regeneration or functional recovery following peripheral nerve injury.

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Retrograde labelling of DRG after sciatic nerve injury. Anatomical analyses of regeneration of sensory axons after sciatic nerve injuries in wild-type versus knockout mice. Figs. 7a-c show composite images made up by merging the entire z-series of optical sections through L5 DRG in wild-type (Fig. 7a) and knockout mice (Fig. 7b) 13 days after sciatic nerve crush and 2 days after injection of DiAsp into the skin of the hindpaw. Fig. 7c shows the control DRG (no sciatic nerve injury) from the same animal as Fig. 7b. Figs. 7d and e show a similar comparison of labelling in wild-type and knockout mice 90 days after sciatic nerve crush. Scale bar = 200 μm and applies to Figs. 7a-e. Figs. 7f and g compare the numbers of labelled cells in L5 DRG 13 days (Fig. 7f) and 90 days (Fig. 7g) after sciatic nerve crush in wild-type (N = 8 + 8; black columns) versus knockout mice (N = 8 + 8; grey columns). The quantitative analyses show that there are no statistically significant differences in the numbers of retrogradely labelled DRG cells (and thus in the extent of sensory axon regeneration) between wild-type and knockout mice at either short or long postoperative survival times.
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Figure 7: Retrograde labelling of DRG after sciatic nerve injury. Anatomical analyses of regeneration of sensory axons after sciatic nerve injuries in wild-type versus knockout mice. Figs. 7a-c show composite images made up by merging the entire z-series of optical sections through L5 DRG in wild-type (Fig. 7a) and knockout mice (Fig. 7b) 13 days after sciatic nerve crush and 2 days after injection of DiAsp into the skin of the hindpaw. Fig. 7c shows the control DRG (no sciatic nerve injury) from the same animal as Fig. 7b. Figs. 7d and e show a similar comparison of labelling in wild-type and knockout mice 90 days after sciatic nerve crush. Scale bar = 200 μm and applies to Figs. 7a-e. Figs. 7f and g compare the numbers of labelled cells in L5 DRG 13 days (Fig. 7f) and 90 days (Fig. 7g) after sciatic nerve crush in wild-type (N = 8 + 8; black columns) versus knockout mice (N = 8 + 8; grey columns). The quantitative analyses show that there are no statistically significant differences in the numbers of retrogradely labelled DRG cells (and thus in the extent of sensory axon regeneration) between wild-type and knockout mice at either short or long postoperative survival times.

Mentions: At 5 and 9 days after sciatic nerve crush (2 animals at each survival time), the contralateral DRG were heavily labelled but there were no labelled cells in ipsilateral L4 or L5 DRG (data not shown) indicating that the crush lesions were complete, that L4 and L5 DRG are not labelled via routes other than injured sciatic nerve, and that regenerating sensory axons had not reached the hindpaw at these survival times. Very few labelled neurons were present in the ipsilateral DRG 13 days after sciatic nerve crush (i.e. 11 days after DiAsp injection into hindpaw), in either wild-type or knockout mice, with an average of 28 labelled neurons per DRG in wild-type mice compared to 14 in knockout mice (p = 0.18 n.s.; Figs. 7a, b and 7f). The appearance of retrograde label in injured DRG neurons 13 days after sciatic nerve crush, but not before, correlates with the onset of functional recovery being 9–10 days after sciatic nerve crush in our other experiments (see Figure 5a and 5b).


Analysis of axonal regeneration in the central and peripheral nervous systems of the NG2-deficient mouse.

Hossain-Ibrahim MK, Rezajooi K, Stallcup WB, Lieberman AR, Anderson PN - BMC Neurosci (2007)

Retrograde labelling of DRG after sciatic nerve injury. Anatomical analyses of regeneration of sensory axons after sciatic nerve injuries in wild-type versus knockout mice. Figs. 7a-c show composite images made up by merging the entire z-series of optical sections through L5 DRG in wild-type (Fig. 7a) and knockout mice (Fig. 7b) 13 days after sciatic nerve crush and 2 days after injection of DiAsp into the skin of the hindpaw. Fig. 7c shows the control DRG (no sciatic nerve injury) from the same animal as Fig. 7b. Figs. 7d and e show a similar comparison of labelling in wild-type and knockout mice 90 days after sciatic nerve crush. Scale bar = 200 μm and applies to Figs. 7a-e. Figs. 7f and g compare the numbers of labelled cells in L5 DRG 13 days (Fig. 7f) and 90 days (Fig. 7g) after sciatic nerve crush in wild-type (N = 8 + 8; black columns) versus knockout mice (N = 8 + 8; grey columns). The quantitative analyses show that there are no statistically significant differences in the numbers of retrogradely labelled DRG cells (and thus in the extent of sensory axon regeneration) between wild-type and knockout mice at either short or long postoperative survival times.
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Figure 7: Retrograde labelling of DRG after sciatic nerve injury. Anatomical analyses of regeneration of sensory axons after sciatic nerve injuries in wild-type versus knockout mice. Figs. 7a-c show composite images made up by merging the entire z-series of optical sections through L5 DRG in wild-type (Fig. 7a) and knockout mice (Fig. 7b) 13 days after sciatic nerve crush and 2 days after injection of DiAsp into the skin of the hindpaw. Fig. 7c shows the control DRG (no sciatic nerve injury) from the same animal as Fig. 7b. Figs. 7d and e show a similar comparison of labelling in wild-type and knockout mice 90 days after sciatic nerve crush. Scale bar = 200 μm and applies to Figs. 7a-e. Figs. 7f and g compare the numbers of labelled cells in L5 DRG 13 days (Fig. 7f) and 90 days (Fig. 7g) after sciatic nerve crush in wild-type (N = 8 + 8; black columns) versus knockout mice (N = 8 + 8; grey columns). The quantitative analyses show that there are no statistically significant differences in the numbers of retrogradely labelled DRG cells (and thus in the extent of sensory axon regeneration) between wild-type and knockout mice at either short or long postoperative survival times.
Mentions: At 5 and 9 days after sciatic nerve crush (2 animals at each survival time), the contralateral DRG were heavily labelled but there were no labelled cells in ipsilateral L4 or L5 DRG (data not shown) indicating that the crush lesions were complete, that L4 and L5 DRG are not labelled via routes other than injured sciatic nerve, and that regenerating sensory axons had not reached the hindpaw at these survival times. Very few labelled neurons were present in the ipsilateral DRG 13 days after sciatic nerve crush (i.e. 11 days after DiAsp injection into hindpaw), in either wild-type or knockout mice, with an average of 28 labelled neurons per DRG in wild-type mice compared to 14 in knockout mice (p = 0.18 n.s.; Figs. 7a, b and 7f). The appearance of retrograde label in injured DRG neurons 13 days after sciatic nerve crush, but not before, correlates with the onset of functional recovery being 9–10 days after sciatic nerve crush in our other experiments (see Figure 5a and 5b).

Bottom Line: Although a substantial body of evidence underpins this hypothesis, it is challenged by recent findings including strong expression of NG2 in regenerating peripheral nerve.In the CNS, we used anterograde tracing with BDA to study corticospinal tract (CST) axons after spinal cord injury and transganglionic labelling with CT-HRP to trace ascending sensory dorsal column (DC) axons after DC lesions and a conditioning lesion of the sciatic nerve.Anatomical correlates of regeneration were assessed by: retrograde labelling of regenerating dorsal root ganglion (DRG) cells with DiAsp; immunostaining with PGP 9.5 to visualise sensory reinnervation of plantar hindpaws; electron microscopic analysis of regenerating axons in tibial and digital nerves; and by silver-cholinesterase histochemical study of motor end plate reinnervation.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Anatomy and Developmental Biology, University College London, Gower Street, London WC1E 6BT, UK. mkhi@hotmail.com

ABSTRACT

Background: The chondroitin sulphate proteoglycan NG2 blocks neurite outgrowth in vitro and has been proposed as a major inhibitor of axonal regeneration in the CNS. Although a substantial body of evidence underpins this hypothesis, it is challenged by recent findings including strong expression of NG2 in regenerating peripheral nerve.

Results: We studied axonal regeneration in the PNS and CNS of genetically engineered mice that do not express NG2, and in sex and age matched wild-type controls. In the CNS, we used anterograde tracing with BDA to study corticospinal tract (CST) axons after spinal cord injury and transganglionic labelling with CT-HRP to trace ascending sensory dorsal column (DC) axons after DC lesions and a conditioning lesion of the sciatic nerve. Injury to these fibre tracts resulted in no difference between knockout and wild-type mice in the ability of CST axons or DC axons to enter or cross the lesion site. Similarly, after dorsal root injury (with conditioning lesion), most regenerating dorsal root axons failed to grow across the dorsal root entry zone in both transgenic and wild-type mice. Following sciatic nerve injuries, functional recovery was assessed by analysis of the toe-spreading reflex and cutaneous sensitivity to Von Frey hairs. Anatomical correlates of regeneration were assessed by: retrograde labelling of regenerating dorsal root ganglion (DRG) cells with DiAsp; immunostaining with PGP 9.5 to visualise sensory reinnervation of plantar hindpaws; electron microscopic analysis of regenerating axons in tibial and digital nerves; and by silver-cholinesterase histochemical study of motor end plate reinnervation. We also examined functional and anatomical correlates of regeneration after injury of the facial nerve by assessing the time taken for whisker movements and corneal reflexes to recover and by retrograde labelling of regenerated axons with Fluorogold and DiAsp. None of the anatomical or functional analyses revealed significant differences between wild-type and knockout mice.

Conclusion: These findings show that NG2 is unlikely to be a major inhibitor of axonal regeneration after injury to the CNS, and, further, that NG2 is unlikely to be necessary for regeneration or functional recovery following peripheral nerve injury.

Show MeSH
Related in: MedlinePlus