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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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Functional recovery after sciatic nerve injury. Histograms illustrating the time taken for the recovery of motor function (assayed by toe spreading reflex) and sensory function (assayed by sensitivity to Von Frey hairs) after peripheral nerve injury in wild-type (black columns) versus knockout mice (grey columns). Group size was N = 6 wild-type and N = 6 knockout for Fig. 5a and b and N = 9 wild-type and N = 9 knockout for Fig. 5c. Standard error of the mean is indicated for each column. The differences between wild-type and knockout mice in time taken to recover sensory and motor function were not significant for any of the lesion groups (sciatic nerve crush, Fig. 5a; sciatic nerve crush and saphenous nerve excision, Fig. 5b; sciatic nerve transection and reanastamosis, Fig. 5c).
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Figure 5: Functional recovery after sciatic nerve injury. Histograms illustrating the time taken for the recovery of motor function (assayed by toe spreading reflex) and sensory function (assayed by sensitivity to Von Frey hairs) after peripheral nerve injury in wild-type (black columns) versus knockout mice (grey columns). Group size was N = 6 wild-type and N = 6 knockout for Fig. 5a and b and N = 9 wild-type and N = 9 knockout for Fig. 5c. Standard error of the mean is indicated for each column. The differences between wild-type and knockout mice in time taken to recover sensory and motor function were not significant for any of the lesion groups (sciatic nerve crush, Fig. 5a; sciatic nerve crush and saphenous nerve excision, Fig. 5b; sciatic nerve transection and reanastamosis, Fig. 5c).

Mentions: Recovery of sensory function, as a measure of the rate and extent of regeneration of sensory axons in the injured sciatic nerve, was assessed by twice-daily monitoring of the withdrawal reflex in response to stimulation of the lateral hindpaw with Von Frey hairs from 1 day following nerve injury to 2 days after full recovery was first observed. The threshold stimulus for eliciting withdrawal was identical for both hindpaws preoperatively and for the control hindpaw postoperatively in both wild-type and knockout mice. After sciatic nerve crush, wild-type mice recovered the preoperative withdrawal reflex threshold after a mean of 10.2 days and knockout mice after a mean of 9.8 days (p = 0.63 n.s.; Fig. 5a). After simultaneous excision of 3 mm of the saphenous nerve, recovery times were not significantly different: the withdrawal reflex was restored by 10.3 days in wild-type mice and by 11.0 days in knockout mice (p = 1.0, n.s. (calculated using a two tailed Fisher exact test because all 6 NG2 knockout mice recovered on the same day); Fig. 5b). These results confirm that recovery times after sciatic nerve injury alone could not be due to reinnervation as a result of sprouting from branches of the saphenous nerve, which, if it occurred, might give rise to false positive results. After sciatic nerve section and reanastamosis, which presents a greater barrier to regeneration than crush injury, recovery times were 16.7 days on average for wild-type and 15.3 days for knockout mice (p = 0.18 n.s.; Fig. 5c).


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)

Functional recovery after sciatic nerve injury. Histograms illustrating the time taken for the recovery of motor function (assayed by toe spreading reflex) and sensory function (assayed by sensitivity to Von Frey hairs) after peripheral nerve injury in wild-type (black columns) versus knockout mice (grey columns). Group size was N = 6 wild-type and N = 6 knockout for Fig. 5a and b and N = 9 wild-type and N = 9 knockout for Fig. 5c. Standard error of the mean is indicated for each column. The differences between wild-type and knockout mice in time taken to recover sensory and motor function were not significant for any of the lesion groups (sciatic nerve crush, Fig. 5a; sciatic nerve crush and saphenous nerve excision, Fig. 5b; sciatic nerve transection and reanastamosis, Fig. 5c).
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC2100060&req=5

Figure 5: Functional recovery after sciatic nerve injury. Histograms illustrating the time taken for the recovery of motor function (assayed by toe spreading reflex) and sensory function (assayed by sensitivity to Von Frey hairs) after peripheral nerve injury in wild-type (black columns) versus knockout mice (grey columns). Group size was N = 6 wild-type and N = 6 knockout for Fig. 5a and b and N = 9 wild-type and N = 9 knockout for Fig. 5c. Standard error of the mean is indicated for each column. The differences between wild-type and knockout mice in time taken to recover sensory and motor function were not significant for any of the lesion groups (sciatic nerve crush, Fig. 5a; sciatic nerve crush and saphenous nerve excision, Fig. 5b; sciatic nerve transection and reanastamosis, Fig. 5c).
Mentions: Recovery of sensory function, as a measure of the rate and extent of regeneration of sensory axons in the injured sciatic nerve, was assessed by twice-daily monitoring of the withdrawal reflex in response to stimulation of the lateral hindpaw with Von Frey hairs from 1 day following nerve injury to 2 days after full recovery was first observed. The threshold stimulus for eliciting withdrawal was identical for both hindpaws preoperatively and for the control hindpaw postoperatively in both wild-type and knockout mice. After sciatic nerve crush, wild-type mice recovered the preoperative withdrawal reflex threshold after a mean of 10.2 days and knockout mice after a mean of 9.8 days (p = 0.63 n.s.; Fig. 5a). After simultaneous excision of 3 mm of the saphenous nerve, recovery times were not significantly different: the withdrawal reflex was restored by 10.3 days in wild-type mice and by 11.0 days in knockout mice (p = 1.0, n.s. (calculated using a two tailed Fisher exact test because all 6 NG2 knockout mice recovered on the same day); Fig. 5b). These results confirm that recovery times after sciatic nerve injury alone could not be due to reinnervation as a result of sprouting from branches of the saphenous nerve, which, if it occurred, might give rise to false positive results. After sciatic nerve section and reanastamosis, which presents a greater barrier to regeneration than crush injury, recovery times were 16.7 days on average for wild-type and 15.3 days for knockout mice (p = 0.18 n.s.; Fig. 5c).

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