Elsevier

Neuroscience

Volume 120, Issue 1, 4 August 2003, Pages 41-56
Neuroscience

Inhibiting cell proliferation during formation of the glial scar: effects on axon regeneration in the CNS

https://doi.org/10.1016/S0306-4522(03)00285-9Get rights and content

Abstract

Following a CNS lesion many glial cell types proliferate and/or migrate to the lesion site, forming the glial scar. The majority of these cells express chondroitin sulphate proteoglycans (CS-PGs), previously shown to inhibit axonal growth. In this study, in an attempt to diminish glial scar formation and improve axonal regeneration, proliferating cells were eliminated from the lesion site. Adult rats received a continuous infusion of 2% cytosine-D-arabinofuranoside (araC) or saline for 7 days over the lesion site, immediately following a unilateral transection of the right medial forebrain bundle. Additional groups of rats that received subdural infusions prior to the lesion, and lesioned rats which received no infusion, were also compared in the analyses. Animals were killed at 4, 7, 12 or 18 days post-lesion (dpl) and immunohistochemistry was used to determine the effects of these treatments on tyrosine hydroxylase (TH)+-lesioned axons, and on the injury response of glial cells. Almost complete elimination of NG2+ oligodendrocyte progenitor cells from the lesion site was seen up to 7 dpl in araC-infused animals; reduced numbers of reactive CD11b+ microglia were also seen but no effects were seen on the injury response of GFAP+ astrocytes. Significantly more TH+ axons were seen distal to the lesion in araC-treated brains, but these numbers dwindled by 18 dpl.

Section snippets

General procedures and animal care

All animal work was carried out in accordance with the UK Animals (Scientific Procedures) Act (1986); all efforts were made to minimise the number of animals used, and their suffering. Adult male Sprague–Dawley rats (200–260 g; n=76) were anaesthetized for 5 min with 5% halothane and 2 L/min O2 prior to surgery. Each animal was positioned into a stereotactic frame where anaesthesia was maintained at 1–2% halothane during surgery (with 0.6 L/min O2 and N2O); the incisor bar was set 2.3 mm below

Results

Groups of animals were examined after five different treatments (see Table 1):

  • 1.

    pre-lesion saline: animals received a continuous subarachnoid infusion of saline for 7 days, prior to transection of the MFB/nigrostriatal tract;

  • 2.

    pre-lesion araC: animals received a continuous subarachnoid infusion of araC for 7 days (after Doetsch et al., 1999), prior to MFB transection;

  • 3.

    lesion only: MFB transection without infusion;

  • 4.

    post-lesion saline: MFB transection immediately followed by infusion of saline for 7

Injury response of OP cells

As in previous studies Levine, 1994, Ong and Levine, 1999, Bu et al., 2001, McTigue et al., 2001, Jones et al., 2002, Tang et al., 2003 we have shown a substantial increase in the number of NG2+ OP cells around CNS lesions, and a large increase in the intensity of NG2 staining on those cells. Expression of NG2 on cells with the morphology of inflammatory cells or astrocytes was not observed in this study (see also below).

Our first experiment was to infuse the antimitotic, araC, over the surface

Conclusions

Infusion of the antimitotic, araC, over the lesion site for 7 days immediately following a unilateral lesion of the nigrostriatal tract significantly reduced the number of OP cells able to participate in the scar formation. The number of CD11b+ microglia around the lesion was also considerably reduced but GFAP+ astrocytes were not affected. The findings imply that the OP cell population was increased around the lesion by intensive local proliferation whilst the astrocyte population was mainly

Acknowledgements

This work was funded by grants from the Medical Research Council, the International Spinal Research Trust, the Wellcome Trust and Internationales Forschungsinstitut für Paraplegiologie (Zurich). The monoclonal antibody to NG2 was generously donated by Joel Levine, and the polyclonal antibody to GAP43 was a gift from Graham Wilkin.

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    Present address: The Miami Project to Cure Paralysis, Miami, FL 33101, USA.

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