Purinergic Signalling (2008) 4:61–71 67 Fig. 2 (continued) injured cords correlated with each animal’s open field walking scores (r =0.72; p< 0.05; Fig. 1f), indicating that guanosine-induced functional recovery may be related to the increase in myelin. Immunostaining for CNS myelin basic protein (MBP; Fig. 1g,h) and a specific marker for Schwann cell myelin, P0 (Fig. 1i,j) showed that Schwann cells from the peripheral nervous system did not enter spinal cord and, therefore, did not contribute to the remyelination. To determine whether existing cells elaborated the myelin or whether it was a product of other cells that proliferated and subsequently elaborated myelin, the number of proliferating cells was determined by counting all BrdU+ nuclei in cross sections of the lesion pen- umbra at different times after treatment. In the first 3 days after treatment, cell proliferation at the injury site in both guanosine-treated and control animals was not different from baseline (immediately before treatment; Fig. 2c), although at 3 days there was a trend toward more BrdU+ cells in guanosine-treated animals than controls (112 ± 25 vs 79 ± 16). At 7 and 14 days after guanosine treatment, the number of BrdU+ cells increased signifi- cantly; there were 382 ± 42 BrdU+ cells per section at 7 days and 477 ± 49 at 14 days compared with only 88 ± 20 at 7 days and 95 ± 22 at 14 days in controls ( p < 0.05; Fig. 2a–c). There were few dividing cells beyond the lesion margins. Oligodendrocytes that survive the demyelinating insult are not thought to be able to contribute to remyelination [41, 42]. Instead, endogenous oligodendrocyte progenitor cells local to the lesion are believed to be the source of new myelinating cells [43]. Previous studies have illustrated that NG2+ cells are the major cycling cell within the resting adult CNS [14, 44] and express many characteristics of oligodendroglial lineage cells, not least their ability to differentiate into oligodendrocytes when isolated into culture. Their role in demyelination and remyelination has been extensively studied and there is evidence that they are the cells responsible for myelin repair [43, 45–48]. To determine whether the proliferating cells were related to NG2+ oligodendroglial progenitor cells, we counted the total number of NG2+ progenitor cells and the number that had proliferated (NG2+/BrdU+) from 2 mm rostral to 2 mm caudal to the lesion site (Fig. 2d,e). There was minimal turnover in the NG2+ cells in the spinal cord during the first 3 days in both guanosine-treated and control animals (13 ±5 in the guanosine group vs 9 ±4 cells per section in controls; Fig. 2f). By 7 days, guanosine significantly stimulated progenitor proliferation (85 ± 18 vs 13 ± 4 NG2+/BrdU+ cells/section; Fig. 2f); these cells were observed throughout the penumbra with the majority localised in the dorsal columns. Enhanced proliferation of the NG2+ progenitors after guanosine persisted, and by 14 days, double-labelled progenitors in the guanosine- treated group were eightfold more than in controls (108 ± 12 vs 13 ±5 NG2+/BrdU+ cells/section; Fig. 2f). The total number of NG2+ cells was also determined. During the first 3 days, there was no significant difference in the numbers of NG2+ cells in cords at baseline after vehicle treatment or guanosine treatment (Fig. 2g). By 7 days, guanosine treatment increased the total NG2+ cells from 96 ±22 cells/section at baseline to 266±38 cells/ section—more than 2 times higher than in the control group (110 ±24 /section; Fig. 2g). By 14 days, the numbers of double-labelled progenitors in the cords of guanosine- treated animals were threefold higher than controls (347 ± 36 NG2+ cells/section for guanosine-treated animals; 115 ± 24 cells/section for controls; Fig. 2g). Mature oligodendrocytes were identified with Rip+ staining (Fig. 2h,i). Guanosine had no effect on mature oligodendroglia 3 days after treatment; at 7 and 14 days, there was a significant increase in Rip+ cells in the penumbra of the cords of animals that had received guanosine compared to controls (7 days: 468 ±40 vs 205 ± 25; and at 14 days: 492±43 vs 219± 23 per section, p< 0.01; Fig. 2j). Guanosine treatment stimulated astrocyte proliferation in the penumbra as early as 3 days and throughout the 14 days of treatment (Fig. 3a,b) and was detected by antibodies to glial fibrillary acidic protein (GFAP). Astrocytes may stimulate oligodendroglial progenitor proliferation or in- duce their migration [49]. Guanosine did not alter the