Purinergic Signalling (2008) 4:61–71 65 Fig. 1 (continued) For quantification of cell proliferation, five to seven sections taken from the penumbra of the lesion and spaced about 100 μm apart were analysed for each animal (n = 7–9 animals per group). Cellular proliferation was determined by counting the total number of BrdU+ nuclei in spinal cord cross sections. Low power sections were digitized and manually outlined using an image analysis system. Any cavities present in the sections were excluded from analysis. Positively labelled nuclei were counted automat- ically. The threshold was set such that only positively labelled profiles of the appropriate size were counted. Data are expressed as cells per section. The total number of NG2+ progenitor cells and the number of those that proliferated (double-labelled for NG2 and BrdU: NG2+/BrdU+) was determined by manual counting. Cell counting was done conservatively, and a positive profile was counted as a cell only when clearly defined borders could be detected completely around and adjacent to the entire nucleus. Data are expressed as the number of immunostained cells per section. Mature oligodendrocytes in injured cords were identified with Rip, a specific marker for mature oligodendrocytes that recognises an unknown epitope [39] and has been used successfully in the CNS to label both oligodendrocyte processes and myelin sheaths. The number of mature oligodendrocyte (Rip+ cells) in the tissue sections was determined by manual counting. Evaluation of exogenous guanosine distribution A guano- sine solution (8 mg/kg) was administered intraperitoneally to rats with spinal cord injury induced 5 weeks previously. Samples of spinal cord were taken from the site of the lesion and immediately above or below the lesion just before the intraperitoneal injection of guanosine and at 7.5, 30 and 60 min after the guanosine injection. There were between three and six animals at each time point. Samples of spinal cord were placed in 0.5 ml of cold 0.4 M perchloric acid solution and immediately homogenized. Samples were then centrifuged (4°C, 10,000 g, 10 min) to remove proteins precipitated by perchloric acid and the supernates containing the soluble molecules were neutral- ized with KOH solution and centrifuged (4°C, 10,000 g, 5 min). Samples were filtered with 0.2-μm filters (Milli- pore, Billerica, MA, USA) and stored at −70°C before high-performance liquid chromatography (HPLC) [40] or capillary electrophoresis (CE) analysis. Tissue concentrations of guanosine and guanine To eval- uate the concentration of guanosine and its principal degradation product, guanine, in spinal cords before and after the intraperitoneal injection of guanosine, either HPLC or CE was used. There was no significant difference in the values obtained by these two methods so the data represent the mean of values obtained by both analyses. HPLC analysis was carried out using an Agilent 1100 series HPLC (Agilent Technologies, Waldbronn, Germany) and an ion-pair technique. Separation was carried out with a reverse-phase analytical column (LiChroCART 125-4 LiChrospher 100 RP-18 5 μm, Merck, Darmstadt, Germany) and the flow rate was 1.5 ml/min. A 15-min linear gradient was applied from 100% buffer A (60 mM KH2PO4 and 5 mM tetrabutyl- ammonium phosphate, pH 6) to 100% buffer B (30% methanol plus 70% buffer A). CE separations were carried out using an HP3DCE capillary electrophoresis system (Agilent Technologies, Waldbronn, Germany) equipped with a diode array detection system. Analysis was performed using a 80.5 cm × 50 μm ID × 375 μm OD uncoated fused silica capillary from Agilent Technologies (Waldbronn, Germany) with an effective length of 72 cm to the detector window. The capillary temperature was kept constant at 37°C and the running buffer was 10 mM borate, pH 9.3. The samples were injected by pressure (50 mb for 40 s) and the separation was conducted at 22 kV. Detection took place at 254 nm and purines were identified by their migration times and UV spectra using the diode array detector (Agilent Technologies, Waldbronn, Germany). Statistical analysis Data are expressed as mean ±SEM. Behavioural scores were analysed by the Kruskal-Wallis non-parametric analysis of variance (ANOVA). Post hoc comparisons were made using the Dunnett’s test. Correla- tion between behavioural and histological outcomes was analysed using regression analysis. Significance was set at p <0.05.