62 HPLC high-performance liquid chromatography MBP myelin basic protein NG2 a chondroitin sulfate proteoglycan NGF nerve growth factor NT-3 neurotrophin-3 OFWT open field walking test PBS phosphate-buffered saline PFA paraformaldehyde PNP purine nucleoside phosphorylase PNS peripheral nervous system SCI spinal cord injury TdT terminal deoxynucleotidyltransferase TGF beta transforming growth factor beta TUNEL terminal deoxynucleotide transferase dUTP nick end labelling Introduction Demyelination contributes to functional deficits in central nervous system (CNS) disorders [1–3]. Traumatic spinal cord injury (SCI) induces local inflammation and demyelination in the white matter around the lesion resulting in disrupted axonal conduction [4–7]. Remyelination is critical for recovery, but does not occur spontaneously [8–11]. Spinal cords contain endogenous progenitors that can proliferate and differentiate into mature oligodendroglia and remyelinate axons under certain circumstances [12–14]. After acute SCI in rats, oligodendrocyte precursors proliferate in the first 2 weeks but are “silent” thereafter [15], possibly because the oligodendroglial precursors cannot differentiate into mature oligodendroglia capable of remyelinating axons. Enhance- ment of proliferation of oligodendrocytes or progenitors may lead to repair and restoration of function after SCI. Neurotrophic factors restore physiological function by promoting oligodendrocyte proliferation and axonal sprouting [16–19], regulating the proliferation of oligodendrocyte progenitors [3] and modulating the differentiation [20–22] and the maturation of oligodendrocyte precursors into myelin-forming cells [23, 24]. Guanosine has trophic effects on many cell types [25– 28], and we have some evidence that guanine acts similarly. For example, it stimulates proliferation of a variety of cells in culture [25, 28] and promotes synthesis and release of several potentially neuroprotective trophic factors from a variety of cells, including neuron growth factor (NGF) from astrocytes as well as basic fibroblast growth factor (bFGF) and transforming growth factor beta (TGF beta) [25, 27]. Extracellular guanosine itself also enhances outgrowth of nerve processes from PC12 cells and from primary cultures of embryonic rat brain neurons [25]. As well, it acts synergistically with NGF to promote outgrowth of neurites [29, 30]. Purinergic Signalling (2008) 4:61–71 Our preliminary data [31] showed that 35 days after moderate traumatic injury of the spinal cord of rats, when the initial spontaneous incomplete recovery of function had plateaued, systemic administration of guanosine (intra- peritoneally 8 mg/kg body weight) for 7 days enhanced locomotor functional recovery that continued to improve throughout the 7-day duration of the treatment and correlated with the increase in myelination. In the present study, we treated rats for up to 14 days to determine whether guanosine-induced functional improvement was due to triggering the endogenous adult progenitors. Mean- while, we also examined the uptake and tissue distribution of systemically administered guanosine. Materials and methods All experiments were performed in compliance with the requirements of the Animals for Research Act of Ontario, Canada and the Guidelines of the Canadian Council on Animal Care, and had been approved by the Animal Research Ethics Board of McMaster University. Spinal cord injury Adult female Wistar rats (280–300 g weight, Charles River) were anaesthetized with isoflurane (3–5%): O2 (1 l/min). Buprenorphine (0.03 mg/kg body weight, subcutaneously) was administered prior to surgery for pain relief. Moderate spinal cord injury was induced by crushing exposed cords with modified coverslip forceps [31–33]. The forceps were closed slowly over 2 s and the compression sustained for 15 s. The muscles and fat pad over the lesion were sutured and the skin was closed with stainless steel clips. Post-operatively, the rats were kept quiet and warm [31, 34, 35]. Behavioural assessment and drug administration Rats were handled daily for 2 weeks pre-operatively to acclimatize them to the experimental procedures and behavioural test. After the spinal injury, locomotor recovery was assessed weekly for 35 days in an open field walking task (OFWT) before treatment. Cagemates (two animals) were placed in the centre of the open field, formed by a child’s circular plastic swimming pool (1.3 m in diameter). They were observed for 5-min periods and scored for general locomotor ability using the Basso- Beattie-Bresnahan (BBB) locomotor rating scale [36, 37]. Rats were rated on a scale of 0 to 21, with 0 being no function and 21 being normal. If the animal stopped moving for a minute, it was placed again in the centre of the open field; otherwise it was left alone for the duration of the 5-min test period. On day 35 following the injury, rats were randomly assigned to three groups. One group of rats was used for