cutaneous nociceptive reflex activity following remote electrical conditioning stimulation [34] while dorsolateral funiculus lesion leads to immediate stretch hyperreflexia [35,36]. As such the evaluation of both tonic and phasic modulation of nociceptive flexor reflex activity may provide important information regarding clinically relevant neuroplasticity within spinal and descending modulatory systems after iSCI [37–40], and in this study treatment effects with novel neurotrophic factors such as Alb-OA. Here we show that local administration of the Alb-OA complex synergistically promoted tonic inhibition of temporal summation of TA noxious reflex activity, de novo phasic descending inhibition of nociceptive reflex activity mediated across the iSCI, and early motor recovery [41]. In addition Alb-OA strongly increased lumbar serotoninergic innervation, well above the normal level observed in animals without SCI, reduced phosphorylation of the NR1 NMDA receptor and effectively blocked microglia reactivity. Preclinical development of the Alb-OA complex for the treatment of cutaneous noxious hyperreflexia, altered spinal nociception, spasticity and deficit of voluntary motor function following iSCI will be discussed. Materials and Methods Ten week old male Wistar rats, with an approximate weight of 250–300 g, were used in this study and maintained in the animal resource unit with food and water ad libitum, following approval by the institutional animal experimentation ethical committee. The experiments adhered to the guidelines of the Committee for Research and Ethical Issues of IASP published in Pain 1983;16:109–110, following approval from the Ethical Committee for Animal Welfare of the ‘‘Hospital Nacional de Paraple´jicos’’ in Toledo (2007; PI07-0806). Animals were subjected to spinal cord injury by moderate T9 contusion. Following SCI, animals were randomly assigned to five groups, each of which was administered with an intrathecal bolus of the following: Saline (Sal, 0.9%, n = 8), Albumin (Alb, 0.4 nanomoles, n = 8), Oleic Acid (OA, 80 nanomoles, n = 8), Albumin-Oleic Acid (Alb-OA, 0.4/80 nano- moles, n = 11) and Albumin-Elaidic Acid (Alb-EA, 0.4/80 nanomoles, n = 11). The compounds were administered in a volume of 10 ml, by the intrathecal route [42], immediately following the SCI and every 3 days thereafter for a total of 28 days. A further group of na¨ıve rats was used as the uninjured control (n = 8), instead of animals with a sham T8 laminectomy because non-significant changes in central sensitization measured by Tibialis Anterior (TA) reflex temporal summation have been identified previously (unpublished data). Preparation of the Albumin-Oleic acid complex The dialysis membrane (D0655-100FT, Sigma-Aldrich, Spain) was prepared by washing it for 4 h under running water under reduced flow. The membrane was then washed, firstly with 0.3% (w/v) solution of sodium sulphide at 80uC for 2 minutes, then with double distilled and deionised water (Milli-Q, Merck) preheated to 60uC for a further 2 minutes, followed with a 0.2% (w/v) solution of sulphuric acid at ambient temperature. Finally, the membrane was washed again with double distilled and deionised water and placed in a receptacle with the same water. Elliot-Calcium buffer [43] was prepared with the final pH adjusted to 7.4 and filtered with a 0.22 mm pore size (SCGPT05RE, Millipore). Before incubation CaCl2.2 H2O was added to prepare 1.3 mM Elliot-Ca buffer. Commercially available lyophilised bovine albumin (D0655, Sigma-Aldrich, Spain) was then dialysed by preparing a 10% solution in Elliot-calcium buffer, which was then placed inside the pre-treated dialysis membrane. The dialysis Albumin-Oleic Acid Treatment of Spinal Cord Injury was performed in agitation at 4uC, with 3 washes at 12 hours, 18 and 24 hours respectively. Finally bovine albumin was filtered with a 0.2 mm pore size (4525, Serum AcrodiscH 37 mm syringe filter, Pall Gelman Laboratory). Aliquots of 1 ml bovine albumin were prepared and stored at 220uC. The albumin-oleic acid complex was prepared with 2% (w/v) bovine albumin solution, by adding oleic acid (O1008, Sigma- Aldrich, Spain) [20]. An albumin-oleic acid (1:1) solution was prepared to a concentration of 80 nanomoles in saline (0.9%). The albumin-elaidic acid complex was similarly prepared to the same concentration (E4637, Sigma-Aldrich, Spain). Experimental animal surgery Wistar rats were anesthetized with pentobarbital (Dolethal, Ve´toquinol, 65 mg/kg, i.p., Ref: 737) and xylazine (Xilagesic 10 mg/kg concentration, i.p. Calier, Ref: 25225), followed by a supplementary 30% dose following aproximately 90 minutes of experimental surgery. In addition 0.1 ml of antibiotic was administered (2.5% Baytril, Enrofloxacin, Bayer) after surgery, followed by daily doses up to 3 days after spinal cord injury. Commercially available rat intrathecal catheters (ALZT7740Z, Charles River Laboratories, Spain) were implanted (see below) and externalized accordingly [42]. Immediately before surgical im- plantation, the catheter was re-sterilised with absolute ethanol, and thoroughly washed with sterile 0.9% saline. Following skin incision and blunt dissection of the muscle layers overlying the vertebrae, a small hemi-laminectomy at the vertebral T10 level was performed (spinal T9). The exposed T9 dura was subjected to a small durectomy with iris-type scissors so that the tip of the intrathecal catheter could be inserted rostrally and medially on top of the spinal cord with a final position just below the intended T9 contusion site. The area was cleaned to permit catheter fixture with acrylate cement to the T11 vertebrae. The percutaneous end of the intrathecal catheter was finally secured by inserting it through a small cutaneous incision at the base of the cranium, whereupon it was filled with 0.9% sterile saline and tapped with a custom-made nylon filament. Following intrathecal catheter implantation a spinal T9 contusion was performed [44]. A bilateral T9 vertebral laminec- tomy enabled spinal contusion by allowing an 11-gram weight to fall from a height of 12 mm onto a cylindrical flat-tipped impactor with a 2.5 mm diameter placed centrally over the exposed spinal cord above the intact dura. Once the contusion was performed, artificial dura mater (Neuropatch, Ref: 106403, B. Braun), the overlying muscle layers were reapposed with a continuous suture stitch and the skin was finally closed with a subdermal suture, both with a 4-0 reabsorbable thread. Rats were carefully observed during recovery, and the bladder was manually expressed daily until recovery of function. Behavioural analysis of evoked voluntary motor function Evoked voluntary motor function before and after T9 contusion injury was analyzed in all experimental groups with the Rotarod test (4600, Ugo Basile), instead of the standard BBB test [45], because of the specific need to document early the maximal evoked capacity for generalised hindlimb-forelimb locomotor function after SCI. Prior to contusion injury, each animal was trained for three days to remain upon a cylindrical surface which rotated at 5 rpm for at least 5 minutes [46]. On the day before SCI control data were obtained by subjecting the rats to the Rotarod test, but with the cylinder rotating at a steadily accelerating speed from 5 to 15 rpm during the 5 minute test duration [46]. Following SCI rats were tested on day 4 and then weekly thereafter up to 28 days to document general motor recovery and the effect of the different treatments. PLoS ONE | www.plosone.org 2 October 2011 | Volume 6 | Issue 10 | e26107