the exception of pNR1 analysis which was performed in lamina V). The OX-42 analysis protocol was performed as previously published [49]. The immunoreactive 5-HT and OX-42 density in addition to the number of cells expessing pNR1 were averaged and presented as group data. The quantification was presented for 5-HT innervation and OX-42 density as the area in mm2, in addition to the number of cells which positively expressed p-NR1 within the dorsal horn. All analytical procedures were performed blind, without knowledge of the experimental conditions. Statistical tests Data were analyzed using a one-way ANOVA for comparisons between groups, while a two-way ANOVA was used for longitudinal analysis such as with the rotarod tests, or for analysis of the temporal summation of TA nociceptive reflex activity. Post-hoc tests were performed with the Students t test (rotarod analysis), the Tukey post- hoc test (electrophysiological data) and the Bonferroni test (immunohistochemistry, Graphpad Prism 4.00, USA). Results All experimental groups demonstrated an increase in body weight during the 28 day study following moderate T9 contusion SCI, suggesting that none of the pharmaceutical treatments were associated with poor health and or adverse drug reactions. Animals treated with saline revealed an increase of 22.965.6% body weight during the one month SCI period, while albumin alone (49.964.8%) and oleic acid alone (48.662.5%) applied via the intrathecal route significantly potentiated weight gain compared to saline alone (p,0.05). Animals treated with albumin-oleic acid also revealed an increase in body weight (39.964.6%). Synergistic early recovery of evoked motor function after iSCI with Alb-OA Four days after T9 contusion SCI in animals treated with saline evoked voluntary motor function as assessed on the rotarod was reduced to 1.060.1% when compared to the pre-lesion control Albumin-Oleic Acid Treatment of Spinal Cord Injury value (100.062.4%, Fig. 1A). Two-way repeated measures ANOVA revealed a difference in recovery rate of voluntary motor function between all experimental groups with different treatments (F = 17.7; d.f. = 4,3; p,0.001). The experimental SCI group treated with albumin-oleic acid demonstrated the greatest recovery of voluntary movement to 50.0614.8% at 28 days compared to the saline-treated control group (F = 8.96, d.f. = 1, p,0.01). However animals treated with albumin alone also revealed recovery of motor function up to 28 days after SCI to 34.9610.3%, when compared to the control group (F = 5.6, d.f. = 1, p,0.05). The effect of albumin-oleic acid upon evoked voluntary motor function was also evident during acute SCI (Fig. 1B). When all data were analyzed between 4–14 and 21–28 days after SCI an early synergistic effect of albumin-oleic acid on motor function recovery was evident. Specifically, motor function measured from 4–14 days after SCI with saline treatment was reduced to 2.460.6%, but was significantly potentiated to 3467% following albumin-oleic acid treatment (Fig. 1B, unpaired two-tailed t-test, t = 4.63; d.f. = 4; p,0.01). Treatment with albumin or oleic acid alone during the acute phase of SCI failed to promote significant motor recovery. The early effect of albumin-oleic acid treatment was maintained during the sub-acute phase of SCI from 21 to 28 days (Fig. 1B). Specifically animals treated with albumin-oleic acid recovered up to 50610% of the pre-lesion control level of motor activity compared to the saline-treated group (t = 25.8; d.f. = 2; p,0.01), while the group treated with albumin also revealed a modest recovery to 3267% (t = 6.5; d.f. = 2; p,0.05). Tonic inhibition of TA reflex activity with Alb, OA and Alb-OA treatment after iSCI Rectified TA reflex EMG activity recorded in response to 16 noxious electrical stimuli was present in na¨ıve (Fig. 2A) and animals with experimental T9 contusion SCI with different treatments (Fig. 2B–2F), illustrating a strong inhibitory effect with albumin (Fig. 2C) or Alb-OA alone (Fig. 2F) on temporal summation. Figure 1. Albumin-Oleic Acid (Alb-OA) Synergistically Promotes Early Recovery of Motor Function following T9 Spinal Cord Injury. Longitudinal analysis of the mean time spent on the rotarod following contusion (Cont) SCI from 4 to 28 days revealed that intrathecal administration of both Alb (0.4 nanomoles, p,0.05) and Alb-OA (80 nanomoles, p,0.01) promoted better motor recovery, compared to the spinal contusion group treated with saline (Sal) alone (Fig. 1A). Treatment with OA (80 nanomoles) alone or Alb-Elaidic acid (Alb-EA, 0.4/80 nanomoles) failed to potentiate locomotor recovery (Fig. 1A). Analysis of voluntary motor function during the early (4–14 day) and subacute (21–28 day) period of contusion SCI demonstrated that Alb-OA (0.4/80 nanomoles) synergistically promoted functional recovery soon after contusion injury when compared to animals treated with saline (Sal) alone, while Alb treatment enhanced the time spent on the rotarod specifically during the late phase (Fig. 1B). Statistical analysis was performed with a two-way (Fig. 1A) and one-way ANOVA (Fig. 1B), supported by a post-hoc Bonferronni test for each experimental group compared with animals with SCI treated with Sal alone (* - p,0.05; ** - p,0.01). doi:10.1371/journal.pone.0026107.g001 PLoS ONE | www.plosone.org 4 October 2011 | Volume 6 | Issue 10 | e26107