Measurement of absolute TA noxious reflex activity amongst the different groups in response to the first stimulus (inset Fig. 2G) or averaged over the second to seventh response (Fig. 2G) revealed no treatment effect on general activity level, further supporting the use of temporal summation as a measure of change in spinal central sensitisation to noxious stimuli. In animals with SCI treated with saline alone the temporal summation of the nociceptive TA flexor reflex was observed up to a maximal value of 11366326% (average of 921685% during the 16 stimuli) when compared to the first reflex response (Fig. 2H). In general temporal summation of the TA noxious flexor reflex was modulated differently between the experimental groups (F = 42.69; d.f. = 4,5; p,0.001). A similar level of maximal TA temporal summation was observed in na¨ıve rats (13046564%, Fig. 2H). Post-hoc analysis revealed that temporal summation of the TA nociceptive reflex was inhibited following albumin (195613%; Tukey post-hoc test, p,0.001, q = 14.36), oleic acid alone (521644%; Tukey post-hoc test, p,0.001, q = 7.91) or albumin-oleic acid (434646%; Tukey post-hoc test, p,0.001, q = 9.62), when compared to the SCI control group treated with saline alone (921685%, Fig. 2G). Interestingly animals treated with albumin alone displayed greater inhibition of TA nociceptive reflex temporal summation compared to those treated with albumin-oleic acid (Tukey p,0.05, q = 4.7). When TA nociceptive reflex data were analyzed during the early (2–7th) or late (8–16th) response (data not shown), intrathecal administration of albumin alone, oleic acid alone and albumin- oleic acid were shown to inhibit both the first and second phase of temporal summation 28 days following contusion SCI when compared to the group treated with saline alone (first phase: 10266117%; second phase: 9416125%). Specifically the first phase of TA temporal summation was inhibited by Alb (232624%, t = 6.56, d.f. = 94, p,0.001), OA (501640%, t = 4.03, d.f. = 88, p,0.001) and Alb-OA (534679%, t = 3.61, d.f. = 112, p,0.001) when compared to the saline treated group with SCI. Similarly the second phase of TA temporal summation was reduced by albumin (181614%, t = 6.56, d.f. = 94, p,0.001), oleic acid (581670%, t = 6.56, d.f. = 133, p,0.05) and albumin- oleic acid (403662%, t = 4.18, d.f. = 169, p,0.001). Synergistic de novo phasic inhibition of TA reflex activity across the iSCI with Alb-OA Percutaneous high-frequency electrical conditioning stimulation applied across the spinal cord above the contusion T9 SCI revealed a strong phasic inhibition of nociceptive TA reflex temporal summation following albumin-oleic acid treatment in animals with SCI. Examination of the rectified TA reflex activity recorded from animals with SCI and treated with albumin-oleic acid before (Pre-Cond, Fig. 3C) and after (Post-Cond, Fig. 3C) high-frequency electrical conditioning stimulation revealed this strong inhibition of temporal summation (Fig. 3I). Synergistic phasic inhibition of TA nociceptive reflex activity was evident in animals treated with albumin-oleic acid from a peak of 6096212% to 3146115% (p,0.001, Fig. 3I) compared to no effect measured following albumin (Fig. 3F) or oleic acid treatment alone (Fig. 3G). Synergistic de novo phasic inhibition of TA nociceptive reflex activity across the SCI following albumin-oleic acid treatment was supported when compared to the presence of normal phasic facilitation following conditioning electrical stimu- lation in na¨ıve animals without SCI from a peak of 13736511% to 17646173% (p,0.05, Fig. 3D). The synergistic de novo phasic inhibition of TA reflex activity following one month of albumin-oleic acid treatment was further supported when data were analyzed for both the early and late phase of temporal summation, where the second phase may Albumin-Oleic Acid Treatment of Spinal Cord Injury provide indirect evidence of descending modulatory mechanisms (see methods, Fig. 4). Specifically albumin-oleic acid treatment inhibited the first phase from 100615% to 4765% (t = 3.43; d.f. = 20; p,0.01, Fig. 4C) and the second phase of TA nociceptive reflex temporal summation from 75612% to 4765% (t = 2.2; d.f. = 20; p,0.05, Fig. 4C). The significant decrease of the second phase of TA nociceptive reflex temporal summation suggested that albumin-oleic acid promoted phasic descending inhibitory control mechanisms (Fig. 4C). Furthermore de novo phasic inhibition of TA temporal summation with Alb-OA was shown to promote general antinociception when compared to noxious reflex activity measured in na¨ıve animals (Fig. 4D). Potent synergistic increase in 5-HT innervation with Alb-OA after iSCI Detailed analysis of the volume of T9 contusion SCI in all the experimental groups failed to identify general neuroprotection either with albumin, oleic acid or with the albumin-oleic acid complex. The volume of the contusion injury as a percentage of the total possible volume measured in na¨ıve animals was 30.563.4% following SCI in the saline treated group. Similar values of SCI contusion volumes were measured with treatment with albumin alone (29.563.0%), oleic acid alone (28.962.1%), albumin-oleic acid (28.962.4%) and albumin-elaidic acid (33.363.1%). However immunohistochemical analysis revealed a potent increase in 5-HT innervation density below the T9 contusion SCI within the lumbar dorsal and ventral horn with albumin-oleic acid treatment. Although 5-HT innervation density was difficult to detect within the lumbar dorsal horn (Fig. 5A and 5B, see detailed photographs in 5D–5F), Alb-OA treatment revealed a notable generalized increase in lumbar dorsal horn 5-HT innervation density below the SCI (Fig. 5C and 5F). An upregulation of serotonin innervation with Alb-OA treatment was also evident in the ventral horn (Fig. 5G–5H) and in the detailed photographs of lamina VII (Fig. 5J–5L) and laminae IX (Fig. 5M–5O). Original microphotographs of 5-HT immunohistochemistry are included after Alb-OA treatment for lamina I–II (Fig. 5R) and lamina VII (Fig. 5S) at 28 days after SCI. Quantification of serotoninergic innervation density measured below the SCI in animals treated with saline and normalised to the na¨ıve level (Fig. 5P dorsal horn: 1.0060.14, Fig. 5Q ventral horn: 1.0060.12) revealed non-significant decreases of 5-HT levels within both the dorsal (0.6560.14, Fig. 5P) and ventral horn (0.7660.10, Fig. 5Q). In general significant differences were identified amongst all experimental groups within the dorsal (F = 6.79, d.f. = 5, p,0.001) and ventral horn (F = 6.96, d.f = 5, p,0.001). Only the albumin-oleic acid treatment significantly increased the normalised 5-HT innervation density levels to a remarkable 4.1761.09 fold (p,0.001, Fig. 5P) within the dorsal horn and 2.2760.34 fold (p,0.001, Fig. 5Q) within the ventral horn, when compared to animals treated with saline alone. Further evidence for a neurotrophic effect of albumin-oleic acid was evident by the significant difference between the observed increase in 5-HT innervation density compared to animals treated with albumin alone (dorsal horn: 1.2660.18, p,0.01; ventral horn: 0.6560.03, p,0.001) or with oleic acid alone (dorsal horn: 1.5360.30, p,0.05; ventral horn: 1.0060.21, p,0.05). The T9 spinal contusion injury increased microglia (OX-42) density within both the dorsal laminae I/II (Fig. 6B) and lamina V (Fig. 6E), accompanied by a generalized increase in the number of cells with phosphorylated NMDA NR1 receptor within the dorsal horn (Fig. 6J) compared to tissue from na¨ıve animals (Fig. 6A, 6D, 6I). Importantly the albumin-oleic acid complex inhibited microglial OX-42 reactivity (Fig. 6C and 6F) and the number of PLoS ONE | www.plosone.org 6 October 2011 | Volume 6 | Issue 10 | e26107