Albumin-Oleic Acid Treatment of Spinal Cord Injury Figure 3. Albumin-Oleic Acid Promotes De Novo Synergistic Descending Inhibition of Tibialis Anterior Reflex Activity after SCI. Examination of rectified Tibialis Anterior (TA) electromyograhic reflex activity (Fig. 3A–3C) revealed strong inhibition of temporal summation in animals with T9 spinal contusion (Cont) treated with albumin-oleic acid (Alb-OA, 0.4/80 nanomoles) following high-frequency conditioning stimulation (Post-Cond) above the SCI when compared to the pre-conditioning (Pre-Cond) response (Fig. 3C, scale bar 10 mV/2.5 s). No inhibition was observed either in na¨ıve (Fig. 3A, scale bar 20 mV/2.5 s) or in animals with contusion SCI treated with saline alone (Fig. 3B, scale bar 20 mV/2.5 s). Quantitative analysis of TA reflex temporal summation (Fig. 3D–3I) demonstrated a phasic facilitation in na¨ıve animals following high-frequency electrical conditioning (Fig. 3D), which was absent in the experimental SCI groups (Fig. 3E–3I). Indeed de novo synergistic phasic inhibition of TA reflex activity mediated across the SCI was only promoted by treatment with albumin-oleic acid (Alb-OA, Fig. 3I, p,0.001), but not with albumin (Alb, Fig. 3F), oleic acid (OA, Fig. 3G) or albumin-elaidic acid (Alb-EA, Fig. 3H). Statistical analysis was performed with a one-way ANOVA for each experimental group (Fig. 3D–3I, * - p,0.05; *** - p,0.001). doi:10.1371/journal.pone.0026107.g003 pNR1-positive cell counts within the dorsal horn to those observed in na¨ıve animals (Fig. 6K). Spinal T9 contusion injury led to an increase in microglial OX- 42 area within the superficial (LI/II: na¨ıve 25.163.2, contusion 81.560.18, p,0.001, Fig. 6G) and deep dorsal horn (LV: na¨ıve 13.361.4, contusion 97.8624.3, p,0.001, Fig. 6H). Compared to microglial reactivity following SCI, the OX-42 area decreased within both the superficial (21.663.9, p,0.001, Fig. 6G) and deep dorsal horn (32.664.80, p,0.01, Fig. 6H) following albumin-oleic acid treatment. When compared to na¨ıve animals, an increase in the number of pNR1-positive cells following SCI was observed within the superficial dorsal horn from 66.9611.1 to 101.9610.1 (p,0.05, Fig. 6L) and deep dorsal horn 110.068.3 to 147.368.1 (p,0.01, Fig. 6M). Treatment with albumin-oleic acid reduced the number of pNR1-positive cells compared to the SCI group treated with saline alone within the superficial (38.262.6, p,0.001, Fig. 6L) and deeper dorsal horn (69.264.5, p,0.001, Fig. 6M). Interest- ingly within the deep dorsal horn, albumin-oleic acid treatment decreased pNR1-positive cells to below that identified in na¨ıve levels (69.264.5 vs. 110.068.3, p,0.01, Fig. 6M), suggesting a clear antinociceptive effect. Discussion The novel effect of intrathecal albumin, oleic acid and especially Alb-OA treatment on spinal and descending inhibition of TA nociceptive reflex activity and early recovery of voluntary motor function following iSCI is described for the first time in this study, and is supported by the simultaneous modulation of three spinal cellular mechanisms at the lumbar level. These include reduction of NR1 NMDA receptor phosphorylation, inhibition of microglial reactivity and a strong increase in 5-HT levels to above normal levels within the dorsal and ventral horn. The specific synergistic effect of the putative neurotrophic factor Alb-OA on promotion of tonic and de novo phasic descending inhibition of spinal noxious reflex excitability across the iSCI is notable. In addition the measurement of Tibialis Anterior reflex activity using standard spinal and a newly developed translational measure of descending function assures the clinical relevance of the effect of Alb-OA as a novel neurotrophic factor in vivo following central neurotrauma. The potentially safe clinical profile of this complex [50] and to the possibility that specific SCI symptoms of spinal change in nociceptive function and spasticity could be treated with Alb-OA is discussed below. Pleiotropic effect of Alb-OA on noxious central sensitisation and paralysis after iSCI Albumin comprises of about half of blood serum protein and has multiple specific binding sites for fatty acids [23,51]. Motor recovery and neuroprotection have been demonstrated with both albumin [21] and omega 3 fatty acids following iSCI [22,49]. Although OA is a known neurotrophic factor in vitro [20], a functional effect of systemic administration of this omega-9 fatty acid has not been demonstrated alone [22], and is supported by the poor motor recovery following SCI observed in our study. In contrast inhibition of flexor reflex activity was identified individually with either Alb or OA, as predicted by other studies [21,52–54]. Surprisingly synergistic early motor recovery and de novo phasic inhibition of spinal nociceptive reflex activity was observed only when both molecules were combined, which suggests that the neurotrophic effect of this complex in vivo is likely to be mediated via collateral sprouting of residual descending modulatory pathways after iSCI [37]. An in vitro study demon- strates that extracellular Alb and OA are incorporated into neurons leading to dendritic growth and GAP-43 upregulation [20]. At the in vivo level CNS injury leads to SCD-1 upregulation, the enzyme responsible for oleic acid synthesis [55], which is also related to normal myelin production [56]. Another in vitro study has demonstrated that oleic acid-mediated upregulation of GAP- 43 is synergistic with the neurotrophic factors NT3 and NT4/5 [57]. Interestingly the role of these factors have been related to the prevention of corticospinal tract atrophy [58] and the promotion of serotoninergic innervation below iSCI [59]. The application of the non-invasive electrical conditioning protocol developed to indirectly assess the role of descending pathways across the iSCI was adapted from Liu et al. (1998), which examined long-term depression (LTD) of C-fibre-evoked field potentials [47]. Interestingly in the original study LTD was shown to convert to long-term potentiation when the spinal cord was transected, suggesting that the activation of descending inhibitory control systems maybe critical for the observation of spinal reflex inhibition in vivo [47]. Although phasic descending of noxious reflex excitability has been demonstrated in uninjured animals with the application of other electrical conditioning stimuli remote to the test site [34], this is the first time that this technique has revealed descending modulatory system plasticity following iSCI following treatment, although the role of altered propriospinal system function through the iSCI cannot be excluded [60,61]. Alb-OA modulates iSCI neuronal excitability, neuroinflammation and serotonin neuroplasticity Albumin-OA mediated a strong increase in serotonin innerva- tion density within both the dorsal and ventral lumbar level, higher than that observed following other experimental treatments [37,62] or indeed to that found in uninjured spinal tissue. Oleic acid is an allosteric factor for the 5-HT7A receptor [63], and this receptor is present within the superficial dorsal horn [64] and may play a role in analgesia [65]. As such tonic inhibition of nociceptive TA reflex temporal summation could be mediated via 5-HT7A receptor activation [66]. Although there are many 5- HT receptor subtypes with mixed inhibitory and facilitatory neuronal effects, enhanced neuroplasticity of residual descending systems with a phenotypic shift from serotoninergic facilitation to inhibition of spinal function [66] would be expected to overcome PLoS ONE | www.plosone.org 8 October 2011 | Volume 6 | Issue 10 | e26107