Albumin-Oleic Acid Treatment of Spinal Cord Injury Figure 5. Albumin-Oleic Acid Increases Serotonin (5-HT) Innervation to Above Normal Levels after SCI. Immunohistochemical images revealed that 5-HT innervation density was increased within both the dorsal and ventral horn following Albumin-Oleic acid treatment (0.4/80 nanomoles, 5C and 5I, 10x) compared to either na¨ıve animals (A and G, 10x) or those with SCI contusion (Cont) treated with saline alone (Sal, B and H, 10x). Detailed photographs of laminae I-II (D-F, note faint medial signal in D), lamina VII (J–L) and lamina IX (M–O) revealed the normal presence of 5- HT fibres with varicosities (D, J, M), following SCI (E, K, N) and in animals with spinal T9 contusion with albumin-oleic acid treatment (F, L, O). Quantification of the increase in lumbar 5-HT innervation levels below the SCI within the dorsal (P, p,0.001) or ventral horn (Q, p,0.001) following Albumin-OA treatment (Alb-OA, 0.4/80 nanomoles) was observed, but not after saline (Sal), albumin (Alb), oleic acid (OA) or albumin-elaidic acid (Alb- EA) administration. Statistical analysis was performed with a one-way ANOVA for each experimental group (P and Q, *** - p,0.001). Detailed original examples of fluorescent images of 5-HT fibres within laminae I–II (R) and laminae VII (S) are included with Alb-OA treatment 28 days after T9 SCI. doi:10.1371/journal.pone.0026107.g005 treatment could mediate a reduction in nociceptive reflex excitability via control of the pNR1 NMDA receptor subtype below the iSCI. Albumin-OA treatment also strongly reduced microglia reac- tivity within the lumbar dorsal horn following SCI. Microglia activation has been identified up to several weeks following SCI in humans [73] and is part of the normal inflammatory response [74]. Furthermore microglia reactivity has been implicated in central sensitisation to noxious stimuli [16] and in patients following SCI with symptoms of spasticity [75]. Oleic acid inhibits the induction of proinflammatory mediators, such as NF-kB, within microglial cell culture [54], and so may have a direct effect against neuroinflammation. In parallel with the hypothesis suggested above for the interaction between descending seroto- ninergic and spinal mechanisms of excitability modulated via the pNR1 NMDA receptor, the application of noradrenergic agonists such as clonidine has been shown to inhibit GFAP upregulation, nuclear factor-kappa B activation, and p38 mitogen- activated protein kinase induction following peripheral nerve injury [76]. As such Albumin-OA complex treatment may control spinal excitability following iSCI following descending 5-HT inhibitory tract neuroplasticity, which in turn would inhibit microglia reactivity and NMDA receptor phosphorylation follow- ing iSCI. Clinical significance of Alb-OA treatment for iSCI and remaining key experiments Simultaneous successful treatment of SCI central sensitisation to noxious stimuli, spasticity, and paralysis below the injury depends on the control of several spinal pathophysiological mechanisms [2,77–80], which can be broadly grouped into processes of neuronal excitability, neuroinflammation and plasticity of senso- rimotor circuits following partial or complete damage to the spinal cord [7–10]. Novel pleiotropic treatments are not only required to modulate these basic mechanisms [9,11–13], but to target the most debilitating symptoms of spasticity and pain [2,81,82], without precipitating secondary effects of somnolence or fatigue [83–85]. Neurophysiological evidence for the modulation of non-invasive translational measures of spinal nociceptive reflex activity such as those used here, combined with immunohistochemical evidence for the role of three cellular mechanisms of action related to the control of spinal sensorimotor function, demonstrates that the Alb- OA complex meets all of these criterion. While Alb has a an established clinical safety profile [86], only indirect evidence exists for OA as a non-toxic agent according to one nutritional study of olive oil administration [50]. Importantly in our study no change in feeding behaviour, body weight or grooming was observed with Alb-OA treatment following iSCI. Albumin most probably provides therapeutic benefits for spinal cord injury when administered alone [21]. Indeed the replacement of cerebrospinal fluid with 8% albumin following experimental ischaemic experimental SCI can reduce motor deficits, although this has been attributed mainly to reduction of intraspinal oedema [87]. However evidence for direct penetrability of Alb-OA into the spinal parenchyma should be addressed in future studies. Functional motor recovery has been identified when Alb is injected directly into the spinal tissue or via the intravenous route [21], but careful analysis should be performed with FITC-albumin injected via the intrathecal route which would address this concern. Indeed a similar study has identified both grey and white spinal matter labelling with FITC-albumin when adminis- tered via the intravenous route, confirming penetration at least into the intraspinal microcirculatory system [87]. Albumin is commonly used a drug delivery system in the clinical setting [88] and specific known binding sites with OA suggest a biological basis for the synergistic action between both molecules observed in this study [89]. As such drug delivery of OA across the blood brain barrier could be mediated by albumin transcytosis [90,91], followed by cellular uptake within the spinal parenchyma [19], and synergistic modulation of sensorimotor function following iSCI. Of course several limitations regarding the preclinical develop- ment and translational application of Alb-OA still remain, including the possibility of its administration via the systemic route and characterisation of the therapeutic window. General neuroprotection following SCI was not observed with Alb-OA in this study, in direct contrast to the effect of omega-3 fatty acids such as docosahexanoic acid alone [22], or in combination with albumin in a stroke model [92]. The neurotrophic effect of Albumin-OA in vivo also needs to be addressed directly through pharmacological blockade of the peroxisome pro- liferator activated receptors, the role of which have been highlighted in an in vitro study [93]. Finally, further evidence for specific cellular in vivo mechanisms of action of Alb-OA should now be extended with invasive neuroanatomical and neurophys- iological techniques to further understand the role of Alb-OA on both extrapyramidal and corticospinal descending control systems across the iSCI. Conclusion The Albumin-Oleic acid complex is a new pharmacological treatment strategy with pleiotropic functional effects for the symptoms of paralysis, spasticity and change in spinal pain processing following iSCI. Initial evidence of specific mechanisms of action include the simultaneous tonic and de novo phasic descending inhibition of spinal reflex excitability mediated by a potent increase in lumbar 5-HT innervation, with a reduction in NR1 NMDA receptor phosphorylation and microglial reactivity. Neurotrophic enhancement of collateral sprouting of residual 5- HT fibres across the iSCI has been demonstrated to mediate locomotor recovery [38,39]. However the effective inhibition by Alb-OA of a translational non-invasive measure of the most debilitating symptoms of spasticity and change in spinal nocicep- tion [81,82], while promoting residual voluntary motor function below the lesion, represents an important clinical goal for subacute rehabilitation of patients with iSCI. PLoS ONE | www.plosone.org 11 October 2011 | Volume 6 | Issue 10 | e26107