Treatment of Rat Spinal Cord Injury with the Neurotrophic Factor Albumin-Oleic Acid: Translational Application for Paralysis, Spasticity and Pain

Sensorimotor dysfunction following incomplete spinal cord injury (iSCI) is often characterized by the debilitating symptoms of paralysis, spasticity and pain, which require treatment with novel pleiotropic pharmacological agents. Previous in vitro studies suggest that Albumin (Alb) and Oleic Acid (OA) may play a role together as an endogenous neurotrophic factor. Although Alb can promote basic recovery of motor function after iSCI, the therapeutic effect of OA or Alb-OA on a known translational measure of SCI associated with symptoms of spasticity and change in nociception has not been studied. Following T9 spinal contusion injury in Wistar rats, intrathecal treatment with: i) Saline, ii) Alb (0.4 nanomoles), iii) OA (80 nanomoles), iv) Alb-Elaidic acid (0.4/80 nanomoles), or v) Alb-OA (0.4/80 nanomoles) were evaluated on basic motor function, temporal summation of noxious reflex activity, and with a new test of descending modulation of spinal activity below the SCI up to one month after injury. Albumin, OA and Alb-OA treatment inhibited nociceptive Tibialis Anterior (TA) reflex activity. Moreover Alb-OA synergistically promoted early recovery of locomotor activity to 50±10% of control and promoted de novo phasic descending inhibition of TA noxious reflex activity to 47±5% following non-invasive electrical conditioning stimulation applied above the iSCI. Spinal L4–L5 immunohistochemistry demonstrated a unique increase in serotonin fibre innervation up to 4.2±1.1 and 2.3±0.3 fold within the dorsal and ventral horn respectively with Alb-OA treatment when compared to uninjured tissue, in addition to a reduction in NR1 NMDA receptor phosphorylation and microglia reactivity. Early recovery of voluntary motor function accompanied with tonic and de novo phasic descending inhibition of nociceptive TA flexor reflex activity following Alb-OA treatment, mediated via known endogenous spinal mechanisms of action, suggests a clinical application of this novel neurotrophic factor for the treatment of paralysis, spasticity and pain

Albumin-Oleic Acid Increases Serotonin (5-HT) Innervation to Above Normal Levels after SCI.

<p>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 naïve 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.</p

Albumin-Oleic Acid (Alb-OA) Synergistically Promotes Early Recovery of Motor Function following T9 Spinal Cord Injury.

<p>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 (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0026107#pone-0026107-g001" target="_blank">Fig. 1A</a>). Treatment with OA (80 nanomoles) alone or Alb-Elaidic acid (Alb-EA, 0.4/80 nanomoles) failed to potentiate locomotor recovery (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0026107#pone-0026107-g001" target="_blank">Fig. 1A</a>). 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 (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0026107#pone-0026107-g001" target="_blank">Fig. 1B</a>). Statistical analysis was performed with a two-way (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0026107#pone-0026107-g001" target="_blank">Fig. 1A</a>) and one-way ANOVA (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0026107#pone-0026107-g001" target="_blank">Fig. 1B</a>), 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).</p

Inhibition of Tibialis Anterior Reflex Activity with Albumin, Oleic Acid and Albumin-Oleic Acid after SCI.

<p>Qualitative analysis of rectified nociceptive Tibialis Anterior (TA) noxious reflex temporal summation at 28 days following spinal contusion (Cont, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0026107#pone-0026107-g002" target="_blank">Fig. 2A–2F</a>, scale bar 20 µV/2.5 s) supported the quantification of reflex inhibition (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0026107#pone-0026107-g002" target="_blank">Fig. 2H</a>) with either albumin (Alb, 0.4 nanomoles, p<0.001), oleic acid (OA, 80 nanomoles, p<0.001) or the complex Alb-OA (0.4/80 nanomoles, p<0.001) when compared to animals with SCI treated with saline alone (Sal). Importantly noxious TA reflex activity following moderate T9 contusion injury was not different for either the averaged response calculated from the second to seventh stimulus (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0026107#pone-0026107-g002" target="_blank">Fig. 2G</a>) or the first reflex response (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0026107#pone-0026107-g002" target="_blank">Fig. 2G</a> inset, same format) amongst the experimental treatment groups when compared to the responses observed in naïve animals or with SCI treated with saline alone. Although no increase in temporal summation for the first to the sixteenth reflex response was identified following contusion SCI (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0026107#pone-0026107-g002" target="_blank">Fig. 2H</a>), significant inhibition of noxious TA temporal summation in animals with contusion SCI was observed with albumin (p<0.001), oleic acid (OA, p<0.001) and albumin-oleic acid (Alb-OA, p<.001) when compared to the group treated with saline alone (Sal, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0026107#pone-0026107-g002" target="_blank">Fig. 2H</a>). The stereoisomer complex albumin-elaidic acid (Alb-EA, 0.4/80 nanomoles) failed to inhibit flexor reflex activity following contusion SCI (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0026107#pone-0026107-g002" target="_blank">Fig. 2H</a>). Statistical analysis was performed with a two-way ANOVA supported by the post-hoc Bonferronni test compared to the SCI group treated with saline alone (*** - p<0.001).</p

Albumin-Oleic Acid Promotes <i>De Novo</i> Synergistic Descending Inhibition of Tibialis Anterior Reflex Activity after SCI.

<p>Examination of rectified Tibialis Anterior (TA) electromyograhic reflex activity (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0026107#pone-0026107-g003" target="_blank">Fig. 3A–3C</a>) 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 (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0026107#pone-0026107-g003" target="_blank">Fig. 3C</a>, scale bar 10 µV/2.5 s). No inhibition was observed either in naïve (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0026107#pone-0026107-g003" target="_blank">Fig. 3A</a>, scale bar 20 µV/2.5 s) or in animals with contusion SCI treated with saline alone (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0026107#pone-0026107-g003" target="_blank">Fig. 3B</a>, scale bar 20 µV/2.5 s). Quantitative analysis of TA reflex temporal summation (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0026107#pone-0026107-g003" target="_blank">Fig. 3D–3I</a>) demonstrated a phasic facilitation in naïve animals following high-frequency electrical conditioning (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0026107#pone-0026107-g003" target="_blank">Fig. 3D</a>), which was absent in the experimental SCI groups (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0026107#pone-0026107-g003" target="_blank">Fig. 3E–3I</a>). Indeed <i>de novo</i> synergistic phasic inhibition of TA reflex activity mediated across the SCI was only promoted by treatment with albumin-oleic acid (Alb-OA, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0026107#pone-0026107-g003" target="_blank">Fig. 3I</a>, p<0.001), but not with albumin (Alb, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0026107#pone-0026107-g003" target="_blank">Fig. 3F</a>), oleic acid (OA, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0026107#pone-0026107-g003" target="_blank">Fig. 3G</a>) or albumin-elaidic acid (Alb-EA, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0026107#pone-0026107-g003" target="_blank">Fig. 3H</a>). Statistical analysis was performed with a one-way ANOVA for each experimental group (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0026107#pone-0026107-g003" target="_blank">Fig. 3D–3I</a>, * - p<0.05; *** - p<0.001).</p

Albumin-Oleic Acid Promotes Phasic Inhibition of Early and Late Tibialis Anterior Reflex Activity following SCI.

<p>Analysis of the first and second phase of Tibialis Anterior (TA) reflex temporal summation in naïve animals (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0026107#pone-0026107-g004" target="_blank">Fig. 4A</a>) and those with SCI 28 days after T9 spinal contusion (Cont) revealed that the synergistic phasic inhibitory effect of Albumin-Oleic acid produced with the high-frequency electrical conditioning stimulation when normalized to the first phase of the pre-conditioned response was present during both the early and late phases of temporal summation (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0026107#pone-0026107-g004" target="_blank">Fig. 4C</a>), but not in the group treated with Saline (Sal, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0026107#pone-0026107-g004" target="_blank">Fig. 4B</a>) or in naïve animals (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0026107#pone-0026107-g004" target="_blank">Fig. 4A</a>). Furthermore activation of phasic inhibition of absolute integrated TA reflex activity measured during both phases of temporal summation revealed that Alb-OA treatment significantly inhibited nociceptive activity to below the normal value observed in naïve animals during the early phase (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0026107#pone-0026107-g004" target="_blank">Fig. 4D</a>). Statistical analysis was performed with a one-way ANOVA supported by a post-hoc Bonferroni test (* - p<0.05; ** - p<0.01, ***-p<0.001).</p