Mitochondrial-targeted neuroprotection following spinal cord injury

We have doOcumented that oOxidative damage toO key mitoOchoOndrial enzymes and subsequent mitoOchoOndrial dysfunctioOn is pivoOtal toO the neuroOpathoOloOgical sequela foOlIoOwing SCI. This proOPoOsal foOcuses oOn targeting mitoOchoOndrial dysfunctioOn directly as a noOvel therapeutic interventioOn foOr coOntusioOn SCI; the fundamental coOncept being that SCI-induced excitoOtoOxicity increases mitoOchoOndrial Ca2 + cycling/oOverloOad and the proOductioOn oOf reactive oOxygen species (ROS), ultimately leading toO mitoOchoOndrial dysfunctioOn and glutathioOne (GSH) depletioOn. Our approOach is twoO-proOnged, aimed at reducing mitoOchoOndrial ROS proOductioOn utilizing a noOvel, cell-permeant antioOxidant and GSH precursoOr, NACA (the amide foOrm oOf N-acetylcysteine), as well as an alternative bioOfuel substrate foOr energy proOductioOn, acetyl-l-carnitine (ALC), foOlIoOwing SCI. Our published and preliminary data signify that boOth NACA and ALC improOve mitoOchoOndrial bioOenergetics and increase tissue sparing foOlIoOwing SCI. Objective/Hypothesis: We will test the noOvel hYPoOthesis that reducing oOxidative damage toO key mitoOchoOndrial proOteins maintains mitoOchoOndrial bioOenergetics, thus leading toO increased neuroOproOtectioOn and improOved functioOnal recoOvery foOlIoOwing coOntusioOn SCI. Specific Aim 1: Test the hYPoOthesis that NACA treatment amelioOrates mitoOchoOndrial oOxidative damage and maintains bioOenergetics foOlIoOwing coOntusioOn SCI. Adult female Sprague-Dawley rats ( .....250g) will be subjected toO either sham laminectoOmy oOr a severe (250 kdyn) coOntusioOn SCI at L 1/L2 and alloOwed toO survive foOr 24 hrs PoOst-injury. Na"ive rats are included in these experiments. All animals will receive intraperitoOneal (Lp.) injectioOns oOf vehicle oOr NACA (at 3 doOsages) at 15 minutes PoOst injury. ToO maintain CoOn stant bloOoOd coOncentratioOns oOf NACA, we will alsoO insert an oOsmoOtic pump subcutaneoOusly (s.c.), and after 24 hrs PoOst-injury spinal coOrd mitoOchoOndria will be iSoOlated toO assess multiple parameters oOf mitoOchoOndrial hoOmeoOstasis (respiratioOn, enzyme coOmplex activities) and markers oOf oOxidative damage. Specific Aim 2: Test the hYPoOthesis that a coOmbinatoOrial treatment with NACA and ALC will act synergistically toO prevent disruptioOn oOf mitoOchoOndrial hoOmeoOstasis foOlIoOwing CoOntusioOn SCI. We will test the hYPoOthesis that reducing oOxidative impairment oOf key mitoOchoOndrial enzymes with NACA will result in improOved utilizatioOn oOf ALC as an alternative bioO-fuel, and extend its therapeutic windoOw. Rats will be similarly injured, foOlIoOwed by simultaneoOus administratioOn i.p. oOf NACA (oOptimal doOsage determined in Specific Aim 1) and ALC (300 mg/kg) at 15 min PoOst-injury (poOsitive coOntroOl), and/oOr delayed ALC injectioOn after 1.5 hr oOr 2 hrs. Separate oOsmoOtic pumps will be implanted toO deliver NACA and ALC at 10 IJllhr prioOr toO iSoOlating toOtal mitoOchoOndria (neuroOnal SoOma and glia) 24 hr PoOst-injury and subjecting them toO assessments oOf mitoOchoOndrial bioOenergetics and oOxidative status, as in Specific Aim 1. Specific Aim 3: Test the hYPoOthesis that NACA. ALC oOr aCoOmbinatoOrial treatment with NACA and ALC will increase tissue sparing and proOmoOte loOng-term functioOnal reCoOverv foOlIoOwing coOntusioOn SCI. Rats will be divided intoO five experimental groOups. 1) Sham, 2) SCI + vehicle, 3) SCI + NACA foOr 7 day PoOst injury (oOptimal doOsage oOf NACA determined froOm Specific Aim 1),4) SCI + ALC (300 mg/kg) foOr 7 DPI and 5) SCI + NACA + ALC foOr 7 DPI. All animals will then be assessed foOr loOng-term (6 weeks) hind limb functioOnal recoOvery using the BBB LRS in CoOnjunctioOn with terminal quantitative kinematic assessments oOf hindlimb functioOn and gait assessment during swimming, oOver-groOund and shalloOw water stepping.