Elevated MMP-9 in the Thoracic and Lumbar Cord Impedes Activity Dependent Plasticity and Recovery Early after SCI in Mice

Abstract

PURPOSE: Spinal cord injury (SCI) disrupts sensory and motor systems resulting in permanent functional deficits. The inability to walk is the most noticeable and debilitating impairment. New advances in activity-based neurorehabilitation has improved recoveryin the clinic, but complete recovery remains elusive.. To further optimize recovery, activity-based therapy like treadmill training should be combined with therapeutics to increase neural plasticity. To date, local cell death and inflammation at the injury site have been important factors in the extent of locomotor recovery after SCI. One explanation for persistent locomotor deficits is that this type of pathology extends beyond the primary injury site distally into the regions of the cord responsible for organizing locomotion. Indeed, we previously found pronounced microglial activation and pro-inflammatory cytokine production in the lumbar cord but the role of remote inflammation on locomotor recovery remains unknown. Here, we explore the role of one of the earliest inflammatory signals, matrix-metalloproteinase-9 (MMP-9), as a regulator of plasticity and locomotor recovery. MMP-9 promotes inflammation by increasing permeability of the blood vessels in the spinal cord, facilitating immune cell access and activating pro-inflammatory cytokines. Primary outcomes for permeability, cell trafficking and cytokine production were collected in the lumbar cord in SCI groups with and without treadmill training. RESEARCH METHOD: Using a mouse SCI model that replicates human pathology, we produced moderate/severe contusions at midthoracic levels (T9) and analyzed the remote lumbar cord. To identify novel mechanisms of remote neuroinflammation, we used ELISA, gel and in situ zymography to measure MMPs and pro-inflammatory cytokines in C57BL/6 (WT) mice. We used immunohistochemistry to describe remote microglia phenotype and anatomy. To prove that early inflammation impedes locomotor plasticity, we examined recovery after SCI in WT and MMP-9 KO mice with or without treadmill (TM) training delivered in acute (2d) or chronic (35d) phases of recovery. FINDINGS: Pro-gelatinase MMP-3 increased significantly at 24 hrs in L4/5 (423.9% Naïve) whileMMP-9 and TNFα protein increased at 7d (511.9% and 197% Naïve, respectively) and 42d. MMP-2 remained at baseline in KO and WT mice in the lumbar enlargement. Reactivity was observed in lumbar endothelia as active MMP-9 localized to vasculature. Microglia displayed an activated phenotype within 24h through 7d in L1-L5 gray matter. TM training delivered in the presence or absence of MMP-9 had differential effects. Neither TM training alone nor MMP-9 depletion alone promoted recovery. In WT mice with SCI, TM training failed to induce locomotor recovery compared to untrained groups and paralysis persisted. However, acute TM training in MMP-9 KO mice resulted in robust recovery. Independent walking was evident in early trained KO mice by 7d (p<.01) and improvements were retained 4 wks after training ended (p<.05). IMPLICATIONS: Midthoracic SCI produces acute pathology at- and distant to the injury site that prevents plasticity and recovery in locomotor networks. Early markers of cellular inflammation included resident microglial activation and reactivity in lumbar endothelia. MMP-9 activity persisted around lumbar vasculature during the first week of SCI alongside increased pro-inflammatory cytokine production within centers for locomotion. Robust locomotor recovery occurred only when MMP-9 deletion was combined with acute TM training. We show for the first time, that locomotor recovery after SCI depends on at-level and remote inflammation. It is likely that neurorehabilitation will be most effective when delivered early but only in a low inflammatory environment.