Macrophage Depletion Therapy for Spinal Cord Injury

Traumatic spinal cord injury (SCI) elicits an intraspinal inflammatory response comprised of resident glia and infiltrating blood leukocytes. Several lines of evidence indicate that acute anti-inflammatory therapies (e.g., steroids, minocycline) can enhance recovery after SCI; however, these generic immune suppressive therapies may also affect reparative aspects of inflammation. Although subsets of resident and recruited immune cells have been implicated in CNS repair, >20 years of experimental data in different animal models of SCI indicate that acute monocyte depletion is consistently neuroprotective [14]. Why then are human clinical trials not being considered to target this immune cell population? It is an important question, especially since FDA-approved drugs already exist that can selectively deplete monocytes. At the recent NIH-sponsored SCI 2020: Launching a Decade for Disruption in SCI Research meeting, investigators, clinicians, and individuals living with SCI voiced frustration at the lack of translation of therapies from rodents to humans. In their report of the meeting, the North American SCI Consortium noted that “funded research portfolios need to reflect the needs of people living with SCI” and that “research is driven so much by innovation that studies needed for translation… don’t get funded.” Therefore, comprehensive preclinical evaluations of potential therapies are needed and outcomes measures that directly correlate to the human condition must be applied. From those discussions, it became clear that before acute monocyte depletion should be tried in human clinical trials, a number of fundamental scientific questions must be answered. What are the long-term consequences of acute monocyte depletion? Does the efficacy of monocyte depletion vary as a function of spinal injury level? Can monocyte depletion improve outcomes with the potential to improve the quality of life for SCI individuals? Therefore, the goals of this proposal are to examine the chronic and levelspecific functional changes after acute monocyte depletion using clinically relevant outcome measures including pain, autonomic dysreflexia, respiration, hindlimb and forelimb/hand function. Aim 1: Determine the effects of acute MD on myelopoiesis, biodistribution, and toxicity after SCI. SCI induces immune deficiency syndrome facilitated in part by monocytes with reduced function [15,16]. Further, monocyte deactivation is dependent upon SCI level [17]. Both SCI and acute MD will trigger the rapid synthesis of new monocytes (i.e., myelopoiesis) in the bone marrow and spleen (sites of monocyte production); however, the effects of MD on subsequent monocyte function and phenotype, as well as, potential side effects after SCI are unknown. Here we will perform dose-response studies to answer these questions while determining the toxicity and biodistribution of clodronate liposomes. Specifically, we will use flow cytometry to compare monocytes isolated from spinal cord, spleen, and bone marrow after MD in models of C5, T3, and T9 contusion SCI while performing organ-specific pathology analyses. Since we observe profound changes in myelopoiesis after SCI across many immune organs, we hypothesize that MD will cause renewal of phenotypically distinct monocytes vs. vehicle controls. Aim 2: Evaluate the effects of acute monocyte depletion (MD) on recovery of locomotor, sensory, and autonomic function in chronic SCI rats. The therapeutic potential of acute MD has been documented using several models and species of pre-clinical SCI; however, the most chronic studies to evaluate recovery of function were analyzed for only 12 weeks post-injury. It was recently reported that MD within the first 48 hours of SCI leads to long-term – up to 10 months – changes in the lesion microenvironment [18]. The magnitude and propensity of those effects on functional outcomes is unknown. Thus, it is important to understand how acute MD affects chronic indices of cell death, tissue repair, and spinal cord plasticity (e.g., axon sprouting, synapse formation). We will evaluate discrete aspects of motor, sensory, and autonomic function for 1 year after SCI. We will utilize T3 and T9 contusion injuries to compare outcomes between vehicle and liposomeencapsulated clodronate (a clinically approved pharmacotherapy for MD) treated animals. Based upon extensive published data showing the therapeutic potential of clodronate on locomotor outcomes, we hypothesize that acute MD will improve autonomic, sensory, and locomotor function chronically after injury. Aim 3: Determine the efficacy of acute MD on recovery of respiratory motor and forelimb function after cervical SCI. In humans, SCI most often occurs at the cervical spinal cord level where the central pattern generators (CPG) and motor pools for respiration and arm/hand function exist. Emerging data indicates that monocytes infiltrate non-traumatized tissue removed from the impact site [19]. Therefore, MD may be neuroprotective by limiting pathology at the site of injury, as well as, in CPG regions proximal and distal to the injury site. Whether acute MD is neuroprotective after cervical SCI has not been tested. Our published and preliminary data indicate that acute clodronate-mediated MD is feasible after unilateral cervical contusion SCI in rats. We will use this model to test our hypothesis that acute MD will protect respiratory motor pools to improve respiratory motor capacity and output. Further, we hypothesize that acute MD depletion will lead to long-term improvements in forelimb/paw motor and sensory function.