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Targeting the potassium-chloride cotransporter, KCC2, to improve sympathetic dysfunction after high-level spinal cord injury
Dissertation

Targeting the potassium-chloride cotransporter, KCC2, to improve sympathetic dysfunction after high-level spinal cord injury

Mariah Jade Wulf
Doctor of Philosophy (Ph.D.), Drexel University
Aug 2026
DOI:
https://doi.org/10.17918/00011532
pdf
Wulf_Mariah_20265.03 MB
PDF Embargoed Access, Embargo ends: 31 Aug 2028

Abstract

Cellular biology Autonomic dysfunction Circuit plasticity Immune dysfunction Sympathetic dysfunction Influenza Spinal Cord Injury
Cardiovascular and immune dysfunction are leading causes of morbidity and mortality in individuals living with high-level, severe, spinal cord injuries (SCI). This is due to the detrimental impact of heightened sympathetic reflexes (i.e., sympathetic hyperreflexia) after SCI on organs receiving sympathetic input. Sympathetic hyperreflexia is driven by changes within the spinal sympathetic reflex (SSR) circuit below the injury that contributes to increased excitability of the circuit. However, the exact mechanisms for the development and progression of sympathetic hyperreflexia remain largely unknown. We sought to elucidate such mechanisms that mediate the development of sympathetic hyperreflexia. The K⁺-Cl⁻ cotransporter type 2 (KCC2), which is expressed solely in central nervous system neurons, helps to maintain low levels of intracellular Cl⁻ that is essential for GABAergic and glycinergic neuronal inhibition. Interestingly, decreased membrane expression of KCC2 has been implicated in a variety of pathologies associated with increased neuronal activity. We hypothesize that spinal cord injury induced loss of KCC2 on the membrane of neurons within the SSR circuit is a mechanism for the development of sympathetic hyperreflexia. We further hypothesize that pharmacologically increasing KCC2 localization to the membrane of SSR circuit neurons with mitigate sympathetic hyperreflexia and associated cardiovascular and immune dysfunctions following spinal cord injury. To test this, we administered CLP290 a known KCC2 enhancer drug, daily to adult, wild-type, mice starting one week following a complete spinal transection at thoracic segment 3 (T3Tx) -- an injury known to reliably result in sympathetic hyperreflexia. Hemodynamic activity was recorded weekly (4 total weeks) after T3Tx to assess spontaneous autonomic dysreflexia (AD) events -- a real-time readout of sympathetic hyperreflexia. Histological analyses of intraspinal plasticity of the SSR circuit in these mice further examines the role KCC2 plays in driving sympathetic dysfunction after injury. Sympathetic hyperreflexia is also associated with loss of leukocytes in the spleen and dysimmunity with infections being the leading cause of mortality within the SCI population. To directly assess if targeting KCC2 enhances immunity after SCI, we intranasally administered influenza to vehicle- or CLP290-treated animals 4 weeks after T3Tx. Seven or ten days after infection, spleens and lungs were collected and processed via flow cytometry to assess immune cell profiling. Similarly, 10-days post infection lungs were collected fresh to analyze viral clearance. The findings presented will demonstrate the critical role KCC2 is playing in the development and progression of sympathetic hyperreflexia and subsequent detrimental secondary consequences. Moreover, this research highlights a potential novel therapeutic target to attenuate sympathetic dysfunction following an injury.

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