Harry G. Goshgarian

Context: Following a spinal cord hemisection at the second cervical segment the ipsilateral hemidiaphragm is paralyzed due to the disruption of the rostral ventral respiratory group (rVRG) axons descending to the ipsilateral phrenic motoneurons (PN). Systemically administered theophylline activates a functionally latent crossed phrenic pathway (CPP) which decussates caudal to the hemisection and activates phrenic motoneurons ipsilateral to the hemisection. The result is return of function to the paralyzed hemidiaphragm. Unfortunately, in humans, systemically administered theophylline at a therapeutic dose produces many unwanted side effects. Design and setting: A tripartite nanoconjugate was synthesized in which theophylline was coupled to a neuronal tracer, wheat germ agglutinin conjugated to horseradish peroxidase (WGA-HRP), using gold nanoparticles as the coupler. Following intradiaphragmatic injection of the nanoconjugate, WGA-HRP selectively targets the theophylline-bound nanoconjugate to phrenic motoneurons initially, followed by neurons in the rVRG by retrograde transsynaptic transport. Participants: (N/A) Interventions: (N/A) Outcome Measures: Immunostaining, Electromyography (EMG). Results: Delivery of the theophylline-coupled nanoconjugate to the nuclei involved in respiration induces a return of respiratory activity as detected by EMG of the diaphragm and a modest return of phrenic nerve activity. Conclusion: In addition to the modest return of phrenic nerve activity, there were many difficulties using the theophylline nanoconjugate because of its chemical instability, which suggests that the theophylline nanoconjugate should not be developed for clinical use as explained herein.

Adenosine operating in the nucleus of the solitary tract (NTS) may inhibit or facilitate neurotransmitter release from nerve terminals and directly inhibit or facilitate central neurons via A₁ and A_2a pre‐ and postsynaptic receptors, respectively. However, adenosine A_2a receptors, may also activate GABA‐ergic neurons/terminals which in turn inhibit glutamatergic transmission in the NTS network. Our previous studies showed that adenosine operating via both A₁ (inhibitor) and A_2a (activator) receptors powerfully inhibits the cardiopulmonary chemoreflex (CCR) at the level of the caudal NTS. A₁ receptors most likely inhibit glutamate release in the CCR network, whereas A_2a receptors facilitate NTS GABA‐ergic mechanisms which in turn inhibit CCR glutamatergic transmission. Therefore, we hypothesized that A_2a receptors are located on NTS GABA‐ergic neurons/terminals whereas A₁ receptors may be located on NTS glutamatergic neurons/terminals. We investigated this hypothesis using double immunofluorescent staining for A_2a or A₁ adenosine receptors and GABA synthesizing enzyme, GAD67, in 30 μm thick, floating, medullary rat sections. We found that A_2a adenosine receptors are localized within the GABA‐ergic cells in the caudal NTS, whereas A₁ adenosine receptors are absent from these neurons. Instead, A₁ receptors were located on non‐GABA‐ergic (likely glutamatergic) neurons/terminals in the caudal NTS. These data support our functional findings and the hypothesis that adenosine A_2a, but not A₁ receptors are located on GABA‐ergic neurons.

Drug delivery to the central nervous system (CNS) is challenging due to the inability of many drugs to cross the blood-brain barrier (BBB). Here, we show that wheat germ agglutinin horse radish peroxidase (WGA-HRP) chemically conjugated to gold nanoparticles (AuNPs) can be transported to the spinal cord and brainstem following intramuscular injection into the diaphragm of rats. We synthesized and determined the size and chemical composition of a three-part nanoconjugate consisting of WGA-HRP, AuNPs and drugs for the treatment of diaphragm paralysis associated with high cervical spinal cord injury (SCI). Upon injection into the diaphragm muscle of rats, we show that the nanoconjugate is capable of delivering the drug at a much lower dose than the unconjugated drug injected systemically to effectively induce respiratory recovery in rats following SCI. This study not only demonstrates a promising strategy to deliver drugs to the CNS bypassing the BBB but also contributes a potential nanotherapy for the treatment of respiratory muscle paralysis resulted from cervical SCI.

Respiratory complications in patients with spinal cord injury (SCI) are common and have a negative impact on the quality of patients' lives. Systemic administration of drugs that improve respiratory function often cause deleterious side effects. The present study examines the applicability of a novel nanotechnology-based drug delivery system, which induces recovery of diaphragm function after SCI in the adult rat model. We developed a protein-coupled nanoconjugate to selectively deliver by transsynaptic transport small therapeutic amounts of an A1 adenosine receptor antagonist to the respiratory centers. A single administration of the nanoconjugate restored 75% of the respiratory drive at 0.1% of the systemic therapeutic drug dose. The reduction of the systemic dose may obviate the side effects. The recovery lasted for 4 weeks (the longest period studied). These findings have translational implications for patients with respiratory dysfunction after SCI.SIGNIFICANCE STATEMENTThe leading causes of death in humans following SCI are respiratory complications secondary to paralysis of respiratory muscles. Systemic administration of methylxantines improves respiratory function but also leads to the development of deleterious side effects due to actions of the drug on nonrespiratory sites. The importance of the present study lies in the novel drug delivery approach that uses nanotechnology to selectively deliver recovery-inducing drugs to the respiratory centers exclusively. This strategy allows for a reduction in the therapeutic drug dose, which may reduce harmful side effects and markedly improve the quality of life for SCI patients.