A hydrogel engineered to deliver minocycline locally to the injured cervical spinal cord protects respiratory neural circuitry and preserves diaphragm function.

We tested a biomaterial-based approach to preserve the critical phrenic motor circuitry that controls diaphragm function by locally delivering minocycline hydrochloride (MH) following cervical spinal cord injury (SCI). MH is a clinically-available antibiotic and anti-inflammatory drug that targets a broad range of secondary injury mechanisms via its anti-inflammatory, anti-oxidant and anti-apoptotic properties. However, MH is only neuroprotective at high concentrations that cannot be achieved by systemic administration, which limits its clinical efficacy. We have developed a hydrogel-based MH delivery system that can be injected into the intrathecal space for local delivery of high concentrations of MH, without damaging spinal cord tissue. Implantation of MH hydrogel after unilateral level-C4/5 contusion SCI robustly preserved diaphragm function, as assessed by in vivo recordings of compound muscle action potential (CMAP) and electromyography (EMG) amplitudes. MH hydrogel also decreased lesion size and degeneration of cervical motor neuron somata, demonstrating its central neuroprotective effects within the injured cervical spinal cord. Furthermore, MH hydrogel significantly preserved diaphragm innervation by the axons of phrenic motor neurons (PhMNs), as assessed by both detailed neuromuscular junction (NMJ) morphological analysis and retrograde PhMN labeling from the diaphragm using cholera toxin B (CTB). In conclusion, our findings demonstrate that local MH hydrogel delivery to the injured cervical spinal cord is effective in preserving respiratory function after SCI by protecting the important neural circuitry that controls diaphragm activation.

Local BDNF Delivery to the Injured Cervical Spinal Cord using an Engineered Hydrogel Enhances Diaphragmatic Respiratory Function.

We developed an innovative biomaterial-based approach to repair the critical neural circuitry that controls diaphragm activation by locally delivering brain-derived neurotrophic factor (BDNF) to injured cervical spinal cord. BDNF can be used to restore respiratory function via a number of potential repair mechanisms; however, widespread BDNF biodistribution resulting from delivery methods such as systemic injection or lumbar puncture can lead to inefficient drug delivery and adverse side effects. As a viable alternative, we developed a novel hydrogel-based system loaded with polysaccharide-BDNF particles self-assembled by electrostatic interactions that can be safely implanted in the intrathecal space for achieving local BDNF delivery with controlled dosing and duration. Implantation of BDNF hydrogel after C4/C5 contusion-type spinal cord injury (SCI) in female rats robustly preserved diaphragm function, as assessed by in vivo recordings of compound muscle action potential and electromyography amplitudes. However, BDNF hydrogel did not decrease lesion size or degeneration of cervical motor neuron soma, suggesting that its therapeutic mechanism of action was not neuroprotection within spinal cord. Interestingly, BDNF hydrogel significantly preserved diaphragm innervation by phrenic motor neurons (PhMNs), as assessed by detailed neuromuscular junction morphological analysis and retrograde PhMN labeling from diaphragm using cholera toxin B. Furthermore, BDNF hydrogel enhanced the serotonergic axon innervation of PhMNs that plays an important role in modulating PhMN excitability. Our findings demonstrate that local BDNF hydrogel delivery is a robustly effective and safe strategy to restore diaphragm function after SCI. In addition, we demonstrate novel therapeutic mechanisms by which BDNF can repair respiratory neural circuitry.SIGNIFICANCE STATEMENT Respiratory compromise is a leading cause of morbidity and mortality following traumatic spinal cord injury (SCI). We used an innovative biomaterial-based drug delivery system in the form of a hydrogel that can be safely injected into the intrathecal space for achieving local delivery of brain-derived neurotrophic factor (BDNF) with controlled dosing and duration, while avoiding side effects associated with other delivery methods. In a clinically relevant rat model of cervical contusion-type SCI, BDNF hydrogel robustly and persistently improved diaphragmatic respiratory function by enhancing phrenic motor neuron (PhMN) innervation of the diaphragm neuromuscular junction and by increasing serotonergic innervation of PhMNs in ventral horn of the cervical spinal cord. These exciting findings demonstrate that local BDNF hydrogel delivery is a safe and robustly effective strategy to maintain respiratory function after cervical SCI.

Differential Response in Novel Stem Cell Niches of the Brain after Cervical Spinal Cord Injury and Traumatic Brain Injury.

Populations of neural stem cells (NSCs) reside in a number of defined niches in the adult central nervous system (CNS) where they continually give rise to mature cell types throughout life, including newly born neurons. In addition to the prototypical niches of the subventricular zone (SVZ) and subgranular zone (SGZ) of the hippocampal dentate gyrus, novel stem cell niches that are also neurogenic have recently been identified in multiple midline structures, including circumventricular organs (CVOs) of the brain. These resident NSCs serve as a homeostatic source of new neurons and glial cells under intact physiological conditions. Importantly, they may also have the potential for reparative processes in pathological states such as traumatic spinal cord injury (SCI) and traumatic brain injury (TBI). As the response in these novel CVO stem cell niches has been characterized after stroke but not following SCI or TBI, we quantitatively assessed cell proliferation and the neuronal and glial lineage fate of resident NSCs in three CVO nuclei-area postrema (AP), median eminence (ME), and subfornical organ (SFO) -in rat models of cervical contusion-type SCI and controlled cortical impact (CCI)-induced TBI. Using bromodeoxyuridine (BrdU) labeling of proliferating cells, we find that TBI significantly enhanced proliferation in AP, ME, and SFO, whereas cervical SCI had no effects at early or chronic time-points post-injury. In addition, SCI did not alter NSC differentiation profile into doublecortin-positive neuroblasts, GFAP-expressing astrocytes, or Olig2-labeled cells of the oligodendrocyte lineage within AP, ME, or SFO at both time-points. In contrast, CCI induced a pronounced increase in Sox2- and doublecortin-labeled cells in the AP and Iba1-labeled microglia in the SFO. Lastly, plasma derived from CCI animals significantly increased NSC expansion in an in vitro neurosphere assay, whereas plasma from SCI animals did not exert such an effect, suggesting that signaling factors present in blood may be relevant to stimulating CVO niches after CNS injury and may explain the differential in vivo effects of SCI and TBI on the novel stem cell niches.