Proinflammatory and Immunomodulatory Gene and Protein Expression Patterns in Spinal Cord and Spleen Following Acute and Chronic High Thoracic Injury.

Introduction: In addition to paralysis and loss of sensation, high-level spinal cord injury (SCI) causes sympathetic dysfunction that can lead to autonomic dysreflexia (AD) and chronic immune suppression involving splenic leukopenia. Evidence has shown that treatment with either gabapentin or blockade of TNFα mitigates maladaptive plasticity and the underlying hemodynamic dysfunction, spleen atrophy, and immune dysfunction associated with AD. Because significant improvements long term was noted following treatments only during acute stages of recovery, we sought to systematically examine changes in proinflammatory and immunomodulatory cytokines to ascertain the reason. Methods: Adult female Wistar rats underwent complete T4 spinal transection before euthanasia at systematic intervals from 3 days to 8 weeks after injury. Using qRT-PCR and meso scale discovery (MSD) assays, the gene and protein expression of TNFα and IFNγ in the spleen, upper thoracic (T4-9) and lumbosacral (L5-S6) spinal cords were analyzed. Results: We found that spleen atrophy occurs in a biphasic manner compared to naïve controls, with significant decreases in the spleen mass noted at 3 days and 8 weeks after injury. Splenic TNFα mRNA and protein levels did not change significantly over time, while IFNγ gene expression dipped acutely with trends for increased protein levels at more chronic time points. TNFα protein increased significantly only in thoracic spinal cord segments from 3 to 14 days post-injury. IFNγ mRNA and protein levels remained unelevated in injured spinal cords over time, with trends for increased protein levels at 2 and 8 weeks in the lumbosacral segments. Discussion: Novel temporal-spatial cytokine expression profiles reveal that TNFα protein levels are increased solely in upper thoracic segments after high thoracic SCI, while IFNγ remains unaltered. Splenic leukopenia and latent systemic immunosuppression are not associated with altered TNFα or IFNγ expression in the spleen or spinal cord.

Delivery of mitoceuticals or respiratory competent mitochondria to sites of neurotrauma.

Herein, we review evidence that targeting mitochondrial dysfunction with 'mitoceuticals' is an effective neuroprotective strategy following neurotrauma, and that isolated exogenous mitochondria can be effectively transplanted into host spinal cord parenchyma to increase overall cellular metabolism. We further discuss control measures to ensure greatest potential for mitochondrial transfer, notably using erodible thermogelling hydrogels to deliver respiratory competent mitochondria to the injured spinal cord.

Erodible thermogelling hydrogels for localized mitochondrial transplantation to the spinal cord.

We developed a thermal-gelling, erodible hydrogel system for localized delivery of viable mitochondria in vivo, as well as labeled transplanted mitochondria with specific dyes and/or genetically modified mitochondria tagged with red fluorescence protein (RFP). We also employed cell lines to optimize a hydrogel composed of methylcellulose and hyaluronic acid designed to preserve bioenergetics while facilitating mitochondrial release. We further investigated how transplantation of allogeneic or xenogeneic mitochondria into respective cell lines affects host cellular metabolism, as measured by MTS assay. We found that 70% of mitochondria are released from the hydrogel within 20 min at 37 °C, that the respiratory capacity of hydrogel-released mitochondria over 60 min was greater than those without gel, and that MTR-labeling of mitochondria is not indelible. RFP-tagged transgenic mitochondria isolated from modified SH-SY5Y human neuroblastoma cells showed effective uptake into both naïve SH-SY5Y cells and rat PC-12 cells, notably when released from hydrogel. The hydrogel both protected the mitochondria at physiological conditions in vitro while solidifying and diffusing within 60 min locally in situ. To assess metabolic effects, both cell lines were transplanted with different concentrations of SH-SY5Y or PC-12 cell line-derived mitochondria and all resulted in significant increases in metabolism at 6- and 24-hour after transplantation. Alternatively, transplanted mitochondria at highest concentration from rat brain and spinal cord tissues reduced metabolic activities after 24-hour. Along with hydrogel refinements, we are further investigating whether such metabolic changes are due to alterations in cell proliferation or the number of exogenous mitochondria incorporated into individual host cells.

Rat Spinal Cord Injury Associated with Spasticity Leads to Widespread Changes in the Regulation of Retained Introns.

To determine molecular changes that correlate with long-term physiological changes after spinal cord injury associated with spasticity, we used a complete transection model with an injury at sacral spinal level S2, wherein tail spasms develop in rats weeks to months post-injury. Using Illumina and nanopore sequencing, we found that from 12,266 expressed genes roughly 11% (1,342) change expression levels in the rats with spasticity. The transcription factor PU.1 (Spi-1 proto-oncogene) and several of its known regulated genes were upregulated during injury, possibly reflecting changes in cellular composition. In contrast to widespread changes in gene expression, only a few changes in alternative exon usage could be detected because of injury. There were more than 1,000 changes in retained intron usage, however. Unexpectedly, most of these retained introns have not been described yet but could be validated using direct RNA nanopore sequencing. In addition to changes from injury, our model allowed regional analysis of gene expression. Comparing the segments rostral and caudal to the injury site in naïve animals showed 525 differentially regulated genes and differential regional use of retained introns. We did not detect changes in the serotonin receptor 2C editing that were implicated previously in this spinal cord injury model. Our data suggest that regulation of intron retention of polyadenylated pre-mRNA is an important regulatory mechanism in the spinal cord under both physiological and pathophysiological conditions.

Mitochondria focused neurotherapeutics for spinal cord injury.

The mitochondrion is a double membrane structured organelle involved in a variety of regulatory functions such as calcium signaling, production of adenosine triphosphate, apoptosis, reactive oxygen species generation, cell growth, and cell cycling. Impaired mitochondrial function is evident in various neurological disorders stemming from both acute and chronic neural injury. Herein, we review the role of mitochondrial regulation in maintaining cellular homeostasis, the consequences of their dysfunction in relation to pathophysiology after neurotrauma, approaches being used to promote their bioenergetic integrity for neuroprotection, and multifaceted methods being used to preserve/rescue their function following both traumatic brain and spinal cord injury.

Intraspinal Plasticity Associated With the Development of Autonomic Dysreflexia After Complete Spinal Cord Injury.

Traumatic spinal cord injury (SCI) leads to disruption of sensory, motor and autonomic function, and triggers structural, physiological and biochemical changes that cause reorganization of existing circuits that affect functional recovery. Propriospinal neurons (PN) appear to be very plastic within the inhibitory microenvironment of the injured spinal cord by forming compensatory circuits that aid in relaying information across the lesion site and, thus, are being investigated for their potential to promote locomotor recovery after experimental SCI. Yet the role of PN plasticity in autonomic dysfunction is not well characterized, notably, the disruption of supraspinal modulatory signals to spinal sympathetic neurons after SCI at the sixth thoracic spinal segment or above resulting in autonomic dysreflexia (AD). This condition is characterized by unmodulated sympathetic reflexes triggering sporadic hypertension associated with baroreflex mediated bradycardia in response to noxious yet unperceived stimuli below the injury to reduce blood pressure. AD is frequently triggered by pelvic visceral distension (bowel and bladder), and there are documented structural relationships between injury-induced sprouting of pelvic visceral afferent C-fibers. Their excitation of lumbosacral PN, in turn, sprout and relay noxious visceral sensory stimuli to rostral disinhibited thoracic sympathetic preganglionic neurons (SPN) that manifest hypertension. Herein, we review evidence for maladaptive plasticity of PN in neural circuits mediating heightened sympathetic reflexes after complete high thoracic SCI that manifest cardiovascular dysfunction, as well as contemporary research methodologies being employed to unveil the precise contribution of PN plasticity to the pathophysiology underlying AD development.