Brainstem-Evoked Transcription of Defensive Genes After Spinal Cord Injury.

The spinal cord after injury shows altered transcription in numerous genes. We tested in a pilot study whether the nucleus raphé magnus, a descending serotonergic brainstem region whose stimulation improves recovery after incomplete spinal cord injury (SCI), can influence these transcriptional changes. Rats received 2 h of low-frequency electrical stimulation in the raphé magnus 3 days after an impact contusion at segment T8. Comparison groups lacked injuries or activated stimulators or both. Immediately following stimulation, spinal cords were extracted, their RNA transcriptome sequenced, and differential gene expression quantified. Confirming many previous studies, injury primarily increased inflammatory and immune transcripts and decreased those related to lipid and cholesterol synthesis and neuronal signaling. Stimulation plus injury, contrasted with injury alone, caused significant changes in 43 transcripts (39 increases, 4 decreases), all protein-coding. Injury itself decreased only four of these 43 transcripts, all reversed by stimulation, and increased none of them. The non-specific 5-HT7 receptor antagonist pimozide reversed 25 of the 43 changes. Stimulation in intact rats principally caused decreases in transcripts related to oxidative phosphorylation, none of which were altered by stimulation in injury. Gene ontology (biological process) annotations comparing stimulation with either no stimulation or pimozide treatment in injured rats highlighted defense responses to lipopolysaccharides and microorganisms, and also erythrocyte development and oxygen transport (possibly yielding cellular oxidant detoxification). Connectivity maps of human orthologous genes generated in the CLUE database of perturbagen-response transcriptional signatures showed that drug classes whose effects in injured rats most closely resembled stimulation without pimozide include peroxisome proliferator-activated receptor agonists and angiotensin receptor blockers, which are reportedly beneficial in SCI. Thus the initial transcriptional response of the injured spinal cord to raphé magnus stimulation is upregulation of genes that in various ways are mostly protective, some probably located in recently arrived myeloid cells.

Cellular Changes in Injured Rat Spinal Cord Following Electrical Brainstem Stimulation.

Spinal cord injury (SCI) is a major cause of disability and pain, but little progress has been made in its clinical management. Low-frequency electrical stimulation (LFS) of various anti-nociceptive targets improves outcomes after SCI, including motor recovery and mechanical allodynia. However, the mechanisms of these beneficial effects are incompletely delineated and probably multiple. Our aim was to explore near-term effects of LFS in the hindbrain's nucleus raphe magnus (NRM) on cellular proliferation in a rat SCI model. Starting 24 h after incomplete contusional SCI at C5, intermittent LFS at 8 Hz was delivered wirelessly to NRM. Controls were given inactive stimulators. At 48 h, 5-bromodeoxyuridine (BrdU) was administered and, at 72 h, spinal cords were extracted and immunostained for various immune and neuroglial progenitor markers and BrdU at the level of the lesion and proximally and distally. LFS altered cell marker counts predominantly at the dorsal injury site. BrdU cell counts were decreased. Individually and in combination with BrdU, there were reductions in CD68 (monocytes) and Sox2 (immature neural precursors) and increases in Blbp (radial glia) expression. CD68-positive cells showed increased co-staining with iNOS. No differences in the expression of GFAP (glia) and NG2 (oligodendrocytes) or in GFAP cell morphology were found. In conclusion, our work shows that LFS of NRM in subacute SCI influences the proliferation of cell types implicated in inflammation and repair, thus providing mechanistic insight into deep brain stimulation as a neuromodulatory treatment for this devastating pathology.

Cells in the adult human spinal cord ependymal region do not proliferate after injury.

In vertebrates that regenerate the injured spinal cord, cells at the ependymal region proliferate and coordinate the formation of bridges between the lesion stumps. In mammals, these cells also proliferate profusely around the central canal after spinal cord injury, although their actual contribution to repair is controversial. In humans, however, the central canal disappears from early childhood in the majority of individuals, being replaced by astrocyte gliosis, ependymocyte clusters, and perivascular pseudo-rosettes. In this human ependymal remnant, cells do not proliferate under normal conditions, but it is not known if they do after a lesion. Here, we studied the human ependymal remnant after traumatic spinal cord injury using samples from 21 individuals with survival times ranging from days to months post-injury. With three different monoclonal antibodies raised against two different proliferation markers (Ki67 and MCM2), we found that the ependymal remnant in adult humans does not proliferate after injury at any time or distance from the lesion. Our results seriously challenge the view of the spinal cord ependymal region as a neurogenic niche in adult humans and suggest that it would not be involved in cell replacement after a lesion. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Some Autonomic Deficits of Acute or Chronic Cervical Spinal Contusion Reversed by Interim Brainstem Stimulation.

Prolonged electrical stimulation of the hindbrain's nucleus raphe magnus (NRM) or of its major midbrain input region, the periaqueductal gray (PAG), was previously found in rats to promote recovery from sensory-motor and histological deficits of acute thoracic spinal cord injury (SCI). Here, some visceral deficits of acute and chronic midline cervical (C5) contusion are similarly examined. Cranially implanted wireless stimulators delivered intermittent 8 Hz, 30-70 µA cathodal pulse trains to a brainstem microelectrode. Injured controls were given inactive stimulators; rats without injuries or implants were also compared. Rectal distension or squeezing of the forepaws caused an exaggerated rise in mean arterial pressure in injured, untreated rats under anesthesia on post-injury week 6, probably reflecting autonomic dysreflexia (AD). These pressor responses became normal when 7 days of unilateral PAG stimulation was started on the injury day. Older untreated injuries (weeks 18-19) showed normal pressor responses, but unexpectedly had significant resting and nociceptive bradycardia, which was reversed by 3 weeks of PAG stimulation started on weeks 7 or 12. Subsequent chronic studies examined gastric emptying (GE), as indicated by intestinal transit of gavaged dye, and serum chemistry. GE and fasting serum insulin were reduced on injury weeks 14-15, and were both normalized by ∼5 weeks of PAG stimulation begun in weeks 7-8. Increases in calcitonin gene-related peptide, a prominent visceral afferent neurotransmitter, measured near untreated injuries (first thoracic segment) in superficial dorsal laminae were reversed by acutely or chronically initiated PAG stimulation. The NRM, given 2-3 weeks of stimulation beginning 2 days after SCI, prevented abnormalities in both pressor responses and GE on post-injury week 9, consistent with its relaying of repair commands from the PAG. The descending PAG-NRM axis thus exhibits broadly restorative influences on visceral as well as sensory-motor deficits, improving chronic as well as acute signs of injury.

Prolonged stimulation of a brainstem raphe region attenuates experimental autoimmune encephalomyelitis.

Multiple sclerosis (MS), a neuroinflammatory disease, has few treatment options, none entirely adequate. We studied whether prolonged electrical microstimulation of a hindbrain region (the nucleus raphe magnus) can attenuate experimental autoimmune encephalomyelitis, a murine model of MS induced by MOG35-55 injection. Eight days after symptoms emerged, a wireless electrical stimulator with an attached microelectrode was implanted cranially, and daily intermittent stimulation was begun in awake, unrestrained mice. The thoracic spinal cord was analyzed for changes in histology (on day 29) and gene expression (on day 37), with a focus on myelination and cytokine production. Controls, with inactive implants, showed a phase of disease exacerbation on days 19-25 that stimulation for >16days eliminated. Prolonged stimulation also reduced numbers of infiltrating immune cells and increased numbers of myelinated axons. It additionally lowered genetic expression of some pro-inflammatory cytokines (interferon gamma and tumor necrosis factor) and platelet-derived growth factor receptor alpha, a marker of oligodendrocyte precursors, while raising expression of myelin basic protein. Studies of restorative treatments for MS might profitably consider ways to stimulate the raphe magnus, directly or via its inputs, or to emulate its serotonergic and peptidergic output.

The genetic audiogenic seizure hamster from Salamanca: The GASH:Sal.

The hamster has been previously described as a paroxysmal dystonia model, but our strain is currently recognized as a model of audiogenic seizures (AGS). The original first epileptic hamster appeared spontaneously at the University of Valladolid, where it was known as the GPG:Vall line, and was transferred to the University of Salamanca where a new strain was developed, named GASH:Sal. By testing auditory brainstem responses, the GASH:Sal exhibits elevated auditory thresholds that indicate a hearing impairment. Moreover, amplified fragment length polymorphism analysis distinguished genetic differences between the susceptible GASH:Sal hamster strain and the control Syrian hamsters. The GASH:Sal constitutes an experimental model of reflex epilepsy of audiogenic origin derived from an autosomal recessive disorder. Thus, the GASH:Sal exhibits generalized tonic-clonic seizures, characterized by a short latency period after auditory stimulation, followed by wild running, a convulsive phase, and finally stupor, with origin in the brainstem. The seizure profile of the GASH:Sal is similar to those exhibited by other models of inherited AGS susceptibility, which decreases after six months of age, but the proneness across generations is maintained. The GASH:Sal can be considered a reliable model of audiogenic seizures, suitable to investigate current antiepileptic pharmaceutical treatments as well as novel therapeutic drugs. This article is part of a Special Issue entitled "Genetic and Reflex Epilepsies, Audiogenic Seizures and Strains: From Experimental Models to the Clinic".