Intraspinal transplantation of subventricular zone-derived neural progenitor cells improves phrenic motor output after high cervical spinal cord injury.

Following spinal cord injury (SCI), intraspinal transplantation of neural progenitor cells (NPCs) harvested from the forebrain sub-ventricular zone (SVZ) can improve locomotor outcomes. Cervical SCI often results in respiratory-related impairments, and here we used an established model cervical SCI (C2 hemisection, C2Hx) to confirm the feasibility of mid-cervical transplantation of SVZ-derived NPCs and the hypothesis that that this procedure would improve spontaneous respiratory motor recovery. NPCs were isolated from the SVZ of enhanced green fluorescent protein (GFP) expressing neonatal rats, and then intraspinally delivered immediately caudal to an acute C2Hx lesion in adult non-GFP rats. Whole body plethysmography conducted at 4 and 8wks post-transplant demonstrated increased inspiratory tidal volume in SVZ vs. sham transplants during hypoxic (P=0.003) or hypercapnic respiratory challenge (P=0.019). Phrenic nerve output was assessed at 8wks post-transplant; burst amplitude recorded ipsilateral to C2Hx was greater in SVZ vs. sham rats across a wide range of conditions (e.g., quiet breathing through maximal chemoreceptor stimulation; P<0.001). Stereological analyses at 8wks post-injury indicated survival of ~50% of transplanted NPCs with ~90% of cells distributed in ipsilateral white matter at or near the injection site. Peak inspiratory phrenic bursting after NPC transplant was positively correlated with the total number of surviving cells (P<0.001). Immunohistochemistry confirmed an astrocytic phenotype in a subset of the transplanted cells with no evidence for neuronal differentiation. We conclude that intraspinal transplantation of SVZ-derived NPCs can improve respiratory recovery following high cervical SCI.

Respiratory outcomes after mid-cervical transplantation of embryonic medullary cells in rats with cervical spinal cord injury.

Respiratory motor output after cervical spinal cord injury (cSCI) is profoundly influenced by spinal serotonin. We hypothesized that intraspinal transplantation of embryonic midline brainstem (MB) cells rich in serotonergic raphé neurons would improve respiratory outcomes after cSCI. One week after hemisection of the 2nd cervical segment (C2Hx) a suspension of either embryonic (E14) MB cells, fetal spinal cord cells (FSC), or media only (sham) was delivered to the dorsal C3 spinal cord of adult male rats. Six weeks later, ventilation was evaluated using plethysmography; phrenic nerve activity was evaluated in a subset of rats. Seven of 12 rats receiving MB-derived grafts had clear histological evidence of serotonin-positive neurons in the C3-4 dorsal white matter. The transplantations had no impact on baseline breathing patterns, but during a brief respiratory challenge (7% inspired CO2) rats with successful MB grafts had increased ventilation compared to rats with failed MB grafts, FSC or sham grafts. Recordings from the phrenic nerve ipsilateral to C2Hx also indicated increased output during respiratory challenge in rats with successful MB grafts. We conclude that intraspinal allografting of E14 MB cells can have a positive impact on respiratory motor recovery following high cSCI.

Rapid diaphragm atrophy following cervical spinal cord hemisection.

A cervical (C2) hemilesion (C2Hx), which disrupts ipsilateral bulbospinal inputs to the phrenic nucleus, was used to study diaphragm plasticity after acute spinal cord injury. We hypothesized that C2Hx would result in rapid atrophy of the ipsilateral hemidiaphragm and increases in mRNA expression of proteolytic biomarkers. Diaphragm tissue was harvested from male Sprague-Dawley rats at 1 or 7 days following C2Hx. Histological analysis demonstrated reduction in cross-sectional area (CSA) of type I and IIa fibers in the ipsilateral hemidiaphragm at 1 but not 7 days. Type IIb/x fibers, however, had reduced CSA at 1 and 7 days. A targeted gene array was used to screen mRNA changes for genes associated with skeletal muscle myopathy and myogenesis; this was followed by qRT-PCR validation. Changes in diaphragm gene expression suggested that profound myoplasticity is initiated immediately following C2Hx including activation of both proteolytic and myogenic pathways. We conclude that an immediate myoplastic response occurs in the diaphragm after C2Hx with atrophy occurring in ipsilateral myofibers within 1 day.

Repeated intravenous doxapram induces phrenic motor facilitation.

Doxapram is a respiratory stimulant used to treat hypoventilation. Here we investigated whether doxapram could also trigger respiratory neuroplasticity. Specifically, we hypothesized that intermittent delivery of doxapram at low doses would lead to long-lasting increases (i.e., facilitation) of phrenic motor output in anesthetized, vagotomized, and mechanically-ventilated rats. Doxapram was delivered intravenously in a single bolus (2 or 6mg/kg) or as a series of 3 injections (2mg/kg) at 5min intervals. Control groups received pH-matched saline injections (vehicle) or no treatment (anesthesia time control). Doxapram evoked an immediate increase in phrenic output in all groups, but a persistent increase in burst amplitude only occurred after repeated dosing with 2mg/kg. At 60min following the last injection, phrenic burst amplitude was 168±24% of baseline (%BL) in the group receiving 3 injections (P<0.05 vs. controls), but was 103±8%BL and 112±4%BL in the groups receiving a single dose of 2 or 6mg/kg, respectively. Following bilateral section of the carotid sinus nerves, the acute phrenic response to doxapram (2mg/kg) was reduced by 68% suggesting that at low doses the drug was acting primarily via the carotid chemoreceptors. We conclude that intermittent application of doxapram can trigger phrenic neuroplasticity, and this approach might be of use in the context of respiratory rehabilitation following neurologic injury.

Recovery of inspiratory intercostal muscle activity following high cervical hemisection.

Anatomical and neurophysiological evidence indicates that thoracic interneurons can serve a commissural function and activate contralateral motoneurons. Accordingly, we hypothesized that respiratory-related intercostal (IC) muscle electromyogram (EMG) activity would be only modestly impaired by a unilateral cervical spinal cord injury. Inspiratory tidal volume (VT) was recorded using pneumotachography and EMG activity was recorded bilaterally from the 1st to 2nd intercostal space in anesthetized, spontaneously breathing rats. Studies were conducted at 1-3 days, 2 wks or 8 wks following C2 spinal cord hemisection (C2HS). Data were collected during baseline breathing and a brief respiratory challenge (7% CO(2)). A substantial reduction in inspiratory intercostal EMG bursting ipsilateral to the lesion was observed at 1-3 days post-C2HS. However, a time-dependent return of activity occurred such that by 2 wks post-injury inspiratory intercostal EMG bursts ipsilateral to the lesion were similar to age-matched, uninjured controls. The increases in ipsilateral intercostal EMG activity occurred in parallel with increases in VT following the injury (R=0.55; P<0.001). We conclude that plasticity occurring within a "crossed-intercostal" circuitry enables a robust, spontaneous recovery of ipsilateral intercostal activity following C2HS in rats.

The phrenic motor nucleus in the adult mouse.

The present study was performed to establish an anatomical context for studies of phrenic motor function in mouse models of central nervous system trauma and disease. Application of cholera toxin ß-subunit to the diaphragm of adult C57BL/6 mice revealed a columnar organization of phrenic motoneurons (PhMNs) which extended from rostral C3 to C6. Injection of Miniruby into the ventrolateral medulla revealed decussating, anterogradely labeled axons in the cervical spinal cord. In addition, application of the transneuronal tracer pseudorabies virus (PRV) to the right hemidiaphragm demonstrated a population of putative pre-phrenic interneurons at the level of the infected PhMN pool. These neuroanatomical features of the mouse phrenic nucleus are consistent with those described in other species and provide a foundation for studies of neuroplasticity and repair in relation to a functionally and anatomically identified spinal network.

Phrenicotomy alters phrenic long-term facilitation following intermittent hypoxia in anesthetized rats.

Intermittent hypoxia (IH) can induce a persistent increase in neural drive to the respiratory muscles known as long-term facilitation (LTF). LTF of phrenic inspiratory activity is often studied in anesthetized animals after phrenicotomy (PhrX), with subsequent recordings being made from the proximal stump of the phrenic nerve. However, severing afferent and efferent axons in the phrenic nerve has the potential to alter the excitability of phrenic motoneurons, which has been hypothesized to be an important determinant of phrenic LTF. Here we test the hypothesis that acute PhrX influences immediate and long-term phrenic motor responses to hypoxia. Phrenic neurograms were recorded in anesthetized, ventilated, and vagotomized adult male rats with intact phrenic nerves or bilateral PhrX. Data were obtained before (i.e., baseline), during, and after three 5-min bouts of isocapnic hypoxia. Inspiratory burst amplitude during hypoxia (%baseline) was greater in PhrX than in phrenic nerve-intact rats (P < 0.001). Similarly, burst amplitude 55 min after IH was greater in PhrX than in phrenic nerve-intact rats (175 + or - 9 vs. 126 + or - 8% baseline, P < 0.001). In separate experiments, phrenic bursting was recorded before and after PhrX in the same animal. Afferent bursting that was clearly observable in phase with lung deflation was immediately abolished by PhrX. The PhrX procedure also induced a form of facilitation as inspiratory burst amplitude was increased at 30 min post-PhrX (P = 0.01 vs. pre-PhrX). We conclude that, after PhrX, axotomy of phrenic motoneurons and, possibly, removal of phrenic afferents result in increased phrenic motoneuron excitability and enhanced LTF following IH.

The Pristionchus HOX gene Ppa-lin-39 inhibits programmed cell death to specify the vulva equivalence group and is not required during vulval induction.

In the two nematode species Caenorhabditis elegans and Pristionchus pacificus the vulva equivalence group in the central body region is specified by the Hox gene lin-39. C. elegans lin-39 mutants are vulvaless and the vulval precursor cells fuse with the surrounding hypodermis, whereas in P. pacificus lin-39 mutants the vulval precursor cells die by apoptosis. Mechanistically, LIN-39 might inhibit non-vulval fate (cell fusion in C. elegans, apoptosis in P. pacificus), promote vulval fate or do both. To study the mechanism of lin-39 function, we isolated P. pacificus cell death mutants and identified mutations in ced-3. Surprisingly, P. pacificus ced-3; lin-39 double mutants form a functional vulva in the absence of LIN-39 activity. Thus, in P. pacificus lin-39 specifies the vulva equivalence group by inhibiting programmed cell death. Furthermore, these data reveal an important difference in a later function of lin-39 between the two species. In C. elegans, LIN-39 specifies vulval cell fates in response to inductive RAS signaling, and in P. pacificus LIN-39 is not required for vulval induction. Thus, the comparative analysis indicates that lin-39 has distinct functions in both species although the gene is acting in a homologous developmental system.