Cervical Spinal Cord Hemisection Research Articles

Cervical spinal cord hemisection impacts sigh and the respiratory reset in male rats.

Cervical spinal cord injury impacts ventilatory and non-ventilatory functions of the diaphragm muscle (DIAm) and contributes to clinical morbidity and mortality in the afflicted population. Periodically, integrated brainstem neural circuit activity drives the DIAm to generate a markedly augmented effort or sigh-which plays an important role in preventing atelectasis and thus maintaining lung function. Across species, the general pattern of DIAm efforts during a normal sigh is variable in amplitude and the extent of post-sigh "apnea" (i.e., the post-sigh inter-breath interval). This post-sigh inter-breath interval acts as a respiratory reset, following the interruption of regular respiratory rhythm by sigh. We examined the impact of upper cervical (C2 ) spinal cord hemisection (C2 SH) on the transdiaphragmatic pressure (Pdi ) generated during sighs and the post-sigh respiratory reset in rats. Sighs were identified in Pdi traces by their characteristic biphasic pattern. We found that C2 SH results in a reduction of Pdi during both eupnea and sighs, and a decrease in the immediate post-sigh breath interval. These results are consistent with partial removal of descending excitatory synaptic inputs to phrenic motor neurons that results from C2 SH. Following cervical spinal cord injury, a reduction in the amplitude of Pdi during sighs may compromise the maintenance of normal lung function.

Cerebrolysin recovers diaphragmatic function and reduces injury-associated astrogliosis following a cervical spinal cord hemi-section injury in rats

BackgroundSpinal cord injury (SCI) is widely considered the most disastrous medical condition. With no available treatment to date, SCI continues to cause disabilities to the patients and affect their own and their caregivers' quality of life. Cerebrolysin (CBL) is a neuropeptide preparation derived from purified brain proteins with suggested neuroprotective and neurotrophic properties. CBL showed earlier the ability to target multiple pathways that helped in the improvement of the recovery following different groups of neurological diseases and injuries, including ischemic stroke, traumatic brain injuries, and even neurodegenerative diseases. In the current study, the neuroprotective effect of CBL following a SCI is called into question using a rat model of spinal cord cervical hemi-section validated earlier by our lab and others. Using 32 rats divided into four groups randomly, CBL treatment was implemented for either 7 or 21 days duration, following the cervical spinal cord hemi-section.ResultsFollowing the CBL treatment, rats with cervical cord hemi-section showed functional improvement of diaphragmatic muscle as recorded by electromyography (EMG). In addition, the histopathological assessment of the spinal cord showed improved neuronal viability and reduced astrogliosis at the site of the injury compared to the non-treated group. 21-day treatment showed significant improvement when compared to the shorter 7-day regimen.ConclusionOur data suggest that CBL is capable of protecting and regenerating anterior horn motor neurons with functional recovery of diaphragmatic muscle functions in rats, suggesting the potential use of CBL for future regenerative and neuroprotective therapy following SCI.

Mitochondria Adapt to Diaphragm Muscle Inactivity Imposed by Cervical Spinal Cord Injury

Type I and IIa diaphragm muscle (DIAm) fibers comprise fatigue resistant slow and fast motor units that are recruited to accomplish breathing. In contrast, type IIx/IIb fibers comprise more fatigable fast motor units that are infrequently recruited only during airway protective and straining behaviors. The activity and energetic demand for type I and IIa DIAm fibers is markedly increased compared to type IIx/IIb fibers. Cervical spinal cord hemisection at C2 (C2SH) removes excitatory drive for inspiratory activity of the DIAm and thereby imposing prolonged inactivity of type I and IIa fibers. We hypothesize that mitochondrial structure and function in type I and IIa DIAm fibers adapt in response to imposed inactivity induced by spinal cord hemisection at C2 (C2SH). After C2SH, inactivity was confirmed by the absence of DIAm EMG, and after 14 days, and the effect of inactivity on mitochondrial structure and function was assessed in type identified fibers. Mitochondria in DIAm fibers were labeled using MitoTracker Green and imaged in 3‐D by fluorescence confocal microscopy. Mitochondrial volume density and morphometry were analyzed using ImageJ. In alternate serial sections of the same DIAm fibers, the maximum velocity of the succinate dehydrogenase reaction (SDHmax) was determined using a quantitative histochemical technique. Overall, mitochondrial volume density was higher in type I and IIa DIAm fibers and mitochondria were more filamentous compared to type IIx/IIb fibers. Inactivity reduced mitochondrial volume density in type I and IIa DIAm fibers and induced mitochondrial fragmentation. The SDHmax values normalized for fiber volume and for mitochondrial volume were both higher in type I and IIa DIAm fibers compared to type IIx/IIb fibers. After 14 days of inactivity induced by C2SH, SDHmax (normalized for fiber volume and for mitochondrial volume) decreased in type I and IIa. These results are consistent with mitochondrial structural and functional adaptions to the marked reduction in the energetic demand of type I and IIa DIAm fibers imposed by C2SH.

Evaluation of Diaphragmatic electromyography of cervical spinal cord hemi section in Rat model

Evaluation of Diaphragmatic electromyography of cervical spinal cord hemi section in Rat model

Neuropathological and Motor Impairments after Incomplete Cervical Spinal Cord Injury in Pigs

Humans, primates, and rodents with cervical spinal cord injury (SCI) show permanent sensorimotor dysfunction of the upper/forelimb as consequence of axonal damage and local neuronal death. This work aimed at characterizing a model of cervical SCI in domestic pigs in which hemisection with excision of 1 cm of spinal cord was performed to reproduce the loss of neural tissue observed in human neuropathology. Posture and motor control were assessed over 3 months by scales and kinematics of treadmill locomotion. Histological measurements included lesion length, atrophy of the adjacent spinal cord segments, and neuronal death. In some animals, the retrograde neural tracer aminostilbamidine was injected in segments caudal to the lesion to visualize propriospinal projection neurons. Neuronal loss extended for 4-6 mm from the lesion borders and was more severe in the ipsilateral, caudal spinal cord stump. Axonal Wallerian degeneration was observed caudally and rostrally, associated with marked atrophy of the white matter in the spinal cord segments adjacent to the lesion. The pigs showed chronic monoplegia or severe monoparesis of the foreleg ipsilateral to the lesion, whereas the trunk and the other legs had postural and motor impairments that substantially improved during the first month post-lesion. Adaptations of the walking cycle such as those reported for rats and humans ameliorated the negative impact of focal neurological deficits on locomotor performance. These results provide a baseline of behavior and histology in a porcine model of cervical spinal cord hemisection that can be used for translational research in SCI therapeutics.

O2-6-08. Mechanism of forelimb motor function restoration after cervical spinal cord hemisection in rats: Electorophysiological verification

The objective of this study was to electrophysiologically assess corticospinal tracts of adult rats and recovery of motor function of their forelimbs after cervical cord hemisection. Of 39 adult rats used, compound muscle action potentials (CMAPs) of forelimbs of 15 rats were evaluated, before they received left C5 segmental hemisection of the spinal cord, by stimulating the pyramid of medulla oblongata on one side using an exciting microelectrode. All 15 rats exhibited contralateral electrical activity, but their CMAPs disappeared after hemisection. The remaining 24 rats received hemisection first, and CMAPs of 12 rats were assessed over time to study their recovery time. All of them exhibited electrical activity of forelimbs in four weeks after surgery. The remaining 12 rats received additional right C2 segmental hemisection, and variation of CMAPs between before and after surgery was examined. The right side of the 12 rats that received the additional hemisection exhibited no electrical activity in response to the stimulation of pyramids on both sides. These results suggest that changes in path between the resected and healthy sides, activation of ventral corticospinal tracts, and propriospinal neurons were involved in the recovery of motor function after cervical cord injury.

Impact of glutamatergic and serotonergic neurotransmission on diaphragm muscle activity after cervical spinal hemisection.

Incomplete cervical spinal cord hemisection at C2 (SH) disrupts descending excitatory drive to phrenic motoneurons, paralyzing the ipsilateral diaphragm muscle. Spontaneous recovery over time is associated with increased phrenic motoneuron expression of glutamatergic N-methyl-d-aspartate (NMDA) and serotonergic 5-HT2A receptors. We hypothesized that NMDA and 5-HT2A receptor-mediated neurotransmission play a role in ipsilateral diaphragm muscle activity post-SH. Adult male Sprague-Dawley rats were implanted with bilateral diaphragm EMG electrodes for chronic EMG recordings up to 28 days post-SH (SH 28D). The extent of recovery was calculated by peak root-mean-square (RMS) EMG amplitude. In all animals, absence of ipsilateral activity was verified at 3 days post-SH. Diaphragm EMG activity was also recorded during exposure to hypoxia-hypercapnia (10% O2-5% CO2). In SH animals displaying recovery of ipsilateral diaphragm EMG activity at SH 28D, cervical spinal cord segments containing the phrenic motor nucleus (C3-C5) were surgically exposed and either the NMDA receptor antagonist d-2-amino-5-phosphonovalerate (d-AP5; 100 mM, 30 μl) or 5-HT2A receptor antagonist ketanserin (40 mM, 30 μl) was instilled intrathecally. Following d-AP5, diaphragm EMG amplitude was reduced ipsilaterally, during both eupnea (42% of pre-d-AP5 value; P = 0.007) and hypoxia-hypercapnia (31% of pre-d-AP5 value; P = 0.015), with no effect on contralateral EMG activity or in uninjured controls. Treatment with ketanserin did not change ipsilateral or contralateral RMS EMG amplitude in SH animals displaying recovery at SH 28D. Our results suggest that spinal glutamatergic NMDA receptor-mediated neurotransmission plays an important role in ipsilateral diaphragm muscle activity after cervical spinal cord injury.NEW & NOTEWORTHY Spontaneous recovery following C2 spinal hemisection (SH) is associated with increased phrenic motoneuron expression of glutamatergic and serotonergic receptors. In this study, we show that pharmacological inhibition of glutamatergic N-methyl-d-aspartate (NMDA) receptors blunts ipsilateral diaphragm activity post-SH. In contrast, pharmacological inhibition of serotonergic 5-HT2A receptors does not change diaphragm EMG activity post-SH. Our results suggest that NMDA receptor-mediated glutamatergic neurotransmission plays an important role in enhancing rhythmic respiratory-related diaphragm activity after spinal cord injury.

Diaphragm Muscle Activity Following High Cervical Spinal Cord Injury (SCI) in Rats

Diaphragm dysfunction occurs following high cervicall spinal cord injury (SCI). As the diaphragm is a primary respiratory muscle its dysfunction often necessitates assited‐ventiation, dramatically impacting quality of life and increasing risk of mortality in injured individuals. Despite these devastating outcomes, functional plasticity and recovery of diaphragm activity, albeit limited, can occur spontaneously over the time. Understanding of the mechanisms underlying this recovery will facilitate developing new therapeutic strategies to treat respiratory impairment post‐SCI. In the current study, a C2 cervical spinal cord hemisection (C2Hx) was used to explore spontaneous plasticity ‐ known as the spontaneous crossed phrenic phenomenon – in awake behaving animals. While previous studies have assessed functional under anesthesia, the present study monitored changes in diaphragm activity using chronically implanted diaphragm EMG electrodes in behaving rats up to 8 weeks. Our EMG recording data show that recovery of diaphragm activity in awake animal occurs in one‐two weeks post injury, but inspiratory bursts on the injured side differ from the contralateral. Even when the amplitude of ipsilateral bursts is comparable with uninjured side, intraburst spike frequency is significantly lower especially during quiet breathing. Recording diaphragm EMG activity under anesthesia (isoflurane or ketamine) can eliminate ipsilateral diaphragm activity up to 100% indicating that recovery seen in behaving rats is mostly attributed to NMDA receptor activation. Ongoing studies are assessing this progressive functional recovery in more detail.Support or Funding InformationSupported by Conquer Paralysis Now (CPN) and the Edward Jekkal Muscular Dystrophy Association Fellowship (Drexel).

Mechanism of Restoration of Forelimb Motor Function after Cervical Spinal Cord Hemisection in Rats: Electrophysiological Verification.

The objective of this study was to electrophysiologically assess the corticospinal tracts of adult rats and the recovery of motor function of their forelimbs after cervical cord hemisection. Of 39 adult rats used, compound muscle action potentials (CMAPs) of the forelimbs of 15 rats were evaluated, before they received left C5 segmental hemisection of the spinal cord, by stimulating the pyramid of the medulla oblongata on one side using an exciting microelectrode. All 15 rats exhibited contralateral electrical activity, but their CMAPs disappeared after hemisection. The remaining 24 rats received hemisection first, and CMAPs of 12 rats were assessed over time to study their recovery time. All of them exhibited electrical activity of the forelimbs in 4 weeks after surgery. The remaining 12 rats received additional right C2 segmental hemisection, and variation of CMAPs between before and after surgery was examined. The right side of the 12 rats that received the additional hemisection exhibited no electrical activity in response to the stimulation of the pyramids on both sides. These results suggest that changes in path between the resected and healthy sides, activation of the ventral corticospinal tracts, and propriospinal neurons were involved in the recovery of motor function after cervical cord injury.

Mechanism of Forelimb Motor Function Restoration after Cervical Spinal Cord Hemisection in Rats: A Comparison of Juveniles and Adults

The aim of this study was to investigate forelimb motor function after cervical spinal cord injury in juvenile and adult rats. Both rats received a left segmental hemisection of the spinal cord after C3-C4 laminectomy. Behavioral evaluation of motor function was monitored and assessed using the New Rating Scale (NRS) and Forelimb Locomotor Scale (FLS) and by measuring the range of motion (ROM) of both the elbow and wrist. Complete left forelimb motor paralysis was observed in both rats. The NRS showed motor function recovery restored to 50.2 ± 24.7% in juvenile rats and 34.0 ± 19.8% in adult rats. FLS was 60.4 ± 26.8% in juvenile rats and 46.5 ± 26.9% in adult rats. ROM of the elbow and wrist were 88.9 ± 20.6% and 44.4 ± 24.1% in juvenile rats and 70.0 ± 29.2% and 40.0 ± 21.1% in adult rats. Thus, the NRS and ROM of the elbow showed a significant difference between age groups. These results indicate that left hemisection of the cervical spinal cord was not related to right-sided motor functions. Moreover, while motor paralysis of the left forelimb gradually recovered in both groups, the improvement was greater in juvenile rats.

TrkB gene therapy by adeno-associated virus enhances recovery after cervical spinal cord injury

Unilateral cervical spinal cord hemisection at C2 (C2SH) interrupts descending bulbospinal inputs to phrenic motoneurons, paralyzing the diaphragm muscle. Recovery after C2SH is enhanced by brain derived neurotrophic factor (BDNF) signaling via the tropomyosin-related kinase subtype B (TrkB) receptor in phrenic motoneurons. The role for gene therapy using adeno-associated virus (AAV)-mediated delivery of TrkB to phrenic motoneurons is not known. The present study determined the therapeutic efficacy of intrapleural delivery of AAV7 encoding for full-length TrkB (AAV-TrkB) to phrenic motoneurons 3days post-C2SH. Diaphragm EMG was recorded chronically in male rats (n=26) up to 21days post-C2SH. Absent ipsilateral diaphragm EMG activity was verified 3days post-C2SH. A greater proportion of animals displayed recovery of ipsilateral diaphragm EMG activity during eupnea by 14 and 21days post-SH after AAV-TrkB (10/15) compared to AAV-GFP treatment (2/11; p=0.031). Diaphragm EMG amplitude increased over time post-C2SH (p<0.001), and by 14days post-C2SH, AAV-TrkB treated animals displaying recovery achieved 48% of the pre-injury values compared to 27% in AAV-GFP treated animals. Phrenic motoneuron mRNA expression of glutamatergic AMPA and NMDA receptors revealed a significant, positive correlation (r2=0.82), with increased motoneuron NMDA expression evident in animals treated with AAV-TrkB and that displayed recovery after C2SH. Overall, gene therapy using intrapleural delivery of AAV-TrkB to phrenic motoneurons is sufficient to promote recovery of diaphragm activity, adding a novel potential intervention that can be administered after upper cervical spinal cord injury to improve impaired respiratory function.

Effects of serotonergic agents on respiratory recovery after cervical spinal injury.

Unilateral cervical spinal cord hemisection (i.e., C2Hx) usually interrupts the bulbospinal respiratory pathways and results in respiratory impairment. It has been demonstrated that activation of the serotonin system can promote locomotor recovery after spinal cord injury. The present study was designed to investigate whether serotonergic activation can improve respiratory function during the chronic injury state. Bilateral diaphragm electromyogram and tidal volume were measured in anesthetized and spontaneously breathing adult rats at 8 wk post-C2Hx or C2 laminectomy. A bolus intravenous injection of a serotonin precursor [5-hydroxytryptophan (5-HTP), 10 mg/kg], a serotonin reuptake inhibitor (fluoxetine, 10 mg/kg), or a potent agonist for serotonin 2A receptors (TCB-2, 0.05 mg/kg) was used to activate the serotonergic system. Present results demonstrated that 5-HTP and TCB-2, but not fluoxetine, significantly increased the inspiratory activity of the diaphragm electromyogram ipsilateral to the lesion for at least 30 min in C2Hx animals, but not in animals that received sham surgery. However, the tidal volume was not increased after administration of 5-HTP or TCB-2, indicating that the enhancement of ipsilateral diaphragm activity is not associated with improvement of the tidal volume. These results suggest that exogenous activation of the serotonergic system can specifically enhance the ipsilateral diaphragmatic motor outputs, but this approach may not be sufficient to improve respiratory functional recovery following chronic cervical spinal injury.

Assessment of Diaphragm EMG Activity Recovery Following Upper Cervical Spinal Cord Injury

Motor units are normally recruited in an orderly manner in order to accomplish ventilatory behaviors. The duration of non‐stationarity in electromyographic (EMG) bursts provides an estimate of the period of motor unit recruitment. Motor unit recruitment also reflects central drive, which can be estimated by the root‐mean‐squared (RMS) EMG value 75 ms after the EMG burst onset (RMS75). Following unilateral diaphragm muscle (DIAm) paralysis induced by upper cervical spinal cord hemisection at C2 (SH), there is gradual spontaneous recovery of ipsilateral EMG activity. Increased activity of the contralateral (uninjured) DIAm is sufficient to maintain ventilation following SH. We hypothesized that changes in central drive can be estimated by the period of non‐stationarity and RMS75 from the contralateral DIAm EMG following ipsilateral paralysis and spontaneous recovery of DIAm EMG activity. We found that the duration of non‐stationarity increased to a greater extent in rats that did not recover ipsilateral EMG activity by 14 days post‐SH (D14) compared to those that did. In addition, we found that RMS75 values following SH were reduced in rats displaying recovery of ipsilateral DIAm EMG activity compared to rats that did not. Collectively, these results suggest that the duration of non‐stationarity and RMS75 can be used to evaluate recovery of ipsilateral DIAm function following spinal cord injury.

Effects of Serotonergic Activation on Respiratory Motor Recovery following Chronic Cervical Spinal Cord Injury

Unilateral cervical spinal cord hemisection (i.e., C2Hx) usually interrupts the bulbospinal respiratory pathways and results in attenuation of the phrenic motor output. It has been shown that activation of serotonergic system can induce partial recovery of phrenic activity after acute C2Hx. The purpose of present study was to examine whether manipulation of the serotonin system can still improve respiratory function during the chronic injury phase. Bilateral diaphragm electromyogram (EMG) and tidal volume were measured in anesthetized and spontaneously breathing adult rats at 8 weeks post‐C2Hx or C2 laminectomy. A bolus intravenous injection of a serotonin precursor (5‐Hydroxytryptophan; 5‐HTP, 10 mg/kg) or a potent agonist for serotonin 2A receptor agonist (TCB‐2, 0.05 mg/kg) was used to activate serotonergic system. Current results demonstrated that both 5‐HTP and TCB‐2 significantly induced a persistent increase in activity of the diaphragm EMG ipsilateral to the lesion but not contralateral diaphragm in C2Hx animals or bilateral diaphragm in uninjured animals. However, the tidal volume was not changed after administration of 5‐HTP or TCB‐2, indicating the enhancement of ipsilateral diaphragm activity is not associated with improvement of the tidal volume. These results suggest that activation of the serotonergic system can specifically increase the ipsilateral diaphragmatic motor output, but it is not sufficient to improve respiratory functional recovery following chronic cervical spinal injury.

Ipsilateral inspiratory intercostal muscle activity after C2 spinal cord hemisection in rats

BackgroundUpper cervical spinal cord hemisection causes paralysis of the ipsilateral hemidiaphragm; however, the effect of C2 hemisection on the function of the intercostal muscles is not clear. We hypothesized that C2 hemisection would eliminate inspiratory intercostal activity ipsilateral to the injury and that some activity would return in a time-dependent manner.MethodsFemale Sprague Dawley rats were anesthetized with urethane and inspiratory intercostal electromyogram (EMG) activity was recorded in control rats, acutely injured C2 hemisected rats, and at 1 and 16 weeks post C2 hemisection.ResultsBilateral recordings of intercostal EMG activity showed that inspiratory activity was reduced immediately after injury and increased over time. EMG activity was observed first in rostral spaces followed by recovery occurring in caudal spaces. Theophylline increased respiratory drive and increased intercostal activity, inducing activity that was previously absent.ConclusionThese results suggest that there are crossed, initially latent, respiratory connections to neurons innervating the intercostal muscles similar to those innervating phrenic motor neurons.

TrkB kinase activity is critical for recovery of respiratory function after cervical spinal cord hemisection

Neuroplasticity following spinal cord injury contributes to spontaneous recovery over time. Recent studies highlight the important role of brain-derived neurotrophic factor (BDNF) signaling via the high-affinity tropomyosin-related kinase (Trk) receptor subtype B (TrkB) in recovery of rhythmic diaphragm activity following unilateral spinal hemisection at C2 (C2SH). We hypothesized that TrkB kinase activity is necessary for spontaneous recovery of diaphragm activity post-C2SH. A chemical-genetic approach employing adult male TrkBF616A mice (n=49) was used to determine the impact of inhibiting TrkB kinase activity by the phosphoprotein phosphatase 1 inhibitor derivative 1NMPP1 on recovery of ipsilateral hemidiaphragm EMG activity. In mice, C2SH was localized primarily to white matter tracts comprising the lateral funiculus. The extent of damaged spinal cord (~27%) was similar regardless of the presence of functional recovery, consistent with spontaneous recovery reflecting neuroplasticity primarily of contralateral spared descending pathways to the phrenic motor pools. Ipsilateral hemidiaphragm EMG activity was verified as absent in all mice at 3days post-C2SH. By 2weeks after C2SH, ipsilateral hemidiaphragm EMG activity was present in 39% of vehicle-treated mice compared to 7% of 1NMPP1-treated mice (P=0.03). These data support the hypothesis that BDNF/TrkB signaling involving TrkB kinase activity plays a critical role in spontaneous recovery of diaphragm activity following cervical spinal cord injury.

Chasing central nervous system plasticity: the brainstem's contribution to locomotor recovery in rats with spinal cord injury.

Anatomical plasticity such as fibre growth and the formation of new connections in the cortex and spinal cord is one known mechanism mediating functional recovery after damage to the central nervous system. Little is known about anatomical plasticity in the brainstem, which contains key locomotor regions. We compared changes of the spinal projection pattern of the major descending systems following a cervical unilateral spinal cord hemisection in adult rats. As in humans (Brown-Séquard syndrome), this type of injury resulted in a permanent loss of fine motor control of the ipsilesional fore- and hindlimb, but for basic locomotor functions substantial recovery was observed. Antero- and retrograde tracings revealed spontaneous changes in spinal projections originating from the reticular formation, in particular from the contralesional gigantocellular reticular nucleus: more reticulospinal fibres from the intact hemicord crossed the spinal midline at cervical and lumbar levels. The intact-side rubrospinal tract showed a statistically not significant tendency towards an increased number of midline crossings after injury. In contrast, the corticospinal and the vestibulospinal tract, as well as serotonergic projections, showed little or no side-switching in this lesion paradigm. Spinal adaptations were accompanied by modifications at higher levels of control including side-switching of the input to the gigantocellular reticular nuclei from the mesencephalic locomotor region. Electrolytic microlesioning of one or both gigantocellular reticular nuclei in behaviourally recovered rats led to the reappearance of the impairments observed acutely after the initial injury showing that anatomical plasticity in defined brainstem motor networks contributes significantly to functional recovery after injury of the central nervous system.

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.

Motoneuron BDNF/TrkB signaling enhances functional recovery after cervical spinal cord injury

A C2 cervical spinal cord hemisection (SH) interrupts descending inspiratory-related drive to phrenic motoneurons located between C3 and C5 in rats, paralyzing the ipsilateral hemidiaphragm muscle. There is gradual recovery of rhythmic diaphragm muscle activity ipsilateral to cervical spinal cord injury over time, consistent with neuroplasticity and strengthening of spared, contralateral descending premotor input to phrenic motoneurons. Brain-derived neurotrophic factor (BDNF) signaling through the tropomyosin related kinase receptor subtype B (TrkB) plays an important role in neuroplasticity following spinal cord injury. We hypothesized that 1) increasing BDNF/TrkB signaling at the level of the phrenic motoneuron pool by intrathecal BDNF delivery enhances functional recovery of rhythmic diaphragm activity after SH, and 2) inhibiting BDNF/TrkB signaling by quenching endogenous neurotrophins with the soluble fusion protein TrkB-Fc or by knocking down TrkB receptor expression in phrenic motoneurons using intrapleurally-delivered siRNA impairs functional recovery after SH. Diaphragm EMG electrodes were implanted bilaterally to verify complete hemisection at the time of SH and 3days post-SH. After SH surgery in adult rats, an intrathecal catheter was placed at C4 to chronically infuse BDNF or TrkB-Fc using an implanted mini-osmotic pump. At 14days post-SH, all intrathecal BDNF treated rats (n=9) displayed recovery of ipsilateral hemidiaphragm EMG activity, compared to 3 out of 8 untreated SH rats (p<0.01). During eupnea, BDNF treated rats exhibited 76±17% of pre-SH root mean squared EMG vs. only 5±3% in untreated SH rats (p<0.01). In contrast, quenching endogenous BDNF with intrathecal TrkB-Fc treatment completely prevented functional recovery up to 14days post-SH (n=7). Immunoreactivity of the transcription factor cAMP response element-binding protein (CREB), a downstream effector of TrkB signaling, increased in phrenic motoneurons following BDNF treatment (n=6) compared to artificial cerebrospinal fluid treatment (n=6; p<0.001). Intrapleural injections of non-sense or TrkB siRNA were administered after SH to specifically target phrenic motoneurons. At 14days post-SH, none out of 9 TrkB siRNA treated rats displayed functional recovery compared to 5 out of 9 non-sense siRNA treated rats. These results indicate that BDNF/TrkB signaling in phrenic motoneuron pool plays a critical role in functional recovery after cervical spinal cord injury.

TrkB kinase activity is necessary for spontaneous recovery of ipsilateral rhythmic phrenic activity following cervical spinal cord hemisection

Following unilateral C2 spinal hemisection (SH), descending excitatory drive to phrenic motoneurons is disrupted causing ipsilateral diaphragm muscle paralysis. After SH, there is gradual recovery of phrenic nerve activity reflecting neuroplasticity. We hypothesized that this neuroplasticity is dependent on BDNF signaling via its TrkB receptor and kinase activity. In this study we used TrkB(F616A) knockin mice, that permit rapid and selective inhibition of TrkB kinase activity by 1NMPP1. Adult TrkB(F616A) mice were treated with 1NMPP1 (25 μM) or vehicle in the drinking water starting 3 days post‐SH. At the terminal experiment, bilateral diaphragm EMG was monitored to determine the proportion of animals displaying recovery of ipsilateral phrenic activity. At 3 days post‐SH, no animals displayed ipsilateral diaphragm EMG activity (0/10). At 14 days post‐SH, 40% (6/15) of TrkB(F616A) mice displayed ipsilateral phrenic activity, although EMG amplitude remained substantially below contralateral levels. Following 1NMPP1 treatment, no animals displayed ipsilateral diaphragm EMG activity (0/10; p&lt;0.05). In control, wild‐type mice, 1NMPP1 treatment (for 11 days) had no evident effect on bilateral diaphragm EMG activity (n=7). These results support an essential role for TrkB kinase activity in the spontaneous recovery of rhythmic phrenic activity post spinal cord injury.Supported by NIH grant HL096750.