Serotonergic innervation of respiratory motor nuclei after cervical spinal injury: Impact of intermittent hypoxia

Abstract

Although cervical spinal cord injury (cSCI) disrupts bulbo-spinal serotonergic projections, partial recovery of spinal serotonergic innervation below the injury site is observed after incomplete cSCI. Since serotonin contributes to functional recovery post-injury, treatments to restore or accelerate serotonergic reinnervation are of considerable interest. Intermittent hypoxia (IH) was reported to increase serotonin innervation near respiratory motor neurons in spinal intact rats, and to improve function after cSCI. Here, we tested the hypotheses that spontaneous serotonergic reinnervation of key respiratory (phrenic and intercostal) motor nuclei: 1) is partially restored 12 weeks post C2 hemisection (C2Hx); 2) is enhanced by IH; and 3) results from sprouting of spared crossed-spinal serotonergic projections below the site of injury. Serotonin was assessed via immunofluorescence in male Sprague Dawley rats with and without C2Hx (12 wks post-injury); individual groups were exposed to 28 days of: 1) normoxia; 2) daily acute IH (dAIH28: 10, 5 min 10.5% O2 episodes per day; 5 min normoxic intervals); 3) mild chronic IH (IH28-5/5: 5 min 10.5% O2 episodes; 5 min intervals; 8 h/day); or 4) moderate chronic IH (IH28-2/2: 2 min 10.5% O2 episodes; 2 min intervals; 8 h/day), simulating IH experienced during moderate sleep apnea. After C2Hx, the number of ipsilateral serotonergic structures was decreased in both motor nuclei, regardless of IH protocol. However, serotonergic structures were larger after C2Hx in both motor nuclei, and total serotonin immunolabeling area was increased in the phrenic motor nucleus but reduced in the intercostal motor nucleus. Both chronic IH protocols increased serotonin structure size and total area in the phrenic motor nuclei of uninjured rats, but had no detectable effects after C2Hx. Although the functional implications of fewer but larger serotonergic structures are unclear, we confirm that serotonergic reinnervation is substantial following injury, but IH does not affect the extent of reinnervation.

Keywords: Cervical hemisection; Intercostal motor neurons; Intermittent hypoxia; Phrenic motor neurons; Raphe; Serotonin; Spinal cord injury.

Figure 1:. Experimental time-line and intermittent hypoxia protocols.

A. Rats were injected with Cholera toxin subunit B (CtB) 2 weeks prior to C2 spinal hemisection (C2Hx). 8 weeks post C2Hx rats began intermittent hypoxia exposures for 28 days. B. Normoxia protocol (Nx28) consisted of 28 days of 21% O₂ for 8 hours. C. Daily acute intermittent hypoxia (dAIH28) consisted of 10, 5 minute episodes of 10.5% O₂ alternating with 5 minute normoxic intervals (~1.5 hours/day) for 28 days. D. Mild chronic intermittent hypoxia (IH28-5/5) consisted of 8 hours per day of 5 minute episodes of 10.5% O₂ alternating with 5 minute normoxic intervals. E. Moderate chronic intermittent hypoxia (IH28-2/2) consisted of 2 minute episodes of 10.5% O₂ alternating with 2 minute normoxic intervals for a total duration of 8 hours/day. Rats were randomly assigned to groups: 1) spinal intact+Nx28 (n=8); 2) spinal intact+dAIH28 (n=10); 3) spinal intact+IH28-5/5 (n=10); 4) spinal intact+IH28-2/2 (n=10); 5) C2Hx+Nx28 (n=9); 6) C2Hx+dAIH28 (n=8); 7) C2Hx+IH28-5/5 (n=12); and 8) C2Hx+IH28-2/2 (n=12).

Figure 2:. Representative images of serotonergic innervation in the phrenic motor nucleus.

Representative images from uninjured rats, and rats 12 weeks post-C2Hx exposed to 28 days of normoxia. Images illustrate serotonin (red), often described as looking like “beads on a string”, and Cholera toxin subunit B-labeled phrenic motor neurons (green). Images from left to right are representative samples from the left and right side of the cervical spinal cord of intact rats, and the ipsilateral and contralateral sides of the cervical spinal cord from rats 12 weeks post-C2Hx. Images were taken at 20x magnification with the phrenic motor nucleus at the center. The white scale bars represent a distance of 100 μm.

Figure 3:. Serotonergic innervation of the phrenic motor nucleus: effects of injury and IH protocol.

Serotonin innervation in the phrenic motor nucleus was quantified in uninjured rats (left) and rats 12 weeks post-C2 hemisection (C2Hx; right) exposed to 28 days of intermittent hypoxia (IH) protocols (Nx28, white; dAIH28, light gray; IH28-5/5, dark gray; IH28-2/2, black). A. Number of serotonin structures. C2Hx significantly reduced the number of serotonin structures ipsi- and contralateral to C2Hx versus uninjured rats (p<0.0001 and p<0.05, respectively). Post-C2Hx, ipsilateral serotonin structure number was also significantly lower vs. contralateral side (p<0.0001). B. Size of serotonin structures. C2Hx significantly increased area of serotonin structures ipsilateral to injury versus uninjured or contralateral side (p<0.0001). There were no IH effects after C2Hx. We averaged the left and right sides of uninjured rats and ran a one-way ANOVA by IH protocol. IH28-5/5 and IH28-2/2 rats had significantly greater areas per structure vs. dAIH28 or Nx28 (IH28-2/2 vs. Nx28 p=0.0024, IH28-2/2 vs. dAIH28 p=0.0034, IH28-5/5 vs. Nx28 p=0.0087, IH28-5/5 vs. dAIH28 p=0.0134). C. Total area of serotonin immunolabeling. C2Hx significantly increased the total area of serotonin immunolabeling ipsilateral to injury versus uninjured rats or contralateral side (p<0.0001). There were no IH effects after C2Hx. We averaged the left and right sides of uninjured rats and ran a one-way ANOVA by IH protocol. IH28-5/5 and IH28-2/2 protocols had significantly greater areas per structure than Nx28 (IH28-2/2 vs. Nx28 p=0.0179, IH28-5/5 vs. Nx28 p=0.0262). All other data were analyzed via repeated measures Mixed Model ANOVA; injury (intact vs. C2Hx) and IH protocol (Nx28, dAIH28, IH28-5/5 and IH28-2/2) were between subject variables; side (left/ipsilateral vs. right/contralateral) was the within subject repeated measure. Differences were considered significant if p<0.05. Bars denote means ± 1 SEM with the individual rats denoted as data points.

Figure 4:. Total area of serotonin immunopositive labeling at cervical central commissures.

A. Representative images of serotonergic fibers near central commissure of cervical spinal sections (left). Spinal intact (top) and C2 hemisection (C2Hx; bottom) rats were exposed to 28 consecutive days of normoxia. Dotted white lines represent gray and white matter border. Serotonin (red) mostly occurs in gray matter and exhibits the typical “beads on a string” morphology. Images were taken at 20x magnification with the central canal at the center. The white scale bars represent a distance of 100 μm. B. Quantitative analysis of serotonergic fibers at central commissures (right). Neither C2Hx nor any IH protocol (Nx28, white; dAIH28, light gray; IH28-5/5, dark gray; IH28-2/2, black) had an effect on total area of serotonergic innervation in cervical central commissures. Data were analyzed via two-way ANOVA (p=0.4648) with injury (intact vs. C2Hx) and IH protocol (Nx28, dAIH28, IH28-5/5, and IH28-2/2) as independent variables. Bars denote means ± 1 SEM with individual rats as individual data points.

Figure 5:. Representative images of serotonergic innervation in intercostal motor nuclei.

Representative images from uninjured rats, and rats 12 weeks post-C2Hx exposed to 28 days of normoxia. Images illustrate serotonin (red), often described as looking like “beads on a string”, and cholera toxin subunit B-labeled phrenic motor neurons (green). Images from left to right are representative samples from the left and right side of the thoracic spinal cord of intact rats, and the ipsilateral and contralateral sides of the thoracic spinal cord from rats 12 weeks post-C2Hx. Images were taken at 20x magnification with the phrenic motor nucleus at the center. The white scale bars represent a distance of 100 μm.

Figure 6:. Serotonergic innervation of the intercostal motor nucleus.

Serotonin innervation in intercostal motor nuclei was quantified in uninjured rats (left) and rats 12 weeks post-C2 hemisection (C2Hx; right) exposed to 28 days of IH protocols (Nx28, white; dAIH28, light gray; IH28-5/5, dark gray; IH28-2/2, black). A. Number of serotonin structures. C2Hx significantly reduced the number of serotonin structures ipsilateral to injury versus uninjured rats and contralateral side (p<0.0001 for all). No IH protocol significantly affected the number of serotonin structures innervating intercostal motor nuclei in uninjured rats or rats 12 weeks post-C2Hx. B. Size of serotonin structures. C2Hx significantly increased the area of individual serotonergic structures ipsilateral to injury versus uninjured rats and contralateral side (p<0.0001 for all). IH had no effects on uninjured rats or rats 12 weeks post-C2Hx. C. Total area of serotonin immunolabeling. C2Hx reduced the total area of serotonin immunolabeling ipsilateral to injury versus uninjured rats or the contralateral side (p<0.0001 for all). There were no IH effects on total area of intercostal motor nuclei in uninjured rats. All data were analyzed with a repeated measures Mixed Model ANOVA; injury (intact vs. C2Hx) and IH protocol (Nx28, dAIH28, IH28-5/5 and IH28-2/2) were between subject variables; side (left/ipsilateral vs. right/contralateral) was the repeated measure. Differences were considered significant if p<0.05. Bars denote means ± 1 SEM with the individual rats as individual data points.

Figure 7:. Immunoreactive serotonin staining intensity within immunopositive structures.

Fluorescent intensity in immunopositive structures of the phrenic motor nucleus was quantified in uninjured rats (left) and rats 12 weeks post-C2 hemisection (C2Hx; right) exposed to 28 days IH protocols (Nx28, white; dAIH28, light gray; IH28-5/5, dark gray; IH28-2/2, black). A. Serotonin labeling intensity in phrenic motor nucleus. C2Hx significantly reduced the intensity of serotonin positive structures ipsilateral to injury versus uninjured rats and contralateral side (p<0.0001). There was no IH effect on staining intensity after C2Hx. We averaged the left and right sides of uninjured rats and ran a one-way ANOVA by IH protocol. In uninjured rats, dAIH28 significantly increased staining intensity per structure versus Nx28 (p=0.0444). B. Serotonin labeling intensity in intercostal motor nuclei. C2Hx significantly reduced the intensity of serotonin staining within structures ipsilateral to C2Hx versus uninjured rats or contralateral side (p<0.0001). There were no IH effects on serotonin structure intensity in uninjured rats or rats post- C2Hx. All data were analyzed with a repeated measures Mixed Model ANOVA; injury (intact vs. C2Hx) and IH protocol (Nx28, dAIH28, IH28-5/5 and IH28-2/2) were between subject variables; side (left/ipsilateral vs. right/contralateral) was the within subject repeated measure. Differences were considered significant if p<0.05. Data are displayed in arbitrary units (A.U.). Bars denote means ± 1 SEM with the individual rats as individual data points.