Repetitive Intermittent Hypoxia Induces Respiratory and Somatic Motor Recovery after Chronic Cervical Spinal Injury

Spinal surgery: C₂HS.

Buprenorphine (50 μg/kg), carpofen (5 mg/kg), enrofloxacin (4 mg/kg), and medetomidine (100 μg/kg) were administered subcutaneously 10–20 min before inducing isoflurane anesthesia in a closed chamber. Rats were intubated, and anesthesia was maintained while rats were mechanically ventilated. A C2 laminectomy and durotomy were performed, and the spinal cord was hemisected just caudal to the C2 dorsal roots as described previously (Fuller et al., 2003, 2006; Golder and Mitchell, 2005); 1 mm of spinal tissue was aspirated to ensure complete hemisection, and the wounds were sutured closed. Sham rats underwent the same procedures without hemisection. Atipamezole (400 μg/kg) was given to reverse medetomidine, the endotracheal tube was removed, and the rats were monitored throughout recovery. Rats received analgesic (buprenorphine, 50 μg/kg) and anti-inflammatory (carpofen, 5 mg/kg) drugs for 2 d after surgery, with enrofloxacin (4 mg/kg) given as needed.

dAIH.

dAIH began 1 week after surgery and lasted for 7 d. Each day, rats were placed in a Plexiglas chamber flushed with a mixture of air/N₂/O₂ (4 L/min) to attain continuous normoxia or intermittent hypoxia (75 s equilibration; 5 min episodes of 10.5% O₂; 5 min intervals of 21% O₂). After 10 hypoxic episodes, or the equivalent duration or normoxia (110 min), animals were returned to their cages until the next exposure.

Ventilatory measurements in unanesthetized rats.

In rats studied 7 d after dAIH (i.e., 14 d after injury), whole-body plethysmography (Buxco) was used to measure V_T, breathing frequency (f), and minute pulmonary ventilation (V̇_E). After a 30 min acclimation period, baseline ventilation was recorded under normoxic (21% O₂) conditions for >30 min. Rats were then exposed to 25 min each of 5% and 7% CO₂ in 21% O₂ (balance N₂), followed by 25 min of 7% CO₂ with hypoxia (10.5% O₂). This procedure enabled an assessment of maximal chemo-reflex stimulated breathing in unanesthetized rats. Gas mixtures flowed continuously through the chamber at 2 L/min to prevent CO₂ buildup and allow rapid control of inspired gas concentrations. Data were analyzed in 5 min bins and are presented as means ± 1 SEM.

Bilateral phrenic nerve recordings in anesthetized rats.

Maximal bilateral phrenic motor output and the efficacy of crossed spinal synaptic pathways to phrenic motor neurons were assessed 1 d after the final dAIH exposure. In these experiments, isoflurane anesthesia was induced in a closed chamber and maintained via nose cone (2.5–3.5% isoflurane in 50% O₂, balance N₂) until a tracheotomy was complete. Rats were then mechanically ventilated [V_T = 2–2.5 ml; Fraction of inspired oxygen (FIO₂) = 0.50; Rodent Respirator model 682; Harvard Apparatus] with isoflurane anesthesia. The rats were then bilaterally vagotomized, and a femoral artery was catheterized to monitor blood pressure (Gould P23ID) and blood gases (ABL-500; Radiometer). Fluids (lactated Ringer's solution/Hetastarch, 4:1 by volume; 2.5 ml/h) and any necessary drugs were administered via tail-vein catheter. Phrenic nerves were isolated via a dorsal approach, cut distally, desheathed, and positioned on bipolar silver electrodes submerged in mineral oil. Rats were then converted slowly from isoflurane to urethane anesthesia (1.6–1.7 mg/kg) and paralyzed with pancuronium bromide (2.5 mg/kg, i.v.). End-tidal CO₂ partial pressure was monitored using a CO₂ analyzer with sufficient response time to measure end-tidal PCO₂ in rats (Capnoguard; Novametrix Medical Systems), and body temperature was maintained with a temperature-controlled table (37.5 ± 1°C).

Nerve signals were amplified (gain of 10,000), filtered (100–10,000 Hz; A-M Systems), rectified, and integrated (time constant, 50 ms; CWE 821 filter; Paynter). Spontaneous inspiratory bursts in both phrenic nerves were digitized, recorded, and analyzed using a WINDAQ data acquisition system (DATAQ Instruments). The CO₂ apneic and recruitment thresholds for inspiratory phrenic nerve activity were determined at least 1 h after conversion to urethane anesthesia. To determine the CO₂ apneic threshold, inspired CO₂ was decreased until inspiratory phrenic nerve activity ceased; inspired CO₂ was then slowly increased until inspiratory activity resumed. The end-tidal CO₂ partial pressure at which inspiratory activity resumed is the CO₂ recruitment threshold. End-tidal PCO₂ was maintained at 2–3 mmHg above the recruitment threshold during baseline measurements (30–60 min). Three graded respiratory challenges were then imposed: (1) 20 mmHg increase in arterial CO₂ partial pressure (PaCO₂) above baseline; (2) 40 mmHg increase in PaCO₂ above baseline; and (3) 40 mmHg increase in PaCO₂ combined with hypoxia (PaO₂ of ∼35–45 mmHg; FIO₂ = 0.11 ± 0.01). Each challenge lasted 5–7 min, and blood samples were drawn (0.3 ml) during each challenge to confirm PaCO₂ and PaO₂ levels. Baseline conditions were then restored.

Evoked phrenic nerve recordings were made as described previously (Golder et al., 2008) in hyperventilated rats (PaCO₂ ≤ 10 mmHg below the apneic CO₂ threshold) to prevent spontaneous inspiratory activity. A monopolar tungsten stimulating electrode (5 MΩ; A-M Systems) was placed in the ventrolateral funiculus below the dorsal root entry zone on the uninjured side rostral to the lesion. Electrode depth was chosen to maximize the amplitude of short-latency evoked potential (≤1 ms) on the uninjured side. The ventrolateral funiculus was stimulated with current pulses from 100 to 1500 μA (0.2 ms duration) to generate a stimulus–response curve (Grass Stimulator model S88 and stimulus isolation unit model PSIU6E; Grass Instruments). Nerve potentials were digitized and analyzed with pClamp software (Molecular Devices).

Data analyses.

Data from plethysmography and spontaneous phrenic nerve recording experiments were analyzed with two-way, repeated-measures ANOVAs using the Student–Newman–Keuls post hoc test. Evoked potentials on the uninjured side were analyzed with a two-way, repeated-measures ANOVA with Student–Newman–Keuls post hoc test. Evoked potentials on the injured side were analyzed using a χ² test with a Mann–Whitney post hoc test. Data are presented as mean ± 1 SEM.

Immunohistochemistry.

Four rat groups (C₂HS or sham surgery treated with normoxia or dAIH, see above; n = 5 per group) were killed and perfused transcardially with ice-cold 0.01 m PBS, pH 7.4, followed by 4% buffered paraformaldehyde. Cervical spinal cords were excised, postfixed overnight, and cryoprotected in 30% sucrose at 4°C until the tissues sank. Transverse sections (40 μm) of C4 and C7 were cut with a freezing microtome (Leica SM 200R). Free-floating sections were washed in 0.1 m Tris-buffered saline with 0.1% Triton X-100 (TBS-Tx; three times for 5 min) and incubated in TBS containing 1% H₂O₂ for 30 min. After washing (three times for 5 min) in TBS-Tx, tissues were blocked with 5% normal goat serum at room temperature (RT) for 60 min. Staining was performed by incubating sections with rabbit polyclonal anti-BDNF (N-20, sc-546; 1:1000; Santa Cruz Biotechnology), rabbit anti-TrkB (H-181, sc-8316; 1:1000; Santa Cruz Biotechnology), or rabbit anti-phospho-TrkB (1:1000; courtesy of Dr. Moses Chao, New York University, New York, NY) at 4°C overnight. The sections were washed in TBS-Tx and incubated in biotinylated secondary goat anti-rabbit antibody (1:1000; Vector Laboratories). Conjugation with avidin–biotin complex (Vecstatin Elite ABC kit; Vector Laboratories) was followed by visualization with 3,3′-diaminobenzidine hydrogen peroxidase (Vector Laboratories) according to the instruction of the manufacturer. Sections were then washed in TBS, placed on gelatin-coated slides, dried, dehydrated in a graded alcohol series, cleared with xylenes, and mounted with Eukitt mounting medium (Electron Microscope Sciences). All images were captured and analyzed with a digital camera (SPOT II; Diagnostic Instruments). Photomicrographs were created with Adobe Photoshop software (Adobe Systems). All images received equivalent adjustments to tone scale, gamma, and sharpness. Negative controls were performed by omitting the primary or secondary antibodies; in addition, we preabsorbed the primary BDNF antibody with a fivefold (by concentration) excess of a specific blocking peptide (sc-546 P; Santa Cruz Biotechnology).

Immunofluorescence.

Gliosis in hippocampal sections was assessed using antibodies targeting a marker for astrocytes (GFAP; 1:1000; Millipore Bioscience Research Reagents) or microglia (OX-42; 1:500; AbD Serotec). Sections were incubated at 4°C overnight and, after washing with TBS-Tx (three times for 5 min), were incubated in conjugated goat anti-rabbit red fluorescent Alexa Fluor 495 (1:200; Invitrogen) at RT for 60 min. Tissue sections were mounted on glass using anti-fade solution (Prolong Gold antifade reagent; Invitrogen) and examined using an epifluorescence microscope (Nikon).

TUNEL assay.

Apoptotic cell death in hippocampal sections was assessed using TUNEL with the In Situ Cell Death Detection Kit (Roche Molecular Biochemical) according to the instructions of the manufacturer. Briefly, hippocampal sections (40 μm) were permeabilized with 1% Proteinase K (in 50 mm Tris/5 mm EDTA buffer) for 15 min, rinsed with PBS, and incubated in TUNEL reaction mixture for 1 h at 37°C. Samples were rinsed, mounted in anti-fade solution (Invitrogen), and analyzed with the epifluorescence microscope. TUNEL (red)-stained tissues were also exposed to anti-NeuN (green) to identify any apoptotic neurons. Cresyl violet staining was used to identify any nonspecific dAIH-induced cell death in separate hippocampal sections.

Quantification and statistical analysis.

Sections were numbered sequentially, and every eighth section was selected for immunohistochemistry. Thus, approximately six sections from each animal at each segmental level were used in this study. The phrenic motor nucleus was identified as a cluster of large neurons in the mediolateral C4 ventral horn (described by Boulenguez et al., 2007; Mantilla et al., 2009). Digital photomicrographs of immunoreactive labeling in the region of phrenic motor neurons were taken with a 20× objective lens (SPOT II; Diagnostic Instruments). Densitometry was performed by circumscribing the phrenic motor nucleus based on their location and large size in the medial–lateral–ventral aspect of the C4 ventral horn (Boulenguez et al., 2007; Mantilla et al., 2009; Dale-Nagle et al., 2011). A similar procedure was used in the C7 ventral horn, although the area of interest was centered on spinal lamina 9 (Paxinos and Watson, 1997). The intensity of BDNF-, TrkB-, and phospho-TrkB-immunoreactive staining in the defined area of interest was quantified using NIH Image J software (National Institutes of Health; http://rsb.info.nih.gov/ij). Images were converted to eight-bit resolution, and the threshold was set between 120 and 160 during all analyses. The optical density (OD) was measured within circumscribed labeled motor neurons and expressed as an averaged of OD per unit area for each individual cell. For each cell, the OD of BDNF, TrkB, and phospho-TrkB immunoreactivity was expressed as a fraction of the average OD of sham animals. Thus, the mean OD in the sham rats is expected to be 1.0, with variance that reflects variation among the group. Average labeling intensity was compared between treatment groups (each n = 5) using one-way ANOVA followed by Fisher's LSD post hoc test (SigmaStat 2.03; SPSS). The numbers of GFAP-, OX-42-, TUNEL- and Nissl-positive cells in the hippocampus (C2 subfield) were counted manually, and normoxia versus dAIH-treated rats were compared using a t test. Differences were considered significant if p < 0.05. All values are expressed as mean ± 1 SEM.

Spinal surgery (partial C₂HS).

Rats were premedicated with an anti-cholinergic drug, glycopyrrolate (0.03 mg/ml, s.c.; Sabex) and a mixture of medetomidine hydrochloride (0.3 mg/kg, i.p.; Damitor; Novartis Animal Health) and buprenorphine (0.05 mg/kg, i.p.; Buprenex; Reckitt Benckiser Pharmaceuticals). A surgical plane of anesthesia was induced and maintained with isoflurane administered by mask. After anesthetic induction, a unilateral dorsolateral funiculotomy was performed as described previously (Webb and Muir, 2003; Muir et al., 2007). Briefly, the skin and muscles over the dorsal cervical spinal cord were incised and bluntly dissected to expose the dorsal spinous processes and spinal laminae of the second and third cervical vertebrae. A laminectomy and durotomy were performed to expose the spinal cord. Using the entry site of the dorsal roots as a landmark, the dorsolateral funiculus at the level of the third cervical spinal segment was transected unilaterally using a modified 25-gauge bevel-tipped needle. An autologous fat graft obtained near the surgical site was placed over the laminectomy site, and the muscles and skin were closed.

dAIH.

Four weeks after surgery, rats were randomly assigned to receive either dAIH or normoxia. The day before treatment onset, rats were acclimated to custom-made Plexiglas chambers (1 rat per chamber; 30 × 17 × 12 cm) under normoxic conditions (FIO₂ = 0.21 ± 0.005). For the next 7 d, rats were placed into the Plexiglas chambers and exposed daily to AIH (n = 7) or normoxia (n = 7). As in the previous groups, dAIH consisted of 10 5-min episodes of hypoxia (FIO₂ = 0.11) interspersed with 5-min normoxic intervals. Sham-treated rats were in the chambers for an equivalent period of time under continually normoxic conditions. Oxygen levels were continuously monitored (AX300–1 Portable Oxygen Analyzer; Teledyne Analytical Instruments). After acclimation to the chambers, both dAIH- and normoxia-treated rats would rest quietly or sleep throughout the exposure period.

Behavioral assessment of skilled locomotion: horizontal ladder test.

The horizontal ladder was used to assess the skilled locomotor ability of the rats as described previously (Poulton and Muir, 2005; Muir et al., 2007; Kanagal and Muir, 2009). Briefly, a ladder consisting of 2-mm-diameter metallic rungs placed 2 cm apart was used. The ladder was placed above a 45 ° angled mirror so that rats could be videotaped simultaneously from both a lateral and ventral view. At each session, a minimum of 10 runs were recorded from each rat using a VHS camera. Videotapes were analyzed field by field (60 fields/s), and the number of footfalls by each limb were counted as the rats walked over the rungs. Data from only one complete stride was sampled from each pass to satisfy statistical requirements for independence between multiple measurements from an individual limb. Data from at least 10 passes from each rat were averaged for each day of data collection. Data were expressed as number of correct steps per total steps for each limb per rat. Assessment of ladder locomotion was performed before surgery and at 4 weeks after surgery on the day before dAIH treatment. Ladder locomotion was also assessed 1 h after AIH treatment on each exposure day and again 1 and 3 weeks after the final AIH treatment.

Analyses.

Group data were averaged and expressed as means ± 1 SEM. Groups were compared using a two-way, repeated-measures ANOVA (SigmaStat; Systat Software). Post hoc analysis was performed using the Holm–Sidak method with p < 0.05 considered significant.