Respiratory abnormalities resulting from midcervical spinal cord injury and their reversal by serotonin 1A agonists in conscious rats

Abstract

Respiratory dysfunction after cervical spinal cord injury (SCI) has not been examined experimentally using conscious animals, although clinical SCI most frequently occurs in midcervical segments. Here, we report a C5 hemicontusion SCI model in rats with abnormalities that emulate human post-SCI pathophysiology, including spontaneous recovery processes. Post-C5 SCI rats demonstrated deficits in minute ventilation (Ve) responses to a 7% CO2 challenge that correlated significantly with lesion severities (no injury or 12.5, 25, or 50 mm x 10 g weight drop; New York University impactor; p < 0.001) and ipsilateral motor neuron loss (p = 0.016). Importantly, C5 SCI resulted in at least 4 weeks of respiratory abnormalities that ultimately recovered afterward. Because serotonin is involved in respiration-related neuroplasticity, we investigated the impact of activating 5-HT1A receptors on post-C5 SCI respiratory dysfunction. Treatment with the 5-HT1A agonist 8-hydroxy-2-(di-n-propylmino)tetralin (8-OH DPAT) (250 microg/kg, i.p.) restored hypercapnic Ve at 2 and 4 weeks after injury (i.e., approximately 39.2% increase vs post-SCI baseline; p < or = 0.033). Improvements in hypercapnic Ve response after single administration of 8-OH DPAT were dose dependent and lasted for approximately 4 h(p < or = 0.038 and p < or = 0.024, respectively). Treatment with another 5-HT1A receptor agonist, buspirone (1.5 mg/kg, i.p.), replicated the results, whereas pretreatment with a 5-HT1A-specific antagonist, 4-iodo-N-[2-[4(methoxyphenyl)-1-piperazinyl]ethyl]-N-2-pyridinyl-benzamide (3 mg/kg, i.p.) given 20 min before 8-OH DPAT negated the effect of 8-OH DPAT. These results imply a potential clinical use of 5-HT1A agonists for post-SCI respiratory disorders.

Figure 1.

Noninvasive measurement of respiratory function in conscious rats using WBP. A, Diagram of a fixed-volume (3.9 L) plethysmograph chamber for WBP testing of unrestrained conscious rats. After acclimation, rats remained calm within the chambers for prolonged periods (up to 5 h). B, WBP flow tracings (ml/s) of an uninjured rat exposed to room air (left) and 7% CO₂ (right). The tracings were converted to respiratory parameters such as f, Vt, and Ve by software.

Figure 2.

More severe grades of C5 hemicontusion injury resulted in greater loss of postinjury body weight and higher degrees of respiratory dysfunction. A, The effect of lesion dose (uninjured control vs 12.5, 25, or 50 mm C5 hemicontusion injury) is shown to correlate inversely with body weight during the 6 week post-SCI course. Note that rats with 50 mm injuries did not recover to control levels, even after 6 weeks (^*p < 0.05; Spearman correlation). ctrl, Control; p.i., postinjury. B, Representative ventilatory parameters at 2 weeks after SCI versus uninjured controls. Error bars represent SEM. ^*p < 0.05; GLM, post hoc multiple linear regression. mod, Moderate injury. Left, Respiratory frequency as a function of lesion dose (uninjured control vs 12.5, 25, or 50 mm C5 hemicontusion injury) and CO₂ exposure (room air, 5% CO₂, or 7% CO₂). The CO₂ level was a significant covariate across all lesion dose groups (p < 0.001). Lesion dose was a significant covariate during room air ventilation (p = 0.037). Rats with more severe lesions had a tendency for greater resting respiratory frequency while breathing room air but had a diminished augmentation of response to hypercapnic stimulus. Middle, Tidal volume as a function of lesion dose and CO₂ exposure. The CO₂ level was a significant covariate across all lesion dose groups (p < 0.001). Lesion dose was a significant covariate during room air ventilation and 5% and 7% CO₂ ventilation (p ≤ 0.017). Right, Minute ventilation as a function of lesion dose and CO₂ exposure. The CO₂ level was a significant covariate across all lesion dose groups (p < 0.001). Rats with all grades of C5 hemicontusion injury, resulting from a combination of higher f and diminished Vt, had resting Ve similar to those of controls while breathing room air. When challenged with hypercapnic stimulus, rats with C5 hemicontusion injury displayed a lesion dose-dependent diminished augmentation of minute ventilation response compared with uninjured controls (p = 0.035 at 5% CO₂; p < 0.001 at 7% CO₂).

Figure 3.

Histopathological changes after C5 hemicontusion injury. a, Solvent blue plus hematoxylin and eosin transverse sections 2 mm rostral from the epicenter (a1, a4), at the epicenter (a2, a5), and 2 mm caudal from the epicenter (a3, a6) in rats representative of mild injury (top; a1-a3) and severe injury (bottom; a4-a6) at 4 weeks after SCI. Scale bar, (in a1) 500 μm. b, Cresyl violet transverse sections 2 mm rostral to the epicenter in a representative severe hemicontusion injury (b1) at 4 weeks after SCI versus an uninjured control (b2) rat. Arrowheads indicate ventral motoneurons. b3, A representative ventral motoneuron (oil immersion) displaying Nissl substance, a euchromatic nucleus, and a distinct nucleolus. Scale bars: b1-b3, 20 μm.

Figure 4.

Forelimb and hindlimb neurobehavioral function declined after C5 hemicontusion injury in lesion dose-dependent manners. Top, Neurobehavioral function of the forelimb (measured by a modified Tarlov scale) on the side ipsilateral to the hemicontused spinal cord correlated inversely with lesion dose (uninjured control vs 12.5, 25, or 50 mm C5 hemicontusion injury) from 1 d to 6 weeks after SCI (^*p < 0.05; Spearman correlation). Forelimb neurobehavioral function on the side opposite of the spinal cord hemicontusion recovered to preoperative levels during the first week after SCI. Bottom, Neurobehavioral function of the hindlimb (measured by a modified BBB scale) on the side ipsilateral to the hemicontused spinal cord correlated inversely with lesion dose (uninjured control vs 12.5, 25, or 50 mm C5 hemicontusion injury) from 1 d to 6 weeks after SCI (^*p < 0.05; Spearman correlation). ctrl, Control; p.i., postinjury.

Figure 5.

Effects of 5-HT_1A agents on minute ventilation in hypercapnic conditions after C5 hemicontusion injury. A, WBP respiratory flow tracings of a representative uninjured rat breathing 7% CO₂ (top) and then breathing 7% CO₂ 2 weeks after severe C5 hemicontusion injury (HCSCI) before (middle) and after administration of 8-OH DPAT (250 μg/kg, i.p.; bottom). B, The effects of 8-OH DPAT on Ve in rats (n = 4) with severe (50 mm) hemicontusion injury at 5 d after SCI. Systemic 8-OH DPAT (250 μg/kg) restored Ve responses to 7% CO₂ stimulus in severely injured rats to a level comparable with preinjury baseline, an effect that lasted up to 4 h after a single intraperitoneal dose (^*p < 0.05, significantly different from predrug levels; repeated-measures ANOVA with post hoc Tukey's tests). C1, Effects of 5-HT_1A agonists and antagonists on respiratory parameters at 2 weeks after SCI in severely injured rats (^*p < 0.05; repeated-measures ANOVA with post hoc Tukey's tests). Note that Ve responses to a hypercapnic stimulus were markedly diminished at 2 weeks after SCI before drug treatment compared with preinjury baselines. 8-OH DPAT treatment resulted in a dose-dependent increase in Ve response; treatment with buspirone also improved Ve response compared with predrug levels. The augmentation of Ve after 8-OH DPAT was blocked by preadministration of the 5-HT_1A antagonist p-MPPI (PMPPI). C2, Histogram of Ve responses to 7% CO₂ stimulus in rats with severe hemicontusion injury (50 mm; n = 12) after 8-OH DPAT (125 μg/kg), 8-OH DPAT (250 μg/kg), p-MPPI (3 mg/kg), p-MPPI plus 8-OH DPAT (250 μg/kg), or buspirone (1.5 mg/kg) treatment at 2 weeks after SCI compared with predrug Ve at 2 weeks after SCI and preoperative baselines. Each black horizontal line within each larger colored line represents the distribution of per-breath Ve responses for each rat during hypercapnic challenge.