Serotonin 1A receptor agonists reverse respiratory abnormalities in spinal cord-injured rats

Abstract

Contusion spinal cord injury (SCI) at T8 produces respiratory abnormalities in conscious rats breathing room air and challenged with CO2. In seeking ways to improve respiration after SCI, we tested drugs that stimulate serotonin 1A (5-HT1A) receptors, based on our previous findings that these agents can counteract respiratory depression produced by morphine overdose. Respiratory function was measured with a head-out plethysmograph system in conscious rats. T8 SCI rats (n = 5) showed decreased tidal volume (Vt; 0.90 +/- 0.02-0.66 +/- 0.03 ml; p < 0.05) and increased respiratory rate (f;91 +/- 3.7-132 +/- 5.7 breaths/min; p < 0.05) with room air ventilation at 24 hr after injury. They also exhibited a diminished response to the respiratory stimulating effect of 7% CO2; minute ventilation increased to 250 +/- 17 ml/min before, but only to 162 +/- 15 ml/min at 24 hr after SCI (p < 0.05). Respiratory deficits during room air ventilation were also observed at 7 d after injury (n = 3). Treatment with the 5-HT1A receptor agonist 8-hydroxy-2-(di-n-propylmino)tetralin (8-OH-DPAT; 250 microg/kg, i.p.) at 24 hr (n = 5) or 7 d (n = 3) after injury normalized Vt, f, and the respiratory response to 7% CO2. Identical results were obtained with another 5-HT1A receptor agonist, buspirone (1.5 mg/kg, i.p.; n = 3). In contrast, intraperitoneal saline vehicle administration (n = 5) showed no beneficial effects on SCI-impaired respiration. Finally, pretreatment with a specific antagonist of 5-HT1A receptors, 4-iodo-N-[2-[4-(methoxyphenyl)-1-piperazinyl]ethyl]-N-2-pyridinyl-benzamide (3 mg/kg, i.p.; n = 3) given 20 min before 8-OH-DPAT, prevented 8-OH-DPAT from restoring respiration to normal. Our results demonstrate that drugs that stimulate 5-HT1A receptors counteract respiratory abnormalities in conscious rats after SCI.

Figure 1.

Noninvasive measurements of respiratory function in conscious rats. A, Schematic presentation of the restrained head-out plethysmograph system for rodents. B, The animals breathe from a funnel fixed in the front wall of a box made of an opaque material. The box surrounds the front two-thirds of the body cylinder of the plethysmograph, and the rear outlet of the box is covered with a piece of bath towel (illustrated by a dashed line). The animals were exposed to room air for baseline recordings and then to a gas mixture containing 7% CO₂ mixed with 60% O₂ and 33% N₂) for 5 min, and recording of respiratory activity was continued for another 2 min (a total recording duration of 7 min). After a new baseline was obtained by allowing the animals to breathe room air for 20 min, the rats were injected with drugs that affect 5-HT_1A receptors, and the recordings were repeated as specified in each experiment (figure modified from Teng et al., 1999).

Figure 2.

Effects of 8-OH-DPAT on SCI-induced respiratory dysfunction at 24 hr after injury. A, Vt is significantly decreased after SCI as compared with preinjury data for the same rats breathing room air or a gas mixture containing 7% CO₂. In addition, the increase in Vt normally elicited by 7% CO₂ is diminished. Administration of saline (vehicle for 8-OH-DPAT) had no effect on SCI-induced alterations in Vt. A baseline was re-established, and then 8-OH-DPAT (250 μg/kg, i.p.) was administered. Twenty-four minutes later, the Vt with room air and with 7% CO₂ was normalized. B, The measurement of f under room air conditions was significantly increased at 24 hr after injury. There was no further increase in f in response to 7% CO₂. Administration of saline had no effect. At 24 min after injection with 8-OH-DPAT, f with room air was reduced to preinjury levels and demonstrated a normal increase when respiration was stimulated with 7% CO₂. C, Ve while breathing room air was not affected by SCI, but there was a decreased response to 7% CO₂. Saline administration had no effect. The response to 7% CO₂ returned to preinjury levels at 24 min after injection with 8-OH-DPAT. Data reported are means ± SEM for five rats and were analyzed by one-way ANOVA followed by Tukey's and Dunn's tests. *Significant difference from preinjury value obtained with the same breathing condition (room air or 7% CO₂); #significant difference compared with value obtained breathing room air.

Figure 3.

The time course of the effect of 8-OH-DPAT on Ve in rats at 24 hr after SCI. Bars represent the average Ve ± SEM for rats (n = 3) before and after SCI, the baseline measure before 8-OH-DPAT administration, and at specified times after the drug injection (250 μg/kg, i.p.) at 24 hr after injury. In this group of rats, SCI resulted in a small drop in baseline Ve when breathing room air, and greatly diminished the increase in Ve in response to 7% CO₂ challenge. 8-OH-DPAT treatment produced a transient increase in Ve with room air conditions at the 3 min point. At 20 min after treatment, Ve returned to a preinjury level, and the increase in Ve in response to 7% CO₂ was similar to that seen before SCI. Data reported are means ± SEM and were analyzed by repeated measures ANOVA followed by Tukey's test. *Significant difference from preinjury value obtained with the same breathing condition (room air or 7% CO₂); #significant difference compared with value obtained breathing room air.

Figure 4.

Respiratory response to 8-OH-DPAT of SCI animals at 7 d after injury. Tidal volume (A) was significantly decreased, and respiratory rate (B) significantly increased at 7 d after SCI as compared with preinjury data for the same rats, whereas Ve (C) was similar to that before injury. Stimulation with a gas mixture containing 7% CO₂ increased Vt, f, and Ve, similar to that seen before injury. Saline injection has no effect. Administration of 8-OH-DPAT (250 μg/kg, i.p.) normalized the Vt with room air conditions, but f and Ve were significantly greater than that before injury with both room air and 7% CO₂ conditions. Data reported were means ± SEM for three rats and were analyzed by one-way ANOVA followed by Tukey's and Dunn's tests. *Significant difference from preinjury value obtained with the same breathing condition (room air or 7% CO₂); #significant difference compared with values obtained breathing room air.

Figure 5.

Effects of 8-OH-DPAT on respiratory function of normal rats. Normal rats breathing room air exhibited a transitory increase in Vt (A) at 4 min and a longer lasting increase in f (B) and Ve (C) at 4 and at 22 min after administration of 8-OH-DPAT (250 μg/kg, i.p.). The values for Vt, f, and Ve obtained with 7% CO₂ conditions were not affected by administration of 8-OH-DPAT. Data reported are means ± SEM for three rats and were analyzed by one-way ANOVA followed by Dunn's tests. *Significant difference from preinjury value obtained with the same breathing condition (room air or 7% CO₂); #significant difference compared with values obtained breathing room air. Administration of saline at the 3 min time point had no effect.

Figure 6.

Effects of buspirone on SCI-induced respiratory dysfunction. At 24 hr after SCI, rats demonstrated reduced Vt (A) and increased f (B) with room air breathing conditions, although Ve (C) was similar to that obtained before injury. Stimulation with 7% CO₂ increased Ve to a lesser extent than before injury. A baseline was re-established, and then buspirone (1.5 mg/kg, i.p.) was administered. Buspirone normalized Vt and f with room air breathing conditions and increased the response of Ve to 7%CO₂ to equal that seen before SCI. Data reported are mean ± SEM for three rats and were analyzed by one-way ANOVA followed by Tukey's and Dunn's tests. *Significant difference from preinjury value obtained with the same breathing condition (room air or 7% CO₂); #significant difference compared with values obtained breathing room air.

Figure 7.

Effects of 8-OH-DPAT on SCI-induced respiratory dysfunction at 24 hr in animals treated with the 5-HT_1A receptor antagonist p-MPPI. Twenty-four hours after SCI, animals exhibited decreased Vt (A), increased f (B), and decreased Ve (C) with room air breathing conditions as well as a reduced response to 7% CO₂. Treatment with p-MPPI (3 mg/kg, i.p.) followed by 8-OH-DPAT (250 μg/kg, i.p.) blocked the normalization of respiration previously seen with 8-OH-DPAT (Fig. 2). Data reported are means ± SEM for three rats and were analyzed by one-way ANOVA followed by Tukey's and Dunn's tests. *Significant difference from preinjury value obtained with the same breathing condition (room air or 7% CO₂); #significant difference compared with values obtained breathing room air.