Alpha2-adrenergic receptor activation reinstates motor deficits in rats recovering from cortical injury

Abstract

Norepinephrine plays an important role in motor functional recovery after a brain injury caused by ferrous chloride. Inhibition of norepinephrine release by clonidine is correlated with motor deficits after motor cortex injury. The aim of this study was to analyze the role of α₂-adrenergic receptors in the restoration of motor deficits in recovering rats after brain damage. The rats were randomly assigned to the sham and injury groups and then treated with the following pharmacological agents at 3 hours before and 8 hours, 3 days, and 20 days after ferrous chloride-induced cortical injury: saline, clonidine, efaroxan (a selective antagonist of α₂-adrenergic receptors) and clonidine + efaroxan. The sensorimotor score, the immunohistochemical staining for α_2A-adrenergic receptors, and norepinephrine levels were evaluated. Eight hours post-injury, the sensorimotor score and norepinephrine levels in the locus coeruleus of the injured rats decreased, and these effects were maintained 3 days post-injury. However, 20 days later, clonidine administration diminished norepinephrine levels in the pons compared with the sham group. This effect was accompanied by sensorimotor deficits. These effects were blocked by efaroxan. In conclusion, an increase in α₂-adrenergic receptor levels was observed after injury. Clonidine restores motor deficits in rats recovering from cortical injury, an effect that was prevented by efaroxan. The underlying mechanisms involve the stimulation of hypersensitive α₂-adrenergic receptors and inhibition of norepinephrine activity in the locus coeruleus. The results of this study suggest that α₂ receptor agonists might restore deficits or impede rehabilitation in patients with brain injury, and therefore pharmacological therapies need to be prescribed cautiously to these patients.

Keywords: alpha₂-adrenoceptors; ambulatory behavior; clonidine; cortical injury; efaroxan; functional recovery; immunohistochemistry; motor deficit; norepinephrine; sensorimotor score.

Figure 1

Experimental timeline. CSF: Cerebrospinal fluid; HPLC: high-performance liquid chromatography; NE: norepinephrine.

Figure 2

Effect of FeCl₂-induced MC injury on the temporal evolution of rat sensorimotor activity at 3 hours pre-injury and 8 hours, 3 days, and 20 days post-injury. Values are presented as means ± SEM (n = 8 per group). Statistical analyses were performed with the nonparametric Kruskal-Wallis test followed by the Mann-Whitney U test to compare the mean rank of the drug treatment groups. *P < 0.05, vs. sham group.

Figure 3

Percent change with respect to basal values obtained for the distance traveled by the sham and injured rats. (A) Effect of FeCl₂-induced MC injury on the distance traveled by rats in each group at 3 hours pre-injury and 8 hours, 3 and 20 days post-injury. (B) Representative maps of the distance traveled under the abovementioned conditions are shown. Values are presented as means ± SEM (n = 8 per group). Statistical analyses were performed with the nonparametric Kruskal-Wallis test followed by the Mann-Whitney U test to compare the mean rank of the drug treatment groups. ***P < 0.001, vs. sham group.

Figure 4

Effects obtained 30 minutes after the systemic administration of clonidine (CL), efaroxan (EF), and the coadministration of CL + EF on the functional recovery of sensorimotor activity at 20 days after FeCl₂-induced cortical injury. Values are presented as means ± SEM (n = 8 per group). Statistical analyses were performed with the nonparametric Kruskal-Wallis test followed by the Mann-Whitney U test to compare the mean rank of the drug treatment groups. ***P < 0.001, vs. sham group.

Figure 5

Percent change with respect to basal values obtained for the distance traveled by sham and injured rats. (A) Effects of the administration of clonidine (CL), efaroxan (EF), and CL + EF on the distance traveled at 20 days post-injury. (B) Representative maps of the distance traveled under the abovementioned conditions are shown. Values are presented as means ± SEM (n = 8 per group). Statistical analyses were performed with the nonparametric Kruskal-Wallis test followed by the Mann-Whitney U test to compare the mean rank of the drug treatment groups. ***P < 0.001, vs. sham group; ###P < 0.001, vs. sham (vehicle) group.

Figure 6

Effects of the administration of clonidine (CL), efaroxan (EF), and CL + EF on pontine NE levels at 20 days after cortical injury (chromatographic analysis). Values are presented as means ± SEM (n = 8 per group). Statistical analyses were performed using a one-way analysis of variance followed by Tukey’s post hoc test. **P < 0.01, vs. sham group. NE: Norepinephrine.

Figure 7

Histological analysis of the locus coeruleus (LC). (A) Nissl staining was performed to show the site of the LC (indicated with a white arrow) that was considered in the analysis. Scale bar: 200 μm. (B, C) Tissues were immunostained with an α_2A antibody (green), and Hoechst was used for core staining (blue). α_2A-adrenergic receptors are indicated by white arrows in the LC. Scale bars: 50 μm. (D) Immunohistochemical analysis of α_2A-adrenergic receptors indicated increased levels in the injured group compared to the sham group (P < 0.05). Values are presented as means ± SEM (n = 5 per group). *P = 0.039, vs. sham group