MK-801 upregulates NR2A protein levels and induces functional recovery of the ipsilateral hemidiaphragm following acute C2 hemisection in adult rats

Abstract

Background: C2 hemisection results in paralysis of the ipsilateral hemidiaphragm. Recent data indicate that an upregulation of the N-methyl-D-aspartate (NMDA) receptor 2A subunit following chronic C2 hemisection is associated with spontaneous hemidiaphragmatic recovery following injury. MK-801, an antagonist of the NMDA receptor, upregulates the NR2A subunit in neonatal rats.

Hypothesis: We hypothesized that administration of MK-801 to adult, acute C2-hemisected rats would result in an increase of NR2A in the spinal cord. Furthermore, we hypothesized that upregulation of NR2A would be associated with recovery of the ipsilateral hemidiaphragm as in the chronic studies.

Design: To develop a dose-response curve, adult rats were treated with varying doses of MK-801 and their spinal cords harvested and assessed for NR2A as well as AMPA GluR1 and GluR2 subunit protein levels. In the second part of this study, C2-hemisected animals received MK-801. Following treatment, the animals were assessed for recovery of the hemidiaphragm through electromyographic recordings and their spinal cords assessed for NR2A, GluR1, and GluR2.

Results: Treatment with MK-801 leads to an increase of the NR2A subunit in the spinal cords of adult noninjured rats. There were no changes in the expression of GluR1 and GluR2 in these animals. Administration of MK-801 to C2-hemisected rats resulted in recovery of the ipsilateral hemidiaphragm, an increase of NR2A, and a decrease of GluR2.

Conclusion: Our findings strengthen the evidence that the NR2A subunit plays a substantial role in mediating recovery of the paralyzed hemidiaphragm following C2 spinal cord hemisection.

Figure 1. Increase in NR2A after treatment with 0.25 or 0.5 mg/kg MK-801 as revealed by Western blot analysis. (A) All optical density values (OD) corresponding to NR2A levels are relative to a standard cortical sample run alongside the samples. After treatment with 0.25 or 0.5 mg/kg MK-801 for 2 consecutive days, the left spinal cord of noninjured animals had a significant increase of NR2A compared to saline-treated control animals (A). There was no significant change in animals treated with 0.125 or 1.0 mg/kg MK-801 (A). No significant change of GluR1 (B) or GluR2 (C) after any dose of MK-801. All values are relative to a standard cortical sample run alongside the samples. After treatment with 0.125, 0.25, 0.5, or 1.0 mg/kg for 2 consecutive days, there were no changes in the levels of GluR1 or GluR2 in the left spinal cord of adult noninjured animals compared to saline-treated controls as revealed by Western blot analysis (B and C). * indicates significance with P < 0.05. Inserts show a representative Western blot.

Figure 2. Significant increase of NR2A following C2 hemisection and treatment with MK-801 as revealed by Western blot analysis (A). All values are relative to a standard cortical sample run alongside the samples. After 2 days of consecutive treatment with either 0.125 or 0.5 mg/kg MK-801, there was a significant increase of NR2A in the left spinal cord of C2-hemisected animals compared to saline-treated controls (A). No significant change of GluR1 (B) and down regulation of GluR2 (C) following C2 hemisection and treatment with MK-801 as revealed by Western blot analysis. All values are relative to a standard cortical sample run alongside the samples. After 2 days of consecutive treatment with either 0.125 or 0.5 mg/kg MK-801, there was no significant change in the levels of GluR1 in C2-hemisected rats compared to saline-treated animals (B). However, treatment with 0.5 mg/kg MK-801 resulted in a significant reduction of GluR2 levels compared to saline-treated animals (C).: * indicates significance with P < 0.05. Representative Western blots shown in the inserts.

Figure 3. Light micrographs showing that treatment of C2-hemisected rats with 0.5 mg/kg MK-801 induces an upregulation of NR2A, no change in GluR1, and a down regulation of GluR2 in C3–C6 ventral horn motor neurons. A, C, and E are C2-hemisected rats treated with 0.5 mg/kg MK-801. B, D, and F are C2-hemisected rats treated with saline. NR2A (A and B), GluR1 (C and D), and GluR2 (E and F) subunits are localized to motor neurons (arrows). Scale bars = 100 μm.

Figure 4. Electromyograms from both the left and right sides of the hemidiaphragm of a saline-treated and the same 0.5 mg/kg MK-801–treated left C2-hemisected rat before and after drug treatment. In both saline- and MK-801–treated rats, there was an absence of EMG activity on the left hemidiaphragm following C2 hemisection and before treatment (first and third pair of traces). Following saline treatment for 2 consecutive days starting 7 days after injury, there was no return of diaphragmatic activity as observed through EMG activity (fourth pair of traces). Following 0.5 mg/kg MK-801 treatment for 2 consecutive days starting 1 week after injury, there was a return of left hemidiaphragmatic activity that was rhythmic and synchronized to the right hemidiaphragm as revealed through bilateral EMG recordings (second pair of traces). Each trace is 7 seconds long. Note difference in scale bars.