Combination Drug Therapy of Pioglitazone and D-cycloserine Attenuates Chronic Orofacial Neuropathic Pain and Anxiety by Improving Mitochondrial Function Following Trigeminal Nerve Injury

Abstract

Objectives: The study aim was to determine how peripheral trigeminal nerve injury affects mitochondrial respiration and to test efficacy of combined treatment with 2 Federal Drug Administration approved drugs with potential for improving mitochondrial bioenergetics, pain and anxiety-related behaviors in a chronic orofacial neuropathic pain mouse model.

Methods: Efficacy of (R)-(+)-4-amino-3-isoxazolidinone (D-cycloserine, DCS), an N-Methyl-D-aspartate antagonist/agonist, and Pioglitazone (PIO), a selective agonist of nuclear receptor peroxisome proliferator-activated receptor gamma was investigate in the trigeminal inflammatory compression (TIC) neuropathic nerve injury mouse model. Combined low doses of these drugs (80 mg/kg DCS and 100 mg/kg PIO) were given as a single bolus or daily for 7 days post-TIC to test ability to attenuate neuropathic nociceptive and associated cognitive dependent anxiety behaviors. In addition, beneficial effects of the DCS/PIO drug combination were explored ex vivo in isolated cortex/brainstem mitochondria at 28 weeks post-TIC.

Results: The DCS/PIO combination not only attenuated orofacial neuropathic pain and anxiety-related behaviors associated with trigeminal nerve injury, but it also improved mitochondrial bioenergetics.

Discussion: The DCS/PIO combination uncoupled mitochondrial respiration in the TIC model to improve cortical mitochondrial dysfunction, as well as reduced nociceptive and anxiety behaviors present in mice with centralized chronic neuropathic nerve injury. Combining these drugs could be a beneficial treatment for patients with depression, anxiety, or other psychological conditions due to their chronic pain status.

Figure 1. TIC Injury Induced Unilateral Whisker Pad Mechanical Allodynia

The 50% mechanical threshold (in gram force) was measured bilaterally on the whisker pads of the mice with TIC injury and the sham mice. The mechanical threshold was decreased on the ipsilateral whisker pad of mice with TIC injury within one week post injury. The mechanical threshold of contralateral whisker pad was unaffected by the surgery. The mechanical threshold of the ipsilateral and contralateral whisker pads of the sham or naïve mice also did not change. TIC n=9; TIC (ipsilateral) vs. TIC (contralateral) **** p<0.0001; two-way ANOVA, Bonferroni post hoc test.

Figure 2. DCS Attenuated Whisker Pad Mechanical Allodynia, but Does Not Reverse Anxiety-Like Behavior in the Mice with TIC Injury

(A) Dose response curve for DCS showed that higher doses of DCS (160 mg/kg and 320 mg/kg) are effective at alleviating the mechanical allodynia on the whisker pad of the mice with TIC injury (n=6). (B) When mice were given a 7-day treatment of lower doses of DCS (s.c.), only the 80 mg/kg dose elevated the mechanical threshold in the mice with TIC injury (n=8). (C) On day 7, two hours post-injection, there was no difference in the light side occupancy times for the mice with TIC injury treated with either the vehicle or with 80 mg/kg dose of DCS in the light-dark box preference test (n=5). A and B: t-test * p<0.05, *** p<0.01; **** p<0.0001; two-way ANOVA, Bonferroni post hoc test. C: Unpaired t-test, n.s.

Figure 3. Combined DCS and PIO Attenuated Whisker Pad Mechanical Allodynia and Anxiety-Like Behavior in Mice with TIC Injury

(A) One-time dose of DCS (80 mg/kg) + PIO (100 mg/kg) elevated mechanical threshold on the whisker pad of the mice with TIC injury. This was significantly different from the effect of a single dose of either DCS (80 mg/kg) or PIO (100 mg/kg) only (n=6–8). The mechanical threshold after the drug combination was also significantly different from the threshold of the vehicle treated group indicating reduced hypersensitivity. (B) A bolus of PIO (100 mg/kg) was given on the last day of the 7 consecutive day DCS (80 mg/kg) treatment course. The whisker pad mechanical threshold was significantly increased in the mice with TIC injury compared to the vehicle treated mice with TIC injury (n=5). (C) Two hours after injection of the PIO bolus on day 7, the drug and the vehicle treated mice with TIC injury were tested with the light-dark box place preference test (n=8). The drug treated group spent significantly more time in the lighted chamber compared to the vehicle treated group A and B: * p<0.05, *** p<0.001; **** p<0.0001; two-way ANOVA, Bonferroni post hoc test. C: * p<0.05; unpaired t-test.

Figure 4. A Mild Mitochondrial Uncoupler Attenuated the Whisker Pad Mechanical Allodynia in the Mice with TIC Injury

The 2,4-DNP (5 mg/kg) significantly increased the 50% mechanical threshold of the whisker pad of mice with TIC injury compared to vehicle treated mice. This effect started at one hour, peaked at 3 hour, and persisted for 5 hours post injection (n=8). * p<0.05, **** p<0.0001; two-way ANOVA, Bonferroni post hoc test.

Figure 5. Isolated Cortical Mitochondria from Mice with TIC Injury Had Increased State III and State IV OCR, but Decreased RCR That was Reversed with Combined DCS + PIO

(A) Cortical mitochondria isolated from mice with TIC injury had a significantly increased oxygen consumption rate (OCR) for State III compared to naïve mice. (B) Cortical mitochondria isolated from mice with TIC injury had an increased State IV respiration compared to naïve mice. (C) The respiratory control ratio (RCR = State III/State IV)) decreased in the cortical mitochondria of mice with TIC injury. However, the drug combination of DCS and PIO decreased the OCR in State IV, and therefore increased the RCR. (n=8/group) * p<0.05, ** p<0.01; two-way ANOVA, Bonferroni post hoc test; # p<0.05, unpaired t-test

Figure 6. Combined DCS + PIO Increased the Brainstem Mitochondrial RCR in Mice With TIC Injury

(A) For isolated brainstem mitochondria there was no significant difference in the oxygen consumption rate (OCR) of State III for mice with TIC injury compared to naïve mice. However, due to the ex vivo drug combination treatment (50 nM DCS+ 50 nM PIO), the OCR for State III increased in brainstem mitochondria from mice with TIC injury compared to treated brainstem mitochondria from naïve mice. (B) There were no significant differences in the oxygen consumption rate (OCR) of State IV. (C) While the respiratory control ratio (RCR) did not change significantly in the isolated brainstem mitochondria from the mice with TIC injury compared to naïve mice, the RCR of the brainstem mitochondria from the mice with TIC injury was significantly increased compared to treated mitochondria from naïve mice. (n=4/group) * p<0.05; two- way ANOVA, Bonferroni post hoc test