Neuropathic Pain Creates an Enduring Prefrontal Cortex Dysfunction Corrected by the Type II Diabetic Drug Metformin But Not by Gabapentin

Abstract

Chronic pain patients suffer from pain-related cognitive deficits, even when taking commonly prescribed analgesics. These deficits are likely related to pain-related maladaptive plasticity in the frontal cortex. We sought to model cognitive deficits in mice with neuropathic pain to examine maladaptive morphological plasticity in the mPFC and to assess the effects of several therapeutics. We used an attentional set-shifting task in mice with spared nerve injury (SNI) who received either a single intrathecal injection of an analgesic dose of clonidine, 7 d of 100 mg/kg gabapentin, or 7 d of 200 mg/kg metformin. Male SNI mice were significantly more impaired in the set-shifting task than females. This deficit correlated with a loss of parvalbumin (PV) and reductions in axon initial segment (AIS) length in layers 5/6 of the infralimbic (IL) cortex. Acute pain relief with clonidine had no effect on set-shifting performance, whereas pain relief via 7 day treatment with gabapentin worsened the impairment in both SNI and sham mice. Gabapentin reversed the PV loss in the IL but had no effect on AIS length. Treatment with the AMPK-activator metformin completely reversed the pain-related cognitive impairment and restored AIS length in the IL but had little effect on PV expression. Our findings reveal that neuropathic pain-related cognitive impairments in male mice are correlated to bilateral morphological changes in PV interneurons and layer 5/6 IL pyramidal neuron AIS. Pain relief with metformin can reverse some of the functional and anatomical changes.SIGNIFICANCE STATEMENT Cognitive impairments are a comorbidity of neuropathic pain but are inadequately addressed by existing therapeutics. We used a neuropathic pain model in mice to demonstrate that male (but not female) mice show a robust pain-related deficit in attentional set-shifting, which is associated with structural plasticity in axon initial segments in the infralimbic cortex. These deficits were completely reversed by 7 day treatment with the antidiabetic drug metformin, suggesting that this drug can be repurposed for the treatment of neuropathic pain and its cognitive comorbidities. Our findings have implications for our understanding of how neuropathic pain causes structural plasticity in the brain, and they point to a marked sexual dimorphism in neuropathic pain mechanisms in mice.

Keywords: analgesics; axon initial segment; chronic pain; cognitive impairment; medial prefrontal cortex; metformin.

Figure 1.

Neuropathic pain impairs cognitive flexibility with greater impairment in male than in female mice. A, Illustration of the attentional set-shifting protocol. Mice were habituated to the maze over 3 d. On day 4, mice were assessed for preference to turn right or left over 7 trials (Turn bias). The following day, mice were trained to turn against their turn bias to retrieve food reward and to ignore the pseudorandomly placed visual cue (Response). Approximately 3 weeks after SNI, the animals were assessed for retention of the task (Retest). The next day, mice were trained to shift strategies so that they now had to turn toward the arm containing the visual cue to obtain food reward (Set-shifting). B, Male and female SNI mice showed no impairment on retest day, indicating that SNI had no negative effect on retention of response training. In contrast, male and female SNI mice required significantly more trials to reach criterion on set-shifting day with males requiring significantly more trials than females. C, Male SNI mice made significantly more NRE and RE compared with female SNI mice. Two-way ANOVA with Bonferroni multiple comparisons: ****p < 0.0001; **p < 0.01; *p < 0.05.

Figure 2.

Clonidine-induced acute analgesia has no effect on the pain-related impairment in cognitive flexibility. A, SNI mice treated with clonidine (10 μg i.t.) 1 h before set-shifting showed no differences in set-shifting performance compared with vehicle-treated SNI animals. B, SNI vehicle and clonidine-treated animals made significantly more NRE and OE type errors compared with vehicle-treated sham controls. C, SNI mice received either vehicle or clonidine (10 μg i.t.) and were tested 1 h later for mechanical hypersensitivity. Two-way ANOVA with Bonferroni multiple comparisons: ****p < 0.0001; ***p < 0.001; **p < 0.01; *p < 0.05.

Figure 3.

Pain relief with gabapentin worsens attentional set-shifting performance in SNI and sham mice. A, Gabapentin was administered twice daily (100 mg/kg) for 7 d, a dose determined to alleviate tactile allodynia without impairing motor function (Figure 3-1). The last injection was delivered ∼1 h before set-shifting. Gabapentin significantly impaired SNI- and Sham-treated animals. B, SNI animals treated with gabapentin made significantly more NRE, RE, and OE compared with vehicle-treated sham controls. C, Gabapentin reversed mechanical withdrawal thresholds when tested on days 3 and 7 of its 7 day administration period. Two-way ANOVA with Bonferroni multiple comparisons: ****p < 0.0001; ***p < 0.001; **p < 0.01; *p < 0.05.

Figure 4.

Metformin reverses pain-related impairment in cognitive flexibility. A, Metformin delivered once daily (200 mg/kg i.p.) for 7 d completely reversed the pain-related deficit in set-shifting performance. B, Metformin-treated SNI animals made significantly less NRE and OE compared with SNI vehicle-treated controls and performed similarly to sham animals. C, Metformin reverses mechanical hypersensitivity with 7 d of daily injections. Two-way ANOVA with Bonferroni. Significant comparisons between the SNI vehicle group and the SNI metformin group: ‡SNI + veh and SNI + metformin. Significantly different compared with sham controls: ****p < 0.0001; **p < 0.01; *p < 0.05.

Figure 5.

PV expression is reduced in the ipsilateral IL of SNI mice. A, Representative 20× images showing PV loss in the IL of SNI male mice. B, Male SNI mice showed a significant loss of PV in the IL ipsilateral to injury (left hemisphere), which was confined to cortical layers 5/6. C, Female SNI mice showed no PV loss in the IL. The PrL (D) and Cg1 (E) cortices showed no loss in PV. F, Gabapentin treatment (100 mg/kg twice daily for 7 d) reversed the PV loss in male SNI mice. G, Metformin treatment (200 mg/kg once daily for 7 d) had no effect on PV loss. This effect was not dependent on when the tissue was collected: 1 or 14 d after treatment (tx). One- or two-way ANOVA with Bonferroni with all groups compared with the SNI + vehicle group: ***p < 0.001; **p < 0.01; *p < 0.05.

Figure 6.

SNI causes a reduction in AIS length in the IL, an effect that is partially reversed by metformin. A, Representative 40× and 100× images showing AIS staining in the IL. B, Male SNI mice had significantly shorter AISs in the ipsilateral and contralateral IL, which was confined to cortical layers 5/6. C, Female SNI mice did not show a significant reduction in AIS length in the IL. There were no changes observed in AIS length in the PrL (D) or Cg1 (E) subregions of male SNI mice. F, Gabapentin treatment had no effect on AIS length in the IL. G, Metformin treatment reversed the loss in the ipsilateral IL, but not contralateral IL, when assessed 1 d after treatment but reversed it in both hemispheres when assessed 14 d after treatment. Two-way ANOVA with Bonferroni with all groups compared with the SNI + vehicle group: **p < 0.01; *p < 0.05.