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BACKGROUND Gut microbiota, a consortium of diverse microorganisms residing in the gastrointestinal tract, has emerged as a key player in neuroinflammatory responses, supporting the functional relevance of the "gut-brain axis." Chronic-constriction injury of the sciatic nerve (CCI) is a commonly used animal model of neuropathic pain with a major input from T cell-mediated immune responses. In this article, we sought to examine whether gut microbiota influences CCI neuropathic pain, and, if so, whether T-cell immune responses are implicated. METHODS We used a mixture of wide-spectrum oral antibiotics to perturbate gut microbiota in mice and then performed CCI in these animals. Nociceptive behaviors, including mechanical allodynia and thermal hyperalgesia, were examined before and after CCI. Additionally, we characterized the spinal cord infiltrating T cells by examining interferon (IFN)-γ, interleukin (IL)-17, and Foxp3. Using a Foxp3-GFP-DTR "knock-in" mouse model that allows punctual depletion of regulatory T cells, we interrogated the role of these cells in mediating the effects of gut microbiota in the context of CCI neuropathic pain. RESULTS We found that oral antibiotics induced gut microbiota changes and attenuated the development of CCI neuropathic pain, as demonstrated by dampened mechanical allodynia and thermal hyperalgesia. Percentages of IFN-γ-producing Th1 cells and Foxp3+ regulatory T cells were significantly different between animals that received oral antibiotics (Th1 mean = 1.0, 95% confidence interval [CI], 0.9-1.2; Foxp3 mean = 8.1, 95% CI, 6.8-9.3) and those that received regular water (Th1 mean = 8.4, 95% CI, 7.8-9.0, P < .01 oral antibiotics versus water, Cohen's d = 18.8; Foxp 3 mean = 2.8, 95% CI, 2.2-3.3, P < .01 oral antibiotics versus water, Cohen's d = 6.2). These T cells characterized a skewing from a proinflammatory to an anti-inflammatory immune profile induced by gut microbiota changes. Moreover, we depleted Foxp3+ regulatory T cells and found that their depletion reversed the protection of neuropathic pain mediated by gut microbiota changes, along with a dramatic increase of IFN-γ-producing Th1 cell infiltration in the spinal cord (before depletion mean = 2.8%, 95% CI, 2.2-3.5; after depletion mean = 9.1%, 95% CI, 7.2-11.0, p < .01 before versus after, Cohen's d = 5.0). CONCLUSIONS Gut microbiota plays a critical role in CCI neuropathic pain. This role is mediated, in part, through modulating proinflammatory and anti-inflammatory T cells.

BACKGROUND: The incremental dose of opioids used in chronic pain management often leads to a reduced opioid analgesic effect, opioid misuse and addiction. Central dopamine dysfunction contributes to the chronicity of pain and a decreased opioid analgesic effect. Methylphenidate (MPH/Ritalin) enhances central dopamine function by inhibiting dopamine re-uptake. In this study, we used a rat model of chronic pain to examine whether combination of MPH with morphine would improve the morphine analgesic effect under a chronic pain condition. METHODS: Tibiotarsal joint Complete Freund’s Adjuvant (CFA) injection in rats was utilized to induce chronic nociception. The analgesic effect of low-dose MPH (0.25 mg/kg), low-dose morphine (2.5 mg/kg), and their combination was examined in CFA rats. Nociceptive behavior was assessed by von Frey test. Conditioned place preference and open field tests were used to examine the rewarding behavior and locomotor activity in rats respectively. RESULTS: Our findings are as follows: (1) in CFA rats with chronic pain, 2.5 mg/kg of morphine had less analgesic effect than 10 mg/kg of morphine at 28 days after injury (95% confidence intervals for difference of means of von Frey threshold in gram: −11.9 [−6.5 to −17.3]); (2) in the one hour time window of 30 to 90 minutes after injection, the combination of MPH (0.25 mg/kg) with morphine (2.5 mg/kg) increased synergistically and prolonged the analgesic effect in CFA rats as compared with MPH or morphine alone (P=0.01 for MPH by morphine interaction, and 95% confidence intervals for difference of means of von Frey threshold in gram: 3.3 [1.37 to 6.12] for the combination versus MPH and 3.2 [1.35 to 5.74] for the combination versus morphine); (3) at the low dose (0.25 mg/kg), MPH did not increase locomotor activity (MOR+MPH versus MOR, P=0.13), nor significantly enhanced morphine reward behavior (MOR+MPH versus MOR, P=0.63) in CFA rats. CONCLUSIONS: Our data suggest that a combination therapy using low-dose MPH and morphine may produce a morphine-sparing effect in chronic pain management.

As opioid abuse and addiction have developed into a major national health crisis, prescription of opioids for pain management has become more controversial. However, opioids do help some patients by providing pain relief and improving the quality of life. To better understand the addictive properties of opioids under chronic pain conditions, we used a conditioned place preference (CPP) paradigm to examine the rewarding properties of morphine in rats with persistent nociception.Spared nerve injury (SNI) model was used to induce persistent nociception in rats. Nociceptive behavior was assessed by von Frey test. CPP test was used to examine the rewarding properties of morphine.Our findings are as follows: (1) SNI rats did not show a difference compared with sham rats in magnitude of morphine-induced CPP 1 day after last morphine injection (2-way analysis of variance; for SNI versus sham, F[1,42] = 0.014, P = .91; and 95% confidence intervals for difference of means, -5.9 [-58 to 46], 0.76 [-51 to 53], and 0.90 [-51 to 53] for 2.5, 5, and 10 mg/kg, respectively); (2) increasing morphine dosage (2.5, 5, and 10 mg/kg) did not further increase the magnitude of CPP in both sham and SNI rats (for dosage: F[2,42] = 0.94, P = .40); and (3) morphine-induced CPP persisted in sham rats but extinguished in SNI rats when tested at 8 days after last morphine injection (for sham versus SNI: Bonferroni correction, P.006 for both 5 and 10 mg/kg doses; and 95% confidence intervals for difference of means, 80.3 [19.7-141] and 87.0 [26.3-148] for 5 and 10 mg/kg, respectively).Our data provide new evidence supporting the notion that the brain's reward circuitry changes in the context of persistent pain. This observational study suggests that future investigation into the neurobiology of opioid reward requires consideration of the circumstances in which opioid analgesics are administered.