Involvement of oxidative stress in orofacial mechanical pain hypersensitivity following neonatal maternal separation in rats.

Patients with persistent pain have sometimes history of physical abuse or neglect during infancy. However, the pathogenic mechanisms underlying orofacial pain hypersensitivity associated with early-life stress remain unclear. The present study focused on oxidative stress and investigated its role in pain hypersensitivity in adulthood following early-life stress. To establish an early-life stress model, neonatal pups were separated with their mother in isolated cages for 2 weeks. The mechanical head-withdrawal threshold (MHWT) in the whisker pad skin of rats received maternal separation (MS) was lower than that of non-MS rats at postnatal week 7. In MS rats, the expression of 8-hydroxy-deoxyguanosine, a marker of DNA oxidative damage, was enhanced, and plasma antioxidant capacity, but not mitochondrial complex I activity, decreased compared with that in non-MS rats. Reactive oxygen species (ROS) inactivation and ROS-sensitive transient receptor potential ankyrin 1 (TRPA1) antagonism in the whisker pad skin at week 7 suppressed the decrease of MHWT. Corticosterone levels on day 14 increased in MS rats. Corticosterone receptor antagonism during MS periods suppressed the reduction in antioxidant capacity and MHWT. The findings suggest that early-life stress potentially induces orofacial mechanical pain hypersensitivity via peripheral nociceptor TRPA1 hyperactivation induced by oxidative stress in the orofacial region.

Neonatal Injury Modulates Incisional Pain Sensitivity in Adulthood: An Animal Study.

Neonatal pain experiences including traumatic injury influence negatively on development of nociceptive circuits, resulting in persistent pain hypersensitivity in adults. However, the detailed mechanism is not yet well understood. In the present study, to clarify the pathogenesis of orofacial pain hypersensitivity associated with neonatal injury, the involvement of the voltage-gated sodium channel (Nav) 1.8 and the C-C chemokine ligand 2 (CCL2)/C-C chemokine receptor 2 (CCR2) signaling in the trigeminal ganglion (TG) in facial skin incisional pain hypersensitivity was examined in 190 neonatal facial-injured and sham male rats. The whisker pad skin was incised on postnatal day 4 and week 7 (Incision-Incision group). Compared to the group without neonatal incision (Sham-Incision group), mechanical hypersensitivity in the whisker pad skin was enhanced in Incision-Incision group. The number of Nav1.8-immunoreactive TG neurons and the amount of CCL2 expressed in the macrophages and satellite glial cells in the TG were increased on day 14 after re-incision in the Incision-Incision group, compared with Sham-Incision group. Blockages of Nav1.8 in the incised region and CCR2 in the TG suppressed the enhancement of mechanical hypersensitivity in the Incision-Incision group. Administration of CCL2 into the TG enhanced mechanical hypersensitivity in the Sham-Sham, Incision-Sham and Sham-Incision group. Our results suggest that neonatal facial injury accelerates the TG neuronal hyperexcitability following orofacial skin injury in adult in association with Nav1.8 overexpression via CCL2 signaling, resulting in the enhancement of orofacial incisional pain hypersensitivity in the adulthood.

Decreased PPARgamma in the trigeminal spinal subnucleus caudalis due to neonatal injury contributes to incision-induced mechanical allodynia in female rats.

Whisker pad skin incision in infancy causes the prolongation of mechanical allodynia after re-incision in adulthood. A recent study also proposed the importance of sex differences in pain signaling in the spinal cord. However, the sex difference in re-incision-induced mechanical allodynia in the orofacial region is not fully understood. In the rats that experienced neonatal injury in the whisker pad skin, the mechanical allodynia in the whisker pad was significantly prolonged after re-incision in adulthood compared to sham injury in infancy. No significant sex differences were observed in the duration of mechanical allodynia. The duration of mechanical allodynia in male rats was shortened by intracisternal administration of minocycline. However, minocycline had no effects on the duration of mechanical allodynia in female rats. In contrast, intracisternal administration of pioglitazone markedly suppressed mechanical allodynia in female rats after re-incision. Following re-incision, the number of peroxisome proliferator-activated receptor gamma (PPARgamma)-positive cells were reduced in the trigeminal spinal subnucleus caudalis (Vc) in female rats that experienced neonatal injury. Immunohistochemical analyses revealed that PPARgamma was predominantly expressed in Vc neurons. Pioglitazone increased the number of PPARgamma-positive Vc neurons in female rats whose whisker pad skin was incised in both infancy and adulthood stages. Pioglitazone also upregulated heme oxygenase 1 and downregulated NR1 subunit in the Vc in female rats after re-incision. Together, PPARgamma signaling in Vc neurons is a female-specific pathway for whisker pad skin incision-induced mechanical allodynia.

Microglial activation in the trigeminal spinal subnucleus interpolaris/caudalis modulates orofacial incisional mechanical pain hypersensitivity associated with orofacial injury in infancy.

PURPOSE: Infantile tissue injury induces sensory deficits in adulthood. Infantile facial incision (IFI) was reported to cause an enhancement of incision-induced mechanical hypersensitivity in adulthood due to acceleration of the trigeminal ganglion neuronal excitability. However, the effects of IFI on activation of microglia in the spinal trigeminal nucleus and its involvement in facial pain sensitivity is not well known. METHODS: A facial skin incision was made in the left whisker pad in infant (IFI) and/or adult rats (AFI). Mechanical head withdrawal threshold and microglial activation in the trigeminal spinal nucleus were analyzed. RESULTS: Mechanical pain hypersensitivity induced by AFI was significantly exacerbated and prolonged by IFI. The number of Iba1-immunoreactive cells in the trigeminal spinal nucleus following AFI was increased by IFI, suggesting that IFI facilitates microglial hyperactivation following AFI. Intraperitoneal administration of minocycline, a microglial activation inhibitor, suppressed the facial incision-induced microglial hyperactivation in the trigeminal spinal nucleus and the exacerbation of the facial mechanical pain hypersensitivity induced by IFI. CONCLUSION: These results suggest that facial trauma in infants causes hyperactivation of microglia in the trigeminal spinal nucleus following AFI, leading to the prolongation of the facial mechanical pain hypersensitivity.

Properties of heat-sensitive neurons in the premotor cortex of conscious monkeys.

To investigate neuronal activity involved in responses to noxious stimuli in conscious monkeys, the animals were subjected to a task that required them to detect a small change in facial skin temperature or light (second temperature: T2, second light: V2) relative to an initial condition (T1 or V1), and to detect changes in V2 along with a heat task. Recordings were obtained from 57 neurons in the ventral premotor cortex (PMv) during the heat or light detection task. T1 neurons and T2 neurons showed increased activity only during T1 or T2, and T1/T2 neurons were activated by both T1 and T2 stimuli. T1/T2 neurons showed an increase in firing at higher T1 temperatures, whereas T1 neurons did not. About half of the non-light/heat-sensitive T1/T2 neurons showed increased firing at higher T2 temperatures, whereas T2 neurons showed no such increase. The heat responses of heat-sensitive PMv neurons were significantly suppressed when monkeys shifted their attention from heat to light. The present findings suggest that heat-sensitive PMv neurons may be involved in motor responses to noxious heat, whereas light/heat-PMv neurons may be involved in emotional and motivational aspects of pain and inappropriate motor responses to allow escape from noxious stimuli.

Suppression of Superficial Microglial Activation by Spinal Cord Stimulation Attenuates Neuropathic Pain Following Sciatic Nerve Injury in Rats.

We evaluated the mechanisms underlying the spinal cord stimulation (SCS)-induced analgesic effect on neuropathic pain following spared nerve injury (SNI). On day 3 after SNI, SCS was performed for 6 h by using electrodes paraspinally placed on the L4-S1 spinal cord. The effects of SCS and intraperitoneal minocycline administration on plantar mechanical sensitivity, microglial activation, and neuronal excitability in the L4 dorsal horn were assessed on day 3 after SNI. The somatosensory cortical responses to electrical stimulation of the hind paw on day 3 following SNI were examined by using in vivo optical imaging with a voltage-sensitive dye. On day 3 after SNI, plantar mechanical hypersensitivity and enhanced microglial activation were suppressed by minocycline or SCS, and L4 dorsal horn nociceptive neuronal hyperexcitability was suppressed by SCS. In vivo optical imaging also revealed that electrical stimulation of the hind paw-activated areas in the somatosensory cortex was decreased by SCS. The present findings suggest that SCS could suppress plantar SNI-induced neuropathic pain via inhibition of microglial activation in the L4 dorsal horn, which is involved in spinal neuronal hyperexcitability. SCS is likely to be a potential alternative and complementary medicine therapy to alleviate neuropathic pain following nerve injury.

Involvement of Satellite Cell Activation via Nitric Oxide Signaling in Ectopic Orofacial Hypersensitivity.

: The mechanical head-withdrawal threshold (MHWT) was significantly reduced following inferior alveolar nerve transection (IANX) in rats. Nitrate and nitrite synthesis was dramatically increased in the trigeminal ganglion (TG) at 6 h after the IANX. The relative number of neuronal nitric oxide synthase (nNOS)-immunoreactive (IR) cells was significantly higher in IANX rats compared to sham-operated and N-propyl-L-arginine (NPLA)-treated IANX rats. On day 3 after NPLA administration, the MHWT recovered considerably in IANX rats. Following L-arginine injection into the TG, the MHWT was significantly reduced within 15 min, and the mean number of TG cells encircled by glial fibrillary acidic protein (GFAP)-IR cells was substantially higher. The relative number of nNOS-IR cells encircled by GFAP-IR cells was significantly increased in IANX rats. In contrast, after NPLA injection into the TG, the relative number of GFAP-IR cells was considerably reduced in IANX rats. Fluorocitrate administration into the TG significantly reduced the number of GFAP-IR cells and prevented the MHWT reduction in IANX rats. The present findings suggest that following IANX, satellite glial cells are activated via nitric oxide (NO) signaling from TG neurons. The spreading satellite glial cell activation within the TG results in mechanical hypersensitivity of face regions not directly associated with the trigeminal nerve injury.

Involvement of TNFα in the enhancement of hypersensitivity in the adulthood-injured face associated with facial injury in infancy.

To evaluate the mechanisms underlying acceleration of hypersensitivity in the adulthood-injured face following facial injury in infants, we developed the rats model with facial skin injury in infants and adulthoods (incision + incision), and facial skin suture in infants and facial skin injury in adulthoods (sham + incision), and analyzed the mechanical head-withdrawal threshold (MHWT) of the facial skin, immunohistochemical analysis of trigeminal ganglion (TG) and the effects of intra-ganglionic administration of neutralizing ant-TNFα antibody and recombinant TNFα on nocifensive behavior. The MHWT became considerably lower in incision + incision rats than in sham + incision rats at 10-14 days after the surgery. We observed many TG neurons encircled by glial fibrillary acidic protein-immunoreactive (GFAP-IR) cells and those exhibited TNFα immunoreactivity. TNFα was also expressed in GFAP-IR cells in incision + inicision TG. TNFα protein levels and the relative number of TNFα-IR cells were significantly higher in incision + incision rats than in sham + incision rats. The MHWT was significantly recovered during the intra-ganglionic administration of neutralizing anti-TNFα antibody 4-14 days after the incision. Furthermore, the MHWT was significantly decreased in sham + incision rats following the intra-ganglionic administration of recombinant TNFα. The present findings suggest that the neuron-satellite glial cell communication via TNFα is a critical mechanism in the enhancement of mechanical hypersensitivity in the adulthood-injured face following facial injury in infants.

Tumor Necrosis Factor Alpha Signaling in Trigeminal Ganglion Contributes to Mechanical Hypersensitivity in Masseter Muscle During Temporomandibular Joint Inflammation.

AIMS: To determine the involvement of tumor necrosis factor alpha (TNFα) signaling in the trigeminal ganglion (TG) in the mechanical hypersensitivity of the masseter muscle during temporomandibular joint (TMJ) inflammation. METHODS: A total of 55 male Sprague-Dawley rats were used. Following injection of Complete Freund's Adjuvant into the TMJ, the mechanical sensitivities of the masseter muscle and the overlying facial skin were measured. Satellite glial cell (SGC) activation and TNFα expression in the TG were investigated immunohistochemically, and the effects of their inhibition on the mechanical hypersensitivity of the masseter muscle were also examined. Student t test or two-way repeated-measures analysis of variance followed by Bonferroni multiple comparisons test were used for statistical analyses. P < .05 was considered to reflect statistical significance. RESULTS: Mechanical allodynia in the masseter muscle was induced without any inflammatory cell infiltration in the muscle after TMJ inflammation. SGC activation and an increased number of TNFα-immunoreactive cells were induced in the TG following TMJ inflammation. Intra-TG administration of an inhibitor of SGC activity or of TNFα-neutralizing antibody depressed both the increased number of TG cells encircled by activated SGCs and the mechanical hypersensitivity of the masseter following TMJ inflammation. CONCLUSION: These findings suggest that persistent masseter hypersensitivity associated with TMJ inflammation was mediated by SGC-TG neuron interactions via TNFα signaling in the TG.