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TRPV1 is a nonselective cation channel in nociceptors. TRPV1 stimulation has been shown to lead to the activation of microglia and astrocytes in the dorsal horn of the spinal cord. However, information on the effect of TRPV1 stimulation on glial activation in the trigeminal nucleus caudalis (TNC) is lacking. Here, we stimulated TRPV1 in the trigeminal afferents by a repetitive injection of 10 mmol/l capsaicin into the whisker pad for 2 days (d2 group), 4 days (d4 group), or 6 days (d6 group). As a control (c group), the vehicle was injected for 2 days. Anti-Iba1 and anti-glial fibrillary acidic protein antibodies were used to immunostain microglia and astrocytes in the TNC, respectively. The ratio of the cross-sectional area immunoreactive for Iba1 to the entire area of the TNC was increased in the d2 group compared with the c group on the injected side. Microglia were recruited to the superficial layers of the TNC. The numbers of microglia were reduced in the d4 group and the d6 group compared with the d2 group. The ratio of the cross-sectional area immunoreactive for glial fibrillary acidic protein to the entire TNC showed a significant increase in d2 group and the d4 group compared with the c group on the injected side. Behavioral analysis indicated that mechanical allodynia began to develop after 2 days of capsaicin treatment and persisted for at least 6 days after the onset of the repetitive capsaicin injection. These data indicate that TRPV1 stimulation activates the microglia and astrocytes in temporally distinct ways and that the development of mechanical allodynia is independent of such glial activation.