Role of Spinal Microglia in Visceral Hyperalgesia and NK1R Up-Regulation in a Rat Model of Chronic Stress

Abstract

Chronic psychological stress is associated with visceral hyperalgesia. Microglia in the central nervous system are a type of nonneuronal cells and play a key role in the initiation or maintenance of hyperalgesia and allodynia, as demonstrated in animal models of chronic pain caused by peripheral inflammation or nerve injury.1 However, the mechanisms of microglia activation and the downstream pathways leading to amplification of the nociceptive signaling are only partially understood. Activated microglia produce and release a range of proinflammatory mediators, such as cytokines (IL-1, IL-6, tumor necrosis factor ), chemokines, nitric oxide, and prostaglandins, all of which can act on afferent nerve terminals and spinal sensory neurons and increase neuronal excitability.2 The authors wanted to identify the role of spinal microglia in the development of visceral hyperalgesia from chronic stress in a rat model. In the study, the authors used adult male Wistar rats with or without water avoidance (WA) stress. Rats were placed on a block (8×8×10 cm) affixed to the center of a Plexiglas cage (25 ×25×45 cm) filled with fresh water at room temperature (25oC) to within 1 cm of the top of the block (WA) or kept empty (sham WA) for a period of one hour daily for 10 consecutive days.3 The rats had a surgically implanted chronic intrathecal (IT) catheter and an osmotic mini-pump. The rats then had a Teflon-coated electrode inserted into the external oblique musculature for electromyographic (EMG) recording.4One group of rats was exposed to chronic WA stress or sham WA for 10 days and were sacrificed on day 11. Samples of lumbar spinal cord (L6S1) were collected and processed for immunohistochemically for P-p38, the microglia marker OX42, or the neuronal marker NeuN. Also, other groups of rats were exposed to WA stress or sham stress for 10 days and given daily injections of minocycline, the mitogen-activated protein kinase (MAPK) p38 inhibitor SB203580, or vehicle (0.9% saline) via osmotic pump connected to an IT catheter. Phosphorylation levels of the kinase p38 (P-p38), the microglia marker OX42, NK1R, and IB were assessed by immunoblotting and/or immunostaining of spinal samples. The authors also performed EMG recordings and baseline recordings of the visceromotor response (VMR) to colorectal distention (CRD) before beginning the treatment (day 0) and then again 24 hours after the last WA or sham WA sessions (day 11). The effects of the microglia-activating factor fractalkine were assessed on the visceral sensitivity in control nonstressed rats exposed to minocycline or vehicle. The immunohistochemistry for the activation marker of mi croglia, P-p38, revealed greater P-p38 staining in the spinal dorsal horn from WA stressed rats compared with controls. The immunoreactivity for P-p38 colocalized with OX42-positive cells (microglia) from WA stressed rats. Western blotting also demonstrated increased levels of P-p38 in WA stressed rats compared with controls. This increase was inhibited by treatment with IT minocycline or with IT SB203580. Western blotting showed increased expression of NK1R in WA stressed rats compared with controls. Stress-induced increased NK1R expression was completely blocked by minocycline but not SB203580. WA stress induced decreased IB expression, and this decrease was partially blocked by minocycline and SB203580. WA stress induced hyperalgesia in stressed rats treated with vehicle. WA induced visceral hyperalgesia was blocked by daily treatment with IT injection of minocycline or SB203580. Fractalkine injection-induced hyperalgesia was blocked by minocycline.