Progesterone Prevents Nerve Injury-Induced Allodynia and Spinal NMDA Receptor Upregulation in Rats

The ovarian hormones progesterone and estrogen have well-established neurotrophic and neuroprotective effects supporting both reproductive function and cognitive health. More recently, it has been recognized that these steroids also regulate metabolic functions sustaining the energetic demands of this neuronal activation. Underlying this metabolic control is an interpretation of signals from diverse environmental sources integrated by receptor-mediated responses converging upon mitochondrial function. In this study, to determine the effects of progesterone (P4) and 17beta-estradiol (E2) on metabolic control via mitochondrial function, ovariectomized rats were treated with P4, E2, or E2 plus P4, and whole-brain mitochondria were isolated for functional assessment. Brain mitochondria from hormone-treated rats displayed enhanced functional efficiency and increased metabolic rates. The hormone-treated mitochondria exhibited increased respiratory function coupled to increased expression and activity of the electron transport chain complex IV (cytochrome c oxidase). This increased respiratory activity was coupled with a decreased rate of reactive oxygen leak and reduced lipid peroxidation representing a systematic enhancement of brain mitochondrial efficiency. As such, ovarian hormone replacement induces mitochondrial alterations in the central nervous system supporting efficient and balanced bioenergetics reducing oxidative stress and attenuating endogenous oxidative damage.

Increasing evidence supports the notion that spinal cord microglia activation plays a causal role in the development of neuropathic pain after peripheral nerve injury; yet the mechanisms for microglia activation remain elusive. Here, we provide evidence that NADPH oxidase 2 (Nox2)-derived ROS production plays a critical role in nerve injury-induced spinal cord microglia activation and subsequent pain hypersensitivity. Nox2 expression was induced in dorsal horn microglia immediately after L5 spinal nerve transection (SNT). Studies using Nox2-deficient mice show that Nox2 is required for SNT-induced ROS generation, microglia activation, and proinflammatory cytokine expression in the spinal cord. SNT-induced mechanical allodynia and thermal hyperalgesia were similarly attenuated in Nox2-deficient mice. In addition, reducing microglial ROS level via intrathecal sulforaphane administration attenuated mechanical allodynia and thermal hyperalgesia in SNT-injured mice. Sulforaphane also inhibited SNT-induced proinflammatory gene expression in microglia, and studies using primary microglia indicate that ROS generation is required for proinflammatory gene expression in microglia. These studies delineate a pathway involving nerve damage leading to microglial Nox2-generated ROS, resulting in the expression of proinflammatory cytokines that are involved in the initiation of neuropathic pain.

Recent studies indicate that reactive oxygen species (ROS) play an important role in neuropathic pain, predominantly through spinal mechanisms. Since the data suggest that ROS are involved in central sensitization, the present study examines the levels of activated N-methyl-d-aspartate (NMDA) receptors in the dorsal horn before and after removal of ROS with a ROS scavenger, phenyl-N-t-butyl nitrone (PBN), in animal models of pain. Tight ligation of the L5 spinal nerve was used for the neuropathic pain model and intradermal injection of capsaicin was used for the inflammatory pain model. Foot withdrawal thresholds to von Frey stimuli to the paw were measured as pain indicators. The number of neurons showing immunoreactivity to phosphorylated NMDA-receptor subunit 1 (pNR1) and the total amount of pNR1 proteins in the spinal cord were determined using immunohistochemical and Western blotting techniques, respectively. Hyperalgesia and increased pNR1 expression were observed in both neuropathic and capsaicin-treated rats. A systemic injection of PBN (100 mg/kg, i.p.) dramatically reduced hyperalgesia and blocked the enhancement of spinal pNR1 in both pain models within 1h after PBN treatment. The data suggest that ROS are involved in NMDA-receptor activation, an essential step in central sensitization, and thus contribute to neuropathic and capsaicin-induced pain.