Background & Aims
Microglia have been shown to be drivers of pain hypersensitivity in the spared nerve injury model of neuropathic pain in mice. Following SNI, microglia in the dorsal horn of the spinal cord proliferate and transition from a homeostatic phenotype to a pro-inflammatory phenotype, potentially in a sex-dependent manner. These microglia have been shown to retain their morphology and pro-inflammatory phenotype from acute to chronic timepoints (>3 months). Cellular changes in microglia are accompanied by behavioural changes as mice develop mechanical and thermal (cold) allodynia, which they retain into the chronic phase. Research has now shown that microglial gene expression patterns, activation states, and physiological function are governed by circadian (24-hour) rhythms, with diverse regional heterogeneity. Clinical studies have also shown that pain can be rhythmic in various chronic pain states. However, it remains unknown whether microglia exhibit circadian rhythmicity in chronic pain.
Methods
To investigate this gap in knowledge, male and female C57BL/6J mice received a spared nerve injury, with tissues collected at 3, 7, 10, 14, 28, and 84 days following injury. Animals were sacrificed at ZT2 (rest phase) and ZT14 (active phase), corresponding to 2 hours after start of the light and dark period on each collection day. Cryostat sections of the spinal cord were immunostained for markers of homeostatic and pro-inflammatory microglia, as well as transcriptional regulators known to be under circadian control. Confocal z-stacks were taken to create 3D surface renderings of cells, with surface renderings analyzed to characterize microglial morphology and activation state. Data collected include changes in cell volume, lysosomal volume, and process length and ramification, which are characteristic changes in microglial response to injury and pain. The experiments were repeated using CX3CR1CreER; Bmal1 flox/flox mice, in which clock gene Bmal1 is specifically ablated in microglia.
Results
We found rhythmic changes in both microglial morphology and activation state in both the naïve and injured state. In the naïve state, microglia exhibited more ramified, extended processes indicative of a homeostatic surveillance phenotype during the dark (active) phase. Meanwhile, microglia had shorter, less ramified processes and morphology indicative of a pro-inflammatory phenotype in the light (rest) phase. During peak periods of microglial activation following SNI, which occur between 7- and 14-days following injury, microglia in the dorsal horn took on an extremely pro-inflammatory phenotype during the light phase, but retained a more homeostatic phenotype during the dark phase relative to the light-phase in injured animals. In mice with Bmal1 conditionally knocked out in microglia, the morphological changes between time points were less distinct.
Conclusions
Our work indicates that in both the naïve and post-SNI spinal cord, microglia undergo changes in their gene expression, morphology, and function over a 24-hour cycle, and that disruption of circadian gene expression machinery affects microglial activation patterns. As microglia have been shown to the development of chronic pain, understanding microglial activation states across male and female mice, and during the circadian cycle, may provide new insight into mechanisms regulating their activity and function in the pathophysiology of chronic neuropathic pain.
References
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