Hippocampal extracellular matrix remodeling and temporally distinct glial reorganization following peripheral injury

Chronic pain is frequently accompanied by anxiety and other affective disturbances, but the molecular and cellular changes linking peripheral injury to persistent alterations in brain function remain incompletely defined. The hippocampal extracellular matrix (ECM) regulates synaptic plasticity, cellular signaling, and neuroimmune interactions and may therefore contribute to pain-related central plasticity. We used a tibial fracture/cast immobilization model to examine temporal changes in hippocampal ECM, glial morphology, and affective behavior following peripheral injury. Behavioral testing was performed at 3, 7, and 20 weeks after injury, while high-dimensional morphometric analysis of microglia and astrocytes was performed at 1, 3, 7, and 20 weeks. Hippocampal ECM associated molecular signals were mapped at the chronic stage using decellularization coupled with MALDI-TOF mass spectrometry imaging. Injured mice developed mechanical hypersensitivity and reduced exploration in two anxiety related paradigms during the early and chronic stages, with these behavioral phenotypes no longer detected at 20 weeks. At 7 weeks, injury was associated with changes in hippocampal ECM-associated peptide signals, including reduced signals corresponding to four ECM-associated proteins. Microglia and astrocytes exhibited distinct, time-dependent changes in density and multidimensional morphology, with some alterations persisting after behavioral measures returned toward control levels. In vitro, BV2 microglia exhibited rigidity-dependent changes in morphology, phagocytic behavior, and calcium signaling. Together, these findings define a temporally evolving hippocampal ECM and glial phenotype following peripheral injury and identify matrix biomechanics as a candidate regulator of microglial function.