CX₃CR1 Fate-Mapping In Vivo Distinguishes Cochlear Resident and Recruited Macrophages After Acoustic Trauma

Cochlear injury activates the resident macrophages (RM) and recruits the blood-circulating monocytes and monocyte-derived macrophages (Mo/Mo-M), but their specific functions in the injured cochlea are unknown. It is well established that the chemokine fractalkine receptor (CX₃CR1), expressed by cochlear macrophages, influences the density of those macrophages and promotes synaptic repair and spiral ganglion neuron survival in the injured cochlea. As CX₃CR1 is expressed on both RM and Mo/Mo-M, it remains unclear if CX₃CR1-expressing RM and Mo/Mo-M are distinct and differentially promote SGN survival after cochlear injury. Here, we demonstrate the use of fate mapping via a tamoxifen-inducible CX₃CR1 mouse model (CX₃CR1^{YFP-CreERT2/wildtype}:R26RFP) wherein CX₃CR1-expressing RM and Mo/Mo-M are endogenously labeled with different fluorescent reporters to define the heterogeneity in cochlear macrophages regarding their origin, turnover, spatiotemporal distribution, morphology, and fate following a loud acoustic trauma. After 60 days of tamoxifen injections at 4 weeks of age, long-lived cochlear RM were YFP+ RFP+ with 98.0 ± 1.7% recombinant efficiency, and short-lived blood-circulating CX₃CR1 lineage (Mo/Mo-M) were YFP+ RFP- with 2.5 ± 1.1% recombinant efficiency. Following an acoustic trauma of 112 dB SPL at 8-16 kHz octave band for 2 hours, morphologically similar RM and Mo/Mo-M were observed in the spiral ganglion, lamina, ligament, and around the sensory epithelium. Quantification of RM and Mo/Mo-M in the spiral lamina and ganglion revealed distinct spatial and temporal distribution patterns. Furthermore, recruited Mo/Mo-M expressed classical monocyte markers such as Ly6C and CCR2. Both RM and Mo/Mo-M were positive for proliferation marker, Ki67, and negative for apoptotic marker, cleaved caspase-3, suggesting that the overall increase in macrophage numbers in the noise-injured cochlea is a contribution of both the proliferation of RM and recruitment of Mo/Mo-M. Probing for blood-clotting protein, fibrinogen, showed its presence in the cochlea after acoustic trauma, suggesting vascular damage that positively and strongly correlated with the time course of recruitment of blood-circulating Mo/Mo-M in the noise-injured cochlea. These data imply that macrophages in the noise-injured cochlea are heterogeneous regarding their ontogeny, distribution, and fate. They offer a robust tool to study the precise roles of resident and recruited macrophages in healthy and pathological ears.