MICROBIAL INFLUENCE ON PERIPHERAL NERVOUS SYSTEM: INSIGHTS FROM GERM-FREE AND RECOLONIZED MICE

Farzin S¹, Pellegrino D¹, Cicconetti C², Bertone F¹, Oliviero S², Basic M³, Bolsega S³, Haastert-Talini K⁴, Cescon M⁵, Gambarotta G¹ and Ronchi G¹ | ¹Department of Clinical and Biological Sciences (DSCB), University of Torino, Orbassano, (Torino), Italy; ²Department of Life Sciences and System Biology, University of Torino, Torino, Italy; ³Institute for Laboratory Animal Science and Central Animal Facility, Hannover Medical School, Hannover, Lower- Saxony, Germany; ⁴Institute of Neuroanatomy and Cell Biology, Hannover Medical School, Hannover, Lower-Saxony, Germany; ⁵Department of Molecular Medicine, University of Padova, Padova, Italy

Emerging evidence links gut microbiota to nervous system development and repair. We investigated how different microbial states affect peripheral nerve morphology and gene expression. Peripheral nerves and dorsal root ganglia (DRG) were collected from four murine models: complex gut microbiota (CGM, control), gnotobiotic (OMM12, mice stably colonized with 12 specifically defined bacterial strains), germ-free (GF, mice bred in sterile environment to prevent microbial exposure) and ex-germ-free (EX-GF, mice which were initially maintained in a germ-free environment but later exposed to environmental microbes) mice. Fecal communities were profiled (species- level relative abundance, α/β diversity). Sciatic nerve and DRG transcriptomes were analysed by bulk RNA-seq. Ultrastructure was examined by transmission electron microscopy to assess myelination, and nodal features. Cohousing recolonized EX-GF mice, but did not reconstitute CGM complexity: CGM samples showed significantly higher α- diversity and separated from EX-GF by β-diversity. RNA-seq revealed a distinct transcriptional program in GF, OMM12 and EX-GF compared to CGM. TEM showed hypermyelination in GF and EX-GF mice: increased myelin thickness reflected a greater number of myelin lamellae rather than altered lamellar spacing. Aberrant Schwann cell-axon interactions (myelin outfoldings, inclusions, sheath splitting) were observed in OMM12, GF and EX-GF groups. Nodal length frequency distribution was similar in CGM and OMM12, and broader in GF and EX-GF, pointing to a longer node of Ranvier. DRG soma and nuclear areas were largely unchanged, in spite of that, injury-associated genes were differentially regulated across microbiota conditions. These results showed that partial microbiota reconstitution in EX-GF mice does not restore CGM-like peripheral nerve transcriptomes or morphology. Microbiota composition strongly influences myelination patterns, suggesting microbial control of nerve development and repair.