Introduction

Escherichia coli K1 is a leading cause of neonatal bacterial meningitis, carrying 10-15% mortality and leaving 30-50% of survivors with lasting neurological damage. The mechanisms underlying this neuropathology remain poorly understood, particularly the innate immune responses involving resident microglia and peripheral immune infiltration.

Aims

This study aimed to characterise neonatal brains after intrauterine E. coli K1 infection, quantifying neuronal and microglial populations as primary outcomes, with exploratory analysis of mast cell infiltration and cerebellar external granule layer (EGL) thickness.

Methods

Fixed P0 brain tissue was obtained directly from the Boyle et al. (2025) cohort (E. coli K1-infected and PBS control C57BL/6 Tyrc-2J mice; n = 3-4 per group), a timepoint not previously examined in that study. All procedures were conducted under UK Home Office Licence PAD4E6357 in accordance with the Animals (Scientific Procedures) Act 1986. Sections were cut at 70 µm using a Leica vibratome. Immunofluorescence was performed for TUJ1 (neurons), IBA1 (microglia), and tryptase (mast cells), imaged on a Zeiss LSM880 confocal microscope. Cerebellar sections were stained with cresyl violet (Nissl) and imaged on a brightfield microscope. Neuronal and mast cell density were quantified by manual counting in ImageJ; microglial density and morphology were assessed by quantification and Sholl analysis. EGL thickness was measured in Fiji. Group differences were assessed using Welch’s t-tests with the individual brain as the statistical unit.

Results

TUJ1-positive neuronal density was reduced by approximately 60% in infected animals (control: 203 ± 11; infected: 81 ± 6 cells per ROI; n = 3 per group; p = 0.0026). IBA1-positive microglial density was significantly elevated across all three cortical regions examined: lower cortical layer (p = 0.0223), upper cortical layer (p = 0.0402), and cortical white matter (p = 0.0453; n = 3 per group). A trend towards reduced process complexity in infected microglia was observed by Sholl analysis (p = 0.0525; n = 4 per group), consistent with a shift towards an activated morphology. Tryptase-positive mast cell density was approximately 5-fold higher in infected animals (control: 4.67 ± 1.45; infected: 23.67 ± 1.76 cells per section; n = 3 per group; p = 0.0013). Cerebellar EGL thickness was reduced by 34% in infected animals (n = 4 per group; p = 0.0054).

Conclusions

E. coli K1 infection drives significant neuronal loss and concurrent activation of both resident (microglia) and peripheral (mast cell) innate immune populations at birth, alongside disruption of cerebellar development. These findings suggest neuropathology is established at birth and identify mast cell infiltration and cerebellar disruption as novel features of this model. Limitations include small sample sizes (n = 3-4) and a single timepoint. Future work should examine longitudinal trajectories and mast cell-microglia interactions as potential therapeutic targets.