Fig. 2

TBI induces enteroendocrine cell loss and decreases EEC differentiation. male C57BL/6 mice were used at the age of 4–6 weeks to induce moderate-severe TBI as previously described [66]. Mice were sacrificed three days later, and the ileum was harvested to investigate the expression of EECs. Intestinal tissues were fixed overnight with 4% paraformaldehyde and processed for paraffin embedding. Subsequently, 5-μm tissue sections were cut from paraffin blocks using a CUT 6062 microtome (SLEE Medical GmbH, D-55129 Mainz, Germany) and stained for DAPI (blue) and chromogranin A (ChgA) (green). ChgA⁺ cells were counted using ImageJ2 software. Scale bars, 50-μm. A–C Representative confocal images showing decreased intestinal ChgA expression in TBI mice when compared to sham on post-injury day (PID) 3. D An illustration of EEC differentiation pathway in the intestine. TBI reduces the expression of key transcription factors implicated in the differentiation of Lgr5⁺ intestinal stem cells into ChgA⁺ mature EECs, including E Notch1, F Atoh1, and G Nuerog3, as measured by quantitative reverse transcription polymerase chain reaction (qRT-PCR). The mRNA levels are expressed as relative to housekeeping gene Rplp0 expression. Statistical significance was determined by student’s t-test using GraphPad Prism 10 software. Each dot on the graph represents a different mouse. Error bars indicate the mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001