Figure 1.
Representative histology from mice with (c,d) and without (a,b) NEC. After 72 h of exposure to the experimental feeding protocol, intestines were collected and embedded in OCT as described in Materials and Methods. Frozen sections were prepared and processed for H&E staining. Sections were scored by a blinded observer based on a well established NEC scoring scale [17].
Figure 2.
TLR4 and iNOS QRT-PCR Analysis.
Colonic TLR4 and iNOS mRNA levels in mice with and without NEC. Following total colonic RNA isolation, cDNA was synthesized, and copy numbers of TLR4 and iNOS were determined using QRT-PCR. Data shown are mean ± SEM of TLR4 and iNOS copy numbers relative to GAPDH. Each column represents n = 4 animals with duplicate measurements.
Figure 3.
Principal Coordinate Analysis.
Results of weighted principal coordinate analysis of the colonic microbiota of mice with NEC (▪N) as compared to mice without NEC (○NN). PC1 = 28.9% and PC2 = 25.1%. This analysis was performed via the mothur analytical pipeline as described for phylotype-based analysis [20].
Figure 4.
Temporal Temperature Gradient Gel Electrophoresis (TTGE) Analysis.
Following total colonic DNA extraction and PCR amplification, PCR products were separated via TTGE to identify bacterial community structures in mice with NEC (e–h) as compared to mice without NEC (a–d).
Figure 5.
Analysis of Colonic Microbiota.
Following total colonic DNA extraction, PCR was performed with 8 bar coded Bact27 and UNIV788 primers to amplify the bacterial 16S rRNA gene. PCR products were purified and underwent subsequent sequencing on the Roche GS-FLX pyrosequencing platform. Taxonomy was assigned to sequences using the Ribosomal Database Project classifier tool. Mice with NEC (a) are compared to mice without NEC (b). Statistical tests for differentially abundant 16S-based taxonomic annotations between populations were performed using Metastats methodology to compute nonparametric p-values. Differences were considered significant at p<0.05. *indicates statistically significantly more in mice with NEC. **indicates statistically significantly more in mice without NEC.