Fig 1.
Impact of Nb infection on small intestinal morphology and motility in BALB/c and dblGATA1 mice.
dblGATA1 mice and wildtype BALB/c controls were infected with 400–500 L3 Nb and sacrificed at the indicated days p.i. Control groups of naïve mice were included. A) Weight and (B) length of the entire small intestine were determined. (C) Intestinal motility was determined using a carmine red dye transit assay. Dye was administered by oral gavage 30 mins prior to sacrifice and motility determined as the distance travelled by dye (as a percentage of total small intestine length) at the time of sacrifice. (D) Number of luminal adult Nb worms was determined using a Baermann assay. For all data, symbols represent individual animals. Data in A-C are pooled from 2–3 independent experiments with n = 6–16 per timepoint for infected groups. Data in D are from n = 4–10 animals per timepoint and are pooled from 2 independent experiments. All data are shown as mean ± SEM and significance determined using a two-way ANOVA with Tukey’s post-hoc analysis.
Fig 2.
Impact of Nb infection on jejunal villous architecture in BALB/c and dblGATA1 mice.
dblGATA1 mice and wildtype BALB/c controls were infected with 400–500 L3 Nb and sacrificed at the indicated days p.i. Control groups of naïve mice were included. (A) Jejunal whole mounted tissue from naïve and Nb infected mice stained with αSMA (magenta) and αVEGFR2 (cyan) antibodies. Images were taken as z-stakes that capture entire villious structures and are presented as a composite image. Jejunal villi length (B) and villi surface area (C) and ileal villi length (D) and villi surface area (E) were measured using FIJI (Image J). Villi length was determined using the line tool and surface area was calculated as the area enclosed by VEGFR2 staining using the polygon tool. Data in A show representative images of single villi from each group. Data in B—E are one representative of two experiments with symbols represent individual villi, using n = 3–5 animals per group with 2–8 villi per animal. All data are shown as mean ± SEM and significance determined using a two-way ANOVA with Tukey’s post-hoc analysis.
Fig 3.
Impact of Nb infection on neuronal structure in the submucosal and myenteric plexus.
dblGATA1 mice and wildtype BALB/c controls were infected with 400–500 L3 Nb and sacrificed day 9 p.i. Control groups of naïve mice were included. Myenteric and submucosal plexus layers were collected as detailed in Materials and Methods and stained with a pan neuronal marker HuC/D. (A) Schematic illustration of the neuroanatomy of the ENS inside the small intestine created with BioRender.com. The size of cells and tissue structures is not to scale. (B) Myenteric neuronal cell density was calculated by counting cell bodies and presented as number/mm2. (C) Representative images of myenteric plexus stained with HuC/D from a single naïve and infected BALB/c mouse. (D) Number of neurons per ganglion in the submucosal plexus. (E) Representative images of submucosal plexus stained with HuC/D from a single naïve and infected BALB/c mouse. Symbols in A and C represent individual animals with data pooled from 2–3 separate experiments, n = 9–11 per group for submucosal plexus and n = 7–8 per group for myenteric plexus. All data are shown as mean ± SEM and significance determined using a two-way ANOVA with Tukey’s post-hoc analysis.
Fig 4.
Impact of Nb infection on neurochemical marker expression in the submucosal and myenteric plexus.
dblGATA1 mice and wildtype BALB/c controls were infected with 400–500 L3 Nb and sacrificed day 9 p.i. Control groups of naïve mice were included. Myenteric and submucosal plexus were collected as mentioned in Materials and Methods and stained for the indicated neurochemical markers using antibodies. (A, C&E) Representative images are shown from a single naïve BALB /c animal for myenteric plexus stained with antibodies directed against (A) HuC/D (red), CalB (green), and CalR (cyan) or (C) HuC/D (red), CalB (green), and nNOS (cyan) and for (E) submucosal plexus stained with antibodies directed against HuC/D (red), VIP (green), and ChAT (cyan). (B, D &F) The percentage of total neurons expressing the indicated neurochemical markers was calculated by counting at least 180 HuC/D+ cell bodies in myenteric plexus and least 100 HuC/D+ cell bodies in submucosal plexus from each animal. Symbols represent individual animals with data pooled from 2–3 separate experiments, n = 9–11 animals per group for submucosal plexus and n = 7–8 animals per group for myenteric plexus. All data are shown as mean ± SEM and significance determined using a two-way ANOVA with Tukey’s post-hoc analysis.
Fig 5.
Nb infection does not influence neurogenic-driven muscle contraction in the jejunum.
BALB/c mice were infected with 400–500 L3 Nb and sacrificed day 6–9 p.i. Control groups of naïve mice were included. Jejunal contractile activities were recorded under physiological conditions in a horizontal organ bath using a video camera, and data analysed with in-house software Scribble 2.0 and Analyse 2. A) An example image of spatiotemporal heatmap showing jejunum contractions at baseline. X-axis on the left indicates the position of the tissue, y-axis indicates time. Each color-coded pixel indicates jejunum diameter (based on the scale on the left) at each position at a specific time, thus each warm coloured band indicates a contraction. All contractions that started from the distal half of the jejunum are recorded as initiations (circles and arrow heads), and initiations that propagated through more than 50% of the tissue are recorded as PCCs (arrow heads). Analysis of the spatiotemporal heatmap provided information on PCC contraction duration and velocity, contraction amplitude, and slow wave frequencies (see also S2 Fig). (B&C) Comparison of (B) contraction amplitudes and (C) contraction velocities recorded in tissues from naïve or infected mice under baseline conditions or after DMPP application. Open and closed symbols indicate recordings taken during baseline conditions (open) or after DMPP application (closed). Symbols represent individual animals (n = 7–8 per group), with each experiment including tissues from one naïve animal and one infected animal kept within the individual chambers of the same organ bath. Tissues that ceased to contract before the end of the recording were excluded from analyses. Data are shown mean ± SEM and significance determined using unpaired student T-tests comparing naïve and infected mice, and paired student T-tests for comparing baseline and DMPP.
Fig 6.
Nb infection drives intestinal smooth muscle hypertrophy and hypercontractility.
dblGATA1 mice and wildtype BALB/c controls were infected with 400–500 L3 Nb and sacrificed at the indicated days p.i. Control groups of naïve mice were included. (A&B) Representative images are shown for jejunal cross-sections from single naïve or Nb infected BALB/c and dblGATA1 mice stained with antibodies directed against the muscle cell marker SMA. Muscle thickness was measured using the line tool from FIJI (Image J) for the ① longitudinal ② circular and ③ total muscularis layers. (C) Muscle thickness was determined as shown in A). 40 measurements were made per muscle layer from each animal, with symbols representing the mean of individual animals. (D-G) jejunal tissues were mounted longitudinally within an organ bath as described in the Materials and Methods and (D) contraction frequency and (E) contraction strength recorded under baseline conditions. Thereafter the same tissues were stimulated using incremental dosages of acetylcholine and (F) the dose-dependent change in contraction strength or (G) the maximum increase in contraction strength. Symbols represent individual animals pooled from 2 independent experiments (n = 7–13 animals/group). Data are shown mean ± SEM and significance determined using a two-way ANOVA with Tukey’s post-hoc analysis.
Fig 7.
Hp infection alters small intestinal motility and smooth muscle hypertrophy.
BALB/c mice were infected with 200 L3 Hp at day 0 for the primary infection group or day 0 and day 37 for the secondary infection group and were sacrificed day 14 p.i. or day 51 p.i., respectively. Control groups of naïve mice were included. (A) Length of the entire small intestine was determined. (B) Intestinal motility was determined using a carmine red dye transit assay. Dye was administered by oral gavage 30 mins prior to sacrifice and motility determined as the distance travelled by dye (as a percentage of total small intestine length) at the time of sacrifice. (C) Number of luminal adult Hp worms were counted. (D) Representative images are shown for jejunal cross-sections from a single naïve, primary Hp infected, or secondary Hp infected BALB/c mice stained with antibodies directed against the muscle cell marker SMA. (E) Longitudinal muscle, circular muscle, and overall muscularis thickness was determined as detailed in Method. Symbols represent individual animals pooled from 2 independent experiments (n = 7–8 animals/group). Data are shown mean ± SEM and significance determined using a one-way ANOVA with Tukey’s post-hoc analysis.
Table 1.
Primary and secondary antibodies used.