Interleukin 10 Restores Gastric Emptying, Electrical Activity, and Interstitial Cells of Cajal Networks in Diabetic Mice

Abstract

Background & aims: Gastroparesis is a complication of diabetes characterized by delayed emptying of stomach contents and accompanied by early satiety, nausea, vomiting, and pain. No safe and reliable treatments are available. Interleukin 10 (IL10) activates the M2 cytoprotective phenotype of macrophages and induces expression of heme oxygenase 1 (HO1) protein. We investigated whether IL10 administration could improve gastric emptying and reverse the associated cellular and electrical abnormalities in diabetic mice.

Methods: Nonobese diabetic mice with delayed gastric emptying were given either IL10 (0.1-1 μg, twice/day) or vehicle (controls). Stomach tissues were isolated, and sharp microelectrode recordings were made of the electrical activity in the gastric muscle layers. Changes to interstitial cells of Cajal (ICC), reduced nicotinamide adenine dinucleotide phosphate diaphorase, and levels and distribution of HO1 protein were determined by histochemical and imaging analyses of the same tissues.

Results: Gastric emptying remained delayed in vehicle-treated diabetic mice but returned to normal in mice given IL10 (n = 10 mice; P < .05). In mice given IL10, normalization of gastric emptying was associated with a membrane potential difference between the proximal and distal stomach, and lower irregularity and higher frequency of slow-wave activity, particularly in the distal stomach. Levels of HO1 protein were higher in stomach tissues from mice given IL10, and ICC networks were more organized, better connected, and more evenly distributed compared with controls.

Conclusions: IL10 increases gastric emptying in diabetic mice and has therapeutic potential for patients with diabetic gastroparesis. This response is associated with up-regulation of HO1 and repair of connectivity of ICC networks.

Keywords: Alternatively Activated Macrophages; CO, carbon monoxide; Electrical Slow Wave; HO1, heme oxygenase 1; Heme Oxygenase 1; ICC, interstitial cells of Cajal; IL10, interleukin 10; MDA, malondialdehyde; NADPH, reduced nicotinamide adenine dinucleotide phosphate; NOD, nonobese diabetic; NOS, nitric oxide synthase; PBS, phosphate-buffered saline.

Figure 1

(A) Experimental design. Gastric emptying (GE) is indicated by larger arrows, treatment is indicated by small arrows. (B) Location of electrical recording and image collection sites.

Figure 2

Effects of IL10 treatment. (A) Glucose levels (bars, medians with interquartile range; whiskers, 5th/95th percentiles). *Higher glucose levels for diabetic vs nondiabetic mice (n = 5; 2-way analysis of variance with Bonferroni post-test). (B) Malondialdehyde levels were associated significantly with delayed gastric emptying and response to IL10 treatment but not diabetes. Circles, values for each mouse; whiskers, means ± SE; dotted lines, 5th/95th percentile of MDA levels in diabetic NOD mice with normal gastric emptying. (C) Gastric emptying. Circles, data of each mouse; whiskers, means ± SE; dotted lines, 5th/95th percentile of T_1/2 values in nondiabetic mice. *P < .05 (n = 5; 1-way analysis of variance with the Tukey post-test). Db, diabetic; GE, gastric emptying.

Figure 3

Electrical activity. Slow-wave activity in the distal antrum of all (A) vehicle-treated and (B) IL10-treated mice. Note the variations in peak amplitude of the slow waves in the vehicle-treated mice that are absent in the IL10-treated mice.

Figure 4

Electrical activity. Response to IL10 treatment. (A) Variability of peak amplitudes in the distal stomach (regions 7–12). Means ± SEM. *P < .05, one-way analysis of variance with Bonferroni correction; n = 5. (B) Quantification of the interevent intervals (IEIs) for slow waves from all areas of all mice. Means ± SEM; n = 5. *P < .05, unpaired t test. (C) Resting membrane potential in the corpus (regions 1–3) and antrum (regions 7–9) of vehicle- and IL10-treated diabetic mice. Each point represents the membrane potential for each mouse. Statistical differences were determined by repeated measures 2-way analysis of variance with a Bonferroni post-test; N = 5 mice. (D) Resting membrane potential. Note the variance in the membrane potential across tissues was different. **P = .03, F-test; means for 12 recording sites; n = 5 mice. Whiskers, medians–interquartile range. Pk, peak.

Figure 5

HO1 expression. Image stacks of HO1 immunoreactivity in (A) vehicle and (B) IL10 mice. Numbers indicate regions. (C) Scores for HO1 immunoreactivity. Means ± SEM; n = 5; *P < .01, t test. (D) HO1-positive macrophages (left) and neurons in a myenteric ganglion (middle) from the gastric body of IL10-treated mouse. Right: a ganglion from an IL10-treated mouse in which HO1 was not detected in the neurons. (E) Image stacks of F4/80, HO1, and CD206 immunoreactivity in the muscularis propria of a diabetic mouse treated with IL10. Data are representative of immunolabeling in 2 different mice.

Figure 6

NADPH-diaphorase activity. Quantification of neurons in the myenteric plexus from diabetic NOD mice with delayed gastric emptying treated with vehicle or 1 μg IL10 twice a day. Positively labeled neurons were counted from 7 high-power fields using a 20× objective from each tissue. A minimum of 174 neurons were counted from each tissue. There was no significant difference in the number of positively labeled neurons between the 2 groups (means ± SEM, t test).

Figure 7

ICC networks. Image stacks from (A) vehicle and (B) IL10 mice. (C) Network density scores. Means ± SD of scores for all fields; n = 5; *P < .05, t test. (D) Network volume from recording sites in antrum. (E) Count of independent connected structures after morphologic opening in reconstructed images. Data are for 75 fields from 5 mice in each group. *P = .026, t test. (F) Three-dimensional (3D) volume-rendered bitmap of the largest connected Kit-positive ICC networks in 2 fields with similar total Kit volumes. Note that most of the Kit-positive structures in the field from the IL10-treated mouse are connected into a single object.

Figure 8

A low dose of IL10 (100 ng) also reversed delayed gastric emptying in diabetic NOD mice. Data are the mean T_1/2 values for gastric emptying ± SEM. P < .001, 1 way analysis of variance. P < .05 vs Db before delay (a); P < .05 vs Db before treatment (b); and not significantly different vs Db before delay (c). Db, diabetic.