A Novel Zebrafish Model to Provide Mechanistic Insights into the Inflammatory Events in Carrageenan-Induced Abdominal Edema

<div><p>A suitable small animal model may help in the screening and evaluation of new drugs, especially those from natural products, which can be administered at lower dosages, fulfilling an urgent worldwide need. In this study, we explore whether zebrafish could be a model organism for carrageenan-induced abdominal edema. The research results showed that intraperitoneal (i.p.) administration of 1.5% λ-carrageenan in a volume of 20 µL significantly increased abdominal edema in adult zebrafish. Levels of the proinflammatory proteins tumor necrosis factor-α (TNF-α) and inducible nitric oxide synthase (iNOS) were increased in carrageenan-injected adult zebrafish during the development of abdominal edema. An associated enhancement was also observed in the leukocyte marker, myeloperoxidase (MPO). To support these results, we further observed that i.p. methylprednisolone (MP; 1 µg), a positive control, significantly inhibited carrageenan-induced inflammation 24 h after carrageenan administration. Furthermore, i.p. pretreatment with either an anti-TNF-α antibody (1∶5 dilution in a volume of 20 µL) or the iNOS-selective inhibitor aminoguanidine (AG; 1 µg) inhibited carrageenan-induced abdominal edema in adult zebrafish. This new animal model is uncomplicated, easy to develop, and involves a straightforward inducement of inflammatory edema for the evaluation of small volumes of drugs or test compounds.</p></div

Comparison of the carrageenan-injected adult zebrafish model to the carrageenan-injected rodent model.

<p>Comparison of the carrageenan-injected adult zebrafish model to the carrageenan-injected rodent model.</p

The time course for the abdominal-edematous effects of i.p. carrageenan (1.5%) in adult zebrafish.

<p>The photographic images show the gross pathology of the abdomen in the lateral view following i.p. injection of vehicle (PBS) plus i.p. injection vehicle (PBS) (A and E) and i.p. injection vehicle plus i.p. injection of 1.5% carrageenan at each time point (B–D and F–H). Images A–D were taken at 0 h after i.p. injection of vehicle or 1.5% carrageenan; images E, F, G, and H were taken at 24 h after i.p. injection of vehicle and 8, 16, and 24 h after 1.5% carrageenan injection. Scale bars: 5 mm for images A–H. The increase of the lateral area of the abdomen was given as a difference change from the basal values by subtracting the basal lateral area from the lateral area measured at each time point. Time course of change of the lateral area of abdomen induced by i.p. injection of vehicle (PBS) or carrageenan in adult zebrafish using a photographic image analysis system (I). Compared with the i.p. vehicle groups, the lateral area of abdomen increased progressively in the i.p. 1.5% carrageenan groups until 24 h. Sections (2 µm) of zebrafish abdominal tissues at 24 h after an i.p. injection of vehicle (J) or carrageenan (K). I.p. carrageenan dramatically induced leukocyte infiltration (arrows) in the intestine. Scale bars: 100 µm for images (J) and (K). Each bar in Figure (I) represents the mean ± SEM of 9 adult zebrafish per group. *<i>P</i><0.05 compared with the same time points in the i.p. vehicle (PBS) group.</p

The anti-edematous effects of i.p. anti-TNF-α antibody and AG in carrageenan-injected adult zebrafish.

<p>The photographic images show the gross pathology of the abdomen in the lateral view in the i.p. vehicle (PBS) plus i.p. vehicle group (A and E), the i.p. vehicle plus i.p. 1.5% carrageenan group (B and F), the i.p. anti-TNF-α antibody (1∶5 dilution in a volume of 20 µL) plus i.p. 1.5% carrageenan group (C and G), and the i.p. AG (1 µg) plus i.p. 1.5% carrageenan group (D and H). Images A–D were taken at 0 h after the second injection of vehicle or carrageenan (as well as 1 h after the first injection of vehicle, anti-TNF-α antibody, or AG); images E–H were taken at 24 h after the second injection of vehicle or carrageenan (as well as 25 h after the first injection of vehicle, anti-TNF-α antibody, or AG injection). Scale bars: 5 mm for all images. Quantification of the lateral area of the abdomen induced by the second injection of vehicle or carrageenan in adult zebrafish using a photographic image analysis system (I). Anti-TNF-α antibody or AG administered 1 h before carrageenan injection markedly reduced carrageenan-induced abdominal edema. Each bar in Figure (I) represents the mean ± SEM of 9 adult zebrafish per group. anti-TNF: anti-TNF-α antibody; AG: aminoguanidine; Ca: carrageenan; <b>*</b><i>P</i><0.05 compared with the i.p. injection of vehicle plus i.p. vehicle group; #<i>P</i><0.05 compared with the i.p. vehicle plus i.p. 1.5% carrageenan group.</p

The anti-edematous effects of i.p. MP in carrageenan-injected adult zebrafish.

<p>The photographic images show the gross pathology of the abdomen in the lateral view from the i.p. injection vehicle (PBS) plus i.p. injection vehicle group (A and D), the i.p. injection vehicle plus i.p. injection 1.5% carrageenan group (B and E), and the i.p. injection MP plus i.p. 1.5% carrageenan group (C and F). Images A–C were taken at 0 h after the second injection (as well as 1 h after the first injection); images D–F were taken at 24 h after the second injection (as well as 25 h after the first vehicle or MP injection). Scale bars: 5 mm (for all images). Quantification of the lateral area of abdomen induced by i.p. injection of vehicle or carrageenan in adult zebrafish using a photographic image analysis system (G). We used 1 µg MP as a positive control. MP administered by i.p. injection 1 h before carrageenan injection significantly inhibited carrageenan-induced abdominal edema. Each bar in Figure G represents the mean ± SEM of 9 adult zebrafish per group. MP: methylprednisolone. <b>*</b><i>P</i><0.05 compared with the i.p. vehicle plus i.p. vehicle group; #<i>P</i><0.05 compared with the i.p. vehicle plus i.p. 1.5% carrageenan group.</p

The dose responses for the whole-body edematous effects of carrageenan in adult zebrafish.

<p>Time course and the percentage change of whole-body volume induced by i.p. injection of vehicle (PBS) or carrageenan in adult zebrafish (A), using a rat paw volume meter. The basal whole-body volume of each adult zebrafish before i.p. injection of vehicle or carrageenan injection was considered as 100%, and the increase in whole-body volume is presented as a percentage change from the basal values by subtracting the basal whole-body volume from the whole-body volume measured at each time point. The absolute basal whole-body volume of adult zebrafish before the injection of vehicle or carrageenan is 593.9±49.3 µL (<i>n</i> = 36). The area under the abdominal-edematous-effect time curve (B), which was calculated from Figure A, for i.p. vehicle or carrageenan (0.75%, 1.5%, and 3%) injection extended from 0 to 8 h after i.p. carrageenan injection and showed that i.p. 0.75–3% carrageenan induced a significant increase in the whole-body volume in adult zebrafish. Each point or bar in all figures represents the mean ± SEM of 9 adult zebrafish per group. AUC: area under the curve; 0.75% Ca: 0.75% carrageenan; 1.5% Ca: 1.5% carrageenan; 3% Ca: 3% carrageenan; <b>*</b><i>P</i><0.05 compared with the same time points after i.p. injection of the vehicle (PBS) group; +<i>P</i><0.05 compared with the same time points after i.p. injection of the carrageenan (0.75%) group; #<i>P</i><0.05 compared with the same time points after i.p. injection of the carrageenan (1.5%) group.</p

The effect of MP on protein expression of MPO, TNF-α, and iNOS in carrageenan-injected zebrafish.

<p>(A) Western blots for MPO, TNF-α, iNOS, and β-actin proteins from adult zebrafish; (B) relative density of the MPO immunoblot; (C) relative density of the TNF-α immunoblot; (D) relative density of the iNOS immunoblot. There are 3 groups: vehicle+vehicle: i.p. injection first vehicle (20 µL PBS) 1 h before i.p. second vehicle (20 µL PBS); vehicle+carra: i.p. vehicle (20 µL PBS) 1 h before i.p. 1.5% carrageenan (20 µL); MP+carra: i.p. 1 µg MP (20 µL) 1 h before i.p. 1.5% carrageenan (20 µL). The abdominal samples from adult zebrafish were collected at 24 h after the second injection (as well as 25 h after the first injection). Relative intensity of the i.p. vehicle plus i.p. 1.5% carrageenan group was taken to be 100%. Band intensities of MPO, TNF-α, and iNOS were quantified by densitometry and are indicated as a percentage change relative to that of the vehicle plus carrageenan group. Western blotting of the abdominal tissue homogenate revealed that injection of carrageenan evoked significant upregulation of MPO, TNF-α, and iNOS proteins at 24 h compared with that of the vehicle plus vehicle group. MP administered at a dose of 1 µg (i.p.) 1 h prior to i.p. 1.5% carrageenan significantly reduced carrageenan-induced upregulation of MPO, TNF-α, and iNOS. Western blotting of β-actin was performed to verify that equivalent amounts of protein were loaded in each lane. These experiments were repeated 3 times. Ca: carrageenan; MP: methylprednisolone; MPO: myeloperoxidase, TNF-α: tumor necrosis factor-α; iNOS: inducible nitric oxide synthase. <b>*</b><i>P</i><0.05 compared with the i.p. vehicle plus i.p. vehicle group; #<i>P</i><0.05 compared with the i.p. vehicle plus i.p. 1.5% carrageenan group.</p