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Effects of the Probiotic Formulation VSL#3 on Colitis in Weanling Rats

Fitzpatrick, Leo R*; Hertzog, Kristy L*; Quatse, Angela L*; Koltun, Walter A*; Small, Jeffrey S*; Vrana, Kent

Journal of Pediatric Gastroenterology and Nutrition: May 2007 - Volume 44 - Issue 5 - p 561–570
doi: 10.1097/MPG.0b013e31803bda51
Original Articles: Gastroenterology
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Background: Only a few studies have used models of inflammatory bowel disease (IBD) with weanling animals. Previously, the effects of probiotics have not been assessed in such IBD models. The objectives of our study were 2-fold: to establish a suitable model of dextran sulfate sodium (DSS)–induced colitis in weanling rats and to determine the effects of the probiotic formulation VSL#3 on DSS-induced colitis in weanling animals.

Materials and Methods: Rats were weaned on postnatal day 21 and administered 2%, 2.5%, or 3% (wt/vol) DSS in drinking water. In subsequent experiments, newly weaned animals were administered vehicle or VSL#3 (0.06, 0.6, or 6 mg) by orogastric gavage. These treatments were given to animals maintained on water (postnatal days 21–28) and then on DSS (postnatal days 28–35). Disease activity indices were determined on a routine basis. On day 35, rats were euthanized. The total colon length was determined. Other parameters of colitis were measured from the distal colon. These parameters included myeloperoxidase (MPO), interleukin (IL)–1β, inhibitory κB-α (IκB-α), and histological assessment of crypt damage and inflammation.

Results: DSS 2% was optimal for inducing colitis in weanling rats without significant morbidity. VSL#3 treatments improved various parameters of 2% DSS–induced colitis in weanling rats. The 0.6- and 6-mg doses of VSL#3 were most effective for attenuating this colitis.

Conclusions: The probiotic formulation VSL#3 improved DSS-induced colitis in weanling rats. This improvement of colitis involved changes in colonic IκB-α, IL-1β, and MPO, which are suggestive of immune modulation by VSL#3.

*Division of Surgery

Department of Pharmacology, Pennsylvania State University College of Medicine, Hershey

Received 12 October, 2006

Accepted 22 January, 2007

Address correspondence and reprint requests to Leo R. Fitzpatrick, PhD, Department of Surgery 500, University Drive, Hershey, PA 17033 (e-mail: lfitzpatrick@psu.edu).

This work was supported in part by a grant from American Lifeline, Inc, as well as funding from the Pennsylvania State University College of Medicine.

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INTRODUCTION

Animal models of inflammatory bowel disease (IBD) are useful for investigating mechanisms involved in the etiology of intestinal inflammation, as well as for testing pharmacological approaches that may effectively treat IBD (1,2). However, most of these IBD models use adult rodents. Adult rodents may have different bacterial floral populations and intrinsic intestinal mucosal properties than younger animals (3,4). Moreover, only a limited number of studies have specifically investigated experimental IBD with weanling animals (4–6).

Previous studies in weanling-age rodents have focused on defining microcirculatory changes following the consumption of dextran sodium sulfate (DSS), linear growth changes after the administration of trinitrobenzene-sulfonic acid (TNBS), or the effects of DSS on intestinal surface hydrophobicity (4–6). In these studies the effects of pharmacological intervention were studied by only one group of investigators (6). Specifically, Ballinger et al found that the administration of insulin-like growth factor-1 (IGF-1) improved linear growth in TNBS-treated rats. However, as determined by myeloperoxidase (MPO) activity, intestinal inflammation was not attenuated by IGF-1 (6). Moreover, generally similar results were found in this study when weanling rats were treated with an elemental diet (6). Hence, studies that have examined potentially useful pharmacological therapies (eg, probiotics) for the treatment of intestinal inflammation in weanling animals are lacking.

Probiotics can be defined as ingestible microorganisms with health benefits (7). Typically, probiotics are various strains of Lactobacillus or Bifidobacterium species. They exist as single entities or as combination products (eg, VSL#3) (8,9). Various probiotics are moving into the mainstream of medical therapy for pouchitis (8,9). Moreover, some evidence of probiotic efficacy has been reported in the treatment of IBD (8,9).

Several different probiotic agents have improved parameters of DSS-induced colitis in adult animals (10–12). However, as alluded to previously, studies that have explicitly examined the efficacy of probiotics in weanling animals are needed. Therefore, the objectives of our study were 2-fold: to establish a suitable model of DSS-induced colitis in weanling rats and to determine the effects of a probiotic formulation, VSL#3, on DSS-induced colitis in weanling animals. From a mechanistic standpoint, VSL#3 can attenuate immune mechanisms in relevant cell culture systems, such as inhibiting the nuclear factor-κB (NF-κB) signaling pathway (6,13).

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MATERIALS AND METHODS

Probiotic Formulation and Other Chemicals

VSL#3 was obtained from Sigma Tau Pharmaceuticals (Gaithersburg, MD). This probiotic formulation consists of 8 live bacterial strains: 3 Bifidobacterium strains (eg, longum, infantis, breve); 4 Lactobacillus strains (acidophilus, casei, bulgaricus, plantarium); and 1 Streptococcus strain (thermophilus). VSL#3 is reported to contain 3 × 1011 colony-forming unit (CFU)/g of viable lyophilized bacteria (14,15).

DSS (molecular weight 36,000–50,000) was obtained from MP Biomedicals (Aurora, OH). Key reagents for the MPO assay were (3,3′,5,5′ tetramethylbenzidine [TMB],N,N-dimethylformamide, hydrogen peroxide, and hexadecyltrimethylammonium bromide [HTAB]) were obtained from Sigma Chemical (St. Louis, MO). The inhibitory κB-α (IκB-α) antibody (sc-847) was obtained from Santa Cruz Biotechnology (Santa Cruz, CA). The rat interleukin (IL)-1β enzyme-linked immunosorbent assay kit was obtained from Pierce Endogen (Rockford, IL).

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Rats

Timed pregnant female Wistar rats were purchased from Charles River Laboratories (Wilmington, MA). Male and female weanling rats were used in the colitis studies. Animals were housed under standard conditions, with a 12-hour light/dark cycle. For the DSS-induced colitis studies, rats were weaned on postnatal day 21 and provided standard rodent laboratory chow throughout the study.

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Dextran Sulfate Sodium–induced Colitis

The pathogenesis of DSS-induced colitis involves a defect in epithelial barrier function, which subsequently leads to the influx of various inflammatory cells, macrophage activation, and proinflammatory cytokine production (16,17). This IBD model has been well characterized in adult animals and is thought to resemble human ulcerative colitis to a certain degree (18,19).

In the initial DSS dose-response experiment, newly weaned rats were randomly divided into 4 groups. Rats were given 0% DSS (Millipore-filtered drinking water), 2% (wt/vol) DSS, 2.5% DSS, or 3% DSS for 7 days. These concentrations of DSS were chosen based on the relevant literature. This literature included previous results obtained with weanling rats, as well as results showing that 40 kD DSS produced more severe colitis in mice than did 5 kD DSS (5,20). Approximately equal numbers of male and female weanling rats were included in the treatment groups. Rats were monitored on a daily basis for changes in body weights. A disease activity index (DAI) was determined on a daily basis by the method described later. On postnatal day 28, rats were euthanized by exposure to carbon dioxide, and the total colon length was measured. Samples of distal colon were collected, fixed in 10% buffered formalin, and used for a colonic histology evaluation (as described later).

The following paradigm was used in the VSL#3 treatment studies. Approximately equal numbers of male and female weanling rats were randomized into 4 different treatment groups. During the first 7 days (postnatal days 21–28), newly weaned rats were given Millipore-filtered drinking water. The body weights were monitored during this period. During this initial 7-day period, specific groups of mice were dosed once daily with vehicle (Millipore-filtered phosphate-buffered saline solution) or 3 doses of VSL#3. The 3 VSL#3 doses were 0.06 mg, 0.6 mg, and 6 mg. This equates to 0 CFU, 0.18 × 108 CFU, 1.8 × 108 CFU, or 1.8 × 109 CFU of VSL#3. The dose range of VSL#3 (0.06–6 mg) was reflective of doses used in previous rodent studies with this probiotic (21–24). For example, Di Giacinto et al used a 2-mg dose of VSL#3 to treat TNBS-induced colitis in mice (21). The dosing was performed by orogastric gavage at a constant dose volume of 0.3 mL. Then, 4 groups of rats were dosed for the next 7 days (postnatal days 28–35) with vehicle or VSL#3 while they also received 2% DSS in their drinking water. One group of vehicle-treated animals received water without DSS. Body weight data were also collected during this phase of the study. DAIs were also determined on a regular basis during this time period. On postnatal day 35, rats were euthanized with carbon dioxide. The total colon length was measured and recorded. Next, the distal 6 cm of colon was divided into three 2-cm segments. The most distal segment was used for a histological evaluation, the middle segment was used for the measurement of IκB-α, and the most proximal segment was used for the determination of MPO and IL-1β. The methods used to measure these biochemical and histological parameters of colitis are described in the following sections.

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DAI Determination

The DAIs were determined from a 5-point severity scale ranging from 0 to 4 (Table 1), slightly modified from the method of Murthy et al (25). DAIs were determined on postnatal days 21, 23, 25, 27, 29, and 30 to 35.

TABLE 1

TABLE 1

The DAI (0–4) was calculated by totaling the scores for stool consistency and occult blood/gross bleeding and then dividing by 2 (25). The presence of occult blood was determined on fecal smears by a hemoccult fecal blood kit (Beckman-Coulter, Fullerton, CA).

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Colonic Histology Evaluation

On postnatal day 35 the distal colonic segment was slit open and fecal material was removed by thorough rinsing in 0.9% saline solution. This 2-cm segment of distal colon was immediately fixed in 10% buffered formalin, and the tissue was subsequently processed for a histological evaluation. Histological damage was determined on a 40-point severity scale. Various investigators have used this scoring system (26–28) in conjunction with the DSS-induced colitis model (Table 2).

TABLE 2

TABLE 2

Total colonic histology scores were determined by multiplying the percent involvement for each of the 3 different histological features by the area of involvement. Using this scoring system, the minimal score was 0 and the maximum score was 40. The percent area of involvement was determined with a 25-mm ocular grid that was attached to an Olympus CH light microscope (Olympus, Center Valley, PA). This histological evaluation was done at a magnification of 400. Six areas on each histology slide were evaluated, and a mean histology score was determined for each slide (28). The evaluation was done on coded slides so the investigator (L.R.F.) was unaware of the treatment group.

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Western Blot Determination of Colonic IκB-α Expression

The Western blot technique was performed basically according to a previously described method (28). Using the middle 2 cm of rat colon, we obtained colonic homogenates. A protein determination of these whole cell colonic extracts was done with the Bio-Rad protein assay (Bio-Rad Laboratories, Hercules, CA). For the Western blot analysis, we used 20 μg of protein per colonic sample. Western blots were performed using a standard technique. Briefly, 4% to 15% Tris-HCl ready gels (Bio-Rad) were used for this protocol. The gels were run at 100 V for approximately 1 hour. Gels were then transferred onto PROTRAN nitrocellulose membranes (Schleicher & Schuell Bioscience, Dassel, Germany). Subsequently, blots are blocked with PBS-Tween containing 5% blotto (nonfat dried milk). Next, the blots were incubated overnight in primary antibody (rabbit polyclonal antibody to IκB-α), washed in PBS-Tween, and then exposed to an appropriate secondary antibody (goat antirabbit). After another series of washes, equal amounts of an oxidizing agent and a luminol reagent (Western Lightning; Perkin-Elmer, Waltham, MA) is applied for 1 minute. Subsequently, the blots were dried and exposed to Kodak Scientific Imaging XB-1 film (Kodak, Rochester, NY). For evaluating the levels of colonic IκB-α expression, a densitometry analysis was performed with a QuantiScan software program (Biosoft, Cambridge, UK).

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Measurement of Colonic MPO and IL-1β

The most proximal 2-cm segment of colon was used to measure MPO and IL-1β from colonic homogenates, as described previously by the principal investigator (28–30). Briefly, the homogenates were centrifuged at 10,000 rpm for 15 minutes. The pellet was retained for measurement of MPO by the tetramethylbenzidine method. The supernatant was divided into aliquots and frozen at −70°C for the subsequent determination of colonic IL-1β. IL-1β was measured using the appropriate rat cytokine enzyme-linked immunosorbent assay kit.

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Statistical Analyses

All of the statistical analyses were performed with GraphPad Prism software (GraphPad, San Diego, CA). Data are presented as means ± SE. Typically, multiple groups were analyzed by 1-way analysis of variance and then individual group comparisons were performed with the Newman-Keuls multiple comparison test. For some data, to confirm differences between 2 treatment groups, the Student t test was used. The day-35 DAI data were not normally distributed. Therefore, it was analyzed by the Mann-Whitney test. The comparison of slopes analyses (DAI time course data) were also performed with Graph-Pad Prism software. Specifically, the comparison of slopes was done by an analysis of covariance method. A difference of P < 0.05 was considered significant for all of these analyses.

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RESULTS

Optimal DSS for Colitis Induction

As shown in Figure 1, no evidence of colitis was found in animals consuming only filtered water (0% DSS). The administration of DSS to weanling rats resulted in a concentration dependent increase in the DAI. Specifically, the administration of 2% DSS resulted in moderately severe colitis. Typically, rats showed some evidence of diarrhea, as well as bloody stools, by postnatal day 28. The mean DAI in the 2% DSS treatment group was 2.6 (Table 3). Animals receiving the larger concentrations of DSS (2.5% and 3%) showed evidence of severe colitis with gross rectal bleeding and severe diarrhea. The mean DAIs in these treatment groups were 3.2 (2.5% DSS) and 3.6 (3% DSS). All weanling rats gained weight during the 7-day treatment period. However, the overall weight gain was less pronounced in the rats consuming 2.5% and 3% DSS (Table 3). Morbidity (defined as animals requiring early euthanasia because of severe symptoms of colitis), or mortality (defined as premature death before postnatal day 28) occurred in 3 of 16 (19%) of the 2.5% DSS-treated weanling rats and 2 of 16 (13%) of the 3% DSS-treated rats. No morbidity or mortality was evident in rats that consumed 2% DSS (Table 3).

FIG. 1

FIG. 1

TABLE 3

TABLE 3

The histological and colon length data (Table 3) were obtained from rats euthanized on postnatal day 28. These colonic parameters were significantly increased (P < 0.05) in all of the DSS treatment groups compared with the values obtained from non–DSS-treated animals. Overall, these clinical, morphometric, and histological data suggested that a moderate to severe level of colitis could be obtained in weanling rats that were administered 2% DSS. More severe colitis was found in animals consuming 2.5% or 3% DSS. However, these concentrations of DSS also resulted in unwanted morbidity and mortality. Therefore, 2% DSS was chosen for the evaluation of VSL#3 on colitis in weanling rats.

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VSL#3 Reduced DAIs in Weanling Rats Given DSS

Figure 2A shows the time course of colitis symptoms in weanling rats treated with vehicle or VSL#3 over a 2-week period (postnatal days 21–35). No evidence of symptoms was found in any of the treatment groups in days 21–28, when animals were administered only filtered water (without DSS). All of the rats gained weight during the 2-week study period. There were no significant differences in weight gain among the treatment groups during the water treatment phase of the study (days 21–28). During the DSS treatment phase of the study (days 29–35), the weight gains were also similar: 45.3 ± 2.6 g (vehicle/water), 40.8 ± 2.5 g (vehicle/DSS), 43.9 ± 2.2 g (VSL#3 0.06 mg/DSS), 43.9 ± 2.6 g (VSL#3 0.6 mg/DSS) and 41.5 ± 2.4 g (VSL#3 6 mg/DSS). As shown by the steadily increasing DAIs from postnatal days 32 through 35, symptoms of colitis clearly occurred in the vehicle/DSS-treated rats (Fig. 2A). By day 35, most vehicle-treated animals showed evidence of diarrhea, as well as bloody stools. In contrast, the VSL#3-treated weanling rats typically showed less severe symptoms (mainly loose stools). This was particularly the case in rats dosed with the 0.6- and 6-mg doses of probiotic. On postnatal day 35, the mean DAIs were 0.1 ± 0.1 (vehicle/water), 2.3 ± 0.2 (vehicle/DSS), 1.4 ± 0.3 (VSL#3 0.06 mg/DSS), 0.9 ± 0.2 (VSL#3 0.6 mg/DSS) and 0.9 ± 0.3 (VSL#3 6 mg/DSS). The reduced DAIs evident with all 3 VSL#3 treatments were statistically significant (P < 0.05 vs vehicle/DSS). Overall, for reducing the DAI, the 0.6- and 6-mg doses were qualitatively better in reducing symptoms than the 0.06-mg dose (Fig. 2A). Also, there was no evidence of mortality in the 0.6- and 6-mg VSL#3 treatment groups, whereas 1 animal in the 0.06-mg treatment group died prematurely of severe symptoms of colitis. These results imply more optimal efficacy of VSL#3 in the range of 0.6–6 mg for attenuating symptoms of DSS-induced colitis.

FIG. 2

FIG. 2

To assess the time-related effects of VSL#3 treatment on symptoms of colitis, we compared the slopes of the DAI time course data in vehicle- and VSL-treated weanling rats. As shown in Figure 2B, the slopes were significantly different (P = 0.0027) in the VSL 0.6 mg and vehicle treatment groups. These results indicate that the evolution of DSS-induced colitis was attenuated in probiotic-treated weanling rats. Similar results were obtained with the other VSL#3 treatment groups (data not shown).

The total colon length was significantly shorter (P < 0.05) in vehicle/DSS–treated rats on postnatal day 35 compared with vehicle/water–treated animals. The administration of VSL#3 increased the total colon lengths in rats consuming DSS. The mean colon length values (in mm) were 14.1 ± 0.5 (vehicle/water), 12.9 ± 0.3 (vehicle/DSS), 13.3 ± 0.3 (VSL#3 0.06 mg/DSS), 14.0 ± 0.3 (VSL#3 0.6 mg/DSS) and 14.2 ± 0.3 (VSL#3 6 mg/DSS). Administering the 2 largest doses of VSL#3 (0.6 and 6 mg) to weanling rats resulted in significantly longer colons (P < 0.05) than in parallel vehicle/DSS–treated animals.

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VSL#3 Improved Histologic Alterations Associated with Colitis

Figure 3 shows representative histological photographs from the distal colon of rats on postnatal day 35. The colonic histological view from a vehicle/water–treated rat (Fig. 3A) showed normal crypts, with scattered leukocytes in the lamina propria. In contrast, the administration of 2% DSS (Fig. 3B) resulted in crypt destruction, with infiltration of leukocytes (black arrows) throughout the lamina propria and extending into the submucosa. The administration of VSL (6 mg) resulted in less prominent crypt damage, although there was some evidence of leukocyte infiltration (black arrow) in the colonic lamina propria and submucosa of this particular rat (Fig. 3C). In some animals, the administration of VSL#3 resulted in a relatively normal histological picture (Fig. 3D), which resembled that seen in rats maintained on water (Fig. 3A). The individual components of the histology scores, as well as the overall mean total histology scores, are shown in Table 2. The total histology scores in all 3 of the VSL treatment groups were significantly lower (P < 0.05) than in the vehicle/DSS treatment group. There were no obvious differences in the histological parameters of colitis among the 3 VSL#3 treatment groups (Table 2).

FIG. 3

FIG. 3

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VSL#3 Treatment Partially Limited IκB-α Degradation in DSS-treated Rats

Figure 4A shows representative colonic IκB-α expression results from postnatal day 35. There was evidence of IκB-α degradation (ie, lower IκB-α expression) in vehicle/DSS–treated rats. However, in some VSL#3-treated animals, there was evidence of enhanced IκB-α expression compared with parallel vehicle/DSS–treated rats (solid arrow in Fig. 4A). The actin results were essentially the same with all of the treatments, which confirmed equal loading of protein in the Western blot analysis. The overall colonic IκB-α expression results are summarized in Figure 4B. These results show reduced IκB-α expression in the colon of vehicle/DSS–treated rats compared with the vehicle/water–treated animals. Those rats treated with the 0.06 mg dose of VSL#3 also had reduced IκB-α expression. The 0.6-mg and 6-mg doses of VSL#3 partially reversed the attenuation in IκB-α expression, which was found in vehicle/DSS–treated rats. However, the overall expression of IκB-α found in these VSL/DSS–treated animals did not reach the level of colonic IκB-α expression found in the vehicle/water–treated rats (Fig. 4B).

FIG. 4

FIG. 4

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VSL#3 Attenuated Colonic MPO and IL-1β in Rats Receiving DSS

As shown in Figure 5A, there was a modest but significant increase (approximately 2-fold) in the colonic MPO activity of rats receiving DSS for a 1-week period. Colonic MPO was partially attenuated in all VSL treatment groups. In a similar fashion, the colonic IL-1β content was increased approximately 2-fold in vehicle/DSS–treated rats (Fig. 5B). Administering all 3 doses of VSL#3 resulted in a reduction in the colonic IL-1β content. This was particularly the case in rats receiving the 6-mg dose of VSL (P < 0.05 vs vehicle/DSS).

FIG. 5

FIG. 5

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DISCUSSION

Earlier studies examining experimental IBD in weanling animals are sparse (4–6). Shimizu et al did examine the effects of DSS administration to weanling rats (5). However, their study differed from the present study in various ways. Specifically, their rats were weaned at 4 weeks of age, whereas we weaned our animals earlier during the postnatal period (ie, at 3 weeks old). Also, our study used weanling animals of both sexes, whereas their study used 4-week-old male rats. Moreover, there were differences in the molecular weights of DSS that were used in the respective studies (5).

Shimizu and colleagues found only mild colitis in weanling rats given 2% DSS, whereas 4% DSS resulted in severe colitis, which was associated with a 20% mortality rate. Overall, a moderate level of colitis was observed in rats administered 3% DSS (5). In contrast, we found that DSS concentrations from 2.5% to 3% were not well tolerated in our cohort of weanling animals. Evidence of morbidity (ie, animals requiring early euthanasia) or early mortality were evident in these rats. These scenarios were related to severe rectal bleeding and diarrhea in these animals. However, weanling rats that were administered 2% DSS showed evidence of moderate to severe colitis, without associated morbidity or mortality. The effects of lower concentrations of DSS (eg, 1.5%) were not tested in our study, and therefore the severity of colitis with these doses of DSS is unknown. Kitajima et al found that colitis was more severe in rats administered 40 kD DSS as opposed to 5 kD DSS (20). We believe that this variable likely accounted for the differences found in our DSS dose-response study and the study conducted by Shimizu and colleagues (5). Based on our results (Table 3), we concluded that 2% was an optimal concentration of DSS for testing the efficacy of VSL#3 in weanling rats.

Various probiotics, including VSL#3, have improved DSS-induced colitis in adult animals (10–12); however, studies that have explicitly examined the efficacy of probiotics in weanling animals are lacking. Our results (Fig. 2) demonstrate that the administration of all 3 VSL#3 treatments reduced the overall degree of colitis symptoms (bloody stool, diarrhea) in weanling rats, and also delayed the evolution (time course) of symptom development.

A reduction in colon length is associated with the DSS-induced colitis model, and is often used as a marker of colonic injury (18,28,30). As anticipated, the mean colon length of our vehicle/DSS–treated animals was significantly shorter than in parallel water-treated rats. This reduction of total colon length was not evident in animals treated with VSL#3 at doses of 0.6 and 6 mg. These data further demonstrate the effectiveness of this probiotic formulation in weanling rats for improving a common parameter of DSS-induced colitis.

After DSS administration to adult rodents, macroscopic and histological manifestations of colitis are more pronounced in the distal colon (18,19). Similar observations were observed in the distal colon of “young rats” administered DSS for 1 week (31). In our initial dose-response study with DSS, we also found more consistent evidence of macroscopic inflammation in the distal portion of the colon. Therefore, when evaluating histological and biochemical parameters of colitis in vehicle- or VSL#3-treated rats, we used the most distal 6 cm of colon for these evaluations.

Overall, our histology results suggested evidence of moderate to severe pathological processes in vehicle/DSS–treated weanling rats (Fig. 3; Table 4). The mean total colonic histology score in vehicle/DSS–treated animals was 25. In this regard 55% of the vehicle-treated rats had histology scores >25, whereas the analogous percentages in DSS-treated rats dosed with VSL#3 were 11% (VSL#3 0.06 mg), 10% (VSL#3 0.6 mg), and 10% (VSL#3 6 mg). Therefore, evidence of histological improvement was found in nearly all of the VSL-treated weanling rats. This histological improvement was evident in all 3 components (crypt damage, inflammation severity, inflammation extent) of the total histology score. Therefore, the mean total histology scores were significantly reduced (P < 0.05) in VSL-treated weanling rats (Table 4).

TABLE 4

TABLE 4

Recently, immune modulation was proposed to be one of the mechanisms of action for probiotics, as related to therapy for intestinal inflammation (9). With regard to this proposed mechanism of action, in vitro data from 2 groups of investigators showed that VSL#3 can inhibit NF-κB by preventing IκB-α degradation (7,13). However, this type of data is somewhat controversial, as Rachmilewitz et al found that the administration of DNA from VSL#3 actually further increased the nuclear binding of NF-κB in isolated mouse macrophages (12). An upregulation of the NF-κB signaling pathway has been shown to occur previously in conjunction with DSS-induced colitis in adult rodents (32,33), Therefore, based on these previous results (7,13,32,33), we chose to evaluate the expression of IκB-α in colonic extracts from our weanling rats. Overall, our data showed that colonic IκB-α expression was attenuated in animals administered DSS as opposed to those not treated with DSS (Fig. 4B). These data are consistent with those from other colitis studies in adult rats, which found IκB-α degradation in the presence of colonic inflammation (34,35). In 20% of weanling rats treated with VSL#3 (0.6 or 6 mg), the mean IκB-α expression value was at least as great as the mean value (0.59) found in animals not receiving DSS. One example of this scenario is shown in the last lane of the Western blot (Fig. 4A). Nevertheless, these 2 doses of VSL#3 overall only partially reversed the attenuated IκB-α found in vehicle/DSS–treated rats (Fig. 4B). In contrast, the 0.06-mg dose of VSL#3 did not show this pattern because these animals had a low overall level of IκB-α expression. These results suggest the possibility that reduced IκB-α degradation may have contributed to the reduction in other parameters of colitis, which were found in this study with the 0.6- and 6-mg doses of VSL#3. However, it is likely that other undetermined mechanisms of action are also operative. Such mechanisms may include the inhibition of p38 mitogen-activated protein kinase by VSL#3, as was suggested by other investigators (13).

Colonic MPO is a marker of neutrophil influx into the intestinal tissue and is often used as a marker of inflammation in conjunction with the DSS-induced colitis model (24,31). Our results (Fig. 5A) showed approximately a 2-fold increase in colonic MPO with DSS treatment in weanling rats. These results are similar to those reported previously by Vicario et al in the colon of young rats (ie, approximately 37 days old) (31). Interestingly, all 3 VSL#3 treatment regimens partially reduced the mean level of colonic MPO compared with vehicle treatment (Fig. 5A).

Interleukin-1β is a proinflammatory cytokine that has been linked to the NF-κB signaling pathway and intestinal inflammation (36–38). Moreover, IL-1β levels are upregulated in the acute DSS-induced colitis model, and this cytokine has been proposed to play a prominent role in the pathogenesis of this colitis (39–41). For this reason, we chose to use the colonic IL-1β level as another marker for the severity of DSS-induced colitis in weanling rats. Similar to the MPO results, there was approximately a 2-fold increase in the mean colonic IL-1β content (Fig. 5B). Based on previous results from our laboratory (28,30), it is possible that even more profound increases in IL-1β and MPO contents would have occurred if these parameters were measured in a more distal segment of colon. Finally, the observed reductions of the IL-1β content with the VSL#3 treatment regimens (Fig. 5B) may have contributed to the reduction in colitis symptoms observed in these probiotic-treated weanling rats (40,41).

In summary, we have established a model of IBD in weanling rats by administering 2% DSS for a 1-week period. Our results with VSL#3 treatment during postnatal days 21–35 showed that this probiotic formulation could improve clinical, morphological, histological, and biochemical parameters of colitis. Overall, doses of VSL#3 in the range of 0.6–6 mg seemed to be most optimal for attenuating these parameters. More studies of the type reported here will be required to determine which probiotic agents are most effective in weanling rats, and to learn more about the operative mechanisms of action for probiotics. These studies could also include examining the effects of probiotics on intestinal flora and permeability in weanling rats. Such studies may also provide a further impetus for the more frequent use of probiotics in the treatment of pediatric IBD.

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Keywords:

Colitis; Probiotics; Weanling rats

© 2007 Lippincott Williams & Wilkins, Inc.