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Effect of bevacizumab on acetic acideinduced ulcerative colitis in rats Zeki Ozsoy, MD,a,* Seyma Ozsoy, MSc,b Fikret Gevrek, MD,c Emre Demir, MSc,d Ismail Benli, PhD,e Emin Daldal, MD,a and Erdinc Yenidogan, MDa a
Faculty of Medicine, Department of General Surgery, Gaziosmanpas‚a University, Tokat, Turkey Faculty of Medicine, Department of Physiology, Gaziosmanpas‚a University, Tokat, Turkey c Faculty of Medicine, Department of Histology and Embryology, Gaziosmanpas‚a University, Tokat, Turkey d Faculty of Medicine, Department of Biostatistic, Hitit University, Corum, Turkey e Faculty of Medicine, Department of Biochemistry, Gaziosmanpas‚a University, Tokat, Turkey b
article info
abstract
Article history:
Background: The aim of this study was to examine the effect of intraperitoneally admin-
Received 30 January 2017
istered bevacizumab on colitis induced by acetic acid.
Received in revised form
Methods: An experimental model of acetic acideinduced colitis was introduced in rats.
5 April 2017
After the induction of colitis, bevacizumab was administered intraperitoneally at two
Accepted 2 May 2017
different daily doses of low (2.5 mg/kg) or high (5 mg/kg) concentration. Control groups
Available online 10 May 2017
were included for colitis and bevacizumab. After 7 d, the rats were sacrificed, and colonic tissues were harvested for macroscopic and microscopic examination of colonic damage.
Keywords:
Tumor necrosis factor alpha, interleukin 1 beta (IL-1b), IL-6, myeloperoxidase, malondial-
Ulcerative colitis
dehyde, glutathione, and superoxidismutase values were measured biochemically.
Bevacizumab
Results: There was no statistically significant macroscopic improvement in damage to the
Treatment
colon tissues (P > 0.05). The severity of inflammation was significantly reduced (0.98 0.22)
Rats
in the low-dose bevacizumabetreated rat group compared with the control group (P < 0.001). The decrease in the inflammation score in the high-dose bevacizumabetreated rat group was not statistically significant (1.40 0.33). In addition, although there was no significant change in the myeloperoxidase levels biochemically, IL-6 and malondialdehyde levels decreased in the low-dose treatment group (P ¼ 0.014, P ¼ 0.002, respectively). A significant decrease was found at both treatment doses in IL-1b (P < 0.001, P ¼ 0.010), tumor necrosis factor alpha (P < 0.001, P ¼ 0.015), superoxidismutase (P ¼ 0.046, P ¼ 0.011), and glutathione (P ¼ 0.012 and P < 0.001) levels. Conclusions: Both treatment doses of bevacizumab were observed to have a protective effect in an experimental colitis model, and the dosage of 2.5 mg/kg bevacizumab was found to have a more prominent effect. ª 2017 Elsevier Inc. All rights reserved.
The manuscript is original. It has not been published or is not under consideration by any other journal. * Corresponding author. Faculty of Medicine, Department of General Surgery, University of Gaziosmanpasa, 60100 Tokat, Turkey. Tel.: þ90 506 5637037; fax: þ90 356 2133179. E-mail address:
[email protected] (Z. Ozsoy). 0022-4804/$ e see front matter ª 2017 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.jss.2017.05.011
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Introduction
Experimental material and laboratory environment
Ulcerative colitis (UC) is a chronic inflammatory disease of the large intestine, and its pathogenesis is not fully known.1 Physiological angiogenesis and the formation of granulation tissue are necessary for the recovery of wounds and ulcers. However, angiogenesis in neoplastic diseases and chronic inflammatory diseases (especially inflammatory intestinal disease, rheumatoid arthritis, atherosclerosis, diabetic retinopathy, and psoriasis) can lead to pathologic conditions.2,3 It results in the formation of blood vessels that leak pathologic angiogenesis in the UC, which are not sufficiently mature and cannot provide adequate wound and ulcer recovery.4 This intestinal pathologic angiogenesis enhances gut inflammation by allowing a higher influx of immune cells into the affected tissue. These newly formed vessels can directly secrete proinflammatory cytokines and chemokines that attract more immune cells, further amplifying the gut inflammation.5 In addition, the formation of free oxygen radicals increases, which leads to oxidative damage in the colonic mucosa.6 Thus, targeting intestinal pathologic angiogenesis may reduce chronic gut inflammation. Vascular endothelial growth factor (VEGF) is an important proangiogenic growth factor, which increases vascular permeability and stimulates inflammatory cell infiltration in UC.7 It is reported that the inhibition of the VEGF in individuals with inflammatory intestinal disease leads to recovery of colonic tissue damage by reducing vascularization and vascular permeability, and thereby contributes to the treatment of inflammation.8 To this end, clinical studies have trialed several antiangiogenic factors and the most widely used and well known one is bevacizumab.8,9 Bevacizumab is a recombinant monoclonal antibody, which targets the VEGF. It is reported to have increased general survival rates when used in combination with chemotherapy regimens in patients with metastatic colorectal cancer.10 Unlike other cancer drugs, it does not directly affect the tumor cells but targets the vessels feeding them. Oxygenation of the tumor is increased by lowering interstitial pressure in the tumor.11 Despite the biological antineoplastic effect of bevacizumab, there have been no studies on the anti-inflammatory efficacy in a rat colitis model. The aim of this study was to examine the effect of intraperitoneally administered bevacizumab on colitis induced by acetic acid (AA).
A total of 42 male Wistar albino rats, each weighing between 250 to 350 g, were used in this experimental study. The rats were housed in wire cages in groups of two or three, under standard laboratory conditions (23 C room temperature, 12-h lightedark cycle) where they could access food and drinking water. Twenty-four hours before the surgical procedures, food was restricted but not water. Through simple random sampling method, the rats were divided into six groups, with seven rats in each group.
Materials and methods
Drug administration
Approval for the study protocol was granted by the Experimental Animals Ethics Committee of Gaziosmanpasa University Medical Faculty (2014-HAYDEK-26). All experimental, surgical, and laboratory procedures were applied in Gaziosmanpasa University Medical Faculty Experimental Research Centre and Gaziosmanpasa University Medical Faculty Biochemistry and Histology Laboratories.
Bevacizumab (Altuzan, Roche 400 mg/16 mL flacon, Basel, Switzerland) was diluted in saline to 0.625 mg/mL for the LDB groups and 1.25 mg/mL for the HDB groups. In the HDB groups, the solution was prepared by the addition of 0.25 mL Altuzan (25 mg bevacizumab/mL) to 4.75 mL saline. Thus, 1 mL solution contained 1.25 mg bevacizumab. For each 25 g weight, 0.1 mL solution was administered. In the LDB groups, the
Surgical procedures and experimental protocol No antibiotics were given to the rats before or after the surgical procedures. All the surgical procedures were performed by the same researcher. Before all the experimental surgical procedures, anesthesia was provided with intramuscular 75 mg/kg ketamine hydrochloride (Ketalar 500 mg flacon; Pfizer, Istanbul, Turkey) and 10 mg/kg xylazine hydrochloride (Rompun 2% flacon; Bayer, Istanbul, Turkey). Before the creation of experimental colitis, the rats were weighed, and their weights were recorded. To create the experimental colitis, a soft 6 mm pediatric-feeding catheter was inserted into the anus of each rat and the tip was advanced to 8 cm. One milliliter of 4% AA (pH 2.3) solution was slowly administered transrectally. Before withdrawal of the catheter, 2 mL of air was injected to make it spread through the colon. Then the catheter was withdrawn slowly to prevent any physical trauma. The rats were held by the tail upside down for 30 s to prevent any leakage of the administered substance.12 The study groups were organized as follows: Group 1 (control saline): saline was administered intrarectally. Group 2 (AA): the experimental colitis model was created with AA. Group 3 (Beva 2.5 saline): the rats were administered saline intrarectally, then 5 min later, low-dose (2.5 mg/kg) bevacizumab (LDB) was administered. The treatment was continued for 6 d. Group 4 (Beva 2.5 AA): 5 min after the induction of colitis, LDB was administered, and the treatment was continued for 6 d. Group 5 (Beva 5 saline): the rats were administered saline intrarectally, then 5 min later, high-dose (5 mg/kg) bevacizumab (HDB) was administered. The treatment was continued for 6 d. Group 6 (Beva 5 AA): 5 min after the induction of colitis, HDB was administered, and the treatment was continued for 6 d.
ozsoy et al effect of bevacizumab on ulcerative colitis
Table 1 e Macroscopic grading scale. Score
Gross morphology
0
No damage
1
Localized hyperemia, but no ulcers
2
Linear ulcers with no significant inflammation
3
Linear ulcer with inflammation at one site
4
Two or more sites of ulceration or inflammation
5
Two or more major sites of inflammation and ulceration or one major site of inflammation and ulceration extending >l cm along the length of the colon
6-10
Site of inflammation and ulceration extending >2 cm along the length of the colon (the score is increased by 1 for every 1 cm damage)
solution was prepared by the addition of 0.125 mL Altuzan (25 mg bevacizumab/mL) to 4.875 mL saline. Thus, 1 mL solution contained 0.625 mg bevacizumab. For each 25 g weight, 0.1 mL solution was administered. The solution was administered by intraperitoneal injection every day at the same time, for a period of 7 d that started on the operation day and finished on the sacrifice day. After the injection, the catheter was removed, and the abdomen was massaged to ensure even spread of the injected material13
Blood and tissue sampling Before sacrifice, the rats were weighed and their weights were recorded. Sacrifice was performed under standard laboratory conditions. Before the procedure, 30 mg/kg hydrochloride and 5 mg/kg xylazine for anesthesia was administered. By opening the abdomen with a midline incision, the heart was reached from the diaphragm, the blood was collected via puncture, and sacrifice was applied with cervical decapitation. Serum was separated and stored at 80 C until the day of analysis. Procedures were repeated twice on all samples. By extending the midline incision as far as the pubis, the rectum was cut from the most distal part that was possible. From this section, the last 8 cm of the colon was removed.
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embedded in paraffin blocks and placed on standard glass slides. The paraffin was melted with a period of approximately 12 h in an incubator at 58 C. The samples were then stained with hematoxylene and eosin according to the protocol. Histologic analyses were performed on 10 sections belonging to seven animals in each group, in 20 random microscopic view areas on the average. The analysis was performed using a computer-assisted light microscope (Nikon Eclıpse 200 Serial No: T1al 944909, Japan) with an integrated camera (Nikon Ds-Fi1, Japan), and the microscopic views were transferred to the monitor for analysis using Nis element program. The microscopic analyses were performed using a coding system by a researcher blinded to the group information. The mucosa, submucosa, and adventitia strata of all the colon tissue section samples were evaluated microscopically in terms of intensity of inflammation. Hence, semiquantitative tissue inflammation severity scoring was applied. The grading of this semiquantitative tissue inflammation was performed using a four-level modified scoring scale.15 In this scoring system the values are indicated as follows: 0, none; 1, giant cells and occasional lymphocytes; 2, giant cells, plasma cells, eosinophils, and neutrophils; and 3, many inflammatory cells and microabscesses.
Biochemical studies The blood samples taken from the rats were placed in blood tubes and left for clotting for approximately 30 min. Serum was separated by centrifugation of the tubes at 4 C, 4000 rpm for 10 min. Then the separated serum samples were stored in Eppendorf tubes at 80 C until analysis. In accordance with the kit manufacturer’s instructions, glutathione (GSH; item no: 7003002; Cayman Chemical, Estonia) and malondialdehyde (MDA; item no: 10009055; Cayman Chemical, Estonia) levels were measured with the colormetric method, and myeloperoxidase (MPO; SEA601Ra; Cloud-CloneCorp, Katy, TX), superoxidismutase (SOD; cat no: YHB2870Hu; YH-Biosearch, China), tumor necrosis factor alpha (TNF-a; cat no: YHB1098Ra; YH-Biosearch, China), interleukin 6 (IL-6; cat no: YHB0630Ra; YH-Biosearch, China), and IL-1b (cat no: YHB0616Ra; YH-Biosearch, China) levels were measured with the enzyme-linked immunosorbent assay method.
Macroscopic colitis evaluation Statistical analysis After removal, the colon was opened longitudinally and cleaned with saline. Mucosal lesions were then scored macroscopically using the Morris scoring system14 (Table 1).
Histopathologic evaluation The histopathologic examination was conducted in the Histology Department of Gaziosmanpasa University Medical Faculty. For microscopic evaluation, samples were taken from the sections of rat colon tissue with highest macroscopic damage. The tissue samples were fixed in 10% buffer neutral formaldehyde solution for a period of 36 h on the average. After the fixation procedure, the tissues were embedded in paraffin blocks according to the routine histology tissue protocol. Using a rotary microtome (Leica RM2135, Germany), sections of 5 mm thickness were cut from the tissue samples
Data were analyzed using Statistical Packages for the Social Sciences (version 22.0, SPSS Inc, Chicago, IL). Descriptive statistics were presented as the mean standard deviation, and median (minemax) values. Distributions of the groups were evaluated with ShapiroeWilk tests. The significance of the difference between two paired groups was evaluated using the paired t-test in cases of normally distributed data and the Wilcoxon signed rank test when data were not normally distributed. The significance of the difference between more than two groups was evaluated using the KruskaleWallis test (nonparametric analysis of variance) because data did not meet the assumptions of the parametric test analysis of variance. Post hoc pairwise comparison tests were used to determine which groups differed significantly. A value of P < 0.05 was considered statistically significant.
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Fig. 1 e Box plots of weight measurements for before and after experiment (A) and weight change means (B) according to groups. (Color version of figure is available online.)
Results No complications or deaths occurred in the rats during or after the surgery.
Macroscopic results The body weight measurements taken before and after the experiment were compared to evaluate weight loss. A statistically significant difference was found between the before and after procedure weights of the control saline and the AA groups (P ¼ 0.018, P ¼ 0.018, respectively). No statistically significant difference was found between the before and after procedure weights of the other groups. The body weight values of the groups before and after the procedures are shown as a box plot graph in Figure 1A. A new variable was defined as the difference in body weight, and the measurements of weight change were
compared between the groups. A statistically significant difference was found between the groups in terms of the change in body weight (P < 0.001). The change in body weight of the groups is shown as a box plot graph in Figure 1B. An analysis of the groups in terms of macroscopic damage scores indicated a statistically significant difference (P < 0.001). The means for macroscopic damage scores of the groups are shown in Table 2. Both treatment doses indicated no statistically significant macroscopic improvement in the colon tissue damage (P > 0.05).
Histopathologic results A statistically significant difference was identified when the groups were analyzed in terms of the mean inflammation scores (P < 0.001). Table 3 demonstrates descriptive statistics about the inflammation scores of the groups. The intensity of inflammatory cells and tissue integrity looked
Table 2 e Comparison of macroscopic damage score means according to groups. Groups
N
Mean SD
Control saline
7
0.14 0.37
AA
7
7.29 0.75
Beva 2.5 saline
7
0.43 0.53
Beva 2.5 AA
7
2.86 0.69
Beva 5 saline
7
0.43 0.53
Beva 5 AA
7
2.29 1.11
Max ¼ maximum; Min ¼ minimum; SD ¼ standard deviation. Values in bold indicate statistical significance. * Statistically significant P < 0.001. y Control saline-Beva 2.5 AA. z Control saline-AA. x Beva 2.5 saline-AA. k Beva 5 saline-AA.
Median (minemax) 0 (0-1) 7 (6-8) 0 (0-1)
P value
