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Comparison of mitomycin-c and heparin affects in experimental corrosive esophagitis on rats
International Journal of Pediatric Otorhinolaryngology 75 (2011) 785–789
Contents lists available at ScienceDirect
International Journal of Pediatric Otorhinolaryngology journal homepage: www.elsevier.com/locate/ijporl
Comparison of mitomycin-c and heparin affects in experimental corrosive esophagitis on rats Ekrem Senturk a,*, Engin Pabuscu a, Serdar Sen a, Cengiz Unsal b a b
Adnan Menderes University, Medicine faculty, General Thoracic Surgery, Aydin, Turkey Adnan Menderes University, Veterinary Faculty, Physiology, Aydin, Turkey
A R T I C L E I N F O
A B S T R A C T
Article history: Received 3 February 2011 Received in revised form 3 March 2011 Accepted 6 March 2011 Available online 1 April 2011
Objective: We comparative effects of mitomycin-c and heparin which have different mechanism of action in a minimal invasive corrosive esophagitis model which was formed by NaOH 40%. Method: The study was performed on forty female Wistar albino rats; were divided into four equal groups each including ten animals. Group C (n = 10); control, the group that esophagus was washed with normal saline, group I (n = 10); injury group; alkali esophagus burn, not treated, group M (n = 10); alkali esophagus burn, mitomycin-c treatment group, group H (n = 10); alkali esophagus burn, heparin treatment group. The study was performed on a minimal invasive model which did not require general anesthesia and abdominal operation. In 28 day, all subjects were killed and their esophagus’s were removed by thoraco-abdominal cut. Total esophagi from oropharynx to stomach were removed and they were examined macroscopically and microscopically and evaluated for esophageal tissue collagen deposition and histopathologic damage score. Results: When group C is compared with each of the other groups, statistically significant weight losses were detected; [(p < 0.005, p < 0.05, p < 0.005), respectively]. Significant inflammation increase was detected in groups I, M and H in comparison to group C [(p < 0.001, p < 0, 0001, p < 0.005)]. When granulation scores of groups were compared; statistically significant granulation increases were detected in groups I, M, and H [(p < 0.05, p < 0.05, p < 0.05) compared to group C]. Significant collagen increase was detected in all 3 layers in groups; I, M and H according to group C [(p < 0.05, p < 0.05, p < 0.05)]. Collagen increase in every 3 layers in groups M and H were significantly less according to group I [(p < 0.05, p < 0.05, p < 0.05)]. Collagen increase in every 3 layers was less in group M than group H (p < 0.05). Conclusion: In corrosive esophagitis due to NaOH, heparin treatment is more effective in inflammation and granulation formation, mitomycin-c treatment is more effective in preventing the collagen accumulation step. Heparin decreases the tissue damage by preventing the inflammation and granulation formation; and prevents collagen accumulation and stricture development. As completing the effect of heparin; mitomycin prevents fibroblastic activity inhibition with direct collagen accumulation and stricture development strongly. ß 2011 Elsevier Ireland Ltd. All rights reserved.
Keywords: Experimental study Rat model Corrosive esophagitis Heparin Mitomycin-c
1. Introduction Corrosive esophagitis is a serious problem in different age groups. While ingestion occurs as an accidental exposure in children, adult exposure is mostly intentional, although it may also occur as an accident. In adults, corrosive esophagitis is usually seen in the 2nd and 3rd decades [1]. Corrosive esophagitis is a serious
* Corresponding author at: Adnan Menderes University, School of Medicine, Thoracic Surgery, 09010 Aydin, Turkey. Tel.: +90 256 4441256; fax: +90 256 2146495. E-mail address: [email protected] (E. Senturk). 0165-5876/$ – see front matter ß 2011 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.ijporl.2011.03.007
problem that occurs after accidental or suicidal ingestion of household bleaches, drain cleaners, sodium hydroxide, and hydrochloric acid. In a study conducted by Atug et al. it was found that lye at pHs < 11.5 had no damaging effects on the esophagus mucosa, whereas lye at pHs 1.5 caused liquefaction necrosis [2]. There still exists a huge debate on approach and treatment searches for corrosive esophagitis nowadays. Late term complications are seen as a common problem particularly in childhood. According to one study, among the people who ingested chemical agents in our country, 83.7% of males and 61% of females ingested them accidentally [3]. Today, the aim of therapy in corrosive esophagitis is to avoid development of perforation,
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fibrosis and stricture formation. Stricture formation can be prevented by suppressing fibrosis and scar formation [4]. Different agents are explored for treatment on various models in this subject. Today some chemical agents, dilatation, stent applications and surgical treatment options are used for treatment [5,6]. In experimental models, some agents such as vitamin C and E, heparin, mitomycin, caffeic acid phenethyl ester, epidermal growth factor, interferon g, sphingosylphosphorylcholine, penicillamine, estradiol and progesterone are found to be successful but a current protocol could not be formed yet [7–14]. Difficulties in application of the experimental agents, limitations in clinical usage and different mechanisms of action are the disadvantages of these models. Besides, mostly and only the injury and stricture located at the distal part of the esophagus is taken account. This situation limits the usage of chemical agents. But the effect of the treatment agents can be definitely demonstrated in a basic corrosive injury model targeting the whole esophagus pathologies arisen in chemical burns of esophagus differ according to anatomical structure, chemical factor and exposure amount and period. Dantas et al. [15]; specified in their study that motility of the esophagus was deteriorated in esophagus burns formed by sodium hydroxide and low amplitude non-peristaltic contractions were frequently observed. Motility disorders in chemical burns exhibit nonhomogenous pathologies ranging from stricture to dilatation in esophagus wall [6,15]. Therefore we planned for search the role of using different agents in the treatment of various non-homogenous pathologies. It is stated that in a minimally invasive corrosive esophagitis model a burn can be achieved throughout the esophagus by 40% NaOH and this model is suitable also for the treatment [16]. With this aim, we compared in our study in a minimally invasive model [16], the effect of mitomycin-c and heparin which have different mechanisms of action on corrosive esophagitis cases caused by 40% NaOH. 2. Material and method Ethical consent was obtained from Adnan Menderes University Experimental Animal Production and Research Laboratory Ethical Council. And the study was performed in Adnan Menderes University Experimental Animal Production and Research Laboratory. 2.1. Work groups The study was performed on forty female Wistar albino rats (average weight 280 60 g, average age 6 months, out-bred production). Rats which were kept in experimental animal production cages of which were closed with plastic on bottom and sides and with wire fence on top were fed by pellet type fabrication feed that is
[()TD$FIG]
produced specially for experimental animals. They were kept in day and night cycle and in room temperature (22 2 8C). Rats were separated into four equal groups each including ten. Group C (n = 10), which was the control group; esophagus was washed with normal saline. Group I (n = 10), which was the injury group; alkali esophagus burn, not treated. Group M (n = 10), which was the alkali esophagus burn, mitomycin-c treatment group. Group H (n = 10), which was the alkali esophagus burn, heparin treatment group. 2.2. Experimental model The study was performed on a minimal invasive model which did not require general anesthesia and abdominal operation [16]. The experiment was done under ether anesthesia and standard animal care with analgesia was provided. A Fogarty catheter inside a guide catheter which were forwarded from oropharynx to the stomach was used (Fig. 1a). Fogarty catheter was inflated inside the stomach so that esophago-gastric passage was closed. Caustic agents and treatment agents could easily be given through the guide catheter having the Fogarthy catheter inside. Also by adding a pressure gauge to the guide catheter, filling of the esophagus was taken into control and overflow and aspiration could be avoided. 0.3 ml of distilled water was given through the catheter to the control group (C) and 0.3 ml 40% NaOH was given to the mitomycin group, injury group and the heparin group from the second entry of the guide catheter and waited for 60 s. Then the fluid was aspirated and the guide catheter was washed with distilled water. After the process, Fogarty catheter balloon was deflated and it was removed together with the guide catheter. This process was done for each rat. After forming an esophagus burn, all subjects were catheterized by the same method. Again 0.3 ml of distilled water for control group, 0.3 ml of mitomycin-c for group M and 0.3 ml of heparin for group H was loaded to esophagus lumen of the subjects and waited for 60 s. After the process was terminated, subjects were not fed orally for 24 h, only water was allowed. In the following 48 h subjects were fed with enteral feeding solutions and they have been treated for pain. We used standard water bottles with a dropper filled with liquid feeding solutions and they were allowed to be fed spontaneously 2 days of the experiment. Then solid food was started to be given. Subjects were observed for 28 days. In the 28th day, all subjects were sacrificed and their esophagi were revealed by thoracoabdominal incision. Total esophagi from oropharynx to stomach were removed and they were examined macroscopically and microscopically. 2.3. Histopathological evaluation Two samples from upper and lower ends were taken from totally removed esophagi of every subject. Tissue samples were
Fig. 1. Rat model used for burn and treatment (a) and normal esophagus section microscopically (b).
E. Senturk et al. / International Journal of Pediatric Otorhinolaryngology 75 (2011) 785–789
fixed in 10% formaline for 24 h. After macroscopic examination, 5 mm sections of paraffin blocks of tissue samples were stained by Hematoxylin–eosin (H&E) and trichrome to evaluate connective tissue changes. The collagen increase by inflammation and granulation occurring in the esophagus was evaluated according to Table 1. Hystopathological evaluation was performed on 3 main groups specified in Table 1. Inflammation, granulation and collagen accumulation were evaluated by scoring.
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inflammation increase was detected in groups I, M and H in comparison to group C [(p < 0.001, p < 0, 0001, p < 0.005)]. The microscopic imaging of the corrosive esophagus segments are shown in Fig. 2a and b. Inflammation was significantly less in group H than group C (p < 0.05). Inflammation was found significantly less in animals treated with heparin than those treated with mitomycin-c (p < 0.05). 3.3. Granulation analysis
2.4. Statistical analysis All the statistical analysis were performed using SPSS statistical software for Windows (release 10.0, SPSS Inc. Chicago, IL, USA). A one-way analysis of variance (ANOVA) was performed to search any differences between the groups in terms of the weight loss. Depending on the homogeneity of the variances, the histological scores were compared between the groups using Kruskall–Wallis variance analysis and Mann–Whitney U-test for non-parametric data. Results were considered statistically significant at level p < 0.05. 3. Results In the study, a rat in the injury group died due to esophagus perforation in the 2nd day. Results are showed in Table 2. Weight 0 280 40/ 290 70/ 260 90/ 290 50/ (gr) day/28 day 330 70 240 80 230 50 280 70
When granulation scores of groups were compared; granulation indexes were found as [SD; I > M > H > C (0)]. Statistically significant granulation increases were detected in groups I, M, and H [(p < 0.05, p < 0.05, p < 0.05) compared to group C]. Statistically significant granulation decrease was detected in groups M and H according to group C (p < 0.05, p < 0.05) compared to group C. Granulation decrease was significantly less in group H than group M compared to group C; (p < 0.05). 3.4. Collagen accumulation analysis Collagen increase scores were found as [SD; I > H > M > C (0)]. Significant collagen increase was detected in all 3 layers in groups; I, M and H according to group C [(p < 0.05, p < 0.05, p < 0.05)]. Collagen increase in every 3 layers in groups M and H were significantly less according to group I [(p < 0.05, p < 0.05, p < 0.05)]. Collagen increase in every 3 layers was less in group M than group H (p < 0.05). The microscopic examination of the esophagi treated with mitomycin-c and heparin is shown in Fig. 3(a–d).
3.1. Weight analysis 4. Discussion In 28 day; group M, H, and I have loses of weight (group I; 50 gr, group H; 30 gr and group M; 10 gr.). But group c has not weight loses. When group C is compared with each of the other groups, statistically significant weight losses were detected [SD; I M > H > C(0)]. Weight losses in group M and H were less than the weight loss in the group I, which were statistically significant was detected [(p < 0.005, p < 0.05, p < 0.005) respectively]. Weight loss in group M and H was statistically less than the group I (p < 0.05, p < 0.05), respectively. When group M and H was compared, it was observed that weight loss in group H was significantly less than group M (p < 0.05) compared to group C. 3.2. Inflammation analysis When inflammation scores of groups were compared; inflammation indexes were found as [SD; I M > H > C (0)]. Significant
Lesions varying from minimal mucosal damage to full necrosis in esophagus wall and resulting with stricture may occur in corrosive esophagus burns [13,17]. After taking corrosive substance, change in superficial necrotic layer occurs 5–7 days after the damage and fibroblastic activity increases. After fibroblastic activity, collagen accumulation is observed in 7th to 28th days. Typical strictures may develop within 4–6 weeks [17,18]. After taking corrosive substance, in 5% of the patients an esophagus damage which will result as a stricture occurs [19]. Still there is no algorithm which is valid and accepted for the treatment of corrosive esophagitis. One important reason for not forming a standard treatment protocol may be the difficulty in treating of non-homogenous burn focuses occurring in different times. We compared affects of heparin and mitomycin-c which have different model of action in the treatment of experimental
Table 1 Histopathological evaluation. Criteria for histopathologic evaluation
Inflammation
Granulation
None Little Mild Marked None Little Mild Marked Submucosa
Increase of collagen deposition Muscularis mucosa Tunica muscularis
Score
None Mild (submucosal collagen at least twice the thickness of the muscularis mucosa) Marked (submucosal collagen more than twice the thickness of the muscularis mucosa) Damage to the Muscularis mucosa; none Damage to the muscularis Mucosa; present Damage and collagen deposition in the tunica Muscularis; none Mild (collagen deposition around the smooth muscle fibers) Marked (same as mild, with collagen deposition replacing some of the fibers)
0 1 2 3 0 1 2 3 0 1 2 0 1 0 1 2
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Table 2 Results for histopathologic evaluation and lose of weight. Groups
Weight (gr) Inflammation Granulation Collagen deposition SD
0 day/28 day SD SD Submucosal Muscularis Mucosa Tunica Muscularis
Control (C) (n = 10)
Injury (I) (n = 9)
Mitomycin (M) (n = 10)
Heparin (H) (n = 10)
280 40/330 70 0 0 0 0 0
290 70/240 80 2.4 0.3 1.9 0.3 1.7 0.2 1.5 0.3 1.6 0.5
260 90/230 50 2.5 0.5 1.7 0.4 0.8 0.4 0.9 0.5 1.0 0.4
290 50/280 70 2.1 0.4 1.0 0.3 1.1 0.3 1.2 0.4 1.2 0.5
[()TD$FIG]
Fig. 2. The sections of corrosive esophagitis with NaOH; dilatation with damage and collagen deposition in the tunica muscularis (a), stricture with damage and collagen deposition in the tunica muscularis (b).
esophagus burns that we have formed with NaOH. It should be remembered that previous experimental models are performed in the model of Gehanno et al. [11] or its modifications. These models which were invasive and focused on the lower end of the esophagus had technical faults and hystopathological deficiencies
[()TD$FIG]
as they did not include entire esophagus [20–22]. We hope that these problems are defeated in this noninvasive model. Measurement of the esophagus volume and constitution of corrosive esophagitis is controversial matter a lot of manuscripts. But it is not problem on our noninvasive model [16]. At the end of the first
Fig. 3. Esophageal section of a rat given mitomycin-c; minimal increase to collagen content (a), esophageal section of a rat given mitomycin-c; with middle treated mucosal structure (b), esophageal section of a rat given heparin; with middle treatment to collagen contents (c) and esophagus microscopy treated with the heparin; re-epitalization is attractively obvious (d).
E. Senturk et al. / International Journal of Pediatric Otorhinolaryngology 75 (2011) 785–789
week in experimental sodium hydroxide burn, esophagus wall is damaged due to various levels of necrosis and intensive inflammatory event. The inflammation may diffuse to the muscle tissue, peripheral tissue and mediastinum; granulation tissue replaces the necrotic tissue in 10th day; after three weeks most of muscle tissue is transformed into fibrous tissue; after fourth week contraction of the granulation tissue results with severe obstruction [23]. Holinger [23] classified esophagus burns as similar to skin burns. According to this classification: first degree burn: the burn is superficial. It is a superficial burn that includes mucosal hyperemia and edema. The second degree burn: there is an infiltration to the esophagus wall. Bulla, ulceration, exudation, mucosal loss and damage to muscularis layer are present. The third degree burn: erosion and perforation reaching up to peripheral tissue of esophagus are present. Agents being searched for the treatment affect in different phases of esophagus burns and in different strength. Encouraging results were taken in many studies for preventing obstructions occurred after corrosive esophagus burn. But it seems hard to treat non-homogenous pathologies which arises from NaOH by a single chemical agent. One of the drugs that we tried in the treatment is heparin. Heparin inhibits smooth muscle proliferation and prevents intimal hyperplasia besides anticoagulant feature. Interruption of the endothelial continuity due to vascular damage, thrombocyte adhesion, mitogenic factor release, medial smooth muscle cell excitation and proliferation and then migration to the intima are the steps for development of intimal hyperplasia. Kologlu et al. [7] declared in their study that heparin significantly decreases collagen storing between submucosa and muscle layers in animal models. Successful results were obtained in clinical and experimental researches related with mitomycin-c, an antimytotic agent which inhibits the fibroblastic activity [8,24]. But differences in severity and degree of burn set forth that one single chemical substance or surgical method cannot solve the problem. We observe in our study that heparin and mitomycin-c application are effective to prevent esophageal obstructions. But we saw that heparin is more effective on inflammation and granulation formation. We determined that effect of mitomycin-c is more than heparin in terms of preventing the collagen formation. Weight loss is less in heparin treatment group than mitomycin-c treatment group. We think that probably pain and obstruction is less in the acute phase in which inflammation and granulation formation decrease significantly by the effect of heparin. Therefore it may be thought that feeding might have been more effective. 5. Conclusion Consequently; in corrosive esophagitis due to NaOH, heparin treatment is effective more in inflammation and granulation formation, besides mitomycin-c treatment is more effective in preventing the collagen accumulation. Heparin decreases the tissue damage by preventing the inflammation and granulation formation; and avoids collagen accumulation and stricture development. As completing the effect of heparin; mitomycin-c strongly prevents direct collagen accumulation and stricture development by inhibiting fibroblastic activity. Alkaline esophageal injuries (for example batteries, accidental or suicidal
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drinking of alkaline fluids) can be treated by common use of heparin and mitomycin-c. We consider that results of this study will pave the way for use of agents which have curable effect with different mechanisms of action.
Conflicts of interest All authors declared to; there is no conflict of interests
References [1] A.L. De Jong, R. Macdonald, S. Ein, et al., Corrosive esophagitis in children: a 30 year review, Int. J. Pediatr. Otorhinolaryngol. 57 (3) (2001) 203–211. [2] O. Atug, A. Dobrucali, R.C. Orlando, Critical pH level of lye (NaOH) for esophageal injury, Dig. Dis. Sci. 54 (May (5)) (2009) 980–987. ¨. O ¨ zu¨temiz, Pet s¸is¸edeki tehlike: Koroziv o¨zefajit, Gu¨ncel Gastroenter[3] E. Kasap, O oloji 10 (2006) 29–35. [4] S. Demirbilek, F. Bernay, R. Rizalar, et al., Effects of estradiol and progesterone on the synthesis of collagen in corrosive esophageal burns in rats, J. Pediatr. Surg. 29 (1994) 1425–1428. [5] J.A. Fulton, R.S. Hoffman, Steroids in second degree caustic burns of the. esophagus: a systematic pooled analysis of fifty years of human data: 1956–2006, Clin. Toxicol. (Phila). 45 (May (4)) (2007) 4028. [6] N.K. Chokshi, Y.S. Guner, F. Ndiforchu, R. Mathis, et al., Combined laparoscopic and thoracoscopic esophagectomy and gastric pull up in a child, J. Laparoendosc. Adv. Surg. Tech. A 19 (1) (2009) 197–200. [7] M. Bingol Kologlu, F.C. Tanyel, S. Muftuoglu, et al., The preventive effect of heparin on stricture formation after caustic esophageal burns, J. Pediatr. Surg. 34 (2) (1999) 291–294. [8] M.K. Heran, R. Baird, G.K. Blair, Skarsgard, ED. Topical mitomycin-c for recalcitrant. esophageal strictures: a novel endoscopic/fluoroscopic technique for safe. endoluminal delivery, J. Pediatr. Surg. 43 (May (5)) (2008) 815–818. [9] U. Koltuksuz, H.M. Mutus, R. Kutlu, et al., Effects of caffeic acid phenethyl ester and epidermal growth factor on the development of caustic esophageal stricture in rats, J. Pediatr. Surg. 36 (10) (2001) 1504–1509. [10] B. Berthet, J. di Costanzo, C. Arnaud, et al., Influence of epidermal growth factor and interferon gamma on healing of esophageal corrosive burns in the rat, Br. J. Surg. 81 (3) (1994) 395–398. [11] P. Gehanno, C. Guedon, Inhibition of experimental esophageal lye strictures by penicillamine, Arch. Otolaryngol. 107 (1981) 145–147. [12] S. Demirbilek, F. Bernay, R. Rizalar, et al., Effects of estradiol and progesterone on the synthesis of collagen in corrosive esophageal burns in rats, J. Pediatr. Surg. 29 (11) (1994) 1425–1428. [13] A. Bautista, R. Tojo, R. Varela, et al., Effects of prednisolone and dexamethasone. on alkali burns of the esophagus in rabbit, J. Pediatr. Gastroenterol. Nutr. 22 (3) (1996) 275–283. [14] A. Yagmurlu, B. Aksu, M. Bingol Kologlu, et al., A novel approach for preventing esophageal stricture formation: sphingosylphosphorylcholine enhanced tissue remodeling, Pediatr. Surg. Int. (2004) 778–782, 20810. [15] R.O. Dantas, R.C. Mamede, Esophageal motility in patients with esophageal caustic injury, Am. J. Gastroenterol. 91 (6) (1996) 1157–1161. [16] E. Senturk, S. Sen, E. Pabuscu, C. Unsal, et al., New experimental corrosive esophagitis model in rats, Pediatr. Surg. Int. 26 (March (3)) (2010) 257–261. [17] E.M. Friedman, Caustic ingestions and foreign bodies in the aerodigestive tract of children, Pediatr. Clin. North Am. 36 (6) (1989) 1403–1410. [18] J.A. Haller Jr., H.G. Andrews, J.J. White, M.A. Tamer, et al., Pathophsysiology and management of acute corrosive burns of the esophagus, Am. Pediatr. Surg. 6 (5) (1971) 578–584. [19] R.W. Postleitwait, Chemical burns of the esophagus, Surg. Clin. North Am. 63 (4) (1983) 915–924. [20] A.J. Liu, A.M. Richardson, Effects of N-acetylcysteine on experimentally induced. esophageal lye injury, Ann. Rhinol. Laryngol. 94 (5 (Pt 1)) (1985) 477–482. [21] F. Yildiz, N. Zeybek, A. Terzi, S. Coban, Accurate esophageal volume of rats: an accurate Gateway to esophageal burn studies, Pediatr. Surg. Int. 24 (8) (2008) 971–972. [22] R. Hebel, M.W. Stromberg, Anatomy of the Laboratory Rat, The Williams& Wilkins Company, Baltimore, 1976, p. 4352. [23] L.D. Holinger, Caustic esophageal burns, in: J.G. Raffensperger (Ed.), Swenson’s Pediatric Surgery, 5th ed., Appleton & Lange, New York, 1990, p. 82731. [24] Z. Turkyilmaz, K. Sonmez, R. Karabulut, O. Gulbahar, et al., Mitomycin-c decreases the rate of stricture formation in caustic esophageal burns in rats, Surgery 145 (February (2)) (2009) 21925.
Contents lists available at ScienceDirect
International Journal of Pediatric Otorhinolaryngology journal homepage: www.elsevier.com/locate/ijporl
Comparison of mitomycin-c and heparin affects in experimental corrosive esophagitis on rats Ekrem Senturk a,*, Engin Pabuscu a, Serdar Sen a, Cengiz Unsal b a b
Adnan Menderes University, Medicine faculty, General Thoracic Surgery, Aydin, Turkey Adnan Menderes University, Veterinary Faculty, Physiology, Aydin, Turkey
A R T I C L E I N F O
A B S T R A C T
Article history: Received 3 February 2011 Received in revised form 3 March 2011 Accepted 6 March 2011 Available online 1 April 2011
Objective: We comparative effects of mitomycin-c and heparin which have different mechanism of action in a minimal invasive corrosive esophagitis model which was formed by NaOH 40%. Method: The study was performed on forty female Wistar albino rats; were divided into four equal groups each including ten animals. Group C (n = 10); control, the group that esophagus was washed with normal saline, group I (n = 10); injury group; alkali esophagus burn, not treated, group M (n = 10); alkali esophagus burn, mitomycin-c treatment group, group H (n = 10); alkali esophagus burn, heparin treatment group. The study was performed on a minimal invasive model which did not require general anesthesia and abdominal operation. In 28 day, all subjects were killed and their esophagus’s were removed by thoraco-abdominal cut. Total esophagi from oropharynx to stomach were removed and they were examined macroscopically and microscopically and evaluated for esophageal tissue collagen deposition and histopathologic damage score. Results: When group C is compared with each of the other groups, statistically significant weight losses were detected; [(p < 0.005, p < 0.05, p < 0.005), respectively]. Significant inflammation increase was detected in groups I, M and H in comparison to group C [(p < 0.001, p < 0, 0001, p < 0.005)]. When granulation scores of groups were compared; statistically significant granulation increases were detected in groups I, M, and H [(p < 0.05, p < 0.05, p < 0.05) compared to group C]. Significant collagen increase was detected in all 3 layers in groups; I, M and H according to group C [(p < 0.05, p < 0.05, p < 0.05)]. Collagen increase in every 3 layers in groups M and H were significantly less according to group I [(p < 0.05, p < 0.05, p < 0.05)]. Collagen increase in every 3 layers was less in group M than group H (p < 0.05). Conclusion: In corrosive esophagitis due to NaOH, heparin treatment is more effective in inflammation and granulation formation, mitomycin-c treatment is more effective in preventing the collagen accumulation step. Heparin decreases the tissue damage by preventing the inflammation and granulation formation; and prevents collagen accumulation and stricture development. As completing the effect of heparin; mitomycin prevents fibroblastic activity inhibition with direct collagen accumulation and stricture development strongly. ß 2011 Elsevier Ireland Ltd. All rights reserved.
Keywords: Experimental study Rat model Corrosive esophagitis Heparin Mitomycin-c
1. Introduction Corrosive esophagitis is a serious problem in different age groups. While ingestion occurs as an accidental exposure in children, adult exposure is mostly intentional, although it may also occur as an accident. In adults, corrosive esophagitis is usually seen in the 2nd and 3rd decades [1]. Corrosive esophagitis is a serious
* Corresponding author at: Adnan Menderes University, School of Medicine, Thoracic Surgery, 09010 Aydin, Turkey. Tel.: +90 256 4441256; fax: +90 256 2146495. E-mail address: [email protected] (E. Senturk). 0165-5876/$ – see front matter ß 2011 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.ijporl.2011.03.007
problem that occurs after accidental or suicidal ingestion of household bleaches, drain cleaners, sodium hydroxide, and hydrochloric acid. In a study conducted by Atug et al. it was found that lye at pHs < 11.5 had no damaging effects on the esophagus mucosa, whereas lye at pHs 1.5 caused liquefaction necrosis [2]. There still exists a huge debate on approach and treatment searches for corrosive esophagitis nowadays. Late term complications are seen as a common problem particularly in childhood. According to one study, among the people who ingested chemical agents in our country, 83.7% of males and 61% of females ingested them accidentally [3]. Today, the aim of therapy in corrosive esophagitis is to avoid development of perforation,
786
E. Senturk et al. / International Journal of Pediatric Otorhinolaryngology 75 (2011) 785–789
fibrosis and stricture formation. Stricture formation can be prevented by suppressing fibrosis and scar formation [4]. Different agents are explored for treatment on various models in this subject. Today some chemical agents, dilatation, stent applications and surgical treatment options are used for treatment [5,6]. In experimental models, some agents such as vitamin C and E, heparin, mitomycin, caffeic acid phenethyl ester, epidermal growth factor, interferon g, sphingosylphosphorylcholine, penicillamine, estradiol and progesterone are found to be successful but a current protocol could not be formed yet [7–14]. Difficulties in application of the experimental agents, limitations in clinical usage and different mechanisms of action are the disadvantages of these models. Besides, mostly and only the injury and stricture located at the distal part of the esophagus is taken account. This situation limits the usage of chemical agents. But the effect of the treatment agents can be definitely demonstrated in a basic corrosive injury model targeting the whole esophagus pathologies arisen in chemical burns of esophagus differ according to anatomical structure, chemical factor and exposure amount and period. Dantas et al. [15]; specified in their study that motility of the esophagus was deteriorated in esophagus burns formed by sodium hydroxide and low amplitude non-peristaltic contractions were frequently observed. Motility disorders in chemical burns exhibit nonhomogenous pathologies ranging from stricture to dilatation in esophagus wall [6,15]. Therefore we planned for search the role of using different agents in the treatment of various non-homogenous pathologies. It is stated that in a minimally invasive corrosive esophagitis model a burn can be achieved throughout the esophagus by 40% NaOH and this model is suitable also for the treatment [16]. With this aim, we compared in our study in a minimally invasive model [16], the effect of mitomycin-c and heparin which have different mechanisms of action on corrosive esophagitis cases caused by 40% NaOH. 2. Material and method Ethical consent was obtained from Adnan Menderes University Experimental Animal Production and Research Laboratory Ethical Council. And the study was performed in Adnan Menderes University Experimental Animal Production and Research Laboratory. 2.1. Work groups The study was performed on forty female Wistar albino rats (average weight 280 60 g, average age 6 months, out-bred production). Rats which were kept in experimental animal production cages of which were closed with plastic on bottom and sides and with wire fence on top were fed by pellet type fabrication feed that is
[()TD$FIG]
produced specially for experimental animals. They were kept in day and night cycle and in room temperature (22 2 8C). Rats were separated into four equal groups each including ten. Group C (n = 10), which was the control group; esophagus was washed with normal saline. Group I (n = 10), which was the injury group; alkali esophagus burn, not treated. Group M (n = 10), which was the alkali esophagus burn, mitomycin-c treatment group. Group H (n = 10), which was the alkali esophagus burn, heparin treatment group. 2.2. Experimental model The study was performed on a minimal invasive model which did not require general anesthesia and abdominal operation [16]. The experiment was done under ether anesthesia and standard animal care with analgesia was provided. A Fogarty catheter inside a guide catheter which were forwarded from oropharynx to the stomach was used (Fig. 1a). Fogarty catheter was inflated inside the stomach so that esophago-gastric passage was closed. Caustic agents and treatment agents could easily be given through the guide catheter having the Fogarthy catheter inside. Also by adding a pressure gauge to the guide catheter, filling of the esophagus was taken into control and overflow and aspiration could be avoided. 0.3 ml of distilled water was given through the catheter to the control group (C) and 0.3 ml 40% NaOH was given to the mitomycin group, injury group and the heparin group from the second entry of the guide catheter and waited for 60 s. Then the fluid was aspirated and the guide catheter was washed with distilled water. After the process, Fogarty catheter balloon was deflated and it was removed together with the guide catheter. This process was done for each rat. After forming an esophagus burn, all subjects were catheterized by the same method. Again 0.3 ml of distilled water for control group, 0.3 ml of mitomycin-c for group M and 0.3 ml of heparin for group H was loaded to esophagus lumen of the subjects and waited for 60 s. After the process was terminated, subjects were not fed orally for 24 h, only water was allowed. In the following 48 h subjects were fed with enteral feeding solutions and they have been treated for pain. We used standard water bottles with a dropper filled with liquid feeding solutions and they were allowed to be fed spontaneously 2 days of the experiment. Then solid food was started to be given. Subjects were observed for 28 days. In the 28th day, all subjects were sacrificed and their esophagi were revealed by thoracoabdominal incision. Total esophagi from oropharynx to stomach were removed and they were examined macroscopically and microscopically. 2.3. Histopathological evaluation Two samples from upper and lower ends were taken from totally removed esophagi of every subject. Tissue samples were
Fig. 1. Rat model used for burn and treatment (a) and normal esophagus section microscopically (b).
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fixed in 10% formaline for 24 h. After macroscopic examination, 5 mm sections of paraffin blocks of tissue samples were stained by Hematoxylin–eosin (H&E) and trichrome to evaluate connective tissue changes. The collagen increase by inflammation and granulation occurring in the esophagus was evaluated according to Table 1. Hystopathological evaluation was performed on 3 main groups specified in Table 1. Inflammation, granulation and collagen accumulation were evaluated by scoring.
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inflammation increase was detected in groups I, M and H in comparison to group C [(p < 0.001, p < 0, 0001, p < 0.005)]. The microscopic imaging of the corrosive esophagus segments are shown in Fig. 2a and b. Inflammation was significantly less in group H than group C (p < 0.05). Inflammation was found significantly less in animals treated with heparin than those treated with mitomycin-c (p < 0.05). 3.3. Granulation analysis
2.4. Statistical analysis All the statistical analysis were performed using SPSS statistical software for Windows (release 10.0, SPSS Inc. Chicago, IL, USA). A one-way analysis of variance (ANOVA) was performed to search any differences between the groups in terms of the weight loss. Depending on the homogeneity of the variances, the histological scores were compared between the groups using Kruskall–Wallis variance analysis and Mann–Whitney U-test for non-parametric data. Results were considered statistically significant at level p < 0.05. 3. Results In the study, a rat in the injury group died due to esophagus perforation in the 2nd day. Results are showed in Table 2. Weight 0 280 40/ 290 70/ 260 90/ 290 50/ (gr) day/28 day 330 70 240 80 230 50 280 70
When granulation scores of groups were compared; granulation indexes were found as [SD; I > M > H > C (0)]. Statistically significant granulation increases were detected in groups I, M, and H [(p < 0.05, p < 0.05, p < 0.05) compared to group C]. Statistically significant granulation decrease was detected in groups M and H according to group C (p < 0.05, p < 0.05) compared to group C. Granulation decrease was significantly less in group H than group M compared to group C; (p < 0.05). 3.4. Collagen accumulation analysis Collagen increase scores were found as [SD; I > H > M > C (0)]. Significant collagen increase was detected in all 3 layers in groups; I, M and H according to group C [(p < 0.05, p < 0.05, p < 0.05)]. Collagen increase in every 3 layers in groups M and H were significantly less according to group I [(p < 0.05, p < 0.05, p < 0.05)]. Collagen increase in every 3 layers was less in group M than group H (p < 0.05). The microscopic examination of the esophagi treated with mitomycin-c and heparin is shown in Fig. 3(a–d).
3.1. Weight analysis 4. Discussion In 28 day; group M, H, and I have loses of weight (group I; 50 gr, group H; 30 gr and group M; 10 gr.). But group c has not weight loses. When group C is compared with each of the other groups, statistically significant weight losses were detected [SD; I M > H > C(0)]. Weight losses in group M and H were less than the weight loss in the group I, which were statistically significant was detected [(p < 0.005, p < 0.05, p < 0.005) respectively]. Weight loss in group M and H was statistically less than the group I (p < 0.05, p < 0.05), respectively. When group M and H was compared, it was observed that weight loss in group H was significantly less than group M (p < 0.05) compared to group C. 3.2. Inflammation analysis When inflammation scores of groups were compared; inflammation indexes were found as [SD; I M > H > C (0)]. Significant
Lesions varying from minimal mucosal damage to full necrosis in esophagus wall and resulting with stricture may occur in corrosive esophagus burns [13,17]. After taking corrosive substance, change in superficial necrotic layer occurs 5–7 days after the damage and fibroblastic activity increases. After fibroblastic activity, collagen accumulation is observed in 7th to 28th days. Typical strictures may develop within 4–6 weeks [17,18]. After taking corrosive substance, in 5% of the patients an esophagus damage which will result as a stricture occurs [19]. Still there is no algorithm which is valid and accepted for the treatment of corrosive esophagitis. One important reason for not forming a standard treatment protocol may be the difficulty in treating of non-homogenous burn focuses occurring in different times. We compared affects of heparin and mitomycin-c which have different model of action in the treatment of experimental
Table 1 Histopathological evaluation. Criteria for histopathologic evaluation
Inflammation
Granulation
None Little Mild Marked None Little Mild Marked Submucosa
Increase of collagen deposition Muscularis mucosa Tunica muscularis
Score
None Mild (submucosal collagen at least twice the thickness of the muscularis mucosa) Marked (submucosal collagen more than twice the thickness of the muscularis mucosa) Damage to the Muscularis mucosa; none Damage to the muscularis Mucosa; present Damage and collagen deposition in the tunica Muscularis; none Mild (collagen deposition around the smooth muscle fibers) Marked (same as mild, with collagen deposition replacing some of the fibers)
0 1 2 3 0 1 2 3 0 1 2 0 1 0 1 2
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Table 2 Results for histopathologic evaluation and lose of weight. Groups
Weight (gr) Inflammation Granulation Collagen deposition SD
0 day/28 day SD SD Submucosal Muscularis Mucosa Tunica Muscularis
Control (C) (n = 10)
Injury (I) (n = 9)
Mitomycin (M) (n = 10)
Heparin (H) (n = 10)
280 40/330 70 0 0 0 0 0
290 70/240 80 2.4 0.3 1.9 0.3 1.7 0.2 1.5 0.3 1.6 0.5
260 90/230 50 2.5 0.5 1.7 0.4 0.8 0.4 0.9 0.5 1.0 0.4
290 50/280 70 2.1 0.4 1.0 0.3 1.1 0.3 1.2 0.4 1.2 0.5
[()TD$FIG]
Fig. 2. The sections of corrosive esophagitis with NaOH; dilatation with damage and collagen deposition in the tunica muscularis (a), stricture with damage and collagen deposition in the tunica muscularis (b).
esophagus burns that we have formed with NaOH. It should be remembered that previous experimental models are performed in the model of Gehanno et al. [11] or its modifications. These models which were invasive and focused on the lower end of the esophagus had technical faults and hystopathological deficiencies
[()TD$FIG]
as they did not include entire esophagus [20–22]. We hope that these problems are defeated in this noninvasive model. Measurement of the esophagus volume and constitution of corrosive esophagitis is controversial matter a lot of manuscripts. But it is not problem on our noninvasive model [16]. At the end of the first
Fig. 3. Esophageal section of a rat given mitomycin-c; minimal increase to collagen content (a), esophageal section of a rat given mitomycin-c; with middle treated mucosal structure (b), esophageal section of a rat given heparin; with middle treatment to collagen contents (c) and esophagus microscopy treated with the heparin; re-epitalization is attractively obvious (d).
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week in experimental sodium hydroxide burn, esophagus wall is damaged due to various levels of necrosis and intensive inflammatory event. The inflammation may diffuse to the muscle tissue, peripheral tissue and mediastinum; granulation tissue replaces the necrotic tissue in 10th day; after three weeks most of muscle tissue is transformed into fibrous tissue; after fourth week contraction of the granulation tissue results with severe obstruction [23]. Holinger [23] classified esophagus burns as similar to skin burns. According to this classification: first degree burn: the burn is superficial. It is a superficial burn that includes mucosal hyperemia and edema. The second degree burn: there is an infiltration to the esophagus wall. Bulla, ulceration, exudation, mucosal loss and damage to muscularis layer are present. The third degree burn: erosion and perforation reaching up to peripheral tissue of esophagus are present. Agents being searched for the treatment affect in different phases of esophagus burns and in different strength. Encouraging results were taken in many studies for preventing obstructions occurred after corrosive esophagus burn. But it seems hard to treat non-homogenous pathologies which arises from NaOH by a single chemical agent. One of the drugs that we tried in the treatment is heparin. Heparin inhibits smooth muscle proliferation and prevents intimal hyperplasia besides anticoagulant feature. Interruption of the endothelial continuity due to vascular damage, thrombocyte adhesion, mitogenic factor release, medial smooth muscle cell excitation and proliferation and then migration to the intima are the steps for development of intimal hyperplasia. Kologlu et al. [7] declared in their study that heparin significantly decreases collagen storing between submucosa and muscle layers in animal models. Successful results were obtained in clinical and experimental researches related with mitomycin-c, an antimytotic agent which inhibits the fibroblastic activity [8,24]. But differences in severity and degree of burn set forth that one single chemical substance or surgical method cannot solve the problem. We observe in our study that heparin and mitomycin-c application are effective to prevent esophageal obstructions. But we saw that heparin is more effective on inflammation and granulation formation. We determined that effect of mitomycin-c is more than heparin in terms of preventing the collagen formation. Weight loss is less in heparin treatment group than mitomycin-c treatment group. We think that probably pain and obstruction is less in the acute phase in which inflammation and granulation formation decrease significantly by the effect of heparin. Therefore it may be thought that feeding might have been more effective. 5. Conclusion Consequently; in corrosive esophagitis due to NaOH, heparin treatment is effective more in inflammation and granulation formation, besides mitomycin-c treatment is more effective in preventing the collagen accumulation. Heparin decreases the tissue damage by preventing the inflammation and granulation formation; and avoids collagen accumulation and stricture development. As completing the effect of heparin; mitomycin-c strongly prevents direct collagen accumulation and stricture development by inhibiting fibroblastic activity. Alkaline esophageal injuries (for example batteries, accidental or suicidal
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drinking of alkaline fluids) can be treated by common use of heparin and mitomycin-c. We consider that results of this study will pave the way for use of agents which have curable effect with different mechanisms of action.
Conflicts of interest All authors declared to; there is no conflict of interests
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