Abstract

Background: This study aims to investigate the effects of an immunostimulant, beta glucan, on the bronchial anastomosis in rabbits that received chemotherapy in preoperative period.

Methods: Forty-five New Zealand male rabbits were used in this study. The rabbits were divided into three groups as group 1 (control), group 2 (chemotherapy), and group 3 (chemotherapy + beta glucan) with 15 rabbits in each group. The left main bronchus was incised completely and then endto- end anastomosis was performed in all groups. Cisplatin was administered on the preoperative first day and etoposide was administered on the preoperative first, second, and third days in group 2. In group 3, in addition to the same chemotherapy protocol, 10 mg/kg/day beta glucan was administered via enteral route starting from seven days before operation until one day before sacrifications. The rabbits were sacrificed at postoperative third, fifth, and seventh days by groups of five. The bronchial anastomosis lines were removed and tissue sections were stained with hematoxylin and eosin.

Results: In pathological analysis, while there were statistically significant differences between group 1 and group 2 and between group 2 and group 3, there was no difference between group 1 and group 3 in terms of bronchial healing.

Conclusion: The use of beta glucan during chemotherapy treatment may improve bronchial healing at postoperative period.

Lung resections have been performed in many diseases, especially in lung cancers for many years. Although these resections can be performed easily in patients who have sufficient pulmonary capacity, parenchyma-saving surgery such as sleeve lobectomy and bronchial sleeve resection come to the agenda in patients with limited lung capacity. Some patients with limited lung capacity are operated after neoadjuvant chemotherapy. However, after the postoperative period, bronchitis may be aggravated due to chemotherapeutic drug since these agents may impair the wound healing in bronchial stump and anastomosis line.[1] This condition may cause bronchopleural or bronchoarterial fistula, prolonged air leak, empyema, and fatal bleeding at postoperative period.[2] Increased bronchial complication rate after neoadjuvant therapy was showed in the literature.[3] Although several techniques supporting the bronchial stump and anastomosis have been described to prevent this complication, a non-surgical technique has not been proposed yet.[4] Therefore, in this experimental study, we aimed to investigate the effects of an immunostimulant, beta glucan, on the bronchial anastomosis in rabbits that received chemotherapy in preoperative period.

Methods

This study was carried out in the Experimental Medicine and Application Center of the Selcuk University between July 2006 and January 2008. Pathology specimens were examined in the Department of Pathology, Meram Medical Faculty, Selcuk University. The study protocol was approved by Selcuk University, Experimental Medicine and Application Center, Laboratory Animals Ethical Board (Approval No: 12.05.2006/22).

In the study, 45 male New Zealand albino rabbits with a mean weight of 302.2±419.75 g were used. These animals were preferred since we would be able to see the bronchial system with more detail and perform the surgical procedure more easily. During the study, ethic principles were respected.

The rabbits were divided into three groups as group 1 (control group), group 2 (chemotherapy group), and group 3 (chemotherapy plus beta glucan group) with 15 rabbits in each group. In addition, each group had three subgroups including five rabbits, which were adjusted according to sacrification periods. All rabbits were fed by standard rabbit diet.

No drug was given to group 1 during pre- and postoperative periods. At the operation day, ketamine 35 mg/kg and xylazine 5 mg/kg were administered intramuscularly, surgical area was shaved and cleaned by povidone-iodine. For prophylaxis, cephazoline sodium 25 mg/kg was given intravenously before surgery. Thereafter, left thoracotomy was performed (Figure 1a). After the left main bronchus was found (Figure 1b), complete incision was performed (Figure 1c). Then, cartilaginous and membranous parts were continuously sutured by 5-0 polypropylene suture (Figure 1d). Finally, thorax was closed and pneumothorax was drained by thoracentesis. After completion of the surgical intervention, same dose cephazoline was given again for prophylaxis and additionally, tramadol 1 mg/kg was administered intravenously for pain control. In the postoperative period, each subgroup was sacrificed at third, fifth, and seventh days, respectively. After the sacrification, anastomosis lines were removed for pathological examination.

Figure 1: (a) Skin incision for left thoracotomy to rabbits lied on their right side. (b) Left main bronchus. (c) Complete incision of left main bronchus. (d) Appearance after left main bronchus anastomosis.

Rabbits in group 2 were given cisplatin 4 mg/kg at preoperative first day and etoposide 4.8 mg/kg at preoperative first, second, and third days through ear vein. Dose of the chemotherapeutics was the same with the dose administered to the rats in the experimental study performed by Inoue et al.[5] Surgical procedure, anesthesia, prophylaxis, and analgesia were identical with group 1. Sacrifications were done at postoperative third, fifth, and seventh days and anastomosis lines were removed for pathological examination.

The same chemotherapy protocol was given to group 3. Additionally, beta glucan 10 mg/kg was enterally given through an orogastric tube starting from seven days before operation until one day before sacrifications. Surgical procedure, anesthesia, prophylaxis, and analgesia were the same with the others groups. Sacrifications were done at postoperative third, fifth, and seventh days and anastomosis lines were removed.

Anastomosis lines were fixed by 10% formaldehyde. The sections were stained with hematoxylin and eosin. Thereafter, the slides were examined using Couraud scoring (Table 1).[6] Grade 1 was considered as five, grade 2a as four, grade 2b as three, grade 3a as two, and grade 3b as one to perform statistical analysis (Figure 2a-d).

Figure 2: (a) Grade 1, complete epithelization and healing (H-E x 40). (b) Grade 2a, near-complete wound healing with epithelization in patches and less inflammatory cell (H-E x 40). (c) Grade 2b, incomplete wound healing without epithelization (H-E x 40). (d) Grade 3a, necrosis on endobronchial surface and mild healing findings (H-E x 40).

Statistical analysis
Statistical Package for the Social Sciences version 13.0 for Windows software program (SPSS Inc., Chicago, IL, USA) was used for statistical analysis. Descriptive statistics were shown in Tables 1, 2, and 3. Groups were compared using Kruskal-Wallis variance analysis and a p value of <0.05 was considered as significance level. Pairwise comparisons were done using Mann-Whitney U test with Bonferroni correction and a p value of <0.05 was considered as significance level.

Table 1: Grading system for bronchial healing[6]

Table 2: Pathological scores of the groups

Table 3: Pairwise comparison of the pathological scoring values between the groups

Results

At postoperative third day, two animals in group 2 developed limited necrosis (Figure 2d), whereas none of the animals in group 1 and group 3 had necrosis. Although we detected better healing in groups 1 and 3, a statistically significant difference was not found (p>0.05). Complete and near-complete epithelization and healing were seen in group 1 and group 3 (Figure 2a, b).

At postoperative fifth day, one animal in group 2 developed limited necrosis, whereas necrosis was not observed in other groups. Incomplete wound healing without epithelization was detected in all groups (Figure 2c). An evaluation at postoperative seventh day revealed that three animals in group 1 and one animal in group 3 healed completely (Figure 2a). In group 2, complete recovery was not observed in any of the postoperative periods.

Minimum, maximum, and median values of the pathological scores of groups obtained at postoperative third, fifth, and seventh days are shown in Table 2. When all groups were collectively evaluated, wound healing was significant at postoperative seventh day (p<0.05). When the groups were dichotomously evaluated, no statistically significant difference was detected between the groups at postoperative third and fifth days (p>0.05). An evaluation at postoperative seventh day showed a significant difference between group 1 and group 2 in terms of bronchial healing. While there was no statistically significant difference between group 1 and group 3 (p>0.05), there was a statistically significant difference between group 2 and group 3 (p<0.05), (Table 3).

Discussion

Thanks to recent technological developments and increasing knowledge, thoracic surgery has been performed more frequently. Meanwhile, lung cancer is increasingly diagnosed due to increased incidence of smoking and environmental exposure. Surgery is still the most widely accepted method for the treatment of operable lung cancer. Chemotherapy, radiotherapy, and supportive therapy are commonly used in the treatment of lung cancer at advanced stages. However, the toxicity of chemotherapy on the tissues remains to be an important problem and data about the effect of the chemotherapy on post-surgical bronchial healing have not been clearly obtained yet.

Although the effect of acute radiotherapy on bronchial anastomosis has been experimentally and clinically demonstrated, there are limited numbers of studies about the effect of the preoperative chemotherapy on postsurgical tracheobronchial wound healing.[7,8] Inoue et al.[5] demonstrated that preoperative chemotherapy impaired bronchial healing in their experimental study.

This condition may be linked with subgroups of leukocytes, which are peripheral blood cells, including neutrophils, monocytes, lymphocytes, eosinophils, and basophils. Macrophages originate from the monocytes. Etoposide and cisplatin may impair the bronchial healing by causing leukopenia and macrophage deficiency in the healing tissue.[9] In our study, we found that group 2 had a significantly impaired bronchial wound healing.

Beta glucan is obtained from the cell wall of Saccharomyces cerevisiae, commonly known as Baker’s yeast. This agent ensures macrophage activation and accelerates wound healing.[10-12] Nevertheless, to our knowledge, there is no study in the literature that investigated the effect of beta glucan on bronchial healing. We investigated the effect of beta glucan on bronchial healing since neoadjuvant chemoradiotherapy, which may increase bronchial complication rates, is considered in patients with lung cancer.

In another experimental study, Breivik et al.[13] demonstrated that soluble beta 1.3/1.6 glucan may prevent periodontal disease. In their study, the ligation of the neck of the second right maxillar molar tooth by sterile silk resulted in the retention of oral microorganisms. This protective effect of beta glucan was attributed to the activation of the immune system and they reported that the group treated with beta glucan had higher level of interleukin 10 compared to control group. Furthermore, beta glucan can increase the macrophage count in the early stage of wound healing and accelerate wound healing in diabetic mice.[11] Oral and systemic intake of beta glucan has a protective effect against lipopolysaccharide-related shock and organ damage in rats.[14]

Similarly with previous experimental animal models, we also performed the sacrifications at postoperative third, fifth, and seventh days and consistently to the reported time interval. Histopathological evaluation was performed using the Couraud scoring system, considering the mucosal healing grade and presence of necrosis (Table 2). At postoperative third day, two animals in group 2 developed limited necrosis, whereas none of the animals in group 1 and in group 3 had necrosis. Although bronchial healing was better in these groups, no statistically significant difference was detected (p>0.05).

At postoperative day 5, one animal in the group 2 showed limited necrosis, whereas necrosis was not observed in other groups. In the evaluation done at postoperative day 7, three animals in group 1 and one animal in the group 3 showed complete healing. In group 2, complete recovery was observed in none of the postoperative periods. When all groups were collectively evaluated, wound healing was significant at postoperative day 7 (p<0.05).

When the groups were dichotomously evaluated, there was not statistically significant difference between the groups at postoperative days 3 and 5 (p>0.05). In the evaluation done at postoperative day 7, there was significant difference between group 1 and group 2 in bronchial healing. While there was no statistically significant difference between group 1 and group 3 (p>0.05), there was a statistically significant difference between the group 2 and group 3 (p<0.05). The facts that necrosis was observed only in group 2 and complete healing was shown in group 1 and group 3 were the strongest findings indicating that chemotherapy histopathologically impairs bronchial healing and beta glucan has a favorable effect on wound recovery.

Similarly, Shirafuji et al.,[9] obtained the same results for chemotherapy. According to the researchers, chemotherapy impaired bronchial healing and this risk may be reduced by increasing the interval between chemotherapy and surgical intervention. Wound healing is impaired in patients with cancer due to the disposition of the disease and chemotherapeutics used, especially by suppressing the inflammatory response.[15,16]

Many favorable results have been reported for beta glucan. It has been used in combination with chemotherapy for the treatment of cancer for its immunostimulant effect. Beta glucan is efficient in the treatment of lymph node metastasis of ovarian cancer and advanced breast cancer as it activates peripheral blood monocytes and stimulates the proliferation.[17,18] Furthermore, beta glucan can improve the survival in non-resectable or recurrent gastric cancers.[19,20]

In conclusion, based on the results we obtained in experimental complete bronchial section similar to sleeve bronchial resection, we concluded that preoperative chemotherapy impairs bronchial healing. On the other hand, concomitant use of chemotherapy and beta-glucan led to statistically significantly improved healing parameters. However, due to the lack of previous studies that investigated the effect of beta glucan on bronchial healing, we believe that our results should be supported by further comprehensive studies with larger sample size.

Acknowledgments
We thank to veterinary surgeon Mehmet Oz from Selcuk University, Experimental Medicine Research Center, and to Mehmet Uyar, MD, from Selcuk University, Meram Medical Faculty, Department of Public Health and Biostatistics, for their contributions to this study.

Declaration of conflicting interests
The authors declared no conflicts of interest with respect to the authorship and/or publication of this article.

Funding
The authors received no financial support for the research and/or authorship of this article.

References

1) Rea F, Marulli G, Schiavon M, Zuin A, Hamad AM, Rizzardi G, et al. A quarter of a century experience with sleeve lobectomy for non-small cell lung cancer. Eur J Cardiothorac Surg 2008;34:488-92.

2) Gómez-Caro A, Boada M, Reguart N, Viñolas N, Casas F, Molins L. Sleeve lobectomy after induction chemoradiotherapy. Eur J Cardiothorac Surg 2012;41:1052-8.

3) Ludwig C, Engel-Riedel W, Stoelben E. Morbidity and mortality after neoadjuvant therapy and sleeve lobectomyin N2-disease. Zhongguo Fei Ai Za Zhi 2008;11:668-71.

4) Yamamoto R, Tada H, Kishi A, Tojo T. Effects of preoperative chemotherapy and radiation therapy on human bronchial blood flow. J Thorac Cardiovasc Surg 2000;119:939-45.

5) Inoue M, Oka T, Shima Y, Shirafuji T, Sumida Y, Yamashita H, et al. Preoperative irradiation combined with chemotherapy impairs healing of bronchial anastomosis during the early postoperative period in rats. Tohoku J Exp Med 2003;199:1-12.

6) Couraud L, Nashef SA, Nicolini P, Jougon J. Classification of airway anastomotic healing. Eur J Cardiothorac Surg 1992;6:496-7.

7) Tsubota N, Simpson WJ, Van Nostrand AW, Pearson FG. The effects of preoperative irradiation on primary tracheal anastomosis. Ann Thorac Surg 1975;20:152-60.

8) Gonzalez M, Litzistorf Y, Krueger T, Popeskou SG, Matzinger O, Ris HB, et al. Impact of induction therapy on airway complications after sleeve lobectomy for lung cancer. Ann Thorac Surg 2013;96:247-52.

9) Shirafuji T, Oka T, Sawada T, Tamura K, Kishimoto K, Yamamoto S, et al. The importance of peripheral blood leukocytes and macrophage infiltration on bronchial wall wound healing in rats treated preoperatively with anticancer agents. Surg Today 2001;31:308-16.

10) Rice PJ, Adams EL, Ozment-Skelton T, Gonzalez AJ, Goldman M P, L ockhart B E, e t a l. O ral d elivery a nd gastrointestinal absorption of soluble glucans stimulate increased resistance to infectious challenge. J Pharmacol Exp Ther 2005;314:1079-86.

11) Berdal M, Appelbom HI, Eikrem JH, Lund A, Zykova S, Busund LT, et al. Aminated beta-1,3-D-glucan improves wound healing in diabetic db/db mice. Wound Repair Regen 2007;15:825-32.

12) Zykova SN, Jenssen TG, Berdal M, Olsen R, Myklebust R, Seljelid R. Altered cytokine and nitric oxide secretion in vitro by macrophages from diabetic type II-like db/db mice. Diabetes 2000;49:1451-8.

13) Breivik T, Opstad PK, Engstad R, Gundersen G, Gjermo P, Preus H. Soluble beta-1,3/1,6-glucan from yeast inhibits experimental periodontal disease in Wistar rats. J Clin Periodontol 2005;32:347-52.

14) Sandvik A, Wang YY, Morton HC, Aasen AO, Wang JE, Johansen FE. Oral and systemic administration of betaglucan protects against lipopolysaccharide-induced shock and organ injury in rats. Clin Exp Immunol 2007;148:168-77.

15) Payne WG, Naidu DK, Wheeler CK, Barkoe D, Mentis M, Salas RE, et al. Wound healing in patients with cancer. Eplasty 2008;8:9. 16- Ekmekci P, Bostancı S. Yara iyileşmesi. T Klin Dermatoloji 2002,12:114-20

17) Fujimoto K, Tomonaga M, Goto S. A case of recurrent ovarian cancer successfully treated with adoptive immunotherapy and lentinan. Anticancer Res 2006;26:4015-8.

18) Demir G, Klein HO, Mandel-Molinas N, Tuzuner N. Beta glucan induces proliferation and activation of monocytes in peripheral blood of patients with advanced breast cancer. Int Immunopharmacol 2007;7:113-6.

19) Nimura H, Mitsumori N, Takahashi N, Kashimura H, Takayama S, Kashiwagi H, et al. S-1 combined with lentinan in patients with unresectable or recurrent gastric cancer. Gan To Kagaku Ryoho 2006;33:106-9. [Abstract]

20) Hamuro J. Anticancer immunotherapy with perorally effective lentinan. Gan To Kagaku Ryoho 2005;32:1209-15. [Abstract]