Research progress and comparison of the establishment of animal models of radiotherapy or chemotherapy-induced oral mucositis

Chen Chen, Ran Yu, Dong-Mei Chen, Chao Deng, Yan-Ni Lou, Li-Qun Jia✉

1.Clinical Medical College of China-Japan Friendship Hospital, Beijing University of Chinese Medicine, Beijing 100029, China

2. China-Japan Friendship Hospital, Beijing 100029, China

Keywords:Radiotherapy Chemotherapy Oral mucositis Animal model Research progress

ABSTRACT The mechanism of radiotherapy or chemotherapy-induced oral mucositis is not yet clear.And model establishment is needed in further study.In order to summarize the methods of model establishment and make a comparison,literature databases including Web of Science、Pubmed and CNKI were searched for related researches from January 2015 to January 2021. Hamsters,mice, rats,guinea pigs and miniature pigs were chosen to be modeling animals and modeling methods could be classified into: chemotherapy, chemotherapy combined with superficial mucosal irritation, radiotherapy, radiotherapy combined with superficial mucosal irritation and chemoradiotherapy. Advantages and disadvantages had been analyzed in this study to provide reference for following studies.

Oral mucositis (OM) is one of the common adverse reactions of radiotherapy and chemotherapy. The main manifestations of the disease are erythema, ulcers and pain of oral mucosa, which may lead to decreased intake and weight loss of patients [1]. OM significantly reduced the quality of life of patients and affected the progress of cancer treatment [2]. Also, it may cause secondary infection, which may be life-threatening. Some data show that more than 80% of patients with head and neck tumor undergoing radiotherapy will have OM [3]. More than 90% of patients receiving concurrent chemoradiotherapy will have OM [4]. At present, there is no recommended prevention or treatment methods based on evidence based medicine for OM related to radiotherapy and chemotherapy. The clinical practice mainly focuses on relieving pain and discomfort [5]. Therefore, it is an urgent problem to find some biology based control methods. In order to find its pathogenesis and prevention methods, it is necessary to build animal models. In this article, we searched the relevant literature in recent 6 years, and then we introduced and compared animal selection and modeling methods, in order to provide reference for further research.

1. Selection and comparison of model animals

The model animals included golden hamster, mouse, rat, guinea pig and miniature pig. Among them, golden hamster, mouse and rat are widely used. The advantages of rodent experimental animals are low cost, easy operation, and easy to carry out large sample experiments. Rodent models are used in most of the related studies,which have been verified more. The disadvantages are as follows:1. The oral structure of rodents and their response to radiation are quite different from that of human. 2. Due to the small size of rodents, it is difficult to completely shield the brain and other important organs when exposed to radiation, and the influence of radiation on important organs is easy to cause death. At the same time, it is difficult to observe the oral mucosa due to its small size,and it is difficult to obtain enough tissue samples. 3. It is difficult to dynamically monitor the changes of blood indexes of rodents [6]. The advantage of miniature pig is that its oral tissue structure is similar to human, and its response to radiation is closer to human. Because of its large size, it is easy to limit the radiation area, collect blood samples for many times, observe oral mucosa and collect tissues for detection. However, due to the high cost, it is difficult to carry out large sample.

Among rodents, golden hamster has the advantages of large buccal pouch, which is easy to operate and observe the oral mucosa. The disadvantages are as follows: 1. The structure of cheek pouch is special, which lack of histocompatibility antigen so it does not have typical representation. 2. The animal lines are not comprehensive and cannot meet the needs of certain strains. The advantages of rats and mice are rich in strains, and the disadvantages are that it is difficult to observe and operate on their oral mucosa. Guinea pigs are at high price, incomplete strains, and the application of research is less.

2. Selection of modeling methods

2.1 Chemotherapy model

2.1.1 Model of chemotherapy combined with mucosa stimulation

2.1.1.1 Model of chemotherapy combined with mechanical stimulation

The model of chemotherapy combined with mechanical stimulation is widely used. In this model, most of the animals are golden hamsters. The chemotherapy method was intraperitoneal injection of 5-fluorouracil (5-FU). 5-FU is widely used in clinical treatment of colon cancer, breast cancer and gastric cancer. The method was originally proposed by Sonis[7].In his study, golden hamsters were injected with 5-FU intraperitoneally at the dose of 60mg/kg in day0, day5 and day10. The study indicated that this dosage ensured the establishment of the model, with the lowest incidence rate of systemic diseases and lowest mortality rate. Besides, The buccal pouch mucosa was stimulated by horizontal superficial scraping with 18 needle twice until the erythema appeared in the buccal pouch.The control group only received the same mechanical stimulation for 3 consecutive days. After injection of 5-FU on the 10th day, the experimental group showed more serious mucositis. The histological characteristics were epithelial destruction and surface necrosis, and the mucosa healed on the 16th day. In the control group, only local mucosal injury and local abscess appeared after mechanical injury,which healed spontaneously within 24 to 48 hours.

Since then, many studies have improved animal selection or model construction by using this method of chemotherapy combined with mechanical stimulation. For example, Lee [8] used golden hamsters and BALB / c mice. The two kinds of animals were divided into chemotherapy + mechanical stimulation group and chemotherapy group, and the differences between the two groups were compared.The mechanical stimulation site of golden hamster was buccal pouch mucosa, and that of mouse was tongue mucosa. The results showed that compared with chemotherapy group, 5-FU + mechanical stimulation group showed severe oral ulcer, OM score increased significantly, and body weight decreased significantly (P ＜ 0.01).The results showed that the effect of 5-FU + mechanical stimulation was better than that of 5-FU alone. Isman [9] used Wistar rats. In the study, rats were injected intraperitoneally with 5-FU of 100 mg/ kg and 65 mg / kg on the first and third day of the experiment.And on the third and fifth day, the left cheek pouch mucosa was stimulated with an 18 needle tip. Antunes [10] and Barbosa [11] and Barbosa [12] and Vilar [13] referred to the modeling method of Sonis[14],in their studies, Golden hamsters were injected intraperitoneally with 5-FU at the doses of 60 mg / kg and 40 mg / kg on the first and second days ,and on the fourth day, the mucosa was mechanically stimulated. The results show that the peak of inflammation appeared on the tenth day of the experiment. The other modeling methods included injecting 60 mg/ kg 5-FU intraperitoneally on day 0, 2 and 4, and mechanical stimulation on day 1, 2 and 4.[15] Because of the special buccal pouch structure of golden hamsters, it is most used as the experimental animal in the model of chemotherapy combined with mechanical stimulation.

2.1.1.2 Model of chemotherapy combined with chemical stimulation

The main chemical stimulation was local stimulation of oral mucosa with high concentration of acetic acid. This method was first put forward by Japanese scholar Kenji in 1998. He used acetic acid to produce ulcers in rabbit oral mucosa in his study. After gradual improvement, it was combined with chemotherapy to establish the animal model of chemotherapy-induced OM. Yosuke

[16] selected ICR mice to establish the model and explored the modeling method. The mice was divided into 5-FU group and 5-FU+ acetic acid group. In the 5-FU group, no oral ulcer was found after intraperitoneal injection of corresponding dose of 5-FU on day -5, -3, - 1 of the experiment. The 60 mg / kg group had higher mortality.For the 5-FU + acetic acid group, on the basis of 5-FU, different doses of 20% acetic acid were injected into the buccal mucosa on day 0, and oral ulcer was observed. In this study, 50mg/kg dose of 5-FU combined with 15µl 20% acetic acid injection was proved to be the best. Takeuchi [17] also carried out dose comparison. Wistar rats were injected intraperitoneally with 5-FU 5 days, 3 days and 1 day before the experiment, and 25µl different concentrations of acetic acid solution were injected into the right cheek pouch on day0 of the experiment. At last, the dosage of 40 mg / kg of 5-FU and the concentration of 25% of acetic acid solution was determined. In addition, Braz [18] and Lima [19] used the method of intraperitoneal injection of 5-FU combined with filter paper soaked in concentrated acetic acid sticking to oral mucosa in Wistar rats, which can also effectively cause oral ulcers.

2.1.2 Chemotherapy alone model

2.1.2.1 Intraperitoneal chemotherapy

Intraperitoneal chemotherapy alone can only induce OM, but not oral ulcers. Ahmed [20] used Wistar rats and established the model by intraperitoneal injection of single dose of methotrexate (20 mg/kg).4 days after injection, the rats were sacrificed. The results showed that the injection of methotrexate could induce OM, and the histological characteristics were consistent with the initial inflammatory/vascular phase, but could not induce oral ulcers. In the study of azri [21], Agouti rats were intraperitoneally injected with a single dose of irinotecan (200mg / kg) and killed within 72 hours.The results showed that the thickness of oral mucosa epithelium reduced, but no ulcers were found. The explanation of this study was that the histology of oral mucosa of rats is different from that of human. However, due to the short observation time, the peak of OM inflammation may have not yet reached, so that the conclusion is not rigorous enough.

2.1.2.2 Intravenous chemotherapy

Few studies used the model of intravenous chemotherapy. M.Bertolini[22] injected 5-FU intravenously to C57 / BL6 mice at a dose of 50 mg / kg every 48 hours for 13 days, and sacrificed the animals on the 14th day. The control group was given intraperitoneal injection of 5-FU at the dose of 100 mg / kg daily for 4 days,and the mice were killed on the 5th day. The results showed that weight loss and enteritis occurred rapidly in the intraperitoneal injection group, but no oral inflammation or higher expression of inflammatory mediators was observed. In the intravenous injection group, the atrophy of oral and esophageal epithelium and the decrease of cell proliferation, together with increase of apoptosis,change of inflammatory mediators, decrease of peripheral blood neutrophils, infiltration of local neutrophils in oral mucosa, and decrease of E-cadherin protein level in oral mucosa were observed.By comparison, this study showed that intraperitoneal injection of chemotherapy drugs alone can not induce OM, and the advantages of intravenous injection over intraperitoneal injection are as follows:1.It can trigger significant inflammatory reaction in oral epithelium,without seriously affecting intestinal functions;2. It simulated the clinical application of 5-FU better. The conclusion of this study on the intraperitoneal injection group is different from other literatures.And the difference may be related to the short observation time.

2.2 Radiotherapy model

2.2.1 Radiotherapy alone modelAs for radiotherapy model, different researches used different methods, including single-dose irradiation and low-dose fractionated irradiation. The irradiation dose and the position of irradiation were also different in different researches. Positions include the snout,buccal mucosa, tongue, etc. According to Han [23], mice were given 8Gy a day for 3 consecutive days to simulate the clinical low-dose fractionated radiation. And the mice developed oral ulcers on the 9th day of the experiment. Kowaliuk [24] and Sylvia [25] made the model of mice by 3Gy for 5 days in per week, and 2 weeks in total. Liang Hu [26] used miniature pigs. After the multi-dose comparative study,the final method of a daily dose of 6Gy for 5 days was determined,which is thought to induce significant OM, and at last the animal weight and oral mucosa can recover spontaneously. Some studies compared single dose and fractionated irradiation. Chao [27] used C57BL/6 mice, and a single dosage of 16.5Gy or 8Gy daily in 3 consecutive days of irradiation was used on the head and neck of mice. The results showed that: the single dose of 16.5Gy irradiation could cause tongue ulcers in all mice, but all mice lost more than 45% of their weight compared with the initial weight, and all mice died within 15 days, which means that the mortality rate of single dose irradiation is higher. Tao [28] used C57/BL6 mice and the study decided that 25Gy was the best dose for rapid induction of OM.Maria [29] used Balb/c mice, and the study chose the dose of 18Gy as the best. With this method, the survival rate can be 100% while the ulcer duration was long enough. Kowaliuk [30] set up a study comparing single dose irradiation and fractionated irradiation using C3H/Neu mice. The ED50 dose of tongue ulcers induced by single dose irradiation was 18.3 ± 4.3Gy. The average incubation period of ulcer was 10.8 ±0.6 days. The average ulcer duration period was 3.5 ± 0.8 days. ED50 of fractionated irradiation (3Gy/day *5 days/week ,and 2 weeks in total) was 9.3 ± 1.8Gy. The average incubation period of ulcer was 7.9±0.6 day. The average ulcer duration period was 4.6±1.1 days. Li [31]established the model by single dose 30Gy irradiation on guinea pigs, which was at an average weight of 300g.According to the studies in the past five years, the single dose of irradiation was 16Gy[32] to 25Gy[28] in mice, and from 15Gy[33] to 20Gy[34] in rats. The best dose of the model was different in different studies. For golden hamster, Watanabe [35] compared the dose of 20Gy,30Gy,40Gy and 50Gy. The result showed that OM became the most serious when the radiation dose reached 40Gy, and when it got more than 40Gy, the severity was no longer related to dose.

2.2.2 Model of radiotherapy combined with mucosa stimulation

Shen [36] used the method of radiotherapy combined with chemical stimulation. In the study, C57 mice were irradiated with a single dose of 16Gy X-ray, and a filter paper soaked in concentrated acetic acid was sticking to the oral mucosa to induce OM. Moura [37] suggested that radiation alone could only cause slight lesions. In his study, a single dose of 35Gy Co-60 irradiation combined with mechanical stimulation after 3 days of irradiation could induce severe OM in golden hamsters.

2.3 Chemoradiotherapy model

Choi [38] used BALB/c mice. On the day0, 5-FU at a dose of 100 mg/kg was intraperitoneally injected. Thirty minutes after injection,radiation at a dose of 20 Gy was given. The radiation was divided into two times, with 10 Gy each time, and an interval of 5 minutes.On the 9th day of the experiment, the rats were killed and the tongue was taken out for tissue analysis. It was confirmed that OM could be induced and no animals died during the experiment.

3. Comparison of the modeling methods

3.1 Chemotherapy model

Injection of chemotherapeutic drugs includes intraperitoneal injection and intravenous injection. Intravenous injection can better simulate the human chemotherapy regimen without affecting the intestinal function, but its operation is more difficult than intraperitoneal injection, and there are fewer studies using this method.

3.1.1 Model of chemotherapy combined with mucosa stimulation

Local stimulation simulates the chronic stimulation process in clinic, which is meant to induce severe OM of oral mucosa so that the OM is easier to be observed and detected. Advantages:Chemotherapy combined with mucosa stimulation can induce serious OM and oral ulcers exactly. The disadvantages are as follows:1.it is difficult to quantify and standardize the stimulation. For example,mechanical stimulation should be ensured to be the same amount of stimulation to different animals, which is difficult to quantify.2.The similarity of simulating clinical practice is low, especially chemical stimulation. 3. The operation is difficult, especially mechanical stimulation, which requires superficial stimulation in oral mucosa without causing mucosal rupture.

3.1.2 Chemotherapy alone modelAdvantages: fewer operations. Disadvantages: it is difficult to induce serious OM and oral ulcers, and the effect is not accurate.

3.2 Radiotherapy model

If the shielding is not complete, it is easy to damage the brain and other important organs and causes death. Radiation can be high-dose single radiation and low-dose fractionated radiation. The location of radiation includes the snout, unilateral buccal mucosa, tongue, and so on. It is better to induce OM with high-dose single irradiation,but the fatality rate is higher and it is different from clinical practice.The fatality rate of low-dose fractionated irradiation is lower, which is more in line with clinical practice. However, its effect of inducing severe OM is relatively worse, and it needs multiple anesthesia,which has a great impact on experimental animals.

3.2.1 Radiotherapy alone model: Advantagessimple operation. Disadvantages: the effect of inducing severe OM and oral ulcers is not exact.

3.2.2 Radiotherapy combined with mucosa stimulation:Advantages

It can exactly induce severe OM and oral ulcers. Disadvantages:mucosal stimulation is difficult to be quantified and standardized,and it is not in line with clinical practice.

3.3 Chemoradiotherapy model

It has great influence on the overall condition of animals, and the effect of inducing severe OM and oral ulcer is not exact.

In addition, there are two problems needed to be paid attention to: 1. Observation of oral mucosa requires multiple anesthesia on animals, which may have great influence on the animals. In order to avoid multiple anesthesia, some studies such as a study carried by mangoni [39] used the lip scoring system established by Parkins [40]to evaluate OM, whose accuracy still needs to be assessed.2.Some models focus on buccal pouch mucosa, and the others may focus on the change of dorsal tongue mucosa. However, the response of buccal pouch mucosa and dorsal tongue mucosa to chemoradiotherapy or drugs may be different.

4. Summary

At present, all kinds of animal models of chemotherapy or radiotherapy induced OM have their shortcomings. The model animals include golden hamster, mouse, rat, guinea pig and miniature pig. It is easy to observe and operate the oral cavity when using golden hamster. Rat and mouse are cheap and rich in strains.Modeling methods include chemotherapy combined with mucosa stimulation, radiotherapy alone, chemotherapy alone, radiotherapy combined with mucosa stimulation, radiotherapy combined with chemotherapy. It is difficult to exactly induce severe OM and oral ulcers by radiotherapy or chemotherapy alone, so that it is necessary to combine it with mucosa stimulation. However, mucosa stimulation is difficult to be quantified and standardized, and it is difficult to ensure its clinical simulation degree. The present methods are all difficult to achieve the goal of simple operation, in line with clinical practice, and exactly inducing severe OM.

The mechanism of chemotherapy or radiotherapy induced OM has not been fully understood, and there is no prevention and treatment methods based on biological evidence. It is necessary to find an ideal animal model for the study of its pathogenesis. Therefore, it is of great significance to compare the above methods.

Author’s contribution

1. CHEN Chen: literature searching, reviewing and article writing 2

. YU Ran: literature rsearching and reviewing

3. CHEN Dong-mei: guidance of model comparative analysis

4. DENG Chao: guidance for animal model analysis

5. LOU Yan-ni: sorting out the ideas of the article

6. JIA Li-qun: sorting out the ideas of the article and guidance on the modification of the article.