1.

Introduction

Head and neck radiotherapy often involves the major salivary glands, which undergo morphological and functional changes, resulting in hyposalivation and xerostomia. Approximately 70% of patients irradiated in the head and neck have a progressive loss of salivary gland function.¹ Quantitative and qualitative salivary changes cause a total or partial loss of taste, pain and burning mouth, dysphagia, dysphonia, increased susceptibility to oral infections and dental caries, among other complications.²

There are no studies that clearly show how radiotherapy affects the function of the salivary glands. Although acinar cells are functionally mature and do not have a high mitotic rate, they respond quickly to radiation.^3–6 The parotid gland accounts for $\sim 60\%$ of the saliva and is considered the most radiosensitive of the major salivary glands.^4,7–9 The acute and late changes observed in irradiated glands include loss and atrophy of acinar cells, decrease in glandular weight, and formation of fibrous tissue.^4,5,10,11

Several studies have demonstrated the role of apoptosis in radiotherapy-induced glandular dysfunction.^4,5 Dysfunction of the salivary glands is directly related to the decrease in acinar cells after radiation.^4,5,12 To assess cell death after radiation therapy, studies have investigated the expression of caspase-3, a protein that plays an important role in cell apoptosis.^3–5 Activation of caspase-3 protein is an acute event of apoptosis, which occurs because the ionizing radiation alters mitochondrial membrane permeability, causing the release of cytochrome C into the cytoplasm.¹³ As a result of radiation therapy, there is also loss of cell homeostasis, production of reactive oxygen species (ROS), and disruption of adenosine triphosphate synthesis. High levels of ROS further increase mitochondrial membrane permeability and activation of caspase-9 and -3.¹⁴

In an attempt to avoid the adverse effects of radiotherapy in the salivary glands, different methods of prevention and treatment of xerostomia have been studied. These include especially cytoprotective agents, growth factors, muscarinic cholinergic agonists, stem/progenitor cell-based therapy, and low-level laser radiation.^4,5,15–20 Low-level laser therapy (LLLT) has been used due to its potential to induce several metabolic and biochemical processes, promoting tissue biomodulation, analgesia, and modulation of the inflammatory process.^21,22 It can regulate the synthesis of nucleic acids and proteins and modulate the levels of cytokines, growth factors, and inflammatory mediators, as well as stimulate cell proliferation and differentiation.²² Simões et al.²³ used LLLT on the major salivary glands of mice and observed an increase in salivary flow at the end of treatment. Simões et al.¹⁹ evaluated the response of the salivary glands to LLLT in patients undergoing radiotherapy. When applied concomitantly with head and neck radiotherapy, LLLT prevented the reduction in salivary flow. Loncar et al.²⁴ applied LLLT to the parotid, submandibular, and sublingual glands of patients with xerostomia, for 10 consecutive days. The authors observed that the amount of saliva produced in the laser group increased gradually during the study. Onizawa et al.,²⁵ in an in vitro study with mouse parotid acinar cells, observed that LLLT-induced cell proliferation and increased expression of the antiapoptotic proteins Bcl-2 and HSP25. Considering the aforementioned evidence, the aim of this study was to evaluate the effect of LLLT on radiotherapy-induced morphological changes and immunodetection of caspase-3 protein in parotids of mice.

2.

Materials and Methods

This study was approved by the Ethics Committee on Animal Use of the Pontifical Catholic University of Rio Grande do Sul (PUCRS), Brazil. The sample consisted of 41 male Swiss mice, weighing 25 to 30 g at the beginning of the experiment. The animals were kept in the Center for Experimental Biological Models of PUCRS in temperature-controlled ($23\pm 1°\mathrm{C}$) chambers equipped with input and output air filters and with a 12-h light-dark cycle. They were housed in cages appropriate for rodents with free access to water and food.

The animals were randomly divided into four groups: control group ($n=5$), radiotherapy group ($n=12$), 2-J (J) laser group ($n=12$), and 4-J laser group ($n=12$). The radiotherapy group and 2- and 4-J laser groups were divided into two experimental times, i.e., six animals were euthanized 48 h after radiotherapy and the other animals, 7 days after receiving the ionizing radiation. The study flow diagram is shown in Fig. 1.

Fig. 1

Flowchart representing the stages of the study.

3.

Results

3.1.

Morphological Analysis

The control group showed normal acinar structure and vascularization [Figs. 2(a) and 2(b)], but some vacuolated cells were seen. The glands of the animals in the radiotherapy group showed marked acinar disorganization, characterized by altered morphology and size of the acini, which was most significant 48 h after radiation. Also in this group, the presence of vacuolated cells was more evident than in the control, mainly 7 days after radiotherapy [Figs. 2(e) and 2(f)].

Fig. 2

Histologic examination of parotid gland structure. (a) Control group, 200x and (b) 400x showing normal acinar structure. (c) 4-J laser group, 48 h after radiotherapy, showing greater vascularization, 200x. (d) 2-J laser group, 7 days after radiotherapy, displaying acinar disorganization, and vacuolated cells (arrow) 400x. (e) Radiotherapy group showing acinar disorganization and (f) vacuolated cells (arrow) 200x.

In the 2- and 4-J laser groups, 48 h after radiotherapy, cytoplasmic vacuolation was similar to that observed in the control group. Acinar disorganization areas were present but were less evident than in the radiotherapy group. Greater vascularization was observed in the glands of the 4-J laser group at this experimental time [Fig. 2(c)].

Seven days after radiotherapy, the 2- and 4-J laser groups had higher vascularization compared to the other groups. At this time, the presence of vacuolated cells was more pronounced compared to the control group but less evident than in the radiotherapy group. As for acinar structure, the pattern in the 4-J laser group at 7 days after radiation was the same as that observed at 48 h. In the 2-J laser group, we observed marked acinar disorganization, resembling that in the radiotherapy group (Table 1) [Fig. 2(d)]. There were no areas of fibrosis, inflammatory infiltrate, or edema in the study groups.

Table 1

Changes in glandular morphology based on descriptive analysis, in the control, 2-J laser, 4-J laser, and radiotherapy groups, at different time points (48 h and 7 days).

Groups		Acinar disorganization	Vacuolation	Vascularization
Control		−	+	+
2 J laser	48 h	+	+	+
	7 days	++	++	++
4 J laser	48 h	+	+	++
	7 days	+	++	++
Radiotherapy	48 h	+++	++	+
	7 days	++	+++	+

(−) absent; (+) slight; (++) moderate; (+++) severe.

3.2.

Immunodetection of Caspase-3

There was no significant difference in caspase-3 immunodetection between groups or between time points (Table 2). However, the data showed a lower percentage in the control group and greater percentage in the radiotherapy group. In the laser groups, percentage of caspase-3 immunodetection was intermediate between the values found in the radiotherapy and control groups [Figs. 3(a) and 3(b)].

Table 2

Percentage (median and percentiles 25 to 75) of caspase-3 immunodetection in the radiotherapy, 2 J laser, 4 J laser, and control groups, at different time points (48 h and 7 days).

	48 h	7 days
Groups	Median (P25–P75)	Median (P25–P75)	$P$a
Radiotherapy	0.64 (0.19–1.41)	0.53 (0.3–1.02)	1.0
2-J laser	0.17 (0.02–0.63)	0.32 (0.09–0.75)	0.42
4-J laser	0.15 (0.07–1.29)	0.26 (0.02–1.46)	1.0
Control	0.05 (0.03–0.1)	0.05 (0.03–0.1)	—
$P$b	0.06	0.085

^aMann–Whitney tests significant at p≤0.05.

^bKruskal–Wallis test.

Fig. 3

(a) Caspase-3 immunostaining in parotid gland in radiotherapy group and (b) 4-J laser group, 7 days after radiotherapy.

4.

Discussion

The dysfunction of the salivary glands is a frequent complication of head and neck radiotherapy and is directly related to structural damage.²⁶ Changes in acinar structure are described within the first 48 and 72 h after radiotherapy.²⁷ The acute effects of ionizing radiation on the salivary glands appear to be due to high levels of cell death, and many studies have suggested that chronic effects may be due to the damage initially produced.^9,27 The present study investigated the effect of LLLT on acute morphological changes and on the detection of caspase-3 protein in parotid glands of irradiated mice.

In this study, in both laser groups, 48 h after radiotherapy, cytoplasmic vacuolation as well as acinar structure appeared similar to that found in the control group. These results indicate that LLLT could have preserved glandular morphology during the first hours after exposure to ionizing radiation. Seven days after radiotherapy, however, there was an increase in cytoplasmic vacuolation and changes in acinar structure, which were more pronounced in the 2-J laser group. However, most of these changes were still less evident than that observed in the radiotherapy group. In our study, LLLT was applied in two sessions, i.e., immediately before and 24 h after radiation. If more sessions of LLLT had been used in this 7-day period, it is possible that the morphological changes found a week after radiotherapy could have been less evident.

Several authors have described the occurrence of cytoplasmic vacuolation in salivary glands of irradiated animals,^6,27–29 evident in the first hours after radiotherapy.^6,27,28 The vacuoles in the cytoplasm represent an active process of autophagy,³⁰ induced by nutritional shortage, infection, or oxidative stress.³¹ Vacuolated cells were more evident in the radiotherapy group than the other groups. However, even the salivary glands in the control group showed some vacuolated cells. The presence of cytoplasmic vacuolation was also recorded by Radfar and Sirois¹¹ in salivary glands of nonirradiated minipigs.

The salivary glands of the laser group had higher vascularization compared to control and radiotherapy groups. Laser stimulation of microcirculation has been described by other authors, when applying LLLT to different tissues.^32,33 On the other hand, the literature demonstrates that salivary glands of irradiated animals display hypovascularization with changes in blood flow and distribution of vessels immediately after radiotherapy.^29,34,35 These alterations were not observed in this study when comparing the radiotherapy and control groups, a result that can be explained by the difference in the methods for analyzing vascularization, since in the literature, other techniques such as immunohistochemistry and doppler have been used.^29,34,35

In this study, there were no areas of fibrosis, inflammatory infiltrate, or edema. These results are consistent with most studies reporting the occurrence of fibrosis as a late effect in irradiated salivary glands, where it is found more often after the 30-day postirradiation.^6,11,36 The presence of inflammatory infiltrate in salivary glands resulting from radiotherapy is rarely mentioned in the literature, and only observed in studies using minipigs and rhesus monkeys as experimental model.^27,37 The occurrence of edema, although not manifesting late, was quoted in the literature only on the 10th day after exposure to ionizing radiation, which would explain why we did not observe this up to the seventh day.⁶

Since apoptosis of acinar cells has been identified as an acute event caused by radiotherapy,^4,5,38,39 which influences the loss of function of the salivary glands, we chose to investigate the immunodetection of caspase-3 protein. Although not significantly different between the groups, the percentage of caspase-3 immunostaining was higher in the radiotherapy group and lower in the control group, while the laser groups showed intermediate values, indicating that LLLT might have influenced acinar cell apoptosis.

In the literature, quantification of caspase-3 in the salivary glands of irradiated mice varied from 2% to 27%, depending on the radiation dose and observation time.^4,5,30,39 These percentages are higher than those found in our study, probably because of the quantification methods used. In these studies, the percentage was obtained by the number of labeled cells. In our study, caspase-3 was quantified by the stained area in relation to the total area of each histological field, because cytoplasmic staining appeared diffuse in some slides and because cell counts alone could produce bias. Another issue to be considered is that studies differ on the peak occurrence of apoptosis. Avila et al.⁴ observed in transgenic animals an increased number of apoptotic cells 48 h after radiation. In the study by Limesand et al.,³⁹ the expression of caspase-3 was higher 24 h after radiotherapy, decreasing drastically when assessed at 48 h. The authors explained this result as being due to the removal of these cells by phagocytosis,³⁰ which could also explain the low percentage of detection of this protein in our study.

The lack of a therapeutic protocol for LLLT made methodological definitions in this study difficult. In the literature, laser parameters differ considerably in studies using LLLT to treat xerostomia.^19,24 There have been differences in wavelength, power, energy, and frequency of sessions.^7,19,24 We opted for an infrared wavelength due to the depth of glandular parenchyma to be irradiated.²⁴ The determination of the energy of 2 J supplied per point was based on the study of Saleh et al.,⁴⁰ and for comparison, we decided to use also an LLLT protocol with energy of 4 J.

Given the methodological limitations of this study, further research should be conducted in irradiated animals, using different LLLT protocols and observing glandular response, not only in the short term but also long term, when the occurrence of late changes in the salivary glands can be analyzed.

5.

Conclusions

The present study investigated the effect of LLLT on radiotherapy-induced morphological changes and immunodetection of caspase-3 protein in parotids of mice. The results suggest that when using the protocols defined in this study, LLLT had a tendency to reduce cell apoptosis by reducing active caspase-3. In addition, LLLT promoted the preservation of acinar structure, with regard to the organization of acini, reduced the occurrence of vacuolation, and even stimulated the vascularization of the parotid glands of irradiated mice. Of the two LLLT protocols studied (2 and 4 J), the one using 4 J of energy showed better results.