Abstract
The aim of this study was to evaluate the effect of hyperbaric oxygen therapy (HBOT) on microvascular tissue and cell proliferation in the oral mucosa. Twenty patients, aged 51–78 years, were allocated randomly to a treatment or a control group. All had a history of radiotherapy (50–70 Gy) to the orofacial region 2–6 years previously. Tissue samples were taken from the irradiated buccal oral mucosa before HBOT and at 6 months after treatment. In the control group, tissue samples were taken on two occasions, 6 months apart. The samples were subjected to immunohistochemistry staining: double staining with CD31 and D2-40 for microvessels, or Ki-67 for the analysis of cell proliferation. Blood vessel density and area were significantly increased after HBOT ( P = 0.002–0.041). D2-40-positive lymphatic vessels were significantly increased in number and area in the sub-epithelial area ( P = 0.002 and P = 0.019, respectively). No significant differences were observed in the control group. There were no significant differences in Ki-67-expressing epithelial cells between the two groups. It is concluded that the density and area of blood and lymphatic vessels in the irradiated mucosa are increased by HBOT 6 months after therapy. Epithelial cell proliferation is not affected by HBOT.
Radiation injury to the normal tissues is an inevitable effect of radiotherapy in the treatment of cancer. In the head and neck region, the skin, mucosa, subcutaneous tissue, bone, and salivary glands may be included in the field of radiation. Late effects in normal tissues may present clinically as skin and mucosal atrophy, fibrosis, bone necrosis, trismus, and xerostomia, thereby negatively affecting patient quality of life.
Histologically, radiated tissue is characterized by atrophy, changes in cellular morphology, increased collagen deposition, a decreased number of blood vessels, and dilatation of the remaining vessels.
The clinical consequences of the late effects of radiotherapy may pose a significant therapeutic challenge. Currently, hyperbaric oxygen treatment (HBOT) is used as a single modality or as an adjunct to surgery to improve wound healing by inducing angiogenesis and increased oxygen tension in hypoxic tissues. The increased partial pressure of oxygen by HBOT in tissue is thought to induce the necessary oxygen gradient to stimulate collagen production by fibroblasts and capillary angiogenesis.
Experimental animal studies by Marx et al. and human studies by the same group have reported significantly increased vascular density in irradiated skin, subcutaneous tissue, periosteum, and bone marrow after treatment with HBOT. The angiogenic effect has been confirmed in bone in other animal models and a human dynamic magnetic resonance imaging (MRI) study, but there is a paucity of data in the literature regarding soft tissue.
The authors of the present study have recently demonstrated an increase in microvascular capacity in irradiated skin and mucosa after HBOT. This may represent improved endothelial function or vascular capacitance and indicates a need for histomorphometric analysis of the microvascular effects of HBOT in soft tissue in order to further elucidate the role of HBOT.
The aim of the present study was to test the null hypothesis of no effect of HBOT on the morphology of the microvasculature in the irradiated oral mucosa.
Materials and methods
Ethics
Participation in the study was based on the written informed consent of each subject. The study protocol was approved by the Regional Committee for Medical Research Ethics in Western Norway (REK Vest) and the Privacy Ombudsman for Research at the Norwegian Social Science Data Services (NSD). The study was conducted in accordance with the Declaration of Helsinki.
Subjects
The subjects comprised 20 patients, 15 men and five women, ranging in age from 51 to 78 years. Patients formerly treated for head and neck cancer and referred to the hyperbaric medical unit of the university hospital in Bergen, Norway, were recruited consecutively and allocated to a treatment group ( n = 12) or a control group ( n = 8). Group assignment was made after enrolment using a predetermined randomized allocation sequence. Inclusion criteria were a history of malignant disease treated with radiotherapy ≥50 Gy to an area including the oral cavity. Exclusion criteria were unwillingness to receive HBOT, previous treatment with HBOT, active malignant disease or other medical conditions precluding HBOT, and inability to attend the scheduled follow-up appointments. Fifty-four patients were invited to participate, giving a participation rate of 37%. Patients were not asked to give any reason for non-participation.
Patient characteristics and the site of former malignant disease, radiation dose given, and time elapsed since radiotherapy are summarized in Table 1 . For all patients, the radiation modality was fractionated three-dimensional conformal radiotherapy with multiple fields. Indications for HBOT were clinical osteoradionecrosis, xerostomia, or as a prophylactic measure before tooth extraction or other surgical procedures. All patients were also part of a formerly reported study. Two of the patients in the former study refused to have a tissue sample taken. This reduced the number of patients in the test group by two compared to the former study.
| Subject | Sex | Age, years | Site of cancer | Radiation dose, Gy | Time since radiation, years |
|---|---|---|---|---|---|
| HBOT group | |||||
| 1 | M | 78 | Tonsil | 64 | 3 |
| 2 | M | 55 | Retromolar | 60 | 6 |
| 3 | M | 66 | Tonsil | 64 | 3 |
| 4 | M | 62 | Floor of mouth | 70 | 3 |
| 5 | F | 69 | Tonsil | 70 | 3 |
| 6 | F | 51 | Tonsil | 70 | 6 |
| 7 | F | 56 | Parotid gland | 70 | 6 |
| 8 | M | 67 | Tongue | 70 | 2 |
| 9 | M | 63 | Origo incerta a | 50 | 6 |
| 10 | M | 70 | Buccal | 70 | 5 |
| 11 | M | 56 | Floor of mouth | 64 | 5 |
| 12 | M | 51 | Tonsil | 64 | 5 |
| Mean (range) | 62 (51–78) | 66 (50–70) | 4 (2–6) | ||
| Controls | |||||
| 1 | M | 65 | Gingiva | 70 | 6 |
| 2 | M | 55 | Retromolar | 64 | 2 |
| 3 | M | 56 | Tonsil | 70 | 3 |
| 4 | M | 63 | Retromolar | 64 | 4 |
| 5 | F | 55 | Tongue | 64 | 5 |
| 6 | F | 73 | Floor of mouth | 64 | 4 |
| 7 | M | 53 | Origo incerta a | 50 | 2 |
| 8 | M | 62 | Floor of mouth | 70 | 4 |
| Mean (range) | 60 (53–73) | 65 (50–70) | 4 (2–6) | ||
Hyperbaric oxygen treatment (HBOT)
Patients received HBOT once daily, 5 days a week, for an average of 29 days (range 20–40 days). The number of treatments was determined by the individual indication for HBOT. The patients were compressed with oxygen in a monoplace hyperbaric chamber to a pressure of 240 kPa within 10–15 min. Oxygen was breathed at this pressure for 90 min, in three cycles of 30 min, with breathing of compressed air from an oronasal mask for 5 min between the cycles. They were decompressed to atmospheric pressure in 7–10 min.
Tissue sampling
Tissue samples were obtained from the buccal oral mucosa within the field of maximum irradiation, according to the dose planning. After peripherally infiltrating approximately 0.5 ml of local anaesthetic (2% Xylocaine Dental with epinephrine 1:100,000; Dentsply Pharmaceutical, York, PA, USA), a sample was taken using a tissue punch 5 mm in diameter. The depth of the sample was approximately 3–4 mm. Repeat samples were taken approximately 5 mm away from the original sample, based on measurements using the parotid duct as a local anatomical landmark. All of the samples were fixed in 10% buffered formalin, and subsequently embedded in paraffin.
In the treatment group, samples were taken before the start of HBOT and at 6 months after treatment. In the control group, the subjects agreed to wait for at least 6 months for the HBOT, and samples were taken on two separate occasions 6 months apart.
Immunohistochemistry
Immunohistochemical staining was performed on 3-μm sections of the samples. All procedures followed a standardized protocol. The sections were deparaffinized in xylene after being heated overnight, not exceeding 58 °C. They were then hydrated with ethanol in decreasing concentration (100%, 96%, and 70%) and then rehydrated in Tris buffer pH 7.6 with 0.1% Tween. The epitopes were then retrieved by heating a solution of Tris and ethylenediaminetetraacetic acid (EDTA), pH 9.0, in a microwave oven to smooth boiling for 8 min. The sections were rinsed in tap water for 5 min and then rinsed in Tris buffer, pH 7.6, twice for 5 min. Endogenous enzymes were blocked by H 2 O 2 for 10 min, followed by rinsing in Tris buffer twice for 5 min. Separate sections from each patient at each time point were then immunostained using either double staining with CD31 and D2-40 for vessels, or Ki-67 for analysis of cell proliferation. Normal human tonsils were treated in the same way and served as positive control tissue.
CD31/D2-40
The sections were placed in a chamber for 60 min with diluted primary antibody CD31 (M0823, 1:35; Dako, Glostrup, Denmark), and then placed in a chamber with the visualization reagent horseradish peroxidase (Dako) for 30 min. Binding of the first antibody was visualized using a DAB Kit (Sigma, St Louis, USA), 1 ml buffer to 1 drop of diaminobenzidine (DAB) for 12 min. After applying Doublestain Block in the EnVision G|2 System (Dako; K5361), the sections were incubated with the second primary antibody D2-40 (M3619, 1:50; Dako) in a humidity chamber for 60 min at room temperature, and then incubated overnight at 4 °C, followed by 60 min at room temperature. A rabbit/mouse link (Dako) was then applied for 30 min, followed by a visualization agent, alkaline phosphatase, for 30 min and Liquid Permanent Red (K0640; Dako) for 11 min. The sections were rinsed in Tris buffer, pH 7.6 with 0.1% Tween, between each step. They were counterstained with haematoxylin, washed with tap water, ethanol, and toluene, and then covered with Eukitt mounting medium (Sigma).
Ki-67
The sections were placed in a chamber with Ki-67 (ready-to-use M7240, Clone MIB-1; Dako) for 60 min, then placed in a chamber with EnVision Mouse (Dako) for 30 min. The staining reactions were developed using a DAB Kit (Sigma), 1 ml buffer to 1 drop of DAB for 10 min. The sections were rinsed in Tris buffer with 0.1% Tween, pH 7.6, between each step. They were counterstained with haematoxylin, washed with tap water, ethanol, and toluene, and then covered with Eukitt mounting medium (Sigma).
Measurements
All microscopic analyses were performed using a Nikon microscope on a computer monitor, with NIS-Elements BR software version 2.30 (Nikon, Japan). All vessels were initially stained brown (CD-31), but after double staining, the lymph vessels presented as red (D2-40). The area of the vessels was calculated using the software after outlining the vessels (magnification 20×). Relative numbers of vessels and the area of vessels were calculated using a grid applied by the software, with squares of 200 μm × 200 μm. The average of at least five fields was reported for vessels/mm 2 and percentage area. The sub-epithelial and connective tissue layer were analyzed separately. Cell proliferation, as indicated by Ki-67-positive cells, was reported as the proliferation index, i.e. the percentage of positive cells in the basal and parabasal cell layers (magnification 60×). The parabasal layer was defined as the two cell layers above the basal layer. The observer performing the measurements was blinded to the treatment received.
Statistical analysis
The data were entered and analyzed using IBM SPSS software version 19.0 (IBM Corp., Armonk, NY, USA). Reproducibility of the measurements was analyzed by intra-class correlation coefficient (ICC) (two-way mixed, random effect model, absolute agreement). Paired differences were analyzed by Wilcoxon signed-rank test. Comparisons between groups were analyzed by Mann–Whitney U -test. The randomized allocation sequence was performed using the random number generator function in SPSS based on a potential enrolment of 50 patients. The level of statistical significance was set at P < 0.05.
Results
To assess the reproducibility of the measurements, 15 sections were re-evaluated after 4 weeks for both microvessel density and cell proliferation. Both measures were highly reproducible with an ICC of 0.98 for microvessel density and 0.99 for cell proliferation. There were no differences between the groups with respect to age ( P = 0.624), radiation dose ( P = 0.734), or time interval since the last radiation session ( P = 0.343).
All biopsy sites had full mucosal coverage at the time of the second harvesting. One patient experienced subcutaneous emphysema after performing the Valsalva manoeuvre shortly after a biopsy procedure. This was treated with prophylactic antibiotic therapy and resolved in a few days. There were no other complications reported by the patients, and no major complications caused by the HBOT were observed. None of the patients presented a marked lymphoedema at baseline. A potential effect on lymph vessels could therefore not be clinically assessed.
Histomorphology
All sections presented a normal stratified squamous epithelium ( Fig. 1 ). Dilated vessels in papillary projections of the connective tissue were seen in some of the sections, but were not a consistent finding. The vessels in the papillary projections were found to be blood vessels ( Fig. 2 ). The connective tissue contained a scarce inflammatory infiltrate both before and after HBOT.
