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Effect of hyperbaric oxygen treatment on irradiated oral mucosa: microvessel density
Int. J. Oral Maxillofac. Surg. 2015; 44: 301–307 http://dx.doi.org/10.1016/j.ijom.2014.12.012, available online at http://www.sciencedirect.com
Research Paper Head and Neck Oncology
Effect of hyperbaric oxygen treatment on irradiated oral mucosa: microvessel density J. Svalestad, S. Hellem, E. Thorsen, A.C. Johannessen: Effect of hyperbaric oxygen treatment on irradiated oral mucosa: microvessel density. Int. J. Oral Maxillofac. Surg. 2015; 44: 301–307. # 2015 International Association of Oral and Maxillofacial Surgeons. Published by Elsevier Ltd. All rights reserved.
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-67expressing 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 0901-5027/030301 + 07
xerostomia,1 thereby negatively affecting patient quality of life.2 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.3–6 The clinical consequences of the late effects of radiotherapy may pose a
J. Svalestad1,2,, S. Hellem1, E. Thorsen3,4, A. C. Johannessen5,6 1
Department of Clinical Dentistry – Oral and Maxillofacial Surgery, University of Bergen, Bergen, Norway; 2Department of Oral and Maxillofacial Surgery, Haukeland University Hospital, Bergen, Norway; 3Hyperbaric Medical Unit, Department of Occupational Medicine, Haukeland University Hospital, Bergen, Norway; 4Department of Clinical Science, University of Bergen, Bergen, Norway; 5Department of Clinical Medicine, The Gade Laboratory for Pathology, University of Bergen, Bergen, Norway; 6 Department of Pathology, Haukeland University Hospital, Bergen, Norway
Key words: angiogenesis; cell proliferation; immunohistochemistry; microvascular tissue; radiotherapy. Accepted for publication 19 December 2014 Available online 17 January 2015
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.7,8 The increased partial pressure of oxygen by HBOT in tissue is thought to induce the necessary oxygen gradient to
# 2015 International Association of Oral and Maxillofacial Surgeons. Published by Elsevier Ltd. All rights reserved.
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stimulate collagen production by fibroblasts9 and capillary angiogenesis.10,11 Experimental animal studies by Marx et al.12 and human studies13 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 models14 and a human dynamic magnetic resonance imaging (MRI) study,15 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.16 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 nonparticipation. 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.16 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. 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
Table 1. Patient characteristics. Subject HBOT group 1 2 3 4 5 6 7 8 9 10 11 12 Mean (range) Controls 1 2 3 4 5 6 7 8 Mean (range)
Radiation dose, Gy
Time since radiation, years
Tonsil Retromolar Tonsil Floor of mouth Tonsil Tonsil Parotid gland Tongue Origo incertaa Buccal Floor of mouth Tonsil
64 60 64 70 70 70 70 70 50 70 64 64 66 (50–70)
3 6 3 3 3 6 6 2 6 5 5 5 4 (2–6)
Gingiva Retromolar Tonsil Retromolar Tongue Floor of mouth Origo incertaa Floor of mouth
70 64 70 64 64 64 50 70 65 (50–70)
6 2 3 4 5 4 2 4 4 (2–6)
Sex
Age, years
Site of cancer
M M M M F F F M M M M M
78 55 66 62 69 51 56 67 63 70 56 51 62 (51–78)
M M M M F F M M
65 55 56 63 55 73 53 62 60 (53–73)
HBOT, hyperbaric oxygen treatment; M, male; F, female.
Hyperbaric oxygen in irradiated tissue 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-mm sections of the samples. All procedures followed a standardized protocol. The sections were deparaffinized in xylene after being heated overnight, not exceeding 58 8C. 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 H2O2 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 Gj2 System
(Dako; K5361), the sections were incubated with the second primary antibody D240 (M3619, 1:50; Dako) in a humidity chamber for 60 min at room temperature, and then incubated overnight at 4 8C, 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).
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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
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 mm 200 mm. The average of at least five fields was reported for vessels/ mm2 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.
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. Microvessel density
Both blood vessel density and blood vessel area were significantly increased after HBOT (P = 0.002–0.041). The effects
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Svalestad et al. Cell proliferation
No significant differences in Ki-67-positive epithelial cells could be found in either the treatment group or the control group (Table 2). Cell proliferation was prominent in the parabasal layer (Fig. 4). Ki-67-positive endothelial cells were an occasional finding after HBOT. Discussion
Fig. 1. Irradiated buccal mucosa before (a) and 6 months after (b) hyperbaric oxygen treatment (HBOT). The sections are stained with haematoxylin and eosin. Scale bar: 200 mm.
were more pronounced in the sub-epithelial area than in the deeper connective tissue (Table 2), and were largely due to an increase in the number of small vessels (diameter <25 mm). A significant increase in the number and area of D2-40-positive
lymphatic vessels could be observed in the sub-epithelial area (P = 0.002 and P = 0.019, respectively). Representative sections are presented in Fig. 3. No significant differences were observed in the control group.
Fig. 2. Proliferating vessels in connective tissue papillae. Scale bar: 200 mm.
A patent network of macro- and microvessels is a prerequisite for exchange of oxygen and nutrients to the healing wound. Therapy stimulating angiogenesis may therefore prove beneficial to support healing in selected clinical situations with compromised microcirculation, e.g. due to radiotherapy. The present study provides evidence of neoangiogenesis by HBOT in human irradiated tissue, and the new vessels were present after 6 months. An increased microvascular density was also reported by Marx et al.13 after a course of 20 HBOT sessions at 2.4 ata. The microvascular density increased from 25–35% to 70– 80% of pre-radiation levels. However, that study did not differentiate between blood vessels and lymphatic vessels, making comparison difficult. Advances made in immunohistochemistry techniques since that study was performed have opened opportunities to easily differentiate between blood and lymph microvessels.17 The present study demonstrated an increased number and area of blood microvessels. This angiogenetic effect has been demonstrated in experimental animal models and involves local oxygen gradients,9,10 up-regulation of growth factors such as vascular endothelial growth factor (VEGF),18 and stimulation of vasculogenic stem cells from the bone marrow.19 Handschel et al.4 reported a marked reduction in microvessels throughout the tissue of the oral mucosa with a compensatory dilation of the deeper vessels at 6– 12 months after radiotherapy. A similar histology with obliterated vessels and some dilated vessels was observed in the present study at baseline, and this may explain the difference in response between the sub-epithelial and deeper connective tissue. An increased tissue oxygenation in skin and vascular capacity in skin and gingival mucosa with the use of HBOT was recently demonstrated in the irradiated tissue in these patients.16 The ability to stimulate an increased blood flow by heat provocation was significantly increased at 3 and 6 months after HBOT. This was suggested
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Hyperbaric oxygen in irradiated tissue Table 2. Measurements of vascularization and cell proliferation (mean SD). HBOT group Baseline Blood vessels Sub-epithelial area MVD 45.4 13.9 MVA 1.5 0.6 Deeper connective tissue MVD 30.4 10.1 MVA 2.5 1.3 Lymph vessels Sub-epithelial area MVD 18.3 8.1 MVA 1.3 0.7 Deeper connective tissue MVD 14.6 7.2 MVA 1.7 1.1 Proliferation index: Ki-67 (%) 23.2 4.5
Controls 6 months
P-value
Baseline
6 months
P-value
98.0 15.9a 4.4 1.9a
0.002 0.003
45.6 15.7 1.5 0.6
49.3 10.5 1.6 0.5
NS NS
45.1 16.4a 3.7 1.3a
0.01 0.041
28.1 9.6 2.2 0.9
34.4 7.8 2.7 1.4
NS NS
36.1 12.6a 2.7 1.8a
0.002 0.019
19.4 6.2 1.2 0.6
16.9 8.8 1.5 0.7
NS NS
16.8 7.0 1.5 0.9
NS NS
13.1 7.0 2.0 0.9
15.0 5.3 1.8 1.1
NS NS
21.8 1.6
NS
19.4 4.0
20.0 4.3
NS
SD, standard deviation; HBOT, hyperbaric oxygen treatment; MVD, microvessel density (vessels/mm2); MVA, microvessel area (%); NS, not significant. a Significantly increased compared to baseline (P < 0.05).
Fig. 3. Irradiated oral mucosa before (a) and 6 months after (b) hyperbaric oxygen treatment (HBOT). The sections are stained with CD31 and D2-40. Blood vessels appear as brown and lymphatic vessels as red. Scale bar: 200 mm.
to be a combined effect of an increased vascular density and improved endothelial function. The present study supports the findings of an increased vascular density, providing improved physiological function of the microvascular network. Teas et al.20 reported a significant increase in lymph angiogenic-associated VEGF-C after HBOT in patients with lymphoedema related to breast cancer treatment. This may also explain the increased lymphatic vessel density in the present study, and this may potentially help alleviate the symptoms related to lymphoedema. Morphological effects on lymphatic vessels in irradiated tissue as a result of HBOT have not been reported previously. There is suggestive evidence in the literature of an effect of HBOT on lymphoedema following radiotherapy.21 This effect may be a result of increased lymphatic clearance by new lymphatic vessels in irradiated tissue. A recent randomized trial evaluating arm lymphoedema after primary surgery and adjuvant radiotherapy for breast cancer could not, however, confirm any benefit of HBOT over best standard care.22 More studies are therefore needed to further explore the potential clinical benefits.21,22 The proliferation index (Ki-67) was high compared to reported values for normal mucosa, but comparable to clinically normal distant cheek mucosa from patients treated for oral squamous cell carcinomas.23,24 All biopsies were taken within the field of maximum irradiation and hence close to a former cancerous mucosa. It has been demonstrated that
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Svalestad et al. Ethical approval
The study protocol was approved by the Regional Committee for Medical Research Ethics in Western Norway (REK Vest), reference 218.05, and the Privacy Ombudsman for Research at the Norwegian Social Science Data Services (NSD). Patient consent
Not required. Acknowledgements. We thank Gunnvor Øijordsbakken for skilful technical assistance and the performance of immunohistochemistry procedures. Fig. 4. Immunostaining with Ki-67. There was prominent cell proliferation in the parabasal layer. Scale bar: 100 mm.
References
such mucosa exhibits a high proliferative status, probably due to genetic alterations according to the field cancerization theory, and that an abnormally high proliferation status is a negative prognostic factor for disease-free survival.23 The proliferation index was not affected by HBOT in the present study. It has been hypothesized that HBOT could lead to higher rates of local recurrence as a result of increased vascularization. An increased proliferation index by HBOT beyond the baseline values in the present study could potentially indicate an increased risk of tumour recurrence. However, such an alteration was not found, and the potential risk of tumour recurrence or potentiation has not been supported by clinical or experimental studies.25 The high proliferation index further indicates a sufficient tissue oxygenation for normal cell homeostasis in irradiated tissue during normal metabolic demand. This is in accordance with clinical experience, as necrosis usually occurs in situations with an increased metabolic demand, e.g. due to a surgical procedure.26 The immunohistochemical methods used in the present study are well established17,27,28 and the methods of assessment were highly reproducible with an ICC of 0.98 and 0.99 for vascular density and the proliferation index, respectively. Taking tissue samples from the buccal oral mucosa proved to be a safe procedure, as all sites healed completely. The subcutaneous emphysema experienced by one patient was probably due to a combination of loose suturing and a faulty technique while performing the Valsalva manoeuvre. Subsequent biopsy sites were sutured watertight, and no further complications were experienced.
1. Sciubba JJ, Goldenberg D. Oral complications of radiotherapy. Lancet Oncol 2006;7:175–83. 2. Abendstein H, Nordgren M, Boysen M, Jannert M, Silander E, Ahlner-Elmqvist. et al. Quality of life and head and neck cancer: a 5 year prospective study. Laryngoscope 2005; 115:2183–92. 3. Stone HB, Coleman CN, Anscher MS, McBride WH. Effects of radiation on normal tissue: consequences and mechanisms. Lancet Oncol 2003;4:529–36. 4. Handschel J, Sunderkotter C, Kruse-Losler B, Prott FJ, Meyer U, Piffko J, et al. Late effects of radiotherapy on oral mucosa in humans. Eur J Oral Sci 2001;109:95–102. 5. Riekki R, Jukkola A, Sassi ML, Hoyhtya M, Kallioinen M, Risteli J, et al. Modulation of skin collagen metabolism by irradiation: collagen synthesis is increased in irradiated human skin. Br J Dermatol 2000;142:874–80. 6. Richter KK, Fink LM, Hughes BM, Sung CC, Hauer-Jensen M. Is the loss of endothelial thrombomodulin involved in the mechanism of chronicity in late radiation enteropathy. Radiother Oncol 1997;44:65–71. 7. Shaw RJ, Butterworth C. Hyperbaric oxygen in the management of late radiation injury to the head and neck. Part II: prevention. Br J Oral Maxillofac Surg 2011;49:9–13. 8. Shaw RJ, Dhanda J. Hyperbaric oxygen in the management of late radiation injury to the head and neck. Part I: treatment. Br J Oral Maxillofac Surg 2011;49:2–8. 9. Hunt TK, Pai MP. Effect of varying ambient oxygen tensions on wound metabolism and collagen synthesis. Surg Gynecol Obstet 1972;135:561–7. 10. Knighton DR, Silver IA, Hunt TK. Regulation of wound-healing angiogenesis—effect of oxygen gradients and inspired oxygen concentration. Surgery 1981;90:262–70. 11. Knighton DR, Hunt TK, Scheuenstuhl H, Halliday BJ, Werb Z, Banda MJ. Oxygentension regulates the expression of angiogenesis factor by macrophages. Science 1983; 221:1283–5.
Oral mucosa is easily available, usually heals uneventfully when no bone is exposed, leaves no visible scars, and is therefore ideal for studies on irradiated human tissue. The patients recruited in the present study represented a heterogeneous sample as there were several indications for HBOT. Although all patients had received substantial radiation doses, the tissue effect might have been variable due to the inter-individual variation in radiosensitivity.29 Severe late effects of radiotherapy are evident in patients with osteoradionecrosis, hence including only patients with osteoradionecrosis might have demonstrated an even more pronounced effect of HBOT. However, the results in the present study reflect the effect on patients referred to a hyperbaric medical unit, and several effects were found significant. Furthermore, as the repeat samples were taken 6 months after HBOT, this allowed the inclusion of possible continuous effects after the completion of therapy, and the results indicate a long-term effect of HBOT. Within the limitations of this study, it is concluded that the density and area of blood and lymphatic microvessels in irradiated mucosa are increased by HBOT at 6 months after therapy. Epithelial cell proliferation is not affected by HBOT. Funding
The present study was supported by grants from the University of Bergen, ColgatePalmolive, and the L. Meltzer Foundation. Competing interests
None declared.
Hyperbaric oxygen in irradiated tissue 12. Marx RE, Ehler WJ, Tayapongsak P, Pierce LW. Relationship of oxygen dose to angiogenesis induction in irradiated tissue. Am J Surg 1990;160:519–24. 13. Marx RE, Johnson RP. Studies in the radiobiology of osteoradionecrosis and their clinical-significance. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 1987;64:379–90. 14. Muhonen A, Haaparanta M, Gronroos T, Bergman J, Knuuti J, Hinkka S, et al. Osteoblastic activity and neoangiogenesis in distracted bone of irradiated rabbit mandible with or without hyperbaric oxygen treatment. Int J Oral Maxillofac Surg 2004;33: 173–8. 15. Store G, Smith HJ, Larheim TA. Dynamic MR imaging of mandibular osteoradionecrosis. Acta Radiol 2000;41:31–7. 16. Svalestad J, Thorsen E, Vaagboe G, Hellem S. Effect of hyperbaric oxygen treatment on oxygen tension and vascular capacity in irradiated skin and mucosa. Int J Oral Maxillofac 2014;43:107–12. 17. Kahn HJ, Bailey D, Marks A. Monoclonal antibody D2-40, a new marker of lymphatic endothelium, reacts with Kaposi’s sarcoma and a subset of angiosarcomas. Mod Pathol 2002;15:434–40. 18. Sheikh AY, Gibson JJ, Rollins MD, Hopf HW, Hussain Z, Hunt TK. Effect of hyperoxia on vascular endothelial growth factor levels in a wound model. Arch Surg 2000; 135:1293–7. 19. Thom SR, Bhopale VM, Velazquez OC, Goldstein LJ, Thom LH, Buerk DG. Stem
20.
21.
22.
23.
24.
cell mobilization by hyperbaric oxygen. Am J Physiol Heart Circ Physiol 2006;290: H1378–86. Teas J, Cunningham JE, Cone L, Jansen K, Raghavan SK, Nitcheva DK, et al. Can hyperbaric oxygen therapy reduce breast cancer treatment-related lymphedema? A pilot study. J Womens Health 2004;13:1008–18. Gothard L, Stanton A, MacLaren J, Lawrence D, Hall E, Mortimer P, et al. Nonrandomised phase II trial of hyperbaric oxygen therapy in patients with chronic arm lymphoedema and tissue fibrosis after radiotherapy for early breast cancer. Radiother Oncol 2004;70:217–24. Gothard L, Haviland J, Bryson P, Laden G, Glover M, Harrison S, et al. Randomised phase II trial of hyperbaric oxygen therapy in patients with chronic arm lymphoedema after radiotherapy for cancer. Radiother Oncol 2010;97:101–7. Montebugnoli L, Badiali G, Marchetti C, Cervellati F, Farnedi A, Foschini MP. Prognostic value of Ki67 from clinically and histologically ‘normal’ distant mucosa in patients surgically treated for oral squamous cell carcinoma: a prospective study. Int J Oral Maxillofac Surg 2009;38:1165–72. Montebugnoli L, Gissi DB, Badiali G, Marchetti C, Cervellati F, Farnedi A, et al. Ki-67 from clinically and histologically ‘normal’ distant mucosa as prognostic marker in early-stage (T1–T2N0) oral squamous cell carcinoma: a prospective study. J Oral Maxillofac Surg 2011;69:2579–84.
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25. Feldmeier J, Carl U, Hartmann K, Sminia P. Hyperbaric oxygen: does it promote growth or recurrence of malignancy. Undersea Hyperb Med 2003;30:1–18. 26. Reuther T, Schuster T, Mende U, Kubler A. Osteoradionecrosis of the jaws as a side effect of radiotherapy of head and neck tumour patients—a report of a thirty year retrospective review. Int J Oral Maxillofac Surg 2003;32:289–95. 27. Lindboe CF, Torp SH. Comparison of Ki-67 equivalent antibodies. J Clin Pathol 2002; 55:467–71. 28. Fukunaga M. Expression of D2-40 in lymphatic endothelium of normal tissues and in vascular tumours. Histopathology 2005;46: 396–402. 29. Tucker SL, Geara FB, Peters LJ, Brock WA. How much could the radiotherapy dose be altered for individual patients based on a predictive assay of normal-tissue radiosensitivity. Radiother Oncol 1996;38:103–13.
Address: Jarle Svalestad Department of Clinical Dentistry Faculty of Medicine and Dentistry University of Bergen A˚rstadveien 19 N-5009 Bergen Norway Tel: +47 55586560; Fax: +47 55586577 E-mail: [email protected]
Research Paper Head and Neck Oncology
Effect of hyperbaric oxygen treatment on irradiated oral mucosa: microvessel density J. Svalestad, S. Hellem, E. Thorsen, A.C. Johannessen: Effect of hyperbaric oxygen treatment on irradiated oral mucosa: microvessel density. Int. J. Oral Maxillofac. Surg. 2015; 44: 301–307. # 2015 International Association of Oral and Maxillofacial Surgeons. Published by Elsevier Ltd. All rights reserved.
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-67expressing 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 0901-5027/030301 + 07
xerostomia,1 thereby negatively affecting patient quality of life.2 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.3–6 The clinical consequences of the late effects of radiotherapy may pose a
J. Svalestad1,2,, S. Hellem1, E. Thorsen3,4, A. C. Johannessen5,6 1
Department of Clinical Dentistry – Oral and Maxillofacial Surgery, University of Bergen, Bergen, Norway; 2Department of Oral and Maxillofacial Surgery, Haukeland University Hospital, Bergen, Norway; 3Hyperbaric Medical Unit, Department of Occupational Medicine, Haukeland University Hospital, Bergen, Norway; 4Department of Clinical Science, University of Bergen, Bergen, Norway; 5Department of Clinical Medicine, The Gade Laboratory for Pathology, University of Bergen, Bergen, Norway; 6 Department of Pathology, Haukeland University Hospital, Bergen, Norway
Key words: angiogenesis; cell proliferation; immunohistochemistry; microvascular tissue; radiotherapy. Accepted for publication 19 December 2014 Available online 17 January 2015
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.7,8 The increased partial pressure of oxygen by HBOT in tissue is thought to induce the necessary oxygen gradient to
# 2015 International Association of Oral and Maxillofacial Surgeons. Published by Elsevier Ltd. All rights reserved.
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stimulate collagen production by fibroblasts9 and capillary angiogenesis.10,11 Experimental animal studies by Marx et al.12 and human studies13 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 models14 and a human dynamic magnetic resonance imaging (MRI) study,15 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.16 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 nonparticipation. 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.16 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. 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
Table 1. Patient characteristics. Subject HBOT group 1 2 3 4 5 6 7 8 9 10 11 12 Mean (range) Controls 1 2 3 4 5 6 7 8 Mean (range)
Radiation dose, Gy
Time since radiation, years
Tonsil Retromolar Tonsil Floor of mouth Tonsil Tonsil Parotid gland Tongue Origo incertaa Buccal Floor of mouth Tonsil
64 60 64 70 70 70 70 70 50 70 64 64 66 (50–70)
3 6 3 3 3 6 6 2 6 5 5 5 4 (2–6)
Gingiva Retromolar Tonsil Retromolar Tongue Floor of mouth Origo incertaa Floor of mouth
70 64 70 64 64 64 50 70 65 (50–70)
6 2 3 4 5 4 2 4 4 (2–6)
Sex
Age, years
Site of cancer
M M M M F F F M M M M M
78 55 66 62 69 51 56 67 63 70 56 51 62 (51–78)
M M M M F F M M
65 55 56 63 55 73 53 62 60 (53–73)
HBOT, hyperbaric oxygen treatment; M, male; F, female.
Hyperbaric oxygen in irradiated tissue 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-mm sections of the samples. All procedures followed a standardized protocol. The sections were deparaffinized in xylene after being heated overnight, not exceeding 58 8C. 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 H2O2 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 Gj2 System
(Dako; K5361), the sections were incubated with the second primary antibody D240 (M3619, 1:50; Dako) in a humidity chamber for 60 min at room temperature, and then incubated overnight at 4 8C, 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).
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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
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 mm 200 mm. The average of at least five fields was reported for vessels/ mm2 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.
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. Microvessel density
Both blood vessel density and blood vessel area were significantly increased after HBOT (P = 0.002–0.041). The effects
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Svalestad et al. Cell proliferation
No significant differences in Ki-67-positive epithelial cells could be found in either the treatment group or the control group (Table 2). Cell proliferation was prominent in the parabasal layer (Fig. 4). Ki-67-positive endothelial cells were an occasional finding after HBOT. Discussion
Fig. 1. Irradiated buccal mucosa before (a) and 6 months after (b) hyperbaric oxygen treatment (HBOT). The sections are stained with haematoxylin and eosin. Scale bar: 200 mm.
were more pronounced in the sub-epithelial area than in the deeper connective tissue (Table 2), and were largely due to an increase in the number of small vessels (diameter <25 mm). A significant increase in the number and area of D2-40-positive
lymphatic vessels could be observed in the sub-epithelial area (P = 0.002 and P = 0.019, respectively). Representative sections are presented in Fig. 3. No significant differences were observed in the control group.
Fig. 2. Proliferating vessels in connective tissue papillae. Scale bar: 200 mm.
A patent network of macro- and microvessels is a prerequisite for exchange of oxygen and nutrients to the healing wound. Therapy stimulating angiogenesis may therefore prove beneficial to support healing in selected clinical situations with compromised microcirculation, e.g. due to radiotherapy. The present study provides evidence of neoangiogenesis by HBOT in human irradiated tissue, and the new vessels were present after 6 months. An increased microvascular density was also reported by Marx et al.13 after a course of 20 HBOT sessions at 2.4 ata. The microvascular density increased from 25–35% to 70– 80% of pre-radiation levels. However, that study did not differentiate between blood vessels and lymphatic vessels, making comparison difficult. Advances made in immunohistochemistry techniques since that study was performed have opened opportunities to easily differentiate between blood and lymph microvessels.17 The present study demonstrated an increased number and area of blood microvessels. This angiogenetic effect has been demonstrated in experimental animal models and involves local oxygen gradients,9,10 up-regulation of growth factors such as vascular endothelial growth factor (VEGF),18 and stimulation of vasculogenic stem cells from the bone marrow.19 Handschel et al.4 reported a marked reduction in microvessels throughout the tissue of the oral mucosa with a compensatory dilation of the deeper vessels at 6– 12 months after radiotherapy. A similar histology with obliterated vessels and some dilated vessels was observed in the present study at baseline, and this may explain the difference in response between the sub-epithelial and deeper connective tissue. An increased tissue oxygenation in skin and vascular capacity in skin and gingival mucosa with the use of HBOT was recently demonstrated in the irradiated tissue in these patients.16 The ability to stimulate an increased blood flow by heat provocation was significantly increased at 3 and 6 months after HBOT. This was suggested
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Hyperbaric oxygen in irradiated tissue Table 2. Measurements of vascularization and cell proliferation (mean SD). HBOT group Baseline Blood vessels Sub-epithelial area MVD 45.4 13.9 MVA 1.5 0.6 Deeper connective tissue MVD 30.4 10.1 MVA 2.5 1.3 Lymph vessels Sub-epithelial area MVD 18.3 8.1 MVA 1.3 0.7 Deeper connective tissue MVD 14.6 7.2 MVA 1.7 1.1 Proliferation index: Ki-67 (%) 23.2 4.5
Controls 6 months
P-value
Baseline
6 months
P-value
98.0 15.9a 4.4 1.9a
0.002 0.003
45.6 15.7 1.5 0.6
49.3 10.5 1.6 0.5
NS NS
45.1 16.4a 3.7 1.3a
0.01 0.041
28.1 9.6 2.2 0.9
34.4 7.8 2.7 1.4
NS NS
36.1 12.6a 2.7 1.8a
0.002 0.019
19.4 6.2 1.2 0.6
16.9 8.8 1.5 0.7
NS NS
16.8 7.0 1.5 0.9
NS NS
13.1 7.0 2.0 0.9
15.0 5.3 1.8 1.1
NS NS
21.8 1.6
NS
19.4 4.0
20.0 4.3
NS
SD, standard deviation; HBOT, hyperbaric oxygen treatment; MVD, microvessel density (vessels/mm2); MVA, microvessel area (%); NS, not significant. a Significantly increased compared to baseline (P < 0.05).
Fig. 3. Irradiated oral mucosa before (a) and 6 months after (b) hyperbaric oxygen treatment (HBOT). The sections are stained with CD31 and D2-40. Blood vessels appear as brown and lymphatic vessels as red. Scale bar: 200 mm.
to be a combined effect of an increased vascular density and improved endothelial function. The present study supports the findings of an increased vascular density, providing improved physiological function of the microvascular network. Teas et al.20 reported a significant increase in lymph angiogenic-associated VEGF-C after HBOT in patients with lymphoedema related to breast cancer treatment. This may also explain the increased lymphatic vessel density in the present study, and this may potentially help alleviate the symptoms related to lymphoedema. Morphological effects on lymphatic vessels in irradiated tissue as a result of HBOT have not been reported previously. There is suggestive evidence in the literature of an effect of HBOT on lymphoedema following radiotherapy.21 This effect may be a result of increased lymphatic clearance by new lymphatic vessels in irradiated tissue. A recent randomized trial evaluating arm lymphoedema after primary surgery and adjuvant radiotherapy for breast cancer could not, however, confirm any benefit of HBOT over best standard care.22 More studies are therefore needed to further explore the potential clinical benefits.21,22 The proliferation index (Ki-67) was high compared to reported values for normal mucosa, but comparable to clinically normal distant cheek mucosa from patients treated for oral squamous cell carcinomas.23,24 All biopsies were taken within the field of maximum irradiation and hence close to a former cancerous mucosa. It has been demonstrated that
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Svalestad et al. Ethical approval
The study protocol was approved by the Regional Committee for Medical Research Ethics in Western Norway (REK Vest), reference 218.05, and the Privacy Ombudsman for Research at the Norwegian Social Science Data Services (NSD). Patient consent
Not required. Acknowledgements. We thank Gunnvor Øijordsbakken for skilful technical assistance and the performance of immunohistochemistry procedures. Fig. 4. Immunostaining with Ki-67. There was prominent cell proliferation in the parabasal layer. Scale bar: 100 mm.
References
such mucosa exhibits a high proliferative status, probably due to genetic alterations according to the field cancerization theory, and that an abnormally high proliferation status is a negative prognostic factor for disease-free survival.23 The proliferation index was not affected by HBOT in the present study. It has been hypothesized that HBOT could lead to higher rates of local recurrence as a result of increased vascularization. An increased proliferation index by HBOT beyond the baseline values in the present study could potentially indicate an increased risk of tumour recurrence. However, such an alteration was not found, and the potential risk of tumour recurrence or potentiation has not been supported by clinical or experimental studies.25 The high proliferation index further indicates a sufficient tissue oxygenation for normal cell homeostasis in irradiated tissue during normal metabolic demand. This is in accordance with clinical experience, as necrosis usually occurs in situations with an increased metabolic demand, e.g. due to a surgical procedure.26 The immunohistochemical methods used in the present study are well established17,27,28 and the methods of assessment were highly reproducible with an ICC of 0.98 and 0.99 for vascular density and the proliferation index, respectively. Taking tissue samples from the buccal oral mucosa proved to be a safe procedure, as all sites healed completely. The subcutaneous emphysema experienced by one patient was probably due to a combination of loose suturing and a faulty technique while performing the Valsalva manoeuvre. Subsequent biopsy sites were sutured watertight, and no further complications were experienced.
1. Sciubba JJ, Goldenberg D. Oral complications of radiotherapy. Lancet Oncol 2006;7:175–83. 2. Abendstein H, Nordgren M, Boysen M, Jannert M, Silander E, Ahlner-Elmqvist. et al. Quality of life and head and neck cancer: a 5 year prospective study. Laryngoscope 2005; 115:2183–92. 3. Stone HB, Coleman CN, Anscher MS, McBride WH. Effects of radiation on normal tissue: consequences and mechanisms. Lancet Oncol 2003;4:529–36. 4. Handschel J, Sunderkotter C, Kruse-Losler B, Prott FJ, Meyer U, Piffko J, et al. Late effects of radiotherapy on oral mucosa in humans. Eur J Oral Sci 2001;109:95–102. 5. Riekki R, Jukkola A, Sassi ML, Hoyhtya M, Kallioinen M, Risteli J, et al. Modulation of skin collagen metabolism by irradiation: collagen synthesis is increased in irradiated human skin. Br J Dermatol 2000;142:874–80. 6. Richter KK, Fink LM, Hughes BM, Sung CC, Hauer-Jensen M. Is the loss of endothelial thrombomodulin involved in the mechanism of chronicity in late radiation enteropathy. Radiother Oncol 1997;44:65–71. 7. Shaw RJ, Butterworth C. Hyperbaric oxygen in the management of late radiation injury to the head and neck. Part II: prevention. Br J Oral Maxillofac Surg 2011;49:9–13. 8. Shaw RJ, Dhanda J. Hyperbaric oxygen in the management of late radiation injury to the head and neck. Part I: treatment. Br J Oral Maxillofac Surg 2011;49:2–8. 9. Hunt TK, Pai MP. Effect of varying ambient oxygen tensions on wound metabolism and collagen synthesis. Surg Gynecol Obstet 1972;135:561–7. 10. Knighton DR, Silver IA, Hunt TK. Regulation of wound-healing angiogenesis—effect of oxygen gradients and inspired oxygen concentration. Surgery 1981;90:262–70. 11. Knighton DR, Hunt TK, Scheuenstuhl H, Halliday BJ, Werb Z, Banda MJ. Oxygentension regulates the expression of angiogenesis factor by macrophages. Science 1983; 221:1283–5.
Oral mucosa is easily available, usually heals uneventfully when no bone is exposed, leaves no visible scars, and is therefore ideal for studies on irradiated human tissue. The patients recruited in the present study represented a heterogeneous sample as there were several indications for HBOT. Although all patients had received substantial radiation doses, the tissue effect might have been variable due to the inter-individual variation in radiosensitivity.29 Severe late effects of radiotherapy are evident in patients with osteoradionecrosis, hence including only patients with osteoradionecrosis might have demonstrated an even more pronounced effect of HBOT. However, the results in the present study reflect the effect on patients referred to a hyperbaric medical unit, and several effects were found significant. Furthermore, as the repeat samples were taken 6 months after HBOT, this allowed the inclusion of possible continuous effects after the completion of therapy, and the results indicate a long-term effect of HBOT. Within the limitations of this study, it is concluded that the density and area of blood and lymphatic microvessels in irradiated mucosa are increased by HBOT at 6 months after therapy. Epithelial cell proliferation is not affected by HBOT. Funding
The present study was supported by grants from the University of Bergen, ColgatePalmolive, and the L. Meltzer Foundation. Competing interests
None declared.
Hyperbaric oxygen in irradiated tissue 12. Marx RE, Ehler WJ, Tayapongsak P, Pierce LW. Relationship of oxygen dose to angiogenesis induction in irradiated tissue. Am J Surg 1990;160:519–24. 13. Marx RE, Johnson RP. Studies in the radiobiology of osteoradionecrosis and their clinical-significance. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 1987;64:379–90. 14. Muhonen A, Haaparanta M, Gronroos T, Bergman J, Knuuti J, Hinkka S, et al. Osteoblastic activity and neoangiogenesis in distracted bone of irradiated rabbit mandible with or without hyperbaric oxygen treatment. Int J Oral Maxillofac Surg 2004;33: 173–8. 15. Store G, Smith HJ, Larheim TA. Dynamic MR imaging of mandibular osteoradionecrosis. Acta Radiol 2000;41:31–7. 16. Svalestad J, Thorsen E, Vaagboe G, Hellem S. Effect of hyperbaric oxygen treatment on oxygen tension and vascular capacity in irradiated skin and mucosa. Int J Oral Maxillofac 2014;43:107–12. 17. Kahn HJ, Bailey D, Marks A. Monoclonal antibody D2-40, a new marker of lymphatic endothelium, reacts with Kaposi’s sarcoma and a subset of angiosarcomas. Mod Pathol 2002;15:434–40. 18. Sheikh AY, Gibson JJ, Rollins MD, Hopf HW, Hussain Z, Hunt TK. Effect of hyperoxia on vascular endothelial growth factor levels in a wound model. Arch Surg 2000; 135:1293–7. 19. Thom SR, Bhopale VM, Velazquez OC, Goldstein LJ, Thom LH, Buerk DG. Stem
20.
21.
22.
23.
24.
cell mobilization by hyperbaric oxygen. Am J Physiol Heart Circ Physiol 2006;290: H1378–86. Teas J, Cunningham JE, Cone L, Jansen K, Raghavan SK, Nitcheva DK, et al. Can hyperbaric oxygen therapy reduce breast cancer treatment-related lymphedema? A pilot study. J Womens Health 2004;13:1008–18. Gothard L, Stanton A, MacLaren J, Lawrence D, Hall E, Mortimer P, et al. Nonrandomised phase II trial of hyperbaric oxygen therapy in patients with chronic arm lymphoedema and tissue fibrosis after radiotherapy for early breast cancer. Radiother Oncol 2004;70:217–24. Gothard L, Haviland J, Bryson P, Laden G, Glover M, Harrison S, et al. Randomised phase II trial of hyperbaric oxygen therapy in patients with chronic arm lymphoedema after radiotherapy for cancer. Radiother Oncol 2010;97:101–7. Montebugnoli L, Badiali G, Marchetti C, Cervellati F, Farnedi A, Foschini MP. Prognostic value of Ki67 from clinically and histologically ‘normal’ distant mucosa in patients surgically treated for oral squamous cell carcinoma: a prospective study. Int J Oral Maxillofac Surg 2009;38:1165–72. Montebugnoli L, Gissi DB, Badiali G, Marchetti C, Cervellati F, Farnedi A, et al. Ki-67 from clinically and histologically ‘normal’ distant mucosa as prognostic marker in early-stage (T1–T2N0) oral squamous cell carcinoma: a prospective study. J Oral Maxillofac Surg 2011;69:2579–84.
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25. Feldmeier J, Carl U, Hartmann K, Sminia P. Hyperbaric oxygen: does it promote growth or recurrence of malignancy. Undersea Hyperb Med 2003;30:1–18. 26. Reuther T, Schuster T, Mende U, Kubler A. Osteoradionecrosis of the jaws as a side effect of radiotherapy of head and neck tumour patients—a report of a thirty year retrospective review. Int J Oral Maxillofac Surg 2003;32:289–95. 27. Lindboe CF, Torp SH. Comparison of Ki-67 equivalent antibodies. J Clin Pathol 2002; 55:467–71. 28. Fukunaga M. Expression of D2-40 in lymphatic endothelium of normal tissues and in vascular tumours. Histopathology 2005;46: 396–402. 29. Tucker SL, Geara FB, Peters LJ, Brock WA. How much could the radiotherapy dose be altered for individual patients based on a predictive assay of normal-tissue radiosensitivity. Radiother Oncol 1996;38:103–13.
Address: Jarle Svalestad Department of Clinical Dentistry Faculty of Medicine and Dentistry University of Bergen A˚rstadveien 19 N-5009 Bergen Norway Tel: +47 55586560; Fax: +47 55586577 E-mail: [email protected]