Journal of Neuro-Oncology 58: 47–52, 2002. © 2002 Kluwer Academic Publishers. Printed in the Netherlands.

Clinical Study

Change of oxygen pressure in glioblastoma tissue under various conditions Takaaki Beppu1 , Katsura Kamada2 , Yuki Yoshida1 , Hiroshi Arai1 , Kuniaki Ogasawara1 and Akira Ogawa1 Department of Neurosurgery, 2 Division of Hyperbaric Medicine, Iwate Medical University, Morioka, Japan

1

Key words: oxygen pressure, glioblastoma, radiosensitivity, hyperbaric oxygen, oxygenation Summary Measurement of oxygen pressure (pO2 ) in tumor tissue is important, because pO2 is a major factor for radiosensitivity in malignant glioma treatment. We attempted to elucidate the changes in pO2 level in glioblastoma tissue of patients under various conditions. Eighteen patients with newly diagnosed glioblastoma were recruited to this study. Disposable Clark-type electrodes were inserted using CT guided stereotactic surgery under local anesthesia and left in the intra- and peritumoral regions. pO2 was measured in patients under conditions of being awake and asleep, inhaling 100% O2 , being administered osmotic diuretics and following hyperbaric oxygen exposure (HBO). Peritumoral tissue had a significantly higher pO2 value in both awake and sleeping patients. O2 inhalation could not significantly increase the pO2 level, whereas administration of osmotic diuretics induced an increase in pO2 levels in peritumoral tissue alone. The pO2 levels were significantly increased in both regions after HBO, and a high pO2 level was maintained until 15 min after HBO in both regions. It is possible that the pO2 level in peritumoral tissue is affected by intracranial pressure, whereas that in the intratumoral tissue is usually low. HBO was the optimal procedure for oxygenation, but its benefit was reduced over time.

Introduction Hypoxic cells evident in various malignant tumors adversely affect the radiosensitivity of tumor cells and have been identified as a major reason for the radioresistance of malignant tumor tissue [1–4]. Some protocols of oxygenation for hypoxic cells have been evaluated in attempts to enhance the radiosensitivity in treatment of malignant glioma cells [5,6]. Accordingly, measurement of the oxygen pressure (pO2 ) is important, because it is related to each patient’s response to radiation therapy and also clarifies the natural environment of malignant gliomas. Some recent studies of the direct measurement of pO2 in patients with brain tumors have been undertaken [7–10]. However, these measurements were performed intraoperatively under general or local anesthesia only, despite the finding that the pO2 level in the brain tumor must be affected by various waking, sleeping and treatment conditions. It is very important to elucidate the pO2 levels in patients with malignant glioma under various conditions, because patients are treated with radiation therapy under conditions without anesthesia. We describe

here the direct stereotactic measurement of pO2 in glioblastoma tissue of patients under various conditions of being awake or asleep in room air, after inhaling 100% O2 , being administered osmotic diuretics, and following hyperbaric oxygen exposure (HBO).

Methods and materials Patients The study protocol was approved by the Ethics Committee of Iwate Medical University, Morioka, Japan. The patients recruited to this study were hospitalized in the Department of Neurosurgery, Iwate Medical University at some point between January 1999 and July 2001. Entry criteria for this study were: (A) a diagnosis of WHO grade 3 or 4 glioma based upon the findings of preoperative neuroimaging, (B) probably requiring greater than 95% resection of tumor bulk and (C) having given informed, written consent. Eighteen patients with newly diagnosed glioblastoma (12 males and 6 females; mean age 55.2 years;

48 age range 42–70 years) subsequently participated in this study. Insertion of electrodes The pO2 level was measured using two disposable Clark-type electrodes (U0E-04TS, Unique Medical Co., Tokyo, Japan [11]) composed at the tip of a sensor (diameter 0.4 mm, length 10 mm of Teflon tube coating) and followed by a 35-mm stainless steel coating. Electrodes were sterilized by immersion in a solution of 2.25 W/V% glutaraldehyde and buffer for 2 h and were subsequently washed with sterilized physiologic saline solution. Each electrode was then connected to a digital pO2 monitor (POG-203, Unique Medical Co., Tokyo, Japan) to calibrate the value of pO2 to 150 mmHg in a physiologic saline solution immediately prior to insertion into the brain. The pO2 measuring materials used in the present study were able to monitor the absolute value differing from other existing products. Two electrodes were inserted during CT-guided stereotactic surgery under local anesthesia; one into the peritumoral region between the enhanced and non-enhanced area of the lesion as visualized on the CT scan and the other into the intratumoral region of the tumor. Enhanced CT scans were performed postoperatively to confirm whether the electrodes were correctly positioned. Measurement of pO2 After stereotactic surgery, pO2 was measured under the following conditions: (a) breathing room air while awake and asleep; (b) after inhalation of 100% O2 at 3 or 6 l/min via a mask for 15 min while awake; (c) during intravenous administration of osmotic diuretics (200 ml of glycerin) at a speed of 200 ml/h; and (d) for 50 min after HBO decompression. The pO2 level was sequentially measured, although it took about 5 min for a stabilized value to be indicated on the monitor. During osmotic diuretic administration, pO2 was measured at 20-ml intervals. HBO occurred in a hyperbaric chamber according to the following schedule: 20 min of compression with air, 60 min of 100% O2 inhalation via a mask at 2.8 atmosphere absolute and 20 min of decompression with 100% O2 inhalation. After decompression of HBO, pO2 was determined at 5-min intervals between 10 and 50 min after HBO. All measurements were carried out with the patients in the supine position.

To confirm that there were no complications such as hemorrhage, sequential CT scans were performed daily until the electrodes were removed. Daily blood counts and serum examinations were also performed so that the condition of each patient could be monitored. Within 7 days of CT-guided stereotactic surgery, a gross resection of the tumor was performed and the electrodes were removed from the brain. Immediately upon removal, the postoperative pO2 value was determined by immersing the microelectrode in a physiological saline solution. When this value was compared with that at 150 mmHg, if there was a lag value, the surveyed pO2 values under various conditions were revised according to the following formula: Revised pO2 value = Surveyed pO2 value − (postoperative pO2 value − 150) × (T2/T1) where T1 (hours) is the interval between the pO2 calibration immediately prior to insertion and the postoperative pO2 value measurement, and T2 (hours) is the interval between pO2 calibration immediately prior to insertion and measurement of the surveyed pO2 value. Statistic analyses The mean pO2 values of all patients under each condition were calculated and compared. Under condition (A), the mean pO2 value for awake and sleeping patients while breathing room air were compared within each target as well as between the 2 targets. Statistically significant differences were then determined using the Mann–Whitney’s U test. Under condition (B), the mean values before and after inhalation of 100% O2 for 15 min at 3 or 6 l/min at each target were compared. Significant differences were determined using the Wilcoxon signed-rank test. Under condition (C), the mean pO2 values for every 20-ml administration was calculated in each target, and the maximum mean value was compared with that before administration using the Wilcoxon signed-rank test. Under condition (D), the mean pO2 values at 5-min intervals were compared with that before HBO using the Wilcoxon signed-rank test. In conditions (C) and (D), differences in the time course were assessed by repeated measures ANOVA. A difference in the values at each time point between the 2 groups was analyzed by the Bonferroni method of multiple comparison tests. A p-value less than 0.05 was considered significant for all statistical analyses.

49 Results Electrodes were inserted into the peritumoral region in all patients. However, only 16 of 18 patients underwent insertion to the intratumoral region, because preoperative angiograms of 2 patients indicated hypervascularity suggestive of a risk of post-insertion hemorrhage. All electrodes were precisely positioned, and there was no mortality, morbidity or side effects following surgery (Figure 1). Blood counts and serum examinations did not indicate the presence of any severe infections or inflammations prior to resection. The electrodes inserted into the peritumoral region acted as a navigator, which could determine the margin of the tumor, during resection of the tumor. pO2 values varied widely among patients, conditions and targets. Under condition (A), the peritumoral region showed a significantly higher mean pO2 value than the intratumoral region, both awake and sleeping while breathing room air. In both regions, the values

under awake were higher than those under sleeping, although the differences were not significant (Table 1). Under condition (B), there were no significant differences in increasing pO2 under 100% O2 inhalation with 3 or 6 l/min for 15 min (Table 2). As the osmotic diuretics were intravenously administered in condition (C), the pO2 values increased gradually in both targets. The maximum mean pO2 values in the peritumoral and intratumoral regions were 15.8 ± 11.2 mmHg after 120 ml had been administered and 9.2 ± 7.9 mmHg after 160 ml, respectively (Figure 2). When the mean Table 1. Mean pO2 values (mmHg) of while awake and sleeping in room air

Peritumoral Intratumoral p-value∗

Awake

Sleeping

p-value∗

17.9 ± 9.3 9.2 ± 5.8 <0.05

13.3 ± 4.7 7.5 ± 5.0 <0.05

NS NS

∗ A p-value less than 0.05 was considered significant. NS = not significant.

Table 2. Comparisons of mean pO2 values between before and after 100% O2 inhalation at 3 or 6 l/min in each region 3 l O2 Peritumoral Intratumoral 6 l O2 Peritumoral Intratumoral

Before

After

p-value∗

14.8 ± 9.3 11.2 ± 10.8

15.6 ± 9.5 12.2 ± 13.4

NS NS

14.0 ± 9.2 10.7 ± 11.1

16.5 ± 9.1 11.0 ± 12.2

NS NS

∗ A p-value less than 0.05 was considered significant. NS = not significant.

Figure 1. CT scan showing electrodes inserted into the intratumoral region (arrowhead) and the peritumoral region (arrow).

Figure 2. Changes in the mean pO2 value during measurement of the administration of osmotic diuretics at 20-ml intervals. ( ) Peritumoral region; (
) intratumoral region.

•

50

Figure 3. Comparison between the mean pO2 value before and after HBO decompression for each region. ( ) Peritumoral region; (
) intratumoral region.

•

pO2 value was compared for the preadministration and maximum value, there was a significant difference in the peritumoral region (p < 0.05), but not in the intratumoral region. Moreover, the pO2 in the peritumoral region of the tumor showed a significant increase over time. Under condition (D), the pO2 levels increased significantly in both regions, and then declined slowly over time. The mean values for both regions reached the pre-HBO value after approximately 40 min (Figure 3). The mean pO2 value for the peritumoral region was significantly higher than that before HBO until 35 min, whereas that for the intratumoral region was until 30 min. The pO2 value in the peritumoral region was significantly higher than that in the intratumoral region until 25 min after HBO decompression. Discussion No complications or side effects were caused by the placement or removal of electrodes in the present study and, in fact, the electrodes provided the additional benefit of acting as a navigator for tumor resection during surgery. Accordingly, it is suggested that the protocol of the present study allowed for the safe measurement of pO2 levels in tumor tissue under various conditions. Some studies have successfully used the same electrodes as in the present study to measure pO2 levels in other human structures [12,13]. It is possible that the electrodes inserted into the brain could lead to brain injury (e.g. brain edema and microhemorrhage) or its effect on tissue oxygenation over

time. However, the effects to pO2 would be very little because the brain edema caused by glioblastoma was very large. In addition, the electrodes used for this study were Teflon coated to avoid adhesion of blood cells. Some previous studies reported direct pO2 measurement in intratumoral tissue of the brain during surgery under local or general anesthesia. Kayama et al. [8] found the mean pO2 values of 16 various brain tumors was 15.3±2.3 mmHg. Moriglane [9] reported pO2 in 12 various brain tumors to range from 36 to 200 mmHg. In a series limited to glioblastoma alone, the median pO2 level was reported as 7.4 mmHg by Rampling et al. [10] and 5.6 mmHg by Collingridge et al. [7]. The present results of 9.2 ± 5.8 and 7.5 ± 5.0 mmHg in awake and sleeping patients, respectively, are therefore similar to the findings of less than 10 mmHg by Rampling et al. [10] and Collingridge et al. [7] (Table 1). We found that the mean pO2 values in the intratumoral region was slightly and significantly lower than that in the peritumoral region in awake and sleeping patients, respectively. Kayama et al. [8] measured intratumoral pO2 during large craniotomy surgery under general anesthesia, and found that the mean pO2 value in the brain tissue around the tumor and in the intratumoral region were 59.8 ± 6.5 and 15.3 ± 2.3 mmHg, respectively. Although the histology differed among the patients in Kayama et al.’s study [8], the hypoxic areas were located in the intratumoral region rather than in the peritumoral region or brain tissue around the tumor. The findings in the present study were not in conflict with their observations. In the present study, the mean pO2 value of sleeping patients was slightly lower than while they were awake in both targets, although there were no significant differences (Table 1). It was originally presumed that these differences between awake and sleeping states were caused by the change in PaO2 in arterial blood and/or intracranial pressure, because PaO2 decreases and intracranial pressure increases during sleeping. Nevertheless, some studies suggested that the intratumoral pO2 level was not associated with PaO2 [7–8]. The present findings that no significant increases were found in 100% O2 inhalation at 3 or 6 l/min for 15 min at least agreed with this hypothesis (Table 2). In contrast, administration of osmotic diuretics reoxygenated only the peritumoral region, which showed a significantly increased pO2 value (Figure 2). These results suggested that the intracranial pressure probably influenced the peritumoral region alone, whereas unsatisfactory oxygenation such as 100% O2 inhalation with

51 3 or 6 l/min did not affect the peritumoral region or the intratumoral region. The presence of hypoxic cells in tumor tissue reportedly consists of both ‘chronic’ and ‘acute’ hypoxic cells; the former state arising because of the distance of functional capillary vessels and the latter because of the intermittent nutrient deprivation due to transient occlusion of capillary vessels [2,14]. Therefore, the pO2 level in tumors is affected largely by blood supply from capillary vessels. In the present study, intracranial pressure decompression using osmotic diuretics might lead to improve transient occlusion of capillary vessels in the peritumoral region, which is attributable to inadequate blood flow from the compression of capillary vessels by both a mass effect from the tumor and peritumoral edema [7,15]. The present hypothesis is supported by the reported correlation between tumor size and frequency of transient occlusion of vessels in an animal experiment [16]. Radiosensitivity decreases significantly when the pO2 level is 30 mmHg or less, and is halved if it falls below approximately 3 mmHg [10,17]. HBO appears to be a valid method to reoxygenate malignant brain tumors [3,5,6] and other cancers [18–21], because HBO strongly increases oxygen dissolving into blood [22]. In the HBO–radiation combination therapy, fractionated radiation has been consistently performed after HBO in an attempt to optimize malignant glioma treatment, and has been rewarded with good results [3,6,23]. The underlying premise of this treatment is that the oxygen consumption rate is reportedly lower in tumors than it is in normal white matter [6,24]. The present finding that the pO2 levels declined slowly in glioblastoma tissues supports this hypothesis, whereas a difference in pO2 consumption between 2 regions was not found (Figure 3). Kohshi et al. [6] reported that the response of HBO–radiation combination therapy was affected by the time between completion of HBO and irradiation. In their study, all responders received radiation within 15 min after HBO, whereas patients irradiated 30 min after HBO showed no tumor regression. However, their interesting findings have not been explained by any investigations of the relationship between the actual pO2 level and the time after HBO in glioma patients. In the present study, the mean pO2 values for both regions were greater than 30 mmHg, obtaining maximum radiosensitivity, at 15 min after HBO, and were less than 30 mmHg at 25 min after HBO. It was suggested that these findings do not conflict with the above-mentioned observations of Kohshi et al. [6].

In conclusion, the present study clarified that the pO2 level changes among various conditions or regions. Furthermore, the peritumoral tissue is affected by intracranial pressure, whereas that in the intratumoral tissue is usually low. Furthermore, HBO allowed for optimal oxygenation of glioblastoma tissue, but its benefit was reduced over time. We believe that the present findings help to elucidate the physiological characteristics of glioblastoma. Acknowledgement This work was supported in part by a Grant-in-Aid for Advanced Medical Science Research from the Ministry of Science, Education, Sports and Culture, Japan. References 1. Dowling S, Fischer JJ, Rockwell S: Fluosol and hyperbaric oxygen as an adjunct to radiation therapy in the treatment of malignant gliomas: a pilot study. Biomater Artif Cells Immobilization Biotechnol 20: 903–905, 1992 2. Horsman MR: Nicotinamide and other benzamide analogs as agents for overcoming hypoxic cell radiation resistance in tumours. A review. Acta Oncol 34(5): 571–587, 1995 3. Kohshi K, Kinoshita Y, Terashima H, Konda N, Yokota A, Soejima T: Radiotherapy after hyperbaric oxygenation for malignant gliomas: a pilot study. J Cancer Res Clin Oncol 122: 676–678, 1996 4. Thomlinson RH, Gray LH: The histological structure of some human lung cancers and the possible implications for radiotherapy. Br J Cancer 9: 539–549, 1995 5. Chang CH: Hyperbaric oxygen and radiation therapy in the management of glioblastoma. Natl Cancer Inst Monogr 46: 163–169, 1977 6. Kohshi K, Kinoshita Y, Imada H, Kunugita N, Abe H, Terashima H, Tokui N, Uemura S: Effects of radiotherapy after hyperbaric oxygenation on malignant gliomas. Br J Cancer 80: 236–241, 1999 7. Collingridge DR, Piepmeier JM, Rockwell S, Knisely JP: Polarographic measurements of oxygen tension in human glioma and surrounding peritumoural brain tissue. Radiother Oncol 53: 127–131, 1999 8. Kayama T, Yoshimoto T, Fujimoto S, Sakurai Y: Intratumoral oxygen pressure in malignant brain tumor. J Neurosurg 74: 55–59, 1991 9. Moriglane JR: Measurement of oxygen partial pressure in brain tumors under stereotactic conditions. Adv Exp Med Biol 345: 471–477, 1994 10. Rampling R, Cruickshank G, Lewis AD, Fitzsimmons SA, Workman P: Direct measurement of pO2 distribution and bioreductive enzymes in human malignant brain tumors. Int J Radiat Oncol Biol Phys 29: 427–431, 1994 11. Clark LC Jr: Monitor and control of blood and tissue oxygen tensions. Trans Amer Soc Art Int 2: 41–46, 1956

52 12. Tanaka M, Hanioka T, Takaya K, Shizukuishi S: Association of oxygen tension in human periodontal pockets with gingival inflammation. J Periodontol 69: 1127–1130, 1998 13. Okubo J, Watanabe K, Harada H, Teramura K, Koyama S, Ishikawa N, Ogawa A, Hanari K, Watanabe I: O2 tension changes in the tympanic cavity and role of the capillary structure in human mastoid cells (in Japanese). Jibirinsho 80: 1521–1527, 1987 14. Brown JM: Evidence for acutely hypoxic cells in mouse tumours, and a possible mechanism of reoxygenation. Brit J Radiol 52: 650–656, 1979 15. Cruickshank GS, Rampling R: Does tumour related oedema contribute to the hypoxic fraction of human brain tumours? Acta Neurochir (Wien) 60: 378–380, 1994 16. Chaplin DJ, Durand RE, Olove PL: Acute hypoxia in tumours: implication for modifiers of radiation effects. Int J Radiat Oncol Biol Phys 12: 1279–1282, 1986 17. Gray LH, Cogner AD, Ebert M, Hornsey S, Scott OCA: The concentration of oxygen dissolved in tissues at the time of irradiation as a factor in radiotherapy. Br J Radiol 26: 638–648, 1953 18. Kalns JE, Piepmeier EH: Exposure to hyperbaric oxygen induces cell cycle perturbation in prostate cancer cells. In Vitro Cell Dev Biol Anim 35: 98–101, 1999 19. Haffty BG, Hurley RA, Peters LG: Carcinoma of the larynx treated with hypofractionated radiation and hyperbaric oxygen: long-term tumor control and complications. Int J Radiat Oncol Biol Phys 45: 13–20, 1999

20. Haffty BG, Hurley R, Peters LJ: Radiation therapy with hyperbaric oxygen at 4 atmospheres pressure in the management of squamous cell carcinoma of the head and neck: results of a randomized clinical trial. Cancer J Sci Am 5: 341–347, 1999 21. Voute PA, van der Kleij AJ, De Kraker J, Hoefnagel CA, Teil-van Buul MM, Van Gennip H: Clinical experience with radiation enhancement by hyperbaric oxygen in children with recurrent neuroblastoma stage IV. Eur J Cancer 31: 596–600, 1995 22. Saunders M, Dische S: Clinical results of hypoxic cell radiosensitization from hyperbaric oxygen to accelerated radiotherapy, carbogen and nicotinamide. Br J Cancer 74(Suppl. XXVII): 271–278, 1996 23. Kinoshita Y, Kohshi K, Kunugita N, Tosaki T, Yokota A: Preservation of tumour oxygen after hyperbaric oxygenation monitored by magnetic resonance imaging. Br J Cancer 82: 88–92, 2000 24. Jamieson D, van den Brenk HAS: Measurement of oxygen tensions in cerebral tissues of rats exposed to high pressures of oxygen. J Appl Physiol 18: 869–876, 1963

Address for offprints: Takaaki Beppu, Department of Neurosurgery, Iwate Medical University, Uchimaru 19-1, Morioka, 020-8505, Japan; Tel.: +81-196-51-5111 (ext.: 6603); Fax: +81-196-25-8799; E-mail: [email protected]