METODOLOGÍA TRADICIONAL DE PLANIFICACIÓN A CORTO

It is well established that the oxygenation o f tumour tissue compared to normal tissue is considerably lower. The data presented in this chapter are in agreement with this observation, with 23 o f the 24 tumours exhibiting median pOi values o f less than 1% oxygen (7.6 mmHg). In contrast, the median pOi for unanaesthetised mouse subcutis averaged approximately 30 mmHg, whilst normal tissue measurements in three strains o f anaesthetised rat indicated median pO: ^ o f greater than 15 mmHg.

The rat P22 tumour had an oxygenation status comparable with that obtained for the liver and kidney in the same strain o f animal. This suggests that it has either a strong angiogenic influence on the host vessels, or it’s metabolic activity is low compared to the other tumours, whose rate may be higher than subcutis. Tumours have been reported to have higher rates o f metabolism and thus oxygen utilisation compared to normal tissue (Vaupel, 1989a), and this may explain low extracellular oxygen tensions. Although the P22 was comparatively well oxygenated, the results did indicate that regions o f cells were existing in radiobiological hypoxia (20% readings <2.5 mmHg).

Oxygen data obtained for the murine RIF-1 tumour provides indirect evidence of angiogenic activity. Tumours are known to release angiogenic factors (e.g. TAP, Folkman, 1971) which can stimulate neovascularisation, and without a developing blood supply solid tumours can not grow beyond 1 - 2 cubic millimetres. Table 2.2 shows pOz values obtained for RIF-1 tumours grown in a nude strain o f mouse and two different sub-strains o f C3H mice. Since it is reasonable to assume that the consumption o f these cells would remain constant irrespective o f the host, the fact that the oxygenation values are not significantly different may suggest a similar vascular supply, i.e. a level of angiogenesis that is dictated by the tumour and not the host.

Data within this chapter highlight the importance o f anaesthesia on tissue oxygenation. All mouse measurements were performed without anaesthetic, however, an assessment o f the effect o f combining diazepam with ketamine (a combination used in some experiments in later chapters) was shown to reduce CBA subcutis median p02 by 17%, whilst the corresponding reduction in the SaF tumour was much higher at 93%.

Diazepam and ketamine have been reported to reduce blood pressure and heart rate in C3H Km male mice, cause radioprotection in the RIF-1 tumour (Cullen and Walker, 1985), but radiosensitise the C3H mouse mammary adenocarcinoma (Tozer et al, 1984). Similar findings have also been reported for DS-carcinosarcomas in Sprague-Dawley rats, were diazepam alone reduced mean arterial pressure and tumour vascular resistance but had little effect on tumour blood flow (Menke and Vaupel, 1988). Studies by Nias et a l (1986, 1988) are in agreement, diazepam and ketamine are shown to have little effect on the perfusion o f C3H tumours, whilst increasing perfusion o f the gut, liver and kidney. Furthermore, the report also provides evidence that tumour metabolism is decreased under anaesthesia, but this may be due to a combination o f both cellular and thermal effects. The two agents are shown to reduce core temperature o f the mouse by 10°C within 60 minutes, and it has been suggested that metabolism decreases by 6% for every °C fall in temperature (Little, 1972). Thus the anaesthetic effect on cellular metabolism may be caused by anaesthesia-induced hypothermia. However, it should be noted that the doses used in the above studies were different to those used in this chapter, and moreover the investigations were performed on different tumours and host animals. Thus the results may not necessarily be comparable.

There are three possible explanations for the effects o f anaesthesia seen in the SaF and CBA subcutis in this chapter. (1) Poiseuille’s law states that blood flow is related to the mean arterial pressure divided by the vascular resistance (Levick, 1992). The studies above showed increases in normal tissue perfusion under diazepam and ketamine, and this is consistent with the fact that some anaesthetics cause vasodilation. As a consequence vascular resistance is decreased, probably to a larger extent in normal tissues because o f the greater abundance o f vasoactive blood vessels. As mean arterial pressure is constant, albeit lower than in unanaesthetised animals, a larger decrease in

normal tissue vascular resistance relative to tumour tissue will result in higher blood flow to the normal tissue. Thus pOz in the tumour will appear lower than in the skin. (2) Nias and colleagues (1988) showed that tumour metabolism was decreased by anaesthesia, however they did not examine the extent o f the reduction in comparison to the normal tissue. Some normal tissues are known to have lower metabolic activity than tumours (Vaupel, 1989a) and thus the effect o f anaesthesia may appear even more pronounced in these tissues. Therefore the oxygen consumption could be less in the normal tissue resulting in an apparently higher extracellular pOz compared to the tumour. (3) Tumours are poorly vascularised compared to normal tissue. For example, rectal adenocarcinoma is 2.5 times less vascularised than normal mucosa (Vaupel, 1989a). Thus a small effect on normal vessel dilation could translate into a larger effect on the tumour, possibly resulting in a vascular-steal reducing pOz relative to the normal tissue.

pOi measurements with needle electrodes may overestimate hypoxia if the tumour contains large regions o f necrosis. Khalil and colleagues (1995) reported that an increase in necrotic fraction o f a mouse mammary carcinoma correlated with increasing tumour size and decreasing tumour oxygenation. A reduction in tumour pO: with increasing tumour size has also been reported in other tumours (Kallinowski et al, 1989a). The necrotic fractions examined in this chapter ranged from 0.1% to 8.3%, and did not significantly alter the proportion o f values less than 2.5 mmHg. The size o f the murine tumours used in all these studies were stringently maintained between 100 - 400 mg, whereas in the Khalil study, the largest necrotic fractions (40 - 50%) were seen in tumours approaching a mass o f 1 g. The five tumour models examined here, have inherently lower necrotic fractions than the C3H tumour, and the fact that all experiments were performed on a narrow range o f tumour sizes is probably the main reason why the influence o f necrosis was not significant.

The accuracy o f the Eppendorf pOi histograph was assessed by performing measurements in nitrogen asphyxiated animals. Readings in the SaF tumour did not exceed -1.5 mmHg, and in the skin, -2.0 mmHg. These values are in agreement with the manufacturers reported error for the machine o f ± 2.5 mmHg. Although this confidence

limit suggests that pOi readings are not absolute, because the error is a constant factor, the measurements retain intercomparability.

2.4 Summary

This chapter demonstrates the use o f the Eppendorf pOz histograph in vivo and data are presented to show that the accuracy o f the machine is within the manufacturers claims. Twenty-four experimental tumours were assessed and shown to contain oxygen tensions compatible with radiobiological hypoxia. Comparatively, subcutis, liver and kidney were shown to be well oxygenated. The influence o f necrosis on oxygen measurements in 5 murine tumours was not significant, but the use o f a general anaesthetic could dramatically reduce tissue pOi It is recommended that pO] measurements should be performed on conscious subjects whenever possible to eliminate a major source o f artefact, which can be variable according to individual anaesthesia schedules.

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Figure 2.4: Oxygen Tension in the SaF tumour (upper panel) and CBA rear dorsum subcutis (lower panel) 10 minutes after nitrogen asphyxiation. Upper panel: N = 5; n = 306; Median -0.1 mmHg (±0.04); values < 2 . 5 mmHg = 100%. Lower panel: N = 5; n = 139; Median = -0.2 mmHg (±0.1); values < 2 . 5 mmHg - 98.7% (±0.8). Numbers in

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