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Acute effects of low-dose cranial irradiation on regional capillary permeability in experimental brain tumors
Journal of the Neurological Sciences, 1989, 90:147-153 Elsevier
147
JNS 03139
Acute effects of low-dose cranial irradiation on regional capillary permeability in experimental brain tumors Jean Yves Delattre, William R. Shapiro and Jerome B. Posner Cotzias Laboratory of Neuro-Oncology and Departments of Neurology, Memorial Sloan-Kettering Cancer Center and Cornell University Medical College, New York, NY (U.S.A.) (Received 27 May, 1988) (Accepted 30 November, 1988)
SUMMARY
To determine the acute effects of low-dose cranial irradiation (CRT) on regional capillary permeability (RCP) of normal brain, brain tumor and damaged brain surrounding the tumor, we used quantitative autoradiography (QAR) to measure regional blood-to-tissue transport (K) of [ ~4C]aminoisobutyric acid (AIB) in experimental C6 brain tumors 3-4 h after a single dose of 3 Gy CRT. K increased 63 ~o in cortex, 30 ~o in basal ganglia and 31 ~o in brain surrounding the tumor (B ST) vs. controls (P < 0.005). K did not change in the tumor or in the brain adjacent to the tumor (BAT), suggesting that capillaries of normal parenchyma are more sensitive to the acute effects of CRT than capillaries of damaged parenchyma or tumor.
Key words: Experimental brain tumor; Cranial irradiation; Blood-brain barrier
Using quantitative autoradiography (QAR) with [~4C]aminoisobutyric acid (AIB), we found that therapeutic doses of cranial irradiation (CRT) 3 Gy in one fraction, or 30 Gy in 10 fractions over 10 days, induced a significant increase in regional bloodto-brain transport of AIB in normal rats, i.e. it "opens" the blood-brain barrier (BBB) (Phillips et al. 1987). AIB is a small neutral amino acid which crosses the normal BBB Correspondence to: Dr. Jerome B. Posner, Department of Neurology, Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, New York, NY 10021, U.S.A. 0022-510X/89/$03.50 © 1989 Elsevier Science Publishers B.V. (Biomedical Division)
148 slowly but is rapidly taken up, concentrated and "trapped" by parenchymal cells (Blasberg et al. 1983). If capillaries of edematous brain around a tumor are more radiosensitive than normal parenchyma, as suggested by Schetler and Shealy (1970), CRT could have potentially useful (selective opening of the BBB, allowing better entry of water-soluble chemotherapeutic agents) or harmful (increased risk of edema) consequences. We undertook to study the acute effects of a single dose of 3 Gy CRT in an experimental model of brain tumor.
MATERIALAND METHODS
Tumor and animal inoculation C6 glioma was purchased from the American Type Culture Collection (ATCC; Rockville, MD) and maintained in tissue culture with McCoy's 5A medium containing 10~o fetal calf serum at 37 °C and 5% CO 2 in a humidified incubator. Cells from confluent flasks were harvested with 0.5 % trypsin and 0.2 ~o EDTA in Hank's balanced salt solution, centrifuged and resuspended in their media without serum but containing 1 ~o agar. Male Wistar rats weighing 300-400 g were anesthetized with chloral hydrate (35 rng/kg intraperitoneally). A 2~o lidocaine gel was applied to the rat's ear and the head was affixed in a stereotaxic apparatus (Model 900, David Kopf Instruments, Tujunga, CA). The skull was exposed by a midline scalp incision, and one shallow 26-gauge needle puncture was made 4 mm to the fight of the sagittal suture and 1 mm posterior to the coronal suture. Ten/21 of a suspension containing 5 x 105 viable C6cells was inoculated to a depth of 6 mm through the pre-bored hole.
Irradiation protocol At day 10, 6 inoculated rats were irradiated while 6 others served as controls. The unanesthetized animals were kept immobile in a holder, The oral cavity and the part of the body situated behind the ears were shielded by a 5-ram thick sheet of lead. 3 Gy in one fraction was delivered to the head using a GE Maxitron x-ray generator (20 mA, HVL 2 mm Cu, FSD 50 cm). The 300 kV energy was delivered at a rate of 1.04 Gy/min to a lateral field. In order to obtain a homogeneous dose distribution in the brain, the animals were turned 180 ° halfway through the exposure.
Quantitative autoradiography (QAR) The rats were anesthetized and the right femoral artery and vein were cannulated. 100 izCi of 0t-[1-1aC]AIB was injected into the femoral vein. Timed blood samples were centrifuged and the radioactivity of 20 #1 of plasma was measured with a scintillation counter. Blood pressure and temperature were monitored (mean 128 + 8 mm Hg and 37.1 + 0.3 ° C, respectively). Arterial blood gases did not change during the experiments (pH = 7.43 + 0.02, pO2 = 97 + 8, pCO 2 = 35.5 + 4). Ninety minutes after [ 14C]AIB injection (3-4 h post CRT), the rats were killed and their brains removed within 1-2 min, placed in Freon-t2, mounted with embedding matrix and stored at - 70 ° C. Frozen sections, 20 #m thick, were cut on a cryostat and
149 either dried at 60°C and exposed to x-ray film (SB5, Kodak Co.) along with [14C]methylmethacrylate standards previously calibrated to reference 20#m brain sections of known radioactivity, or fixed for staining with hematoxylin-eosin. Individual brain sections on the autoradiograms were digitized along with the standards and a curve relating optical density to tissue radioactivity was generated for each ftlm. For estimating the permeability-surface area product of the tissue capillaries, a unidirectional blood-to-spinal cord transfer constant, K (#l/g per rain), was calculated by: K = Ci(T) - 0.02 Cp (90 rain)/05 T Cp(t)dt where Ci(T) is the tissue concentration of [ 14C]AIB at the end of the experimental time (T) and Cp is the concentration of AIB in the arterial plasma. Regional measurements of K were made in the normal cortex and striatum on both sides and in the tumor. In addition, the K values of 2 consecutive rims of tissue, brain adjacent to the tumor (BAT, 500 #m wide) and brain surrounding the tumor (BST, 1 mm wide) were also measured. BAT contained a variable number of tumor cells: BST was tumor free.
RESULTS
An intraparenchymal tumor developed in 11/12 injected rats. One irradiated rat bad no tumor and therefore only the K values for cortex and striatum could be measured. No significant difference in mean tumor cross-sectional area was observed between control and radiated animals [control, 8.5 ram2+ 2.3 (SEM), radiated, 7.5 mm 2 + 2.1 (SEM)]. The quantitative results are depicted in Table 1 and illustrated in Fig. 1A, B. When compared with control animals, K in the radiated group increased 63 ~o in the cortex (P < 0.001, 2 tailed Student's t test) and 30~o in the striatum (P < 0.005), without
TABL E 1 T R A N S F E R RATE C O N S T A N T OF ENTRY (K, pl/g/min) F O R [14C]AIB IN C O N T R O L A N D I R R A D I A T E D RATS Values are expressed as means +__SEM.
Control a Radiated A% b
a b c d e
Cortex
Striatum
B ST c
BAT c
Tumor
2.57 _+ 0.07 4.19 + 0.26 +63~ a
1.58 + 0.10 2.06 _+ 0.11 +30% e
2.29 + 0.12 3.00 + 0.20 + 31% c
5.90 + 0.I1 6.27 + 0.42 +6~
22.0 + 1.99 22.4 + 3.64 +2%
n = 6 in each group, except BST, BAT, and tumor in irradiated rats (n = 5). A%, % increase of K in irradiated versus control rats. BST, brain surrounding the tumor; BAT, brain around the tumor. p < 0.001. p < 0.005.
150
Fig. 1. [~4C]AIB autoradiographs in coronal section, functionalized to display K values (~d/g/min), and adjacent histologic section (stained with hematoxylin-eosin) in a control rat (A) and 3 h after cranial irradiation (B). Note increase of K in normal brain after irradiation. No change was observed in the tumor and brain tissue around the tumor.
151 difference between non tumor and tumor bearing hemispheres. In the B ST, K increased 31 ~o in the radiated group (P < 0.005). Radiation produced no significant change of K values in the tumors and the brain adjacent to the tumor.
DISCUSSION
Three to 4 h after irradiation of C6 glioma with a single dose of 3 Gy, there is a significant increase of the AIB transport constant (K) in normal brain but not in the tumor or BAT. Four possible sources of experimental error must be considered: first, if the tumors were significantly larger in the control group, values of K would be greater (Hiesiger et al. 1986), causing one to miss a true increase in K after irradiation. However, this was not the case as the mean tumor cross-sectional areas were not different. Second, if there were no blood-tumor barrier at all in C6 gliomas, no further increase in capillary permeability would be possible. However, even very "leaky" tumors are not completely permeable to AIB (Molnar et al. 1983) and certainly BAT retains a partial blood-brain barrier. Third, if radiation profoundly increased the cerebral blood volume, a higher value of K would not reflect a real change in capillary permeability. However, a more than 10-fold increase of cerebral blood volume would be necessary to explain the changes of K values that we observed, and this would likely be incompatible with animal survival and normal histological appearance of the brain. Finally, if radiation dramatically reduced the cerebral blood flow, K would estimate flow rather than the permeability surface product. However, even if radiation reduced the blood flow to 1/5 of its normal value (assuming a cerebral blood flow of 50 ml/min per 100 g), K would deviate from the permeability surface product by only 5 ~ . Furthermore, Spence et al. (1987), using even higher doses of CRT (20 Gy in one fraction) did not observe any significant acute change in cerebral blood volume or blood flow. Thus, the increase of regional capillary permeability in normal parenchyma and the failure of CRT to acutely increase permeability in the tumor and BAT seem true findings. Acute complications of CRT generally occur a few hours after the first treatment in patients with prior evidence of intracranial hypertension due to tumor and are more frequent and severe after higher doses (Young et al. 1974). The clinical picture usually consists of transient headaches, nausea, vomiting, fever and somnolence, worsening of already present neurological signs and, rarely, progression to cerebral herniation and death. The pathophysiology is unknown but the clinical course and anecdotal reports of a beneficial effect of steroids suggest a primary role for radiation-induced edema (Rubin and Casarett 1968). The development of acute change in the BBB following high-dose CRT has been demonstrated experimentally (Klatzo et al. 1961; Nair and Roth 1964) but the effect of doses in the therapeutic range is less certain (Clemente and Hoist 1954). Our results demonstrate that even low dose CRT, similar to that used clinically, can induce an acute increase in regional capillary permeability to small molecules in normal brain and BST, confirming the previous observations of Levin et al. (1979). Whether these capillary changes can lead to vasogenic edema remains speculative since we did not measure the water content of the brains.
152 The rationale for the use of chemotherapy (particularly alkylating agents) during radiation therapy is that the 2 modalities might have a synergistic effect on brain tumors. However, it is also important to minimize damage to the normal brain. The entry of water-soluble chemotherapeutic agents into the tumor and normal brain is limited by the blood-tumor barrier (BTB) and the BBB, respectively. A major therapeutic challenge is, therefore, to "open" the barrier to water-soluble drugs in the tumoral and peritumoral area without doing so in the normal brain, i.e. to increase the tumor-tonormal brain permeability ratio. AIB is believed to be a good model for water-soluble anti-cancer drugs (Blasberg et al. 1983). This study was motivated in part by our previous finding that K increased by more than 100~ in the normal cortex 2-3 h after a single dose of 3 Gy CRT (Phillips et al. 1987). We speculated that if a change of this order or more could be obtained in the tumor and peritumoral areas, RT which can be administered focally to the tumoral area, could be a useful tool to selectively open the BTB to water-soluble chemotherapeutic agents. Our results do not support this hypothesis. Although K increases by 63 ~ in the cortex, it did not increase in the tumor and the result of radiation was a reduction of the tumor-to-normal brain permeability ratio. If similar conditions exist in the clinical setting, the risk of acute neurotoxicity for water-soluble chemotherapeutic agents might be increased without therapeutic benefit. The increased risk of severe neurotoxicity (including acute encephalopathy) when CRT is administered simultaneously to methotrexate (MTX) is well recognized (Bleyer 1981). If one considers that high-dose MTX by itself can increase capillary permeability (Phillips et al. 1987) and therefore that the normal endothelial cell is a potential target for both treatment modalities, our results suggest that one should avoid potentially neurotoxic water-soluble drugs during a course of CRT. Using QAR and AIB in a rat glioma model, Spence et al. (1987) found that K in the tumor actually decreased 50 ~o 24 h after 20 Gy CRT, while K did not change in the normal brain, resulting also in a reduction the tumor-to-normal brain permeability ratio. The reasons why our results in the normal parenchyma differ from those of Spence et al. are unclear but may be related to the fact that we used very different dose (3 Gy vs. 20 Gy). The pathophysiology of the acute effect of CRT on capillary permeability is unknown. One of the primary effects of RT is the formation of free radicals (Rodney Withers 1987). Chan et al. (1984) have demonstrated that the formation of free radicals in the brain leads to vascular leakage of fluorescent dye and suggested that the endothelial cells are highly susceptible to oxygen derived free radicals through lipid peroxidations and degradation of membrane phospholipids. Capillaries of damaged brain and tumor might be more resistant to the acute effects of CRT than normal parenchyma. It is interesting that tumor capillaries also appear to be resistant to osmotic manipulation (Hiesiger et al. 1986). It is also possible that relative hypoxia in the tumor and compressed'BAT makes these structures and their capillary network more resistant to the acute effects of RT than normal parenchyma. These results in acute studies do not preclude the secondary development of permeability changes in the tumor and BAT when subacute or delayed effects of CRT are considered (Brooks et al. 1986). However, we have previously reported that
153
capillary permeability progressively increases in normal brain, at least until 1 month following 30 Gy CRT delivered in 10 fractions over 10 clays (Phillips et al. 1987). Further studies are needed to document the time course of the tumor-to-normal brain permeability relationship after therapeutic doses of CRT.
REFERENCES Blasberg, R. G., J. D. Fenstermacher and C. S. Patlak (1983) Transport of alpha-aminoisobutyric acid across brain capillaries and cellular membranes. J. Cereb. Blood Flow Metab., 3: 8-22. Bleyer, W.A. (1981) Neurologic sequelae of methotrexate and ionizing radiation: a new classification. Cancer Treat. Rep., 65: 89-98. Brooks, D.J., R.P. Beaney and D.G.T. Thomas (1986) The role of positron emission tomography in the study of cerebral tumors. Semin. Oncol., 13: 83-93. Chan, P.H., J.W. Sehmidley, R.A. Fishman and S.M. Longar (1984) Brain injury, edema, and vascular permeability changes induced by oxygen-derived free radicals. Neurology, 34: 315-320. Clemente, C.D. and E.A. Holst (1954) Pathological changes in neurons, neuroglia and blood-brain barrier induced by X-irradiation of heads of monkeys. Arch. Neurol. Psychiat., 71: 66-79. Hiesiger, E. M., R. M. Voorhies, G.A. Basler, L. E. Lipshutz, J. B. Posner and W. R. Shapiro (1986) Opening the blood-tumor barriers in experimental brain tumors: the effect of intracarotid hyperosmolar mannitol on capillary permeability and blood flow. Ann. Neurol., 19: 50-59. Klatzo, I., J. Miquel, C. Tobias and W. Haymaker (1961) Effect of alpha particle radiation on the rat brain including vascular permeability and glycogen studies. J. Neuropath. Exp. Neurol., 20: 459-483. Levin, V.A., M.S. Edwards and A. Byrd (1979) Quantitative observations of the acute effects of X-irradiation on brain capillary permeability. Int. J. Radiat. Oncol. Biol. Phys., 5: 1627-1631. Molnar, P., R.G. Blasberg, M. Horowitz, B. Smith and J. Fenstermacher (1983) Regional blood-to-tissue transport in RT-9 brain tumors. J. Neurosurg., 58: 874-884. Nair, V. and L.J. Roth (1964) Effect of X-irradiation and certain other treatments on blood-brain barrier permeability. Radiat. Res., 23: 249-264. Phillips, P. C., J.Y. Delattre, C.A. Berger and D.A. Rottenberg (1987a) Early and progressive increase in regional brain capillary permeability following single and fractionated dose cranial irradiation in the rat. Neurology, 37 (Suppl. 1): 301. Phillips, P. C., V. Dhawan, S.C. Strother, J.J. Sidtis, A.C. Evans, J. C. Allen and D.A. Rottenberg (1987b) Reduced cerebral glucose metabolism and increased brain capillary permeability following high-dose methotrexate chemotherapy: a positron emission tomographic study. Ann. Neurol., 21: 59-63. Rodney Withers, H. (1987) Biologic basis of radiation. In: Perez, C.A. and L.W. Brady (Eds.), Radiation Ontology, J.B. Lippincott, Philadelphia, PA, pp. 67-99. Rubin, P. and G.W. Casarett (1968) Clinical Radiation Pathology. W.B. Saunders, Philadelphia, PA, pp. 609-661. Schetler, T. and C.N. Shealy (1970) Experimental selective alterations of the blood-brain barrier by irradiation. J. Neurosurg., 32: 89-94. Spence, A.M., M.M. Graham, L.A. O'Gorman, M. Muzi, G.L. Abbott and T.K. Lewellen (1987) Regional blood-to-tissue transport in an irradiated rat glioma model. Radiat. Res., 111: 225-236. Young, D.F., J.B. Posner, F. Chu and L. Nisce (1974) Rapid course radiation therapy of cerebral metastases: results and complications. Cancer, 34: 1069-1076.
147
JNS 03139
Acute effects of low-dose cranial irradiation on regional capillary permeability in experimental brain tumors Jean Yves Delattre, William R. Shapiro and Jerome B. Posner Cotzias Laboratory of Neuro-Oncology and Departments of Neurology, Memorial Sloan-Kettering Cancer Center and Cornell University Medical College, New York, NY (U.S.A.) (Received 27 May, 1988) (Accepted 30 November, 1988)
SUMMARY
To determine the acute effects of low-dose cranial irradiation (CRT) on regional capillary permeability (RCP) of normal brain, brain tumor and damaged brain surrounding the tumor, we used quantitative autoradiography (QAR) to measure regional blood-to-tissue transport (K) of [ ~4C]aminoisobutyric acid (AIB) in experimental C6 brain tumors 3-4 h after a single dose of 3 Gy CRT. K increased 63 ~o in cortex, 30 ~o in basal ganglia and 31 ~o in brain surrounding the tumor (B ST) vs. controls (P < 0.005). K did not change in the tumor or in the brain adjacent to the tumor (BAT), suggesting that capillaries of normal parenchyma are more sensitive to the acute effects of CRT than capillaries of damaged parenchyma or tumor.
Key words: Experimental brain tumor; Cranial irradiation; Blood-brain barrier
Using quantitative autoradiography (QAR) with [~4C]aminoisobutyric acid (AIB), we found that therapeutic doses of cranial irradiation (CRT) 3 Gy in one fraction, or 30 Gy in 10 fractions over 10 days, induced a significant increase in regional bloodto-brain transport of AIB in normal rats, i.e. it "opens" the blood-brain barrier (BBB) (Phillips et al. 1987). AIB is a small neutral amino acid which crosses the normal BBB Correspondence to: Dr. Jerome B. Posner, Department of Neurology, Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, New York, NY 10021, U.S.A. 0022-510X/89/$03.50 © 1989 Elsevier Science Publishers B.V. (Biomedical Division)
148 slowly but is rapidly taken up, concentrated and "trapped" by parenchymal cells (Blasberg et al. 1983). If capillaries of edematous brain around a tumor are more radiosensitive than normal parenchyma, as suggested by Schetler and Shealy (1970), CRT could have potentially useful (selective opening of the BBB, allowing better entry of water-soluble chemotherapeutic agents) or harmful (increased risk of edema) consequences. We undertook to study the acute effects of a single dose of 3 Gy CRT in an experimental model of brain tumor.
MATERIALAND METHODS
Tumor and animal inoculation C6 glioma was purchased from the American Type Culture Collection (ATCC; Rockville, MD) and maintained in tissue culture with McCoy's 5A medium containing 10~o fetal calf serum at 37 °C and 5% CO 2 in a humidified incubator. Cells from confluent flasks were harvested with 0.5 % trypsin and 0.2 ~o EDTA in Hank's balanced salt solution, centrifuged and resuspended in their media without serum but containing 1 ~o agar. Male Wistar rats weighing 300-400 g were anesthetized with chloral hydrate (35 rng/kg intraperitoneally). A 2~o lidocaine gel was applied to the rat's ear and the head was affixed in a stereotaxic apparatus (Model 900, David Kopf Instruments, Tujunga, CA). The skull was exposed by a midline scalp incision, and one shallow 26-gauge needle puncture was made 4 mm to the fight of the sagittal suture and 1 mm posterior to the coronal suture. Ten/21 of a suspension containing 5 x 105 viable C6cells was inoculated to a depth of 6 mm through the pre-bored hole.
Irradiation protocol At day 10, 6 inoculated rats were irradiated while 6 others served as controls. The unanesthetized animals were kept immobile in a holder, The oral cavity and the part of the body situated behind the ears were shielded by a 5-ram thick sheet of lead. 3 Gy in one fraction was delivered to the head using a GE Maxitron x-ray generator (20 mA, HVL 2 mm Cu, FSD 50 cm). The 300 kV energy was delivered at a rate of 1.04 Gy/min to a lateral field. In order to obtain a homogeneous dose distribution in the brain, the animals were turned 180 ° halfway through the exposure.
Quantitative autoradiography (QAR) The rats were anesthetized and the right femoral artery and vein were cannulated. 100 izCi of 0t-[1-1aC]AIB was injected into the femoral vein. Timed blood samples were centrifuged and the radioactivity of 20 #1 of plasma was measured with a scintillation counter. Blood pressure and temperature were monitored (mean 128 + 8 mm Hg and 37.1 + 0.3 ° C, respectively). Arterial blood gases did not change during the experiments (pH = 7.43 + 0.02, pO2 = 97 + 8, pCO 2 = 35.5 + 4). Ninety minutes after [ 14C]AIB injection (3-4 h post CRT), the rats were killed and their brains removed within 1-2 min, placed in Freon-t2, mounted with embedding matrix and stored at - 70 ° C. Frozen sections, 20 #m thick, were cut on a cryostat and
149 either dried at 60°C and exposed to x-ray film (SB5, Kodak Co.) along with [14C]methylmethacrylate standards previously calibrated to reference 20#m brain sections of known radioactivity, or fixed for staining with hematoxylin-eosin. Individual brain sections on the autoradiograms were digitized along with the standards and a curve relating optical density to tissue radioactivity was generated for each ftlm. For estimating the permeability-surface area product of the tissue capillaries, a unidirectional blood-to-spinal cord transfer constant, K (#l/g per rain), was calculated by: K = Ci(T) - 0.02 Cp (90 rain)/05 T Cp(t)dt where Ci(T) is the tissue concentration of [ 14C]AIB at the end of the experimental time (T) and Cp is the concentration of AIB in the arterial plasma. Regional measurements of K were made in the normal cortex and striatum on both sides and in the tumor. In addition, the K values of 2 consecutive rims of tissue, brain adjacent to the tumor (BAT, 500 #m wide) and brain surrounding the tumor (BST, 1 mm wide) were also measured. BAT contained a variable number of tumor cells: BST was tumor free.
RESULTS
An intraparenchymal tumor developed in 11/12 injected rats. One irradiated rat bad no tumor and therefore only the K values for cortex and striatum could be measured. No significant difference in mean tumor cross-sectional area was observed between control and radiated animals [control, 8.5 ram2+ 2.3 (SEM), radiated, 7.5 mm 2 + 2.1 (SEM)]. The quantitative results are depicted in Table 1 and illustrated in Fig. 1A, B. When compared with control animals, K in the radiated group increased 63 ~o in the cortex (P < 0.001, 2 tailed Student's t test) and 30~o in the striatum (P < 0.005), without
TABL E 1 T R A N S F E R RATE C O N S T A N T OF ENTRY (K, pl/g/min) F O R [14C]AIB IN C O N T R O L A N D I R R A D I A T E D RATS Values are expressed as means +__SEM.
Control a Radiated A% b
a b c d e
Cortex
Striatum
B ST c
BAT c
Tumor
2.57 _+ 0.07 4.19 + 0.26 +63~ a
1.58 + 0.10 2.06 _+ 0.11 +30% e
2.29 + 0.12 3.00 + 0.20 + 31% c
5.90 + 0.I1 6.27 + 0.42 +6~
22.0 + 1.99 22.4 + 3.64 +2%
n = 6 in each group, except BST, BAT, and tumor in irradiated rats (n = 5). A%, % increase of K in irradiated versus control rats. BST, brain surrounding the tumor; BAT, brain around the tumor. p < 0.001. p < 0.005.
150
Fig. 1. [~4C]AIB autoradiographs in coronal section, functionalized to display K values (~d/g/min), and adjacent histologic section (stained with hematoxylin-eosin) in a control rat (A) and 3 h after cranial irradiation (B). Note increase of K in normal brain after irradiation. No change was observed in the tumor and brain tissue around the tumor.
151 difference between non tumor and tumor bearing hemispheres. In the B ST, K increased 31 ~o in the radiated group (P < 0.005). Radiation produced no significant change of K values in the tumors and the brain adjacent to the tumor.
DISCUSSION
Three to 4 h after irradiation of C6 glioma with a single dose of 3 Gy, there is a significant increase of the AIB transport constant (K) in normal brain but not in the tumor or BAT. Four possible sources of experimental error must be considered: first, if the tumors were significantly larger in the control group, values of K would be greater (Hiesiger et al. 1986), causing one to miss a true increase in K after irradiation. However, this was not the case as the mean tumor cross-sectional areas were not different. Second, if there were no blood-tumor barrier at all in C6 gliomas, no further increase in capillary permeability would be possible. However, even very "leaky" tumors are not completely permeable to AIB (Molnar et al. 1983) and certainly BAT retains a partial blood-brain barrier. Third, if radiation profoundly increased the cerebral blood volume, a higher value of K would not reflect a real change in capillary permeability. However, a more than 10-fold increase of cerebral blood volume would be necessary to explain the changes of K values that we observed, and this would likely be incompatible with animal survival and normal histological appearance of the brain. Finally, if radiation dramatically reduced the cerebral blood flow, K would estimate flow rather than the permeability surface product. However, even if radiation reduced the blood flow to 1/5 of its normal value (assuming a cerebral blood flow of 50 ml/min per 100 g), K would deviate from the permeability surface product by only 5 ~ . Furthermore, Spence et al. (1987), using even higher doses of CRT (20 Gy in one fraction) did not observe any significant acute change in cerebral blood volume or blood flow. Thus, the increase of regional capillary permeability in normal parenchyma and the failure of CRT to acutely increase permeability in the tumor and BAT seem true findings. Acute complications of CRT generally occur a few hours after the first treatment in patients with prior evidence of intracranial hypertension due to tumor and are more frequent and severe after higher doses (Young et al. 1974). The clinical picture usually consists of transient headaches, nausea, vomiting, fever and somnolence, worsening of already present neurological signs and, rarely, progression to cerebral herniation and death. The pathophysiology is unknown but the clinical course and anecdotal reports of a beneficial effect of steroids suggest a primary role for radiation-induced edema (Rubin and Casarett 1968). The development of acute change in the BBB following high-dose CRT has been demonstrated experimentally (Klatzo et al. 1961; Nair and Roth 1964) but the effect of doses in the therapeutic range is less certain (Clemente and Hoist 1954). Our results demonstrate that even low dose CRT, similar to that used clinically, can induce an acute increase in regional capillary permeability to small molecules in normal brain and BST, confirming the previous observations of Levin et al. (1979). Whether these capillary changes can lead to vasogenic edema remains speculative since we did not measure the water content of the brains.
152 The rationale for the use of chemotherapy (particularly alkylating agents) during radiation therapy is that the 2 modalities might have a synergistic effect on brain tumors. However, it is also important to minimize damage to the normal brain. The entry of water-soluble chemotherapeutic agents into the tumor and normal brain is limited by the blood-tumor barrier (BTB) and the BBB, respectively. A major therapeutic challenge is, therefore, to "open" the barrier to water-soluble drugs in the tumoral and peritumoral area without doing so in the normal brain, i.e. to increase the tumor-tonormal brain permeability ratio. AIB is believed to be a good model for water-soluble anti-cancer drugs (Blasberg et al. 1983). This study was motivated in part by our previous finding that K increased by more than 100~ in the normal cortex 2-3 h after a single dose of 3 Gy CRT (Phillips et al. 1987). We speculated that if a change of this order or more could be obtained in the tumor and peritumoral areas, RT which can be administered focally to the tumoral area, could be a useful tool to selectively open the BTB to water-soluble chemotherapeutic agents. Our results do not support this hypothesis. Although K increases by 63 ~ in the cortex, it did not increase in the tumor and the result of radiation was a reduction of the tumor-to-normal brain permeability ratio. If similar conditions exist in the clinical setting, the risk of acute neurotoxicity for water-soluble chemotherapeutic agents might be increased without therapeutic benefit. The increased risk of severe neurotoxicity (including acute encephalopathy) when CRT is administered simultaneously to methotrexate (MTX) is well recognized (Bleyer 1981). If one considers that high-dose MTX by itself can increase capillary permeability (Phillips et al. 1987) and therefore that the normal endothelial cell is a potential target for both treatment modalities, our results suggest that one should avoid potentially neurotoxic water-soluble drugs during a course of CRT. Using QAR and AIB in a rat glioma model, Spence et al. (1987) found that K in the tumor actually decreased 50 ~o 24 h after 20 Gy CRT, while K did not change in the normal brain, resulting also in a reduction the tumor-to-normal brain permeability ratio. The reasons why our results in the normal parenchyma differ from those of Spence et al. are unclear but may be related to the fact that we used very different dose (3 Gy vs. 20 Gy). The pathophysiology of the acute effect of CRT on capillary permeability is unknown. One of the primary effects of RT is the formation of free radicals (Rodney Withers 1987). Chan et al. (1984) have demonstrated that the formation of free radicals in the brain leads to vascular leakage of fluorescent dye and suggested that the endothelial cells are highly susceptible to oxygen derived free radicals through lipid peroxidations and degradation of membrane phospholipids. Capillaries of damaged brain and tumor might be more resistant to the acute effects of CRT than normal parenchyma. It is interesting that tumor capillaries also appear to be resistant to osmotic manipulation (Hiesiger et al. 1986). It is also possible that relative hypoxia in the tumor and compressed'BAT makes these structures and their capillary network more resistant to the acute effects of RT than normal parenchyma. These results in acute studies do not preclude the secondary development of permeability changes in the tumor and BAT when subacute or delayed effects of CRT are considered (Brooks et al. 1986). However, we have previously reported that
153
capillary permeability progressively increases in normal brain, at least until 1 month following 30 Gy CRT delivered in 10 fractions over 10 clays (Phillips et al. 1987). Further studies are needed to document the time course of the tumor-to-normal brain permeability relationship after therapeutic doses of CRT.
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