Increased capillary permeability in rat brain induced by factors secreted by cultured C6 glioma cells: role in peritumoral brain edema.

To investigate whether brain tumors secrete a factor(s) responsible for peritumoral brain edema, we studied the effect of conditioned medium from cultured C6 glioma cells on rat brain capillary permeability. Three different fractions of conditioned medium were obtained. SUP-N was a culture supernatant incubated 4 hours in serum-free medium. SUP-C was the 60-100 fold concentrated fraction obtained by dialysis-concentration of SUP-N; it contained 950 micrograms/ml of protein greater than 10 k-daltons from 3 x 10(8) cells. SUP-L was a water-dispersible lipid fraction from SUP-N; the major components of SUP-L were neutral lipids and free fatty acids. The supernatant fractions and their corresponding control solutions were infused into normal rat brain, and capillary permeability was determined using quantitative autoradiography by measuring the unidirectional entry constant, K (micrograms l/g.min), of 14C-alpha-aminoisobutyric acid (14C-AIB) into brain tissue. SUP-C and SUP-L significantly increased capillary permeability of normal brain; the effect of SUP-C was more intense and extensive than that of SUP-L. The highest mean K value (Kmax) of SUP-C was 10.83 +/- 0.99 and that of the control was 2.53 +/- 0.22 (p less than 0.001). The Kmax of SUP-L was 5.61 +/- 0.23 and that of the control was 2.67 +/- 0.36 (p less than 0.01). A time-course study after infusion of SUP-C demonstrated that more than 1.5 hours is required for the supernatant fraction to open the barrier and that the effect of SUP-C was reversible. The increase of capillary permeability induced by SUP-C was significantly inhibited by pretreatment of rats with dexamethasone (10 mg/kg, ip) 1 hour before intracerebral infusion of SUP-C (Kmax (untreated): 8.30 +/- 0.82, Kmax (treated): 1.33 +/- 0.64, p less than 0.001). These results indicate that experimental brain tumors secrete at least two different diffusible factors responsible for capillary endothelial leakage in normal brain. One is a protein of molecular weight greater than 10 k-daltons, whose effect is inhibited by glucocorticoids, and the other is a waterdispersible lipid.

Capillary permeability factor secreted by malignant brain tumor. Role in peritumoral brain edema and possible mechanism for anti-edema effect of glucocorticoids.

Conditioned media from two human malignant gliomas, C6 rat glioma, Walker 256 carcinosarcoma, and normal human glia were concentrated 50-fold to create a culture supernatant (SUP-C). The effect of SUP-C on rat brain capillary permeability was investigated by measuring the entry of 14C-aminoisobutyric acid (14C-AIB) by means of quantitative autoradiography. The SUP-C contained proteins with a molecular weight of 10 kD or greater. The SUP-C from all tumor cells markedly increased brain capillary permeability, indicating the presence of a permeability factor, whereas that from normal glial cells did not. Glioma cells produced more factor after incubation for 20 hours than 4 hours. The activity of capillary permeability factor in the SUP-C was inhibited by pretreatment of animals with BW755C (lipoxygenase inhibitor), but not with indomethacin (cyclo-oxygenase inhibitor). Pretreatment of animals with dexamethasone prior to intracerebral infusion of tumor SUP-C significantly reduced the factor-induced increase in capillary permeability. On the other hand, coincubating glioma cells with dexamethasone produced SUP-C with a permeability activity that was about one and a half times greater than that without dexamethasone. These results indicate that glucocorticoids produce their anti-edema effects by directly acting on capillary endothelial cells, possibly through the inhibition of phospholipase A2 activity, resulting in a decrease of lipoxygenase rather than cyclo-oxygenase products. The production of capillary permeability factor by tumor cells was not inhibited, but rather enhanced, by administration of glucocorticoids.

Pharmacokinetics of tumor cell exposure to [14C]methotrexate after intracarotid administration without and with hyperosmotic opening of the blood-brain and blood-tumor barriers in rat brain tumors: a quantitative autoradiographic study.

Using quantitative autoradiography, we investigated the entry over 90 min of [14C]methotrexate (MTX) into C6 gliomas implanted bilaterally into Wistar rat brains. The [14C]MTX was administered into the right carotid artery, yielding ipsilateral "arterial" brain and tumor concentrations and contralateral "systemic" concentrations. In a separate group of tumor-bearing rats, mannitol 1.6 M was given into the right carotid artery prior to administering the [14C]MTX to disrupt the blood-brain barrier on the ipsilateral side. [14C]MTX tissue concentrations were measured in regions of 50 x 50 x 20 microns in tumor, peritumoral brain tissue (brain adjacent to tumor), and cerebral cortex. In the nonmannitol experiments, tissue concentrations from the rats at each time interval were fitted using a nonlinear curve fitting program, and the pharmacokinetic values of influx and efflux of [14C]MTX into the three compartments were calculated. The influx rate constant K1 for [14C]MTX ranged from 1.3 to 8.2 microliters/g/min in the tumor. Influx rate constants in the cortex were 1.3-1.9 microliters/g/min and in the brain adjacent to tumor were 1.7-2.8 microliters/g/min. The efflux rate constant k2 was approximated for each tissue but was less reliable than the K1 values. The k2 for tumor, brain adjacent to tumor, and cortex was always higher than the corresponding K1. Peak [14C]MTX concentrations in the tumor were highest after arterial infusion with hyperosmolar barrier disruption, lower after arterial administration without barrier modification, and lowest after systemic administration. However, cortical [14C]MTX concentration was also highest after arterial administration with barrier modification and higher than the highest tumor concentration. Furthermore, tissue exposure (concentration x time) was also highest in the cortex after barrier disruption. The [14C]MTX concentration x time (micrograms/min/g x 90 min +/- SEM) ratio between tumor and cortex after systemic administration was 33.4 +/- 4.1:15.7 +/- 1.9; after arterial administration it was 96.3 +/- 11.7:30.3 +/- 3.1; after arterial administration with barrier disruption it was 266.6 +/- 28.8:311.2 +/- 15.9. The greatest tumor:cortex ratio (3.1:1) occurred with arterial drug administration without barrier disruption. Disrupting the barrier enough to permit increased tumor exposure actually increased cortical exposure to a greater degree. The resulting poorer therapeutic ratio would not appear to support this technique in humans, at least for neurotoxic drugs.