J Neurosurg 77:40%410, 1992
Selective blood-tumor barrier disruption by leukotrienes CHUNG-CHING CHIO, M.D., TAKEHIKO BABA, M.D., AND KEITH L. BLACK, M.D.
The Brain Research Institute, The Jonsson Cancer Center, Division of Neurosurgery, University of California Medical Center, Los Angeles, California v" The authors have previously reported that intracarotid infusion of 5 ug leukotriene C4 (LTC4) selectively increases blood-tumor barrier permeability in rat RG-2 tumors. In this study, rats harboring RG-2 tumors were given 15-minute intracarotid infusions of LTC4 at concentrations ranging from 0.5 ug to 50.0 #g (seven rats in each dose group). Blood-tumor and blood-brain barrier permeability were determined by quantitative autoradiography using '4C aminoisobutyric acid. The transfer constant for permeability (K0 within the tumors was increased twofold by LTC4 doses of 2.5, 5.0, and 50.0 ug compared to vehicle alone (90.00 + 21.14, 92.68 _+ 15.04, and 80.17 _+ 16.15 vs. 39.37 _+6.45 ul/gm/min, respectively; mean _ standard deviation; p < 0.01). No significant change in K~ within the tumors was observed at the 0.5-ug LTC4 dose. Blood-brain barrier permeability was selectively increased within the tumors. At no dose in this study did leukotrienes increase permeability within normal brain. To determine the duration of increased opening of the blood-tumor barrier by LTC, administration, Ki was measured at 15, 30, and 60 minutes after termination of a 15-minute LTC4 infusion (seven rats at each time point). The mean K~ value was still high at 15 minutes (92.68 ___ 15.04 ul/gm/min), but declined at 30 minutes (56.58 _+ 12.50 ul/gm/min) and 60 minutes (55.40 + 8.10 ul/gm/min) after the end of LTC4 infusion. Sulfidopeptide leukotrienes LTC4, LTD4, LTE4 and LTF4 were infused to compare their potency in opening the blood-tumor barrier. The mean leukotriene E4 was the most potent, increasing the permeability value 389 fold compared with vehicle alone (139.86 + 23.95 vs. 39.37 _+6.45 ~l/gm/min). KEy WOADS
T
9
leukotriene
9
brain neoplasm
HE blood-tumor barrier within the capillaries of
primary brain tumors is considered to be an obstacle to delivering water-soluble antineoplastic agents to brain-tumor tissue. 1~ One approach to brain tumor therapy has therefore focused on methods that open or modify the brain-tumor barrier and increase drug delivery. In a previous study, we demonstrated that leukotriene C4 (LTC4) infused into the carotid artery ipsilateral to experimental RG-2 glial tumors in rats increased the unidirectional transfer constant for permeability (Ki) twofold within the tumors, while no effect on permeability was seen in normal brain. 6 Based on this observation, we suggested that LTC4 may be useful clinically in selectively increasing the delivery of antineoplastic agents to tumors. The purpose of this study was to determine the optimal dose and the duration of opening of the blood-tumor barrier by LTC4 administration. The potencies of various sulfidopeptide leukotrienes (LTC4, LTD4, LTE4, and LTF4) were also compared. Materials and Methods
Eighty-four female Wistar rats, each weighing 130 to 170 gm, were used for this study. The sulfidopeptide
J. Neurosurg. / Volume 77/September, 1992
9
blood-brain barrier
9 rat
leukotrienes* were obtained at a concentration of 0.1 mg/ml in a 1:1 phosphate-buffered saline (PBS)-ethanol solution. Aliquots of LTCa, LTDa, LTE4, and LTFa were diluted to experimental concentrations in PBS and the final ethanol concentration was adjusted to 2.5%. Vials containing the leukotrienes were sealed under nitrogen and stored at -80"C. Alpha-[ 1-14C]-aminoisobutyric acid (AIB) (57.6 mCi/mmol)t was used for quantitative autoradiographic study.
Tumor Inoculation The RG-2 glioma cells were maintained in a monolayer culture in F-12 medium with 10% calf serum., The rats were anesthetized with intraperitoneal pentobarbital sodium (Nembutal, 40 mg/kg). Tumor cells (5 • 105 in a 5-ul solution) were stereotactically implanted by means of a Hamilton syringe into the right cerebral
* Leukotrienes supplied by Cayman Chemicals, Ann Arbor, Michigan. ~"Alpha-[ 1-~4C]-aminoisobutyricacid supplied by New England Nuclear, Boston, Massachusetts. F-12 medium and 10% calf serum supplied by GIBCO, Grand Island, New York. 407
C. C. Chio, T. Baba, and K. L. Black hemisphere 5 mm lateral to the bregma and 5 mm deep to the dural surface.
Dose Response Study Ten to 11 days after tumor implantation, the rats were again anesthetized and a polyethylene (PE-50) catheterw was inserted retrograde through the external carotid artery to the common carotid artery bifurcation ipsilateral to the tumor. The external carotid artery was then ligated, and both femoral arteries and one femoral vein were cannulated. Body temperature was mainmined at 37"C. Arterial blood gas levels, blood pressure, and hematocrit were monitored. Animals with abnormal physiological parameters were eliminated from the study. Infusions of LTC4 at concentrations of 0.5, 2.5, 5.0, and 50.0 ug/0.8 ml or 0.8 ml of vehicle (2.5% ethanol in PBS, pH 7.1) were administered into the fight carotid artery of seven rats in each dose group using a constant-infusion pump]l at a rate of 53.3 ul/ min for 15 minutes. Five minutes after the start of the intracarotid infusion, an intravenous bolus of 100 uCi/ kg of ~4C-AIB was delivered. A peristaltic withdrawal pump* was used to withdraw femoral arterial blood at a constant rate of 0.083 ml/min immediately after injection of ~4C-AIB for determination of serum radioactivity. Fifteen minutes after the start of intracarolid infusions, the animals were killed by decapitation and the brains were rapidly removed and frozen.
Time Course Study Ten to 11 days after tumor implantation, the rats were cannulated and physiological parameters were measured as described above. An intracarotid infusion of 0.8 ml LTC4 at a dose of 5 ug was delivered for 15 minutes, and K~was measured at 15, 30, and 60 minutes after the termination of LTC4 infusion.
Potency of Leukotrienes Various sulfidopeptide leukotrienes (LTC4, LTD4, LTE4, and LTFa), at a dose of 5 ug, were infused for 15 minutes into seven rats in each leukotriene group and the Ki value was measured as described above.
Autoradiography The frozen rat brains were mounted onto pedestals with M-1 embedding matrix,t and 20-urn coronal sections were cut using a cryotome. The sections were thaw-mounted onto coverslips, and autoradiograms were generated by coexposing the sections on Kodak XAR-5 film with tissue-calibrated 14C standards~ for 2 wCatheter manufactured by Clay Adams, Parsippany, New York. II Infusion pump manufactured by Harvard Apparatus, Inc., Millis, Massachusetts. * Withdrawal pump manufactured by Gibson Medical Electronics, Inc., Middleton, Wisconsin. t M-1 embedding matrix supplied by Lipshaw Manufacturing, Detroit, Michigan. 14C standards obtained from Amersham Corp., Arlington Heights, Illinois. 408
weeks. Quantitative analysis was performed using a computer-assisted digital image analyzer at the Laboratory of Neuro-Imaging, University of California School of Medicine, Los Angeles) ~A unidirectional blood-tobrain transfer constant (K~) was calculated using the method of Blasberg, et al.,7 and was expressed in ul/ gm/min.
Definition of Regions For K~ measurements, the tumor periphery was defined as the outer half of the tumor radius, the tumor center as the inner half of the tumor radius, and brain adjacent to tumor as an area of the adjacent basal ganglia in the vascular territory of the lenticular striate vessels.
Data Analysis The K~ values for the experiments were calculated by measuring the three regions of interest described above in three consecutive sections. Analysis of variance and unpaired Student's t-test were applied to the mean values from separate experiments. Results
Physiological Status Arterial blood gas levels were measured in all rats used in the experiments. The mean pH, PaCO2, and PaO2 were 7.394 _+ 0.056, 37.6 4- 5.5 mm Hg, and 96.8 _+ 12.2 mm Hg, respectively (_+ standard deviation). The mean arterial pressure was 132 + 16.8 mm Hg and the hematocrit was 36.2% __ 6.2%. The physiological parameters were not altered after leukotriene infusion.
Brain Tumor Size The size of the rat brain tumors was measured in the histological section with the largest tumor area. The average size was 9.2 _+ 3.1 sq mm, and there was no statistically significant difference among the subgroups.
Dose Response Study An infusion of LTC4 at a dose of 0.5 #g failed to demonstrate any significant change in mean Ki (4standard error of the mean) within the tumors compared to vehicle (36.99 4- 11.26 vs. 39.37 4- 6.45 ~1/ gm/min). The mean Ki values in the 2.5-, 5.0-, and 50.0-#g LTC4 dose groups all increased approximately twofold compared to vehicle alone (90.00 _ 21.14, 92.68 4- 15.04, and 80.17 4- 16.15 vs. 39.37 4- 6.45 ul/ gm/min, respectively; p < 0.01). There was no significant mean change in blood-brain barrier (BBB) permeability of the ipsilateral frontal cortex (4.03 + 4.46, 5.33 4- 3.27, and 3.36 4- 2.44 vs. 3.22 4- 1.97 gl/gm/ rain), contralateral frontal cortex (3.47 _+ 4.72, 3.79 _+ 3.65, and 2.61 4- 2.44 vs. 2.84 + 1.86 ul/gm/min), ipsilateral basal ganglion (4.95 _ 4.15, 4.65 + 3.73, and 3.82 _+ 3.02 vs. 3.11 _+ 1.82 gl/gm/min), and contralateral basal ganglion (3.47 4- 4.72, 3.79 4- 3.65, 2.81 42.73 vs. 2.74 4- 2.02 ul/gm/min) (Fig. 1). No acute side effects were seen in the rats, even in those receiving the
J. Neurosurg. / Volume 77 / September, 1992
Leukotrienes and blood-tumor barrier permeability
FIG. 1. Graph showing permeability (Ki) values of various doses of leukotriene C4 intracarotid infusion. The values are expressed as the mean _+standard error of the mean (vertical bars). Abbreviations: peri = periphery; BAT = brain adjacent to tumor; ipsi = ipsilateral; contra = contralateral; Fr Cx = frontal cortex; BGG = basal ganglion; n = number of rats in each dose group; * = statistically significant (p < 0.01) compared with vehicle.
highest dose. An infusion of 50 ug LTC4 did not, however, elevate the Ki value more than recorded with an infusion of 5 ug.
Time Course Study Fifteen minutes after the termination of LTC4 infusion, the mean Ki value remained elevated by almost twofold compared with vehicle alone (92.68 _+ 15.04 vs. 39.37 + 6.45 ul/gm/min). The mean Ki value significantly declined after 30 and 60 minutes (56.58 _+ 12.50 and 55.40 ___8.10 ul/gm/min, respectively) compared with that after 15 minutes (p < 0.01) (Fig. 2).
FIG. 2. Graph showing permeability (Ki) values at various time intervals after a 5-~g intracarotid infusion of leukotriene C4 (LTC4). The values are expressed as the mean -+ standard error of the mean (vertical bars). Abbreviations: peri = periphery; BAT = brain adjacent to tumor; ipsi Fr Cx = ipsilateral frontal cortex; n = number of rats in each time interval group.
mors. Leukotrienes have also been suggested to be biochemical mediators of ischemic brain edema5 "s,9'~4 In a prior study, we incorrectly suggested that the intracerebral injection of high doses of leukotrienes in rats increased BBB permeability in normal brain. 3'4 Intracerebral injections of leukotrienes in these studies did not in fact increase permeability in normal brain; however, permeability did increase under the area where drilling for the burr hole had occurred, suggesting that injury to brain is prerequisite for leukotrienes to increase permeability. Subsequently, we have demonstrated that intracarot-
Potency of Leukotrienes All sulfidopeptide leukotrienes (LTC4, LTD4, LTE4, and LTF4) increased the mean Ki value within the tumors significantly compared to vehicle alone (92.68 _+ 21.14, 84.19 + 22.16, 139.86 + 23.95, and 100.35 _+ 11.00 vs. 39.37 +_ 6.45 ul/gm/min, respectively; p < 0.01). The most potent was LTE4, which increased the Ki value within the tumors about 389 (Fig. 3).
Discussion Leukotrienes are metabolites of arachidonic acid generated via the 5-1ipoxygenase pathway. Levels of leukotrienes in brain tissue are increased during postischemic reperfusion ~5 and in brain tumors, 5 subaraehnoid hemorrhage, and concussive brain injury. ~ We previously showed a correlation between LTC4 levels and brain edema surrounding tumors in man? Gaetani, et al., ~j also demonstrated that perilesional brain tissue has a significant capacity for an ex vivo synthesis of eicosanoids, and the capacity to synthesize LTC4 is significantly correlated with the extent of edema, particularly in cases of neuroepithelial and metastatic tu-
J. Neurosurg. / Volume 77 / September, 1992
FIG. 3. Graph showing permeability (K0 values of various sulfidopeptide leukotrienes (LTC4, LTD4, LTE4, and LTF4) after intracarotid infusion. The values are expressed as the mean _+ standard error of the mean (vertical bars). Abbreviations: peri = periphery; BAT = brain adjacent to tumor; ipsi = ipsilateral; contra = contralateral; Fr Cx = frontal cortex; n = number of rats in each leukotriene subgroup; * = statistically significant (p < 0.01) compared with vehicle. 409
C. C. C h i o , T. B a b a , a n d K. L. B l a c k id infusion of L]'C4 selectively increases capillary permeability in tumors and ischemic brain but not in normal brain, z,6 This may be due in part to the fact that normal brain capillaries, unlike systemic capillaries, contain high concentrations of the enzyme gamma glutamyl transpeptidase. This enzyme inactivates LTC4 and LTE4 and is lost in brain tumor capillaries and in brain capillaries after ischemia. 26 In effect, normal brain capillaries, unlike capillaries within brain-tumor tissue or ischemic brain, appear to have an "enzymatic barrier" to protect them against the vasoactive effects of leukotrienes. Neuwelt and coworkers 17-2~have shown that osmotic barrier disruption with hypertonic mannitol will nonselectively increase the delivery of chemotherapeutic agents and monoclonal antibodies to brain tissue in humans. However, osmotic disruption of the BBB results in a large increase in drug delivery to normal brain. 16One advantage of leukotrienes selectively opening the blood-tumor barrier is that it leaves the barrier in normal brain intact, 6 protecting normal brain from the adverse effect of antitumor compounds. This study extends our prior report, which demonstrated the ability of LTC4 to selectively open the bloodtumor barrier. The optimal dose range of LTC4 infusion for increasing permeability in rat RG-2 tumors is 2.5 ug or 5.0 #g. Opening of the blood-tumor barrier is reversible and decreases significantly 30 minutes after termination of LTC4 infusions. Leukotriene E4 appears to be the most potent sulfidopeptide leukotriene to open the blood-tumor barrier. Acknowledgments We thank Arthur W. Toga, Ph.D., for use of the NeuroImaging Laboratory and Ms. Gabriele Pollinger for technical assistance. References 1. Asano T, Shigeno T, Johshita H, et al: A novel concept on the pathogenetic mechanism underlying ischaemic brain oedema: relevance of free radicals and eicosanoids. Acta Neuroehir Suppl 41:85-94, 1987 2. Baba T, Black KL, Ikezaki K, et al: Intracarotid infusion ofleukotriene C4 selectively increases blood-brain barrier permeability after focal ischemia in rats. J Cereb Blood Flow Metab 11:638-643, 1991 3. Black KL, Hoff JT: Leukotrienes and blood-brain barrier permeability. J Cereh Blood Flow Metab 5 (Suppl 1): 263-264, 1985 4. Black KL, Hoff JT: Leukotfienes increase blood-brain barrier permeability following intraparenchymal injections in rats. Ann Neurol 18:349-351, 1985 5. Black KL, Holt JT, McGillicuddy JE, et al: Increased leukotriene C4 and vasogenic edema surrounding brain tumors in humans. Ann Neurol 19:592-595, 1986 6. Black KL, King WA, Ikezaki K: Selective opening of the blood-tumor barrier by intracarotid infusion of leukotriene Ca. J Neurosurg 72:912-916, 1990 7. Blasberg RG, Patlak CS, Jehle JW, et al: An autoradiographic technique to measure the permeability of nor-
410
real and abnormal brain capillaries. Neurology 28:363, 1978 (Abstract) 8. Dempsey RJ, Combs DT, Maley ME, et al: Moderate hypothermia reduces postischemic edema development and leukotfiene production. Neurosurgery 21:177-181, 1987 9. Dempsey RJ, Roy MW, Cowen DE, et al: Lipoxygenase metabolites of arachidonic acid and the development of ischaemic cerebral oedema. Neurol Res 8:53-56, 1986 10. Fenstermacher JD, Cowles AL: Theoretic limitations of intracarotid infusions in brain tumor chemotherapy. Cancer Treat Rep 61:519-526, 1977 11. Gaetani P, Rodriguez y Baena R, Marzatico F, et al: "Ex vivo" release of eicosanoid from human brain tissue: its relevance in the development of brain edema. Neurosurgery 28:853-858, 1991 12. Groothuis DR, Fischer JM, Lapin G, et al: Permeability of different experimental brain tumor models to horseradish peroxidase. J Neuropathol Exp Neurol 41: 164-185, 1982 13. Kiwak KJ, Moskowitz MA, Levine L: Leukotriene production in gerbil brain after ischemic insult, subarachnoid hemorrhage, and concussive injury. J Neurosurg 62: 865-869, 1985 14. Minamisawa H, Terashi A, Katayama Y, et al: Brain eicosanoid levels in spontaneously hypertensive rats after ischemia with reperfusion: leukotriene C~ as a possible cause of cerebral edema. Stroke 19:372-377, 1988 15. Moskowitz MA, Kiwak KJ, Hekimian K, et al: Synthesis of compounds with properties of leukotrienes (?4 and D4 in gerbil brains after ischemia and reperfusion. Science 224:886-889, 1984 16. Nakagawa H, Groothuis D, Blasberg RG: The effect of graded hypertonic intracarotid infusions on drug delivery to experimental RG-2 gliemas. Neurology 34:1571-1581, 1984 17. Neuwelt EA, Barnett PA, Bigner DD, et al: Effects of adrenal cortical steroids and osmotic blood-brain barrier opening on methotrexate delivery to gliomas in the rodent: the factor of the blood-brain barrier. Proc Natl Acad Sci USA 79:4420-4423, 1982 18. Neuwelt EA, Barnett PA, McCormick CI, et al: Osmotic blood-brain barrier modification: monoclonal antibody, albumin, and methotrexate delivery to cerebrospinal fluid and brain. Neurosurgery 17:419-423, 1985 19. Neuwelt EA, Hill SA, Frenkel EP: Osmotic blood-brain barrier modification and combination chemotherapy: concurrent tumor regression in areas of barrier opening and progression in brain regions distant to barrier opening. Neurosurgery 15:362-366, 1984 20. Neuwelt EA, Rapoport SI: Modification of the bloodbrain barrier in the chemotherapy of malignant brain tumors. Fed Proc 43:214-219, 1984 21. Toga AW, Santori EM, Samaie M: Regional distribution of flunitrazepam binding constants: visualizing Ka and Bm,x by digital image analysis. J Neurosci 6:2747-2756, 1986
Manuscript received December 18, 1991. Accepted in final form March 4, 1992. This work was supported by National Institutes of Health Grants 1R29NS26523 and IPO1NS2554. Address reprint requests to."Keith L. Black, M.D., Division of Neurosurgery, 74-140 CHS, University of California Medical Center, Los Angeles, California 90024.
J. Neurosurg. / Volume 77/September, 1992
Selective blood-tumor barrier disruption by leukotrienes CHUNG-CHING CHIO, M.D., TAKEHIKO BABA, M.D., AND KEITH L. BLACK, M.D.
The Brain Research Institute, The Jonsson Cancer Center, Division of Neurosurgery, University of California Medical Center, Los Angeles, California v" The authors have previously reported that intracarotid infusion of 5 ug leukotriene C4 (LTC4) selectively increases blood-tumor barrier permeability in rat RG-2 tumors. In this study, rats harboring RG-2 tumors were given 15-minute intracarotid infusions of LTC4 at concentrations ranging from 0.5 ug to 50.0 #g (seven rats in each dose group). Blood-tumor and blood-brain barrier permeability were determined by quantitative autoradiography using '4C aminoisobutyric acid. The transfer constant for permeability (K0 within the tumors was increased twofold by LTC4 doses of 2.5, 5.0, and 50.0 ug compared to vehicle alone (90.00 + 21.14, 92.68 _+ 15.04, and 80.17 _+ 16.15 vs. 39.37 _+6.45 ul/gm/min, respectively; mean _ standard deviation; p < 0.01). No significant change in K~ within the tumors was observed at the 0.5-ug LTC4 dose. Blood-brain barrier permeability was selectively increased within the tumors. At no dose in this study did leukotrienes increase permeability within normal brain. To determine the duration of increased opening of the blood-tumor barrier by LTC, administration, Ki was measured at 15, 30, and 60 minutes after termination of a 15-minute LTC4 infusion (seven rats at each time point). The mean K~ value was still high at 15 minutes (92.68 ___ 15.04 ul/gm/min), but declined at 30 minutes (56.58 _+ 12.50 ul/gm/min) and 60 minutes (55.40 + 8.10 ul/gm/min) after the end of LTC4 infusion. Sulfidopeptide leukotrienes LTC4, LTD4, LTE4 and LTF4 were infused to compare their potency in opening the blood-tumor barrier. The mean leukotriene E4 was the most potent, increasing the permeability value 389 fold compared with vehicle alone (139.86 + 23.95 vs. 39.37 _+6.45 ~l/gm/min). KEy WOADS
T
9
leukotriene
9
brain neoplasm
HE blood-tumor barrier within the capillaries of
primary brain tumors is considered to be an obstacle to delivering water-soluble antineoplastic agents to brain-tumor tissue. 1~ One approach to brain tumor therapy has therefore focused on methods that open or modify the brain-tumor barrier and increase drug delivery. In a previous study, we demonstrated that leukotriene C4 (LTC4) infused into the carotid artery ipsilateral to experimental RG-2 glial tumors in rats increased the unidirectional transfer constant for permeability (Ki) twofold within the tumors, while no effect on permeability was seen in normal brain. 6 Based on this observation, we suggested that LTC4 may be useful clinically in selectively increasing the delivery of antineoplastic agents to tumors. The purpose of this study was to determine the optimal dose and the duration of opening of the blood-tumor barrier by LTC4 administration. The potencies of various sulfidopeptide leukotrienes (LTC4, LTD4, LTE4, and LTF4) were also compared. Materials and Methods
Eighty-four female Wistar rats, each weighing 130 to 170 gm, were used for this study. The sulfidopeptide
J. Neurosurg. / Volume 77/September, 1992
9
blood-brain barrier
9 rat
leukotrienes* were obtained at a concentration of 0.1 mg/ml in a 1:1 phosphate-buffered saline (PBS)-ethanol solution. Aliquots of LTCa, LTDa, LTE4, and LTFa were diluted to experimental concentrations in PBS and the final ethanol concentration was adjusted to 2.5%. Vials containing the leukotrienes were sealed under nitrogen and stored at -80"C. Alpha-[ 1-14C]-aminoisobutyric acid (AIB) (57.6 mCi/mmol)t was used for quantitative autoradiographic study.
Tumor Inoculation The RG-2 glioma cells were maintained in a monolayer culture in F-12 medium with 10% calf serum., The rats were anesthetized with intraperitoneal pentobarbital sodium (Nembutal, 40 mg/kg). Tumor cells (5 • 105 in a 5-ul solution) were stereotactically implanted by means of a Hamilton syringe into the right cerebral
* Leukotrienes supplied by Cayman Chemicals, Ann Arbor, Michigan. ~"Alpha-[ 1-~4C]-aminoisobutyricacid supplied by New England Nuclear, Boston, Massachusetts. F-12 medium and 10% calf serum supplied by GIBCO, Grand Island, New York. 407
C. C. Chio, T. Baba, and K. L. Black hemisphere 5 mm lateral to the bregma and 5 mm deep to the dural surface.
Dose Response Study Ten to 11 days after tumor implantation, the rats were again anesthetized and a polyethylene (PE-50) catheterw was inserted retrograde through the external carotid artery to the common carotid artery bifurcation ipsilateral to the tumor. The external carotid artery was then ligated, and both femoral arteries and one femoral vein were cannulated. Body temperature was mainmined at 37"C. Arterial blood gas levels, blood pressure, and hematocrit were monitored. Animals with abnormal physiological parameters were eliminated from the study. Infusions of LTC4 at concentrations of 0.5, 2.5, 5.0, and 50.0 ug/0.8 ml or 0.8 ml of vehicle (2.5% ethanol in PBS, pH 7.1) were administered into the fight carotid artery of seven rats in each dose group using a constant-infusion pump]l at a rate of 53.3 ul/ min for 15 minutes. Five minutes after the start of the intracarotid infusion, an intravenous bolus of 100 uCi/ kg of ~4C-AIB was delivered. A peristaltic withdrawal pump* was used to withdraw femoral arterial blood at a constant rate of 0.083 ml/min immediately after injection of ~4C-AIB for determination of serum radioactivity. Fifteen minutes after the start of intracarolid infusions, the animals were killed by decapitation and the brains were rapidly removed and frozen.
Time Course Study Ten to 11 days after tumor implantation, the rats were cannulated and physiological parameters were measured as described above. An intracarotid infusion of 0.8 ml LTC4 at a dose of 5 ug was delivered for 15 minutes, and K~was measured at 15, 30, and 60 minutes after the termination of LTC4 infusion.
Potency of Leukotrienes Various sulfidopeptide leukotrienes (LTC4, LTD4, LTE4, and LTFa), at a dose of 5 ug, were infused for 15 minutes into seven rats in each leukotriene group and the Ki value was measured as described above.
Autoradiography The frozen rat brains were mounted onto pedestals with M-1 embedding matrix,t and 20-urn coronal sections were cut using a cryotome. The sections were thaw-mounted onto coverslips, and autoradiograms were generated by coexposing the sections on Kodak XAR-5 film with tissue-calibrated 14C standards~ for 2 wCatheter manufactured by Clay Adams, Parsippany, New York. II Infusion pump manufactured by Harvard Apparatus, Inc., Millis, Massachusetts. * Withdrawal pump manufactured by Gibson Medical Electronics, Inc., Middleton, Wisconsin. t M-1 embedding matrix supplied by Lipshaw Manufacturing, Detroit, Michigan. 14C standards obtained from Amersham Corp., Arlington Heights, Illinois. 408
weeks. Quantitative analysis was performed using a computer-assisted digital image analyzer at the Laboratory of Neuro-Imaging, University of California School of Medicine, Los Angeles) ~A unidirectional blood-tobrain transfer constant (K~) was calculated using the method of Blasberg, et al.,7 and was expressed in ul/ gm/min.
Definition of Regions For K~ measurements, the tumor periphery was defined as the outer half of the tumor radius, the tumor center as the inner half of the tumor radius, and brain adjacent to tumor as an area of the adjacent basal ganglia in the vascular territory of the lenticular striate vessels.
Data Analysis The K~ values for the experiments were calculated by measuring the three regions of interest described above in three consecutive sections. Analysis of variance and unpaired Student's t-test were applied to the mean values from separate experiments. Results
Physiological Status Arterial blood gas levels were measured in all rats used in the experiments. The mean pH, PaCO2, and PaO2 were 7.394 _+ 0.056, 37.6 4- 5.5 mm Hg, and 96.8 _+ 12.2 mm Hg, respectively (_+ standard deviation). The mean arterial pressure was 132 + 16.8 mm Hg and the hematocrit was 36.2% __ 6.2%. The physiological parameters were not altered after leukotriene infusion.
Brain Tumor Size The size of the rat brain tumors was measured in the histological section with the largest tumor area. The average size was 9.2 _+ 3.1 sq mm, and there was no statistically significant difference among the subgroups.
Dose Response Study An infusion of LTC4 at a dose of 0.5 #g failed to demonstrate any significant change in mean Ki (4standard error of the mean) within the tumors compared to vehicle (36.99 4- 11.26 vs. 39.37 4- 6.45 ~1/ gm/min). The mean Ki values in the 2.5-, 5.0-, and 50.0-#g LTC4 dose groups all increased approximately twofold compared to vehicle alone (90.00 _ 21.14, 92.68 4- 15.04, and 80.17 4- 16.15 vs. 39.37 4- 6.45 ul/ gm/min, respectively; p < 0.01). There was no significant mean change in blood-brain barrier (BBB) permeability of the ipsilateral frontal cortex (4.03 + 4.46, 5.33 4- 3.27, and 3.36 4- 2.44 vs. 3.22 4- 1.97 gl/gm/ rain), contralateral frontal cortex (3.47 _+ 4.72, 3.79 _+ 3.65, and 2.61 4- 2.44 vs. 2.84 + 1.86 ul/gm/min), ipsilateral basal ganglion (4.95 _ 4.15, 4.65 + 3.73, and 3.82 _+ 3.02 vs. 3.11 _+ 1.82 gl/gm/min), and contralateral basal ganglion (3.47 4- 4.72, 3.79 4- 3.65, 2.81 42.73 vs. 2.74 4- 2.02 ul/gm/min) (Fig. 1). No acute side effects were seen in the rats, even in those receiving the
J. Neurosurg. / Volume 77 / September, 1992
Leukotrienes and blood-tumor barrier permeability
FIG. 1. Graph showing permeability (Ki) values of various doses of leukotriene C4 intracarotid infusion. The values are expressed as the mean _+standard error of the mean (vertical bars). Abbreviations: peri = periphery; BAT = brain adjacent to tumor; ipsi = ipsilateral; contra = contralateral; Fr Cx = frontal cortex; BGG = basal ganglion; n = number of rats in each dose group; * = statistically significant (p < 0.01) compared with vehicle.
highest dose. An infusion of 50 ug LTC4 did not, however, elevate the Ki value more than recorded with an infusion of 5 ug.
Time Course Study Fifteen minutes after the termination of LTC4 infusion, the mean Ki value remained elevated by almost twofold compared with vehicle alone (92.68 _+ 15.04 vs. 39.37 + 6.45 ul/gm/min). The mean Ki value significantly declined after 30 and 60 minutes (56.58 _+ 12.50 and 55.40 ___8.10 ul/gm/min, respectively) compared with that after 15 minutes (p < 0.01) (Fig. 2).
FIG. 2. Graph showing permeability (Ki) values at various time intervals after a 5-~g intracarotid infusion of leukotriene C4 (LTC4). The values are expressed as the mean -+ standard error of the mean (vertical bars). Abbreviations: peri = periphery; BAT = brain adjacent to tumor; ipsi Fr Cx = ipsilateral frontal cortex; n = number of rats in each time interval group.
mors. Leukotrienes have also been suggested to be biochemical mediators of ischemic brain edema5 "s,9'~4 In a prior study, we incorrectly suggested that the intracerebral injection of high doses of leukotrienes in rats increased BBB permeability in normal brain. 3'4 Intracerebral injections of leukotrienes in these studies did not in fact increase permeability in normal brain; however, permeability did increase under the area where drilling for the burr hole had occurred, suggesting that injury to brain is prerequisite for leukotrienes to increase permeability. Subsequently, we have demonstrated that intracarot-
Potency of Leukotrienes All sulfidopeptide leukotrienes (LTC4, LTD4, LTE4, and LTF4) increased the mean Ki value within the tumors significantly compared to vehicle alone (92.68 _+ 21.14, 84.19 + 22.16, 139.86 + 23.95, and 100.35 _+ 11.00 vs. 39.37 +_ 6.45 ul/gm/min, respectively; p < 0.01). The most potent was LTE4, which increased the Ki value within the tumors about 389 (Fig. 3).
Discussion Leukotrienes are metabolites of arachidonic acid generated via the 5-1ipoxygenase pathway. Levels of leukotrienes in brain tissue are increased during postischemic reperfusion ~5 and in brain tumors, 5 subaraehnoid hemorrhage, and concussive brain injury. ~ We previously showed a correlation between LTC4 levels and brain edema surrounding tumors in man? Gaetani, et al., ~j also demonstrated that perilesional brain tissue has a significant capacity for an ex vivo synthesis of eicosanoids, and the capacity to synthesize LTC4 is significantly correlated with the extent of edema, particularly in cases of neuroepithelial and metastatic tu-
J. Neurosurg. / Volume 77 / September, 1992
FIG. 3. Graph showing permeability (K0 values of various sulfidopeptide leukotrienes (LTC4, LTD4, LTE4, and LTF4) after intracarotid infusion. The values are expressed as the mean _+ standard error of the mean (vertical bars). Abbreviations: peri = periphery; BAT = brain adjacent to tumor; ipsi = ipsilateral; contra = contralateral; Fr Cx = frontal cortex; n = number of rats in each leukotriene subgroup; * = statistically significant (p < 0.01) compared with vehicle. 409
C. C. C h i o , T. B a b a , a n d K. L. B l a c k id infusion of L]'C4 selectively increases capillary permeability in tumors and ischemic brain but not in normal brain, z,6 This may be due in part to the fact that normal brain capillaries, unlike systemic capillaries, contain high concentrations of the enzyme gamma glutamyl transpeptidase. This enzyme inactivates LTC4 and LTE4 and is lost in brain tumor capillaries and in brain capillaries after ischemia. 26 In effect, normal brain capillaries, unlike capillaries within brain-tumor tissue or ischemic brain, appear to have an "enzymatic barrier" to protect them against the vasoactive effects of leukotrienes. Neuwelt and coworkers 17-2~have shown that osmotic barrier disruption with hypertonic mannitol will nonselectively increase the delivery of chemotherapeutic agents and monoclonal antibodies to brain tissue in humans. However, osmotic disruption of the BBB results in a large increase in drug delivery to normal brain. 16One advantage of leukotrienes selectively opening the blood-tumor barrier is that it leaves the barrier in normal brain intact, 6 protecting normal brain from the adverse effect of antitumor compounds. This study extends our prior report, which demonstrated the ability of LTC4 to selectively open the bloodtumor barrier. The optimal dose range of LTC4 infusion for increasing permeability in rat RG-2 tumors is 2.5 ug or 5.0 #g. Opening of the blood-tumor barrier is reversible and decreases significantly 30 minutes after termination of LTC4 infusions. Leukotriene E4 appears to be the most potent sulfidopeptide leukotriene to open the blood-tumor barrier. Acknowledgments We thank Arthur W. Toga, Ph.D., for use of the NeuroImaging Laboratory and Ms. Gabriele Pollinger for technical assistance. References 1. Asano T, Shigeno T, Johshita H, et al: A novel concept on the pathogenetic mechanism underlying ischaemic brain oedema: relevance of free radicals and eicosanoids. Acta Neuroehir Suppl 41:85-94, 1987 2. Baba T, Black KL, Ikezaki K, et al: Intracarotid infusion ofleukotriene C4 selectively increases blood-brain barrier permeability after focal ischemia in rats. J Cereb Blood Flow Metab 11:638-643, 1991 3. Black KL, Hoff JT: Leukotrienes and blood-brain barrier permeability. J Cereh Blood Flow Metab 5 (Suppl 1): 263-264, 1985 4. Black KL, Hoff JT: Leukotfienes increase blood-brain barrier permeability following intraparenchymal injections in rats. Ann Neurol 18:349-351, 1985 5. Black KL, Holt JT, McGillicuddy JE, et al: Increased leukotriene C4 and vasogenic edema surrounding brain tumors in humans. Ann Neurol 19:592-595, 1986 6. Black KL, King WA, Ikezaki K: Selective opening of the blood-tumor barrier by intracarotid infusion of leukotriene Ca. J Neurosurg 72:912-916, 1990 7. Blasberg RG, Patlak CS, Jehle JW, et al: An autoradiographic technique to measure the permeability of nor-
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real and abnormal brain capillaries. Neurology 28:363, 1978 (Abstract) 8. Dempsey RJ, Combs DT, Maley ME, et al: Moderate hypothermia reduces postischemic edema development and leukotfiene production. Neurosurgery 21:177-181, 1987 9. Dempsey RJ, Roy MW, Cowen DE, et al: Lipoxygenase metabolites of arachidonic acid and the development of ischaemic cerebral oedema. Neurol Res 8:53-56, 1986 10. Fenstermacher JD, Cowles AL: Theoretic limitations of intracarotid infusions in brain tumor chemotherapy. Cancer Treat Rep 61:519-526, 1977 11. Gaetani P, Rodriguez y Baena R, Marzatico F, et al: "Ex vivo" release of eicosanoid from human brain tissue: its relevance in the development of brain edema. Neurosurgery 28:853-858, 1991 12. Groothuis DR, Fischer JM, Lapin G, et al: Permeability of different experimental brain tumor models to horseradish peroxidase. J Neuropathol Exp Neurol 41: 164-185, 1982 13. Kiwak KJ, Moskowitz MA, Levine L: Leukotriene production in gerbil brain after ischemic insult, subarachnoid hemorrhage, and concussive injury. J Neurosurg 62: 865-869, 1985 14. Minamisawa H, Terashi A, Katayama Y, et al: Brain eicosanoid levels in spontaneously hypertensive rats after ischemia with reperfusion: leukotriene C~ as a possible cause of cerebral edema. Stroke 19:372-377, 1988 15. Moskowitz MA, Kiwak KJ, Hekimian K, et al: Synthesis of compounds with properties of leukotrienes (?4 and D4 in gerbil brains after ischemia and reperfusion. Science 224:886-889, 1984 16. Nakagawa H, Groothuis D, Blasberg RG: The effect of graded hypertonic intracarotid infusions on drug delivery to experimental RG-2 gliemas. Neurology 34:1571-1581, 1984 17. Neuwelt EA, Barnett PA, Bigner DD, et al: Effects of adrenal cortical steroids and osmotic blood-brain barrier opening on methotrexate delivery to gliomas in the rodent: the factor of the blood-brain barrier. Proc Natl Acad Sci USA 79:4420-4423, 1982 18. Neuwelt EA, Barnett PA, McCormick CI, et al: Osmotic blood-brain barrier modification: monoclonal antibody, albumin, and methotrexate delivery to cerebrospinal fluid and brain. Neurosurgery 17:419-423, 1985 19. Neuwelt EA, Hill SA, Frenkel EP: Osmotic blood-brain barrier modification and combination chemotherapy: concurrent tumor regression in areas of barrier opening and progression in brain regions distant to barrier opening. Neurosurgery 15:362-366, 1984 20. Neuwelt EA, Rapoport SI: Modification of the bloodbrain barrier in the chemotherapy of malignant brain tumors. Fed Proc 43:214-219, 1984 21. Toga AW, Santori EM, Samaie M: Regional distribution of flunitrazepam binding constants: visualizing Ka and Bm,x by digital image analysis. J Neurosci 6:2747-2756, 1986
Manuscript received December 18, 1991. Accepted in final form March 4, 1992. This work was supported by National Institutes of Health Grants 1R29NS26523 and IPO1NS2554. Address reprint requests to."Keith L. Black, M.D., Division of Neurosurgery, 74-140 CHS, University of California Medical Center, Los Angeles, California 90024.
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