Regional blood flow and capillary permeability in the ethylnitrosourea-induced rat glioma

Transcription

1 J Neurosurg 55: , 1981 Regional blood flow and capillary permeability in the ethylnitrosourea-induced rat glioma KAZUO YAMADA, M.D., TORU HAYAKAWA, M.D., YUKITAKA USHIO, M.D., NORIO ARITA, M.D., AMAMI KATO, M.D., AND HEITARO MOGAMI, M.D. Department of Neurosurgery, Osaka University Medical School, Osaka, Japan ~/ Regional cerebral blood flow and capillary permeability of rat brains bearing ethylnitrosourea-induced gliomas of various size were investigated with 14C-antipyrine autoradiography and Evans blue staining. In the small tumors (< 2 mm in diameter), blood flow was uniformly reduced when compared to the adjacent brain. Even in tiny tumors (0.3 to 0.4 mm in diameter), reduction in blood flow was evident. In the medium (2 to 4 mm in diameter) and large (> 4 mm in diameter) tumors, the blood flow increased or decreased depending on the part of the tumor examined. The necrotic center and peripheral edge had low blood flows, whereas the viable portion adjacent to the necrotic center had high blood flows. Blood flow in the brain tissue adjacent to medium and large tumors was lower than control brain tissue, probably due to local edema. Leakage of intravenous Evans blue in the tissue was only evident in the large tumors with central necrosis. The present findings suggest that neovascularization of the tumor may occur when the tumor reaches a certain size, and leaky new vessels may be the cause of brain edema associated with tumor. KEY WORDS 9 regional cerebral blood flow 9 capillary permeability 9 experimental glioma 9 antipyrine autoradiography 9 tumor angiogenesis R EGIONAL cerebral blood flow (rcbf) in the tumor and adjacent brain tissue has been reported to be different from that in normal brains?,n,la In brain tumors, rcbf may increase or decrease depending upon the type of tumor, n In the brain tissue adjacent to tumors, rcbf often decreases, probably due to peritumorous brain edema and subsequent increase in local tissue pressure. 13 Those findings were originally described in clinical studies using a xenon-133 clearance method with external gamma detectors. However, rcbf of deeply seated tumors may not be calculated accurately by this method. Attempts have been made to circumvent this disadvantage by 14C-antipyrine or 14C-iodoantipyrine autoradiographic techniques in experimental animals, 3,7 and by dynamic positron emission tomography in clinical investigations. L~6 By these techniques, rcbf changes in the tumor and adjacent brain tissue can be visualized and correlated with histological sections or computerized tomography (CT) scans. In the present study, rcbf changes in various-sized rat gliomas induced by ethylnitrosourea (ENU) were investigated with ~4C-antipyrine autoradiography. Changes of capillary permeability in the tumor and adjacent brain tissue were evaluated by the extravascular leakage of intravenously administered Evans blue stain in the same sections as were used for rcbf measurement. Using these techniques, we were able to evaluate rcbf and changes in the blood-brain barrier in primary rat brain tumors without any mechanical injury to the brain. Materials and Methods A single dose of ENU (50 mg/kg) was given subcutaneously to Sprague-Dawley newborn rats on the 3rd day after birth. The rats were fed ad libitum and were used for experiment at 377 to 417 days (mean 400 days) of age. Three hours before each experiment, Evans blue (2%, 1 ml/kg body weight) was injected intravenously to evaluate disruption of the bloodbrain barrier in the tumor and adjacent brain tissue. Measurement of rcbf Eight ENU-treated rats were used for rcbf measurements. The rcbf was measured with 14C-anti- 922 J. Neurosurg. / Volume 55 / December, 1981

2 Blood flow and permeability in experimental gliomas pyrine autoradiographic technique as originally described by Reivich, et al. ~2 Under ether anesthesia, animals were placed on a wood platform with their extremities flxed by rubber bands. The right femoral artery, left femoral vein, and right jugular vein were catheterized with Silastic tubes.* Animals were allowed to recover from anesthesia for at least 1 hour. The arterial catheter was attached to a transducer for blood pressure monitoring. Rectal temperature was monitored and kept between 36 ~ to 37~ by cooling or warming as required. Arterial blood gases were measured periodically. The mean values (+ SEM) for po2, pco2 and ph were mm Hg, mm Hg, and , respectively, at the time of rcbf measurements. After those preliminary recordings, 75/~Ci/kg body weight of ~4C-antipyfine (52 mci/mmol)j- dissolved in 1 ml of normal saline was injected at a constant rate for 1 minute through the catheter placed in the left femoral vein. Arterial blood samples were withdrawn from the arterial catheter every 10 seconds during a 1-minute injection of 14C-antipyrine. The blood samples were placed in preweighed counting vials, reweighed, suspended in l0 ml of scintillation phosphor, and radioassayed in a liquid scintillation counter.~ The blood samples generally weighed 0.05 to 0.08 gin. The volume of the blood sample was calculated assuming the specific gravity of blood as 1.05 gm/ml. TM After the injection, animals were sacrificed by the rapid intravenous injection of saturated potassium chloride solution through the catheter placed in the jugular vein. The skull was opened, and the brain was quickly removed, frozen in isopentane suspended in a bath of dry ice, and stored at -40~ until sectioned. Brain sections were cut 40/~m thick in a cryostatw at -20~ placed on cover glass, and dried on a hot plate at 60~ within l0 seconds. The cover glasses were flxed on thick paper with adhesive tape and attached to Kodak no-screen x-ray film (NS-5T) with brain sections facing the emulsion of x-ray film in the dark room. Plastic standard plates containing known amounts of 14C-methyl methacrylate[[ were also placed on x-ray film and fixed with adhesive tapes. These standards had previously been calibrated for their autoradiographic equivalence to the ~4C concentrations in the brain sections (40/zm in thickness) prepared as described above. The method described by * Silastic tubes manufactured by Dow Coming Corp., Midland, Michigan. J-~4C-antipydne obtained from New England Nuclear, Boston, Massachusetts. Mark III scintillation counter manufactured by Tracor Analytic, Chicago, Illinois. w Cryostat manufactured by South London Equipment Co., Ltd., London, England. II 14C-methy I methacrylate manufactured by The Radiochemical Cemre, Amersham, England. TABLE 1 Tumors induced by ethylnitrosourea in 12 rats Description No. of of Tumors Tumors large tumors (> 4 ram) 11 medium tumors (2--4 ram) 14 in cortex 9 in white matter 5 small tumors (< 2 nun) 32 in cortex 16 in white matter 6 in thalamus 7 in pons 3 Reivich, et al, az has been employed for calibration of the standards. The x-ray film with cover glass and plastic standard plates were placed in the x-ray film cassette. After exposure for 3 weeks, films were removed, separated from cover glass and standard plates, and developed. The densitometric measurements of the autoradiographs were made with a densitometer equipped with a 0.5-ram aperture.* A calibration curve of the relationship between optical density and tissue 14C concentration for each film was obtained by densitometric measurements of the standards. The concentration of 14C-antipyrine in the various regions of the brain and tumor was determined from the calibration curve and the optical densities of the film in the corresponding sites. Regional CBF was calculated according to the equation: Ci(T) = ~ki f~ Cai e -kl (T - t) dt, where Ci(T) is the concentration of the tracer in the tissue at time T; ~ is the tissue-blood partition coefficient for ~4C-antipyfine; ki is the rate of blood flow per unit weight of tissue multiplied by the reciprocal of the partition coefficient; and Cai is the arterial concentration of the tracer. A digital computer~- was used for calculation. Capillary Permeability Pictures of the sections of the frozen brain were taken serially to evaluate extravascular leakage of Evans blue. Brain sections on cover glasses were stained with hematoxylin and eosin for macroscopic and microscopic observation of the tumor. Tumor-Blood and Brain-Blood Partition Coefficients Four ENU-treated rats were used to determine tumor-blood and brain-blood partition coefficients of antipyrine. Rats were anesthetized with pentobarbital * Sakura PDA-15 densitometer manufactured by Konishiroku, Tokyo, Japan. J-Canon digital computer, Model BX-1, manufactured by Canon Co., Ltd., Tokyo, Japan. J. Neurosurg. / Volume 55 / December,

3 TABLE 2 Tissue-blood partition coefficients for 14 C-antipyrine in rat brain and ENU-induced gliomas Tissue cortex white matter thalamus ENU-induced glioma necrotic center t viable part ~ periphery ~ small tumors ~: * Mean values SD in 20 measurements of four rats having ethylnitrosourea (ENU)-induced glioma. t" Statistically different (p < 0.01) from the other parts of the tumor. No statistical differences were noted, and a mean value of 0.92 was used for calculation. (30 mg/kg intraperitoneally). Both renal arteries, the hepatic artery, superior mesenteric artery, and portal vein were cauterized and cut under an operating microscope in order to obtain a steady state. The right femoral artery and vein were catheterized with Silastic tubes. Blood pressure was monitored continuously during each 1-hour experiment. A bolus injection of 14C-antipyrine (20 #Ci) was given through a venous catheter. After 1 hour, each animal was sacrificed by cervical dislocation. A blood sample was obtained by cardiac puncture. The blood concentration of 14Cantipyrine was determined by the same method as described above. The brains were removed quickly and cut coronally, and samples of gray and white matter were obtained for direct measurement of 14Cantipyrine concentration. The samples were weighed in counting vials, dissolved with tissue solubilizer,:~ and radioassayed as described above. The other part of the brain was prepared for autoradiography in the same technique as described above. Results A total of 57 tumors induced by ENU were found in 12 rats. A description of the tumors is given in Table 1. Tissue-Blood Partition Coefficient Tumor-blood and brain-blood partition coefficients are listed in Table 2. Brain-blood partition coefficient was No differences were noted in values in the cortex, white matter, and thalamus. In the ENU-induced gliomas, the tumor-blood partition coefficient varied depending on the part of the tumor sampled. In the necrotic center, partition coefficient was 1.02, which was significantly different from the other parts of the tumor. The viable part, the periphery, and K. Yamada, et al. TABLE 3 Blood flow in the ethylnitrosourea-induced gliomas Blood Flow Partition Tumor Tissue No. of (ml/min/100 gm) Tumors Coefficients* Mean _ SD Range large tumors (> 4 mm) necrotic center "t 4-28 viable part periphery ~" * medium tumors (2 to 4 mm) viable part *t periphery * small tumors (< 2 mm) * t * Significantly different (p < 0.01) from normal cortex values. t Significantly different (p < 0.01) from white matter values. small tumors had partition coefficients of 0.93, 0.92 and 0.92, respectively. However, no statistical differences were noted among those tissues, and a mean value of 0.92 was used for calculation. Blood Flow Blood flow data in the ENU-induced gliomas are summarized in Fig. 1 and Table 3. In the small tumors (< 2 mm in diameter), blood flow was uniformly reduced. Although most small tumors were located in the cerebral cortex and thalamus, blood flow data of these tumors were similar to, or lower than, those of the white matter. There was a tendency for blood flow to decrease as tumors grew larger. Typical autoradiographic and histological appearances of small tumors are shown in Figs. 2 and 3. In the medium-sized tumors (2 to 4 mm in diameter), blood flow always showed the same pattern (Fig. 3). The viable center had relatively high blood flow and the periphery had low flow (Fig. 1 and Table 3). NCS tissue solubilizer supplied by Amersham Corp., Arlington Heights, Illinois. FIG. 1. Blood flows of various-sized ethylnitrosoureainduced rat gliomas. 924 J. Neurosurg. / Volume 55 / December, 1981

4 Blood flow and permeability in experimental gliomas FIG. 2. Autoradiographic (left) and histological (right) appearance of a small ethylnitrosourea-induced glioma (arrow). This tiny tumor (0.5 mm in diameter) had about 40% reduction in blood flow compared to the adjacent thalamus. Histological examination revealed that the center of the tumor was highly cellular without any necrosis. The peripheral edge was less cellular, with tumor cells invading surrounding brain tissue. In large tumors (> 4 mm in diameter), the center of the tumor was necrotic. Adjacent to the central necrosis, was an area with histologically viable tumor cells (viable part); this area showed high blood flow. Conversely, low blood flow was found in the periphery where tumor cells were invading surrounding brain tissue. Representative autoradiography of large tumors is shown in Figs. 3 and 4. Blood flows in the brain tissue adjacent to the tumors are listed in Table 4. In the brain tissue adjacent to small and medium-sized tumors, only the cortex was affected by the tumor and showed blood flow significantly lower than normal cortex (Table 4). In the brain adjacent to large tumors, both the cortex and white matter were affected and showed blood flow significantly lower than normal brain. Capillary Permeability Leakage of intravenously administered Evans blue into the tissue was seen only in tumors larger than 4 mm in diameter. A tumor stained by Evans blue is shown in Fig. 5. Although there are two other tumors, one medium and one small, in this section (the same section as shown in Fig. 3) they are not stained and Fro. 3. Autoradiograph (left) and histological picture (right) of small, medium, and large rat gliomas. The small tumor (arrow) had uniform reduction in blood flow. Blood flow in the medium and large tumors varied depending upon the part of the tumors. J. Neurosurg. / Volume 55 / December,

5 K. Yamada, et al. FIG. 4. Autoradiographic (left) and histological (right) appearance of a large right frontal glioma. The necrotic center and periphery have low blood flows, whereas the viable part has a high blood flow. TABLE 4 Regional cerebral blood flow in the brain tissue adjacent to ethylnitrosourea-induced gliomas Brain Tissue No. of Blood Flow Measure- (ml/min/100 gm) ments Mean SD Range normal brain tissue cortex white matter thalamus adjacent brain tissue adjacent to large tumors cortex * white matter t adjacent to medium tumors cortex * white matter adjacent to small tumors cortex " white matter * Significantly different (p < 0.01) from normal cortex values. 1" Significantly different (p < 0.01) from white matter values. are not visible in Fig. 5. Even in the large tumors, leakage of Evans blue was seen mainly in the necrotic center and viable part. Brain tissue at the periphery and adjacent to the tumor was apparently not stained by Evans blue, indicating that the blood-brain barrier was partly preserved in those areas. Discussion Quantitative autoradiography has currently been employed to study rcbf in the various lesions of experimental animals. Blasberg, et al, 3 reported that metastatic Walker 256 tumors in the rat brain 15 decrease blood flow to various degrees. They found that a decrease in blood flow was not uniform in the large tumors. The center of the tumor was often necrotic and showed low blood flow, whereas at the periphery, where viable cells were invading adjacent brain tissue, blood flows were high) Although the present model involves chemically induced gliomas, the results obtained have certain similarities to those of metastatic tumors. In the early stage of the tumors (> 2 mm in diameter), blood flow was always decreased when compared to that in the surrounding brain tissue. Of the tumors with diameters of less than 2 mm, there was a tendency that the larger the tumor was, the lower was the blood flow. The finding may suggest that in the early stage of tumor growth, the proliferating tumor cells do not have their own blood vessels but nutrition is supplied by diffusion from surrounding brain tissue. On the other hand, of the tumors with diameters of more than 2 ram, the center had higher blood flow than the periphery. This may indicate that the center had a direct blood supply from surrounding brain tissue through new blood vessels. This phenomenon has been explained by Folkman as "angiogenesis. ''6 In experiments with subcutaneously transplanted astrocytoma cells, he found that angiogenesis occurred when tumors became larger than 2 to 3 mm in diameter. 5 This observation would support our view, and it is suggested that angiogenesis in the ENU-induced rat glioma seemed to occur when the tumor increased to 2 mm in diameter. When tumors were more than 4 mm in diameter, the center underwent necrotic change, indicating that neovascularization was not enough to supply all of the nutritional demands of the tumor. The viable part of large tumors tended to have higher blood flow than the same part of medium-sized tumors. Histological examination revealed that the viable portion of large tumors was more anaplastic than that of small or medium-sized tumors. These 926 J. Neurosurg. / Volume 55 / December, 1981

6 Blood flow and permeability in experimental gliomas new blood vessels in the center of the tumor do not have tight junctions, a and capillary permeability increased in that area. This increase in capillary permeability may apparently produce edema in the surrounding brain tissue. 2,s The present findings indicate that neovascularization of the tumor might be the key factor for tumor growth and production of brain edema associated with tumor. Acknowledgments The authors wish to thank Mr. K. Sakakibara for collaboration in the radioactive assay. Miss R. Fujita and Mrs. N. Yamada kindly assisted in the autoradiography studies. Fie. 5. Typical Evans blue staining of rat gliomas (the same section as shown in Fig. 3). Evans blue staining was found only in the viable and necrotic part of the large tumor. Although one medium and one small tumor (arrows) were located in this section, no staining with Evans blue was noted. There is also no staining at the periphery of the large tumor or in the adjacent brain. findings suggest that there may be a correlation between malignancy of the tumor and amount of blood flow. The present study also indicates that the brain tissue adjacent to the tumors had decreased blood flows in various degrees. This reduction in blood flow was more apparent in the cortex than in the white matter, and may be partly due to the fact that most small and medium-sized tumors were located in the cerebral cortex and thalamus. In large tumors, brain edema became histologically apparent both in the cortex and in the white matter adjacent to the tumors. In those edematous brain tissues, blood flow was reduced (Table 4) probably due to increased local tissue pressure. 1~ The reduction in rcbf adjacent to the tumor may be partly attributable to the appearance of focal neurological deficits. Capillary permeability of the tumor and adjacent brain tissue has been studied by several authors. 2,a,8 However, many of these experiments were undertaken by direct transplantation of the tumor cells to the brain, and the effect of surgical manipulation on changes in the capillary permeability could not be excluded. In the present study, the tumor was induced without any mechanical injury to the brain, and changes of the capillary permeability might be more physiological than those of a transplantation model. In the early stage of tumor growth, no increase in capillary permeability was noted in the tumor tissue. When the tumor became larger and necrosis occurred, leakage of Evans blue was evident mainly in the necrotic center and viable portion. This indicates that References 1. Ackerman RH, Subramanyam R, Correia JA, et al: Positron imaging of cerebral blood flow during continuous inhalation of C1502. Stroke 11:45--49, Ausman JI, Levin VA, Brown WE, et al: Brain-tumor chemotherapy. Pharmacological principles derived from a monkey brain-tumor model. J Neurosurg 46: , Blasberg R, Patlak C, Shapiro W, et al: Metastatic brain tumors: local blood flow and capillary permeability. Neurology 29:.547, 1979 (Abstract) 4. Endo H, Larsen B, Lassen NA: Regional cerebral blood flow alterations remote from the site of intracranial tumors. J Neurosurg 46: , Folkman J: Anti-angiogenesis: new concept for therapy of solid tumors. Ann Surg 175: , Folkman J: Tumor angiogenesis: a possible control point in tumor growth. Ann Intern Meal 82:96-100, Hossmann KA, Niebuhr I, Tamura M: Blood flow and metabolism in the rat brain during experimental tumor development. Acta Neuroi Stand 60 (Suppl 72): , Levin VA, Freeman-Dove M, Landahl HD: Permeability characteristics of brain adjacent to tumors in rats. Arch Neurol 32: , Long DM: Capillary ultrastructure and the blood-brain barrier in human malignant brain tumors. J Neurosurg 32: , Marmarou A, Shulman K, Shapiro K, et al: The time course of brain tissue pressure and local CBF in vasogenic edema, in Pappius HM, Feindel W (eds): Dynamics of Brain Edema. Berlin/Heidelberg/New York: Springer-Verlag, 1976, pp P~ilv61gyi R: Regional cerebral blood flow in patients with intracranial tumors. J Neurosurg 31: , Reivich M, Jehle J, Sokoloff L, et al: Measurement of regional cerebral blood flow with antipyrine-14c in awake cats. J Appl Physiol 27: , Reulen HJ, Hadjidimos A, Schiirmann K: The effect of dexamethasone on water and electrolyte content and on rcbf in perifocal brain edema in man, in Reulen H J, Schiirmann K (eds): Steroids and Brain Edema. Berlin/ Heidelberg/New York: Springer-Verlag, 1972, pp Sakurada O, Kennedy C, Jehle J, et al: Measurement J. Neurosurg. / Volume 55 / December,

7 K. Yamada, et al. of local cerebral blood flow with iodo[14c]antipyrine. Am J Physiol 234:H59-H66, Ushio Y, Chernik NL, Shapiro WR, et al: Metastatic tumor of the brain: development of an experimental model. Ann Neurol 2:20-29, Yamamoto YL, Thompson CJ, Meyer E, et al: Dynamic positron emission tomography for study of cerebral hemodynamics in a cross section of the head using positron-emitting 68Ga-EDTA and 77Kr. J Comput Assist Tomogr 1:43-56, 1977 Manuscript received September 15, Accepted in final form July 20, This work was supported in part by a grant-in-aid for fundamental scientific research from the Ministry of Education and for cancer research from the Ministry of Health and Welfare. Address reprint requests to: Kazuo Yamada, M.D., Department of Neurosurgery, Osaka University Medical School, , Fukushima, Osaka 553, Japan. 928 J. Neurosurg. / Volume 55 / December, 1981