Quantitative observations of the subacute effects of X irradiation on brain capillary permeability: Part II

Male Fischer 344 rats received cerebral irradiation at 300 rad/day, four days/wk for three wk, to a total dose of 3600 rad. The capillary permeability coefficients for galactitol, urea, and sodium determined at 6, 10 and 12 wk postirradiation were not statistically different when they were compared to control rats. We found no evidence for the “early” delayed effects of X irradiation based on alteration of brain capillary permeability. We feel this syndrome is the result of alteration in the processes that control the maintenance of myelin. Further investigation of cellular and biochemical effects of irradiation would be more informative than additional capillary membrane studies.     

Early and late response of the mouse limb to multifractionated X-irradiation.

Studies have been made of the response of C3H mouse hind limbs to various schedules of fractionated irradiation to elucidate the relative roles of repair and repopulation in determining early and late reactions.Early response has been measured in terms of desquamation and healing of skin covering the foot: late response has been evaluated in terms of deformity of the limb existing at 12 months after treatment.It was found that with 10 equal dose fractions, there was very little difference in response(approximately 6%) for early and late reactions, if treatments were given once, twice or four times per day. If the first nine treatments were given as small doses (200 rad compared with 400–700 rad per fraction) then the extent by which the dose must be decreased in reducing the fraction interval from 24 to 6 or 12 hr is 11% for the early reaction and 17% for the late response. In both cases the increased sensitivity changing from 24 hr to 6 hr fractionation intervals was greater (but not statistically significantly so) for the late than for the early reaction.Radiation given as 10, 20 or 30 “daily” fractions showed that, as overall treatment time increased: (a) the acute skin response (particularly the peak reaction) became less severe than predicted by the Ellis¹² formula, apparently because of rapid proliferation. (b) Late deformity reactions also became less severe at longer treatment times than predicted by the Ellis¹² formula although not to the same extent as did the mean early reaction. (c) The critical parameter determining tolerance as far as the early reaction is concerned is the dose per fraction rather than total dose for treatments given over longer times.A comparison of early versus late reactions showed that, at the longer treatment times, the late reaction was significantly more severe than was predicted from the early skin reaction.     

The effects of adriamycin and X-irradiation on the murine duodenum.

The effects of a single dose of X-irradiation at various times following a single dose of Adriamycin have been evaluated at the cellular level (duodenal crypt cell survival) and whole-animal level (LD_{$\text{50}\text{6}$}; the dose required to kill 50% of the animals in 6 days). Qualitatively, the 10-clone doses (the X-ray dose required to reduce crypt survival to 10) parallel the data for the effects of Adriamycin on the rate of DNA synthesis. In contrast, changes in the slopes (D_o) of the survival curves for the combined treatments do not correlate with the ADR-induced inhibition of DNA synthesis. The (LD_{$\text{50}\text{6}$} doses for the combined treatments also parallel the cellular toxicity data initially but for intervals from 2 to 14 days, when the cellular parameters are back to normal, there is still evidence for an Adriamycin-enhanced radiotoxicity. The discrepancy between the Adriamycin-enhanced duodenal cellular radiotoxicity and whole-body radiotoxicity is not surprising with a systemic drug like Adriamycin; however, neither the specific tissue(s) affected nor the specific underlying molecular and cellular mechanism(s) are evident in the studies to date.     

Sequential change of capillary permeability in the rat brain after surgical removal of an experimental brain tumor

Summary Experimental brain tumors were excised from rats for sequential observation of changes in local capillary permeability during the postsurgical period. Experimental brain tumor-bearing rats were prepared by stereotaxic transplantation of cultured tumor cells and the resultant tumor was delineated by administration of a dye. Following excision of the stained tumor by craniotomy, sequential changes in local capillary permeability were quantitatively followed-up by autoradiography, using¹⁴C-amino-isobutyric acid as a tracer. Capillary permeability was enhanced following surgery, reaching a maximum both in the extent and degree on the third day. After undergoing a gradual reduction, it showed a marked increase for the second time in a very small area on the 10th postoperative day. A recurrence of the tumor was responsible for this late but marked increase. For a control group, the caudate nucleus was excised from normal rats, followed by observation of the sequential changes in the local capillary permeability. Due to surgical procedure, capillary permeability reached a maximum both in the extent and degree on the 5th postoperative day (slightly later than in the tumor group). This change in capillary permeability was less pronounced than in the tumor group. The difference in the conditions of surgery — tumor excision and partial excision of a normal brain tissue — appeared to explain this difference. The results of this study indicated that it is more desirable to give water-soluble antineoplastic agents early during the postoperative period for chemotherapy of a malignant brain tumor after surgery.     

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

Summary 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 µ g/ml of protein > 10 k-daltons from 3 × 10⁸ 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 (µ 1/g · min), of ¹⁴C-alpha-aminoisobutyric acid (¹⁴C-AlB) 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 (K_max) of SUP-C was 10.83 ± 0.99 and that of the control was 2.53 ± 0.22 (p < 0.001). The Km. of SUP-L was 5.61 ± 0.23 and that of the control was 2.67 ± 0.36 (p < 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 (K_max (untreated): 8.30 ± 0.82, K_max (treated): 1.33 ± 0.64, p < 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.Presented in part at the meeting of the American Association for Cancer Research, Atlanta, GA, May 1987.     

Effects of induced hypertension on blood flow and capillary permeability in rats with experimental brain tumors

Summary To study the possibility of enhancing the delivery of antineoplastic agents to tumor tissue, we conducted an experimental study using induced hypertension with angiotensin 11 in rats with experimental brain tumors. Drug delivery was evaluated by measuring local cerebral blood flow (LCBF) and regional cerebral capillary permeability with quantitative autoradiography.There was no significant difference of LCBF in the central region of tumor tissue between the control group and the induced hypertension group. LCBF in the peripheral region of tumor tissue in the induced hypertension group was significantly higher than that in the control group. On the other hand, despite induced hypertension, no significant changes in the regional cerebral capillary permeability were observed between the groups.These results indicate that delivery of the lipid-soluble antineoplastic agents, which depend upon cerebral blood flow, can be enhanced by induced hypertension.     

The effects of dexamethasone on experimental brain tumors: I. Transcapillary transport and blood flow in RG-2 rat gliomas

Summary Dexamethasone dramatically improves cerebral edema associated with malignant gliomas. Although the pathophysiology of this effect is not clearly understood, many investigators have postulated that tumor capillary permeability is reduced by dexamethasone. We studied blood-to-tissue transport and blood flow in 178 RG-2 transplanted gliomas in a control group and four groups given dexamethasone at doses of 3, 6, 9, and 12 mg/kg for four days.¹⁴C- aminoisobutyric acid (AIB) was used to study blood-to-tissue transport in 31 animals; in an additional 27 animals¹⁴C-AIB and¹³¹I-iodoantipyrine (IAP) were used in double label experiments to study blood-to-tissue transport and blood flow. Regional measurements of the transfer constant (K) of AIB and blood flow (F) were made with quantitative autoradiography. There were significant differences between the control and dexamethasone-treated groups with regard to weight loss and plasma glucose. However, there wasno significant effect of dexamethasone on values of K or F, regardless of the tumor or brain region examined, and regardless of the dose of dexamethasone administered. Analysis of the profiles of the transfer constant of AIB in the brain around tumor showed that the K of AIB decreased within 0.5 mm of the tumor edge in direct relationship to the dexamethasone dose. These results do not support the hypothesis that dexamethasone reduces brain tumor capillary permeability, and suggest that dexamethasone may decrease tumor-associated cerebral edema by effects on bulk flow away from the tumor margin.     

The acute effects of CMF-based chemotherapy on maxillary periodontal microcirculation

Purpose  A high incidence of oral complications is associated with chemotherapy (CT) treatment in cancer patients; however, while knowledge into molecular mechanisms of their pathobiology continue to evolve, the direct physiological effects of CT on oral tissue perfusion remain unexplored. The aim of this investigation was to assess the acute effects of CT on gingival microcirculation perfusion by measuring gingival capillary density. Methods  Twenty female specific-pathogen free New Zealand White rabbits were randomly divided into four groups receiving four different intravenous dose levels of cyclophosphamide, methotrexate, and fluorouracil (CMF). Noninvasive measurements of gingival capillary density were performed using sidestream dark-field (SDF) imaging prior to and 30 min after CT treatment. Four rabbits receiving saline solution were used as control animals. Results  Baseline gingival capillary density was 58 ± 11 cpll/mm², no significant differences in baseline capillary densities between the groups were found. From low to high dose CT, capillary density 30 min after CMF treatment increased in each group by 1 ± 7, 5 ± 7, 13 ± 18 and 20 ± 13 cpll/mm², respectively. Capillary density increase was significant in the high-dose group. No change in gingival capillary density was found in the control group. Conclusions  Periodontal microcirculation perfusion had increased 30 min after CT treatment as indicated by a rise in gingival capillary density. Our results support the idea that CT-induced microcirculatory response not only diligently delivers but also saturates peripheral oral tissues with antineoplastic agents by increasing surface area exposure. This functional response of the microcirculation to CT drugs may play a role in contribution to oral complications and the treatment of oral tumors.     

The effect of time between X-irradiation and chemotherapy on the growth of three solid mouse tumors. V. Bleomycin.

Experiments have been carried out to determine the effect of different time intervals between the administration of X-radiation (1200 rod) and bleomycin (20 mg/kg) on the growth delay produced in three mouse tumors. The tumors used were the EMT6 tumor in BALB/c mice and the KHT and RIF-1 sarcomas in C3H mice. All tumors were grown intramuscularly in the gastrocnemius muscle and treatment was carried out at a mean tumor weight of 450 mg. Time to reach 2X (for KHT) or 4X (for EMT6 and RIF-1) treatment volume was used as the endpoint of response. The drug was administered by the intraperitoneal route either 24, 6, or 2 hr before radiation, immediately before the start of radiation, or 3, 6, or 24 hr after radiation. All irradiations were carried out in unanesthetized mice.For a single administration at this dose level, bleomycin alone did not produce a significant growth delay in any of the tumors. In the RIF-1 tumor, growth delays following combination treatments were equal to the addition of the single agent growth delays. In two experiments with EMT6, contrary results were obtained, one producing longer delays following combination treatments than predicted and the other producing shorter delays. This is apparently due to the variability in the growth delay after treatment with radiation alone for this tumor. For the KHT tumor, only small differences from the addition of single agent delays were seen.     

The effect of time between X-irradiation and chemotherapy on the growth of three solid mouse tumors. IV. Actinomycin-D

Experiments have been carried out to determine the effect of different intervals between the administration of X-radiation (1200 rod) and actinomycin-D (200 μg/kg) on the growth delay produced in three mouse tumors. The tumors used were the EMT6 tumor in BALB/c mice and the KHT and RIF-1 sarcomas in C3H mice. All tumors were grown intramuscularly in the gastrocnemius muscle, and treatment was carried out at a mean tumor weight of 450 mg. Time to reach 2 × (for KHT) or 4 × (for EMT6 and REF-1) treatment volume was used as the endpoint of response. The drug was administered intraperitoneally either 24, 6, or 2 hr before radiation, immediately before the start of radiation, or 3, 6, or 24 hr after radiation. All irradiations were carried out in unanesthetized mice.For a single administration at this dose level (close to the maximum tolerated dose) actinomycin-D did not produce a significant delay in the growth of any of the tumors. For the RIF-1 and KHT tumors, the growth delays produced by drug/radiation combinations generally were not significantly greater than that produced by irradiation alone. For the EMT6 tumor, great variability in the growth delays of combined modality groups was seen, with mean growth delays significantly longer than predicted by the radiation alone data. No consistent dependence on timing between irradiation and drug administration was seen.     

The effect of time between X-irradiation and chemotherapy on the growth of three solid mouse tumors. III. Cis-diamminedichloroplatinum

Experiments have been carried out to detemine the effect of different time intervals between the administration of X-irradiation (1200 rad) and cis-diamminedichloroplatinum (cis-DDP) (7 mg/kg) on the growth delay produced in three mouse tumors. The tumors used were the EMT6 tumor in BALB/c mice and the KHT and RIF-1 sarcomas in C3H mice. All tumors were grown intramuscularly in the gastrocnemius muscle and treatment was carried out at a mean tumor weight of 450 mg. Time to reach 2X (for KHT) or 4X (for EMT6 and RIF-1) treatment volume was used as the endpoint of response. The drug was administered by the intraperitoneal route either 24, 6, or 2 hr before radiation, immediately before the start of radiation, or 3, 6, or 24 hr after radiation. All irradiations were carried out in unanesthetized mice.The growth delays due to the drug alone were 2, 10, and 2 days for the EMT6, RIF-1, and KHT tumors, respectively. In the RIF-1 and KHT tumors, the combined modality groups tend to show longer growth delays than predicted by the addition of the growth delays for the single agents. For the EMT6 tumor, however, the trend is in the opposite direction. There is no particular timing between irradiation and drug administration which appears to produce consistently longer or shorter growth delays from system to system.     

The effect of time between X-irradiation and chemotherapy on the growth of three solid mouse tumors. VI. BCNU

Experiments have been carried out to determine the effect of different time intervals between the administration of X-irradiation (1200 rad) and BCNU (15 mg/kg) on the growth delay produced in three mouse tumors. The tumors used were the EMT6 tumor in BALB/c mice and the KHT and RIF-1 sarcomas in C3H mice. All tumors were grown intramuscularly in the gastrocnemius muscle and treatment was carried out at a mean tumor weight of 450 mg. Time to reach 2X (for KHT) or 4X (for EMT6 and RIF-1) treatment volume was used as the endpoint of response. The drug was administered by the intraperitoneal route either 24, 6, or 2 hr before radiation. All irradiations were carried out in unanesthetized mice. The growth delays due to the drug alone were 2, 6, and 11 days for the RIF-1, EMT6, and KHT tumors, respectively.No consistent general pattern emerged from the results of combination treatments. For the RIF-1 tumor, the growth delays following combination treatments were generally less than predicted by the simple addition of the growth delays for the single modalities. For EMT6 this was true when BCNU was administered immediately before X-rays, but not for other timings. In the KHT tumor an unexpectedly high incidence of long-term tumor controls was seen in the group which received BCNU at 2 hr before X-rays.In addition to the single dose studies (above), fractionated regimens in which radiation and BCNU were combined in several different ways were tested with the RIF-1 tumor. None of the combination schedules tested showed a greater-than-additive effect.     

The effect of time between X-irradiation and chemotherapy on the growth of three solid mouse tumors--I. Adriamycin

Experiments have been carried out to determine the effect of different time intervals between X-irradiation (1200 rad) and the administration of adriamycin (ADR) (6 mg/kg) on the growth delay produced in 3 mouse tumors. The tumors used were the EMT6/St/i.d. tumor in BALB/c mice and the KHT and RIF-1 sarcomas in C3H mice. All tumors were grown intramuscularly in the gastrocnemius muscle and treatment was carried out at a mean tumor weight of 450 mg. Time to reach 2X (for KHT) or 4X (for EMT6/St/i.d. and RIF-1) treatment volume was used as the endpoint of response. The drug was administered by the intraperitoneal route either 24, 6, or 2 hr before radiation, immediately before the start of radiation, or 3, 6, or 24 hr after radiation. All irradiations were carried out in unanesthetized mice.For a single administration at this dose level (close to the maximum tolerated dose), ADR alone produced only minimal growth delay in any of the tumors. Furthermore, the growth delays produced by drug/radiation combinations were not significantly different from the addition of the growth delays produced by the single modalities.     

The effects of preoperative x-irradiation on the survival and blood flow of pedicle skin flaps in the pig

Pedicle skin flaps have been raised from pre-irradiated sites on the flanks of pigs. Radiation treatment was given as a single dose, 6 fractions in 18 days or 30 fractions in 39 days. Surgery was performed at 12, 52 or 104 weeks after irradiation. Control flaps were raised from normal skin on the other flank. The length of flap remaining viable after surgery was shorter in the irradiated than the control flaps. This reduction in flap survival was the same at the three time periods at which it was assessed and for each of the radiation doses selected for the different treatment groups. Clearance rates of an isotope (^99mTechnetium) injected intradermally in the distal surviving regions of irradiated and normal flaps were compared. Clearance changes were related to those recorded in normal and irradiated skin before surgery. Isotope clearance in normal flaps was impaired after surgery (days 1–3) but then became faster than in intact skin (days 5–14). A similar pattern of changes was recorded in irradiated flaps only when the pre-operative isotope clearance rates in irradiated skin were similar to that in normal skin (i.e. for all treatment groups at 52 and 104 weeks after treatment). However, when pre-operative clearance was already slower in irradiated than in normal skin (i.e. for a single dose and 6 fraction/ 18 days after 12 weeks), surgery in the irradiated site did not have the usual effect of slowing the clearance rate.     

MCNU effectiveness on brain tumor. Part I: Antitumor activity in vitro on human glioma and neuroblastoma cell lines

A new water-soluble nitrosourea ( MCNU ) was tested for its antitumor activity against fourteen human glioma cell lines and two neuroblastoma cell lines. Four experiments were performed to determine its antitumor activity: inhibition of cell growth, comparison with ACNU, morphological observation, and analysis of DNA histogram with flowcytometry . Seven out of 14 gliomas (50%) and one neuroblastoma cell lines showed more than 50% inhibition of cell growth in vitro, appearance of giant multinucleated cell morphologically, and DNA accumulation in G2+M and/or S phase of cell cycle in the medium of 10 micrograms/ml MCNU . Antitumor activity and spectrum of MCNU against human brain tumors were almost the same as with ACNU.     

Capillary permeability in experimental rat glioma and effects of intracarotid CDDP administration on tumor drug delivery

We have examined the capillary permeability within and adjacent to experimental rat gliomas, and the effect of intracarotid administration of cisplatin in this model. Permeability to¹⁴C-sucrose was 25-fold and six-fold higher within the brain tumor and in the tumor periphery and adjacent tissue respectively than in the normal cortex. Intracarotid administration of cisplatin resulted in ten-fold increase in experimental gliomas and also a significant increase in the tumor periphery and adjacent tissue, when compared to the intravenous route. Intracarotid administration of cisplatin to rat did not cause acute vascular damage in either the brain or brain tumor at the dosage used.     

Long term effects on the immune system following local radiation therapy for breast cancer. I. Cellular composition of the peripheral blood lymphocyte population

Local radiation therapy for breast cancer depletes the blood of various subsets of lymphocytes. Previous studies showed that the recovery is still incomplete at 30 months. To further elucidate the recovery we examined blood lymphocyte counts of 138 disease-free women and various lymphocyte subsets in 102 of these patients. These patients, 5-6 and 10-11 years earlier, had entered a clinical trial in which preoperative irradiation (45 Gy) was evaluated against postoperative irradiation (45 Gy) or surgery only. Patients who had undergone surgery only served as controls. Total lymphocyte counts of the irradiated patients were still significantly reduced 10-11 years after treatment. This reduction was mainly attributable to a subnormal level of T-cells as determined by the monoclonal antibody Leu-1 and the ability to form rosettes with sheep erythrocytes, whereas the number of non-T cells, expressing C'3 receptors, did not differ significantly from the controls. Within the T-cell population a subset with helper/inducer phenotypes, detected by Leu-3a antibodies, was significantly reduced even 10-11 years after irradiation. T-cells with suppressor/cytotoxic phenotypes, stainable with Leu-2a antibodies, however, had already recovered 5-6 years after irradiation. The duration of the radiation induced reductions of different lymphocyte subsets may be related to the physiological turn-over of the cells or a changed distribution of cells in the body.     

A study on the biological behavior of human brain tumors Part I. Arachidonic acid metabolism and DNA content

Summary The study of proliferative characteristics and biochemical aspects seem to be of great importance in order to define brain neoplastic behavior.The purpose of this study is to verify the existence of any possible correlation between Arachidonic Acid (AA) metabolism and proliferative characteristics in 30 meningiomas and 30 neuroepithelial tumors. The most represented metabolite in neuroepithelial tumors is TxB₂, while 6-Keto-PGF₁ is the lowest represented product. Unimodal DNA distribution was observed in 66% of neuroepithelial tumors and in 87% of meningiomas. Aneuploidy was more frequent in glioblastomas and anaplastic meningiomas as previously reported; AA overall synthesis capacity and profile were similar between unimodal and bimodal cases of neuroepithelial tumors. Total AA metabolite, as well as TxB₂ and PGD₂, synthesis capacity are significantly higher in cases with S-phase cell percentage 3% than in cases with S-phase % < 3%.Total production of AA metabolites via the cyclooxygenase pathway is significantly higher in meningiomas with bimodal DNA distribution than in cases with unimodal DNA content; when considering S-phase cell percentage, similarly to what observed in neuroepithelial tumors, meningiomas with S% > 3% shows a significantly higher overall synthesis capacity for AA. AA metabolism capacity well correlates with proliferative patterns in neuroepithelial tumors: the relationship depends preferentially on TxB₂ and PGD₂ synthesis capacity. In cases of meningiomas, the amount of AA metabolites seem to be related to DNA content and proliferative activity when anaplastic features are histologically demonstrated.     

Inhibitory effects of AcSDKP on the mixed lymphocyte reaction (MLR). Part I. MLR with mouse spleen cells.

AcSDKP (inhibitor of entry into cycle of pluripotent hemopoietic stem cells) is able to decrease mixed lymphocyte reaction intensity when H-2 incompatible allogeneic spleen cells are used as stimulators. This is a first approach to determining whether AcSDKP has potential therapeutic value for clinical bone marrow transplantation.