Microvessel organization and structure in experimental brain tumors: microvessel populations with distinctive structural and functional properties.

We studied microvessel organization in five brain tumor models (ENU, MSV, RG-2, S635cl15, and D-54MG) and normal brain, including microvessel diameter (LMVD), intermicrovessel distance (IMVD), microvessel density (MVD), surface area (S(v)), and orientation. LMVD and IMVD were larger and MVD was lower in tumors than normal brain. S(v) in tumors overlapped normal brain values and orientation was random in both tumors and brain. ENU and RG-2 tumors and brain were studied by electron microscopy. Tumor microvessel wall was thicker than that of brain. ENU and normal brain microvessels were continuous and nonfenestrated. RG-2 microvessels contained fenestrations and endothelial gaps; the latter had a maximum major axis of 3.0 microm. Based on anatomic measurements, the pore area of RG-2 tumors was estimated at 7.4 x 10(-6) cm(2) g(-1) from fenestrations and 3.5 x 10(-5) cm(2) g(-1) from endothelial gaps. Increased permeability of RG-2 microvessels to macromolecules is most likely attributable to endothelial gaps. Three microvessel populations may occur in brain tumors: (1) continuous nonfenestrated, (2) continuous fenestrated, and (3) discontinuous (with or without fenestrations). The first group may be unique to brain tumors; the latter two are similar to microvessels found in systemic tumors. Since structure-function properties of brain tumor microvessels will affect drug delivery, studies of microvessel function should be incorporated into clinical trials of brain tumor therapy, especially those using macromolecules.

Absence of host-site influence on angiogenesis, blood flow, and permeability in transplanted RG-2 gliomas.

The host site is believed to regulate tumor angiogenesis, which could result in site-dependent drug delivery parameters, greatly affecting experimental tumor research. In RG-2 rat gliomas we measured cellular proliferation; cell cycle time was the same for RG-2 cells in brain and s.c. tumors (25 h), and was the same for endothelial cells in these tumors (46 h). We measured the transcapillary transfer constant (K) of alpha-aminoisobutyric acid and blood flow (F) with iodoantipyrine in RG-2 gliomas transplanted into brain, liver, kidney, muscle, s.c. tissue, and into the abdominal cavity. Data was evaluated by quantitative autoradiography and direct tissue sampling. The variation of F (12.6-84.0 ml/g/min) and K (26.1-49.2 microl/g/min) in RG-2 tumors in the different host sites was less than in surrounding tumor-free tissue (F = 20-1500 ml/g/min and K = 1.6-700 microl/g/min). In contrast to other models, RG-2 does not result in tumors with host site-dependent behavior. The RG-2 tumor cells appear to participate in, if not dominate, the angiogenesis process regardless of the host site. Values of F and K were more dependent on tumor topography (center versus periphery) and local histological features (necrosis versus viable tumor) than host site. We believe that the methods used for data acquisition may introduce as much variability in Results as the tumors themselves and that to better understand how tumor angiogenesis affects the vascular phenotype, comparative studies are needed to validate the results obtained with newer methodologies.

Changes in blood-brain barrier permeability associated with insertion of brain cannulas and microdialysis probes.

Blood-brain barrier (BBB) transcapillary transport was studied after insertion of cannulas and microdialysis probes into the brains of three groups of rats. Quantitative autoradiography was used to measure changes in BBB permeability around the insertion site. In the first group, BBB function was measured with 14C-sucrose at times from immediately, and up to 28 days, after insertion of a microdialysis probe. BBB function was disrupted biphasically: a 19-fold increase in the influx constant (K1) of sucrose occurred immediately after insertion with a second 17-fold increase at 2 days, followed by a slow decline to 5 times normal values at 28 days. In the second group, 14C-dextran (70 kDa) was used to measure BBB transcapillary transport; K1 was increased 90-fold after probe insertion. In the 3rd group, 14C-AIB (alpha-aminoisobutyric acid) was used to evaluate BBB transport after insertion of a 27 gauge cannula, which was used to infuse 1 microliter of saline over 5 min. The K1 of AIB was increased 25 times control values. We conclude that BBB transcapillary transport function is disturbed in response to insertion of brain cannulas and/or microdialysis probes, that BBB dysfunction is maximal at the cannula or probe tip, varies with time after insertion, may persist for at least 28 days after insertion, and occurs over a wide molecular range of solutes. These results suggest caution when using microdialysis as a method to study normal BBB function, and suggest that microdialysis may overestimate the rate of transfer into and out of the brain.

The effect of L-amino acid oxidase on activity of melphalan against an intracranial xenograft.

We have previously shown that diet restriction-induced depletion of large neutral amino acids (LNAAs) in murine plasma to 46% of control significantly enhances intracranial delivery of melphalan without enhancing delivery to other organs. Studies have now been conducted to determine whether more substantial LNAA depletion could further enhance intracranial delivery of melphalan. Treatment with L-amino acid oxidase (LOX) significantly depleted murine plasma LNAAs: phenylalanine, leucine, and tyrosine (> 95%); methionine (83%); isoleucine (70%); and valine (46%). Experiments evaluating the intracellular uptake of melphalan and high-pressure liquid chromatography quantitation of melphalan metabolites revealed, however, that melphalan is rapidly degraded in the presence of LOX, and that the timing of the administration of melphalan following the use of LOX to deplete LNAAs is crucial. Conditions were found under which LOX-mediated degradation of melphalan was minimized and LNAA depletion was maximized, resulting in a potentiation of the antitumor effect of melphalan on human glioma xenografts in nude mice. Such potentiation could not be obtained using diet restriction alone.

Incidence and treatment of visual dysfunction in traumatic brain injury.

The incidence of visual dysfunction and effectiveness of visual exercises in acute traumatically brain injured inpatients in a rehabilitation programme were studied. Vision evaluation norms were established on 23 hospital staff. The evaluation was then administered to 51 inpatients within days after admission. An additional 21 patients were unable to participate, usually due to decreased cognition or agitation. Thirty of 51 (59%) scored impaired in one or more of the following: pursuits, saccades, ocular posturing, stereopsis, extra-ocular movements, and near/far eso-exotropia. For patients having dysfunction in pursuits or saccades, a 2-week baseline was followed by vision exercises. During the baseline interval patients were evaluated by an optometrist to verify therapists' findings. Six patients who participated in several weeks of treatment were evaluated at 2-week intervals by an independent rater. Progress is graphically illustrated. Conclusions were that the suitability of an inpatient vision programme, from our experience, is questionable. However, an initial evaluation proved valuable for informing staff of patients' visual status and for referral to an optometrist/ophthalmologist for further treatment.

The effect of an amino acid-lowering diet on the rate of melphalan entry into brain and xenotransplanted glioma.

Melphalan (L-phenylalanine mustard, L-PAM, alkeran; molecular weight, 305,000) is transported across tumor cell membranes and the blood-brain barrier by the large neutral amino acid (LNAA) transport system. Normally, plasma LNAA levels are high enough and the affinity low enough that this system does not transport much melphalan into the brain. However, plasma amino acids can be reduced by fasting and protein-free diet. We used this method to reduce competition and to increase melphalan transport into brain tumors. In nude mice fasted for 12 h and then fed a protein-free diet for 2 and 6 h, mean plasma LNAA levels were 46% and 42% of control values. Nude mice with xenotransplanted D-54MG human gliomas were used to study tissue distribution and uptake kinetics of [3H]melphalan in a control group and a diet group (after a 12-h fast and 2 h of a 0% protein diet). The K1 (blood-to-tissue transfer constant) of melphalan, determined by graphical analysis and by nonlinear fitting to a 2-compartment model, was higher in the diet group in all tumor regions except the necrotic center of subcutaneous tumors; the increase was significant in the tumor periphery of brain and s.c. tumors. The ratio of K1s (diet to control) varied from 1.2 to 1.3 in brain tumors, 1.9 to 2.1 in subcutaneous tumors, and 1.8 to 3.1 in tumor-free brain. The apparent [3H]melphalan distribution space was significantly higher in the tumor periphery of both brain and subcutaneous tumors of the 15- and 30-min diet group. We also measured blood-brain barrier transport of [alpha-14C]aminoisobutyric acid and blood flow (with [131I]iodoantipyrine): the K1 of [alpha-14C]aminoisobutyric acid was 28.1 +/- 6.6 (SE) in brain tumors and 24.3 +/- 8.9 microliters/g/min in subcutaneous tumors. Blood flow was 58.2 --> 3.9 in brain tumors and 5.2 +/- 0.4 ml/100 g/min in subcutaneous tumors. Fasting, when combined with a protein-free diet, reduces plasma amino acid levels and thereby reduces competition between melphalan and LNAAs. This may increase the amount of melphalan that can enter a brain tumor without increasing the administered drug dose and suggests a therapeutic manipulation that can be used to increase the delivery of melphalan.

Rate of buthionine sulfoximine entry into brain and xenotransplanted human gliomas.

Buthionine sulfoximine (BSO) is an inhibitor of glutathione synthesis and can be used to potentiate the effects of chemotherapeutic alkylating agents and radiotherapy. We examined the rates of influx and efflux of [35S]BSO administered to athymic mice with and without xenografted D-54MG human gliomas. Three analytic approaches were applied to the experimental data to obtain values of the blood-to-tissue influx constant, K1, of BSO. Multiple time point experiments in tumor-bearing mice were analyzed with a two-compartment model and nonlinear fitting routines, and by graphical analysis which assumed no backflux of BSO from tissue to blood. A third approach used single time point data in nontumor-bearing mice and assumed no backflux. Calculated values of the K1 of BSO ranged from 0.23 to 1.35 microliters/g/min in tumor-free cortex, and from 5.3 to 6.3 microliters/g/min in the D-54MG gliomas. The tissue-to-blood efflux constant, k2, was zero in both cortex and tumor, suggesting that BSO entered cells and was trapped once it crossed the blood-brain barrier. Estimates of plasma vascular space (Vp) ranged from 2 to 20 microliters/g in cortex, and from 103 to 169 microliters/g in tumor. Another set of experiments, done in normal mice with different doses of BSO, suggested that BSO competes for neutral amino acid transport sites at the blood-brain barrier, but that the capacity of the carrier-mediated transport system is low and saturates at administered doses of about 0.5 mmol/kg (corresponding to plasma concentrations of about 12 mumol/ml). The rate of entry into brain was proportional to the octanol/water partition coefficient and molecular weight of BSO, which also supports passive diffusion as the means of entry. Consequently, although the rate of BSO entry into D-54MG gliomas was between 4 and 30 times higher than the rate of entry into tumor-free cortex, the results of these experiments suggest that most of the BSO that enters brain tumors in the doses commonly used in experimental situations will cross capillaries by passive diffusion.