Boron neutron capture therapy of brain tumors: enhanced survival following intracarotid injection of either sodium borocaptate or boronophenylalanine with or without blood-brain barrier disruption

The purpose of the present study was to determine whether the efficacy of boron neutron capture therapy could be enhanced by means of intracarotid (i.c.) injection of sodium borocaptate (BSH) or boronophenylalanine (BPA) with or without blood-brain barrier disruption (BBB-D). For biodistribution studies, F98 glioma-bearing rats were injected i.v. or i.c. with either BSH (30 mg of boron/kg of body weight) or BPA (24 mg of boron/kg of body weight) with or without mannitol-induced, hyperosmotic BBB-D and killed 2.5 h later. The highest tumor boron concentrations for BSH and BPA were attained following i.c. injection with BBB-D (48.6 and 94.0 microg/g, respectively) compared to i.c. (30.8 and 42.7 microg/g) and i.v. injection (12.9 and 20.8 microg). Using the same doses of BSH and BPA, therapy experiments were initiated 14 days after intracerebral implantation of F98 glioma cells. Animals were irradiated 2.5 h after i.v. or i.c. administration of the capture agent with or without BBB-D using a collimated beam of thermal neutrons at the Brookhaven Medical Research Reactor. The median survival times of rats given BSH or BPA i.c. were 52 and 69 days, respectively, for rats with BBB-D; 39 and 48 days for rats without BBB-D; 33 and 37 days for i.v. injected rats; 29 days for irradiated controls; and 24 days for untreated controls. i.c. injection of either BSH or BPA resulted in highly significant enhancement (P = 0.01 and P = 0.0002, respectively) of survival times compared to i.v. injection, and this was further augmented by BBB-D (P = 0.02 and P = 0.04, respectively) compared to i.c. injection. Normal brain tissue tolerance studies were carried out with non-tumor-bearing rats, which were treated in the same way as tumor-bearing animals. One year after irradiation, the brains of these animals showed only minimal radiation-induced changes in the choroid plexus, but no differences were discernible between irradiated controls and those that had BBB-D followed by i.c. injection of either BSH or BPA. Our data clearly show that the route of administration, as well as BBB-D, can enhance the uptake of BSH and BPA, and, subsequently, the efficacy of boron neutron capture therapy.     

The blood-brain barrier disruption controversy: does it hold water?

OBJECTIVES: A defect in mitochondrial energy conservation is strongly suggested to be involved in the pathogenesis of Leber's hereditary optic neuropathy (LHON). The authors therefore compared the energy charge in lymphocytes among patients with LHON, their asymptomatic maternal lineages, and normal control subjects. MATERIALS AND METHODS: Blood samples were obtained from 7 patients, 10 asymptomatic maternal relatives, and 16 normal subjects. Molecular analysis confirmed that all had the homoplasmic 11,778 point mutation in the mtDNA of their blood cells. The concentrations of adenosine triphosphate (ATP), diphosphate (ADP), and monophosphate (AMP) were determined by high-performance liquid chromatography. The energy charge was calculated as (ATP + 1/2 ADP)/(ATP + ADP + AMP). RESULTS: The mean energy charges of lymphocytes were 0.871 +/- 0.049 in patients with LHON, 0.884 +/- 0.061 in their asymptomatic maternal relatives, and 0.885 +/- 0.061 in normal controls, respectively. No statistically significant difference was found among the three groups. CONCLUSIONS: Although the study did not find the anticipated change in energy charge in peripheral blood cells, this neither confirms nor rejects the notion that a defect in the mitochondrial oxidative phosphorylation system is involved in the pathogenesis of LHON.     

Inflammatory response of the spinal cord to multiple episodes of blood-brain barrier disruption and toxic demyelination in Wistar rats

Multiple episodes of blood-brain barrier disruption were induced by sequential intraspinal injections of ethidium bromide. In addition to the barrier disruption, there was toxic demyelination and exposure of myelin components to the immune system. Twenty-seven 3-month-old Wistar rats received 2, 3 or 4 injections of 1 microliter of either 0.1% ethidium bromide in normal saline (19 rats) or 0.9% saline (8 rats) at different levels of the spinal cord. The time intervals between the injections ranged from 28 to 42 days. Ten days after the last injection, all rats were perfused with 2.5% glutaraldehyde. The spinal sections were evaluated macroscopically and by light and transmission electron microscopy. All the lesions demonstrated a mononuclear phagocytic infiltrate apparently removing myelin. Lymphocytes were not conspicuous and were found in only 34% of the lesions. No perivascular cuffings were detected. In older lesions (38 days and older) they were found only within Virchow-Robin spaces. This result suggests that multiple blood-brain barrier disruptions with demyelination and exposure of myelin components to the immune system were not sufficient to induce an immune-mediated reaction in the central nervous system.     

Vulnerability to cerebral hypoxic-ischemic insult in neonatal but not in adult rats is in parallel with disruption of the blood-brain barrier

BACKGROUND AND PURPOSE: Vulnerability to cerebral hypoxic-ischemic (H-I) insult and its relation to disruption of the blood-brain barrier were investigated in postnatal rats. METHODS: Pups of postnatal day (P) 7, P14, and P21 underwent ligation of a unilateral carotid artery and were exposed to hypoxic conditions. For the detection of early-phase deterioration, brains were perfusion-fixed 24 hours after H-I insult and examined by argyrophil III method. For the detection of later infarction, animals were fixed at 72 hours after the H-I insult. RESULTS: In either case, tissue damage was detected in the striatum, parietal cortex, and hippocampus. The vulnerability of P7 and P21 rats was remarkable, as compared with P14 rats. Although the developmental status of the vasculature was not significantly different at each age, the permeability of IgG after H-I injury was prominent in P7 rats and to a lesser extent in P14 rats. In P21 rats, however, there was little IgG leakage even 24 hours after the insult. Dexamethasone pretreatment blocked the extravasation of IgG and reduced the damaged tissue in P7 and P14 rats but not in P21 rats. Percentages of reduction in infarcted areas by the dexamethasone became smaller in proportion to ages. CONCLUSIONS: The results suggest that in younger rats vulnerability to H-I insult was in parallel with permeability of the blood-brain barrier, whereas in adults in might be more dependent on cellular vulnerability.     

Technique of, and complications attributable to, repeated hyperosmotic blood-brain barrier disruption in dogs

OBJECTIVE: To design a repeatable technique for reversible, hyperosmotic blood-brain barrier disruption (BBBD) in dogs and evaluate clinical effects of multiple BBBD. ANIMALS: 10 healthy adult dogs. PROCEDURE: Using fluoroscopic guidance, an arterial catheter was directed into the internal carotid artery via the femoral artery of 10 dogs. Blood-brain barrier disruption was achieved in 5 dogs, using intracarotid infusion of mannitol. Five control dogs received only saline solution. After recovery, dogs were monitored for clinical signs of disease before a second, nonsurvival procedure was performed 2 to 3 weeks later. BBBD was estimated, using computed tomographic (CT) densitometry values, as well as Evan's blue dye staining on necropsy specimens. RESULTS: Seven dogs completed the entire study. Two treatment dogs were lost after the first infusion because of deteriorating neurologic function attributed to CNS edema and increased intracranial pressure. One control dog was lost because of vessel wall damage during catheterization. The remaining dogs had only transient neurologic, ocular, and vasculature injuries. Successful BBBD was documented in all treated dogs by use of CT and Evan's blue dye evaluation. CONCLUSION: Repeated catheterization of the internal carotid artery and disruption of the blood- brain barrier is possible in dogs. CLINICAL RELEVANCE: Refinement of this technique would be useful not only for improved delivery of chemotherapeutic agents in patients with brain tumors, but also would allow further investigation of new treatments involving genetically engineered retroviruses and monoclonal antibodies.     

Long-term effects of pentobarbital anesthesia on the atrial natriuretic peptide system in rats

The midline fascial splitting approach is a modified midline approach to the iliac crest for bone graft that takes advantage of the anatomic planes between layers of the dorsal lumbar fascia. Two hundred consecutive grafts were taken by this technique with one superficial infection, two cases of serous hematoma, and three patients with significant postoperative pain at the harvest site, for an overall complication rate of 3%. In comparison, bone grafts were harvested from 200 consecutive patients by the midline subcutaneous approach to the iliac crest with 2 deep infections, 1 cluneal nerve injury, 15 patients with severe and disabling pain at the harvest site, and 12 patients with a serous hematoma, for an overall complication rate of 15%. The midline fascial splitting approach significantly reduced the incidence of postoperative serous hematoma (P < 0.007) as well as the incidence of significant and disabling pain (P < 0.001). In addition, the approach is simple, straightforward, anatomic, and decreases trauma to soft tissues.     

Activation of protein kinase C does not participate in disruption of the blood-brain barrier to albumin during acute hypertension

The blood-brain barrier minimizes the entry of macromolecules into brain tissue. During acute increases in arterial blood pressure, disruption of the blood-brain barrier occurs primarily in cerebral venules and veins. Mechanisms by which increases in cerebral venous pressure produce disruption of the blood-brain barrier during acute hypertension are not clear. The goal of this study was to determine the role of activation of protein kinase C in disruption of the blood-brain barrier during acute hypertension. We examined the microcirculation of the cerebrum in vivo. Permeability of the blood-brain barrier was quantitated by the formation of venular leaky sites and clearance of fluorescent-labeled albumin (FITC-albumin) before and during phenylephrine-induced acute hypertension. In addition, we examined changes in pial arteriolar and pial venular pressure before and during phenylephrine-induced acute hypertension. We compared responses of the blood-brain barrier to acute hypertension in control (untreated) rats and in rats treated with inhibitors of protein kinase C; calphostin C (0.1 microM) or sphingosine (1.0 microM). Under control conditions, no venular leaky sites were visible and clearance of FITC-albumin was minimal in all groups. Phenylephrine infusion increased systemic arterial, pial arteriolar and pial venular pressures, and increased the formation of venular leaky sites and clearance of FITC-albumin by a similar magnitude in all groups. The findings of the present study suggest that inhibition of protein kinase C does not significantly alter the formation of venular leaky sites and/or clearance of FITC-albumin during acute hypertension. Thus, disruption of the blood-brain barrier during acute hypertension does not appear to be influenced by activation of protein kinase C.     

Role of blood-brain barrier in cerebral homeostasis

A detailed cytogenetic map was constructed around the chromosomal breakpoint of t(8;13) observed in a patient with multiple exostoses. The order of seven loci defined by cosmid clones mapped to 8q23 was determined by means of two-color fluorescence in situ hybridization (FISH) on elongated prophase chromosomes, and localizations of these markers relative to the breakpoint were examined. The results indicated that loci defined by cC18-553 and cC18-1512 flank the breakpoint. By pulsed-field gel electrophoresis of DNA digested with BssHII and Southern hybridization with cC18-1512, DNA from the patient showed a band which was not observed in DNA isolated from either parent. As the normal size of this BssHII fragment is 600 kb, the chromosomal breakpoint probably lies less than 600 kb away from cC18-1512.     

The effect of photon irradiation on blood-brain barrier permeability to methotrexate in mice

Methotrexate was administered by intraperitoneal injection (100 mg/kg) to unirradiated mice, and to mice receiving varying doses of cranial irradiation. The animals were sacrificed 24 hours after injection, and methotrexate assays were performed on brain tissue. No methotrexate was detected in the brains of the unirradiated animals. Detectable levels of methotrexate were present after 2000 rad cranial irradiation, but not after 500 rad, 1000 rad, or 1500 rad. The implications of these findings are discussed.     

Electron microscopy of the blood-brain barrier in disease

The anatomical site of the blood-brain barrier (BBB) is at the capillary endothelium mainly, with some contribution from astrocytes. Electron microscopic observations of endothelial cells and perivascular astrocytes comprising the BBB in brain edema and other pathological conditions are reviewed in this article. The tight junctions of cerebral endothelial cells open under several conditions such as infusion of hyperosmolar solutions. Pinocytotic vesicles increase under various pathological conditions and fenestrae appear in blood vessels of certain brain tumors and several non-neoplastic lesions. Inflammatory cells penetrate between or through endothelial cells. In long standing lesions, endothelial cells containing various tubular structures such as Weibel-Palade bodies proliferate. Other alterations include surface infoldings of endothelial cells and fluid diffusion through damaged endothelium. Astrocytic alterations include abnormal junctions between astrocytic processes in certain gliomas. In vivo and in vitro studies suggest that astrocytes maintain or develop certain functions of BBB. As the BBB is disrupted, edema fluid infiltrates the brain parenchyma. Because the white matter consists of nerve fibers without demonstrable junctions, it invades between nerve fibers. In the gray matter, expansion of the fluid is limited by complicated anatomical structures. In myelinated nerve fibers, edema fluid accumulates in five separate compartments of extracellular space.     

Beneficial effects of S-adenosyl-L-methionine on blood-brain barrier breakdown and neuronal survival after transient cerebral ischemia in gerbils

In the present study, the effect of bradykinin on basal and precontracted mouse-isolated trachea was investigated. In basal conditions mouse-isolated tracheal rings do not respond to bradykinin. However, when the tracheal rings were precontracted with carbachol (10(-7) M) a relaxation with bradykinin (3 x 10(-9)-3 x 10(-7)) was found. The maximal response amounted 69.7+/-4.1% (n=15) with a pD2 value of 7.2+/-0.21. The selective bradykinin B2 receptor antagonist HOE 140 (10(-10)-10(-8) M) antagonized the bradykinin-induced relaxation, while the bradykinin B1 receptor antagonist des-Arg9-Leu8-bradykinin (10(-6) M) had no influence. The selective bradykinin B1 receptor agonist des-Arg9-bradykinin (10(-6) M) caused a small relaxation (8.4+/-2.5%, n=6), which could be antagonized completely by the selective bradykinin B1 receptor antagonist des-Arg9-Leu8-bradykinin (10(-6) M) while addition of the selective bradykinin B2 receptor antagonist HOE 140 (10(-8) M) was without effect. In the presence of indomethacin (10(-6) M) the relaxation of bradykinin was completely abolished. Pretreatment of the tracheal rings with capsaicin, or the presence of the selective NK1 receptor antagonist RP 67851 (10(-6) M) or the presence of the nitric oxide synthase inhibitor L-NAME (3 x 10(-4) M) had no effect on the bradykinin-induced relaxation. In conclusion, these results demonstrate that the mouse-isolated tracheal is a preparation in which bradykinin exerts a relaxant response via stimulation of bradykinin B2 receptors. This response is probably mediated by prostaglandins.     

The pathophysiology of the blood-brain barrier dysfunction induced by severe hypercapnia and by epileptic brain activity

Antiserum to fentanyl was obtained in rabbits repeatedly injected with carboxyfentanyl conjugated to bovine serum albumin. Using the antiserum, a highly sensitive radioimmunoassay has been developed, based on the dextran-coated charcoal method. It proved possible to assay the drug directly in plasma, in amounts as small as 30 picogram in 0.5 ml. The antibody was highly specific for fentanyl and no cross-reaction was observed with its major metabolites. This sensitive and specific radioimmunoassay method was employed to determine fentanyl in plasma from six volunteers after an intravenous bolus of 0.2 mg, and in plasma from dogs treated both intravenously and subcutaneously with 0.02 mg/kg. The plasma level of fentanyl could be followed for up to 6 h after a therapeutic dose in dogs and man.     

Differential blood-brain barrier breakdown and leucocyte recruitment following excitotoxic lesions in juvenile and adult rats

Acute neuronal degeneration can be induced by intracerebral injections of the glutamate receptor agonists kainic acid (KA) and NMDA (N-methyl-D-aspartate). It is accompanied by an inflammatory response that has not yet been fully investigated. We have previously demonstrated that the juvenile rat brain is more susceptible to an inflammatory challenge when compared to adult rat brain. This study set out to investigate whether this also applied to the inflammatory response associated with acute neuronal degeneration. NMDA and kainic acid were injected into the rat striatum and lesion size, leucocyte recruitment, and blood-brain barrier (BBB) breakdown were assessed after 4, 8, 12, 24, 72, and 168 h. Both NMDA and KA induced lesions of similar volume at either age and apoptotic and necrotic nuclei could be detected. NMDA induced cellular loss by 4 h, whereas KA-injected rats did not show signs of neuronal loss until 8-12 h. The inflammatory response was characterized by an infiltration of neutrophils followed by macrophages. Juvenile rats showed a greater susceptibility to leucocyte recruitment compared to adult rats. BBB breakdown in response to NMDA injection occurred in the absence of cellular recruitment at 4 h in juveniles and was significantly greater in juvenile compared to adult rats at 8 h. BBB breakdown was minimal in KA-injected animals while at 7 days an influx of serum IgG coincided with a loss of astrocytic GFAP staining within the lesion.     

CNS drug design based on principles of blood-brain barrier transport

Lipid-soluble small molecules with a molecular mass under a 400-600-Da threshold are transported readily through the blood-brain barrier in vivo owing to lipid-mediated transport. However, other small molecules lacking these particular molecular properties, antisense drugs, and peptide-based pharmaceuticals generally undergo negligible transport through the blood-brain barrier in pharmacologically significant amounts. Therefore, if present day CNS drug discovery programs are to avoid termination caused by negligible blood-brain barrier transport, it is important to merge CNS drug discovery and CNS drug delivery as early as possible in the overall CNS drug development process. Strategies for special formulation that enable drug transport through the blood-brain barrier arise from knowledge of the molecular and cellular biology of blood-brain barrier transport processes.     

The effects of prostaglandins on the blood-retinal barrier

The effects of prostaglandin (PG), a novel PG-related compound, and epinephrine on the blood-retinal barrier (BRB) in the rabbit eye were examined by ophthalmoscopy, fundus photography, fluorescein angiography (FAG), vitreous fluorophotometry (VFPM), light and electron microscopy, and the horseradish peroxidase tracer. Intravitreal injection of PGE2 produced retinal vasodilation and large increase in a vitreous fluorescein leakage in VFPM. Intravitreal injection of PGF2 alpha produced a small increase in vitreous fluorescein leakage in VFPM, but no retinal vasodilation. Intravitreal injection of epinephrine produced retinal vasodilation and a small increase in vitreous fluorescein leakage in VFPM. But intravitreal injection of latanoprost (PhXA41) produced no retinal vasodilation and no increase in vitreous fluorescein leakage in VFPM. After intravitreal injection of PGE2, morphological changes in the retina were found, but intravitreal injection of PhXA41 did not induce morphological changes in the BRB or the retina. PhXA41 was less destructive to the BRB and the retina than PGE2, PGF2 alpha, and epinephrine.     

Sleep induction by microinjection of pentobarbital into the medial preoptic area in rats

In a previous study we have shown that microinjection of the benzodiazepine hypnotic triazolam into the medial preoptic area increases sleep in rats. In order to determine whether this effect is specific to benzodiazepines, or whether it occurs with hypnotic medications from other pharmacologic classes, we have microinjected pentobarbital (1 and 100 micrograms) and vehicle in random sequence into rats and performed two hour sleep studies in the daytime with the lights on. Both doses significantly decreased sleep latency and increased nonREM and total sleep. The amount of REM sleep, REM latency, and intermittent waking time were not significantly altered. These data are consistent with the hypothesis that the medial preoptic area may be involved in sleep induction by both benzodiazepine and barbiturate hypnotic medications.     

Morphological and functional characterization of an in vitro blood-brain barrier model

Cell culture models have been extensively used for studies of blood-brain barrier (BBB) function. However, several in vitro models fail to reproduce some, if not most, of the physiological and morphological properties of in situ brain microvascular endothelial cells. We have recently developed a dynamic, tridimensional BBB model where endothelial cells exposed to intraluminal flow form a barrier to ions and proteins following prolonged co-culturing with glia. We have further characterized this cell culture model to determine whether these barrier properties were due to expression of a BBB phenotype. Endothelial cells of human, bovine or rodent origin were used. When co-cultured with glia, intraluminally grown endothelial cells developed features similar to in vivo endothelial cells, including tight junctional contacts at interdigitating processes and a high transendothelial resistance. This in vitro BBB was characterized by the expression of an abluminal, ouabain-sensitive Na/K pump, and thus favored passage of potassium ions towards the lumen while preventing K+ extravasation. Similarly, the in vitro BBB prevented the passage of blood-brain barrier-impermeant drugs (such as morphine, sucrose and mannitol) while allowing extraluminal accumulation of lipophylic substances such as theophylline. Finally, expression of stereo-selective transporters for Aspartate was revealed by tracer studies. We conclude that the in vitro dynamic BBB model may become an useful tool for the studies of BBB-function and for the testing of drug passage across the brain endothelial monolayer.     

Asymmetries in the effect of side of seizure onset on recognition memory following intracarotid amobarbital injection

OBJECTIVE: To report gross anatomic examination of the canine myocardial bridge (MB), a muscular band found above the coronary artery (CA), with respect to its occurrence, location, vascularization, and innervation. SAMPLE POPULATION: 629 canine hearts obtained within 1 to 3 hours after euthanasia. PROCEDURE: After an incision was made at the left fifth intercostal space, the pericardial sac was cut open, and if an MB was present, the heart, lungs, and annexed structures were removed together and subsequently subjected to macroscopic examination of MB musculature and innervation after formalin fixation. Vascular casting was performed by use of methyl methacrylate perfusion. RESULTS: Of the 629 canine hearts examined, 189 (30%) had MB, occurrence of which was independent of sex, age, and breed. Among 13 MB-containing specimens examined in detail, there was great variation in thickness (0.11 to 2.24 mm; mean, 0.45 mm) of MB and distance (24 to 236 microns; mean, 103 microns) between the MB and the paraconal interventricular branch of the left CA (PIBL). One pair or 2 pairs of blood vessels from the PIBL supplied the MB muscle. Venous blood returned to the coronary circulation via the branches of the great coronary vein coursing on both sides of the PIBL, in close contact with the PIBL and the groove wall. The 2 veins rejoined at the upper portion of the PIBL and passed obliquely to the coronary groove under the left auricle, and finally drained the blood through the coronary sinus into the right ventricle. Innervation to the MB muscle was derived from nerve branches of the middle cervical ganglion and left vagus nerve. CONCLUSION: Prevalence and localization of MB in dogs and human beings are similar. Vascularization of the MB muscle originates from the PIBL. The cervical ganglion and vagus nerve control the MB muscle.