Transient focal ischemia results in persistent and widespread neuroinflammation and loss of glutamate NMDA receptors.

Stroke is accompanied by neuroinflammation in humans and animal models. To examine the temporal and anatomical profile of neuroinflammation and NMDA receptors (NMDAR) in a stroke model, rats (N=17) were subjected to a 90 min occlusion of the middle cerebral artery (MCAO) and compared to sham (N=5) and intact (N=4) controls. Striatal and parietal cortical infarction was confirmed by MRI 24h after reperfusion. Animals were killed 14 or 30-40 days later and consecutive coronal cryostat sections were processed for quantitative autoradiography with the neuroinflammation marker [(3)H]PK11195 and the NMDAR antagonist [(3)H]MK801. Significantly increased specific binding of [(3)H]PK11195 relative to non-ischemic controls was observed in the ipsilateral striatum (>3 fold, p<0.0001), substantia innominata (>2 fold) with smaller (20%-80%) but statistically significant (p=0.002-0.04) ipsilateral increases in other regions partially involved in the infarct such as the parietal and piriform cortex, and in the lateral septum, which was not involved in the infarct. Trends for increases in PBR density were also observed in the contralateral hemisphere. In the same animals, NMDAR specific binding was significantly decreased bilaterally in the septum, substantia innominata and ventral pallidum. Significant decreases were also seen in the ipsilateral striatum, accumbens, frontal and parietal cortex. The different anatomical distribution of the two phenomena suggests that neuroinflammation does not cause the observed reduction in NMDAR, though loss of NMDAR may be locally augmented in ipsilateral regions with intense neuroinflammation. Persistent, bilateral loss of NMDAR, probably reflecting receptor down regulation and internalization, may be responsible for some of the effects of stroke on cognitive function which cannot be explained by infarction alone.

Molecular targeting of the epidermal growth factor receptor for neutron capture therapy of gliomas.

Success of boron neutron capture therapy (BNCT) is dependent on cellular and molecular targeting of sufficient amounts of boron-10 to sustain a lethal (10)B (n, alpha) (7)Li capture reaction. The purpose of the present study was to determine the efficacy of boronated epidermal growth factor (EGF) either alone or in combination with boronophenylalanine (BPA) as delivery agents for an epidermal growth factor receptor (EGFR) -positive glioma, designated F98(EGFR). A heavily boronated precision macromolecule [boronated starburst dendrimer (BSD)] was chemically linked to EGF by heterobifunctional reagents. Either F98 wild-type (F98(WT)) receptor (-) or EGFR gene-transfected F98(EGFR) cells, which expressed 5 x 10(5) receptor sites/cell, were stereotactically implanted into the brains of Fischer rats, and 2 weeks later biodistribution studies were initiated. For biodistribution studies rats received an intratumoral (i.t.) injection of (125)I-labeled BSD-EGF and were euthanized either 6 or 24 h later. At 6 h, equivalent amounts of BSD-EGF were detected in F98(EGFR) and F98(WT) tumors. Persistence of the bioconjugate in F98(EGFR) tumors was specifically determined by EGFR expression. By 24 h 33.2% of injected dose/g of EGF-BSD was retained by F98(EGFR) gliomas compared with 9.4% % of injected dose/g in F98(WT) gliomas, and the corresponding boron concentrations were 21.1 microg/g and 9.2 microg/g, respectively. Boron concentrations in normal brain, blood, liver, kidneys, and spleen all were at nondetectable levels (<0.5 microg/g). On the basis of these results, BNCT was initiated at the Brookhaven National Laboratory Medical Research Reactor. Two weeks after implantation of 10(3) F98(EGFR) or F98(WT) tumor cells, rats received an i.t. injection of BSD-EGF (approximately 60 microg (10)B/approximately 15 microg EGF) either alone or in combination with i.v. BPA (500 mg/kg). Rats were irradiated at the Brookhaven Medical Research Reactor 24 h after i.t. injection, which was timed to coincide with 2.5 h after i.v. injection of BPA for those animals that received both capture agents. Untreated control rats had a mean survival time (MST) +/- SE of 27 +/- 1 day, and irradiated controls had a MST of 31 +/- 1 day. Animals bearing F98(EGFR) gliomas, which had received i.t. BSD-EGF and BNCT, had a MST of 45 +/- 5 days compared with 33 +/- 2 days for animals bearing F98(WT) tumors (P = 0.0032), and rats that received i.t. BSD-EGF in combination with i.v. BPA had a MST of 57 +/- 8 days compared with 39 +/- 2 days for i.v. BPA alone (P = 0.016). Our data are the first to show in vivo efficacy of BNCT using a high molecular weight boronated bioconjugate to target amplified EGFR expressed on gliomas, and they provide a platform for the future development of combinations of high and low molecular weight agents for BNCT.

Response of rat intracranial 9L gliosarcoma to microbeam radiation therapy.

Radiotherapeutic doses for malignant gliomas are generally palliative because greater, supposedly curative doses would impart clinically unacceptable damage to nearby vital CNS tissues. To improve radiation treatment for human gliomas, we evaluated microbeam radiation therapy, which utilizes an array of parallel, microscopically thin (<100 microm) planar beams (microbeams) of synchrotron-generated X rays. Rats with i.c. 9L gliosarcoma tumors were exposed laterally to a single microbeam, 27 pm wide and 3.8 mm high, stepwise, to produce irradiation arrays with 50, 75, or 100 microm of on-center beam spacings and 150, 250, 300, or 500 Gy of in-slice, skin-entrance, single-exposure doses. The resulting array size was 9 mm wide and 10.4 mm high (using three 3.8-mm vertical tiers); the beam's median energy was -70 keV. When all data were collated, the median survival was 70 days; no depletion of nerve cells was observed. However, when data from the highest skin-entrance dose and/or the smallest microbeam spacings were excluded, the median survival time of the subset of rats was 170 days, and no white matter necrosis was observed. Others have reported unilateral single-exposure broad-beam irradiation of i.c. 9L gliosarcomas at 22.5 Gy with a median survival of only -34 days and with severe depletion of neurons. These results suggest that the therapeutic index of unidirectional microbeams is larger than that of the broad beams and that an application for microbeam radiation therapy in treating certain malignant brain tumors may be found in the future.

Combination of boron neutron capture therapy and external beam radiotherapy for brain tumors.

PURPOSE: Boron neutron capture therapy (BNCT) has been used clinically as a single modality treatment for high-grade gliomas and melanomas metastatic to the brain. The purpose of the present study was to determine whether its efficacy could be enhanced by an X-ray boost administered after BNCT. Two brain tumor models were used, the F98 glioma as a model for primary brain tumors and the MRA 27 human melanoma as a model for metastatic brain tumors. METHODS AND MATERIALS: For biodistribution studies, either 10(5) F98 glioma cells were implanted stereotactically into the brains of syngeneic Fischer rats or 10(6) MRA 27 melanoma cells were implanted intracerebrally into National Institutes of Health (NIH)-rnu nude rats. Biodistribution studies were performed 11-13 days after implantation of the F98 glioma and 20-24 days after implantation of the MRA 27 melanoma. Animals bearing the F98 glioma received a combination of two boron-containing drugs, sodium borocaptate at a dose of 30 mg/kg and boron phenylalanine (BPA) at a dose of 250 mg/kg. MRA 27 melanoma-bearing rats received BPA (500 mg/kg) containing an equivalent amount of 10B (27 mg B/kg). The drugs were administered by either intracarotid or i.v. injection. RESULTS: The tumor boron concentration after intracarotid injection was approximately 50% greater in the F98 glioma and MRA 27 melanoma after intracarotid injection (20.8 and 36.8 microg/g, respectively) compared with i.v. injection (11.2 and 19.5 microg/g, respectively). BNCT was carried out at the Brookhaven National Laboratory Medical Research Reactor approximately 14 days after tumor implantation of either the F98 glioma or the MRA 27 melanoma. Approximately 7-10 days after BNCT, subsets of animals were irradiated with 6-MV photons, produced by a linear accelerator at a total dose of 15 Gy, delivered in 5-Gy daily fractions. F98 glioma-bearing rats that received intracarotid or i.v. sodium borocaptate plus BPA, followed 2.5 h later by BNCT and 7-10 days later by X-rays, had similar mean survival times (61 days and 53 days, respectively, p = 0.25), and the non X-irradiated, BNCT-treated animals had a mean survival time of 52 and 40 days, respectively, for intracarotid vs. i.v. injection; the latter was equivalent to that of the irradiated animals. The corresponding survival time for MRA 27 melanoma-bearing rats that received intracarotid or i.v. BPA, followed by BNCT and then X-irradiation, was 75 and 82 days, respectively (p = 0.5), 54 days without X-irradiation (p = 0.0002), 37 days for X-irradiation alone, and 24 days for untreated controls. In contrast to the data obtained with the F98 glioma, MRA 27 melanoma-bearing rats that received i.v. BPA, followed by BNCT, had a highly significant difference in mean survival time compared with the irradiated controls (54 vs. 37 days, p = 0.008). CONCLUSION: Our data are the first to suggest that a significant therapeutic gain may be obtained when BNCT is combined with an X-ray boost. Additional experimental studies are required to determine the optimal combination of X-radiation and neutron doses and whether it is more advantageous to administer the photon boost before or after BNCT.

Neutron capture therapy of intracerebral melanoma: enhanced survival and cure after blood-brain barrier opening to improve delivery of boronophenylalanine.

PURPOSE: Multicentric cerebral metastases of melanoma represent an important clinical problem for which there currently is no satisfactory treatment. We previously developed a model for melanoma metastatic to the brain employing nude rats bearing intracerebral implants of the human MRA27 melanoma. The purpose of the present study was to determine if the efficacy of boron neutron capture therapy (BNCT) could be improved by either Cereport (RMP-7) mediated modulation of blood-brain barrier (BBB) permeability or hyperosmotic mannitol-induced BBB disruption using boronophenylalanine (BPA) as the capture agent. METHODS AND MATERIALS: Biodistribution studies were carried out at 0.5, 2.5, and 4 h after intracarotid administration of Cereport (1.5 microg/kg) and intracarotid or i.v. administration of BPA (500 mg/kg). Peak tumor boron concentrations (65.4 microg/g) and the best composite tumor:brain (6.1:1) and tumor:blood (6.3:1) ratios were observed at 2.5 h after intracarotid administration. BNCT was initiated at the Brookhaven Medical Research Reactor 13-14 days after intracerebral implantation of 10(6) MRA27 cells. RESULTS: Untreated control rats had a median survival time (MeST) of 22 days and for irradiated controls, it was 30 days. Rats that received i.v. or intracarotid BPA without Cereport followed by BNCT 2.5 h later had MeSTs of 41 days and 57 days, respectively, with 20% long-term survivors (>180 days) in the latter group. Rats that received intracarotid BPA with Cereport had an MeST of 86 days with 36% long-term survivors, which was very close to that of rats that had hyperosmotic mannitol-induced disruption of the BBB (85 days with 25% long-term survivors). When these two groups were combined, and survival times were compared, using the Wilcoxon rank sum test, to those of rats that received intracarotid BPA without blood-brain barrier disruption, these differences were significant at the level p = 0.01. CONCLUSIONS: Our data show that optimizing the delivery of BPA by means of intracarotid injection combined with opening the BBB by infusing Cereport or a hyperosmotic solution of mannitol significantly enhanced survival times and produced long-term cures of MRA27 melanoma-bearing rats. These observations are relevant to future clinical studies using BNCT for the treatment of intracerebral melanoma.

A new look at glutamate and ischemia: NMDA agonist improves long-term functional outcome in a rat model of stroke.

Ischemic stroke triggers a massive, although transient, glutamate efflux and excessive activation of NMDA receptors (NMDARs), possibly leading to neuronal death. However, multiple clinical trials with NMDA antagonists failed to improve, or even worsened, stroke outcome. Recent findings of a persistent post-stroke decline in NMDAR density, which plays a pivotal role in plasticity and memory formation, suggest that NMDAR stimulation, rather than inhibition, may prove beneficial in the subacute period after stroke. AIM: This study aims to examine the effect of the NMDAR partial agonist d-cycloserine (DCS) on long-term structural, functional and behavioral outcomes in rats subjected to transient middle cerebral artery occlusion, an animal model of ischemic stroke. MATERIALS #ENTITYSTARTX00026; METHODS: Rats (n = 36) that were subjected to 90 min of middle cerebral artery occlusion were given a single injection of DCS (10 mg/kg) or vehicle (phosphate-buffered saline) 24 h after occlusion and followed up for 30 days. MRI (structural and functional) was used to measure infarction, atrophy and cortical activation due to electrical forepaw stimulation. Memory function was assessed on days 7, 21 and 30 postocclusion using the novel object recognition test. A total of 20 nonischemic controls were included for comparison. RESULTS: DCS treatment resulted in significant improvement of somatosensory and cognitive function relative to vehicle treatment. By day 30, cognitive performance of the DCS-treated animals was indistinguishable from nonischemic controls, while vehicle-treated animals demonstrated a stable memory deficit. DCS had no significant effect on infarction or atrophy. CONCLUSION: These results support a beneficial role for NMDAR stimulation during the recovery period after stroke, most likely due to enhanced neuroplasticity rather than neuroprotection.

Synthesis, toxicity and biodistribution of two 5,15-di[3,5-(nido-carboranylmethyl)phenyl]porphyrins in EMT-6 tumor bearing mice.

The total synthesis of a 5,15-di[3,5-(o-carboranylmethyl)phenyl]porphyrin 5, its zinc(II) complex 6, and the corresponding nido-carboranylporphyrins 7 and 8 are reported. The molecular structures of porphyrin 6 and of potassium nido-carborane were obtained and are described. The biodistribution of nido-carboranylporphyrins 7 and 8 in BALB/c mice bearing EMT-6 mammary tumors are presented and compared. Both compounds are effective tumor localizers and delivered therapeutic concentrations of boron to tumors (mean+/-standard deviation): 32.5+/-7.1 and 54.3+/-14 microg/g for 7 and 8, respectively, 2 days after the last of 3 injections of a total boron dose of 23 mg/kg body weight. The zinc(II) complex 8 was found to deliver 1.2-1.7 times higher amounts of boron to tumors than 7, with lower tumor-to-blood boron concentration ratios (9.8/1 and 4.7/1 for 7 and 8, respectively, 2 days after injections). The tumor-to-brain boron concentration ratios were >100/1 for both porphyrins 2 days after administration. Both nido-carboranylporphyrins 7 and 8 were well-tolerated at the concentrations used (75 and 78 mg/kg body weight, respectively) and no morbidity or mortality were observed in these studies.