Sustained functional improvement by hepatocyte growth factor-like small molecule BB3 after focal cerebral ischemia in rats and mice.

Hepatocyte growth factor (HGF), efficacious in preclinical models of acute central nervous system injury, is burdened by administration of full-length proteins. A multiinstitutional consortium investigated the efficacy of BB3, a small molecule with HGF-like activity that crosses the blood-brain barrier in rodent focal ischemic stroke using Stroke Therapy Academic Industry Roundtable (STAIR) and Good Laboratory Practice guidelines. In rats, BB3, begun 6 hours after temporary middle cerebral artery occlusion (tMCAO) reperfusion, or permanent middle cerebral artery occlusion (pMCAO) onset, and continued for 14 days consistently improved long-term neurologic function independent of sex, age, or laboratory. BB3 had little effect on cerebral infarct size and no effect on blood pressure. BB3 increased HGF receptor c-Met phosphorylation and synaptophysin expression in penumbral tissue consistent with a neurorestorative mechanism from HGF-like activity. In mouse tMCAO, BB3 starting 10 minutes after reperfusion and continued for 14 days improved neurologic function that persisted for 8 weeks in some, but not all measures. Study in animals with comorbidities and those exposed to common stroke drugs are the next steps to complete preclinical assessment. These data, generated in independent, masked, and rigorously controlled settings, are the first to suggest that the HGF pathway can potentially be harnessed by BB3 for neurologic benefit after ischemic stroke.

Metalloporphyrins as therapeutic catalytic oxidoreductants in central nervous system disorders.

SIGNIFICANCE: Metalloporphyrins, characterized by a redox-active transitional metal (Mn or Fe) coordinated to a cyclic porphyrin core ligand, mitigate oxidative/nitrosative stress in biological systems. Side-chain substitutions tune redox properties of metalloporphyrins to act as potent superoxide dismutase mimics, peroxynitrite decomposition catalysts, and redox regulators of transcription factor function. With oxidative/nitrosative stress central to pathogenesis of CNS injury, metalloporphyrins offer unique pharmacologic activity to improve the course of disease. RECENT ADVANCES: Metalloporphyrins are efficacious in models of amyotrophic lateral sclerosis, Alzheimer's disease, epilepsy, neuropathic pain, opioid tolerance, Parkinson's disease, spinal cord injury, and stroke and have proved to be useful tools in defining roles of superoxide, nitric oxide, and peroxynitrite in disease progression. The most substantive recent advance has been the synthesis of lipophilic metalloporphyrins offering improved blood-brain barrier penetration to allow intravenous, subcutaneous, or oral treatment. CRITICAL ISSUES: Insufficient preclinical data have accumulated to enable clinical development of metalloporphyrins for any single indication. An improved definition of mechanisms of action will facilitate preclinical modeling to define and validate optimal dosing strategies to enable appropriate clinical trial design. Due to previous failures of "antioxidants" in clinical trials, with most having markedly less biologic activity and bioavailability than current-generation metalloporphyrins, a stigma against antioxidants has discouraged the development of metalloporphyrins as CNS therapeutics, despite the consistent definition of efficacy in a wide array of CNS disorders. FUTURE DIRECTIONS: Further definition of the metalloporphyrin mechanism of action, side-by-side comparison with "failed" antioxidants, and intense effort to optimize therapeutic dosing strategies are required to inform and encourage clinical trial design.

Superoxide dismutase mimic, MnTE-2-PyP(5+) ameliorates acute and chronic proctitis following focal proton irradiation of the rat rectum.

Radiation proctitis, an inflammation and damage to the lower part of colon, is a common adverse event of the radiotherapy of tumors in the abdominal and pelvic region (colon, prostate, cervical). Several Mn(III) porphyrin-based superoxide dismutase mimics have been synthesized and successfully evaluated in preclinical models as radioprotectants. Here we report for the first time the remarkable rectal radioprotection of frequently explored Mn(III) meso-tetrakis(N-ethylpyridinium-2-yl)porphyrin, MnTE-2-PyP(5+). A batch prepared in compliance with good manufacturing practice (GMP), which has good safety/toxicity profile, was used for this study. MnTE-2-PyP(5+) was given subcutaneously at 5 mg/kg, either 1 h before or 1 h after irradiation, with additional drug administered at weekly intervals thereafter. MnTE-2-PyP(5+) ameliorated both acute and chronic radiation proctitis in male Sprague-Dawley rats irradiated with 20-30 Gy protons delivered to 2.5 cm span of rectum using spread-out Bragg peak of a proton treatment beam. Focal irradiation of the rectum produced acute proctitis, which healed, followed by chronic rectal dilation and symptomatic proctitis. MnTE-2-PyP(5+) protected rectal mucosa from radiation-induced crypt loss measured 10 days post-irradiation. Significant effects were observed with both pre- and post-treatment regimens. However, only MnTE-2-PyP(5+) pre-treatment, but not post-treatment, prevented the development of rectal dilation, indicating that proper dosing regimen is critical for radioprotection. The pre-treatment also prevented or delayed the development of chronic proctitis depending on the radiation dose. Further work aimed at developing MnTE-2-PyP(5+) and similar drugs as adjunctive agents for radiotherapy of pelvic tumors is warranted. The present study substantiates the prospects of employing this and similar analogs in preserving normal tissue during cancer radiation as well as any other radiation exposure.

Small ubiquitin-like modifier 1-3 conjugation [corrected] is activated in human astrocytic brain tumors and is required for glioblastoma cell survival.

Small ubiquitin-like modifier (SUMO1-3) constitutes a group of proteins that conjugate to lysine residues of target proteins thereby modifying their activity, stability, and subcellular localization. A large number of SUMO target proteins are transcription factors and other nuclear proteins involved in gene expression. Furthermore, SUMO conjugation plays key roles in genome stability, quality control of newly synthesized proteins, proteasomal degradation of proteins, and DNA damage repair. Any marked increase in levels of SUMO-conjugated proteins is therefore expected to have a major impact on the fate of cells. We show here that SUMO conjugation is activated in human astrocytic brain tumors. Levels of both SUMO1- and SUMO2/3-conjugated proteins were markedly increased in tumor samples. The effect was least pronounced in low-grade astrocytoma (WHO Grade II) and most pronounced in glioblastoma multiforme (WHO Grade IV). We also found a marked rise in levels of Ubc9, the only SUMO conjugation enzyme identified so far. Blocking SUMO1-3 conjugation in glioblastoma cells by silencing their expression blocked DNA synthesis, cell growth, and clonogenic survival of cells. It also resulted in DNA-dependent protein kinase-induced phosphorylation of H2AX, indicative of DNA double-strand damage, and G(2) /M cell cycle arrest. Collectively, these findings highlight the pivotal role of SUMO conjugation in DNA damage repair processes and imply that the SUMO conjugation pathway could be a new target of therapeutic intervention aimed at increasing the sensitivity of glioblastomas to radiotherapy and chemotherapy.

Xenon neuroprotection in experimental stroke: interactions with hypothermia and intracerebral hemorrhage.

BACKGROUND: Xenon has been proven to be neuroprotective in experimental brain injury. The authors hypothesized that xenon would improve outcome from focal cerebral ischemia with a delayed treatment onset and prolonged recovery interval. METHODS: Rats were subjected to 70 min temporary focal ischemia. Ninety minutes later, rats were treated with 0, 15, 30, or 45% Xe for 20 h or 0 or 30% Xe for 8, 20, or 44 h. Outcome was measured after 7 days. In another experiment, after ischemia, rats were maintained at 37.5° or 36.0°C for 20 h with or without 30% Xe. Outcome was assessed 28 days later. Finally, mice were subjected to intracerebral hemorrhage with or without 30% Xe for 20 h. Brain water content, hematoma volume, rotarod function, and microglial activation were measured. RESULTS: Cerebral infarct sizes (mean±SD) for 0, 15, 30, and 45% Xe were 212±27, 176±55, 160±32, and 198±54 mm, respectively (P=0.023). Neurologic scores (median±interquartile range) followed a similar pattern (P=0.002). Infarct size did not vary with treatment duration, but neurologic score improved (P=0.002) at all xenon exposure durations (8, 20, and 44 h). Postischemic treatment with either 30% Xe or subtherapeutic hypothermia (36°C) had no effect on 28-day outcome. Combination of these interventions provided long-term benefit. Xenon improved intracerebral hemorrhage outcome measures. CONCLUSION: Xenon improved focal ischemic outcome at 7, but not 28 days postischemia. Xenon combined with subtherapeutic hypothermia produced sustained recovery benefit. Xenon improved intracerebral hemorrhage outcome. Xenon may have potential for clinical stroke therapy under carefully defined conditions.

A metalloporphyrin antioxidant alters cytokine responses after irradiation in a prostate tumor model.

The goal of this study was to evaluate cytokine secretion capacity in a mouse model of prostate cancer, both with and without metalloporphyrin antioxidant and radiation treatment. C57BL/6 mice with subcutaneous RM-9 tumors were treated daily for 12 days with MnTE-2-PyP(5+) [Mn (III) tetrakis (N-ethylpyridinium-2-yl) porphyrin], beginning 1 day after injection of RM-9 cells; a 10-Gy tumor-localized dose of (60)Co gamma rays was administered in a single fraction on day 7. Spleen, tumors and plasma were collected on day 12. T cells in the spleen were activated with anti-CD3 antibody and supernatants were collected. Twenty-two cytokines were quantified in spleen supernatants, five in tumor homogenates, and three in plasma using multiplex bead array technology and ELISA. The presence of a tumor had significant effects on many of the cytokines quantified (P < 0.05). Tumor-induced depression was evident for eight spleen cytokines (TNF-alpha, G-CSF, GM-CSF, IFN-gamma, IL10, IP-10, MIP-1alpha and mKC), whereas only three were enhanced (IL1beta, IL6 and MCP-1). Radiotherapy resulted in enhanced splenocyte capacity to produce IL4 and IL13 and increased IL4, MCP-1 and VEGF in tumors (P < 0.05). Addition of MnTE-2-PyP(5+) to radiation decreased the concentrations of IL4, IL13 and TGF-beta1 in spleen supernatants and IL4 and VEGF in tumors (P < 0.05 compared to radiation alone). Some differences were also noted in plasma cytokines. Overall, the findings suggest that administration of MnTE-2-PyP(5+) together with radiotherapy may enhance anti-tumor immune responsiveness and decrease the risk for radiation-induced normal tissue toxicities.

Metalloporphyrin antioxidants ameliorate normal tissue radiation damage in rat brain.

PURPOSE: We examined the effects of manganese (III) meso-tetrakis (diethyl-2-5-imidazole) porphyrin, a metalloporphyrin antioxidant (MPA), on neural tissue radiation toxicity in vivo and on tumour cell radiosensitivity in vitro. MATERIALS AND METHODS: MPA was administered directly into the right lateral ventricle of young adult, male Sprague-Dawley rats (0 or 3.4 microg) 3 h before treatment with a single fraction, 100 Gy radiation dose delivered to the left brain hemisphere. The effects of treatment on radiation responses were assessed at different time points following irradiation. RESULTS: MPA treatment prior to brain irradiation protected against acute radiation-induced apoptosis and ameliorated delayed damage to the blood-brain barrier and radiation necrosis, but without producing a discernible increase in tissue superoxide disumtase (SOD) activity. In vitro, MPA pretreatment protected against radiation-induced apoptosis in primary neuronal cultures and increased clonogenic survival of irradiated rat glioma C6 cells, but had no discernible effect on radiation-induced DNA double-strand breaks. MPA, a low molecular weight SOD mimic, significantly increased mitochondrial SOD activity in C6 cells, but not total cellular SOD activity. MPA up-regulated C6 expression of heme-oxygenase 1 (HO-1), an endogenous radioprotectant, but had no effect on HO-1 levels in human astrocytoma U-251 cells, human prostatic carcinoma LNCaP cells, or primary rat brain microvascular endothelial cells in vitro, nor on brain tissue HO-1 expression levels in vivo. CONCLUSIONS: Metalloporphyrin antioxidants merit further exploration as adjunctive radioprotectants for cranial radiotherapy/radiosurgery applications, although the potential for tumour protection must be carefully considered.

Long-term neuroprotection from a potent redox-modulating metalloporphyrin in the rat.

Sustained oxidative stress is a known sequel to focal cerebral ischemia. This study examined the effects of treatment with a single dose or sustained infusion of the redox-modulating MnPorphyrin Mn(III)TDE-2-ImP(5+) on outcome from middle cerebral artery occlusion (MCAO) in the rat. Normothermic rats were subjected to 90 min MCAO followed by 90 min reperfusion and then were treated with a single intracerebroventricular dose of Mn(III)TDE-2-ImP(5+). Neurologic and histologic outcomes were assessed at 1 or 8 weeks postischemia. A single dose of Mn(III)TDE-2-ImP(5+) caused a dose-dependent improvement in histologic and neurologic outcome when assessed 1 week postischemia. Mn(III)TDE-2-ImP(5+) afforded preservation of brain aconitase activity at 5.5 h after reperfusion onset, consistent with its known antioxidant properties. Mn(III)TDE-2-ImP(5+) also attenuated postischemic NF-kappaB activation. Evidence for effects on cerebral infarct size and neurologic function had completely dissipated when rats were allowed to survive for 8 weeks postischemia. In contrast, a 1-week continuous intracerebroventricular Mn(III)TDE-2-ImP(5+) infusion caused persistent and substantive reduction in both cerebral infarct size and neurologic deficit at 8 weeks postischemia. Pharmacologic modulation of postischemic oxidative stress is likely to require sustained intervention for enduring efficacy in improving neurologic and histologic outcome from a transient focal ischemic insult.

Effect of a metalloporphyrin antioxidant (MnTE-2-PyP) on the response of a mouse prostate cancer model to radiation.

BACKGROUND: Metalloporphyrin antioxidants can protect tissues against radiation-induced damage. However, for effective use in radiotherapy as normal tissue radioprotectants, they must not protect the cancer. The major objectives were to evaluate the effects of Mn (III) tetrakis (N-ethylpyridinium-2-yl) porphyrin (MnTE-2-PyP) on tumor response to radiation and to explore mechanisms responsible for the observed effects. MATERIALS AND METHODS: C57BL/6 mice were subcutaneously (s.c.) injected with RM-9 prostate tumor cells on day 0 and grouped according to treatment with MnTE-2-PyP (s.c. 6 mg/kg/day beginning on day 1 for 16 maximum days), 10 Gray (Gy) single fraction radiation on day 7, a combination of both or neither. Subsets per group and non-tumor bearing controls were evaluated for leukocyte populations, red blood cell (RBC) and platelet characteristics and cytokines on day 12; the remaining mice were followed for tumor growth. RESULTS: Although radiation alone significantly slowed tumor growth and the addition of MnTE-2-PyP resulted in slightly slower tumor progression, the difference between radiation and radiation plus drug was not statistically significant. However, the treatment with drug alone significantly elevated T (helper, Th and cyotoxic, Tc) and natural killer (NK) cells in the spleen, B-cells in the blood and spleen, and the capacity to produce interleukin-2. The addition of the drug to radiation did not ameliorate the depression seen in all the major leukocyte types, but did protect against radiation-induced decreases in RBC counts, hemoglobin and hematocrit. Vascular endothelial growth factor (VEGF) increased in the plasma from both the irradiated groups and a trend for increased transforming growth factor-beta1 (TGF-beta1) was noted with radiation alone. CONCLUSION: MnTE-2-PyP did not protect RM-9 prostate tumors against radiation damage and was not toxic under the conditions used. The drug-induced enhancement of certain immune parameters suggests that MnTE-2-PyP may be beneficial not only as a normal tissue radioprotectant, but also as a facilitator of antitumor immunity.

Infrared imaging of trauma patients for detection of acute compartment syndrome of the leg.

OBJECTIVE: Early compartment syndrome is difficult to diagnose, and a delay in the diagnosis can result in amputation or death. Our objective was to explore the potential of infrared imaging, a portable and noninvasive technology, for detecting compartment syndrome in the legs of patients with multiple trauma. We hypothesized that development of compartment syndrome is associated with a reduction in surface temperature in the involved leg and that the temperature reduction can be detected by infrared imaging. DESIGN: Observational clinical study. SETTING: Level I trauma center between July 2006 and July 2007. PATIENTS: Trauma patients presenting to the emergency department. INTERVENTIONS: Average temperature of the anterior surface of the proximal and distal region of each leg was measured in the emergency department with a radiometrically calibrated, 320 x 240, uncooled microbolometer infrared camera. MEASUREMENTS AND MAIN RESULTS: The difference in surface temperature between the thigh and foot regions (thigh-foot index) of the legs in trauma patients was determined by investigators blinded to injury pattern using thermographic image analysis software. The diagnosis of compartment syndrome was made intraoperatively. Thermographic images from 164 patients were analyzed. Eleven patients developed compartment syndrome, and four of those patients had bilateral compartment syndrome. Legs that developed compartment syndrome had a greater difference in proximal vs. distal surface temperature (8.80 +/- 2.05 degrees C) vs. legs without compartment syndrome (1.22 +/- 0.88 degrees C) (analysis of variance p < .01). Patients who developed unilateral compartment syndrome had a greater proximal vs. distal temperature difference in the leg with (8.57 +/- 2.37 degrees C) vs. the contralateral leg without (1.80 +/- 1.60 degrees C) development of compartment syndrome (analysis of variance p < .01). CONCLUSIONS: Infrared imaging detected a difference in surface temperature between the proximal and distal leg of patients who developed compartment syndrome. This technology holds promise as a supportive tool for the early detection of acute compartment syndrome in trauma patients.

Technical note: rapid prototyping of 3D grid arrays for image guided therapy quality assurance.

Three dimensional grid phantoms offer a number of advantages for measuring imaging related spatial inaccuracies for image guided surgery and radiotherapy. The authors examined the use of rapid prototyping technology for directly fabricating 3D grid phantoms from CAD drawings. We tested three different fabrication process materials, photopolymer jet with acrylic resin (PJ/AR), selective laser sintering with polyamide (SLS/P), and fused deposition modeling with acrylonitrile butadiene styrene (FDM/ABS). The test objects consisted of rectangular arrays of control points formed by the intersections of posts and struts (2 mm rectangular cross section) and spaced 8 mm apart in the x, y, and z directions. The PJ/AR phantom expanded after immersion in water which resulted in permanent warping of the structure. The surface of the FDM/ABS grid exhibited a regular pattern of depressions and ridges from the extrusion process. SLS/P showed the best combination of build accuracy, surface finish, and stability. Based on these findings, a grid phantom for assessing machine-dependent and frame-induced MR spatial distortions was fabricated to be used for quality assurance in stereotactic neurosurgical and radiotherapy procedures. The spatial uniformity of the SLS/P grid control point array was determined by CT imaging (0.6 x 0.6 x 0.625 mm3 resolution) and found suitable for the application, with over 97.5% of the control points located within 0.3 mm of the position specified in CAD drawing and none of the points off by more than 0.4 mm. Rapid prototyping is a flexible and cost effective alternative for development of customized grid phantoms for medical physics quality assurance.

Radiation and a metalloporphyrin radioprotectant in a mouse prostate tumor model.

BACKGROUND: Antioxidants have the potential to protect normal tissues against radiation-induced damage, but must not protect tumor cells during radiotherapy. The major objectives were to determine whether a metalloporphyrin antioxidant affects prostate tumor response to radiation and identify possible mechanisms of interaction. MATERIALS AND METHODS: C57BL/6 mice with RM-9 tumor were treated with manganese (III) meso-tetrakis (1,3-diethylimidazolium-2-yl) porphyrin (MnTDE-2-ImP) and 10 gray (Gy) radiation. Tumor volume was quantified and a subset/group was evaluated for hypoxia-inducible factor-1alpha (HIF-1alpha), bone marrow-derived cell populations and cytokines. RESULTS: The addition of MnTDE-2-ImP transiently increased tumor response compared to radiation alone. The group receiving drug plus radiation had reduced intratumoral HIF-1alpha and decreased capacity to secrete TNF-alpha, whereas production of IL-4 was increased. There were no toxicities associated with combination treatment. CONCLUSION: The results demonstrate that MnTDE-2-ImP did not protect the RM-9 prostate tumor against radiation; instead, radiation effectiveness was modestly increased. Possible mechanisms include reduction of radiation-induced HIF-1alpha and an altered cytokine profile.

Isoflurane provides long-term protection against focal cerebral ischemia in the rat.

BACKGROUND: Long-term neuroprotection by isoflurane has been questioned. The authors examined factors in experimental models potentially critical to definition of enduring isoflurane neuroprotection. METHODS: Rats were prepared for temporary middle cerebral artery occlusion (MCAO). Pericranial normothermia was maintained. Neurologic deficits (range, 0-48; 0=no deficit) and cerebral infarct volumes were measured. In experiment 1, rats underwent 50 or 80 min MCAO while awake or anesthetized with 1.8% isoflurane. Blood pressure was controlled with phenylephrine. Outcome was evaluated 2 weeks later. In experiment 2, rats underwent 50 min MCAO while awake or anesthetized with isoflurane, with outcome evaluated 8 weeks later. In experiment 3, rats underwent 50 min MCAO while awake or anesthetized with isoflurane and 2 weeks recovery. Effects of phenylephrine and the mitochondrial adenosine triphosphate-sensitive K channel antagonist 5-hydroxydecanoate were studied. In experiment 4, isoflurane-anesthetized rats underwent 50 min MCAO with permanent or temporary common carotid artery occlusion, with outcome evaluated 2 weeks later. RESULTS: In experiment 1, isoflurane reduced neurologic deficit (median+/-interquartile range; awake vs. isoflurane: 11+/-12 vs. 8+/-6 for 80 min and 13+/-4 vs. 3+/-9 for 50 min; P=0.0006) and infarct size (160+/-97 vs. 84+/-62 mm for 80 min and 169+/-78 vs. 68+/-61 mm for 50 min; P<0.0001). In experiment 2, isoflurane protection persisted at 8 weeks after ischemia. In experiment 3, there was no effect of phenylephrine or 5-hydroxydecanoate. In experiment 4, permanent common carotid ligation increased infarct size threefold versus temporary occlusion. CONCLUSIONS: Isoflurane repeatedly improved long-term neurologic and histologic outcome from focal ischemia independent of ischemia duration, perfusion pressure, or pretreatment with 5-hydroxydecanoate.

NMDA-induced apoptosis in mixed neuronal/glial cortical cell cultures: the effects of isoflurane and dizocilpine.

In animal models of severe ischemia, it has not been uniformly observed that anesthetics are protective. However, anesthetics have not been evaluated in the presence of a mild excitotoxic insult. We hypothesized that in the presence of a mild excitotoxic insult, 3 microm N-methyl-D-aspartate (NMDA), isoflurane may prevent apoptotic cell death. Primary mixed neuronal/glial cultures were prepared from fetal rat brains. Mature cultures were exposed to dissolved isoflurane [0 mM, 0.4 mM (1.8 minimum alveolar concentration) or 1.6 mM (7 minimum alveolar concentration)] or dizocilpine (10 microM), and NMDA (0 or 3 microM) at 37 degrees C for 30 minutes. Apoptosis was assessed using terminal-deoxy-nucleotidyl end-nick labeling oligonucleosomal DNA fragmentation enzyme-linked immunosorbent assay, and caspases-3 and -9 activation assays. NMDA (3 muM) induced apoptosis in mixed neuronal/glial cell cultures. Apoptosis induced by 3 microm NMDA was caspase-3 but not caspase-9 mediated. In the presence of a mild excitotoxic insult, this investigation showed an attenuation of apoptotic cell death by dizocilpine, but not isoflurane.

Selective gamma-aminobutyric acid type A receptor antagonism reverses isoflurane ischemic neuroprotection.

BACKGROUND: Isoflurane provides protection against severe forebrain ischemia in the rat. The authors hypothesized that this is attributable to interaction with the gamma-aminobutyric acid type A (GABAA) receptor resulting in altered time to onset of ischemic hippocampal depolarization. METHODS: Organotypic hippocampal slices were subjected to oxygen-glucose deprivation in the presence of isoflurane and combinations of GABAA (bicuculline) and GABAB (phaclofen) receptor antagonists. Cell death was measured. Rats were subjected to severe forebrain ischemia while anesthetized with fentanyl-nitrous oxide or 1.4% isoflurane. In the isoflurane group, rats also received intravenous bicuculline (0, 1, or 2 mg/kg). Neurologic and histologic outcomes and time to depolarization were assessed. RESULTS: In slices, 2% isoflurane caused near-complete protection against oxygen-glucose deprivation. This was unaffected by coadministration of phaclofen but largely reversed by bicuculline. The GABAA agonist muscimol was also protective, having an effect equivalent to 1% isoflurane. In rats, isoflurane (0 mg bicuculline) improved neurologic and histologic outcome versus fentanyl-nitrous oxide (CA1 percentage of alive neurons: fentanyl-nitrous oxide, 15 +/- 7; isoflurane, 61 +/- 24). The isoflurane effect was reversed in a dose-dependent manner by bicuculline (CA1 percentage alive: 1 mg/kg, 44 +/- 22; 2 mg/kg, 21 +/- 15). Time to depolarization was delayed with isoflurane versus fentanyl-nitrous oxide (137 vs. 80 s) but was not affected by bicuculline (149 s). In contrast, postischemic time to repolarization was more rapid with fentanyl-nitrous oxide or isoflurane plus bicuculline versus isoflurane alone. CONCLUSIONS: These studies are consistent with the hypothesis that the GABAA receptor serves as a major site of action for isoflurane neuroprotection both in vitro and in vivo. However, the mechanism by which this interaction confers in vivo protection cannot be attributed to effects on the duration of ischemic depolarization.

Assessing neurocognitive dysfunction in cranial radiotherapy: can cognitive event-related potentials help?

Cognitive changes are common sequelae of cancer and cancer treatment, particularly in patients receiving cranial radiotherapy (RT). These effects are typically assessed by subjective clinical examination or using objective neuropsychological tests. Biologically based neurophysiological methods have been increasingly applied to the study of cognitive processing in neuropsychiatric and neurological disorders and as objective measures of cognitive status for patients with dementia. These methods detect the activation of neural circuits that directly mediate cognitive function in the human brain and include metabolic and electrophysiology based techniques. Neuroimaging procedures such as 18FDG PET and more recently fMRI, which detect metabolic activation associated with cognitive processing, provide excellent spatial resolution and can be directly correlated with neuroradiological findings associated with cranial RT neurotoxicity. Clinical electrophysiology procedures such as cognitive event-related potentials (ERP), which detect the neuronal electrical activity associated with cognitive processing, offer excellent temporal resolution at low cost. Cognitive ERP techniques are already being used to assess severity and progression of cognitive dysfunction in patients with vascular and degenerative dementias, but have been largely overlooked in studies of radiation-related cognitive impairments. We review these various electrophysiological methods in the context of their relevance to assessing cranial RT effects on cognitive function, and provide recommendations for a neurophysiological approach to supplement current neuropsychological tests for RT cognitive impairments. This technology is well suited for clinical assessment of neurocognitive sequelae of cancer and should provide new insights into the mechanism of RT-related cognitive dysfunction.

Effects of a manganese (III) porphyrin catalytic antioxidant in a mouse closed head injury model.

Closed head injury induces cerebral oxidative stress. The efficacy of a Mn (III) porphyrin catalytic antioxidant was assessed in a mouse closed head injury model. Mice were subjected to closed head injury and treated 15 min later with an i.v. bolus of vehicle or 3 mg/kg MnTE-2-PyP5+. Aconitase activity, Fluoro-Jade staining, glial fibrillary acidic protein immunoreactivity, and rotarod falling latencies were measured. Closed head injury altered all variables. MnTE-2-PyP5+ had no effect on any variable with the exception of attenuation of aconitase inactivation at 2 h post-closed head injury. In a second experiment, mice received 3 mg/kg or 6 mg/kg MnTE-2-PyP5+ or vehicle i.v. 15 min post-closed head injury. Rotarod and Morris water maze latencies were measured. Closed head injury altered performance in both tests. No statistically significant effect of MnTE-2-PyP5+ was observed. We conclude that single dose MnTE-2-PyP5+ does not alter outcome in this mouse closed head injury model.

Isoflurane-induced neuronal degeneration: an evaluation in organotypic hippocampal slice cultures.

Prolonged exposure of postnatal day (PND) 7 rat pups to anesthetics, which act via N-methyl-D-aspartate antagonism and/or gamma-amino butyric acid enhancement, causes neurodegeneration and persistent behavioral deficits. We studied these findings in vitro and determined whether the age of rat pups used for study or duration of anesthetic exposure modulates resultant neurodegeneration. Organotypic hippocampal slices (OHSs) were prepared from rat pups on PNDs 4, 7, and 14 and cultured 7 or 14 days in vitro. The slices were exposed to 1.5% isoflurane or fresh gas for durations of 1, 3, or 5 h. Hippocampal CA1, CA3, and dentate gyrus neuronal survival was assessed 3 days later. Neuronal cell death was greatest in OHSs prepared from PND 7 rat pups (P < 0.001) and was most evident after 5 h exposure to isoflurane (P < 0.001). By eliminating variables such as hemodynamics, nutrition, oxygenation, and carbon dioxide elimination, this in vitro investigation supports both an age- and duration-dependent relationship between 1.5% isoflurane exposure and perinatal neuronal death.

Apoptosis is not enhanced in primary mixed neuronal/glial cultures protected by isoflurane against N-methyl-D-aspartate excitotoxicity.

Volatile anesthetics reduce acute excitotoxic cell death in primary neuronal/glial cultures. We hypothesized that cells protected by isoflurane against N-methyl-d-aspartate (NMDA)-induced necrosis would instead become apoptotic. Primary mixed neuronal/glial cultures prepared from fetal rat brain were exposed to dissolved isoflurane (0 mM, 0.4 mM [1.8 minimum alveolar anesthetic concentration], or 1.6 mM [7 minimum alveolar anesthetic concentration]) and NMDA (0 or 100 microM) at 37 degrees C for 30 min. Dizocilpine (10 microM) plus 100 microM NMDA served as a positive control. Necrosis and apoptosis were assessed at 24 and/or 48 h after exposure by using Hoechst/propidium iodide staining, terminal-deoxynucleotidyl transferase end-nick labeling, DNA fragmentation enzyme-linked immunoabsorbence, and caspase-3 activity assays. NMDA increased the number of necrotic cells. Isoflurane (1.6 mM) and dizocilpine partially reduced cellular necrosis but did not increase the number of morphologically apoptotic or apoptotic-like cells resulting from exposure to 100 microM NMDA at 24 h. At 48 h, no evidence was found to indicate that cells protected by isoflurane had become apoptotic or apoptotic-like. However, cells protected by dizocilpine against necrosis showed evidence of caspase-3-mediated apoptosis. These in vitro data do not support the hypothesis that isoflurane protection against acute excitotoxic necrosis results in apoptosis.

Hemodilution during cardiopulmonary bypass increases cerebral infarct volume after middle cerebral artery occlusion in rats.

Although the optimal hematocrit during cardiopulmonary bypass (CPB) is not defined, excessive hemodilution may lead to organ ischemia via a reduction in oxygen-carrying capacity uncompensated by autoregulatory and/or rheologic increases in organ blood flow. As a result, the consequences of hemodilution in patients at risk for cerebral ischemia are not clearly understood. We designed this study to evaluate the effects of hemodilution in the setting of focal cerebral ischemia during CPB. Wistar rats surgically prepared for CPB were randomized to either hemodilution (hemoglobin (Hb), 6 g/dL; n = 9) or control (Hb, 11 g/dL; n = 8) groups and subsequently exposed to focal cerebral ischemia induced by middle cerebral artery occlusion (MCAO). Immediately after the onset of MCAO (maintained for 90 min), 65 min of hypothermic (28 degrees C) CPB was initiated. Twenty-four hours later, functional neurological outcome and cerebral infarct volume were determined. Compared with controls, the hemodilution group had worse neurological performance (new score = 8 [2], hemodilution; versus 10 [2], control; P = 0.030) and larger total cerebral infarct volumes (182 +/- 84 mm(3), hemodilution; versus 103 +/- 58 mm(3), control; P = 0.043). In this experimental model of CPB with reversible MCAO-induced focal cerebral ischemia, hemodilution worsened neurological function and increased cerebral infarct volume.