Akt and mTOR mediate programmed necrosis in neurons.

Necroptosis is a newly described form of regulated necrosis that contributes to neuronal death in experimental models of stroke and brain trauma. Although much work has been done elucidating initiating mechanisms, signaling events governing necroptosis remain largely unexplored. Akt is known to inhibit apoptotic neuronal cell death. Mechanistic target of rapamycin (mTOR) is a downstream effector of Akt that controls protein synthesis. We previously reported that dual inhibition of Akt and mTOR reduced acute cell death and improved long term cognitive deficits after controlled-cortical impact in mice. These findings raised the possibility that Akt/mTOR might regulate necroptosis. To test this hypothesis, we induced necroptosis in the hippocampal neuronal cell line HT22 using concomitant treatment with tumor necrosis factor α (TNFα) and the pan-caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone. TNFα/zVAD treatment induced cell death within 4 h. Cell death was preceded by RIPK1-RIPK3-pAkt assembly, and phosphorylation of Thr-308 and Thr473 of AKT and its direct substrate glycogen synthase kinase-3ß, as well as mTOR and its direct substrate S6 ribosomal protein (S6), suggesting activation of Akt/mTOR pathways. Pretreatment with Akt inhibitor viii and rapamycin inhibited Akt and S6 phosphorylation events, mitochondrial reactive oxygen species production, and necroptosis by over 50% without affecting RIPK1-RIPK3 complex assembly. These data were confirmed using small inhibitory ribonucleic acid-mediated knockdown of AKT1/2 and mTOR. All of the aforementioned biochemical events were inhibited by necrostatin-1, including Akt and mTOR phosphorylation, generation of oxidative stress, and RIPK1-RIPK3-pAkt complex assembly. The data suggest a novel, heretofore unexpected role for Akt and mTOR downstream of RIPK1 activation in neuronal cell death.

Invited article: searching for oracles? Blood biomarkers in acute stroke.

Emerging data suggest that a wide array of measurable biomarkers in blood may provide a novel window into the pathophysiology of stroke. In this review, we survey the state of progress in the field. Three specific questions are assessed. Can biomarkers augment the clinical examination and powerful brain imaging tools to enhance the accuracy of the diagnostic process? Can biomarkers be used to help triage patients for thrombolytic therapy? Can biomarkers help predict patients who are most susceptible to malignant infarction? Many encouraging molecular candidates have been found that appear to match the known cascades of neurovascular injury after stroke. However, whether these putative biomarkers may indeed have direct clinical utility remains to be quantitatively validated. Larger clinical trials are warranted to establish the sensitivity and specificity of biomarkers for routine use in clinical stroke.

alphaNAC depletion as an initiator of ER stress-induced apoptosis in hypoxia.

Accumulation of unfolded proteins triggers endoplasmic reticulum (ER) stress and is considered a part of the cellular responses to hypoxia. The nascent polypeptide-associated complex (NAC) participates in the proper maturation of newly synthesized proteins. However, thus far, there have been no comprehensive studies on NAC involvement in hypoxic stress. Here, we show that hypoxia activates glycogen synthase kinase-3beta (GSK-3beta) and that the activated GSK-3beta destabilizes alphaNAC with the subsequent apoptosis of the cell. Hypoxia of various cell types and the mouse ischemic brain was associated with rapid downregulation of alphaNAC and ER stress responses involving PERK, ATF4, gamma-taxilin, elF2alpha, Bip, and CHOP. Depletion of alphaNAC by RNA interference specifically activated ER stress responses and caused mitochondrial dysfunction, which resulted in apoptosis through caspase activation. Interestingly, we found that the hypoxic conditions activated GSK-3beta, and that GSK-3beta inhibition prevented alphaNAC protein downregulation in hypoxic cells and rescued the cells from apoptosis. In addition, alphaNAC overexpression increased the viability of hypoxic cells. Taken together, these results suggest that alphaNAC degradation triggers ER stress responses and initiates apoptotic processes in hypoxic cells, and that GSK-3beta may participate upstream in this mechanism.

Low density lipoprotein as a targeted carrier for doxorubicin in nude mice bearing human hepatoma HepG2 cells.

Doxorubicin (DOX) was coupled to human low density lipoprotein (LDL) to form a complex (LDL-DOX). When injected into mice, LDL-DOX was more accumulated in liver than free DOX. In contrast, LDL-DOX was less accumulated in heart than free DOX. In in vitro studies on human hepatoma (HepG2) cells, although the cellular uptake of LDL-DOX was higher than that of DOX, the anti-proliferative effect of LDL-DOX on these tumor cells was smaller than that of LDL. However, when LDL-DOX or DOX was administered to nude mice bearing HepG2 cells implanted on the shoulder, the anti-proliferative effects on the tumor cells of both drugs were similar. Histological analyses indicated that organization of myocardial filaments was disrupted and vacuolization was observed in DOX-treated group when compared with control group whereas LDL-DOX-treatment did not exhibit any damage in the host's heart. Enzymatic analyses also demonstrated that plasma lactate dehydrogenase activity, which is a common indicator of heart damage, was elevated in DOX-treated group when compared with control group whereas the activity of this enzyme was unchanged in LDL-DOX-treated group. The results in present study indicate that LDL can be used as a targeted carrier for DOX because LDL-DOX can exhibit similar anti-proliferative effect as DOX on tumor but reduce the DOX-induced cardiotoxicity in the host.

Tissue type plasminogen activator amplifies hemoglobin-induced neurotoxicity in rat neuronal cultures.

Tissue type plasminogen activator (tPA) is clinically used as a form of thrombolytic therapy for acute ischemic stroke. However, recent data suggest that there may be negative effects associated with tPA. Experimental studies show that tPA amplifies excitotoxic neuronal damage and clinical trials show that some stroke patients suffer from hemorrhage after tPA therapy. Since hemoglobin is the major component of blood, we tested the hypothesis that tPA can amplify hemoglobin-induced neurotoxicity. PC12 cells and primary cortical rat neurons were exposed to either hemoglobin alone or hemoglobin plus tPA. Hemoglobin induced dose-dependent cytotoxicity. The addition of tPA significantly increased hemoglobin-induced cell death. These results raise the important possibility that tPA may worsen outcomes after hemorrhage.

Delivery of imaging agents into brain.

Delivery of diagnostic agents to the central nervous system (CNS) poses several challenges as a result of the special features of CNS blood vessels and tissue fluids. Diffusion barriers exist between blood and neural tissue, in the endothelium of parenchymal vessels (blood-brain barrier, BBB), and in the epithelia of the choroid plexuses and arachnoid membrane (blood-CSF barriers), which severely restrict penetration of several diagnostic imaging agents. The anatomy of large vessels can be imaged using bolus injection of X-ray contrast agents to identify sites of malformation or occlusion, and blood flow measured using MRI and CT, while new techniques permit analysis of capillary perfusion and blood volume. Absolute quantities can be derived, although relative measures in different CNS regions may be as useful in diagnosis. Local blood flow, blood volume, and their ratio (mean transit time) can be measured with high speed tomographic imaging using MRI and CT. Intravascular contrast agents for MRI are based on high magnetic susceptibility agents such as gadolinium, dysprosium and iron. Steady-state imaging using agents that cross the BBB including (123)I- and (99m)Tc-labelled lipophilic agents with SPECT, gives a 'snapshot' of perfusion at the time of injection. Cerebral perfusion can also be measured with PET, using H(2)(15)O, (11)C- or (15)O-butanol, and (18)F-fluoromethane, and cerebral blood volume measured with C(15)O. Recent advances in MRI permit the non-invasive 'labelling' of endogenous water protons in flowing blood, with subsequent detection as a measure of blood flow. Imaging the BBB most commonly involves detecting disruptions of the barrier, allowing contrast agents to leak out of the vascular system. Gd-DTPA is useful in imaging leaky vessels as in some cerebral tumors, while the shortening of T(1) by MR contrast agents can be used to detect more subtle changes in BBB permeability to water as in cerebral ischemia. Techniques for imaging the dynamic activity of the brain parenchyma mainly involve PET, using a variety of radiopharmaceuticals to image glucose transport and metabolism, neurotransmitter binding and uptake, protein synthesis and DNA dynamics. PET methods permit detailed analysis of regional function by comparing resting and task-related images, important in improving understanding of both normal and pathological brain function.

Alterations in K+ evoked profiles of neurotransmitter and neuromodulator amino acids after focal ischemia-reperfusion.

Secondary elevations in extracellular amino acids occur during reperfusion after transient cerebral ischemia. The delayed accumulation of excitatory amino acids may contribute to the progressive development of neuronal injury. In this study, we explored the mechanisms that may be involved in this phenomenon. Microdialysis samples from probes located in rabbit cortex were analysed with a chiral amino acid procedure. Concentrations of neurotransmitters (L-Glu, GABA), N-methyl-D-aspartate receptor modulators (D-Ser, Gly), an inhibitory neuromodulator (Tau), the lipid component phosphoethanolamine, and L-Gln, L-Ser and L-Ala were measured. Depolarization via perfusion with potassium was used to assess the status of release/reuptake systems at 2 and 4 h reperfusion after 2 h transient focal ischemia. Background experiments classified potassium evoked responses as calcium dependent or calcium-independent by inclusion of 30 microM omega-conopeptide MVIIC or by inclusion of 20 mM magnesium and ommision of calcium. During ischemia, large elevations of almost all amino acids occurred. During reperfusion, secondary elevations in transmitter amino acids (L-Glu, GABA) and N-methyl-D-aspartate receptor modulators (D-Ser, Gly) occurred. Tau remained slightly elevated whereas the lipid component phosphoethanolamine remained high and stable during reperfusion. Reperfusion significantly potentiated the potassium response for amino acids with calcium-dependent responses (L-Glu and GABA). In contrast, calcium-independent responses (Tau, phosphoethanolamine, L-Gln) were significantly attenuated. Intermediate behavior was observed with Gly, while no potassium responses were observed for D-Ser, L-Ser or L-Ala. These data demonstrate that perturbations in evoked amino acid profiles after ischemia-reperfusion are selective. Reduction of calcium-independent responses implicate a general decline in efficacy of transporter mechanisms that restore transmembrane gradients of ions and transmitters. Decreased efficacy of transporter systems may reduce transmitter reuptake and account for the amplified release of L-Glu and GABA, thus contributing to progressive neural dysfunction after cerebral ischemia.

A theoretical analysis of hemodynamic and biomechanical alterations in intracranial AVMs after radiosurgery.

PURPOSE: Stereotactic radiosurgery is being increasingly used to treat intracranial arteriovenous malformations (AVMs). However, successful radiosurgery may involve latent periods of 1-2 years prior to AVM obliteration. This latent period include states of altered flow patterns that may or may not influence hemorrhage probabilities. The probability of hemorrhage is likely to be related to the degree of biomechanical stress across the AVM shunt walls. This paper describes a theoretical analysis of the altered hemodynamics and biomechanical stresses within AVM shunts post-radiosurgery. METHODS AND MATERIALS: The mathematical model is comprised of linked flow compartments that represent the AVM and adjacent normal vasculature. As obliteration of the irradiated shunts occur, changes in flow rates and pressure gradients are calculated based on first order fluid dynamics. Stress on the AVM shunt walls is calculated based on tangential forces due to intramural pressure. Two basic models are presented: a distribution of shunts with fixed thin walls subject to step-function obliteration (Model I), and a distribution of shunts subject to luminal obliteration from slowly thickening walls (Model II). Variations on these models are analyzed, including sequential, selective and random shunt obliteration, and uniform or Poisson distributions of shunt radii. RESULTS: Model I reveals that the range of pressure alterations in the radiosurgically-treated AVM include the possibility of transient increases in the total biomechanical stress within the shunt walls prior to obliteration. Model II demonstrates that uniform luminal narrowing via thickened walls should lead to reduced transmural stresses. The precise temporal pattern of AVM flow decrease and biomechanical stress reduction depends on the selection of shunts that are obliterated. CONCLUSION: (a) The hemodynamic and biomechanical changes appear to be relatively independent of the shunt distribution but highly dependent on the temporal pattern of the obliterative process, (b) uniformly thickened shunt walls should uniformly decrease biomechanical stresses in the latent period prior to complete obliteration, but if uniform obliteration is not achieved, (c) transient alterations in pressure versus stress relationships may lead to temporarily increased biomechanical stress prior to complete obliteration, and (d) reduction in stress may not reach significant levels until the AVM is almost completely obliterated.

High-dose single-fraction brain irradiation: MRI, cerebral blood flow, electrophysiological, and histological studies.

Radiation-induced alterations in cerebrovascular and metabolic function form the basis for the radiosurgical treatment of selected intracranial vascular malformations and tumors in human patients. However, the underlying mechanisms, temporal progression, and modifying factors involved in the radiosurgical obliteration of these intracranial lesions as well as the risks of delayed radiation injury to surrounding normal brain remain poorly understood. In this report, the rabbit brain was used as an animal model to examine the effects of high-dose single-fraction X-irradiation on magnetic resonance imaging (MRI) appearance, neurophysiologic function, and histological integrity. At approximately 10 weeks following left-hemisphere irradiation with 60 Gy (225 kVp) X rays, MRI studies showed radiation-induced changes including blood-brain barrier (BBB) perturbations in the white matter regions and the hippocampus. Significant reductions in regional cerebral blood flow (rCBF) ratios were found in the hippocampus and certain regions of the cortex in irradiated animals. However, no changes in somatosensory evoked potentials (SEP) were observed. Histological studies demonstrated telangiectatic vessels, spreading edema in the white matter, and focal regions of necrosis and hemorrhage in the irradiated cortices and hippocampi. These results demonstrate that the irradiated rabbit brain may be used as an experimental model to correlate the spatiotemporal pattern of functional changes with radiologic and histological changes in delayed radiation injury.

MRI and PET of delayed heavy-ion radiation injury in the rabbit brain.

Magnetic resonance imaging (MRI) and positron emission tomography (PET) techniques were used to obtain in vivo scans of delayed (30 GyE helium ion, 230 MeV/u) radiation injury in rabbit brain. T2-weighted (T2W) MRI scans demonstrated alterations that were restricted primarily to the white matter tracts and the deep perithalamic and thalamic regions. Quantitative measurements of T2 and T1 values demonstrated wide variations in absolute values. However, paired comparisons in hemibrain-irradiated rabbits revealed significant increases in T2 (p less than 0.001) and T1 (p less than 0.01) in irradiated versus unirradiated brain. Gadolinium DTPA (GdDTPA) enhanced MRI and 82Rubidium (82Rb) PET detected focal regions of blood-brain barrier (BBB) disruption restricted to the deep white matter and thalamic regions. Sequential GdDTPA enhanced MRI scans showed the spreading of the tracer from the initial site of contrast enhancement. 18Fluorodeoxyglucose (18FDG) PET studies demonstrated the markedly depressed metabolic profiles of irradiated brain. Histological findings of tissue edema and necrosis correlated well with the in vivo imaging abnormalities. These initial studies demonstrate that the irradiated rabbit brain is a suitable animal model for examining the delayed effects of radiation injury in the brain.

An experimental compartmental flow model for assessing the hemodynamic response of intracranial arteriovenous malformations to stereotactic radiosurgery.

Stereotactic radiosurgery has proven to be an effective method of treating selected inaccessible or inoperable arteriovenous malformations (AVMs) of the brain. Radiation-induced obliteration of successfully-treated AVMs, however, occurs only after some latent period after treatment, depending on size, location, and dose. An experimental compartmental flow model is proposed to describe the hemodynamic alterations in the AVM as a result of the pathophysiological changes after radiosurgery, and to analyze temporal alterations in AVM blood flow rates and pressure gradients before complete obliteration. In representative small (low-flow, 150 ml/min) and large (high-flow, 440 ml/min) AVMs, it is found that increases in pressure gradients across certain vascular structures within the AVM occur during the normal course of radiation-induced flow decrease and AVM obliteration. The magnitude of these pressure alterations, however, may be within the normal physiological variations in cerebrovascular blood pressure. The effects of partial-volume irradiation of the AVM is examined by limiting radiosurgical treatment to varying portions of the flow compartments within the model. It is found that alterations in pressure gradients persist in unirradiated vascular shunts, even after complete obliteration of the treated AVM volume. These pressure alterations may increase the probability of hemorrhage from the untreated shunts of the AVM and cause redistribution of regional cerebral blood flow resulting in increased flow through these untreated shunts.

Delayed biologic reactions to stereotactic charged-particle radiosurgery in the human brain.

Over 350 patients have been treated for inoperable intracranial arteriovenous malformations with charged-particle radiosurgery. Focussed accelerated helium ion beams derived from charged-particle cyclotrons are stereotactically directed into the brain to obliterate abnormal shunts. Treated patients demonstrate delayed changes in brain anatomy and function that occur months to years after radiosurgery. The underlying mechanisms of the brain's delayed reaction to charged-particle radiosurgery involve complex perturbations in cerebrovascular and metabolic function. This report describes the wide range of delayed reactions that may occur in the brain after radiosurgery, including hemodynamic changes, blood-brain barrier disruption and vasogenic edema, metabolic suppression, and parenchymal necrosis. These delayed reactions to injury in the brain involve potential target cells that include cerebral endothelial cells, oligodendroglia and astrocytes.

Cerebrovascular and metabolic perturbations in delayed heavy charged particle radiation injury.

Focal heavy charged particle irradiation of the rabbit brain created defined lesions which were observable by nuclear magnetic resonance (NMR) and positron emission tomography (PET) imaging techniques. The lesions appeared approximately 9-11 months after left partial hemibrain irradiation with 30 Gy (230 MeV/u helium ions), and were restricted to the white matter tracts and deep perithalamic and thalamic regions. 82Rubidium PET and Gadolinium DTPA enhanced NMR imaging were used to detect blood-brain barrier perturbations. 18Fluordeoxyglucose PET studies demonstrated widespread decreases in cerebral glucose uptake in the cortex and thalamus of the irradiated hemisphere. NMR and PET imaging results correlated well with histological findings. Rabbits irradiated with 15 Gy did not demonstrate any abnormalities in the brain with sequential NMR scans through 14 months post-irradiation.

Study on the surface polypeptides isolated from human lymphoblastoid ce-ls by ionic shock.

Ionic shock treatment in the presence of 10% glycerol is an efficient and selective method for extracting cell surface components from Raji cells and effecting the solubilization of up to 22 polypeptides. The majority of these shock-released polypeptides are accessible to lactoperoxidase radioiodination. Sera from rabbits immunized against these soluble extracts are reactive against Raji cell surface as indicated by indirect membrane immunofluorescence and agglutination assays.