Interventional magnetic resonance imaging-guided cell transplantation into the brain with radially branched deployment.

Intracerebral cell transplantation is being pursued as a treatment for many neurological diseases, and effective cell delivery is critical for clinical success. To facilitate intracerebral cell transplantation at the scale and complexity of the human brain, we developed a platform technology that enables radially branched deployment (RBD) of cells to multiple target locations at variable radial distances and depths along the initial brain penetration tract with real-time interventional magnetic resonance image (iMRI) guidance. iMRI-guided RBD functioned as an "add-on" to standard neurosurgical and imaging workflows, and procedures were performed in a commonly available clinical MRI scanner. Multiple deposits of super paramagnetic iron oxide beads were safely delivered to the striatum of live swine, and distribution to the entire putamen was achieved via a single cannula insertion in human cadaveric heads. Human embryonic stem cell-derived dopaminergic neurons were biocompatible with the iMRI-guided RBD platform and successfully delivered with iMRI guidance into the swine striatum. Thus, iMRI-guided RBD overcomes some of the technical limitations inherent to the use of straight cannulas and standard stereotactic targeting. This platform technology could have a major impact on the clinical translation of a wide range of cell therapeutics for the treatment of many neurological diseases.

Hypohaptoglobinemia associated with familial epilepsy.

In select kindreds afflicted with familial idiopathic epilepsy, most individuals suffering seizures also have low levels of the plasma hemoglobin-binding protein, haptoglobin. This hypohaptoglobinemia may be causally associated with a tendency to develop epilepsy. Our experimental results indicate that artificially-induced hypohaptoglobinemia in mice causes retarded clearance of free hemoglobin from the central nervous system, and that such free hemoglobin may engender the peroxidation of brain lipids. We hypothesize that hypohaptoglobinemia, either inherited, or acquired via traumatic processes, may prevent efficient clearance of interstitial hemoglobin from the central nervous system, thereby predisposing these people to encephalic inflammation and the appearance of seizure disorders.

The role of excitatory amino acids and NMDA receptors in traumatic brain injury.

Brain injury induced by fluid percussion in rats caused a marked elevation in extracellular glutamate and aspartate adjacent to the trauma site. This increase in excitatory amino acids was related to the severity of the injury and was associated with a reduction in cellular bioenergetic state and intracellular free magnesium. Treatment with the noncompetitive N-methyl-D-aspartate (NMDA) antagonist dextrophan or the competitive antagonist 3-(2-carboxypiperazin-4-yl)propyl-1-phosphonic acid limited the resultant neurological dysfunction; dextrorphan treatment also improved the bioenergetic state after trauma and increased the intracellular free magnesium. Thus, excitatory amino acids contribute to delayed tissue damage after brain trauma; NMDA antagonists may be of benefit in treating acute head injury.

Hemoglobin potentiates central nervous system damage.

Iron and iron compounds--including mammalian hemoglobins--catalyze hydroxyl radical production and lipid peroxidation. To determine whether hemoglobin-mediated lipid peroxidation might be important in hemorrhagic injuries to the central nervous system (CNS), we studied the effects of purified hemoglobin on CNS homogenates and injected hemoglobin into the spinal cords of anesthetized cats. Hemoglobin markedly inhibits Na/K ATPase activity in CNS homogenates and spinal cords of living cats. Hemoglobin also catalyzes substantial peroxidation of CNS lipids. Importantly, the potent iron chelator, desferrioxamine, blocks these adverse effects of hemoglobin, both in vitro and in vivo. Because desferrioxamine is not known to interact with heme iron, these results indicate that free iron, derived from hemoglobin, is the proximate toxic species. Overall, our data suggest that hemoglobin, released from red cells after trauma, can promote tissue injury through iron-dependent mechanisms. Suppression of this damage by desferrioxamine suggests a rational therapeutic approach to management of trauma-induced CNS injury.

Phosphorylation of lens membranes with a cyclic AMP-dependent protein kinase purified from the bovine lens.

We report the phosphorylation of lens membranes with a cAMP-dependent protein kinase isolated from bovine lenses. The holoenzyme was eluted from DEAE agarose at less than 100 mM NaCl and from gel filtration columns with a relative molecular weight of 180 000. The regulatory subunit was identified with the affinity label 8-azido-[32P]cAMP. Four focusing variants with relative molecular weights of 49 000 were seen on two-dimensional gels. The catalytic subunit was purified approx. 5000-fold and migrated at 42 000 Mr on SDS gels. Based on these observations, the enzyme is classified as a Type I cAMP-dependent protein kinase. Purified lens plasma membranes were incubated with the holoenzyme or its catalytic subunit in the presence of 32P-labeled ATP. Several membrane proteins, including the major lens membrane polypeptide, MP26, were shown to be substrates for the kinase in this reaction. MP26 appears to be the major component of intercellular junctions in the lens. Studies with protease treatments on labeled membranes appeared to localize the phosphorylation sites to the cytoplasmic side of the membrane.

Bacterial endotoxin (lipopolysaccharide) stimulates the rate of iron oxidation.

Bacterial endotoxin (lipopolysaccharide) has affinity for a number of cations, including iron. Previous investigations have demonstrated that lipopolysaccharide can affect the oxidation rate of iron; heme-bound ferrous iron in hemoglobin is oxidized to ferric iron when hemoglobin binds lipopolysaccharide. In the present study, we directly examined the interaction between lipopolysaccharide and iron. Lipopolysaccharide caused a concentration-dependent increase in the rate of iron oxidation, with up to a 23-fold increase in oxidation in the presence of 200 microg/ml Escherichia coli lipopolysaccharide. This effect was seen both with several carbohydrate-rich smooth lipopolysaccharides and also with carbohydrate-poor rough lipopolysaccharide. Extensively deacylated rough lipopolysaccharide had no effect, suggesting a role of the fatty acid components of lipopolysaccharide in this process. Purified lipid A produced inconsistent results: some preparations stimulated iron oxidation and others did not. A series of sugars, starches and a preparation of purified O-chain polysaccharide (the carbohydrate portion of the lipopolysaccharide macro-molecule) had no effect on the rate of iron oxidation, whereas phospholipid-enriched brain tissue extracts (similar to the lipid A component of lipopolysaccharide) stimulated oxidation. We conclude that the lipid moiety of bacterial lipopolysaccharide is responsible for the stimulation of iron oxidation. This process may contribute to the ability of lipopolysaccharide to cause oxidation of heme-bound iron in hemoglobin.

Heme oxygenase-1 is induced in glia throughout brain by subarachnoid hemoglobin.

The heme oxygenase-1 gene, HO-1, induced by heme, ischemia, and heat shock, metabolizes heme to biliverdin, free iron, and carbon monoxide. Though the distribution of HO-1 has been described in normal rat brain, little is known about how extracellular heme proteins in the subarachnoid space distribute in brain. To address this issue, hemoglobin was injected into the cisterna magna of adult rats. Expression of HO-1 in these animals was compared with saline-injected, BSA-injected, and uninjected controls. Western blot analysis showed that 24 hours after injection oxyhemoglobin increased HO-1 levels approximately four- to fivefold in all brain regions studied compared with saline-injected and BSA-injected controls. In the brain, HO-1 immunoreactivity was evident at 4 hours and peaked at 24 hours after oxyhemoglobin injections, returning to control levels by 4 to 8 days. This HO-1 induction was detected mainly in cells with small, rounded somas bearing two to four truncated processes, a morphology consistent with that of microglia. These cells were double-stained with the microglial marker, OX42, in every brain region examined. It is proposed that subarachnoid hemoglobin may be taken up into microglia wherein heme induces HO-1.

Expression of a specific protein in spontaneously metastatic fibrosarcoma cell lines and its enhanced synthesis by growth on laminin or fibronectin.

A panel of tumor cell lines and clones was generated by selecting for different metastatic capacity in 3AM fibrosarcoma cells. These cell lines were exposed to substrata of various purified extracellular matrix (ECM) proteins and labeled in culture with [35S]methionine. Following analysis by two-dimensional polyacrylamide gel electrophoresis the profiles of total cellular proteins produced by the cell lines displaying different phenotypes were examined. The presence of a specific protein, p54, was related to the cellular metastatic potential, and the synthesis of p54 was influenced by growth on extracellular matrix components. The amount of p54 was minimal and non-inducible in tumor cell lines exhibiting two different phenotypes: (1) experimentally metastatic (EM) and (2) transformed, non-tumorigenic (NTT) cell types. In contrast, all of the five different cell lines capable of both spontaneous and experimental metastasis (SEM), produced p54 either constitutively or through induction by growth on ECM protein substrata of either laminin of fibronectin, but not collagen type IV. These data suggest that the p54 protein may be a unique biochemical marker associated with spontaneous metastatic cell types.

Reduced plasma haptoglobin and urinary taurine in familial seizures identified through the multisib strategy.

The multisib (MS) sampling strategy was used for detecting possible genetic influences on a complex and heterogeneous disorder. The MS strategy increases the likelihood of selecting pedigrees for single genetic factors and allows the efficient analysis of data. The collection of complete pedigrees will procure additional data, but at a large marginal expense. The MS ascertainment procedure was applied to seizure disorders by examination of taurine excretion levels and by conducting two-dimensional gel electrophoresis of plasma proteins. Reduced taurine excretion (proposed to be genetically controlled) was found to associate with seizures, particularly in those individuals with a generalized spike and wave (GSW) EEg, or in families with a GSW-seizure history. Examination of two-dimensional gels showed hypohaptoglobinemia in several seizure patients [Panter et al, 1984]. The frequency of hypohaptoglobinemia is greatly increased in familial seizure cases, and may also be genetically controlled. Thus the MS strategy has proven successful in identifying kindreds in which specific physiological alterations may contribute toward the complex phenotype of seizures.

Anti-oxidants prevent focal rat brain injury as assessed by induction of heat shock proteins (HSP70, HO-1/HSP32, HSP47) following subarachnoid injections of lysed blood.

The initial aim of this study was to determine if the HSP70 (the main inducible heat shock protein), HO-1 (heme oxygenase-1, HSP32) and HSP47 (a collagen chaperone) stress proteins were induced in the same focal regions of rat brain following experimental subarachnoid hemorrhage (SAH). The next objective was to determine whether anti-oxidants prevented the stress gene expression in the focal regions. Lysed blood (150 microliter) was injected into the subarachnoid space of adult, female Sprague-Dawley rats via the cisterna magna. Animals were sacrificed 24 h later. Immunocytochemistry showed focal regions of stress gene induction in most animals (13/21), HSP70 and HO-1 proteins being expressed in neurons, microglia and astrocytes and HSP47 being expressed in microglia. Co-induction of the same three stress proteins was observed in focal areas in the striatum and cerebellum as well. In the 13 animals with focal regions of stress gene induction there were 8.1+/-1.8 foci in cortex, 5.5+/-0.9 foci in striatum, and 11.7+/-7.3 foci in cerebellum in the brain of each animal. The focal regions of stress gene induction varied in size from 200 micrometer to 7 mm in diameter. Systemic administration of the tirilazad-like anti-oxidants U101033E (n=8) and U74389G (n=7) completely blocked stress protein induction in focal brain regions normally produced by cisternal injections of lysed blood. There were fewer drug treated animals (0/15) with focal areas of stress gene induction compared to non-drug (13/21) treated animals following the cisternal lysed blood injections (p<0.01 using Fisher's probability test). This study shows that anti-oxidants prevent focal regions of injury as assessed by heat shock protein expression in a rat model of SAH.

Hemoglobin potentiates excitotoxic injury in cortical cell culture.

Excessive activation of glutamate receptors may contribute to neuronal loss after a traumatic or ischemic central nervous system insult. Such injuries are often associated with hemorrhage and extravasation of hemoglobin, a prooxidant and putative neurotoxin. In this study, we investigated the effect of nontoxic concentrations of hemoglobin on the neurotoxicity of the synthetic glutamate receptor agonists NMDA, AMPA, and kainate in primary murine cortical cultures. Continuous exposure to each excitotoxin alone for 24-28 h produced concentration-dependent neuronal death (EC(50) about 12 mu M for AMP(+)A, 50 mu M for kainate, and 12 mu M for NMDA). Hemoglobin 0.25-1.0 mu M consistently potentiated the neurotoxicity of low concentrations of AMPA and kainate, increasing neuronal loss by about 150% at 6 mu M AMPA and by about 90% at 30 mu M kainate. This effect was attenuated by the iron chelator deferoxamine and the alpha-tocopherol analogue trolox. Hemoglobin coexposure had less impact on slowly triggered NMDA neurotoxicity, significantly increasing neuronal death only at agonist concentrations that alone produced little or no injury. Hemoglobin pretreatment had no effect on the rapidly triggered excitotoxicity induced by brief exposure to high concentrations of NMDA. These results suggest that hemoglobin may contribute to neuronal loss after CNS hemorrhage by exacerbating excitotoxicity. At moderate levels of agonist exposure, this effect may be somewhat selective for the AMPA/kainate component of injury.

Dextran-coupled deferoxamine improves outcome in a murine model of head injury.

Tissue damage involving oxygen-derived free radicals may be greatly exacerbated by free, reactive iron, which acts as a catalyst in oxidative reactions. The effects of free iron can be attenuated by the administration of deferoxamine (DFO), an iron chelator. However, DFO has limited therapeutic utility because it has a short plasma half-life (approximately 5.5 min in mice) and produces profound hypotension upon intravenous infusion. These negative attributes have been circumvented by the covalent attachment of DFO to large polymers, such as dextran or hydroxyethyl starch. The ability of the dextran-conjugated DFO (DEX-DFO) to inhibit iron-catalyzed reactions with lipids was compared to that of the native molecule in an in vitro model of CNS lipid degradation in the presence of 200 microM ferrous iron. There was no difference between native DFO and the modified form. Modified and unmodified DFO were also compared for therapeutic efficacy in a murine model of head injury. Using a previously described "grip test" as a measure of neurologic impairment following injury, DEX-DFO, native DFO, and dextran were administered intravenously 3-5 min after injury. Dextran-DFO significantly decreased the incidence of severe neurologic impairment at dosage levels of 0.1 (n = 92), 1.0 (n = 76), and 10.0 (n = 80) mg/kg. Administration of native DFO or dextran had no effect at the same dosages and concentrations. These results suggest that the murine model of head injury contains a significant iron-dependent component that should be assessed in other models of neural injury.

Traumatic neuronal injury in cortical cell culture is attenuated by 21-aminosteroids.

The effect of the 21-aminosteroids U74500A and U74389F, alone and in combination with the NMDA receptor antagonist MK-801, on traumatic neuronal injury was quantitatively assessed in murine neocortical cell cultures. Consistent with prior observations, a mechanical insult to the culture monolayer resulted in widespread neuronal death over the following 24 h. Treatment with either U74500A or U74389F provided moderate protection, reducing neuronal death as measured by lactate dehydrogenase release by 25-50%. This effect was most consistent when these agents were preincubated for 2 h prior to injury. Combined treatment with a 21-aminosteroid plus the NMDA receptor antagonist MK-801 reduced injury more than either drug alone. Approximately 40% of the neuronal death occurring in the presence of MK-801 was blocked by concomitant treatment with 10 microM U74500A or U74389F. These results suggest that free radicals may contribute to cell death in this in vitro model of traumatic neuronal injury.

Ultrastructure of excitotoxic neuronal death in murine cortical culture.

Ischemic and traumatic brain injury are likely to involve neuronal injury triggered by glutamate receptor overactivation. Although excitotoxic neuronal injury has been widely studied in the setting of primary culture, the extent to which these in vitro injury paradigms resemble in vivo ischemic injury morphologically has not previously been well studied. We studied glutamate receptor mediated neuronal death by transmission electron microscopy and light microscopy. Morphologic characteristics of neurons injured by 10 min exposure to 500 microM glutamate include rapid swelling of mitochondria and endoplasmic reticulum, and cytoplasmic and nuclear lucency. Both alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid and kainic acid caused vacuolation, dilatation of the endoplasmic reticulum, cytoplasmic condensation and random condensation of chromatin with preserved mitochondria. None of these injuries was ameliorated by cycloheximide or actinomycin D; all were significantly lessened by aurintricarboxylic acid. Gel electrophoresis showed no increase in DNA fragmentation over control. The morphologic changes seen with alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid and kainate are distinct from the changes induced by glutamate. Excitotoxic injury in this system due to high concentrations of glutamate resembles necrosis while the other agonists produce a different form of cell death which is neither necrosis nor apoptosis.

Induction of heme oxygenase-1 (HO-1) in glia after traumatic brain injury.

In this study we examined the induction of heme oxygenase-1 (HO-1) in glia in the traumatized rat brain. HO-1 was immunolocalized in fixed sections of brain 3 h to 5 days after injury. Induction of this enzyme in astrocytes, microglia/macrophages, and oligodendrocytes was evaluated using immunofluorescent double labeling with monoclonal antibodies to glial fibrillary acidic protein, complement C3bi receptor, and myelin basic protein. Induction of HO-1 was apparent in the injured hemisphere and cerebellum as early as 24 h postinjury. The protein was likewise noted in similar regions of the brain at 72 h postinjury but appeared to be more widespread in its distribution. At 5 days postinjury, there was a notable decline in the degree of immunostaining for HO-1. HO-1 was typically induced in astrocytes in the cerebral cortex at the site of impact, in the deep cortical layers adjacent to the hemorrhagic lesions, and in the hippocampus. HO-1 was induced in Bergmann glia in the vermis of cerebellum. In addition, HO-1 was also induced in microglia/macrophages scattered throughout the ipsilateral cerebral cortex, cerebellum and subarachnoid space. These findings demonstrate prolonged glial induction of HO-1 in the traumatized brain. Such a response may reflect a protective role of these cells against secondary insults including oxidative stress.

Heme-oxygenase-1 induction in glia throughout rat brain following experimental subarachnoid hemorrhage.

The heme released following subarachnoid hemorrhage is metabolized by heme-oxygenase (HO) to biliverdin and carbon monoxide (CO) with the release of iron. The HO reaction is important since heme may contribute to vasospasm and increase oxidative stress in cells. HO is comprised of at least two isozymes, HO-2 and HO-1. HO-1, also known as heat shock protein HSP32, is inducible by many factors including heme and heat shock. HO-2 does not respond to these stresses. To begin to examine HO activity following subarachnoid hemorrhage (SAH), the expression of HO-1 and HO-2 was investigated after experimental SAH in adult rats. Immunocytochemistry for HO-1, HO-2 and HSP70 proteins was performed at 1, 2, 3 and 4 days after injections of lysed blood, whole blood, oxyhemoglobin and saline into the cisterna magna. A large increase in HO-1 immunoreactivity was seen in cells throughout brain following injections of lysed blood, whole blood, and oxyhemoglobin but not saline. Lysed blood, whole blood and oxyhemoglobin induced HO-1 in all of the cortex, hippocampus, striatum, thalamus, forebrain white matter and in cerebellar cortex. HO-1 immunoreactivity was greatest in those regions adjacent to the basal subarachnoid cisterns where blood and oxyhemoglobin concentrations were likely highest. Double immunofluorescence studies showed the HO-1 positive cells to be predominately microglia, though HO-1 was induced in some astrocytes. HO-1 expression resolved by 48 h. HO-2 immunoreactivity was abundant but did not change following injections of blood. A generalized induction of HSP70 heat shock protein was not observed following injections of lysed blood, whole blood, oxyhemoglobin, or saline. These results suggest that HO-1 is induced in microglia throughout rat brain as a general, parenchymal response to the presence of oxyhemoglobin in the subarachnoid space and not as a stress response. This microglial HO-1 response could be protective against the lipid peroxidation and vasospasm induced by hemoglobin, by increasing heme clearance and iron sequestration, and enhancing the production of the antioxidant bilirubin.

Induction of heme oxygenase-1 after hyperosmotic opening of the blood-brain barrier.

The induction of the stress protein heme oxygenase-1 (HO-1) was studied in the rat brain after intracarotid administration of hyperosmolar mannitol. HO-1 was immunolocalized in fixed sections of brain 24 h to 7 days after injection. Immunoglobulin G (IgG) was immunolocalized in adjacent sections to demonstrate areas of breakdown of the blood-brain barrier. Induction of HO-1 was also evaluated by Western immunoblots, performed at 24 h after the insult. Immunofluorescent double labelling with monoclonal antibodies to HO-1 and either glial fibrillary acidic protein or the complement C3bi receptor was used to determine if glia/macrophages expressed HO-1. There was pronounced, widespread induction of HO-1 in the ipsilateral hemisphere and cerebellum by 24 h both by immunocytochemistry and by Western blots. This induction was markedly attenuated at later times. HO-1 was induced in astrocytes and microglia/macrophages in the ipsilateral hemisphere. In addition, the protein was induced in Bergmann glia and scattered microglia/macrophages in the cerebellum. The mechanism of induction of HO-1 in glia after opening of the blood-brain barrier could include exposure to heme proteins, denatured proteins and other plasma constituents known to induce HO-1. This glial induction may reflect a protective response of these cells.

Neurotoxicity of hemoglobin in cortical cell culture.

Hemoglobin (Hb) has been demonstrated to be neurotoxic when injected into the cerebral cortex in vivo. However, associated systemic factors such as ischemia and epileptogenesis have limited investigations of Hb toxicity in the intact central nervous system (CNS). In this study, the neurotoxicity of human Hb was assessed in mixed neuronal and glial neocortical cell cultures derived from fetal mice. Exposure of cultures to Hb for 24-28 h produced widespread and concentration-dependent neuronal death (EC50 1-2.5 microM), without injuring glia. Brief exposures (1-2 h) were not toxic. Neuronal death was completely blocked by the 21-aminosteroid U74500A, the antioxidant Trolox, and the ferric iron chelator deferoxamine. The results of these experiments suggest that, in this system, Hb is a potent neurotoxin, and that Hb neurotoxicity may contribute to secondary injury processes after trauma and intracranial hemorrhage.

Pretreatment with NMDA antagonists limits release of excitatory amino acids following traumatic brain injury.

After central nervous system (CNS) trauma, there are marked elevations in the extracellular levels of excitatory amino acids (EAA), which are believed to contribute to delayed tissue damage. Administration of N-methyl-D-aspartate (NMDA) receptor antagonists reduces injury severity after brain or spinal cord trauma, presumably by blocking the postsynaptic NMDA receptor. In the present studies, levels of extracellular amino acids were monitored by microdialysis during, and after, a moderately severe fluid-percussion brain injury to rats. Pretreatment (15 min prior to injury) with the non-competitive NMDA antagonist dextrorphan or the competitive NMDA antagonist CGS 19755 significantly attenuated the post-traumatic increase in extracellular glutamate. Pretreatment with dextrorphan attenuated the post-traumatic increase in extracellular levels of aspartate; although these differences did not reach significance when examined as absolute values, they were significant when analyzed as percent increase over pre-trauma baseline levels. These results are consistent with recent experiments and suggest that NMDA antagonists may limit the release of glutamate and aspartate after trauma through a presynaptic mechanism.

NMDA neurotoxicity in murine cortical cell cultures is not attenuated by hemoglobin or inhibition of nitric oxide synthesis.

The role of nitric oxide in N-methyl-D-aspartate (NMDA) neurotoxicity was investigated in murine cortical cell cultures. Exposure of cultures to 300 microM NMDA for 5 min resulted in death of 50-80% of neurons over the subsequent 24 h. This injury was not attenuated by hemoglobin, the nitric oxide synthase (NOS) inhibitors NG monomethyl-L-arginine (MMA) or N omega-nitro-L-arginine (NA), or L-arginine depletion. Hemoglobin and NOS inhibitors consistently prevented the increase in cyclic guanosine monophosphate (cGMP) seen after NMDA exposure. These results suggest that NMDA neurotoxicity in this cell culture system is mediated, at least in part, by mechanisms other than NOS activation.