IL-1ß and HMGB1 are anti-neurogenic to endogenous neural stem cells in the sclerotic epileptic human hippocampus.

BACKGROUND: The dentate gyrus exhibits life-long neurogenesis of granule-cell neurons, supporting hippocampal dependent learning and memory. Both temporal lobe epilepsy patients and animal models frequently have hippocampal-dependent learning and memory difficulties and show evidence of reduced neurogenesis. Animal and human temporal lobe epilepsy studies have also shown strong innate immune system activation, which in animal models reduces hippocampal neurogenesis. We sought to determine if and how neuroinflammation signals reduced neurogenesis in the epileptic human hippocampus and its potential reversibility. METHODS: We isolated endogenous neural stem cells from surgically resected hippocampal tissue in 15 patients with unilateral hippocampal sclerosis. We examined resultant neurogenesis after growing them either as neurospheres in an ideal environment, in 3D cultures which preserved the inflammatory microenvironment and/or in 2D cultures which mimicked it. RESULTS: 3D human hippocampal cultures largely replicated the cellular composition and inflammatory environment of the epileptic hippocampus. The microenvironment of sclerotic human epileptic hippocampal tissue is strongly anti-neurogenic, with sustained release of the proinflammatory proteins HMGB1 and IL-1ß. IL-1ß and HMGB1 significantly reduce human hippocampal neurogenesis and blockade of their IL-1R and TLR 2/4 receptors by IL1Ra and Box-A respectively, significantly restores neurogenesis in 2D and 3D culture. CONCLUSION: Our results demonstrate a HMGB1 and IL-1ß-mediated environmental anti-neurogenic effect in human TLE, identifying both the IL-1R and TLR 2/4 receptors as potential drug targets for restoring human hippocampal neurogenesis in temporal lobe epilepsy.

In vitro CNS tissue analogues formed by self-organisation of reaggregated post-natal brain tissue.

In this paper, we report the characterization of 'Hi-Spot' cultures formed by the re-aggregation of dissociated postnatal CNS tissue grown at an air-liquid interface. This produces a self-organised, dense, organotypic cellular network. Western blot, immunohistochemical, viral transfection and electron microscopy analyses reveal neuronal and glial populations, and the development of a synaptic network. Multi-electrode array recordings show synaptically driven network activity that develops through time from single unit spiking activity to global network bursting events. This activity is blocked by tetanus toxin and modified by antagonists of glutamatergic and GABAergic receptors suggesting tonic activity of excitatory and inhibitory synaptic signaling. The tissue-like properties of these cultures has been further demonstrated by their relative insensitivity to glutamate toxicity. Exposure to millimolar concentrations of glutamate for hours is necessary to produce significant excitotoxic neuronal death, as in vivo. We conclude that 'Hi-Spots' are biological analogues of CNS tissue at a level of complexity that allows for detailed functional analyses of emergent neuronal network properties.

The long-term survival of in vitro engineered nervous tissue derived from the specific neural differentiation of mouse embryonic stem cells.

Embryonic stem cells (ESCs) offer attractive prospective as potential source of neurons for cell replacement therapy in human neurodegenerative diseases. Besides, ESCs neural differentiation enables in vitro tissue engineering for fundamental research and drug discovery aimed at the nervous system. We have established stable and long-term three-dimensional (3D) culture conditions which can be used to model long latency and complex neurodegenerative diseases. Mouse ESCs-derived neural progenitor cells generated by MS5 stromal cells induction, result in strictly neural 3D cultures of about 120-mum thick, whose cells expressed mature neuronal, astrocytes and myelin markers. Neurons were from the glutamatergic and gabaergic lineages. This nervous tissue was spatially organized in specific layers resembling brain sub-ependymal (SE) nervous tissue, and was maintained in vitro for at least 3.5 months with great stability. Electron microscopy showed the presence of mature synapses and myelinated axons, suggesting functional maturation. Electrophysiological activity revealed biological signals involving action potential propagation along neuronal fibres and synaptic-like release of neurotransmitters. The rapid development and stabilization of this 3D cultures model result in an abundant and long-lasting production that is compatible with multiple and productive investigations for neurodegenerative diseases modeling, drug and toxicology screening, stress and aging research.

Sodium selenate specifically activates PP2A phosphatase, dephosphorylates tau and reverses memory deficits in an Alzheimer's disease model.

Neurofibrillary tangles composed of abnormally hyperphosphorylated tau protein are a hallmark of Alzheimer's disease (AD) and related tauopathies. Tau hyperphosphorylation is thought to promote aggregation with subsequent tangle formation. Reducing tau phosphorylation by boosting the activity of the key phosphatase/s that mediate dephosphorylation of tau could be a viable clinical strategy in AD. One of the key phosphatases implicated in regulating tau protein phosphorylation is the serine-threonine phosphatase PP2A. We have determined that sodium selenate can act as a specific agonist for PP2A, significantly boosting phosphatase activity. Acute treatment of either neuroblastoma cells or normal aged mice with sodium selenate rapidly reduced tau protein phosphorylation. Sodium selenate-treated transgenic TAU441 mice had significantly lower levels of phospho- and total tau levels in the hippocampus and amygdala compared with controls and exhibited significantly improved spatial learning and memory on the Morris Water Maze task. Sodium selenate is a specific activator of PP2A with excellent oral bioavailability, and favourable central nervous system penetrating properties. Clinical studies in patients with AD are envisaged in the near future.

Stretch-induced injury in organotypic hippocampal slice cultures reproduces in vivo post-traumatic neurodegeneration: role of glutamate receptors and voltage-dependent calcium channels.

The relationship between an initial mechanical event causing brain tissue deformation and delayed neurodegeneration in vivo is complex because of the multiplicity of factors involved. We have used a simplified brain surrogate based on rat hippocampal slices grown on deformable silicone membranes to study stretch-induced traumatic brain injury. Traumatic injury was induced by stretching the culture substrate, and the biological response characterized after 4 days. Morphological abnormalities consistent with traumatic injury in humans were widely observed in injured cultures. Synaptic function was significantly reduced after a severe injury. The N-methyl-D-aspartate (NMDA) receptor antagonist MK-801 attenuated neuronal damage, prevented loss of microtubule-associated protein 2 immunoreactivity and attenuated reduction of synaptic function. In contrast, the NMDA receptor antagonists 3-[(R)-2-carboxypiperazin-4-yl]-propyl-1-phosphonic acid (CPP) and GYKI53655, were neuroprotective in a moderate but not a severe injury paradigm. Nifedipine, an L-type voltage-dependent calcium channel antagonist was protective only after a moderate injury, whereas omega-conotoxin attenuated damage following severe injury. These results indicate that the mechanism of damage following stretch injury is complex and varies depending on the severity of the insult. In conclusion, the pharmacological, morphological and electrophysiological responses of organotypic hippocampal slice cultures to stretch injury were similar to those observed in vivo. Our model provides an alternative to animal testing for understanding the mechanisms of post-traumatic delayed cell death and could be used as a high-content screen to discover neuroprotective compounds before advancing to in vivo models.

An in vitro model of traumatic brain injury utilising two-dimensional stretch of organotypic hippocampal slice cultures.

Traumatic brain injury (TBI) is caused by rapid deformation of the brain, resulting in a cascade of pathological events and ultimately neurodegeneration. Understanding how the biomechanics of brain deformation leads to tissue damage remains a considerable challenge. We have developed an in vitro model of TBI utilising organotypic hippocampal slice cultures on deformable silicone membranes, and an injury device, which generates tissue deformation through stretching the silicone substrate. Our injury device controls the biomechanical parameters of the stretch via feedback control, resulting in a reproducible and equi-biaxial deformation stimulus. Organotypic cultures remain well adhered to the membrane during deformation, so that tissue strain is 93 and 86% of the membrane strain in the x- and y-axis, respectively. Cell damage following injury is positively correlated with strain. In conclusion, we have developed a unique in vitro model to study the effects of mechanical stimuli within a complex cellular environment that mimics the in vivo environment. We believe this model could be a powerful tool to study the acute phases of TBI and the induced cell degeneration could provide a good platform for the development of potential therapeutic approaches and may be a useful in vitro alternative to animal models of TBI.

Temporal development of hippocampal cell death is dependent on tissue strain but not strain rate.

Deformation of brain tissue in response to mechanical loading of the head is the root-cause of traumatic brain injury (TBI). Even below ultimate failure limits, deformation activates pathophysiological cascades resulting in delayed cell death. Injury response of soft tissues, such as the chest and spinal cord, is dependent on the product of deformation and velocity, a parameter termed the viscous criterion. We set out to test if hippocampal cell death could be predicted by a similar combination of strain and strain rate and if the viscous criterion was valid for hippocampus. Quantitative prediction of the brain's biological response to mechanical stimuli is difficult to achieve in animal models of TBI, so we utilized an in vitro model of TBI based on hippocampal slice cultures. We quantified the temporal development of cell death after precisely controlled deformations for 30 combinations of strain (0.05-0.50) and strain rate (0.1-50s(-1)) relevant to TBI. Loading conditions for a subset of cultures were verified by analysis of high-speed video. Cell death was found to be significantly dependent on time-post injury, on strain magnitude, and to a lesser extent, on anatomical region by a repeated-measures, three-way ANOVA. The responses of the CA1 and CA3 regions of the hippocampus were not statistically different in contrast to some in vivo TBI studies. Surprisingly, cell death was not dependent on strain rate leading us to conclude that the viscous criterion is not a valid predictor for hippocampal tissue injury. Given the large data set and extensive combinations of biomechanical parameters, predictive mathematical functions relating independent variables (strain, region, and time post-injury) to the resultant cell death were defined. These functions can be used as tolerance criteria to equip finite element models of TBI with the added capability to predict biological consequences.

Time window and pharmacological characterisation of kainate-mediated preconditioning in organotypic rat hippocampal slice cultures.

Tolerance to normally neurotoxic insults can be induced by prior a preconditioning exposure to a sublethal insult. Kainate toxicity can be attenuated by prior exposure to very low concentrations of kainate both in vivo and in vitro. Using organotypic hippocampal slice cultures from rats we have shown that 5 microM kainate induces a selective lesion in the CA3 region and this can be significantly attenuated by 1 microM kainate administered 1-5 days earlier. The time window for this effect was affected by the length of time in culture, and preconditioning was blocked by NBQX but not the selective AMPA receptor antagonist GYKI53655. These data demonstrate a role for kainate receptors in preconditioning for the first time and show that organotypic cultures can be used as a model to investigate long-term preconditioning mechanisms.

Intraischaemic hypothermia reduces free radical production and protects against ischaemic insults in cultured hippocampal slices.

Hypothermia has been demonstrated to be an effective neuroprotective strategy in a number of models of ischaemic and excitotoxic neurodegeneration in vitro and in vivo. Reduced glutamate release and free radical production have been postulated as potential mechanisms underlying this effect but no definitive mechanism has yet been reported. In the current study, we have used oxygen-glucose deprivation in organotypic hippocampal slice cultures as an in vitro model of cerebral ischaemia. When assessed by propidium iodide fluorescence, reducing the temperature during oxygen-glucose deprivation to 31-33 degrees C was significantly neuroprotective but this effect was lost if the initiation of hypothermia was delayed until the post-insult recovery period. The neuroprotective effects of hypothermia were associated with a significant decrease in both nitric oxide production, as assessed by 3-amino-4-aminomethyl-2',7'-difluorofluorescein fluorescence, and superoxide formation. Further, hypothermia significantly attenuated NMDA-induced nitric oxide formation in the absence of hypoxia/hypoglycaemia. We conclude that the neuroprotective effects of hypothermia are mediated through a reduction in nitric oxide and superoxide formation and that this effect is likely to be downstream of NMDA receptor activation.

Characterisation of a novel class of polyamine-based neuroprotective compounds.

Prolonged cerebral ischaemia initiates complex intra- and inter-cellular signalling cascades ultimately resulting in neuronal death. Well-characterised mediators of ischaemic cell death are glutamate, free radicals and nitric oxide. Many drugs that block these mechanisms are neuroprotective in vitro, but have unfavourable side-effect profiles in man. We have recently demonstrated that the compound L-arginyl-3,4-spermidine (L-Arg3,4) is neuroprotective in vitro through an interaction with several of these mechanisms, and prevents ischaemic neurodegeneration in vivo with no gross side effects. In this study, we have used solid-phase combinatorial chemistry, to synthesise a number of analogues of L-Arg3,4, and investigate the structure-activity relationship using an in vitro, organotypic hippocampal slice culture model of cerebral ischaemia. A number of molecular features were identified which were essential for the neuroprotective activity including the requirement for a positive charge and an amino acid in the L-configuration. Relatively minor alterations to both the terminal arginine and polyamine moieties significantly attenuated neuroprotective efficacy. Our data implies that these compounds are neuroprotective through a currently undefined mechanism rather than non-specific ionic interactions described previously for other polyamine-containing compounds.

7-Hydroxylated epiandrosterone (7-OH-EPIA) reduces ischaemia-induced neuronal damage both in vivo and in vitro.

Recent evidence suggests that steroids such as oestradiol reduce ischaemia-induced neurodegeneration in both in vitro and in vivo models. A cytochrome P450 enzyme termed cyp7b that 7-hydroxylates many steroids is expressed at high levels in brain, although the role of 7-hydroxylated steroids is unknown. We have tested the hypothesis that the steroid-mediated neuroprotection is dependent on the formation of 7-hydroxy metabolites. Organotypic hippocampal slice cultures were prepared from Wistar rat pups and maintained in vitro for 14 days. Cultures were then exposed to 3 h hypoxia and neuronal damage assessed 24 h later using propidium iodide fluorescence as a marker of cell damage. Neurodegeneration occurred primarily in the CA1 pyramidal cell layer. The steroids oestradiol, dehydroepiandrosterone and epiandrosterone (EPIA) were devoid of neuroprotective efficacy when present at 100 nM pre-, during and post-hypoxia. The 7-hydroxy metabolites of EPIA, 7alpha-OH-EPIA and 7beta-OH-EPIA significantly reduced neurotoxicity at 100 nM and 10 nM. 7beta-OH-EPIA was also neuroprotective in two in vivo rat models of cerebral ischaemia: 0.1 mg/kg 7beta-OH-EPIA significantly reduced hippocampal cell loss in a model of global forebrain ischaemia, whereas 0.03 mg/kg was neuroprotective in a model of focal ischaemia even when administration was delayed until 6 h after the onset of ischaemia. Taken together, these data demonstrate that 7-hydroxylation of steroids confers neuroprotective efficacy, and that 7beta-OH-epiandrosterone represents a novel class of neuroprotective compounds with potential for use in acute neurodegenerative diseases.

Neuropeptide Y is neuroproliferative for post-natal hippocampal precursor cells.

New neurones are produced in the adult hippocampus throughout life and are necessary for certain types of hippocampal learning. Little, however, is known about the control of hippocampal neurogenesis. We used primary hippocampal cultures from early post-natal rats and neuropeptide Y Y1 receptor knockout mice as well as selective neuropeptide Y receptor antagonists and agonists to demonstrate that neuropeptide Y is proliferative for nestin-positive, sphere-forming hippocampal precursor cells and beta-tubulin-positive neuroblasts and that the neuroproliferative effect of neuropeptide Y is mediated via its Y1 receptor. Immunohistochemistry confirmed Y1 receptor staining on both nestin-positive cells and beta-tubulin-positive cells in culture and short pulse 5-bromo-2-deoxyuridine studies demonstrated that neuropeptide Y has a proliferative effect on both cell types. These studies suggest that the proliferation of hippocampal neuroblasts and precursor cells is increased by neuropeptide Y and, therefore, that hippocampal learning and memory may be modulated by neuropeptide Y-releasing interneurones.

Synthesis and evaluation of fluorescent probes for the detection of calpain activity.

Two new probes for the detection of calpain I activity based on fluorescence resonance energy transfer technology have been synthesized and evaluated. The probes incorporated the cleavage site present in alpha-spectrin, a naturally occurring substrate of calpain I. The design of the internally quenched substrates is such that the calpain-sensitive bond of the peptides (between the Tyr-Gly residues) is located centrally between the donor and the quencher chromophores. The calpain assay protocol is capable of detecting enzymatic activity in the nanomolar region.

A tissue level tolerance criterion for living brain developed with an in vitro model of traumatic mechanical loading.

Traumatic brain injury (TBI) is caused by brain deformations resulting in the pathophysiological activation of cellular cascades which produce delayed cell damage and death. Understanding the consequences of mechanical injuries on living brain tissue continues to be a significant challenge. We have developed a reproducible tissue culture model of TBI which employs organotypic brain slice cultures to study the relationship between mechanical stimuli and the resultant biological response of living brain tissue. The device allows for the independent control of tissue strain (up to 100%) and strain rate (up to 150 s-1) so that tolerance criteria at the tissue level can be developed for the interpretation of computational simulations. The application of texture correlation image analysis algorithms to high speed video of the dynamic deformation allows for the direct calculation of substrate strain and strain rate which was found to be equi-biaxial and independent of radial position. Precisely controlled, mechanical injuries were applied to organotypic hippocampal slice cultures, and resultant cell death was quantified. Cell death was found to be dependent on both strain magnitude and rate and required several days to develop. An immunohistological examination of injured cultures with antibodies to amyloid precursor protein revealed the presence of traumatic axonal injury, suggesting that the model closely replicates in vivo TBI but with advantages gained in vitro. We anticipate that a combined in vitro approach with optical strain mapping will provide a more detailed understanding of the dependence of brain cell injury and death on strain and strain rate.

Lactate and glucose as energy substrates during, and after, oxygen deprivation in rat hippocampal acute and cultured slices.

The effects of raised brain lactate levels on neuronal survival following hypoxia or ischemia is still a source of controversy among basic and clinical scientists. We have sought to address this controversy by studying the effects of glucose and lactate on neuronal survival in acute and cultured hippocampal slices. Following a 1-h hypoxic episode, neuronal survival in cultured hippocampal slices was significantly higher if glucose was present in the medium compared with lactate. However, when the energy substrate during the hypoxic period was glucose and then switched to lactate during the normoxic recovery period, the level of cell damage in the CA1 region of organotypic cultures was significantly improved from 64.3 +/- 2.1 to 74.6 +/- 2.1% compared with cultures receiving glucose during and after hypoxia. Extracellular field potentials recorded from the CA1 region of acute slices were abolished during oxygen deprivation for 20 min, but recovered almost fully to baseline levels with either glucose (82.6 +/- 10.0%) or lactate present in the reperfusion medium (108.1 +/- 8.3%). These results suggest that lactate alone cannot support neuronal survival during oxygen deprivation, but a combination of glucose followed by lactate provides for better neuroprotection than either substrate alone.

L-arginyl-3,4-spermidine is neuroprotective in several in vitro models of neurodegeneration and in vivo ischaemia without suppressing synaptic transmission.

1. Stroke is the third most common cause of death in the world, and there is a clear need to develop new therapeutics for the stroke victim. To address this need, we generated a combinatorial library of polyamine compounds based on sFTX-3.3 toxin from which L-Arginyl-3,4-Spermidine (L-Arg-3,4) emerged as a lead neuroprotective compound. In the present study, we have extended earlier results to examine the compound's neuroprotective actions in greater detail. 2. In an in vitro ischaemia model, L-Arg-3,4 significantly reduced CA1 cell death when administered prior to induction of 60 min of ischaemia as well as when administered immediately after ischaemia. Surprisingly, L-Arg-3,4 continued to prevent cell death significantly when administration was delayed for as long as 60 min after ischaemia. 3. L-Arg-3,4 significantly reduced cell death in excitotoxicity models mediated by glutamate, NMDA, AMPA, or kainate. Unlike glutamate receptor antagonists, 300 microM L-Arg-3,4 did not suppress synaptic transmission as measured by evoked responses in acute hippocampal slices. 4. L-Arg-3,4 provided significant protection, in vitro, in a superoxide mediated injury model and prevented an increase of superoxide production after AMPA or NMDA stimulation. It also decreased nitric oxide production after in vitro ischaemia and NMDA stimulation, but did so without inhibiting nitric oxide synthase directly. 5. Furthermore, L-Arg-3,4 was significantly neuroprotective in an in vivo model of global forebrain ischaemia, without any apparent neurological side-effects. 6. Taken together, these results demonstrate that L-Arg-3,4 is protective in several models of neurodegeneration and may have potential as a new therapeutic compound for the treatment of stroke, trauma, and other neurodegenerative diseases.