1,720,984 research outputs found
Thinking inside the box: to cope with an increasing disease burden, drug discovery needs biologically relevant and predictive testing systems
Full text linkA method for characterising cell death in vitro by combining propidium iodide staining with immunohistochemistry
No full textThe fluorescent exclusion dye propidium iodide (PI) is widely used as a vital dye in tissue culture systems and labels the nucleus in dying cells which lack an intact plasma membrane. We have developed a method, which allows the preservation of the PI signal in paraformaldehyde-fixed tissue, enabling subsequent immunohistochemical characterisation of labelled cells. We have tested this method in a model of ischemia based on oxygen and glucose deprivation in organotypic hippocampal slice cultures, in combination with immunocytochemical detection of calpain-I mediated spectrin breakdown products (BDPs). Using confocal laser microscopy it was possible to correlate at the single cell level which cells were PI positive and which cells expressed BDPs. This method can also be used with other immunocytochemical markers to determine the phenotype of cells, which accumulate PI in vitro. By fixing tissue at different times after insults, it is possible to obtain a ‘snapshot’ of viability at any time during the experimental protocol and subsequently characterise those cells which had accumulated PI at the time of fixation. The technique may also prove useful in characterising cell death in other in vitro and in vivo systems
Synthesis and evaluation of fluorescent probes for the detection of calpain activity
No full textTwo 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 ?-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
Temporal development of hippocampal cell death is dependent on tissue strain but not strain rate
No full textDeformation 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 (0.1-50 s(-1)) relevant to TBI. Loading conditions for a subset of cultures were verified by analysis of highspeed 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
Organotypic cultures as tools for functional screening in the CNS
No full textScreening gets more complex with organotypic culture systems.A major challenge for the pharmaceutical industry is the development of relevant model systems in which knowledge gained from high-throughput, genomic and proteomic approaches can be integrated to study function. Animal models are still the main choice for such studies but over the past few years powerful new in vitro systems have begun to emerge as useful tools to study function. Organotypic cultures made from slices of explanted tissue represent a complex multi-cellular in vitro environment with the potential to assess biological function and are uniquely placed to act as an important link between high-throughput approaches and animal models
Intraischaemic hypothermia reduces free radical production and protects against ischaemic insults in cultured hippocampal slices
No full textHypothermia 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 31–33C 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
Time window and pharmacological characterisation of kainate-mediated preconditioning in organotypic rat hippocampal slice cultures
No full textTolerance 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 ?M kainate induces a selective lesion in the CA3 region and this can be significantly attenuated by 1 ?M 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
An in vitro model of traumatic brain injury utilising two-dimensional stretch of organotypic hippocampal slice cultures
No full textTraumatic 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
Characterisation of a novel class of polyamine-based neuroprotective compounds
No full textProlonged 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
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