Citation: Salvador, E., Neuhaus, W., Foerster, C. Stretch in Brain Microvascular Endothelial Cells (cEND) as an In Vitro Traumatic Brain Injury Model of the Blood Brain Barrier. J. Vis. Exp. (80), e50928, doi:10.3791/50928 (2013). AbstractDue to the high mortality incident brought about by traumatic brain injury (TBI), methods that would enable one to better understand the underlying mechanisms involved in it are useful for treatment. There are both in vivo and in vitro methods available for this purpose. In vivo models can mimic actual head injury as it occurs during TBI. However, in vivo techniques may not be exploited for studies at the cell physiology level. Hence, in vitro methods are more advantageous for this purpose since they provide easier access to the cells and the extracellular environment for manipulation.Our protocol presents an in vitro model of TBI using stretch injury in brain microvascular endothelial cells. It utilizes pressure applied to the cells cultured in flexible-bottomed wells. The pressure applied may easily be controlled and can produce injury that ranges from low to severe. The murine brain microvascular endothelial cells (cEND) generated in our laboratory is a well-suited model for the blood brain barrier (BBB) thus providing an advantage to other systems that employ a similar technique. In addition, due to the simplicity of the method, experimental set-ups are easily duplicated. Thus, this model can be used in studying the cellular and molecular mechanisms involved in TBI at the BBB.
In order to optimize the lung-protective potential of high-frequency oscillatory ventilation (HFOV), it is currently recommended to maximize oscillatory frequencies. However, very high frequencies may lead to insufficient CO(2) elimination with severe respiratory acidosis. Arteriovenous extracorporeal lung assist (av-ECLA) allows near total CO(2) removal, thereby allowing for maximization of the lung-protective potential of HFOV. The aim of this study was to determine the impact of HFOV and av-ECLA on lung inflammation and function compared to conventional lung-protective ventilation. In a porcine surfactant depletion model of lung injury, the authors randomly assigned 16 female pigs to conventional lung-protective ventilation and HFOV/ECLA. Both strategies were combined with an "open-lung" approach. Gas exchange and hemodynamic parameters were measured at intervals during the 24-hour study period. Postmortem, lung tissue was analyzed to determine histological damage and lung inflammation. The authors found that the combination of HFOV and av-ECLA (1) allows significant reductions in mean and peak airway pressures; and (2) reduces histological signs of lung inflammation in the basal regions of the lung. HFOV/av-ECLA reduces histological signs of lung inflammation compared to conventional lung-protective ventilation strategies. Thus, combination of HFOV and av-ECLA might be a further lung-protective tool if conventional ventilation strategies are failing.
Endothelial cells (ECs) from different vascular beds not only display common characteristics but are also quite heterogeneous in terms of expression and secretion of neuro-angiogenic factors, which may help explain some of their distinct physiological roles. We investigated by RT-PCR the gene expression, by PC12 bioassay the neurotropic activity, and by ELISAs the levels of NGF and FGF-2 using conditioned medium collected from cultures of ECs derived from myocardial and cerebral capillaries. While NGF was expressed and released by both cell types, FGF-2 was expressed and released solely by the brain but not heart ECs. Oxygen-glucose deprivation (ischemic) insult blocked NGF secretion from heart and brain ECs and inhibited by 70% the secretion of FGF-2 from brain ECs. We propose that the differential expression of NGF and FGF-2 in heart and brain EC cultures reflect heterogeneity on demand of the microcapillary components and the surrounding microenvironment for a proper tissue-specific homeostasis.
2551 Background: The greatest hurdle, which even potent and effective drugs targeting central nervous system (CNS) tumors and other disorders face, is the blood brain barrier (BBB). The inability to cross the tight regulatory mechanism renders these drugs futile. Of late, administration of tumor treating fields (TTFields) as part of a combined treatment modality for glioblastoma demonstrated increased overall patient survival. Still, the effects of TTFields on the BBB have not yet been investigated. Here, we report the potential of TTFields application to open up the BBB. Methods: Murine brain endothelial cells were treated with 100-300 kHz TTFields for 24-96 h. Cells were also allowed to recover from 24-96 h after treatment. Subsequently, changes in cell morphology, integrity, and permeability were observed via staining of intercellular junction proteins (IJP) as well as transendothelial electrical resistance (TEER)and permeability assays. In vivo, rats were treated with 100 kHz TTFields or heat for 72 h after which they were IV injected with Evan´s Blue (EB)/ TRITC-dextran (TD) which was later quantified from the brain. Rat brain cryosections were also stained for IJPs as well as immunoglobulin G (IgG) to assess vessel structure. Finally, serial dynamic contrast-enhanced (DCE) MRI with gadolinium (Gd) contrast agent was performed pre- and post- TTFields. Results: Upon TTFields application, IJPs such as claudin-5 were delocalized from the cell membrane to the cytoplasm with maximal effects at 100 kHz. In addition, BBB integrity was significantly reduced and permeability for 4 kDa molecules was significantly increased. Cell morphology recovery was first observed at 48 h post-treatment and completely restored to normal after 96 h, indicating a reversibility of the TTFields effect on the BBB. In addition, EB and TD permeated the rat brain post-TTFields treatment. Brain cryosections displayed IJPs delocalization as well as IgG accumulation in the brain parenchyma. Confirming these observations, increased Gd in the brain was shown by DCE-MRI post-TTFields application. A reversion to normal conditions was detected 96 h after end of treatment, which was demonstrated by no difference in contrast enhancement between control and treated rats. Conclusions: TTFields application both in vitro and in vivo points towards its ability to transiently open the BBB. This presents TTFields as a novel aid for drug delivery geared towards treatment of CNS tumors and other related diseases. Hence, it is indicative of the possibility of an enhanced and more effective combinatorial therapeutic strategy.
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