Barclay Morrison

Ferroptosis: An Iron-Dependent Form of Nonapoptotic Cell Death

Nonapoptotic forms of cell death may facilitate the selective elimination of some tumor cells or be activated in specific pathological states. The oncogenic RAS-selective lethal small molecule erastin triggers a unique iron-dependent form of nonapoptotic cell death that we term ferroptosis. Ferroptosis is dependent upon intracellular iron, but not other metals, and is morphologically, biochemically, and genetically distinct from apoptosis, necrosis, and autophagy. We identify the small molecule ferrostatin-1 as a potent inhibitor of ferroptosis in cancer cells and glutamate-induced cell death in organotypic rat brain slices, suggesting similarities between these two processes. Indeed, erastin, like glutamate, inhibits cystine uptake by the cystine/glutamate antiporter (system x(c)(-)), creating a void in the antioxidant defenses of the cell and ultimately leading to iron-dependent, oxidative death. Thus, activation of ferroptosis results in the nonapoptotic destruction of certain cancer cells, whereas inhibition of this process may protect organisms from neurodegeneration.

Bioorthogonal chemical imaging of metabolic activities in live mammalian hippocampal tissues with stimulated Raman scattering

Brain is an immensely complex system displaying dynamic and heterogeneous metabolic activities. Visualizing cellular metabolism of nucleic acids, proteins, and lipids in brain with chemical specificity has been a long-standing challenge. Recent development in metabolic labeling of small biomolecules allows the study of these metabolisms at the global level. However, these techniques generally require nonphysiological sample preparation for either destructive mass spectrometry imaging or secondary labeling with relatively bulky fluorescent labels. In this study, we have demonstrated bioorthogonal chemical imaging of DNA, RNA, protein and lipid metabolism in live rat brain hippocampal tissues by coupling stimulated Raman scattering microscopy with integrated deuterium and alkyne labeling. Heterogeneous metabolic incorporations for different molecular species and neurogenesis with newly-incorporated DNA were observed in the dentate gyrus of hippocampus at the single cell level. We further applied this platform to study metabolic responses to traumatic brain injury in hippocampal slice cultures, and observed marked upregulation of protein and lipid metabolism particularly in the hilus region of the hippocampus within days of mechanical injury. Thus, our method paves the way for the study of complex metabolic profiles in live brain tissue under both physiological and pathological conditions with single-cell resolution and minimal perturbation.

Flexible and stretchable micro-electrodes for in vitro and in vivo neural interfaces

Microelectrode arrays (MEAs) are designed to monitor and/or stimulate extracellularly neuronal activity. However, the biomechanical and structural mismatch between current MEAs and neural tissues remains a challenge for neural interfaces. This article describes a material strategy to prepare neural electrodes with improved mechanical compliance that relies on thin metal film electrodes embedded in polymeric substrates. The electrode impedance of micro-electrodes on polymer is comparable to that of MEA on glass substrates. Furthermore, MEAs on plastic can be flexed and rolled offering improved structural interface with brain and nerves in vivo. MEAs on elastomer can be stretched reversibly and provide in vitro unique platforms to simultaneously investigate the electrophysiological of neural cells and tissues to mechanical stimulation. Adding mechanical compliance to MEAs is a promising vehicle for robust and reliable neural interfaces.

Permeability of Endothelial and Astrocyte Cocultures: In Vitro Blood–Brain Barrier Models for Drug Delivery Studies

The blood–brain barrier (BBB) is a major obstacle for drug delivery to the brain. To seek for in vitro BBB models that are more accessible than animals for investigating drug transport across the BBB, we compared four in vitro cultured cell models: endothelial monoculture (bEnd3 cell line), coculture of bEnd3 and primary rat astrocytes (coculture), coculture with collagen type I and IV mixture, and coculture with Matrigel. The expression of the BBB tight junction proteins in these in vitro models was assessed using RT-PCR and immunofluorescence. We also quantified the hydraulic conductivity (Lp), transendothelial electrical resistance (TER) and diffusive solute permeability (P) of these models to three solutes: TAMRA, Dextran 10K and Dextran 70K. Our results show that Lp and P of the endothelial monoculture and coculture models are not different from each other. Compared with in vivo permeability data from rat pial microvessels, P of the endothelial monoculture and coculture models are not significantly different from in vivo data for Dextran 70K, but they are 2–4 times higher for TAMRA and Dextran 10K. This suggests that the endothelial monoculture and all of the coculture models are fairly good models for studying the transport of relatively large solutes across the BBB.

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.

Molecules of Various Pharmacologically-Relevant Sizes Can Cross the Ultrasound-Induced Blood-Brain Barrier Opening in vivo

Focused ultrasound (FUS) is hereby shown to noninvasively and selectively deliver compounds at pharmacologically relevant molecular weights through the opened blood-brain barrier (BBB). A complete examination on the size of the FUS-induced BBB opening, the spatial distribution of the delivered agents and its dependence on the agents molecular weight were imaged and quantified using fluorescence microscopy. BBB opening in mice (n=13) was achieved in vivo after systemic administration of microbubbles and subsequent application of pulsed FUS (frequency: 1.525MHz, peak-rarefactional pressure in situ: 570 kPa) to the left murine hippocampus through the intact skin and skull. BBB-impermeant, fluorescent-tagged dextrans at three distinct molecular weights spanning over several orders of magnitude were systemically administered and acted as model therapeutic compounds. First, dextrans of 3 and 70 kDa were delivered trans-BBB while 2000 kDa dextran was not. Second, compared with 70 kDa dextran, a higher concentration of 3 kDa dextran was delivered through the opened BBB. Third, the 3 and 70 kDa dextrans were both diffusely distributed throughout the targeted brain region. However, high concentrations of 70 kDa dextran appeared more punctated throughout the targeted region. In conclusion, FUS combined with microbubbles opened the BBB sufficiently to allow passage of compounds of at least 70 kDa, but not greater than 2000 kDa into the brain parenchyma. This noninvasive and localized BBB opening technique could, thus, provide a unique means for the delivery of compounds of several magnitudes of kDa that include agents with shown therapeutic promise in vitro but whose in vivo translation has been hampered by their associated BBB impermeability. (E-mail: ek2191@columbia.edu).

In Vitro Models of Traumatic Brain Injury

In vitro models of traumatic brain injury (TBI) are helping elucidate the pathobiological mechanisms responsible for dysfunction and delayed cell death after mechanical stimulation of the brain. Researchers have identified compounds that have the potential to break the chain of molecular events set in motion by traumatic injury. Ultimately, the utility of in vitro models in identifying novel therapeutics will be determined by how closely the in vitro cascades recapitulate the sequence of cellular events that play out in vivo after TBI. Herein, the major in vitro models are reviewed, and a discussion of the physical injury mechanisms and culture preparations is employed. A comparison between the efficacy of compounds tested in vitro and in vivo is presented as a critical evaluation of the fidelity of in vitro models to the complex pathobiology that is TBI. We conclude that in vitro models were greater than 88% predictive of in vivo results.

Organotypic cultures as tools for functional screening in the CNS

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.

Traumatic injury induces differential expression of cell death genes in organotypic brain slice cultures determined by complementary DNA array hybridization

The expression of a large panel of selected genes hypothesized to play a central role in post-traumatic cell death was shown to be differentially altered in response to a precisely controlled, mechanical injury applied to an organotypic slice culture of the rat brain. Within 48 h of injury, the expression of nerve growth factor messenger RNA was significantly increased whereas the levels of bcl-2, alpha-subunit of calcium/calmodulin-dependent protein kinase II, cAMP response element binding protein, 65,000 mol. wt isoform of glutamate decarboxylase, 1beta isoform of protein kinase C, and ubiquitin messenger RNA were significantly decreased. Because the expression levels of a number of other messenger RNAs such as the neuron-specific amyloid precursor protein, beta(2) microglobulin, bax, bcl(xl), brain-derived neurotrophic factor, cyclooxygenase-2, interleukin-1beta, interleukin-6, tumor necrosis factor-alpha, receptor tyrosine kinase A, and receptor tyrosine kinase B were unaffected, these selective changes may represent components of an active and directed response of the brain initiated by mechanical trauma. Interpretation of these co-ordinated alterations suggests that mechanical injury to the central nervous system may lead to disruption of calcium homeostasis resulting in altered gene expression, an impairment of intracellular cascades responsible for trophic factor signaling, and initiation of apoptosis via multiple pathways. An understanding of these transcriptional changes may contribute to the development of novel therapeutic strategies to enhance beneficial and blunt detrimental, endogenous, post-injury response mechanisms.

Age-dependent regional mechanical properties of the rat hippocampus and cortex.

Age-dependent outcomes following traumatic brain injury motivate the study of brain injury biomechanics in experimental animal models at different stages of development. Finite element models of the rat brain are used to better understand the mechanical mechanisms behind these age-dependent outcomes; however, age- and region-specific rat brain tissue mechanical properties are required for biofidelity in modeling. Here, we have used the atomic force microscope (AFM) to measure region-dependent mechanical properties for subregions of the cortex and hippocampus in P10, P17, and adult rats. Apparent elastic modulus increased nonlinearly with indentation strain, and a nonlinear Ogden hyperelastic model was used to fit the force-deflection data. Subregional heterogeneous distributions of mechanical properties changed significantly with age. Apparent elastic modulus was also found to increase overall with age, increasing by >100% between P10 and adult rats. Unconfined compression tests (epsilon=-0.3) were performed on whole slices of the hippocampus and cortex of P10, P17, and adult rats to verify the mechanical properties measured with the AFM. Mean apparent elastic modulus at an indentation strain of 30% from AFM measurements for each region and age correlated well with the long-term elastic modulus measured from 30% unconfined compression tests (slope not significantly different from 1, p>0.05). Protein, lipid, and sulfated glycosaminoglycan content of the brain increased with age and were positively correlated with tissue stiffness, whereas water content decreased with age and was negatively correlated with tissue stiffness. These correlations can be used to hypothesize mechanistic models for describing the mechanical behavior of brain tissue as well as to predict relative differences between brain tissue mechanical properties of other species, at different ages, and for different regions based on differences in tissue composition.