Predicting changes in cortical electrophysiological function after in vitro traumatic brain injury.

Finite element (FE) models of traumatic brain injury (TBI) are capable of predicting injury-induced brain tissue deformation. However, current FE models are not equipped to predict the biological consequences of tissue deformation, which requires the determination of tolerance criteria relating the effects of mechanical stimuli to biologically relevant functional responses. To address this deficiency, we present functional tolerance criteria for the cortex for alterations in neuronal network electrophysiological function in response to controlled mechanical stimuli. Organotypic cortical slice cultures were mechanically injured via equibiaxial stretch with a well-characterized in vitro model of TBI at tissue strains and strain rates relevant to TBI (up to Lagrangian strain of 0.59 and strain rates up to 29 [Formula: see text]. At 4-6 days post-injury, electrophysiological function was assessed simultaneously throughout the cortex using microelectrode arrays. Electrophysiological parameters associated with unstimulated spontaneous network activity (neural event rate, duration, and magnitude), stimulated evoked responses (the maximum response [Formula: see text], the stimulus current necessary for a half-maximal response [Formula: see text], and the electrophysiological parameter [Formula: see text] that is representative of firing uniformity), and evoked paired-pulse ratios at varying interstimulus intervals were quantified for each cortical slice culture. Nonlinear regression was performed between mechanical injury parameters as independent variables (tissue strain and strain rate) and each electrophysiological parameter as output. Parsimonious best-fit equations were determined from a large pool of candidate equations with tenfold cross-validation. Changes in electrophysiological parameters were dependent on strain and strain rate in a complex manner. Compared to the hippocampus, the cortex was less spontaneously active, less excitable, and less prone to significant changes in electrophysiological function in response to controlled deformation (strain or strain rate). Our study provides functional data that can be incorporated into FE models to improve their predictive capabilities of the in vivo consequences of TBI.

Functional tolerance to mechanical deformation developed from organotypic hippocampal slice cultures.

In this study, we measured changes in electrophysiological activity after mechanical deformation of living organotypic hippocampal brain slice cultures at tissue strains and strain rates relevant to traumatic brain injury (TBI). Electrophysiological activity was measured throughout the hippocampus with a 60-electrode microelectrode array. Electrophysiological parameters associated with unstimulated spontaneous activity (neural event firing rate, duration, and magnitude), stimulated evoked responses (the maximum response [Formula: see text], the stimulus current necessary for a half-maximal response [Formula: see text], and the electrophysiological parameter m that is representative of firing uniformity), and paired-pulse responses (paired-pulse ratio at varying interstimulus intervals) were quantified for each hippocampal region (CA1, CA3, and DG). We present functional tolerance criteria for the hippocampus in the form of mathematical relationships between the input tissue-level injury parameters (strain and strain rate) and altered neuronal network function. Most changes in electrophysiology were dependent on strain and strain rate in a complex fashion, independent of hippocampal anatomy, with the notable exception of [Formula: see text]. Until it becomes possible to directly measure brain tissue deformation in vivo, finite element (FE) models will be necessary to simulate and predict the in vivo consequences of TBI. One application of our study is to provide functional relationships that can be incorporated into these FE models to enhance their biofidelity of accident and collision reconstructions by predicting biological outcomes in addition to mechanical responses.

Alterations in Hippocampal Network Activity after In Vitro Traumatic Brain Injury.

Traumatic brain injury (TBI) alters function and behavior, which can be characterized by changes in electrophysiological function in vitro. A common cognitive deficit after mild-to-moderate TBI is disruption of persistent working memory, of which the in vitro correlate is long-lasting, neuronal network synchronization that can be induced pharmacologically by the gamma-aminobutyric acid A antagonist, bicuculline. We utilized a novel in vitro platform for TBI research, the stretchable microelectrode array (SMEA), to investigate the effects of TBI on bicuculline-induced, long-lasting network synchronization in the hippocampus. Mechanical stimulation significantly disrupted bicuculline-induced, long-lasting network synchronization 24 h after injury, despite the continued ability of the injured neurons to fire, as revealed by a significant increase in the normalized spontaneous event rate in the dentate gyrus (DG) and CA1. A second challenge with bicuculline 24 h after the first challenge significantly decreased the normalized spontaneous event rate in the DG. In addition, we illustrate the utility of the SMEA for TBI research by combining multiple experimental paradigms in one platform, which has the potential to enable novel investigations into the mechanisms responsible for functional consequences of TBI and speed the rate of drug discovery.

Attenuation of astrocyte activation by TAT-mediated delivery of a peptide JNK inhibitor.

Astrocyte activation contributes to the brain's response to disease and injury. Activated astrocytes generate harmful radicals that exacerbate brain damage including nitric oxide, peroxides and superoxides. Furthermore, reactive astrocytes hinder regeneration of damaged neural circuits by secreting neuro-developmental inhibitors and glycosaminoglycans (GAGs), which physically block growth cone extension. Therefore, targeted therapeutic strategies to limit astrocyte activation may enhance recovery from many neurodegenerative states. Previously, we demonstrated that the HIV-1 TAT cell-penetrating peptide, a short non-toxic peptide from the full-length TAT protein, delivered a protein cargo to astrocytes in a process dependent on cell-surface GAG. Since activated astrocytes produce GAG, in this study we tested whether TAT could transduce activated astrocytes, deliver a biologically active cargo, and produce a physiological effect. Astrocyte activation was induced by IL-1ß, lipopolysaccharide (LPS), or mechanical stretch injury, and quantified by increased GAG and nitrite content. TAT-mediated delivery of a mock therapeutic protein, GFP, increased significantly after activation. Nitrite production, GAG expression, and GFP-TAT transduction were significantly attenuated by inhibitors of JNK, p38, or ERK. TAT fused to a peptide JNK inhibitor delivered the peptide inhibitor to activated astrocytes and significantly reduced activation. Our study is the first to report significant and direct modulation of astrocyte activation with a peptide JNK inhibitor. Our promising in vitro results warrant in vivo follow-up, as TAT-mediated protein delivery may have broad therapeutic potential for preventing astrocyte activation with the possibility of limiting off-target, negative side effects.

TAT is not capable of transcellular delivery across an intact endothelial monolayer in vitro.

Developing delivery vehicles capable of penetrating cell barriers is critical for drug delivery to the brain due to the presence of the blood-brain barrier (BBB). Cell-penetrating peptides (CPPs) are one potential solution since they can enter cells; however, it is unclear whether CPPs can pass through cell barriers. In this study, the ability of the TAT CPP to cross an endothelial barrier without disrupting the integrity of its tight junctions was investigated. Endothelial cell monolayers (bEnd.3) were exposed to the TAT peptide, and cell integrity was quantified by zona occludens-1 immunofluorescence, trans-endothelial electrical resistance, and hydraulic conductivity. None of these parameters were significantly altered following exposure to TAT. To evaluate the passage of TAT through the monolayer, the permeability of a green fluorescent protein (GFP)-TAT fusion protein was not significantly different from the permeability of GFP or fluorescent dextrans of similar sizes. Furthermore, GFP-TAT was unable to significantly transduce astrocytes on the opposite side of the bEnd.3 monolayer. We conclude, therefore, that although TAT may not be an efficient delivery vehicle for trans-BBB delivery, our TAT construct may have utility in delivering therapeutic cargos to endothelial cells or to the brain parenchyma after BBB disruption.

Increased delivery of TAT across an endothelial monolayer following ischemic injury.

There is a great need for the development of vehicles capable of delivering therapeutic cargoes across the blood-brain barrier (BBB) and into brain cells. Cell-penetrating peptides (CPPs), such as TAT, present one such solution, and have been used successfully in vivo to deliver neuroprotective cargoes to the brain in models of stroke and seizure. However, a significant discrepancy exists in the literature, as other groups have not had the same success. One commonality between the successful studies is a compromised BBB. In this study, we hypothesized that ischemic injury increases the transport of TAT across an endothelial monolayer (comprised of bEnd.3 cells) in vitro and, consequently, increases TAT-mediated delivery into astrocytes on the other side. In the 24h following in vitro ischemia (oxygen-glucose deprivation), transendothelial electrical resistance (TEER) significantly decreased, indicating disruption of BBB integrity. Concomitantly, the transport of a green fluorescent protein (GFP)-TAT fusion protein significantly increased, and the transduction of GFP-TAT into astrocytes cultured on the other side of the endothelial monolayer significantly increased. These results explain why TAT-mediated delivery of therapeutic cargoes is successful in the ischemic brain but not in the uninjured brain with an intact BBB, highlighting the necessity for continued development of delivery vehicles. We conclude that although TAT may not be an efficient vehicle for trans-BBB delivery across an intact BBB, it may have utility in clinical situations when the BBB is disrupted.

TAT-mediated intracellular protein delivery to primary brain cells is dependent on glycosaminoglycan expression.

Although some studies have shown that the cell penetrating peptide (CPP) TAT can enter a variety of cell lines with high efficiency, others have observed little or no transduction in vivo or in vitro under conditions mimicking the in vivo environment. The mechanisms underlying TAT-mediated transduction have been investigated in cell lines, but not in primary brain cells. In this study we demonstrate that transduction of a green fluorescent protein (GFP)-TAT fusion protein is dependent on glycosaminoglycan (GAG) expression in both the PC12 cell line and primary astrocytes. GFP-TAT transduced PC12 cells and did so with even higher efficiency following NGF differentiation. In cultures of primary brain cells, TAT significantly enhanced GFP delivery into astrocytes grown under different conditions: (1) monocultures grown in serum-containing medium; (2) monocultures grown in serum-free medium; (3) cocultures with neurons in serum-free medium. The efficiency of GFP-TAT transduction was significantly higher in the monocultures than in the cocultures. The GFP-TAT construct did not significantly enter neurons. Experimental modulation of GAG content correlated with alterations in TAT transduction in PC12 cells and astrocyte monocultures grown in the presence of serum. In addition, this correlation was predictive of TAT-mediated transduction in astrocyte monocultures grown in serum free medium and in coculture. We conclude that culture conditions affect cellular GAG expression, which in turn dictates TAT-mediated transduction efficiency, extending previous results from cell lines to primary cells. These results highlight the cell-type and phenotype-dependence of TAT-mediated transduction, and underscore the necessity of controlling the phenotype of the target cell in future protein engineering efforts aimed at creating more efficacious CPPs.