Increased Retention of Cardiac Cells to a Glass Substrate through Streptavidin-Biotin Affinity.

In vitro analysis of primary isolated adult cardiomyocyte physiological processes often involves optical imaging of dye-loaded cells on a glass substrate. However, when exposed to rapid solution changes, primary cardiomyocytes often move to compromise quantitative measures. Improved immobilization of cells to glass would permit higher throughput assays. Here, we engineer the peripheral membrane of cardiomyocytes with biotin to anchor cardiomyocytes to borosilicate glass coverslips functionalized with streptavidin. We use a rat cardiac myoblast cell line to determine general relationships between processing conditions, ligand density on the cell and the glass substrate, cellular function, and cell retention under shear flow. Use of the streptavidin-biotin system allows for more than 80% retention of cardiac myoblasts under conventional rinsing procedures, while unmodified cells are largely rinsed away. The adhesion system enables the in-field retention of cardiac cells during rapid fluid changes using traditional pipetting or a modern microfluidic system at a flow rate of 160 mL/min. Under fluid flow, the surface-engineered primary adult cardiomyocytes are retained in the field of view of the microscope, while unmodified cells are rinsed away. Importantly, the engineered cardiomyocytes are functional following adhesion to the glass substrate, where contractions are readily observed. When applying this adhesion system to cardiomyocyte functional analysis, we measure calcium release transients by caffeine induction at an 80% success rate compared to 20% without surface engineering.

Therapeutic window analysis of the neuroprotective effects of cyclosporine A after traumatic brain injury.

Mitochondrial dysfunction plays a pivotal role in secondary cell death mechanisms following traumatic brain injury (TBI). Several reports have demonstrated that inhibition of the mitochondrial permeability transition pore with the immunosuppressant drug cyclosporine A (CsA) is efficacious. Accordingly, CsA is being moved forward into late-stage clinical trials for the treatment of moderate and severe TBI. However, several unknowns exist concerning the optimal therapeutic window for administering CsA at the proposed dosages to be used in human studies. The present study utilized a moderate (1.75 mm) unilateral controlled cortical impact model of TBI to determine the most efficacious therapeutic window for initiating CsA therapy. Rats were administered an IP dose of CsA (20 mg/kg) or vehicle at 1, 3, 4, 5, 6, and 8 h post-injury. Immediately following the initial IP dose, osmotic mini-pumps were implanted at these time points to deliver 10 mg/kg/d of CsA or vehicle. Seventy-two hours following the initiation of treatment the pumps were removed to stop CsA administration. Quantitative analysis of cortical tissue sparing 7 days post-injury revealed that CsA treatment initiated at any of the post-injury initiation times out to 8 h resulted in significantly less cortical damage compared to animals receiving vehicle treatment. However, earlier treatment begun in the first 3 h was significantly more protective than that begun at 4 and 8 h. Treatment initiated at 1 h post-injury (∼68% decrease) was not significantly different than that seen at 3 h (∼46% decrease), but resulted in significantly greater cortical tissue sparing compared to CsA treatment initiated at least 4 h post-injury (28% decrease). Together these results illustrate the importance of initiating therapeutic interventions such as CsA as soon as possible following TBI, preferably within 4 h post-injury, to achieve the best possible neuroprotective effect. However, the drug appears to retain some protective efficacy even when initiated as late as 8 h post-injury.

Post-Injury Administration of Mitochondrial Uncouplers Increases Tissue Sparing and Improves Behavioral Outcome following Traumatic Brain Injury in Rodents.

Following experimental traumatic brain injury (TBI), a rapid and significant necrosis occurs at the site of injury which coincides with significant mitochondrial dysfunction. The present study is driven by the hypothesis that TBI-induced glutamate release increases mitochondrial Ca(2+)cycling/overload, ultimately leading to mitochondrial dysfunction. Based on this premise, mitochondrial uncoupling during the acute phases of TBI-induced excitotoxicity should reduce mitochondrial Ca(2+) uptake (cycling) and reactive oxygen species (ROS) production since both are mitochondrial membrane potential dependent. In the present study, we utilized a cortical impact model of TBI to assess the potential use of mitochondrial uncouplers (2,4-DNP, FCCP) as a neuroprotective therapy. Young adult male rats were intraperitoneally administered vehicle (DMSO), 2,4-DNP (5 mg/kg), or FCCP (2.5 mg/kg) at 5 min post-injury. All animals treated with the uncouplers demonstrated a significant reduction in the amount of cortical damage and behavioral improvement following TBI. In addition, mitochondria isolated from the injured cortex at 3 or 6 h post-injury demonstrated that treatment with the uncouplers significantly improved several parameters of mitochondrial bioenergetics. These results demonstrate that post-injury treatment with mitochondrial uncouplers significantly (p < 0.01) increases cortical tissue sparing ( approximately 12%) and significantly (p< 0.01) improves behavioral outcome following TBI. The mechanism of neuroprotection most likely involves the maintenance of mitochondrial homeostasis by reducing mitochondrial Ca(2+) loading and subsequent mitochondrial dysfunction. These results further implicate mitochondrial dysfunction as an early event in the pathophysiology of TBI and that targeting acute mitochondrial events can result in long-term neuroprotection and improve behavioral outcome following brain injury.