Platelet and White Cell Reactivity to Top-Load Intravenous Perfluorocarbon Infusion in Healthy Sheep.

BACKGROUND: Perfluorocarbon emulsions (PFCs) are intravenous artificial oxygen carriers with enhanced gas solubility. As lipid micelle nanoparticle emulsions, PFCs may have a class effect that causes degrees of thrombocytopenia. Understanding the extent of the platelet effects, including mechanism and potential inflammation after PFC infusion, is important for safe human trials. METHODS: Normal sheep (Dorper) were infused with 5 mL/kg of Oxygent (w/v 60% PFC) or Perftoran (w/v 20% PFC). Controls received 6% Hetastarch or were naive. Blood samples were analyzed from baseline, time 0 (the end of infusion), 3 and 24 hours, and 4 and 7 days. Platelet count, plateletcrit, mean platelet volume, platelet distribution width, and CD-62p (a platelet activation-dependent membrane protein) were measured. Neutrophils, monocytes, and total white blood cell counts were analyzed. RESULTS: In these inflammatory cell lines, there were no consistent changes or cellular activation after PFC infusion. A decrease (<10% from baseline and naive controls) in platelet count was seen on day 4 after Oxygent infusion (3 g/kg), which recovered by day 7. No platelet effect was seen in Perftoran (1 g/kg). Plateletcrit, mean platelet volume, and platelet distribution width did not change significantly at any time point among the groups. CD-62p, ADP, and collagen aggregometry showed no significant change in platelet function. CONCLUSION: There was no evidence of overall reduction in platelet number, or any correlation with the change in platelet activation or inhibition. Therefore, the risk of increased thrombosis/bleeding after PFC intravenous infusion is low in this non-trauma sheep model.

Neuronal-Specific Inhibition of Endoplasmic Reticulum Mg2+/Ca2+ ATPase Ca2+ Uptake in a Mixed Primary Hippocampal Culture Model of Status Epilepticus.

Loss of intracellular calcium homeostasis is an established mechanism associated with neuronal dysfunction and status epilepticus. Sequestration of free cytosolic calcium into endoplasmic reticulum by Mg2+/Ca2+ adenosinetriphosphatase (ATPase) is critical for maintenance of intracellular calcium homeostasis. Exposing hippocampal cultures to low-magnesium media is a well-accepted in vitro model of status epilepticus. Using this model, it was shown that endoplasmic reticulum Ca2+ uptake was significantly inhibited in homogenates from cultures demonstrating electrophysiological seizure phenotypes. Calcium uptake was mainly neuronal. However, glial Ca2+ uptake was also significantly inhibited. Viability of neurons exposed to low magnesium was similar to neurons exposed to control solutions. Finally, it was demonstrated that Ca2+ uptake inhibition and intracellular free Ca2+ levels increased in parallel with increasing incubation in low magnesium. The results suggest that inhibition of Mg2+/Ca2+ ATPase-mediated endoplasmic reticulum Ca2+ sequestration contributes to loss of intracellular Ca2+ homeostasis associated with status epilepticus. This study describes for the first time inhibition of endoplasmic reticulum Mg2+/Ca2+ ATPase in a mixed primary hippocampal model of status epilepticus. In combination with animal models of status epilepticus, the cell culture model provides a powerful tool to further elucidate mechanisms that result in inhibition of Mg2+/Ca2+ ATPase and downstream consequences of decreased enzyme activity.

The effect of intravenous perfluorocarbon emulsions on whole-body oxygenation after severe decompression sickness.

INTRODUCTION: Decompression sickness (DCS) results from a decrease in ambient pressure leading to supersaturation of tissues with inert gas and bubble formation. Perfluorocarbons (PFCs) are able to dissolve vast amounts of non-polar gases. Intravenous (IV) PFC emulsions reduce both morbidity and mortality associated with DCS, but the mechanism of this protective effect has not yet been demonstrated. METHODS: Juvenile Dorper-cross sheep (n = 31) were anaesthetised and instrumented for physiological monitoring, IV fluid administration and blood sampling. Animals were compressed in air in a hyperbaric chamber to 608 kPa for 30 minutes and then rapidly decompressed. Upon decompression, animals were randomly assigned to receive 6 mmol per L of PFC or saline over 10 minutes beginning immediately after chamber exit. Arterial and mixed venous bloods were drawn at 5, 10, 15, 30, 60 and 90 minutes to examine pH, partial pressures of oxygen and carbon dioxide, oxygen saturation and electrolytes. RESULTS: Compared to saline, PFC administration increased arterial oxygen content (16.33 ± 0.28 vs. 14.68 ± 0.26 ml per dL, P < 0.0001), mixed venous oxygen content (12.56 ± 0.28 vs. 11.62 ± 0.26 ml per dL, P = 0.0167), oxygen delivery (14.83 ± 0.28 vs. 13.39 ± 0.26 mmol per L kg, P = 0.0003) and tissue oxygen consumption (3.30 ± 0.15 vs. 2.78 ± 0.13 mmol per L kg, P = 0.0149) but did not increase extraction ratio (0.22 ± 0.012 vs. 0.21 ± 0.011, P = 0.5343). CONCLUSIONS: It is likely that the improved oxygenation explains, at least in part, the previously-observed therapeutic effects of PFCs in DCS.

Retinal angiography: noninvasive, real-time bubble assessment from the ocular fundus.

Formation of bubbles in tissue and vasculature from a sudden reduction in ambient pressure is likely an underlying cause of the clinical symptoms of decompression sickness (DCS). Thus, tools detecting bubbles in the vasculature may be important for evaluating DCS. Sheep were air-compressed to 6.0 ATA (30 minutes bottom time) then rapidly decompressed to the surface. A fundus camera was quickly positioned for continuous observation of the retinal vasculature. Bubbles were observed in the retinal vasculature of 25.8% (n = 31) of the sheep. Bubble onset time ranged from 5-22 minutes post-chamber and lodge time ranged from 0-70+ minutes. Bubbles were visualized mostly in the arteries of the retinal circulation. Severe vasoconstriction was captured using red-free angiography in two sheep. In two other sheep, fluorescein angiography demonstrated occluded blood flow caused by arterial gas emboli. This study demonstrates that retinal angiography is a practical tool for real-time, noninvasive detection of bubbles in the retinal circulation, a visible window to the cerebral circulation. Thus retinal angiography may prove invaluable in the early detection of arterial gas emboli in the cerebral circulation, the resolution of which is imperative to favorable neurological outcomes. This study also presents for the first time images of bubbles in the retinal circulation associated with DCS captured by a fundus camera.

A significant increase in both basal and maximal calcineurin activity following fluid percussion injury in the rat.

Calcineurin, a neuronally enriched, calcium-stimulated phosphatase, is an important modulator of many neuronal processes, including several that are physiologically related to the pathology of traumatic brain injury. This study examined the effects of moderate, central fluid percussion injury on the activity of this important neuronal enzyme. Animals were sacrificed at several time-points postinjury and cortical, hippocampal, and cerebellar homogenates were assayed for calcineurin activity by dephosphorylation of p-nitrophenol phosphate. A significant brain injury-dependent increase was observed in both hippocampal and cortical homogenates under both basal and maximally-stimulated reaction conditions. This increase persisted 2-3 weeks post-injury. Brain injury did not alter substrate affinity, but did induce a significant increase in the apparent maximal dephosphorylation rate. Unlike the other brain regions, no change in calcineurin activity was observed in the cerebellum following brain injury. No brain region tested displayed a significant change in calcineurin enzyme levels as determined by Western blot, demonstrating that increased enzyme synthesis was not responsible for the observed increase in activity. The data support the conclusion that fluid percussion injury results in increased calcineurin activity in the rat forebrain. This increased activity has broad physiological implications, possibly resulting in altered cellular excitability or a greater likelihood of neuronal cell death.

Neuronal-specific endoplasmic reticulum Mg(2+)/Ca(2+) ATPase Ca(2+) sequestration in mixed primary hippocampal culture homogenates.

Endoplasmic reticulum Mg(2+)/Ca(2+) ATPase Ca(2+) sequestration is crucial for maintenance of neuronal Ca(2+) homeostasis. The use of cell culture in conjunction with modern Ca(2+) imaging techniques has been invaluable in elucidating these mechanisms. While imaging protocols evaluate endoplasmic reticulum Ca(2+) loads, measurement of Mg(2+)/Ca(2+) ATPase activity is indirect, comparing cytosolic Ca(2+) levels in the presence or absence of the Mg(2+)/Ca(2+) ATPase inhibitor thapsigargin. Direct measurement of Mg(2+)/Ca(2+) ATPase by isolation of microsomes is impossible due to the minuscule amounts of protein yielded from cultures used for imaging. In the current study, endoplasmic reticulum Mg(2+)/Ca(2+) ATPase Ca(2+) sequestration was measured in mixed homogenates of neurons and glia from primary hippocampal cultures. It was demonstrated that Ca(2+) uptake was mediated by the endoplasmic reticulum Mg(2+)/Ca(2+) ATPase due to its dependence on ATP and Mg(2+), enhancement by oxalate, and inhibition by thapsigargin. It was also shown that neuronal Ca(2+) uptake, mediated by the type 2 sarco(endo)plasmic reticulum Ca(2+) ATPase isoform, could be distinguished from glial Ca(2+) uptake in homogenates composed of neurons and glia. Finally, it was revealed that Ca(2+) uptake was sensitive to incubation on ice, extremely labile in the absence of protease inhibitors, and significantly more stable under storage conditions at -80 degrees C.

Status epilepticus-induced changes in the subcellular distribution and activity of calcineurin in rat forebrain.

This study was performed to determine the effect of prolonged status epilepticus on the activity and subcellular location of a neuronally enriched, calcium-regulated enzyme, calcineurin. Brain fractions isolated from control animals and rats subjected to pilocarpine-induced status epilepticus were subjected to differential centrifugation. Specific subcellular fractions were tested for both calcineurin activity and enzyme content. Significant, status epilepticus-induced increases in calcineurin activity were found in homogenates, nuclear fractions, and crude synaptic membrane-enriched fractions isolated from both cortex and hippocampus. Additionally, significant increases in enzyme levels were observed in crude synaptic fractions as measured by Western analysis. Immunohistochemical studies revealed a status epilepticus-induced increase in calcineurin immunoreactivity in dendritic structures of pyramidal neurons of the hippocampus. The data demonstrate a status epilepticus-induced increase in calcineurin activity and concentration in the postsynaptic region of forebrain pyramidal neurons.

Calcium/calmodulin-dependent kinase II phosphorylation of the GABAA receptor alpha1 subunit modulates benzodiazepine binding.

gamma-Aminobutyric acid (GABA) is the primary neurotransmitter that is responsible for the fast inhibitory synaptic transmission in the central nervous system. A major post-translational mechanism that can rapidly regulate GABAAR function is receptor phosphorylation. This study was designed to test the effect of endogenous calcium and calmodulin-dependent kinase II (CaM kinase II) activation on both allosteric modulator binding and GABAA receptor subunit phosphorylation. Endogenous CaM kinase II activity was stimulated, and GABAA receptors were subsequently analyzed for bothallosteric modulator binding properties and immunoprecipitated and analyzed for subunit phosphorylation levels. A significant increase in allosteric-modulator binding of the GABAAR was observed under conditions maximal for CaM kinase II activation. In addition, CaM kinase II activation resulted in a direct increase in phosphorylation of the GABAA receptor alpha1 subunit. The data suggest that the CaM kinase II-dependent phosphorylation of the GABAA receptor alpha1 subunit modulated allosteric modulator binding to the GABAA receptor.