SUMMARY New strategies for introducing genetically encoded activity indicators into animal models facilitate the investigation of nervous system function. We have developed the PC::G5-tdT mouse line that expresses the GCaMP5G calcium indicator in a Cre-dependent fashion. Instead of targeting the ROSA26 locus, we inserted the reporter cassette nearby the ubiquitously expressed Polr2a gene without disrupting locus integrity. The indicator was tagged with IRES-tdTomato to aid detection of positive cells. This reporter system is effective in a wide range of developmental and cellular contexts. We recorded spontaneous cortical calcium waves in intact awake newborns and evaluated concentration-dependent responses to odorants in the adult olfactory bulb. Moreover, PC::G5-tdT effectively reports intracellular calcium dynamics in somas and fine processes of astrocytes and microglial cells. Through electrophysiological and behavioral analyses, we determined that GCaMP5G expression had no major impact on nervous system performance. PC::G5-tdT will be instrumental for a variety of brain mapping experiments.
Complex interactions between networks of astrocytes and neurons are beginning to be appreciated, but remain poorly understood. Transgenic mice expressing fluorescent protein reporters of cellular activity, such as the GCaMP family of genetically encoded calcium indicators (GECIs), have been used to explore network behavior. However, in some cases, it may be desirable to use long-established rat models that closely mimic particular aspects of human conditions such as Parkinson's disease and the development of epilepsy following status epilepticus. Methods for expressing reporter proteins in the rat brain are relatively limited. Transgenic rat technologies exist but are fairly immature. Viral-mediated expression is robust but unstable, requires invasive injections, and only works well for fairly small genes (<5 kb). In utero electroporation (IUE) offers a valuable alternative. IUE is a proven method for transfecting populations of astrocytes and neurons in the rat brain without the strict limitations on transgene size. We built a toolset of IUE plasmids carrying GCaMP variants 3, 6s, or 6f driven by CAG and targeted to the cytosol or the plasma membrane. Because low baseline fluorescence of GCaMP can hinder identification of transfected cells, we included the option of co-expressing a cytosolic tdTomato protein. A binary system consisting of a plasmid carrying a piggyBac inverted terminal repeat (ITR)-flanked CAG-GCaMP-IRES-tdTomato cassette and a separate plasmid encoding for expression of piggyBac transposase was employed to stably express GCaMP and tdTomato. The plasmids were co-electroporated on embryonic days 13.5–14.5 and astrocytic and neuronal activity was subsequently imaged in acute or cultured brain slices prepared from the cortex or hippocampus. Large spontaneous transients were detected in slices obtained from rats of varying ages up to 127 days. In this report, we demonstrate the utility of this toolset for interrogating astrocytic and neuronal activity in the rat brain.
The current literature demonstrated that orthopaedic surgeries, with appropriate indication, in patients with haemophilic ankle arthropathy result in good postoperative results comparable to those observed in non-haemophiliacs. The surgical treatment should be performed in a setting with the ability to have multidisciplinary management, including expertise in haematology.
The development of adult use right ventricular assist devices (RVADs) and pediatric left ventricular assist devices (pediatric LVADs) have significantly lagged behind compared to adult use left ventricular assist devices (LVADs). The HeartWare ventricular assist device (HVAD) intended to be used for adult’s systemic support, is increasingly used off-label for adult pulmonary and pediatric systemic support. Due to different hemodynamics and physiology, however, the HVAD’s hemocompatibility profiles can be drastically different when used in adult pulmonary circulation or in children, compared to its intended usage state, which could have a direct clinical and developmental relevance. Taking these considerations in mind, we sought to conduct in vitro hemocompatibility testing of HVAD in adult systemic, pediatric systemic and adult pulmonary support conditions. Two HVADs coupled to custom-built blood circulation loops were tested for 6 hours using bovine blood at 37°C under adult systemic, pediatric systemic, and adult pulmonary flow conditions (flow rate = 5.0, 2.5, and 4.5 L/min; differential pressure = 100, 69, and 20 mm Hg, respectively). Normalized index of hemolysis for adult systemic, pediatric systemic, and adult pulmonary conditions were 0.0083, 0.0039, and 0.0017 g/100 L, respectively. No significant difference was seen in platelet activation for these given conditions. High molecular weight von Willebrand factor multimer degradation was evident in all conditions (p < 0.05). In conclusion, alterations in the usage mode produce substantial differences in hemocompatibility of the HVAD. These findings would not only have clinical relevance but will also facilitate future adult use RVAD and pediatric LVAD development.
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