Isoflurane is a regularly used anesthetic in translational research. Isoflurane facilitates invasive surgery and a rapid recovery. Specifically, in the pathology of stroke, controversy has surrounded isoflurane's intrinsic neuroprotective abilities, affecting apoptosis, excitotoxicity, and blood brain barrier disruption. Due to the intrinsic neuroprotective nature and lack of standardized guidelines for the use of isoflurane, research has shifted away from this gas in most animal models. Antagonistically, studies have also reported that no neuroprotective effects are observed when a surgery is accompanied with isoflurane exposure under 20 minutes. Isoflurane affects the pathophysiology in stroke patients by altering critical pathways in endothelial, neuronal, and microglial cells. Current studies have elucidated isoflurane neuroprotection to be time dependent and may be minimized in experimental designs if the exposure time is limited to a specific window. Therefore, with detailed and extensive literature on anesthetics, we can hypothesize that isoflurane exposure under the 20-minute benchmark, behavior and molecular pathways can be evaluated at any time-point following ischemic insult without confounding artifacts from isoflurane; however, If the exposure to isoflurane exceeds 20 minutes, the acute neuroprotective effects are evident for 2 weeks in the model, which should be accounted for in molecular and behavioral assessments, with either isoflurane inhibitors or a control group at 2 weeks post middle cerebral artery occlusion. The purpose of this review is to suggest a detailed and standardized outline for interventions and behavioral assessments after the use of isoflurane in experimental designs.
Understanding hypoxia/hyperoxia exposure requires either a high-altitude research facility or a chamber in which gas concentrations are precisely and reproducibly controlled. Hypoxia-induced conditions such as hypoxic-ischemic encephalopathy (HIE), obstructive or central apneas, and ischemic stroke present unique challenges for the development of models with acute or chronic hypoxia exposure. Many murine models exist to study these conditions; however, there are a variety of different hypoxia exposure protocols used across laboratories. Experimental equipment for hypoxia exposure typically includes flow regulators, nitrogen concentrators, and premix oxygen/nitrogen tanks. Commercial hypoxia/hyperoxia chambers with environmental monitoring are incredibly expensive and require proprietary software with subscription fees or highly expensive software licenses. Limitations exist in these systems as most are single animal systems and not designed for extended or intermittent hypoxia exposure. We have developed a simple hypoxia chamber with off-the-shelf components, and controlled by open-source software for continuous data acquisition of oxygen levels and other environmental factors (temperature, humidity, pressure, light, sound, etc.). Our chamber can accommodate up to two mouse cages and one rat cage at any oxygen level needed, when using a nitrogen concentrator or premixed oxygen/nitrogen tank with a flow regulator, but is also scalable. Our system uses a Python-based script to save data in a text file using modules from the sensor vendor. We utilized Python or R scripts for data analysis, and we have provided examples of data analysis scripts and acquired data for extended exposure periods (≤7 days). By using FLOS (Free-Libre and open-source) software and hardware, we have developed a low-cost and customizable system that can be used for a variety of exposure protocols. This hypoxia/hyperoxia exposure chamber allows for reproducible and transparent data acquisition and increased consistency with a high degree of customization for each experimenter’s needs.
Gliosarcomas are an aggressive brain cancer that can become metastatic. Previously, we used a monoclonal antibody, designated H5, to isolate a tumor-associated antigen (TAA) expressed by 9L, a gliosarcoma line isolated from Fischer 344 (F344) rats. This TAA was tentatively identified as vimentin via mass spectrometry. In this project, we compared the level of vimentin mRNA in 9L cells to that of three types of glial cells: microglial cells, astrocytes, and oligodendrocytes isolated from F344 rats via qPCR. The level of vimentin mRNA in 9L cells was significantly upregulated as compared to the expression levels in all three types of glial cells. Similar research on carcinomas has identified vimentin as being involved in metastatic tumors and is now used as a genetic marker of this specific type of cancer. This research suggests that vimentin mRNA in 9L undergoes similar upregulation to that of carcinomas.
High‐altitude long term hypoxia (LTH) during gestation compromises the development and function of the pulmonary vasculature in the fetus and later in the newborn. This may manifest as fetal growth restriction as well as pulmonary hypertension following birth. While the physiological regulation regarding the effects of hypoxia are well‐known, the dysregulation that occurs at the cellular and molecular level is not yet completely understood. Exploring the effects of LTH using a metabolomic approach is advantageous in deciphering the etiology associated with the development of disease. LTH is well regarded for inducing oxidative stress and causing inflammation. A number of possible regulators associated with LTH stress include oxylipins and endocannabinoids, products of polyunsaturated fatty acid (PUFA) oxidation. We hypothesized that high‐altitude LTH would reduce the levels of oxylipins and endocannabinoids of pulmonary arteries and plasma in fetal sheep. To test the hypothesis, we obtained samples of plasma and pulmonary arteries from fetal normoxic and hypoxic sheep raised at 3,800 m. altitude starting on gestation day 30. Metabolite levels were quantified using ultra performance liquid chromatography‐tandem mass spectrometry (UPLC‐MS/MS). Chemical similarity enrichment analyses and visualization by complex pathway analyses was performed on the datasets. Our results support our hypothesis that select oxylipin concentrations were diminished in both venous plasma and pulmonary arteries. Omega‐3 PUFAs alpha linolenic acid (ALA) and eicosapentaenoic acid (EPA) were reduced in both plasma and pulmonary arteries. 15‐HEPE and 12‐HEPE, derivatives of EPA and important anti‐inflammatory mediators, were also significantly reduced by LTH in plasma and pulmonary arterial samples from fetal animals. We believe that our studies provide insight toward identifying key biomarkers of LTH stress that will afford effective and prompt diagnosis of the neonate's physiological response. This is important when it comes to treating diseases associated with gestational hypoxia, which affects numerous mothers and newborns worldwide.Support or Funding InformationThis material is based upon work supported by NIH grants P01HD083132 (LZ) and 1U24DK097154 through a pilot project grant to SMW. VL is a recipient of the American Physiological Society's Short‐Term Research Education Program to Increase Diversity in Health‐Related Research (STRIDE) Fellowship funded by the APS and a grant from the National Heart, Lung and Blood Institute R25HL115473.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
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