Endotracheal intubation is a vital component of many rat in vivo experiments to secure the airway and allow controlled ventilation. Even in the hands of experienced researchers, however, the procedure remains technically challenging. The safest and most reliable way for human intubation is by video laryngoscopy. Previous attempts to apply this technique in rodents have been complicated and expensive. We, hereby, describe a novel, noninvasive method to safely intubate rats orally by video laryngoscopy, thus avoiding the need for a surgical tracheostomy. By repurposing a commercially available ear wax removal device, visualization of the rat larynx can be significantly enhanced. Because of its small diameter, integrated illumination, and a powerful camera with adequate focal length, the device has all of the necessary properties for exploring the upper airway of a rat. After identifying the vocal cords by video laryngoscopy, the insertion of an endotracheal tube (a 14G intravenous catheter) into the trachea under constant visual control is facilitated by using PE50 polyethylene tubing as a stylet (Seldinger technique). The procedure has been performed more than 60 times in our laboratory; all intubations were successful on the first attempt, and no adverse events were observed. We conclude that the described procedure is a simple and effective way to intubate a rat noninvasively, using inexpensive and commercially available equipment.
INTRODUCTIONTraumatic brain injury (TBI) continues to be a leading cause of death and disability, not only in the US but worldwide. Approaches to mimic the injury mechanisms underlying TBI (e.g., stretch, shear and compression) include various models; in‐vivo models better simulate clinical effects, while in‐vitro models focus on the biomechanics of TBI. The effect of the injury in‐vitro can be examined without influence of systemic confounders, and the experiments are well‐controlled, reproducible and isolated from environmental impacts. Therefore, our objective was to establish an in‐vitro TBI model using mouse brain microvascular endothelial cells (MBEC), focusing on compression injury, to further understand underlying pathomechanisms and to test potential treatments. We compared 1) normoxic cells ± compression vs hypoxic cells ± compression and 2) varied hypoxia exposure times.METHODSMBEC cultures were grown to confluency and placed into either normoxic (complete media; 5% CO2, 95% air [21% O2]; 37°C) or hypoxic (glucose‐, serum‐free media; 0.01% O2, 5% CO2, N2 balance; 37°C) conditions. Hypoxia times of 5h, 7.5h, 10h, 12.5h vs 15h were compared. In addition, during the first hour of normoxia/hypoxia, compression (1kg/0.16cm²) was added. After 2h of reperfusion in normoxic conditions, following normoxia/hypoxia, samples were assayed for cell number, cytotoxicity (lactate dehydrogenase [LDH] release), and metabolic activity. Statistics: Data expressed as mean ± SEM. Kruskal‐Wallis one‐way analysis of variance (ANOVA) on Ranks, Dunn's Method; p <0.05, * vs normoxia (each timepoint), Ϯ vs hypoxia (prior timepoint).RESULTSCompared to normoxic conditions, a significant decrease in cell number and metabolic activity, as well as an increase in LDH release, was seen after exposing cells to hypoxia at all durations; except for 12.5h hypoxia, where there was no significant difference in metabolic activity. Compression added significant damage to hypoxia‐exposed MBEC by further decreasing cell number and metabolic activity. However, the effect of compression during hypoxia decreased with increasing hypoxia time. Furthermore, there were significant differences in cell number among the different hypoxia times. Surprisingly, there was only a significant difference for LDH release at 7.5h vs 10h and 12.5h vs 15h of hypoxia and for metabolic activity at 5h vs 7.5h and 12.5h vs 15h of hypoxia.CONCLUSIONOur data show that 5h hypoxia with compression is sufficient to cause significant injury in MBEC cultures. Extending hypoxia time leads to an even greater increase in damage; however, with increasing hypoxia time, the effect of compression is reduced. Therefore, 5h hypoxia with 1h compression is a suitable in‐vitro MBEC TBI model for testing potential treatments.Support or Funding InformationThis work was supported by institutional funds, NIH grant (5R01 HL123227), and a Merit Review Award (I01 BX003482) from the U.S. Department of Veterans Affairs Biomedical Laboratory R&D Service.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Introduction: Traumatic brain injury (TBI), a major cause of severe disability and death, can lead to disruption of the vascular endothelial system and cell membrane integrity, threatening the survival of multiple cell types. Reducing these could improve the clinical outcome of TBI. Poloxamer 188 (P188) has been shown to protect different cell types against ischemia/reperfusion (IR) injury. Hypothesis: P188 protects mouse brain microvascular endothelial cells (MBECs) against injury in an in-vitro compression-type TBI model. Methods: Confluent MBEC cultures were exposed to normoxic (complete media; 21% O 2 , 5% CO 2 , 74% N 2 ; 37°C) or hypoxic (glucose-, serum-free media; 0.01% O 2 , 5% CO 2 , N 2 balance; 37°C) conditions for 5 hrs, with compression (9.81 N / 0.16 cm 2 ) added during the first hour of normoxia/hypoxia. All MBECs then underwent 2 hrs of reoxygenation in normoxic conditions ± P188 (10 μM, 100 μM, 1 mM). Samples were assayed for cell number, cytotoxicity (lactate dehydrogenase [LDH] release), and metabolic activity. Statistics: Data are mean ± SEM. Kruskal-Wallis one-way analysis of variance on Ranks, Dunn’s Method; p <0.05, * vs normoxia, † vs hypoxia, ** vs normoxia + compression, †† vs hypoxia + compression; n = 11-19 experiments/group. Results: Compared to normoxic cells without compression, cell number and metabolic activity decreased and cytotoxicity increased in cells exposed to hypoxic conditions +/- compression followed by reoxygenation. In hypoxic cells, 1 mM P188 increased cell number and metabolic activity and decreased cytotoxicity, while 100 μM only increased metabolic activity and decreased cytotoxicity and 10 μM only increased metabolic activity. In hypoxic compressed cells, no concentration of P188 improved cell number, however, 10 μM and 100 μM P188 increased metabolic activity, while 1 mM increased metabolic activity and decreased cytotoxicity. There was no difference between normoxic compressed and non-compressed cells in any assay, although all concentrations of P188 tested increased metabolic activity in normoxic compressed cells. Conclusion: P188, present during reoxygenation, provides protection to MBECs exposed to simulated IR injury, as well as compression-type TBI.
Myocardial infarction is a leading cause for morbidity and mortality worldwide. The only viable treatment for the ischemic insult is timely reperfusion, which further exacerbates myocardial injury. Maintaining mitochondrial function is crucial in preserving cardiomyocyte function in ischemia reperfusion (IR) injury. Poloxamer (P) 188 has been shown to improve cardiac IR injury by improving cellular and mitochondrial function. The aim of this study was to show if P188 postconditioning has direct protective effects on mitochondrial function in the heart. Langendorff prepared rat hearts were subjected to IR injury ex-vivo and reperfused for 10 min with 1 mM P188 vs. vehicle. Cardiac mitochondria were isolated with 1 mM P188 vs. 1 mM polyethylene glycol (PEG) vs. vehicle by differential centrifugation. Mitochondrial function was assessed by adenosine triphosphate synthesis, oxygen consumption, and calcium retention capacity. Mitochondrial function decreased significantly after ischemia and showed mild improvement with reperfusion. P188 did not improve mitochondrial function in the ex-vivo heart, and neither further P188 nor PEG induced direct mitochondrial protection after IR injury in this model.
Introduction Despite best efforts of cardiopulmonary resuscitation (CPR), many patients still die or suffer severe cardio‐cerebral damage following cardiac arrest (CA). The quality of manual chest compressions (CC) rarely adheres to current guidelines with regards to CC rate, depth and fraction of time spent during CPR. Automated mechanical CC devices offer a reliable improvement in both the clinical arena and in research models. We describe the mechanical and programming aspects of the development of a depth controller for a pneumatic CC device for CPR in rodent CA model. Methods & Results In order to electronically control the amplitude/depth of the vertical CC piston (Weil Institute of Critical Care Medicine, Palm Springs, CA; Fig 1) that is pneumatically displaced between 0 and maximally 2 cm 200 times min−1 (duty cycle 1:1), a horizontally moving wedge was connected to a rack gearbox driven by a low‐voltage motor (Fischertechnik, Waldachtal, Germany) along a fixed rack. The unit was connected to a horizontally sliding potentiometer to monitor the exact position of the wedge and, therefore, the piston displacement which itself was monitored through a second, vertically sliding potentiometer. Both potentiometers were connected to an USB 6343 data acquisition system (DAQ, National Instruments, Austin, TX). The motor was connected to two analogue outputs of the DAQ (for movement and direction) via a commercially available low‐voltage amplifier. Stepwise movement of the wedge in either direction could be controlled by a momentary rocker switch connected to two analogue input channels of the DAQ (for deeper and shallower compressions) and its LabVIEW software which was programmed to allow precise control of the wedge’s position. Conclusions We report the design and construction of an inexpensive, LabVIEW‐based control unit to electronically adjust CC depth in a rodent model of CA and CPR. Neither the hardware, nor the software required to perform this highly specialized application were commercially available. This custom‐designed and ‐built device achieved its purpose of allowing electronic control and recording of the CC depth in this rodent model with high reliability and precision. Moreover, it constitutes the necessary basis of pre‐programmed or feedback‐assisted adjustments of variable CC depth in the future to improve neurologically favorable outcome after CA. Support or Funding Information This work was supported, in part, by institutional funds, and a Merit Review Award (I01 BX003482) from the U.S. Department of Veterans Affairs Biomedical Laboratory R&D Service. Pneumatic chest compression device for rodents with added control unit for electronic depth adjustment.
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