P219. Mitochondrial Transplantation is Feasible in an In Vitro Neuronal Cell Model and Ameliorates Ischemia-Reperfusion Injury
Presented During: POD Abstracts (Available for viewing in the exhibit hall for the duration of the meeting)
University of Colorado Anschutz
Denver, CO
United States - Contact Me
Adam Carroll is a current PGY-3 surgical resident at the University of Colorado. Adam attended medical school at the University of Colorado and has been involved in research with the cardiothoracic surgery department throughout medical school and residency. He has interest specifically in endovascular and transcatheter aortic interventions, as well as neurologic outcomes in aortic research. He is currently in the aortic surgery research labaratory in the Department of Cardiothoracic Surgery at the University of Colorado. He plans to pursue a career in cardiothoracic surgery following his general surgery residency.
Thursday, April 25, 2024: 5:38 PM - 7:00 PM
Sheraton Times Square
Room: Central Park
Description
Objective
Neurologic injury due to ischemia-reperfusion injury (IRI) is a severe outcome of aortic arch surgery, with no pharmacologic treatment available. Mitochondria (MT) play a key role IRI, with initial oxygen-glucose deprivation depleting adenosine triphosphate, and subsequent production of reactive oxygen and nitrogen species leading to cell death. MT transplantation (MTR) has shown promise in other tissue models. We sought to develop an in vitro model of MTR for neuronal cells with the goal of application to aortic surgery.
Methods
MT were harvested from male mice at four different tissue sites and homogenized. MT were isolated and resuspended in PBS. MT concentration was detected via BCA and adjusted to three concentrations (1mg/ml, 0.01mg/ml, and 0.0002mg/ml) and stained.
Ischemia-Reperfusion Model
HT-22, a mouse hippocampal cell line, was cultured in 96 well plates. Cells were pre-cultured for 24 hours prior to ischemia. Ischemia was simulated via an oxygen-glucose deprived (OGD) cell medium and placement into a hypoxia chamber for 18 hours resulting in 30-50% remaining cell viability. Control cells were placed in new culture medium and returned to the incubator. Following OGD exposure, cells were placed in new culture medium and returned to the incubator for a period of 24 hours. At 24 hours, cell viability was assessed via MTS assay.
Transplantation
MTR was performed at two time points: during the pre-culture phase and during reperfusion simultaneous to addition of culture medium. The above three concentrations of MT were added at ratios of 1:5, 1:20 or 1:100 (cells:MT).
Results
BCA demonstrated excellent MT yield from all tissues, with the highest yield from brain and liver tissues. Co-culture with stained neuronal cells demonstrated excellent exogenous MT incorporation.
Incorporation into IRI model
For both control and OGD cells MTR during the reperfusion phase resulted in a significant dose-dependent increase in both control and OGD cell death. MTR performed during the pre-culture phase increased OGD cell viability for the 1mg/ml concentration at 1:100, and for the 0.01mg/ml for all ratios depending on tissue selected. MTR performed at the higher concentrations resulted in an increase in cell death for both groups of cells.
Conclusion
MTR is feasible in a neuronal cell model. In an IRI model, MTR prior to ischemia can preserve cell viability at optimal concentrations. MTR performed during reperfusion results in a dose-dependent increase in cell death.
Authors
Adam Carroll (1), Linling Cheng (1), William Riley Keeler (1), Bo Chang Wu (1), Anastacia Garcia (1), Muhammad Aftab (1), T. Brett Reece (1)
Institutions
(1) University of Colorado Anschutz, Denver, CO
Neurologic injury due to ischemia-reperfusion injury (IRI) is a severe outcome of aortic arch surgery, with no pharmacologic treatment available. Mitochondria (MT) play a key role IRI, with initial oxygen-glucose deprivation depleting adenosine triphosphate, and subsequent production of reactive oxygen and nitrogen species leading to cell death. MT transplantation (MTR) has shown promise in other tissue models. We sought to develop an in vitro model of MTR for neuronal cells with the goal of application to aortic surgery.
Methods
MT were harvested from male mice at four different tissue sites and homogenized. MT were isolated and resuspended in PBS. MT concentration was detected via BCA and adjusted to three concentrations (1mg/ml, 0.01mg/ml, and 0.0002mg/ml) and stained.
Ischemia-Reperfusion Model
HT-22, a mouse hippocampal cell line, was cultured in 96 well plates. Cells were pre-cultured for 24 hours prior to ischemia. Ischemia was simulated via an oxygen-glucose deprived (OGD) cell medium and placement into a hypoxia chamber for 18 hours resulting in 30-50% remaining cell viability. Control cells were placed in new culture medium and returned to the incubator. Following OGD exposure, cells were placed in new culture medium and returned to the incubator for a period of 24 hours. At 24 hours, cell viability was assessed via MTS assay.
Transplantation
MTR was performed at two time points: during the pre-culture phase and during reperfusion simultaneous to addition of culture medium. The above three concentrations of MT were added at ratios of 1:5, 1:20 or 1:100 (cells:MT).
Results
BCA demonstrated excellent MT yield from all tissues, with the highest yield from brain and liver tissues. Co-culture with stained neuronal cells demonstrated excellent exogenous MT incorporation.
Incorporation into IRI model
For both control and OGD cells MTR during the reperfusion phase resulted in a significant dose-dependent increase in both control and OGD cell death. MTR performed during the pre-culture phase increased OGD cell viability for the 1mg/ml concentration at 1:100, and for the 0.01mg/ml for all ratios depending on tissue selected. MTR performed at the higher concentrations resulted in an increase in cell death for both groups of cells.
Conclusion
MTR is feasible in a neuronal cell model. In an IRI model, MTR prior to ischemia can preserve cell viability at optimal concentrations. MTR performed during reperfusion results in a dose-dependent increase in cell death.
Authors
Adam Carroll (1), Linling Cheng (1), William Riley Keeler (1), Bo Chang Wu (1), Anastacia Garcia (1), Muhammad Aftab (1), T. Brett Reece (1)
Institutions
(1) University of Colorado Anschutz, Denver, CO
Presentation Duration
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