TY - JOUR

T1 - Increasing cellular lifespan with a flow system in organotypic culture of the Laterodorsal Tegmentum (LDT)

AU - Leguizamon, Cesar Ramon Romero

AU - Elnagar, Mohamed

AU - Kristiansen, Uffe

AU - Kohlmeier, Kristi Anne

PY - 2019/2/6

Y1 - 2019/2/6

N2 - Organotypic brain culture is an experimental tool widely used in neuroscience studies. One majordrawback of this technique is reduced neuronal survival across time, which is likely exacerbated by theloss of blood flow. We have designed a novel, tube flow system, which is easily incorporated into thecommonly-used, standard semi-permeable membrane culture methodology which has significantlyenhanced neuronal survival in a brain stem nucleus involved in control of motivated and arousal states:the laterodorsal tegmental nucleus (LDT). Our automated system provides nutrients and removeswaste in a comparatively aseptic environment, while preserving temperature, and oxygen levels. Usingimmunohistochemistry and electrophysiology, our system was found superior to standard techniquesin preserving tissue quality and survival of LDT cells for up to 2 weeks. In summary, we provideevidence for the first time that the LDT can be preserved in organotypic slice culture, and further, ourtechnical improvements of adding a flow system, which likely enhanced perfusion to the slice, wereassociated with enhanced neuronal survival. Our perfusion system is expected to facilitate organotypicexperiments focused on chronic stimulations and multielectrode recordings in the LDT, as well asenhance neuronal survival in slice cultures originating from other brain regions.

AB - Organotypic brain culture is an experimental tool widely used in neuroscience studies. One majordrawback of this technique is reduced neuronal survival across time, which is likely exacerbated by theloss of blood flow. We have designed a novel, tube flow system, which is easily incorporated into thecommonly-used, standard semi-permeable membrane culture methodology which has significantlyenhanced neuronal survival in a brain stem nucleus involved in control of motivated and arousal states:the laterodorsal tegmental nucleus (LDT). Our automated system provides nutrients and removeswaste in a comparatively aseptic environment, while preserving temperature, and oxygen levels. Usingimmunohistochemistry and electrophysiology, our system was found superior to standard techniquesin preserving tissue quality and survival of LDT cells for up to 2 weeks. In summary, we provideevidence for the first time that the LDT can be preserved in organotypic slice culture, and further, ourtechnical improvements of adding a flow system, which likely enhanced perfusion to the slice, wereassociated with enhanced neuronal survival. Our perfusion system is expected to facilitate organotypicexperiments focused on chronic stimulations and multielectrode recordings in the LDT, as well asenhance neuronal survival in slice cultures originating from other brain regions.

U2 - 10.1038/s41598-018-37606-3

DO - 10.1038/s41598-018-37606-3

M3 - Journal article

C2 - 30728375

SP - 1

EP - 11

JO - Scientific Reports

JF - Scientific Reports

SN - 2045-2322

ER -