Localized microinjection of intact Drosophila melanogaster larva to investigate the effect of serotonin on heart rate
Localized microinjection of intact Drosophila melanogaster larva to investigate the effect of serotonin on heart rate
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DOI：
10.1039/c9lc00963a
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发表时间：
2020-01-21
期刊：
影响因子：
6.1
通讯作者：
Rezai, Pouya
中科院分区：
文献类型：
--
作者：
Zabihihesari, Alireza;Hilliker, Arthur J.;Rezai, Pouya
在本文中，我们提出了一种新的混合微流控装置的本地化显微注射和心脏监测的完整的果蝇幼虫在不同的发展阶段。果蝇幼虫期的心脏已被用作心脏疾病研究的模型。然而，先前的药理学和毒理学心脏研究仅限于解剖（半完整）的果蝇幼虫，不能用于给药后研究。与完整幼虫的显微注射相关的挑战包括个体幼虫的精细处理，微针穿透的适当方向，局部显微注射控制量的化学品进入血淋巴和可逆固定注射后表型研究，所有这些都由我们的微流体装置解决。幼虫装载和定向通过集成到PDMS微流体装置中的玻璃毛细管实现。在心脏活动记录之前，使用侧吸通道进行固定。局部显微注射实现了一个自由度的微针和定制的压力驱动的试剂输送系统，对心率和动物的生存能力没有任何不利影响。用我们的微流体装置证明了局部注射到靠近心脏腔室或脂肪体的体腔中的精确性。基于MATLAB的心跳定量技术被用来调查5-羟色胺（5-羟色胺），神经递质，对完整的果蝇幼虫的心率的剂量依赖性效应，为第一次。注射浓度>= 0.01 mM的40 nL 5-羟色胺使3龄幼虫的心率显著增加21 +/-7%（SEM）。注射浓度为0.01 mM的5 nL 5-羟色胺使2龄幼虫的心率显著增加12 +/-3%（SEM）。所提出的微流控注射和心跳监测技术可用于染料血管造影和血淋巴循环研究，以及使用整个动物果蝇在体内筛选静脉内药物。
In this paper, we present a novel hybrid microfluidic device for localized microinjection and heart monitoring of intact Drosophila melanogaster larvae at different developmental stages. Drosophila heart at the larval stage has been used as a model for cardiac disorder studies. However, previous pharmacological and toxicological cardiac studies are limited to dissected (semi-intact) Drosophila larvae which cannot be used for post-treatment studies. Challenges associated with microinjection of intact larvae include delicate handling of individual larvae, proper orientation for microneedle penetration, localized microinjection with controlled amount of chemicals into the hemolymph and reversible immobilization for post-injection phenotypic studies, all addressed by our microfluidic device. Larva loading and orientation were achieved by glass capillaries integrated into the PDMS microfluidic device. Side suction channels were used for immobilization prior to heart activity recording. Localized microinjection was achieved with a one degree-of-freedom microneedle and a custom-made pressure driven reagent delivery system, without any adverse effect on heart rate and animal viability. Precision in localized injection into the body cavity close to the heart chamber or the fat body was demonstrated with our microfluidic device. A MATLAB-based heartbeat quantification technique was used to investigate the dose-dependent effect of serotonin (5-hydroxytryptamine), a neurotransmitter, on the heart rate of intact Drosophila larvae, for the first time. Injection of 40 nL serotonin with >= 0.01 mM concentration significantly increased the heart rate of 3(rd) instar larvae by 21 +/- 7% (SEM). Injection of 5 nL serotonin with a concentration of 0.01 mM significantly increased the heart rate of 2(nd) instar larvae by 12 +/- 3% (SEM). The proposed microfluidic injection and heartbeat monitoring technique can be used for dye angiography and hemolymph circulation studies as well as screening intravenous drugs in vivo using the whole-animal Drosophila melanogaster.