2018 Fiscal Year Research-status Report
変性網膜内層機能の最適化とiPSC由来網膜移植時のシナプス形成の促進に関する研究
| Project/Area Number |
17K16994
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| Research Institution | Institute of Physical and Chemical Research |
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Principal Investigator |
Tu HungYa 国立研究開発法人理化学研究所, 生命機能科学研究センター, 訪問研究員 (10780835)
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| Project Period (FY) |
2017-04-01 – 2020-03-31
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| Keywords | retinal transplantation / multi-electrode arrary |
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| Outline of Annual Research Achievements |
The present study has been proposed to clarify and optimize the host bipolar cell properties in the degenerated retinas for a more efficient synaptogenesis with the transplanted photoreceptors. A part of the present study is to test the possibility and effect of reducing inner retinal hyperactivity on transplantation of stem cell-derived retina into degenerated retinas, aiming to increase the connectivity between host rod bipolar cells and transplanted photoreceptors. In addition, the suppression of light-independent, spontaneous activities exhibited by cone bipolar cells and downstream ganglion cells would lead to a higher signal-to-noise ratio of the restored light signaling.The preliminary results show that a long-term, low dose of flupirtine treatment to transplanted mice may lead to increased graft-originated responsiveness detected by multi-electrode array (MEA) recording, However, a locally controlled release method restricted intraocularly is required, instead of the systematic application of flupirtine maleate dissolved in DMSO, to efficiently raise the working concentration of flupirtine right to the targeted AII amacrine cells of the host retina. On the other hand, the established MEA recording and analysis protocol has also been utilized in other transplantation preparations. showing its power in evaluating transplanted retinal tissues that exhibit degenerating, regenerating, mature and immature properties simultaneously.
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| Current Status of Research Progress |
Current Status of Research Progress
2: Research has progressed on the whole more than it was originally planned.
Reason
The pharmacological suppression of hyperactivity is expected to facilitate the host rod bipolar cells to better form synaptic connection with the transplanted photoreceptors, as well as the cone bipolar cells and downstream ganglion cells to exhibit less light-independent, spontaneous activities as noises.The preliminary results show that a lower dose of flupirtine treated to transplanted mice may lead to increased graft-originated responsiveness detected by MEA recording, while the higher doses failed with a drop of responsiveness, which is very likely due to the thus increased concentration of DMSO as the solvent.
On the other hand, the quantitative evaluation of transplantation by MEA recording has been utilized in parallel on many other preparations, including mouse allo-transplantation and human-rat xeno-transplantation aside from the present study. The protocol of recording and analysis has been much improved during the past year and invited to be published as a methodology book chapter.
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| Strategy for Future Research Activity |
An inevitable elevation of DMSO concentration happens when applying higher flupirtine dosage. Also, a recent study indicates that the metabolic rate of flupirtine in postnatal rats (similar to mice in size) is much higher than in larger mammals (horses and humans) that i.v. injection of flupirtine results the peak of concentration observation after only a few hours in various organs including eyes. A locally controlled release method restricted intraocularly is therefore required instead of the systematic application of flupirtine maleate dissolved in DMSO. On the other hand, the direct/indirect elevation of host rod bipolar cell excitability has to be further tested by targeted patch clamp recording. The dual patch clamp recording of transplanted photoreceptor-host bipolar cell pairs may also be conducted to test the direct synaptic inputs, taking the advantage of genetic labeling of both transplants and host retinas.
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| Causes of Carryover |
The travel expense has been lower than expected because of the generous coverage by other funding sources and also the travel grant provided by the international conference committee. Also, the expected expense for conducting targeted patch clamp recording has been postponed. Besides the planned expenses, the residual amount will partially be used for development of controlled release method of flupirtine or other channel blockers that may suppress the inner retinal hyperactivity of degenerated retinas.
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