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Retinal synaptic regeneration via microfluidic guiding channels.

Su PJ, Liu Z, Zhang K, Han X, Saito Y, Xia X, Yokoi K, Shen H, Qin L - Sci Rep (2015)

Bottom Line: The rate of RSR decreased significantly below 100 μM of inhibitor glycine and then approached to a relatively constant level at higher concentrations.Furthermore, RSR was enhanced by chemical stimulation with potassium chloride.Collectively, the microfluidic synaptic regeneration chip provides a novel tool for high-throughput investigation of RSR at the cellular level and may be useful in quality control of retinal precursor cell transplantation.

View Article: PubMed Central - PubMed

Affiliation: Department of Nanomedicine, Houston Methodist Research Institute, Houston, TX 77030, USA.

ABSTRACT
In vitro culture of dissociated retinal neurons is an important model for investigating retinal synaptic regeneration (RSR) and exploring potentials in artificial retina. Here, retinal precursor cells were cultured in a microfluidic chip with multiple arrays of microchannels in order to reconstruct the retinal neuronal synapse. The cultured retinal cells were physically connected through microchannels. Activation of electric signal transduction by the cells through the microchannels was demonstrated by administration of glycinergic factors. In addition, an image-based analytical method was used to quantify the synaptic connections and to assess the kinetics of synaptic regeneration. The rate of RSR decreased significantly below 100 μM of inhibitor glycine and then approached to a relatively constant level at higher concentrations. Furthermore, RSR was enhanced by chemical stimulation with potassium chloride. Collectively, the microfluidic synaptic regeneration chip provides a novel tool for high-throughput investigation of RSR at the cellular level and may be useful in quality control of retinal precursor cell transplantation.

No MeSH data available.


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Induction of functional synaptic communication by glycinergic factors and potassium chloride (KCl).(a) Immunostaining image of phosphorylated extracellular-related kinase (pERK) in retinal cells treated with glycine at concentrations of 0, 10 and 1000 μM. Scale bar, 50 μm. (b) Histogram of fluorescence intensity of pERK and glycine concentrations at 0, 10 and 1000 μM. *p < 0.05. (c–d) Effect of microchannel width (4 μm and 3 μm) with channel length of 100 μm (c) and 50 μm (d) on retinal synaptic regeneration at glycine concentrations of 0, 50, 100, 500 and 1000 μM. W: channel width. L: Channel length. (e) Dynamics of chemically induced retina synaptic regeneration via the stimulation of KCl, compared to control sample without KCl treatment on days 3, 5 and 8. The data in b and c represents the mean ± s.e.m. with n = 3.
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f3: Induction of functional synaptic communication by glycinergic factors and potassium chloride (KCl).(a) Immunostaining image of phosphorylated extracellular-related kinase (pERK) in retinal cells treated with glycine at concentrations of 0, 10 and 1000 μM. Scale bar, 50 μm. (b) Histogram of fluorescence intensity of pERK and glycine concentrations at 0, 10 and 1000 μM. *p < 0.05. (c–d) Effect of microchannel width (4 μm and 3 μm) with channel length of 100 μm (c) and 50 μm (d) on retinal synaptic regeneration at glycine concentrations of 0, 50, 100, 500 and 1000 μM. W: channel width. L: Channel length. (e) Dynamics of chemically induced retina synaptic regeneration via the stimulation of KCl, compared to control sample without KCl treatment on days 3, 5 and 8. The data in b and c represents the mean ± s.e.m. with n = 3.

Mentions: We next examined the retinal synaptic function of the inhibitory neurotransmitter receptors and determined whether the oriented synaptic network connected through the microchannels was active. Glutamate is a major neurotransmitter transported through the retina via the vertical pathway from photoreceptors to bipolar cells and then to ganglion cells16. Electrical stimulation of the glutamatergic retinal signal transduction pathway triggers the phosphorylation cascade that leads to activation of ERK1319. R28 cells were treated with glycine, and pathway activity was assessed by fluorescence imaging of pERK. Glycine is one of the major inhibitory neurotransmitters in mammalian retina20. R28 cells were treated with 0 μM, 10 μM, and 1000 μM glycine (Fig. 3a). We found that the phosphorylation of ERK was increased by 20% at a low dose of 10 μM compared with the high dose of 1000 μM (Fig. 3b).


Retinal synaptic regeneration via microfluidic guiding channels.

Su PJ, Liu Z, Zhang K, Han X, Saito Y, Xia X, Yokoi K, Shen H, Qin L - Sci Rep (2015)

Induction of functional synaptic communication by glycinergic factors and potassium chloride (KCl).(a) Immunostaining image of phosphorylated extracellular-related kinase (pERK) in retinal cells treated with glycine at concentrations of 0, 10 and 1000 μM. Scale bar, 50 μm. (b) Histogram of fluorescence intensity of pERK and glycine concentrations at 0, 10 and 1000 μM. *p < 0.05. (c–d) Effect of microchannel width (4 μm and 3 μm) with channel length of 100 μm (c) and 50 μm (d) on retinal synaptic regeneration at glycine concentrations of 0, 50, 100, 500 and 1000 μM. W: channel width. L: Channel length. (e) Dynamics of chemically induced retina synaptic regeneration via the stimulation of KCl, compared to control sample without KCl treatment on days 3, 5 and 8. The data in b and c represents the mean ± s.e.m. with n = 3.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4551994&req=5

f3: Induction of functional synaptic communication by glycinergic factors and potassium chloride (KCl).(a) Immunostaining image of phosphorylated extracellular-related kinase (pERK) in retinal cells treated with glycine at concentrations of 0, 10 and 1000 μM. Scale bar, 50 μm. (b) Histogram of fluorescence intensity of pERK and glycine concentrations at 0, 10 and 1000 μM. *p < 0.05. (c–d) Effect of microchannel width (4 μm and 3 μm) with channel length of 100 μm (c) and 50 μm (d) on retinal synaptic regeneration at glycine concentrations of 0, 50, 100, 500 and 1000 μM. W: channel width. L: Channel length. (e) Dynamics of chemically induced retina synaptic regeneration via the stimulation of KCl, compared to control sample without KCl treatment on days 3, 5 and 8. The data in b and c represents the mean ± s.e.m. with n = 3.
Mentions: We next examined the retinal synaptic function of the inhibitory neurotransmitter receptors and determined whether the oriented synaptic network connected through the microchannels was active. Glutamate is a major neurotransmitter transported through the retina via the vertical pathway from photoreceptors to bipolar cells and then to ganglion cells16. Electrical stimulation of the glutamatergic retinal signal transduction pathway triggers the phosphorylation cascade that leads to activation of ERK1319. R28 cells were treated with glycine, and pathway activity was assessed by fluorescence imaging of pERK. Glycine is one of the major inhibitory neurotransmitters in mammalian retina20. R28 cells were treated with 0 μM, 10 μM, and 1000 μM glycine (Fig. 3a). We found that the phosphorylation of ERK was increased by 20% at a low dose of 10 μM compared with the high dose of 1000 μM (Fig. 3b).

Bottom Line: The rate of RSR decreased significantly below 100 μM of inhibitor glycine and then approached to a relatively constant level at higher concentrations.Furthermore, RSR was enhanced by chemical stimulation with potassium chloride.Collectively, the microfluidic synaptic regeneration chip provides a novel tool for high-throughput investigation of RSR at the cellular level and may be useful in quality control of retinal precursor cell transplantation.

View Article: PubMed Central - PubMed

Affiliation: Department of Nanomedicine, Houston Methodist Research Institute, Houston, TX 77030, USA.

ABSTRACT
In vitro culture of dissociated retinal neurons is an important model for investigating retinal synaptic regeneration (RSR) and exploring potentials in artificial retina. Here, retinal precursor cells were cultured in a microfluidic chip with multiple arrays of microchannels in order to reconstruct the retinal neuronal synapse. The cultured retinal cells were physically connected through microchannels. Activation of electric signal transduction by the cells through the microchannels was demonstrated by administration of glycinergic factors. In addition, an image-based analytical method was used to quantify the synaptic connections and to assess the kinetics of synaptic regeneration. The rate of RSR decreased significantly below 100 μM of inhibitor glycine and then approached to a relatively constant level at higher concentrations. Furthermore, RSR was enhanced by chemical stimulation with potassium chloride. Collectively, the microfluidic synaptic regeneration chip provides a novel tool for high-throughput investigation of RSR at the cellular level and may be useful in quality control of retinal precursor cell transplantation.

No MeSH data available.


Related in: MedlinePlus