Name: methodology for biomimetic chemical neuromodulation of rat retinas with the neurotransmitter glutamate in vitro
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Description: Watch this Scientific Journal Video about Methodology for Biomimetic Chemical Neuromodulation of Rat Retinas with the Neurotransmitter Glutamate In Vitro at JoVE.com

Det arbejde fremlagt i papiret blev støttet af National Science Foundation, nye grænser inden for forskning og Innovation (NSF-EFRI) program giver nummer 0938072. Indholdet af dette dokument er udelukkende ansvarlig for forfattere og repræsenterer ikke nødvendigvis de officielle synspunkter af NSF. Forfatterne ønsker også at takke Dr. Samsoon Inayat for hans arbejde, designe og afprøve den indledende eksperimentelle opsætning for kemiske stimulation og Mr. Ashwin Raghunathan for hans arbejde designe, opdigte og evaluere multiport mikrofluid enheden bruges i denne undersøgelse.

Alle dyreforsøg blev gennemført i overensstemmelse med retningslinjerne skitseret af National Research Council's Guide til pleje og anvendelse af forsøgsdyr. Animalske håndtering og aktiv dødshjælp protokoller blev gennemgået og godkendt af institutionelle Animal Care og brug udvalg (IACUC) af University of Illinois i Chicago.
1. animalske modeller
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		<li>Skaffe en 24-32 daggamle vildtype lang Evans hætteklædte rotte af beg...

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	<li>Pascolini, D., Mariotti, S. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global+estimates+of+visual+impairment:+2010.">Global estimates of visual impairment: 2010.</a> Br J Ophthalmol. , (2011).</li><li>Fritsche, L. G., Fariss, R. N., Stambolian, D., Abecasis, G. R., Curcio, C. A., Swaroop, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Age-Related+Macular+Degeneration:+Genetics+and+Biology+Coming+Together.">Age-Related Macular Degeneration: Genetics and Biology Coming Together.</a> Annu Rev Genomics Hum Genet. 15, 151-171 (2014).</li><li>Marc, R. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+reprogramming+in+retinal+degeneration.">Neural reprogramming in retinal degeneration.</a> Invest Ophthalmol Vis Sci. 48, 3364-3371 (2007).</li><li>Jones, B. W., Kondo, M., Terasaki, H., Lin, Y., McCall, M., Marc, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retinal+remodeling.">Retinal remodeling.</a> Jpn J Ophthalmol. 56, 289-306 (2012).</li><li>Soto, F., Kerschensteiner, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synaptic+remodeling+of+neuronal+circuits+in+early+retinal+degeneration.">Synaptic remodeling of neuronal circuits in early retinal degeneration.</a> Front Cell Neurosci. 9, (2015).</li><li>Trenholm, S., Awatramani, G. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Origins+of+spontaneous+activity+in+the+degenerating+retina.">Origins of spontaneous activity in the degenerating retina.</a> Front Cell Neurosci. 9, (2015).</li><li>Euler, T., Schubert, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiple+Independent+Oscillatory+Networks+in+the+Degenerating+Retina.">Multiple Independent Oscillatory Networks in the Degenerating Retina.</a> Front Cell Neurosci. 9, (2015).</li><li>Boye, S. E., Boye, S. L., Lewin, A. S., Hauswirth, W. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Comprehensive+Review+of+Retinal+Gene+Therapy.">A Comprehensive Review of Retinal Gene Therapy.</a> Mol Ther. 21, 509-519 (2013).</li><li>Schwartz, S. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+embryonic+stem+cell-derived+retinal+pigment+epithelium+in+patients+with+age-related+macular+degeneration+and+Stargardt's+macular+dystrophy:+follow-up+of+two+open-label+phase+1/2+studies.">Human embryonic stem cell-derived retinal pigment epithelium in patients with age-related macular degeneration and Stargardt's macular dystrophy: follow-up of two open-label phase 1/2 studies.</a> The Lancet. 385, 509-516 (2015).</li><li>Reh, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photoreceptor+Transplantation+in+Late+Stage+Retinal+Degeneration.">Photoreceptor Transplantation in Late Stage Retinal Degeneration.</a> Invest Ophthalmol Vis Sci. 57, (2016).</li><li>Zrenner, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fighting+blindness+with+microelectronics.">Fighting blindness with microelectronics.</a> Sci Transl Med. 5, (2013).</li><li>Humayun, M. S., de Juan, E., Dagnelie, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Bionic+Eye:+A+Quarter+Century+of+Retinal+Prosthesis+Research+and+Development.">The Bionic Eye: A Quarter Century of Retinal Prosthesis Research and Development.</a> Ophthalmol. 123, S89-S97 (2016).</li><li>Cruz, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Argus+II+epiretinal+prosthesis+system+allows+letter+and+word+reading+and+long-term+function+in+patients+with+profound+vision+loss.">The Argus II epiretinal prosthesis system allows letter and word reading and long-term function in patients with profound vision loss.</a> Br J Ophthalmol. 97, 632-636 (2013).</li><li>Zrenner, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Subretinal+electronic+chips+allow+blind+patients+to+read+letters+and+combine+them+to+words.">Subretinal electronic chips allow blind patients to read letters and combine them to words.</a> P R Soc B. 278, 1489-1497 (2011).</li><li>Stronks, H. C., Dagnelie, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+functional+performance+of+the+Argus+II+retinal+prosthesis.">The functional performance of the Argus II retinal prosthesis.</a> Expert Rev Med Devices. 11, 23-30 (2014).</li><li>Stingl, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Artificial+vision+with+wirelessly+powered+subretinal+electronic+implant+alpha-IMS.">Artificial vision with wirelessly powered subretinal electronic implant alpha-IMS.</a> P R Soc B. 280, (2013).</li><li>Rizzo, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Update+on+retinal+prosthetic+research:+the+Boston+Retinal+Implant+Project.">Update on retinal prosthetic research: the Boston Retinal Implant Project.</a> J Neuroophthalmol. 31, 160-168 (2011).</li><li>Ayton, L. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=First-in-Human+Trial+of+a+Novel+Suprachoroidal+Retinal+Prosthesis.">First-in-Human Trial of a Novel Suprachoroidal Retinal Prosthesis.</a> PLoS ONE. 9, e115239 (2014).</li><li>Chuang, A. T., Margo, C. E., Greenberg, P. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retinal+implants:+a+systematic+review.">Retinal implants: a systematic review.</a> Br J Ophthalmol. 98, 852-856 (2014).</li><li>Cai, C., Twyford, P., Fried, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+response+of+retinal+neurons+to+high-frequency+stimulation.">The response of retinal neurons to high-frequency stimulation.</a> J Neural Eng. 10, 036009 (2013).</li><li>Eiber, C. D., Lovell, N. H., Suaning, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Attaining+higher+resolution+visual+prosthetics:+a+review+of+the+factors+and+limitations.">Attaining higher resolution visual prosthetics: a review of the factors and limitations.</a> J Neural Eng. 10, 011002 (2013).</li><li>Humayun, M., Propst, R., de Juan, E., McCormick, K., Hickingbotham, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bipolar+surface+electrical+stimulation+of+the+vertebrate+retina.">Bipolar surface electrical stimulation of the vertebrate retina.</a> Arch Ophthalmol. 112, 110-116 (1994).</li><li>Zrenner, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Can+subretinal+microphotodiodes+successfully+replace+degenerated+photoreceptors?.">Can subretinal microphotodiodes successfully replace degenerated photoreceptors?.</a> Vision Res. 39, 2555-2567 (1999).</li><li>Majji, A. B., Humayun, M. S., Weiland, J. D., Suzuki, S., D'Anna, S. A., de Juan, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-Term+Histological+and+Electrophysiological+Results+of+an+Inactive+Epiretinal+Electrode+Array+Implantation+in+Dogs.">Long-Term Histological and Electrophysiological Results of an Inactive Epiretinal Electrode Array Implantation in Dogs.</a> Invest Ophthalmol Vis Sci. 40, 2073-2081 (1999).</li><li>Peterman, M. C., Noolandi, J., Blumenkranz, M. S., Fishman, H. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Localized+chemical+release+from+an+artificial+synapse+chip.">Localized chemical release from an artificial synapse chip.</a> PNAS. 101, 9951-9954 (2004).</li><li>Finlayson, P. G., Iezzi, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glutamate+stimulation+of+retinal+ganglion+cells+in+normal+and+s334ter-4+rat+retinas:+a+candidate+for+a+neurotransmitter-based+retinal+prosthesis.">Glutamate stimulation of retinal ganglion cells in normal and s334ter-4 rat retinas: a candidate for a neurotransmitter-based retinal prosthesis.</a> Invest Ophthalmol Vis Sci. 51, 3619-3628 (2010).</li><li>Inayat, S., Rountree, C. M., Troy, J. B., Saggere, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemical+stimulation+of+rat+retinal+neurons:+feasibility+of+an+epiretinal+neurotransmitter-based+prosthesis.">Chemical stimulation of rat retinal neurons: feasibility of an epiretinal neurotransmitter-based prosthesis.</a> J Neural Eng. 12, 016010 (2015).</li><li>Rountree, C. M., Inayat, S., Troy, J. B., Saggere, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+stimulation+of+the+retina+with+subretinally+injected+exogenous+neurotransmitter:+A+biomimetic+alternative+to+electrical+stimulation.">Differential stimulation of the retina with subretinally injected exogenous neurotransmitter: A biomimetic alternative to electrical stimulation.</a> Sci Rep. 6, 38505 (2016).</li><li>Ray, A., Sun, G. J., Chan, L., Grzywacz, N. M., Weiland, J., Lee, E. -. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphological+alterations+in+retinal+neurons+in+the+S334ter-line3+transgenic+rat.">Morphological alterations in retinal neurons in the S334ter-line3 transgenic rat.</a> Cell Tissue Res. 339, 481-491 (2010).</li><li>Martinez-Navarrete, G., Seiler, M. J., Aramant, R. B., Fernandez-Sanchez, L., Pinilla, I., Cuenca, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retinal+degeneration+in+two+lines+of+transgenic+S334ter+rats.">Retinal degeneration in two lines of transgenic S334ter rats.</a> Exp Eye Res. 92, 227-237 (2011).</li><li>. Sigma Aldrich Ames Medium Product Information Sheet Available from: <a target="_blank" href="https://www.sigmaaldrich.com/content/dam/sigma-aldrich/docs/Sigma/Product_Information_Sheet/1/a1420pis.pdf">https://www.sigmaaldrich.com/content/dam/sigma-aldrich/docs/Sigma/Product_Information_Sheet/1/a1420pis.pdf</a> (2017)</li><li>Reinhard, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Step-By-Step+instructions+for+retina+recordings+with+perforated+multi+electrode+arrays.">Step-By-Step instructions for retina recordings with perforated multi electrode arrays.</a> PLoS ONE. 9, e106148 (2014).</li><li>Izumi, Y., Kirby, C. O., Benz, A. M., Olney, J. W., Zorumski, C. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=M&#252;ller+cell+swelling,+glutamate+uptake,+and+excitotoxic+neurodegeneration+in+the+isolated+rat+retina.">M&#252;ller cell swelling, glutamate uptake, and excitotoxic neurodegeneration in the isolated rat retina.</a> Glia. 25, 379-389 (1999).</li><li>Tunnicliff, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glutamate+uptake+by+chick+retina.">Glutamate uptake by chick retina.</a> Biochem J. 150, 297-299 (1975).</li><li>Schwartz, E. A., Tachibana, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrophysiology+of+glutamate+and+sodium+co-transport+in+a+glial+cell+of+the+salamander+retina.">Electrophysiology of glutamate and sodium co-transport in a glial cell of the salamander retina.</a> J Physiol (Lond). 426, 43-80 (1990).</li><li>Muller, A., Maurin, L., Bonne, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Free+radicals+and+glutamate+uptake+in+the+retina.">Free radicals and glutamate uptake in the retina.</a> Gen Pharmacol- Vasc S. 30, 315-318 (1998).</li><li>Dhingra, N. K., Kao, Y. -. H., Sterling, P., Smith, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Contrast+threshold+of+a+brisk-transient+ganglion+cell+in+vitro.">Contrast threshold of a brisk-transient ganglion cell in vitro.</a> J of Neurophysiol. 89, 2360-2369 (2003).</li><li>Ahlers, M. T., Ammerm&#252;ller, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+system+for+precise+temperature+control+of+isolated+nervous+tissue+under+optical+access:+Application+to+multi-electrode+recordings.">A system for precise temperature control of isolated nervous tissue under optical access: Application to multi-electrode recordings.</a> J of Neurosci Methods. 219, 83-91 (2013).</li><li>Feke, G. T., Tagawa, H., Deupree, D. M., Goger, D. G., Sebag, J., Weiter, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blood+flow+in+the+normal+human+retina.">Blood flow in the normal human retina.</a> Invest Ophthalmol Vis Sci. 30, 58-65 (1989).</li><li>Purves, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Retina.">The Retina.</a> Neuroscience. , (2001).</li></ol>

Metoden præsenteres her viser en unik neurale stimulation paradigme, hvori retinal neuroner kemisk stimuleres ved at indsprøjte indfødte neurotransmitter kemikalier i undergrunden af nethinden in vitro. Denne kemiske stimulation teknik tilbyder flere fordele over den konventionelle elektriske stimulation teknik, herunder selektivitet og høj fokale specificitet af target neuroner. Protokol over detaljer hvordan lille volumen pneumatisk injektioner af neurotransmitteren glutamat leveret nær målet retinal neu...

Fotoreceptor degenerative sygdomme, som retinitis pigmentosa og aldersrelateret makuladegeneration, er førende arvelige årsager til tab af synet og er i øjeblikket uhelbredelig1,2. Selv om disse sygdomme opstår fra en række specifikke genetiske mutationer, er fotoreceptor degenerative sygdomme karakteriseret som en gruppe af progressive tab af fotoreceptor celler i nethinden, som i sidste ende forårsager blindhed. Tab af fotoreceptorer udløser udbredt remodeling hele nethinden men overlevende retinal neuroner, herunder bipolar celler og RGCs, forblive intakt og relativt funktionelle selv i fremskredne stadier af fotoreceptor degeneration3 ,4,5,6,7.
De mekanismer og patologier af disse sygdomme er blevet godt præget3,4,5,6,7 , men en effektiv behandling er fortsat undvigende. I de seneste tre årtier, har forskere fra hele verden undersøgt en række terapeutiske behandlinger for at genoprette vision til de berørte med fotoreceptor degenerative sygdomme herunder gen terapi8, stamcelle behandling9, retinal transplantation10, og kunstig stimulation11,12 af de overlevende retinal neuroner. Af disse er den mest klinisk tilgængelige retinal proteser, som er kunstige neurostimulation enheder, der traditionelt har udnyttet en række elektroder til elektrisk stimulere enten bipolar celler eller RGCs i bestemte mønstre med mål at at skabe kunstige visuelle opfattelser i patienter11. Nuværende generation elektriske proteser, som Argus II13 og Alpha-IMS14 enheder, har opnået kliniske godkendelse og indledende undersøgelser har vist, at de kan forbedre livskvaliteten for patienter ved at genskabe en foranstaltning af vision ved hjælp af både epiretinal (forsiden af nethinden) og subretinal (bagsiden af nethinden) implanterede enheder15,16. Forskergrupper verden over arbejder på fremrykning retinal proteser ud over de gode resultater af disse førstegenerations enheder17,18,19,20 , men har stået over for vanskeligheder designe en elektrisk protese i stand til at genskabe høj skarphed vision under niveauet, juridiske blindhed til patienter. Nylige undersøgelser har vist, at opnå højere rumlige opløsning end der er aktiveret ved den nuværende generation elektrisk-baserede proteser er udfordrende afgift injektion begrænsning, som nødvendiggør brug af store elektroder til sikkert stimulere retinal neuroner på bekostning af rumlige opløsning, dvs synsstyrke11,21. Øvrigt elektrisk stimulation er yderligere begrænset, fordi det typisk stimulerer alle nærliggende celler og derfor fremkalder unaturlige og forvirrende opfattelser hos patienter, hovedsagelig fordi det er en ifølge sagens natur unaturligt stimulation paradigme21. Ikke desto mindre, de tidlige succeser af elektrisk stimulation har vist, at kunstige neurostimulation kan være en effektiv behandling for fotoreceptor degenerative sygdomme. Dette fører til at hypotesen om, at en endnu mere effektiv behandling kan opnås ved at stimulere nethinden med neurotransmitter kemikalier, naturlige agenter for kommunikation på kemiske synapser. Formålet med metoden præsenteret i dette papir er at udforske de terapeutiske gennemførligheden af kemiske stimulation, som sigter mod at efterligne den naturlige system af synaptic kommunikation mellem retinal neuroner, som en biomimetiske alternativ til elektrisk stimulation for en retinal protese.
Oversættelse af begrebet terapeutisk kemiske stimulation til en kemisk retinal protesen er baseret på kemisk aktivering target retinal neuroner med små mængder af indfødte neurotransmittere, såsom glutamat, udgivet gennem en mikrofluid enhed bestående af en bred vifte af microports som svar på visuel stimulation. På denne måde, ville en kemisk retinal protesen hovedsagelig være en biomimetiske kunstige fotoreceptor laget, der oversætter fotoner naturligvis at nå frem til nethinden til kemiske signaler. Da disse kemiske signaler bruger de samme neurotransmittere udnyttes i almindelige retinale signalering og stimulere de overlevende retinal neuroner i en degenererede nethinden gennem den samme synaptic veje bruges af normale vision veje, den resulterende visuelle opfattelse opnås gennem en kemisk retinal protesen kunne være mere naturlig og forståelig i forhold til en fremkaldt gennem en elektrisk protesen. Desuden, da microports hvorigennem neurotransmittere er frigivet kan gøres meget små og klædt i høj tæthed, i modsætning til elektroderne, en potentiel kemiske proteser kan være stand til at opnå mere fokal stimulation og højere rumlige opløsning end en elektrisk protesen. Således tilbyder baseret på disse potentielle fordele, en kemisk retinal protesen en meget lovende alternativ til elektriske proteser.
Kemiske stimulation af nethinden, dog har været relativt lidt udforsket indtil for nylig. Mens elektrisk stimulation af nethinden har været godt præget over årtiers arbejde gennem in vitro22,23, i vivo23,24, og kliniske undersøgelser13 ,14, undersøgelser af kemiske stimulation har været begrænset udelukkende til et par in vitro- værker25,26,27,28. Iezzi og Finlayson26 og Jens Jørgen et al. 27 viste epiretinal kemiske stimulation af nethinden i vitro ved hjælp af en enkelt elektrode og en multielectrode array (MEA), henholdsvis at registrere glutamat fremkaldte svar af retinale neuroner. Mere nylig, Rountree et al. 28 demonstreret differential stimulering af den og slukkes retinal veje ved hjælp af glutamat fra den subretinal side og en MEA for at optage de neuronale reaktioner fra flere steder på nethinden.Selv om disse værker har foreløbigt fastslået muligheden for kemiske stimulation, yderligere undersøgelser er nødvendige for at undersøge mange aspekter af denne tilgang ud over dem, adresseret hidtil25,26,27 , 28, og finjustere parametrene terapeutiske stimulering i både in vitro- og i vivo dyremodeller før oversætte dette koncept til en kemisk retinal protese, som beskrevet ovenfor. Men i øjeblikket er der ingen etableret metode for varetagelse af kemiske stimulation af nethinden i litteraturen og de metoder, der anvendes i de foregående værker ikke er beskrevet så detaljeret som vil være afgørende for replicative undersøgelser. Begrundelsen for denne metoder papir er derfor at give en velafgrænset ramme for udførelse af in vitro- kemiske stimulation af nethinden for dem interesseret i enten gentager vores tidligere undersøgelser27, efterforskere 28 eller yderligere fremme denne spirende begrebet kemiske neurostimulation.
Her viser vi en metode til at foretage in vitro- kemiske stimulation af retinale neuroner i wholemount nethinder vildtype rotter og en fotoreceptor degenererede rotte model, der nøje efterligner progression af fotoreceptor degenerative sygdomme hos mennesker. Rationalet bag udvikle denne stimulation-metoden i in vitro- modeller er at evaluere de terapeutiske områder af forskellige parametre, stimulation og studere neurale reaktion karakteristika, det ville være umuligt eller svært at observere i i vivo modeller, især under de indledende undersøgelser fokuseret på evaluere gennemførligheden af denne fremgangsmåde. I denne procedure vise vi både single-site og samtidige multi-site kemiske stimulering af nethinder ved at levere små mængder af 1 mM glutamat nær målet retinal neuroner via kommercielt tilgængelige single-port glas Mikropipetter og en brugerdefineret micromachined multi-port mikrofluid enhed, henholdsvis. Mens både single-site og multi-site stimulering udrette det grundlæggende formål at undersøge den terapeutiske gennemførligheden af kemiske Neuromodulationsbehandling, tjener hver et særskilt formål med en unik fordel. Single-site stimulation, som kan opnås med kommercielt tilgængelige pre trak glas Mikropipetter, kan bruges til at injicere kemikalier direkte i undergrunden af nethinden på et enkelt websted og tjener til at undersøge hvis observerbare RGC spike sats svar, der svarer til visuelt evoked lys svar kan være fremkaldt focally under injektionsstedet. På den anden side multi-site stimulation, som kræver et specielt fabrikerede multiport mikrofluid enhed, kan bruges til at injicere kemikalier rumligt på flere websteder over overfladen af nethinden og tjener til at undersøge hvor godt glutamat-fremkaldte Risikokapital respons mønstre svarer til glutamat injektion mønstre i mønster stimulation undersøgelser.

<table>
	<tbody>
		<tr>
			<td>Microelectrode array, perforated layout</td>
			<td>Multi Channel Systems, GmbH</td>
			<td>60pMEA200/30iR-Ti-pr</td>
			<td>http://www.multichannelsystems.com/products/microelectrode-arrays/60pmea20030ir-ti</td>
		</tr>
		<tr>
			<td>MEA amplifier</td>
			<td>Multi Channel Systems, GmbH</td>
			<td>MEA1060-Inv</td>
			<td>http://www.multichannelsystems.com/products/mea1060-inv</td>
		</tr>
		<tr>
			<td>Bottom perfusion groundplate for pMEA</td>
			<td>Multi Channel Systems, GmbH</td>
			<td>MEA1060-Inv-(BC)-PGP</td>
			<td>http://www.multichannelsystems.com/products/mea1060-inv-bc-pgp</td>
		</tr>
		<tr>
			<td>3-axis Motorized Micromanipulator</td>
			<td>Sutter Instruments, Novato, CA</td>
			<td>MP-285</td>
			<td>https://www.sutter.com/MICROMANIPULATION/mp285.html</td>
		</tr>
		<tr>
			<td>Micromanipulator Control System</td>
			<td>Sutter Instruments, Novato, CA</td>
			<td>MPC-200</td>
			<td>https://www.sutter.com/MICROMANIPULATION/mpc200.html</td>
		</tr>
		<tr>
			<td>Gantry style micromanipulator stand with linear slide</td>
			<td>Sutter Instruments, Novato, CA</td>
			<td>MT-75/LS</td>
			<td>https://www.sutter.com/STAGES/mt75.html</td>
		</tr>
		<tr>
			<td>8-channel Programmable Multichannel Pressure Injector</td>
			<td>OEM: MicroData Instrument, S. Plainfield, NJ 
			Vendor: Harvard Apparatus UK</td>
			<td>PM-8000 or PM-8</td>
			<td>OEM: http://www.microdatamdi.com/pm8000.htm 
			Vendor: https://www.harvardapparatus.co.uk/webapp/wcs/stores/servlet/product_11555_10001_39808_-1_HAUK_ProductDetail</td>
		</tr>
		<tr>
			<td>Axopatch 200A Integrating Patch Clamp Amplifier</td>
			<td>Molecular Devices, Sunnyvale, CA</td>
			<td>Axopatch 200A</td>
			<td>Axopatch 200A has been replaced with a newer model Axopatch 200B: 
			https://www.moleculardevices.com/systems/axon-conventional-patch-clamp/axopatch-200b-amplifier</td>
		</tr>
		<tr>
			<td>Patch clamp headstage</td>
			<td>Molecular Devices, Sunnyvale, CA</td>
			<td>CV 201A</td>
			<td>http://mdc.custhelp.com/app/answers/detail/a_id/16554/~/axopatch-200a%3A-selection-cv-headstage</td>
		</tr>
		<tr>
			<td>Vacuum waste kit</td>
			<td>ALA Scientific Instruments, Farmingdale, NY</td>
			<td>VMK</td>
			<td>http://alascience.com/product/vacuum-waste-kit/</td>
		</tr>
		<tr>
			<td>Pipette holder</td>
			<td>Warner Instruments, Hamden, CT</td>
			<td>QSW-A10P</td>
			<td>https://www.warneronline.com/product_info.cfm?id=915</td>
		</tr>
		<tr>
			<td>Pre-pulled 10 &#956;m tip diameter glass micropipettes</td>
			<td>World Precision Instruments, Sarasota, FL</td>
			<td>TIP10TW1</td>
			<td>https://www.wpiinc.com/products/laboratory-supplies/make-selection-pre-pulled-glass-pipettes-plain/</td>
		</tr>
		<tr>
			<td>Zoom stereomicroscope</td>
			<td>Nikon, Tokyo, Japan</td>
			<td>SMZ-745T</td>
			<td>https://www.nikoninstruments.com/Products/Stereomicroscopes-and-Macroscopes/Stereomicroscopes/SMZ745</td>
		</tr>
		<tr>
			<td>Microscope boom stand with dual linear ball bearing arm</td>
			<td>Old School Industries, Inc., Dacono, CO</td>
			<td>OS1010H-16BB</td>
			<td>http://www.osi-incorp.com/productdisplay/dual-linear-ball-bearing-arm</td>
		</tr>
		<tr>
			<td>Zoom Stereo Microscope with C-LEDS Hybrid LED Stand</td>
			<td>Nikon, Tokyo, Japan</td>
			<td>SMZ-445</td>
			<td>https://www.nikoninstruments.com/Products/Stereomicroscopes-and-Macroscopes/Stereomicroscopes/SMZ445</td>
		</tr>
		<tr>
			<td>Inverted microscope system</td>
			<td>Nikon, Tokyo, Japan</td>
			<td>Eclipse Ti-E</td>
			<td>https://www.nikoninstruments.com/Products/Inverted-Microscopes/Eclipse-Ti-E</td>
		</tr>
		<tr>
			<td>Ames medium</td>
			<td>Sigma-Aldrich, St. Louis, MO</td>
			<td>A1420</td>
			<td>http://www.sigmaaldrich.com/catalog/product/sigma/a1420</td>
		</tr>
		<tr>
			<td>L-Glutamic Acid (Glutamate)</td>
			<td>Sigma-Aldrich, St. Louis, MO</td>
			<td>G5667</td>
			<td>http://www.sigmaaldrich.com/catalog/product/mm/100291</td>
		</tr>
		<tr>
			<td>Sodium bicarbonate</td>
			<td>Sigma-Aldrich, St. Louis, MO</td>
			<td>S8761</td>
			<td>http://www.sigmaaldrich.com/catalog/product/sigma/s8761</td>
		</tr>
		<tr>
			<td>60 mm Petri dish (10 mm tall)</td>
			<td>Fischer Scientific, Waltham, MA</td>
			<td>FB0875713A</td>
			<td>60 mm clear petri dish; https://www.fishersci.com/shop/products/fisherbrand-petri-dishes-clear-lid-12/fb0875713a</td>
		</tr>
		<tr>
			<td>Jewelers #5 Forceps</td>
			<td>World Precision Instruments, Sarasota, FL</td>
			<td>555227F</td>
			<td>https://www.wpiinc.com/products/laboratory-supplies/555227f-jewelers-5-forceps-11cm-straight-titanium/</td>
		</tr>
		<tr>
			<td>Standard Scalpel Blad #24</td>
			<td>World Precision Instruments, Sarasota, FL</td>
			<td>500247</td>
			<td>https://www.wpiinc.com/products/laboratory-supplies/500247-standard-scalpel-blade-24/</td>
		</tr>
		<tr>
			<td>Scalpel Handle #4</td>
			<td>World Precision Instruments, Sarasota, FL</td>
			<td>500237</td>
			<td>https://www.wpiinc.com/products/laboratory-supplies/500237-scalpel-handle-4-14cm/</td>
		</tr>
		<tr>
			<td>Vannas Tubingen Dissection Scissors</td>
			<td>World Precision Instruments, Sarasota, FL</td>
			<td>503378</td>
			<td>https://www.wpiinc.com/products/laboratory-supplies/503378-vannas-tubingen-scissors-8cm-straight-german-steel/</td>
		</tr>
		<tr>
			<td>Nylon mesh kit</td>
			<td>Warner Instruments, Hamden, CT</td>
			<td>NYL/MESH</td>
			<td>https://www.warneronline.com/product_info.cfm?id=1173</td>
		</tr>
		<tr>
			<td>Harp slice grid</td>
			<td>ALA Scientific Instruments, Farmingdale, NY</td>
			<td>HSG-5AD</td>
			<td>http://alascience.com/product/standard-harp-slice-grids/</td>
		</tr>
		<tr>
			<td>Ag/AgCl reference electrode pellet</td>
			<td>Multi Channel Systems, GmbH</td>
			<td>P1060</td>
			<td>http://www.multichannelsystems.com/products/p1060</td>
		</tr>
		<tr>
			<td>4 Channel Valve Controlled Gravity Perfusion System</td>
			<td>ALA Scientific Instruments, Farmingdale, NY</td>
			<td>VC3-4xG</td>
			<td>http://alascience.com/product/4-channel-valve-controlled-gravity-perfusion-system/</td>
		</tr>
		<tr>
			<td>Zyla 5.5 sCMOS microscope camera</td>
			<td>Andor Technology, Belfast, UK</td>
			<td>Zyla 5.5 sCMOS</td>
			<td>http://www.andor.com/scientific-cameras/neo-and-zyla-scmos-cameras/zyla-55-scmos</td>
		</tr>
		<tr>
			<td>Silver wire (50 &#956;m diameter)</td>
			<td>Fischer Scientific, Waltham, MA</td>
			<td>AA44461G5</td>
			<td>https://www.fishersci.com/shop/products/silver-wire-0-05mm-0-002-in-dia-annealed-99-99-metals-basis-3/aa44461g5</td>
		</tr>
		<tr>
			<td>Tygon microbore tubing (1.6 mm diameter)</td>
			<td>Cole Parmer, Vernon Hills , IL</td>
			<td>EW-06419-01</td>
			<td>https://www.coleparmer.com/i/tygon-microbore-tubing-0-020-x-0-060-od-100-ft-roll/0641901</td>
		</tr>
		<tr>
			<td>Tilting Tool Holder with Steel Cannula</td>
			<td>ALA Scientific Instruments, Farmingdale, NY</td>
			<td>TILTPORT</td>
			<td>One each of these were utilized for top perfusion and suction; http://alascience.com/product/tilting-tool-holder-with-steel-cannula/</td>
		</tr>
		<tr>
			<td>Roscolux #26 Light Red Filter Sheet</td>
			<td>Rosco Laboratories Inc., 52 Harbor View, Stamford, CT</td>
			<td>R2611</td>
			<td>Manufacturer: http://us.rosco.com/en/products/catalog/roscolux 
			Vendor: https://www.bhphotovideo.com/c/product/43957-REG/Rosco_RS2611_26_Filter_Light.html</td>
		</tr>
		<tr>
			<td>Smith &#38; Wesson Galaxy Red Flashlight</td>
			<td>Smith &#38; Wesson, 2100 Roosevelt Avenue, Springfield, MA</td>
			<td>4588</td>
			<td>Manufacturer: https://www.smith-wesson.com/ 
			Vendor: http://www.mypilotstore.com/mypilotstore/sep/4588</td>
		</tr>
		<tr>
			<td>MC_Rack Software</td>
			<td>Multi Channel Systems, GmbH</td>
			<td>MC_Rack</td>
			<td>http://www.multichannelsystems.com/software/mc-rack</td>
		</tr>
		<tr>
			<td>Labview Software</td>
			<td>National Instruments, Austin, TX</td>
			<td>LabVIEW</td>
			<td>http://www.ni.com/labview/</td>
		</tr>
		<tr>
			<td>NIS-Elements: Basic Research Software</td>
			<td>Nikon, Tokyo, Japan</td>
			<td>NIS-Elements BR</td>
			<td>https://www.nikoninstruments.com/Products/Software/NIS-Elements-Basic-Research</td>
		</tr>
	</tbody>
</table>

methodology for biomimetic chemical neuromodulation of rat retinas with the neurotransmitter glutamate in vitro

Fotoreceptor degenerative sygdomme medføre uoprettelige blindhed gennem den gradvise tab af fotoreceptor celler i nethinden. Retinal proteser er en spirende behandling af fotoreceptor degenerative sygdomme, der søger at genoprette vision af kunstigt stimulere de overlevende retinal neuroner i håb om at fremkalde forståelige visuel perception i patienter. Nuværende retinal proteser har demonstreret succes med at genoprette begrænset vision til patienter, som anvender en vifte af elektroder til elektrisk stimulere nethinden, men står over for betydelige fysiske barrierer i genoprette høj skarphed, naturlige vision til patienter. Kemiske neurostimulation ved hjælp af indfødte neurotransmittere er en biomimetiske alternativ til elektrisk stimulation og kunne omgå de grundlæggende begrænsninger i forbindelse med retinal proteser ved hjælp af elektrisk neurostimulation. Specifikt, har kemiske neurostimulation potentiale til at gendanne mere naturlige vision med sammenlignelige eller bedre visual acuities til patienter ved at indsprøjte meget små mængder af neurotransmittere, de samme naturlige agenter for kommunikation anvendes af retinal kemiske synapser på meget finere opløsning end nuværende elektriske proteser. Men som en relativt uudforskede stimulation paradigme, der er ikke etableret protokol for at opnå kemisk stimulation af nethinden i vitro. Formålet med dette arbejde er at give en detaljeret ramme for varetagelse af kemiske stimulation af nethinden for efterforskere, der ønsker at studere den potentielle af kemiske Neuromodulationsbehandling af nethinden eller lignende neurale væv in vitro. I dette arbejde, vi beskriver eksperimentel opsætning og metodologi for fremkalde retinal ganglion celler (RGC) spike svar ligner visual lys svar i wild-type og fotoreceptor-degenereret wholemount rotte nethinder ved at indsprøjte kontrollerede mængder neurotransmitteren glutamat i subretinal plads ved hjælp af glas Mikropipetter og en brugerdefineret multiport mikrofluid enhed. Denne metode og protokol er generelle nok til at være tilpasset til Neuromodulationsbehandling ved hjælp af andre neurotransmittere eller endog andre neurale væv.

Denne protokol kan bruges til kemisk stimulere både normale, wild-type nethinder samt fotoreceptor degenereret nethinder, trods den betydelige cellulære remodeling forårsaget af tabet af fotoreceptorer. Før begyndelsen eksperimenter med enten fotoreceptor degenereret eller vildtype nethinder, optagelse og stimulation udstyr (figur 1 og figur 2) skal være klar og pMEA (figur 5) skal rengøres for...

Watch this Scientific Journal Video about Methodology for Biomimetic Chemical Neuromodulation of Rat Retinas with the Neurotransmitter Glutamate In Vitro at JoVE.com

Methodology for Biomimetic Chemical Neuromodulation of Rat Retinas with the Neurotransmitter Glutamate In Vitro

Denne protokol beskriver en roman metode for at undersøge en form for kemisk neurostimulation wholemount rotte nethinder i vitro med neurotransmitteren glutamat. Kemiske neurostimulation er en lovende alternativ til den konventionelle elektrisk neurostimulation retinal neuroner til behandling af uoprettelige blindhed forårsaget af fotoreceptor degenerative sygdomme.

Metodologi for biomimetiske kemiske Neuromodulationsbehandling af rotte nethinder med neurotransmitteren glutamat In Vitro

Photoreceptor degenerative diseases cause irreparable blindness through the progressive loss of photoreceptor cells in the retina. Retinal prostheses are ...

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Bioengineering

Methodology for Biomimetic Chemical Neuromodulation of Rat Retinas with the Neurotransmitter Glutamate In Vitro

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