Methylene blue pa kafukufuku wa zebrafish: chowonjezera posamalira nsomba komanso chosokoneza zoyesera

Madzi a mazira a zebrafish nthawi zambiri amakhala abuluu pang'ono, ndipo pafupifupi palibe amene amalemba chifukwa chake. Utotowo ndi methylene blue, wowonjezeredwa kuti uletse mafangasi, ndipo ndondomeko zambiri zimauona ngati chinthu chosafunika kuchiganizira. Kuuona choncho n'kulakwa. Ndi bwino kuuona ngati chinthu cha kuyesera chomwe sichinatchulidwe: chimakhala m'mbale za gulu limene likuyesedwa koma sichikhala m'magulu oyerekezera, chimachitapo kanthu m'thupi pa kuchuluka kumene amagwiritsa ntchito, ndipo chingasinthe miyeso yeniyeni imene kuyeserako kumafufuza.

Kusiyanitsa kumeneku ndiko maziko a zonse zimene zili pansipa. Kusamalira nsomba mwa kuletsa mafangasi ndi nkhani imodzi. Koma ngati chowonjezeracho sichisintha zotsatira za mayeso anu ndi nkhani ina. Kafukufuku waposachedwa wa zebrafish amapereka yankho lolimbikitsa pa nkhani yoyamba, koma lodetsa nkhawa pa yachiwiri.

Zimene malangizo amanena kwenikweni

Palibe muyezo umodzi wokha, ndipo zimenezi ndi mbali ya vuto. Malangizo a Canadian Council on Animal Care amalola 0.5 mg/L, kapena 0.00005%, m'madzi onyamulira mazira ndi ana a nsomba kuti achepetse kukula kwa mafangasi. Malangizo a ku Ulaya okhudza nyama za m'ma labotale amafotokoza miluza yosungidwa m'madzi amene nthawi zambiri amakhala ndi 0.00005% yomweyo. Njira yopangira E3 ya Cold Spring Harbor imatchula kuchuluka kowirikiza kawiri, 1 mg/L kapena 0.0001%, ngati chimodzi mwa zosakaniza. Koma pali chinthu choonekeratu chimene sichinaphatikizidwe: malangizo a OECD a mayeso a kuopsa kwa mankhwala pa miluza ya nsomba amatchula madzi ochepetsera mphamvu a mankhwala opangidwanso ndi mankhwala amene akuyesedwa, popanda kulangiza methylene blue kaya m'buku lake la 2013 kapena m'kusintha kwake kwapambuyo. Choncho labotale ina imakulitsa miluza ya zebrafish m'madzi abuluu okhala ndi methylene blue kuyambira maola oyambirira a moyo, pamene ina imachita mayeso a miluza otsatira malamulo m'madzi oyera opanda utoto, ndipo onse amatcha madzi awo kuti ndi abwinobwino. Aliyense woyerekezera zotsatira za m'ma labotale osiyanasiyana, kapena wophatikiza zotsatira zakale za magulu oyerekezera, amatengera kusagwirizana kumeneku.

Kuti timvetse kuchuluka kwake, 0.00005% ndi pafupifupi 1.56 micromolar ndipo 0.0001% ndi pafupifupi 3.13 micromolar. Ziwerengerozi ndi zochepa, ndipo n'zosavuta kuganiza kuti ndi zochepa kwambiri moti sizingakhudze chilichonse. Kafukufuku amene ali pansipa amayesa lingaliro limeneli, pogwiritsa ntchito kuchuluka komweku.

Owerenga ochokera ku ntchito yosamalira nsomba m'malo mwa labotale amakumana ndi molekyulu yomweyo m'njira ina, yofotokozedwa mu buku la malangizo a methylene blue pa nsomba za m'matanki. Kapangidwe ka mankhwalawo n'kofanana; kufotokoza mmene utotowo umasamutsira maelekitironi m'maselo kumasonyeza chifukwa chake mankhwala ochita nawo kusinthana kwa maelekitironi amapezeka mobwerezabwereza m'maphunziro a kagayidwe. Zimene zikutsatira zikukhudza makamaka madzi a mazira a zebrafish okhala ndi methylene blue.

Kagayidwe: zotsatira zimasintha mbali malinga ndi gawo la kukula

Kafukufuku wopereka zambiri kwambiri anaika zebrafish zomwe zinkakula mwachibadwa m'madzi okhala ndi utoto kuyambira tsiku loyamba la moyo mpaka masiku 5 pambuyo pa kukumana kwa dzira ndi umuna, ndipo anayeza kagayidwe kogwiritsa ntchito okosijeni pa gawo lililonse, monga lipoti lofalitsidwa la zoyesera za kagayidwe ka miluza limafotokozera. Ndondomeko ya zotsatirazi iyenera kufotokozedwa mosamala chifukwa mbali yake imasinthana.

Pa tsiku 1 pambuyo pa kukumana kwa dzira ndi umuna, kugwiritsidwa ntchito kwa okosijeni pa mkhalidwe wamba kunakwera m'miluza yoikidwa m'madzi okhala ndi utoto pa kuchuluka konse kuwiri kogwiritsidwa ntchito posamalira nsomba. Pa masiku 2, kusiyana kwa mphamvu yamagetsi pa nembanemba za mitochondria m'maselo a pamwamba pa ana a nsomba kunakwera pa 0.00005%, 0.0001%, ndi 0.0003%. Pofika masiku 4 mpaka 5, pambuyo potuluka m'dzira, mbali ya zotsatira inali itasintha: kugwiritsa ntchito mphamvu kudzera mu kagayidwe kogwiritsa ntchito okosijeni kunachepa, kaya nsombazo zinakhala mu utotowo mosalekeza kuyambira pamene dzira linakumana ndi umuna kapena zinangokhalamo kwa nthawi yochepa kuyambira tsiku 3 mpaka tsiku 4. Pa nthawi yomweyo, zolemba za RNA za zigawo za magulu opumitsira a mitochondria zinachepa pa masiku 1 ndi 4, pamodzi ndi zolemba za RNA zokhudzana ndi kupanga ma steroid ndi kupanga glucose kuchokera ku zinthu zosakhala chakudya cha mtundu wa carbohydrate.

Zinthu zina ziwiri zimathandiza kumvetsa bwino chithunzichi. Kutuluka m'dzira kunachitika msanga pang'ono pa kuchuluka kwakukulu mwa miyezo iwiri yosamalirira nsomba, ndipo miluza ya m'madzi okhala ndi utoto inali patsogolo pa masiku 2, koma ya m'magulu oyerekezera inafika pamlingo womwewo pofika tsiku 3. Ndipo dongosolo la kuyankha kupsinjika linaoneka kuti likugwira ntchito bwino: ana a nsomba a masiku 5 atapatsidwa kupsinjika kochokera ku kuzunguliridwa m'madzi, cortisol m'thupi lonse inakwera mwachibadwa m'magulu onse awiri. Choncho pa kuchuluka kumeneku, utotowo suli poizoni wosokoneza kukula konse, ndipo suletsa mayankho onse a ntchito za thupi. Umasintha kagayidwe ka mphamvu malinga ndi gawo la kukula: kumakwera asanatuluke m'dzira ndipo kumatsika atatuluka.

Khalidwe: chifukwa chake kafukufuku awiri sakugwirizana, komanso chifukwa chake onse angakhale olondola

Kafukufuku wa khalidwe amaoneka wotsutsana mpaka njira zoikira nsomba m'madzi okhala ndi utoto zitayerekezera. Kafukufuku wina wa chitetezo pa kukula ndi khalidwe anaika miluza m'madzi okhala ndi utoto kuyambira pafupifupi maola 6 pambuyo pa kukumana kwa dzira ndi umuna, pa kuchuluka kofika 10 micromolar. Anasintha madziwo tsiku lililonse mpaka tsiku 4, kenako anasamutsira ana a nsomba m'madzi opanda utoto pa masiku 5 ndi 6, ndipo anayesa kuyenda kwawo m'mayeso a kuwala ndi mdima pa tsiku 6. Zotsatira zake: panalibe kusintha kwakukulu pa kufa, kutuluka m'dzira, kapena maonekedwe ndi kapangidwe ka thupi, ndipo kuikidwa mu utoto sikunakhudze kuyenda. Olembawo anali ndi chifukwa chomveka cholangizira 10 micromolar ngati malire apamwamba pa mikhalidwe imene anagwiritsa ntchito.

Gulu lachiwiri, limene linali ndi olemba ena omwe analinso mu kafukufuku wa kagayidwe, linaika nsomba mu utoto monga mmene zimachitikira posamalira nsomba: utoto unali m'madzi kuyambira maola 2 mpaka 3 pambuyo pa kukumana kwa dzira ndi umuna, ndipo gawo la madzi linkasinthidwa tsiku lililonse. Khalidwe linayesedwa pamene nsombazo zinkakhalabe mu utoto kapena zitangochotsedwamo posachedwa. Pa kuchuluka komweku kogwiritsidwa ntchito nthawi zonse, ana a nsomba anayenda pang'ono m'mayeso a maola 24, ndipo zotsatirazi zinapezekanso m'mitundu ya chibadwa ndi m'ma labotale osiyanasiyana. Kafukufuku wa kusokoneza mayeso a khalidwe anapeza kuti zotsatirazo zinali zogwirizana ndi mlingo wa ntchito ya dongosolo la serotonin, mogwirizana ndi kuthekera kodziwika kwa utotowo koletsa monoamine oxidase. Chofunika kwambiri n'chakuti madzi a miluza okhala ndi methylene blue anabisa kusintha kwa kuyenda komwe kunkayembekezeredwa chifukwa cha fluoxetine, mankhwala a mtundu wa SSRI. Choncho mayeso owunika zinthu ambiri ochitidwa m'madzi abuluu angalephere kuona mphamvu ya mankhwala yomwe ikanawoneka m'madzi opanda utoto.

Palibe kutsutsana kwenikweni. Mankhwala angakhale opanda zotsatira zotsalira zooneka masiku awiri atachotsedwa m'mayeso ena, koma n'kukhalabe ochita kanthu pamene alipo m'mayeso ena. Tanthauzo lothandiza n'lakuti kusapezeka kwa zotsatira kumathandiza kukhazikitsa malire apamwamba a kuchuluka, osati kunena kuti utotowo susintha chilichonse. Koma kupezeka kwa zotsatira kumafotokoza chiopsezo chofunika: kukhalapo kwa utoto wochitapo kanthu panthawi yoyezera, m'njira yeniyeni imene ma labotale ambiri amagwiritsa ntchito.

Utoto wosinthana maelekitironi m'mayeso osinthana maelekitironi

Msampha winanso ukuyenera kukhala ndi ndime yake chifukwa umasokoneza njira yoyezera m'malo mwa nyama. Mayeso a Alamar Blue oyeza kugwiritsa ntchito mphamvu ndiwo okha njira ya kusinthana kwa maelekitironi: resazurin imalandira maelekitironi n'kusanduka resorufin yotulutsa kuwala. Methylene blue imachitanso kusinthana kwa maelekitironi. M'magulu oyerekezera opanda maselo, kuchuluka konse kuwiri kogwiritsidwa ntchito posamalira nsomba kunachepetsa pang'ono koma momveka mwa ziwerengero kuwala kwa Alamar Blue pambuyo pa maola 24. M'zoyesera za nyama, miluza yoikidwa mu utoto inatsukidwa osachepera katatu m'madzi opanda utoto asanayesedwe, ndipo kusintha kwa ntchito za thupi kwa ana a nsomba otuluka m'dzira kunali kwakukulu kuposa kusokoneza komwe kunaoneka popanda maselo. Choncho zonsezi ndi zoona: nyama zotsukidwa zimakhalabe ndi kusintha kwenikweni kwa kagayidwe, ndipo utoto wosatsukidwa umawonjezerapo kusintha kwa muyeso kochokera mwachindunji ku mankhwalawo. Magulu oyerekezera ayenera kusiyanitsa zinthu ziwirizi; apo ayi palibe chiwerengero chimene chingakhale ndi tanthauzo. Malangizo onse a kutanthauzira zotsatira za mayeso ogwiritsa ntchito utoto ndi ofunika kwambiri pano.

Ndondomeko ya magawo a kukula

Ichi ndi chinthu choyambirira chopangidwira nkhaniyi. Kuchuluka kwake ndi miyezo iwiri yogwiritsidwa ntchito posamalira nsomba, 0.00005% ndi 0.0001%, pokhapokha patatchulidwa zina.

Choyenera kukumbukira n'chakuti miyeso ya kagayidwe imakwera koyambirira n'kutsika pambuyo pake, ndipo kusintha kwa khalidwe kumakhalapo panthawi yokhala mu utoto koma sikuoneka utoto utachotsedwa m'thupi, m'mayeso amodzi amene anafufuza zimenezi.

Mndandanda wowunikira kapangidwe ka magulu oyerekezera

Ichi ndi chinthu chachiwiri choyambirira chopangidwira nkhaniyi. Chigwiritseni ntchito pamene njira zogwiritsa ntchito methylene blue pa zebrafish zikuphatikizidwa ndi kuyesera.

1. Fotokozani utotowo ngati mankhwala a kuyesera, osati ngati madzi. Tchulani kuchuluka kwake pa percent komanso micromolar, nthawi yoyambira ndi yomaliza m'maola kapena masiku pambuyo pa kukumana kwa dzira ndi umuna, ndondomeko yosinthira madzi, ndi nthawi yochotsera utoto musanatenge muyeso uliwonse.
2. Pangani magulu oyerekezera ogwirizana ndi funso la kafukufuku. Kafukufuku wa mankhwala amene akuyesedwa amafuna magulu oyerekezera opanda utoto; kafukufuku wa kusamalira nsomba amafuna magulu opanda utoto oyerekezera ndi okhala ndi utoto, ndipo zotsatira za kuletsa mafangasi ziyenera kulembedwa, chifukwa palibe kafukufuku amene watchulidwa amene anayeza mphamvu yoletsa mafangasi.
3. Tsukani musanachite mayeso osinthana maelekitironi, kenako tsimikizirani. Tsukani osachepera katatu m'madzi opanda utoto ndipo nthawi yomweyo yesani gulu loyerekezera lopanda maselo, lokhala ndi utoto pamodzi ndi mankhwala a mayesowo, chifukwa utotowo umachepetsa mwachindunji chizindikiro cha Alamar Blue.
4. Onani miyeso ya khalidwe ndi monoamine ngati yokhala ndi chiopsezo chachikulu chosokonezedwa. Kafukufuku wa kuyenda, kuyankha podzidzimuka, khalidwe lofanana ndi nkhawa, mankhwala ochepetsa kuvutika maganizo, ndi mankhwala oletsa MAO ayenera kupewa utoto panthawi yoyezera kapena kusonyeza kuti utotowo susintha muyeso umene ukufufuzidwawo. Kuchotsa utoto kwa tsiku limodzi mpaka awiri kunachotsa zotsatira zina pa kuyenda, koma sikunatsimikiziridwe pa njira zina za mayeso.
5. Onani miyeso ya kagayidwe ndi mitochondria ngati yokhala ndi chiopsezo chachikulu chosokonezedwa pa gawo lililonse. Kugwiritsa ntchito okosijeni, kugwiritsa ntchito mphamvu, kusiyana kwa mphamvu yamagetsi pa nembanemba, ndi zolemba za RNA za unyolo wopumitsira zimasintha pa kuchuluka kogwiritsidwa ntchito posamalira nsomba, koma zimasintha m'njira zotsutsana asanatuluke m'dzira ndi atatuluka.
6. Musaganize kuti kusapezeka kwa zotsatira mu kapangidwe kena ka mayeso kumagwiranso ntchito mu kena. Kusapezeka kwa zotsatira utoto utachotsedwa sikutanthauza kuti palibe zotsatira pamene ukadalipo; kusapezeka kwa kusintha kwa maonekedwe ndi kapangidwe ka thupi sikutanthauza kuti palibe kusintha kwa kagayidwe kapena kugwirizana ndi mankhwala ena; zotsatira za mtundu umodzi wa chibadwa, kutentha kumodzi, kapena nthawi imodzi ya mayeso sizigwira ntchito pa zina.
7. Tchulani malangizo amene munatsatira komanso njira ina yosiyana nawo. Tchulani momveka kuchuluka kwa CCAC, malangizo a ku Ulaya, kapena Cold Spring Harbor, ndipo fotokozani kumene mayeso a miluza a OECD saphatikiza utoto, kuti wowerenga amvetse mikhalidwe imene nyama zanu zinakhalamo.

Zonsezi sizikutsutsa kugwiritsa ntchito utoto kuti mazira akhalebe ndi moyo. Zikutsutsa kulola chisankho chosamalira nsomba kulowa m'zotsatira za kafukufuku popanda kuunikidwa. Buluu wa m'mbalemo ndi molekyulu imene imachitapo kanthu m'thupi, ndipo zoyesera zomwe zingasokonezedwe kwambiri ndi mayeso a kagayidwe ndi khalidwe lokhudzana ndi dongosolo la mitsempha omwe amawunika zinthu zambiri mwachangu, amene tsopano amakonda kugwiritsa ntchito zebrafish zazing'ono. Lembani kugwiritsa ntchito kwake, konzekerani magulu oyerekezera, chotsani utotowo musanayeze, ndipo fufuzani mayeso amene ungakhale ukuchitamo kanthu mosaonekera.