Shuɗin methylene a binciken zebrafish: ƙarin sinadarin kiwo da abin da ke rikita gwaji

Ruwan ƙwai na zebrafish sau da yawa yana da ɗan launin shuɗi, kuma kusan babu wanda ke rubuta dalilin hakan. Sinadarin launin shi ne shuɗin methylene, wanda ake ƙarawa don hana naman gwari, kuma yawancin ƙa'idojin aiki suna ɗaukarsa a matsayin wani ɓangare na yanayin gwaji kawai. Wannan ba shi ne fahimtar da ta dace ba. Abin da ya fi dacewa shi ne a ɗauke shi a matsayin wani abin gwaji da ba a lissafa ba: wani abu da ke cikin kwanonin da aka yi wa magani amma babu shi a rukunin kwatanci, yana da tasirin magunguna a matakan taro da ake amfani da su, kuma yana iya sauya ainihin ma'aunan sakamakon da gwajin ke aunawa.

Wannan bambanci ne ya tsara duk abin da ke ƙasa. Amfani da shi yadda aka saba wajen kiwo don hana naman gwari tambaya ce guda. Ko ƙarin sinadarin ba ya shafar gwajin aunawar ku kuwa wata tambaya ce dabam. Wallafe-wallafen binciken zebrafish na baya-bayan nan sun amsa ta farko ta hanyar da ke kwantar da hankali, amma ta biyun ta hanyar da ke tayar da damuwa.

Abin da jagororin suka tanada a zahiri

Babu ƙa'ida guda ɗaya da kowa ke bi, kuma wannan wani ɓangare ne na matsalar. Jagorar Canadian Council on Animal Care tana amincewa da 0.5 mg/L, ko 0.00005%, a ruwan jigilar ƙwai da larvae don rage haɓakar naman gwari. Jagorar Turai kan dabbobin ɗakin gwaje-gwaje ta bayyana cewa ana ajiye tayin a ruwan reno wanda sau da yawa ke ɗauke da wannan 0.00005%. Tsarin haɗa ruwan E3 na Cold Spring Harbor ya ƙayyade ninki biyu na wannan, 1 mg/L ko 0.0001%, a matsayin sinadarin haɗin. Amma akwai wani muhimmin abu da aka bari: jagorar OECD ta gwajin guba a tayin kifi ta ƙayyade ruwan tsarma da aka sake haɗawa da sinadarin gwaji, ba tare da shawarar amfani da shuɗin methylene ba a sigarta ta 2013 ko a gyaran da ya biyo baya. Saboda haka, wani ɗakin gwaje-gwaje yana renon tayin zebrafish da shuɗin methylene a ruwan shuɗi tun daga sa'o'in farko na rayuwarsu, wani kuma yana gudanar da gwajin tayin na bin ƙa'idoji a ruwa marar launi, kuma dukansu suna kiran ruwan renonsu na yau da kullum. Duk wanda ke kwatanta sakamako tsakanin ɗakunan gwaje-gwaje, ko haɗa bayanan tsofaffin rukunin kwatanci, zai gaji wannan rashin daidaiton.

Don fahimtar matakan taron sinadarin, 0.00005% ya kai kusan 1.56 micromolar, 0.0001% kuma kusan 3.13 micromolar. Waɗannan ƙananan lambobi ne, kuma mutum zai iya ɗaukarsu a matsayin ƙanƙanta fiye da yadda za su iya yin tasiri. Binciken da ke ƙasa ya gwada ainihin wannan zato, a waɗannan matakan taro ɗin daidai.

Masu karatu da suka zo daga harkar kiwon kifi maimakon ɗakin gwaje-gwaje suna haɗuwa da wannan ƙwayar sinadari a wani yanayi dabam, wanda aka tattauna a jagorar shuɗin methylene don akwatin kiwon kifi. Sinadarin iri ɗaya ne; bayanin yadda sinadarin launin ke jigilar electrons a cikin ƙwayoyin halitta ya bayyana dalilin da ya sa sinadari mai shiga halayen redox yake yawan bayyana a takardun binciken metabolism. Abin da ke tafe ya shafi ruwan ƙwai na zebrafish mai shuɗin methylene ne musamman.

Metabolism: alkiblar tasirin tana sauyawa da matakin girma

Binciken da ya fi bayar da bayani ya fallasa zebrafish masu girma yadda aka saba ga sinadarin daga ranar farko ta rayuwa har zuwa kwanaki 5 bayan haɗuwar maniyyi da ƙwai, kuma ya auna metabolism mai amfani da oxygen a kowane mataki, kamar yadda rahoton da aka wallafa kan gwaje-gwajen metabolism na tayin ya bayyana. Ya dace a bayyana tsarin sakamakon a hankali saboda alkiblarsa tana juyawa.

A rana 1 bayan haɗuwar maniyyi da ƙwai, amfani da oxygen na asali ya ƙaru a tayin da aka fallasa a dukkan matakan taro biyu na kiwo. A kwanaki 2, bambancin ƙarfin lantarki a membrane na mitochondria a shimfiɗar saman jikin larvae ya ƙaru a matakan fallasa na 0.00005%, 0.0001%, da 0.0003%. Zuwa kwanaki 4 zuwa 5, bayan ƙyanƙyashewa, alkiblar tasirin ta juya: kashe kuzari ta hanyoyin amfani da oxygen ya ragu, ko fallasar ta kasance mai ci gaba tun daga haɗuwar maniyyi da ƙwai ko kuma ta ɗan gajeren lokaci daga rana 3 zuwa rana 4 kawai. A lokaci guda, kwafin RNA na sassan rukunonin numfashin mitochondria sun ragu a kwanaki 1 da 4, tare da kwafin RNA masu alaƙa da samar da steroid da samar da glucose daga abubuwan da ba carbohydrate ba.

Ƙarin abubuwa biyu da aka lura da su sun ƙara fayyace hoton. Ƙyanƙyashewa ta ɗan yi wuri a matakin taro mafi girma na kiwo, inda tayin da aka fallasa suka yi gaba a kwanaki 2, amma rukunin kwatanci ya cim musu zuwa rana 3. Kuma tsarin martani ga damuwa kansa ya bayyana bai lalace ba: lokacin da aka sanya larvae masu kwanaki 5 cikin wani yanayin juyawa da ke haifar da damuwa, cortisol na dukkan jiki ya ƙaru yadda aka saba a rukunonin biyu. Saboda haka, sinadarin launin ba guba ce da ke cutar da ci gaban jiki gaba ɗaya a waɗannan adadin ba, kuma ba ya rage kowace amsar aikin jiki. Yana sauya metabolism na kuzari bisa matakin girma: yana ƙaruwa kafin ƙyanƙyashewa kuma yana raguwa bayanta.

Halayya: dalilin da ya sa bincike biyu suka bambanta, da yadda dukansu za su iya zama daidai

Wallafe-wallafen binciken halayya suna kama da masu saɓani har sai an kwatanta tsarin fallasar ɗaya da na ɗaya. Wani binciken aminci na ci gaban jiki da halayya ya fallasa tayin daga kusan sa'o'i 6 bayan haɗuwar maniyyi da ƙwai ga matakan taro har zuwa 10 micromolar, ya sabunta ruwan sinadarin kowace rana har zuwa rana 4, sannan ya mayar da larvae zuwa ruwan reno marar sinadarin launin a kwanaki 5 da 6, ya kuma gwada motsinsu a gwajin haske da duhu a rana 6. Sakamakon: babu wani muhimmin tasiri kan mace-mace, ƙyanƙyashewa, ko siffar jiki, kuma babu tasirin sinadarin kan motsi. Marubutan sun bayar da shawarar 10 micromolar a matsayin iyaka ta sama cikin dalilai masu ma'ana a ƙarƙashin yanayin bincikensu.

Rukuni na biyu, wanda wasu marubutansa su ma suka shiga binciken metabolism, ya gudanar da fallasar kamar yadda ake yi a kiwo a zahiri: sinadarin launin yana nan daga sa'o'i 2 zuwa 3 bayan haɗuwar maniyyi da ƙwai, ana sabunta wani ɓangaren ruwan kowace rana, kuma ana auna halayya yayin da fallasar ke ci gaba ko jim kaɗan bayanta. A waɗannan matakan taro na yau da kullum ɗin, larvae sun nuna raguwar motsi a gwajin sa'o'i 24, wanda aka iya maimaita shi a nau'o'in zuriyar kifi da ɗakunan gwaje-gwaje daban-daban, kuma binciken rikita sakamakon halayya ya danganta tasirin da matakin aikin tsarin serotonin, daidai da sanannen hana aikin monoamine oxidase da sinadarin launin ke yi. Abin da ya fi fito da matsalar shi ne, ruwan renon tayin mai shuɗin methylene ya ɓoye martanin motsi da ake tsammani ga fluoxetine, wanda yake SSRI. Saboda haka, gwajin tantancewa da aka gudanar a ruwan shuɗi na iya kasa gano tasirin magani wanda ruwa marar launi zai bayyana.

Babu saɓani na gaske. Sinadari na iya daina barin wani tasirin da za a iya ganowa kwanaki biyu bayan an cire shi a wani gwajin aunawa, amma ya ci gaba da yin tasiri yayin da yake nan a wani gwajin. Abin da wannan ke nufi a aikace shi ne, sakamakon da bai nuna tasiri ba yana ba da hujjar iyakar taro ta sama, ba da'awar rashin tasiri ba; sakamakon da ya nuna tasiri kuma yana bayyana haɗarin da ya fi muhimmanci: fallasa ga sinadari mai aiki a lokacin aunawa, a daidai tsarin da yawancin ɗakunan gwaje-gwaje ke amfani da shi.

Sinadarin launi na redox a cikin gwajin redox

Wani tarko guda ya cancanci sakin layi na kansa saboda yana gurɓata hanyar aunawa maimakon dabbar. Gwajin kashe kuzari na Alamar Blue kansa halayen sinadarai na redox ne: ana rage resazurin zuwa resorufin mai fitar da hasken fluorescence. Shuɗin methylene ma yana shiga halayen redox. A gwaje-gwajen kwatanci marasa ƙwayoyin halitta, dukkan matakan taro biyu na kiwo sun ɗan rage hasken fluorescence na Alamar Blue bayan sa'o'i 24, amma raguwar tana da muhimmancin ƙididdiga. A gwaje-gwajen dabbobi, an wanke tayin da aka fallasa aƙalla sau uku a ruwan reno marar sinadarin launin kafin gwajin aunawa, kuma tasirin halittu a larvae da suka ƙyanƙyashe ya zarce tsoma bakin sinadarin da aka gani ba tare da ƙwayoyin halitta ba. Saboda haka, abubuwan biyu gaskiya ne: dabbobin da aka wanke suna da tasirin metabolism na gaske, kuma sinadarin launin da ba a wanke ba yana ƙara wani karkatar da ma'auni ta hanyar sinadarai kai tsaye. Dole gwaje-gwajen kwatanci su ware waɗannan biyun, in ba haka ba babu ɗaya daga cikin ƙididdigar da zai ba da ma'ana. Jagorar gaba ɗaya kan fahimtar sakamakon gwaje-gwajen aunawa masu amfani da sinadarin launi tana da matuƙar muhimmanci a nan.

Jadawalin matakan ci gaban jiki

Kayan bayani na asali na wannan labarin. Matakan taron sinadarin su ne ƙimomi biyu na kiwo da aka saba amfani da su, 0.00005% da 0.0001%, sai inda aka nuna wani dabam.

Tsarin da ya kamata a tuna shi ne ƙaruwa da wuri da raguwa daga baya a ma'aunan sakamakon metabolism, tare da tasirin halayya yayin fallasa wanda ba a iya ganowa bayan fitar da sinadarin daga jiki a gwajin aunawa guda ɗaya da ya gwada hakan.

Jerin dubawa don tsara rukunin kwatanci

Kayan bayani na asali na biyu. Yi amfani da shi lokacin da ƙa'idojin amfani da shuɗin methylene a zebrafish suka shiga cikin wani gwaji.

1. Bayyana sinadarin launin a matsayin reagent na gwaji, ba a matsayin ruwa ba. Faɗi taronsa a kashi ɗari da micromolar, lokacin farawa da tsayawa a sa'o'i ko kwanaki bayan haɗuwar maniyyi da ƙwai, jadawalin sabuntawa, da tazarar cire sinadarin kafin kowace aunawa.
2. Daidaita rukunin kwatanci da tambayar binciken. Binciken sinadarin da ake gwadawa yana buƙatar rukunin kwatanci marar sinadarin launin; binciken kiwo yana buƙatar kwatanta rukuni marar sinadarin launin da rukuni mai shi, tare da rubuta sakamakon hana naman gwari, domin babu ɗaya daga cikin binciken biyu da aka ambata da ya auna ingancin hana naman gwari.
3. Yi wanka kafin gwaje-gwajen redox, sannan ka tabbatar. Kurkura aƙalla sau uku a ruwan reno marar sinadarin launin, sannan ka gudanar da gwajin kwatanci marar ƙwayoyin halitta mai sinadarin launin da reagent a gefe, saboda sinadarin launin yana rage siginar Alamar Blue kai tsaye.
4. Ɗauki ma'aunan halayya da monoamine a matsayin masu babban haɗarin karkatar da sakamako. Binciken motsi, firgita, halayyar da ta yi kama da damuwa, magungunan rage baƙin ciki, da masu hana MAO ya kamata ya guji sinadarin launin yayin aunawa ko ya nuna rashin tasirinsa ga takamaiman ma'aunin sakamakon; cire sinadarin na kwana ɗaya zuwa biyu ya kawar da wani tasirin motsi, amma ba a tabbatar da hakan a wasu tsare-tsaren gwaji ba.
5. Ɗauki ma'aunan metabolism da mitochondria a matsayin masu babban haɗarin karkatar da sakamako a kowane mataki. Amfani da oxygen, kashe kuzari, bambancin ƙarfin lantarki na membrane, da kwafin RNA na sarkar numfashi suna sauyawa a matakan taro na kiwo, zuwa alkibloyi masu kishiyantar juna kafin da bayan ƙyanƙyashewa.
6. Kada ka ɗauki rashin tasiri a wani tsarin gwaji a matsayin hujja ga wani tsarin dabam. Sakamakon rashin tasiri bayan cire sinadarin ba ya tabbatar da rashin tasiri yayin fallasa mai ci gaba; rashin tasiri kan siffar jiki ba ya tabbatar da rashin tasiri kan metabolism ko hulɗa da magani; sakamako a wata zuriyar kifi, zafin jiki, ko tsawon gwajin aunawa ba ya tabbatar da abin da zai faru a wasu.
7. Faɗi jagorar da ka bi da madadinta. Ambaci matakan taro na CCAC, na Turai, ko na Cold Spring Harbor a sarari, kuma nuna inda gwajin tayin na OECD ya bar sinadarin launin, domin mai karatu ya iya fahimtar yanayin da aka reni dabbobinka.

Babu ɗaya daga cikin waɗannan bayanan da ke nuna cewa bai kamata a yi amfani da sinadarin launin don kiyaye ƙwai da rai ba. Suna nuna cewa bai kamata a bar zaɓin hanyar kiwo ya shiga cikin bayanan gwaji ba tare da bincikensa ba. Shuɗin da ke cikin kwano ƙwayar sinadari ce mai tasirin magunguna, kuma gwaje-gwajen da suka fi yiwuwar samun karkataccen sakamako su ne ainihin gwaje-gwajen tantance metabolism da halayyar tsarin jijiyoyi masu sarrafa samfurori da yawa da sauri, waɗanda yanzu aka fi son amfani da ƙananan zebrafish a cikinsu. Rubuta shi, tsara kwatanci dominsa, wanke shi kafin aunawa, kuma bincika gwajin aunawar da wataƙila yake shiga a ɓoye.