Name: intestinal epithelial regeneration in response to ionizing irradiation
Uploaded: 2022-07-27T14:05:00
Description: Watch this Scientific Journal Video about Intestinal Epithelial Regeneration in Response to Ionizing Irradiation at JoVE.com

Forfatterne ønsker å anerkjenne Stony Brook Cancer Center Histology Research Core for eksperthjelp med vevsprøvepreparering og Divisjon for laboratoriedyrressurser ved Stony Brook University for hjelp med dyrepleie og håndtering. Dette arbeidet ble støttet av tilskudd fra National Institutes of Health DK124342 tildelt Agnieszka B. Bialkowska og DK052230 til Dr. Vincent W. Yang.

Alle musene ble plassert i Division of Laboratory Animal Resources (DLAR) ved Stony Brook University. Stony Brook University Institutional Animal Care and Use Committee (IACUC) godkjente alle studier og prosedyrer som involverte dyreforsøk. Eksperimenter som involverte dyr ble utført strengt i samsvar med den godkjente dyrehåndteringsprotokollen (IACUC #245094).
MERK: Musestammer B6;129-Bmi1 tm1(cre/ERT)Mrc/J (Bmi1-Cre ER) og B6.129X1-Gt(ROSA)26Sortm1(EYFP)Cos/J (Ros...

Denne protokollen beskriver en robust og reproduserbar stråleskademodell. Det muliggjør presis analyse av endringene i tarmepitelet i løpet av 7 dager etter skaden. Det er viktig at de valgte tidspunktene gjenspeiler viktige stadier av skade og er preget av tydelige endringer i tarmen (skade-, apoptose-, regenererings- og normaliseringsfaser)60. Denne bestrålingsmodellen er etablert og nøye vurdert, og demonstrerer en egnet manifestasjon av skade for å etterligne den som oppleves av pasiente...

Det menneskelige tarmepitelet ville dekke omtrent overflaten av en halv badmintonbane hvis det ble plassert helt flatt1. I stedet komprimeres dette enkeltcellelaget som skiller mennesker fra innholdet i tarmen til en serie fingerlignende fremspring, villi og innrykk, krypter som maksimerer tarmens overflateareal. Cellene i epitelet skiller seg langs en krypt-villus-akse. Villus består primært av næringsabsorberende enterocytter, slimutskillende begerceller og de hormonproduserende enteroendokrine cellene, mens kryptene primært består av defensinproduserende Paneth-celler, aktive og reservestamceller og stamceller 2,3,4,5. Videre genererer den toveis kommunikasjonen disse cellene har med stromale og immunceller i det underliggende mesenkymale rommet og mikrobiota av lumen et komplekst nettverk av interaksjoner som opprettholder tarmhomeostase og er kritisk for utvinning etter skade 6,7,8.
Tarmepitelet er det raskest selvfornyende vevet i menneskekroppen, med en omsetningshastighet på 2-6 dager 9,10,11. Under homeostase deler aktive stamceller ved basen av tarmkrypter (kryptbasesøyleceller), preget av ekspresjonen av leucinrik repetisjonsholdig G-proteinkoblet reseptor 5 (LGR5), raskt og gir stamceller som differensierer i alle andre tarmepitellinjer. På grunn av deres høye mitotiske hastighet er imidlertid aktive stamceller og deres umiddelbare forfedre spesielt følsomme for gammastrålingsskade og gjennomgår apoptose etter bestråling 5,12,13,14. Ved tapet gjennomgår reservestamceller og ikke-stamceller (subpopulasjon av forfedre og noen terminalt differensierte celler) i tarmkrypter aktivering og fyller opp det basale kryptrommet, som deretter kan rekonstituere cellepopulasjoner av villi og dermed regenerere tarmepitelet15. Ved hjelp av avstamningssporingsteknikker har flere forskningsgrupper vist at reservestamceller (hvilende) er i stand til å støtte regenerering ved tap av aktive stamceller 13,16,17,18,19,20,21,22. Disse cellene er karakterisert ved tilstedeværelsen av polycomb kompleks protein 1 onkogen (Bmi1), mus telomerase revers transkriptase gen (mTert), Hop homeobox (Hopx) og leucinrikt repetisjonsprotein 1 gen (Lrig1). I tillegg har det vist seg at ikke-stamceller er i stand til å fylle tarmkrypter ved skade 23,24,25,26,27,28,29,30,31. Spesielt har det vist seg at forfedre til sekretoriske celler og enterocytter gjennomgår dedifferensiering ved skade, går tilbake til stamlignende celler og støtter regenerering av tarmepitelet. Nylige studier har identifisert celler som uttrykker flere markører som har kapasitet til å skaffe seg stammelignende egenskaper ved skade (for eksempel DLL +, ATOH1 +, PROX1 +, MIST1 +, DCLK1 +) 32,33,34,35,36. Overraskende nok viste Yu et al. at selv modne Paneth-celler (LYZ +) kan bidra til intestinal regenerering37. Videre, i tillegg til å forårsake apoptose av tarmepitelceller og forstyrre epitelbarrierefunksjonen, resulterer bestråling i dysbiose av tarmfloraen, immuncelleaktivering og initiering av en proinflammatorisk respons, og aktivering av mesenkymale og stromale celler38,39.
Gammastråling er et verdifullt terapeutisk verktøy i kreftbehandling, spesielt så for kolorektal svulster40. Imidlertid påvirker bestråling signifikant intestinal homeostase ved å indusere skade på cellene, noe som fører til apoptose. Strålingseksponering forårsaker flere forstyrrelser som reduserer pasientens utvinning og er preget av slimhinneskade og betennelse i den akutte fasen og diaré, inkontinens, blødning og magesmerter på lang sikt. Denne panoply av manifestasjoner er referert til som gastrointestinal stråling toksisitet. I tillegg kan stråleindusert progresjon av transmural fibrose og / eller vaskulær sklerose bare manifestere år etter behandlingen 38,41. Samtidig med selve skaden induserer stråling en reparasjonsrespons i tarmceller som aktiverer signalveier som er ansvarlige for å initiere og orkestrere regenerering42. Stråleindusert tarmsykdom kan stamme fra strålebehandling av bekken eller buk gitt til andre organer (som livmorhals, prostata, bukspyttkjertel, endetarm)41,43,44,45,46. Tarmbestrålingsskade er dermed et betydelig klinisk problem, og en bedre forståelse av den resulterende patofysiologien vil sannsynligvis fremme utviklingen av tiltak for å lindre gastrointestinale komplikasjoner forbundet med strålebehandling. Det finnes andre teknikker som gjør det mulig å undersøke det regenerative formålet med tarmepitelet bortsett fra stråling. Transgene og kjemiske murinmodeller for å studere inflammasjon og regenerering deretter er utviklet47. Dextran natriumsulfat (DSS) induserer betennelse i tarmen og fører til utvikling av egenskaper som ligner på inflammatorisk tarmsykdom48. En kombinasjon av DSS-behandling med den kreftfremkallende forbindelsen azoksymetan (AOM) kan resultere i utvikling av kolittassosiert kreft48,49. Iskemi reperfusjonsindusert skade er en annen metode som brukes til å studere det regenerative potensialet i tarmepitelet. Denne teknikken krever erfaring og kirurgisk kunnskap50. Videre forårsaker de nevnte teknikkene forskjellige typer skader enn stråling og kan føre til involvering av forskjellige regenereringsmekanismer. I tillegg er disse modellene tidkrevende, mens strålingsteknikken er ganske kort. Nylig har in vitro-metoder som benytter enteroider og kolonoider generert fra tarm og tykktarm blitt brukt i kombinasjon med strålingsskade for å studere mekanismene for intestinal regenerering51,52. Disse teknikkene rekapitulerer imidlertid ikke fullt ut orgelet de modellerer53,54.
Protokollen som presenteres inkluderer beskrivelsen av en murinmodell for gammastrålingsskade i kombinasjon med en genetisk modell som etter tamoksifenbehandling tillater sporing av linjer som stammer fra reservestamcellepopulasjonen (Bmi1-CreER; Rosa26eYFP). Denne modellen benytter en 12 Gy helkroppsbestråling, som induserer betydelig nok tarmskade til å aktivere reservestamceller, samtidig som den tillater den påfølgende undersøkelsen av intestinal regenerativ evne innen 7 dager etter skade55.

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intestinal epithelial regeneration in response to ionizing irradiation

Tarmepitelet består av et enkelt lag av celler, men inneholder likevel flere typer terminalt differensierte celler, som genereres ved aktiv spredning av intestinale stamceller som ligger i bunnen av tarmkryptene. Under hendelser med akutt tarmskade gjennomgår imidlertid disse aktive intestinale stamcellene celledød. Gammabestråling er en mye brukt kolorektal kreftbehandling, som, selv om den er terapeutisk effektiv, har bivirkningen av å tømme det aktive stamcellebassenget. Faktisk opplever pasienter ofte gastrointestinalt strålingssyndrom mens de gjennomgår strålebehandling, delvis på grunn av aktiv stamcelleuttømming. Tapet av aktive intestinale stamceller i tarmkrypter aktiverer et basseng av typisk hvilende reserve intestinale stamceller og induserer dedifferensiering av sekretoriske og enterocyttforløperceller. Hvis ikke for disse cellene, ville tarmepitelet mangle evnen til å gjenopprette fra strålebehandling og andre slike store vevsfornærmelser. Nye fremskritt innen avstamningssporingsteknologier tillater sporing av aktivering, differensiering og migrasjon av celler under regenerering og har blitt brukt til å studere dette i tarmen. Denne studien tar sikte på å skildre en metode for analyse av celler i musens tarmepitel etter strålingsskade.

Bruk av 12 Gy helkroppsbestråling (TBI) i kombinasjon med genetisk avstamningssporing muliggjør en grundig analyse av konsekvenser av stråleskade i tarmen. For å starte, Bmi1-CreER; Rosa26eYFP-mus mottok en enkelt tamoxifen-injeksjon, som induserer forbedret gult fluorescerende protein (EYFP) -uttrykk i en Bmi1 + reserve stamcellepopulasjon. To dager etter tamoxifen-injeksjonen gjennomgikk musene bestråling eller skambestråling. Tre timer før avlivning ble musene injisert ...

Watch this Scientific Journal Video about Intestinal Epithelial Regeneration in Response to Ionizing Irradiation at JoVE.com

Intestinal Epithelial Regeneration in Response to Ionizing Irradiation

Mage-tarmkanalen er et av de mest følsomme organene for skade ved radioterapeutisk kreftbehandling. Det er samtidig et organsystem med en av de høyeste regenerative kapasitetene etter slike fornærmelser. Den presenterte protokollen beskriver en effektiv metode for å studere regenerativ kapasitet i tarmepitelet.

Intestinal epitelial regenerering som respons på ioniserende bestråling

The intestinal epithelium consists of a single layer of cells yet contains multiple types of terminally differentiated cells, which are generated by the ...

Gamma bestråling er en mye brukt behandling for kolorektal kreft. Og mens det er terapeutisk effektivt, har det negative effekter på mage-tarmkanalen. Den presenterte protokollen beskriver en effektiv metode for å studere aktivering, differensiering og migrasjon av celler under regenerering etter strålingsskade.Denne protokollen beskriver en robust og reproduserbar stråleskademodell. Det er viktig at denne modellen gjør det mulig å spore tarmcelleskjebnen etter skade og gir dermed bedre forståelse av regenerativ kapasitet til spesifikk cellepopulasjon. Denne teknikken kan benytte forskjellige avstamningssporende dyremodeller for å studere sanntidsadferd og samspillet mellom ulike underpopulasjoner av celler etter skade.Mindre modifikasjoner i denne protokollen bør tillate undersøkelse av forholdet mellom mikrobiota og forskjellige epitel-, stromal- og immuncellepopulasjoner ved stråleskade. For å begynne, resuspendere tamoxifenpulver i steril maisolje i en konsentrasjon på 30 mikrogram per mikroliter. Sonikere i tre minutter i 30 sekunder on-off sykluser med 60% amplitude.Bland ved rotasjon i mørket i en time ved romtemperatur. Forbered deretter EdU ved å tilsette sterilt DMSO til EdU-pulver. Tilsett 1/5 av totalvolumet, og tilsett sakte det gjenværende volumet av sterilt ultrarent vann.Når det er helt oppløst, aliquot og lagre ved 20 grader Celsius. Forbered reagenser for vevsinnsamling og fiksering, for eksempel 70% etanol i vann, DPBS kjølt til fire grader Celsius, Bouins fiksative buffer og 10% bufret formalin. Forbered deretter nødvendig utstyr, for eksempel et musdisseksjonssett, petriskåler, en 16-gauge gavage nål festet til en 10 milliliter sprøyte og histologiske kassetter.To dager før gammabestrålingseksponering, klargjør forsøksdyrene for tamoxifeninjeksjon. Vei hvert dyr, og beregne den nødvendige doseringen. Desinfiser bukområdet med 70% etanol.Deretter administreres en enkelt dose tamoxifen intraperitonealt ved hjelp av en 27-gauge nål festet til en en-milliliter sprøyte for å indusere Cre-mediert rekombinasjon. Observer dyr i de neste 48 timene for å utelukke potensiell tamoksifentoksisitet. Etter 48 timer, overfør dyrene til bestrålingsrommet.Desinfiser prøvekammeret i gamma-bestråleren med 70% etanoloppløsning. Plasser den absorberende matten og dyrene inne i prøvekammeret. Plasser lokket, og lukk kammeret.Programmer gammabestråleren til å utsette dyr for 12 grå helkroppsbestråling. Vri deretter nøkkelen til startposisjonen, og trykk på start for å starte eksponeringen. Forlat rommet for aktiv eksponeringstid.Etter at den forhåndsinnstilte tiden er gått, stopper maskinen automatisk og begynner å pipe. Slå av maskinen ved å vri nøkkelen i stoppposisjon. Åpne prøvekammeret, og ta av lokket.Overfør dyrene til buret, og desinfiser prøvekammeret med 70% etanol. Overfør dyrene tilbake til det konvensjonelle boligrommet, og observer tilstanden deres etter behandling. Overvåk dyrets vekt hver dag.Tre timer før den planlagte eutanasien, desinfiser mageområdet og injiser musene intraperitonealt med 100 mikroliter EdU-oppløsning ved hjelp av en 28-gauge insulinsprøyte. Etter å ha avlivet dyret, samle de proksimale tarmene på forskjellige tidspunkter etter bestråling. Dissekere den proksimale delen av tynntarmen, og fjern vedlagte vev.Skyll vevet med kalde DPBS ved hjelp av en 16-gauge rett fôringsnål festet til en 10-milliliter sprøyte, og fest vevet med Bouins fiksative buffer. Klipp tarmene åpne i lengderetningen, og rull de proksimale tarmene ved hjelp av Swiss-roll-teknikken. Plasser vevet i en histologisk kassett i en beholder med 10% bufret formalin, og inkuber det i 24 til 48 timer ved romtemperatur.Etter inkubering, overfør de histologiske kassettene til 70% etanol, og legg vevet i paraffin. Etter innebygging, legg de histologiske kassettene på is i minst en time. Deretter kutter du fem mikron tykke horisontale seksjoner ved hjelp av en mikrotom.Overfør vevsseksjonene til et vannbad oppvarmet opp til 45 grader Celsius, og plasser seksjonene på ladede lysbilder. La lysbildene stå på et stativ over natten ved romtemperatur for å tørke. Hematoksylin- og eosinbilder av tynntarmsprøver viste et homeostatisk tarmepitel ved null time, etterfulgt av tap av celler i kryptrom i den apoptotiske fasen.Dette ble etterfulgt av den regenerative fasen der svært proliferative celler befolket kryptene, noe som førte til normaliseringsfasen etter dag syv. Avstamningssporing av EYFP viste at under homeostase var cellene som stammer fra Bmi1 positive reservestamceller begrenset til pluss fire til pluss seks posisjoner i krypter. EdU- og Ki-67-farging viste normal intestinal homeostase hos sham bestrålte mus, som strakte seg fra aktive stamceller gjennom transittforsterkende sone.Under den apoptotiske fasen ble antallet EYFP-positive celler redusert, ledsaget av tap av proliferative celler forårsaket av strålingsskader. I regenerativ fase ble det observert aktivering av reservestamceller og rask proliferasjon av Bmi1-positive celler. Videre spredning og migrasjon gjenopprettet integriteten til tarmepitelet.TUNEL-farging viste at apoptotiske celler er fraværende under homeostase. En økning i TUNEL-farging ble observert sent i den apoptotiske fasen. Under den regenerative fasen ble TUNEL-fargingen stadig redusert i de regenererende kryptene og var nesten fraværende da kryptene normaliserte seg.Denne teknikken kan kombineres med RNA-sekvensering, enkeltcelle RNA-sekvensering, fluorescensaktivert cellesortering eller proteomisk analyse for å skaffe mer inngående kunnskap om rollen til spesifikke cellelinjer under intestinal regenerering etter skade. Forskerne kan undersøke samspillet mellom mikrobiota og epitel-, stromal- og immuncellepopulasjoner i tarmregenerering for å utvide forståelsen av tarmepitelets regenerative evne.

intestinal-epithelial-regeneration-in-response-to-ionizing-irradiation

Research

JoVE Journal

Biology

Cortical Neurogenesis: Transitioning from Advances in the Laboratory to Cell-Based Therapies

Quantification of &gamma;H2AX Foci in Response to Ionising Radiation

DNA double-strand breaks (DSBs), which are induced by either endogenous metabolic processes or by exogenous sources, are one of the most critical DNA lesions with respect to survival and preservation of genomic integrity. An early response to the induction of DSBs is phosphorylation of the H2A histone variant, H2AX, at the serine-139 residue, in the highly conserved C-terminal SQEY motif, forming &gamma;H2AX1. Following induction of DSBs, H2AX is rapidly phosphorylated by the phosphatidyl-inosito 3-kinase (PIKK) family of proteins, ataxia telangiectasia mutated (ATM), DNA-protein kinase catalytic subunit and ATM and RAD3-related (ATR)2. Typically, only a few base-pairs (bp) are implicated in a DSB, however, there is significant signal amplification, given the importance of chromatin modifications in DNA damage signalling and repair. Phosphorylation of H2AX mediated predominantly by ATM spreads to adjacent areas of chromatin, affecting approximately 0.03% of total cellular H2AX per DSB2,3. This corresponds to phosphorylation of approximately 2000 H2AX molecules spanning ~2 Mbp regions of chromatin surrounding the site of the DSB and results in the formation of discrete &gamma;H2AX foci which can be easily visualized and quantitated by immunofluorescence microscopy2. The loss of &gamma;H2AX at DSB reflects repair, however, there is some controversy as to what defines complete repair of DSBs; it has been proposed that rejoining of both strands of DNA is adequate however, it has also been suggested that re-instatement of the original chromatin state of compaction is necessary4-8. The disappearence of &gamma;H2AX involves at least in part, dephosphorylation by phosphatases, phosphatase 2A and phosphatase 4C5,6. Further, removal of &gamma;H2AX by redistribution involving histone exchange with H2A.Z has been implicated7,8. Importantly, the quantitative analysis of &gamma;H2AX foci has led to a wide range of applications in medical and nuclear research. Here, we demonstrate the most commonly used immunofluorescence method for evaluation of initial DNA damage by detection and quantitation of &gamma;H2AX foci in &gamma;-irradiated adherent human keratinocytes9.

Quantification of &amp;#947;H2AX Foci in Response to Ionising Radiation

Quantification of DNA double-strand streaks using &gamma;H2AX formation as a molecular marker has become an invaluable tool in radiation biology. Here we demonstrate the use of an immunofluorescence assay for quantification of &gamma;H2AX foci after exposure of cells to radiation.

DNA double-strand breaks (DSBs), which are induced by either endogenous metabolic processes or by exogenous sources, are one of the most critical DNA ...

Evaluation of the Spatial Distribution of &gamma;H2AX following Ionizing Radiation

An early molecular response to DNA double-strand breaks (DSBs) is phosphorylation of the Ser-139 residue within the terminal SQEY motif of the histone H2AX1,2. This phosphorylation of H2AX is mediated by the phosphatidyl-inosito 3-kinase (PI3K) family of proteins, ataxia telangiectasia mutated (ATM), DNA-protein kinase catalytic subunit and ATM and RAD3-related (ATR)3. The phosphorylated form of H2AX, referred to as &gamma;H2AX, spreads to adjacent regions of chromatin from the site of the DSB, forming discrete foci, which are easily visualized by immunofluorecence microscopy3. Analysis and quantitation of &gamma;H2AX foci has been widely used to evaluate DSB formation and repair, particularly in response to ionizing radiation and for evaluating the efficacy of various radiation modifying compounds and cytotoxic compounds4.
Given the exquisite specificity and sensitivity of this de novo marker of DSBs, it has provided new insights into the processes of DNA damage and repair in the context of chromatin. For example, in radiation biology the central paradigm is that the nuclear DNA is the critical target with respect to radiation sensitivity. Indeed, the general consensus in the field has largely been to view chromatin as a homogeneous template for DNA damage and repair. However, with the use of &gamma;H2AX as molecular marker of DSBs, a disparity in &gamma;-irradiation-induced &gamma;H2AX foci formation in euchromatin and heterochromatin has been observed5-7. Recently, we used a panel of antibodies to either mono-, di- or tri- methylated histone H3 at lysine 9 (H3K9me1, H3K9me2, H3K9me3) which are epigenetic imprints of constitutive heterochromatin and transcriptional silencing and lysine 4 (H3K4me1, H3K4me2, H3K4me3), which are tightly correlated actively transcribing euchromatic regions, to investigate the spatial distribution of &gamma;H2AX following ionizing radiation8. In accordance with the prevailing ideas regarding chromatin biology, our findings indicated a close correlation between &gamma;H2AX formation and active transcription9. Here we demonstrate our immunofluorescence method for detection and quantitation of &gamma;H2AX foci in non-adherent cells, with a particular focus on co-localization with other epigenetic markers, image analysis and 3D-modeling.

Evaluation of the Spatial Distribution of &amp;#947;H2AX following Ionizing Radiation

Microscopic analysis of &gamma;H2AX foci, which form following the phosphorylation of H2AX at Ser-139 in response to DNA double-strand breaks, has become an invaluable tool in radiation biology. Here we used an antibody to mono-methylated histone H3 at lysine 4 as an epigenetic marker of actively transcribing euchromatin, to evaluate the spatial distribution of radiation-induced &gamma;H2AX formation within the nucleus.

An early molecular response to DNA double-strand breaks (DSBs) is phosphorylation of the Ser-139 residue within the terminal SQEY motif of the histone ...

A System for Culturing Iris Pigment Epithelial Cells to Study Lens Regeneration in Newt

Salamanders like newt and axolotl possess the ability to regenerate many of its lost body parts such as limbs, the tail with spinal cord, eye, brain, heart, the jaw 1. Specifically, newts are unique for its lens regeneration capability. Upon lens removal, IPE cells of the dorsal iris transdifferentiate to lens cells and eventually form a new lens in about a month 2,3. This property of regeneration is never exhibited by the ventral iris cells. The regeneration potential of the iris cells can be studied by making transplants of the in vitro cultured IPE cells. For the culture, the dorsal and ventral iris cells are first isolated from the eye and cultured separately for a time period of 2 weeks (Figure 1). These cultured cells are reaggregated and implanted back to the newt eye. Past studies have shown that the dorsal reaggregate maintains its lens forming capacity whereas the ventral aggregate does not form a lens, recapitulating, thus the in vivo process (Figure 2) 4,5. This system of determining regeneration potential of dorsal and ventral iris cells is very useful in studying the role of genes and proteins involved in lens regeneration.

In newt, the lens regenerates always from the dorsal iris by transdifferentiation of the iris pigmented epithelial cells (IPEs). Here we describe a procedure to culture dorsal and ventral newt IPE cells and their implantation to the newt eye. The implanted cells are then studied by tissue sectioning and immunohistochemistry.

Salamanders like newt and axolotl possess the ability to regenerate many of its lost body parts such as limbs, the tail with spinal cord, eye, brain, ...

Laser Ablation of the Zebrafish Pronephros to Study Renal Epithelial Regeneration

Acute kidney injury (AKI) is characterized by high mortality rates from deterioration of renal function over a period of hours or days that culminates in renal failure1. AKI can be caused by a number of factors including ischemia, drug-based toxicity, or obstructive injury1. This results in an inability to maintain fluid and electrolyte homeostasis. While AKI has been observed for decades, effective clinical therapies have yet to be developed. Intriguingly, some patients with AKI recover renal functions over time, a mysterious phenomenon that has been only rudimentally characterized1,2. Research using mammalian models of AKI has shown that ischemic or nephrotoxin-injured kidneys experience epithelial cell death in nephron tubules1,2, the functional units of the kidney that are made up of a series of specialized regions (segments) of epithelial cell types3. Within nephrons, epithelial cell death is highest in proximal tubule cells. There is evidence that suggests cell destruction is followed by dedifferentiation, proliferation, and migration of surrounding epithelial cells, which can regenerate the nephron entirely1,2. However, there are many unanswered questions about the mechanisms of renal epithelial regeneration, ranging from the signals that modulate these events to reasons for the wide variation of abilities among humans to regenerate injured kidneys.
The larval zebrafish provides an excellent model to study kidney epithelial regeneration as its pronephric kidney is comprised of nephrons that are conserved with higher vertebrates including mammals4,5. The nephrons of zebrafish larvae can be visualized with fluorescence techniques because of the relative transparency of the young zebrafish6. This provides a unique opportunity to image cell and molecular changes in real-time, in contrast to mammalian models where nephrons are inaccessible because the kidneys are structurally complex systems internalized within the animal. Recent studies have employed the aminoglycoside gentamicin as a toxic causative agent for study of AKI and subsequent renal failure: gentamicin and other antibiotics have been shown to cause AKI in humans, and researchers have formulated methods to use this agent to trigger kidney damage in zebrafish7,8. However, the effects of aminoglycoside toxicity in zebrafish larvae are catastrophic and lethal, which presents a difficulty when studying epithelial regeneration and function over time. Our method presents the use of targeted cell ablation as a novel tool for the study of epithelial injury in zebrafish. Laser ablation gives researchers the ability to induce cell death in a limited population of cells. Varying areas of cells can be targeted based on morphological location, function, or even expression of a particular cellular phenotype. Thus, laser ablation will increase the specificity of what researchers can study, and can be a powerful new approach to shed light on the mechanisms of renal epithelial regeneration. This protocol can be broadly applied to target cell populations in other organs in the zebrafish embryo to study injury and regeneration in any number of contexts of interest.

Acute kidney injury (AKI) in humans is a common clinical problem caused by damage to the epithelial cells that comprise kidney nephrons, and AKI is associated with high mortality rates of 50-70%1. Following epithelial cell destruction, nephrons have a limited ability to regenerate, though the mechanisms and limitations that guide this phenomenon remain poorly understood. In this video article, we describe our technique for targeted laser ablation of kidney nephron cells in the zebrafish embryo kidney, or pronephros. Our new method can be used to complement nephrotoxicity-induced models of AKI and gain a high-resolution understanding of the cell and molecular alterations that are associated with epithelial regeneration in the kidney nephron.

Acute kidney injury (AKI) is characterized by high mortality rates from deterioration of renal function over a period of hours or days that culminates in ...

Chromatin Isolation by RNA Purification (ChIRP)

Long noncoding RNAs are key regulators of chromatin states for important biological processes such as dosage compensation, imprinting, and developmental gene expression 1,2,3,4,5,6,7. The recent discovery of thousands of lncRNAs in association with specific chromatin modification complexes, such as Polycomb Repressive Complex 2 (PRC2) that mediates histone H3 lysine 27 trimethylation (H3K27me3), suggests broad roles for numerous lncRNAs in managing chromatin states in a gene-specific fashion 8,9. While some lncRNAs are thought to work in cis on neighboring genes, other lncRNAs work in trans to regulate distantly located genes. For instance, Drosophila lncRNAs roX1 and roX2 bind numerous regions on the X chromosome of male cells, and are critical for dosage compensation 10,11. However, the exact locations of their binding sites are not known at high resolution. Similarly, human lncRNA HOTAIR can affect PRC2 occupancy on hundreds of genes genome-wide 3,12,13, but how specificity is achieved is unclear. LncRNAs can also serve as modular scaffolds to recruit the assembly of multiple protein complexes. The classic trans-acting RNA scaffold is the TERC RNA that serves as the template and scaffold for the telomerase complex 14; HOTAIR can also serve as a scaffold for PRC2 and a H3K4 demethylase complex 13.
Prior studies mapping RNA occupancy at chromatin have revealed substantial insights 15,16, but only at a single gene locus at a time. The occupancy sites of most lncRNAs are not known, and the roles of lncRNAs in chromatin regulation have been mostly inferred from the indirect effects of lncRNA perturbation. Just as chromatin immunoprecipitation followed by microarray or deep sequencing (ChIP-chip or ChIP-seq, respectively) has greatly improved our understanding of protein-DNA interactions on a genomic scale, here we illustrate a recently published strategy to map long RNA occupancy genome-wide at high resolution 17. This method, Chromatin Isolation by RNA Purification (ChIRP) (Figure 1), is based on affinity capture of target lncRNA:chromatin complex by tiling antisense-oligos, which then generates a map of genomic binding sites at a resolution of several hundred bases with high sensitivity and low background. ChIRP is applicable to many lncRNAs because the design of affinity-probes is straightforward given the RNA sequence and requires no knowledge of the RNA's structure or functional domains.

Chromatin Isolation by RNA Purification ChIRP

ChIRP is a novel and rapid technique to map genomic binding sites of long noncoding RNAs (lncRNAs). The method takes advantage of the specificity of anti-sense tiling oligonucleotides to allow the enumeration of lncRNA-bound genomic sites.

Kromatin Isolering av RNA Purification (kvitre)

Long noncoding RNAs are key regulators of chromatin states for important biological processes such as dosage compensation, imprinting, and developmental ...

Rapid Genetic Analysis of Epithelial-Mesenchymal Signaling During Hair Regeneration

Hair follicle morphogenesis, a complex process requiring interaction between epithelia-derived keratinocytes and the underlying mesenchyme, is an attractive model system to study organ development and tissue-specific signaling. Although hair follicle development is genetically tractable, fast and reproducible analysis of factors essential for this process remains a challenge. Here we describe a procedure to generate targeted overexpression or shRNA-mediated knockdown of factors using lentivirus in a tissue-specific manner. Using a modified version of a hair regeneration model 5, 6, 11, we can achieve robust gain- or loss-of-function analysis in primary mouse keratinocytes or dermal cells to facilitate study of epithelial-mesenchymal signaling pathways that lead to hair follicle morphogenesis. We describe how to isolate fresh primary mouse keratinocytes and dermal cells, which contain dermal papilla cells and their precursors, deliver lentivirus containing either shRNA or cDNA to one of the cell populations, and combine the cells to generate fully formed hair follicles on the backs of nude mice. This approach allows analysis of tissue-specific factors required to generate hair follicles within three weeks and provides a fast and convenient companion to existing genetic models.

Tissue-specific analysis of a hair follicle regeneration model using lentivirus to mediate gain- or loss-of-function.

Rapid Genetic Analysis of epitelial-mesenchymale signaleringsprotokoll Under Hair Regeneration

Hair follicle morphogenesis, a complex process requiring interaction between epithelia-derived keratinocytes and the underlying mesenchyme, is an ...

Measuring Glutathione-induced Feeding Response in Hydra

Hydra is among the most primitive organisms possessing a nervous system and chemosensation for detecting reduced glutathione (GSH) for capturing the prey. The movement of prey organisms causes mechanosensory discharge of the stinging cells called nematocysts from hydra, which are inserted into the prey. The feeding response in hydra, which includes curling of the tentacles to bring the prey towards the mouth, opening of the mouth and consequent engulfing of the prey, is triggered by GSH present in the fluid released from the injured prey. To be able to identify the molecular mechanism of the feeding response in hydra which is unknown to date, it is necessary to establish an assay to measure the feeding response. Here, we describe a simple method for the quantitation of the feeding response in which the distance between the apical end of the tentacle and mouth of hydra is measured and the ratio of such distance before and after the addition of GSH is determined. The ratio, called the relative tentacle spread, was found to give a measure of the feeding response. This assay was validated using a starvation model in which starved hydra show an enhanced feeding response in comparison with daily fed hydra.

Here we describe a simple assay for the quantification of the feeding response in hydra induced by the reduced form of glutathione. This assay relies on measuring the distance between the apical end of the tentacle and mouth of hydra.

Måling glutation-indusert Fôring Response i Hydra

Hydra is among the most primitive organisms possessing a nervous system and chemosensation for detecting reduced glutathione (GSH) for capturing the prey. ...

In Vitro and In Vivo Approaches to Determine Intestinal Epithelial Cell Permeability

The intestinal barrier defends against pathogenic microorganism and microbial toxin. Its function is regulated by tight junction permeability and epithelial cell integrity, and disruption of the intestinal barrier function contributes to progression of gastrointestinal and systemic disease. Two simple methods are described here to measure the permeability of intestinal epithelium. In vitro, Caco-2BBe cells are plated in tissue culture wells as a monolayer and transepithelial electrical resistance (TER) can be measured by an epithelial (volt/ohm) meter. This method is convincing because of its user-friendly operation and repeatability. In vivo, mice are gavaged with 4 kDa fluorescein isothiocyanate (FITC)-dextran, and the FITC-dextran concentrations are measured in collected serum samples from mice to determine the epithelial permeability. Oral gavage provides an accurate dose, and therefore is the preferred method to measure the intestinal permeability in vivo. Taken together, these two methods can measure the permeability of the intestinal epithelium in vitro and in vivo, and hence be used to study the connection between diseases and barrier function.

In Vitro and In Vivo Approaches to Determine Intestinal Epithelial Cell Permeability

Two methods are presented here to determine intestinal barrier function. An epithelial meter (volt/ohm) is used for measurements of transepithelial electrical resistance of cultured epithelia directly in tissue culture wells. In mice, the FITC-dextran gavage method is used to determine the intestinal permeability in vivo.

In Vitro og i Vivo tilnærminger til å bestemme Intestinal tarmepitelet permeabilitet

The intestinal barrier defends against pathogenic microorganism and microbial toxin. Its function is regulated by tight junction permeability and ...

Dosimetry for Cell Irradiation using Orthovoltage (40-300 kV) X-Ray Facilities

The importance of dosimetry protocols and standards for radiobiological studies is self-evident. Several protocols have been proposed for dose determination using low energy X-ray facilities, but depending on the irradiation configurations, samples, materials or beam quality, it is sometimes difficult to know which protocol is the most appropriate to employ. We, therefore, propose a dosimetry protocol for cell irradiations using low energy X-ray facility. The aim of this method is to perform the dose estimation at the level of the cell monolayer to make it as close as possible to real cell irradiation conditions. The different steps of the protocol are as follows: determination of the irradiation parameters (high voltage, intensity, cell container etc.), determination of the beam quality index (high voltage-half value layer couple), dose rate measurement with ionization chamber calibrated in air kerma conditions, quantification of the attenuation and scattering of the cell culture medium with EBT3 radiochromic films, and determination of the dose rate at the cellular level. This methodology must be performed for each new cell irradiation configuration as the modification of only one parameter can strongly impact the real dose deposition at the level of the cell monolayer, particularly involving low energy X-rays.

Dosimetry for Cell Irradiation using Orthovoltage 40-300 kV X-Ray Facilities

This document describes a new dosimetry protocol for cell irradiations using low energy X-ray equipment. Measurements are performed in conditions simulating real cell irradiation conditions as much as possible.