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 at anerkende Stony Brook Cancer Center Histology Research Core for eksperthjælp med forberedelse af vævsprøver og Division of Laboratory Animal Resources ved Stony Brook University for hjælp til dyrepleje og håndtering. Dette arbejde blev støttet af tilskud fra National Institutes of Health DK124342 tildelt Agnieszka B. Bialkowska og DK052230 til Dr. Vincent W. Yang.

Alle mus blev anbragt i Division of Laboratory Animal Resources (DLAR) ved Stony Brook University. Stony Brook University Institutional Animal Care and Use Committee (IACUC) godkendte alle undersøgelser og procedurer, der involverede dyreforsøgspersoner. Forsøg med dyr blev udført i nøje overensstemmelse med den godkendte dyrehåndteringsprotokol (IACUC # 245094).
BEMÆRK: Musstammer B6;129-Bmi1 tm1(cre/ERT)Mrc/J (Bmi1-Cre ER) og B6.129X1-Gt(ROSA)26Sortm1(EYFP)Cos/J (<...

Denne protokol beskriver en robust og reproducerbar strålingsskademodel. Det giver mulighed for præcis analyse af ændringerne i tarmepitelet i løbet af 7 dage efter skaden. Det er vigtigt, at de valgte tidspunkter afspejler afgørende stadier af skade og er kendetegnet ved tydelige ændringer i tarmen (skade, apoptose, regenerering og normaliseringsfaser)60. Denne bestrålingsmodel er blevet fastlagt og omhyggeligt vurderet, hvilket viser en passende manifestation af efterligningsskade som den...

Det menneskelige tarmepitel ville dække omtrent overfladen af en halv badmintonbane, hvis den blev placeret helt fladt1. I stedet komprimeres dette enkelt cellelag, der adskiller mennesker fra indholdet af deres tarme, til en række fingerlignende fremspring, villi og fordybninger, krypter, der maksimerer tarmens overfladeareal. Cellerne i epitelet differentierer langs en krypt-villusakse. Villus består primært af næringsstofabsorberende enterocytter, slimudskillende bægerceller og de hormonproducerende enteroendokrine celler, mens krypterne primært består af defensinproducerende Paneth-celler, aktive og reservestamceller og stamceller 2,3,4,5. Desuden genererer den tovejskommunikation, disse celler har med stromale og immunceller i det underliggende mesenkymale rum og lumens mikrobiota, et komplekst netværk af interaktioner, der opretholder tarmhomeostase og er afgørende for genopretning efter skade 6,7,8.
Tarmepitelet er det hurtigst selvfornyende væv i menneskekroppen med en omsætningshastighed på 2-6 dage 9,10,11. Under homeostase deler aktive stamceller i bunden af tarmkrypter (kryptbasesøjleceller), der er kendetegnet ved ekspressionen af leucinrige gentagne holdige G-proteinkoblede receptor 5 (LGR5), hurtigt og tilvejebringer stamceller, der adskiller sig i alle andre intestinale epitellinjer. På grund af deres høje mitotiske hastighed er aktive stamceller og deres umiddelbare forfædre imidlertid særligt følsomme over for gammastrålingsskader og gennemgår apoptose efter bestråling 5,12,13,14. Efter deres tab gennemgår reservestamceller og ikke-stamceller (subpopulation af forfædre og nogle terminalt differentierede celler) i tarmkrypter aktivering og genopfylder basalkryptrummet, som derefter kan rekonstituere cellepopulationer af villi og dermed regenerere tarmepitelet15. Ved hjælp af afstamningssporingsteknikker har flere forskergrupper vist, at reservestamceller (hvilende) er i stand til at understøtte regenerering ved tab af aktive stamceller 13,16,17,18,19,20,21,22. Disse celler er karakteriseret ved tilstedeværelsen af polycomb kompleks protein 1 onkogen (Bmi1), mus telomerase revers transkriptase gen (mTert), Hop homeobox (Hopx) og leucin-rige gentage protein 1 gen (Lrig1). Derudover har det vist sig, at ikke-stamceller er i stand til at genopbygge tarmkrypter ved skade 23,24,25,26,27,28,29,30,31. Det har især vist sig, at forfædre til sekretoriske celler og enterocytter gennemgår dedifferentiering ved skade, vender tilbage til stamlignende celler og understøtter regenerering af tarmepitelet. Nylige undersøgelser har identificeret celler, der udtrykker flere markører, der har kapacitet til at erhverve stammelignende egenskaber ved skade (såsom DLL+, ATOH1+, PROX1+, MIST1+, DCLK1+)32,33,34,35,36. Overraskende viste Yu et al., at selv modne Paneth-celler (LYZ +) kan bidrage til intestinal regenerering37. Ud over at forårsage apoptose af intestinale epitelceller og forstyrre epitelbarrierefunktionen resulterer bestråling desuden i dysbiose af tarmfloraen, immuncelleaktivering og initiering af et proinflammatorisk respons samt aktivering af mesenkymale og stromale celler38,39.
Gammastråling er et værdifuldt terapeutisk værktøj i kræftbehandling, især for kolorektal tumorer40. Bestråling påvirker imidlertid signifikant intestinal homeostase ved at fremkalde skade på cellerne, hvilket fører til apoptose. Strålingseksponering forårsager flere forstyrrelser, der bremser patientens opsving og er præget af slimhindeskade og betændelse i den akutte fase og diarré, inkontinens, blødning og mavesmerter på lang sigt. Denne panoply af manifestationer kaldes gastrointestinal strålingstoksicitet. Derudover kan strålingsinduceret progression af transmural fibrose og/eller vaskulær sklerose først manifestere sig år efter behandlingen38,41. Samtidig med selve skaden inducerer stråling et reparationsrespons i tarmceller, der aktiverer signalveje, der er ansvarlige for at initiere og orkestrere regenerering42. Strålingsinduceret tyndtarmssygdom kan stamme fra bækken- eller abdominal strålebehandling, der leveres til andre organer (såsom livmoderhalsen, prostata, bugspytkirtel, endetarm)41,43,44,45,46. Tarmbestrålingsskader er således et væsentligt klinisk problem, og en bedre forståelse af den resulterende patofysiologi vil sandsynligvis fremme udviklingen af interventioner til lindring af gastrointestinale komplikationer forbundet med strålebehandling. Der er andre teknikker, der gør det muligt at undersøge det regenerative formål med tarmepitelet bortset fra stråling. Transgene og kemiske murinmodeller til undersøgelse af inflammation og regenerering derefter er blevet udviklet47. Dextran natriumsulfat (DSS) inducerer betændelse i tarmen og fører til udvikling af egenskaber svarende til inflammatoriske tarmsygdomme48. En kombination af DSS-behandling med den pro-kræftfremkaldende forbindelse azoxymethan (AOM) kan resultere i udvikling af colitis-associeret cancer48,49. Iskæmi reperfusionsinduceret skade er en anden metode, der anvendes til at studere det regenerative potentiale i tarmepitelet. Denne teknik kræver erfaring og kirurgisk viden50. Desuden forårsager ovennævnte teknikker andre typer skader end stråling og kan føre til involvering af forskellige regenereringsmekanismer. Derudover er disse modeller tidskrævende, mens strålingsteknikken er ret kort. For nylig er in vitro-metoder, der anvender enteroider og colonoider genereret fra tarm og tyktarm, blevet anvendt i kombination med strålingsskade til at studere mekanismerne for intestinal regenerering51,52. Disse teknikker rekapitulerer imidlertid ikke fuldt ud det organ, de modellerer53,54.
Den fremlagte protokol indeholder beskrivelsen af en murinmodel af gammastrålingsskade i kombination med en genetisk model, der efter tamoxifenbehandling muliggør sporing af slægter, der stammer fra reservestamcellepopulationen (Bmi1-CreER; Rosa26eYFP). Denne model anvender en 12 Gy totalkropsbestråling, som inducerer signifikant nok tarmskade til at aktivere reservestamceller, mens den stadig giver mulighed for efterfølgende undersøgelse af tarmregenerativ kapacitet inden for 7 dage efter skade55.

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intestinal epithelial regeneration in response to ionizing irradiation

Tarmepitelet består af et enkelt lag af celler, men indeholder flere typer terminalt differentierede celler, som genereres ved aktiv spredning af intestinale stamceller placeret i bunden af tarmkrypter. Under tilfælde af akut tarmskade gennemgår disse aktive tarmstamceller imidlertid celledød. Gammabestråling er en meget anvendt kolorektal cancerbehandling, som, selvom den er terapeutisk effektiv, har den bivirkning, at den nedbryder den aktive stamcellepulje. Faktisk oplever patienter ofte gastrointestinalt strålingssyndrom, mens de gennemgår strålebehandling, delvis på grund af aktiv stamcelleudtømning. Tabet af aktive tarmstamceller i tarmkrypter aktiverer en pulje af typisk hvilende reserve intestinale stamceller og inducerer dedifferentiering af sekretoriske og enterocytprecursorceller. Hvis ikke for disse celler, ville tarmepitelet mangle evnen til at komme sig efter strålebehandling og andre sådanne store vævsfornærmelser. Nye fremskridt inden for afstamningssporingsteknologier muliggør sporing af aktivering, differentiering og migration af celler under regenerering og er med succes blevet anvendt til at studere dette i tarmen. Denne undersøgelse har til formål at skildre en metode til analyse af celler i musens tarmepitel efter strålingsskade.

Anvendelsen af 12 Gy totalkropsbestråling (TBI) i kombination med sporing af murin genetisk afstamning muliggør en grundig analyse af konsekvenserne af strålingsskader i tarmen. For at starte, Bmi1-CreER; Rosa26eYFP-mus modtog en enkelt tamoxifeninjektion, som inducerer forbedret gult fluorescerende protein (EYFP) ekspression inden for en Bmi1+ reservestamcellepopulation. To dage efter tamoxifeninjektionen gennemgik musene bestråling eller skinbestråling. Tre timer før euta...

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

Mave-tarmkanalen er et af de mest følsomme organer for skade ved stråleterapeutiske kræftbehandlinger. Det er samtidig et organsystem med en af de højeste regenerative kapaciteter efter sådanne fornærmelser. Den præsenterede protokol beskriver en effektiv metode til at studere tarmepithelets regenerative kapacitet.

Intestinal epitelregenerering som reaktion 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 ...

Gammabestråling er en meget anvendt behandling for kolorektal cancer. Og selvom det er terapeutisk effektivt, har det negative virkninger på mave-tarmkanalen. Den præsenterede protokol beskriver en effektiv metode til at studere aktivering, differentiering og migration af celler under regenerering efter strålingsskade.Denne protokol beskriver en robust og reproducerbar strålingsskademodel. Det er vigtigt, at denne model gør det muligt at spore tarmcelleskæbne efter skade og dermed giver bedre forståelse af regenerativ kapacitet hos specifik cellesubpopulation. Denne teknik kan anvende forskellige afstamningssporingsdyremodeller til at studere realtidsadfærd og interaktionen mellem forskellige underpopulationer af celler efter skade.Mindre ændringer af denne protokol bør gøre det muligt at undersøge forholdet mellem mikrobiota og forskellige epitel-, stromale og immuncellepopulationer ved strålingsskade. Til at begynde med resuspenderes tamoxifenpulver i steril majsolie i en koncentration på 30 mikrogram pr. Mikroliter. Sonikere i tre minutter i 30 sekunder on-off cyklusser med 60% amplitude.Bland ved rotation i mørke i en time ved stuetemperatur. Forbered derefter EdU ved at tilsætte steril DMSO til EdU-pulver. Tilsæt 1/5 af det samlede volumen, og tilsæt langsomt det resterende volumen sterilt ultrarent vand.Når den er helt opløst, alikvote og opbevares ved 20 grader Celsius. Forbered reagenser til vævsopsamling og fiksering, såsom 70% ethanol i vand, DPBS kølet til fire grader Celsius, Bouins fikseringsbuffer og 10% bufret formalin. Forbered derefter det nødvendige udstyr, såsom et musedissektionssæt, petriskåle, en 16-gauge sondenål fastgjort til en 10 ml sprøjte og histologiske kassetter.To dage før gammabestråling skal forsøgsdyrene forberedes til tamoxifeninjektion. Væg hvert dyr, og beregn den krævede dosis. Desinficer abdominalområdet med 70% ethanol.Administrer derefter en enkelt dosis tamoxifen intraperitonealt ved hjælp af en 27-gauge nål fastgjort til en milliliter sprøjte for at inducere Cre-medieret rekombination. Overhold dyr i de næste 48 timer for at udelukke potentiel tamoxifentoksicitet. Efter 48 timer overføres dyrene til bestrålingsrummet.Prøvekammeret desinficeres i gammabestrålingsanlægget med 70 % ethanolopløsning. Den absorberende måtte og dyrene anbringes inde i prøvekammeret. Placer låget, og luk kammeret.Programmér gammabestråleren til at udsætte dyr for 12 grå kropsbestråling. Drej derefter nøglen til startpositionen, og tryk på start for at starte eksponeringen. Forlad rummet for aktiv eksponeringstid.Når den forudindstillede tid er gået, stopper maskinen automatisk og begynder at bippe. Sluk for maskinen ved at dreje nøglen i stopposition. Åbn prøvekammeret, og tag låget af.Overfør dyrene til buret, og desinficer prøvekammeret med 70% ethanol. Overfør dyrene tilbage til det konventionelle opstaldningsrum, og overhold deres tilstand efter behandlingen. Overvåg dyrenes vægt hver dag.Tre timer før den planlagte eutanasi desinficeres abdominalområdet og injicerer musene intraperitonealt med 100 mikroliter EdU-stamopløsning ved hjælp af en 28-gauge insulinsprøjte. Efter euthanisering af dyret skal du samle de proksimale tarme på forskellige tidspunkter efter bestråling. Disseker den proksimale del af tyndtarmen, og fjern vedhæftede væv.Skyl vævet med kold DPBS ved hjælp af en 16-gauge lige fodringsnål fastgjort til en 10 ml sprøjte, og fastgør vævet med Bouins fikseringsbuffer. Skær tarmene åbne i længderetningen, og rul de proksimale tarme ved hjælp af schweizerrulleteknikken. Anbring vævene i en histologisk kassette i en beholder med 10% bufret formalin, og inkuber det i 24 til 48 timer ved stuetemperatur.Efter inkubation overføres de histologiske kassetter til 70% ethanol, og vævet indlejres i paraffin. Efter indlejring anbringes de histologiske kassetter på is i mindst en time. Skær derefter fem mikron tykke vandrette sektioner ved hjælp af et mikrotom.Overfør vævssektionerne til et vandbad opvarmet til 45 grader Celsius, og placer sektionerne på ladede dias. Lad diasene stå på et stativ natten over ved stuetemperatur for at tørre. Hæmatoxylin- og eosinbilleder af tyndtarmsprøver viste et homeostatisk tarmepitel ved nul time efterfulgt af tab af celler i kryptrum under den apoptotiske fase.Dette blev efterfulgt af den regenerative fase, hvor stærkt proliferative celler befolkede krypterne, hvilket førte til normaliseringsfasen på dag syv. Sporing af afstamning foretaget af EYFP viste, at cellerne, der stammer fra Bmi1-positive reservestamceller, under homeostase var begrænset til plus fire til plus seks positioner i krypter. EdU- og Ki-67-farvning viste normal intestinal homeostase hos falske bestrålede mus, der strækker sig fra aktive stamceller gennem transit-amplificerende zone.Under den apoptotiske fase faldt antallet af EYFP-positive celler ledsaget af et tab af proliferative celler forårsaget af strålingsskader. I den regenerative fase blev aktivering af reservestamceller og hurtig proliferation af Bmi1-positive celler observeret. Yderligere spredning og migration genoprettede tarmepitelintegritet.TUNEL-farvning viste, at apoptotiske celler er fraværende under homeostase. En stigning i TUNEL-farvning blev observeret sent i den apoptotiske fase. I den regenerative fase faldt TUNEL-farvningen støt inden for de regenererende krypter og var næsten fraværende, da krypterne normaliserede sig.Denne teknik kan kombineres med RNA-sekventering, enkeltcelle RNA-sekventering, fluorescensaktiveret cellesortering eller proteomisk analyse for at få mere dybdegående viden om rollen som specifikke cellelinjer under tarmregenerering efter skade. Forskerne kan undersøge samspillet mellem mikrobiota og epitel-, stromale og immuncellepopulationer i tarmregenerering for at udvide forståelsen af tarmepithelets 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.

Chromatin Isolering af RNA Oprensning (chirp)

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.

Hurtig genetisk analyse af Epithelial-mesenkyme Signaling 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 Glutathion-induceret Fodring respons 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 In Vivo tilgange til at bestemme Intestinal epitelcelle 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.