Murina kolit modellering med hjälp av dextransulfat Natrium (DSS)

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Summary

Dextransulfat natrium (DSS) administreras i dricksvatten är en etablerad mus-inflammatoriska skador modell av akut kolit. Detta protokoll föreslås en metod för DSS behandling och beredning av vävnader.

Cite this Article

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Whittem, C. G., Williams, A. D., Williams, C. S. Murine Colitis Modeling using Dextran Sulfate Sodium (DSS). J. Vis. Exp. (35), e1652, doi:10.3791/1652 (2010).

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Abstract

Kolit kan uppstå från virus eller bakteriella infektioner, ischemisk förolämpning eller autoimmuna sjukdomar, främst ulcerös kolit och kolon variant av Crohns sjukdom - Crohns kolit. Akut kolit kan innebära med buksmärtor och dilatation, malabsorption, diarré, Hematochezia och slem i avföringen. Vi börjar förstå de komplexa sambanden mellan miljö, genetik och epitelceller barriär dysfunktion vid inflammatorisk tarmsjukdom och djurmodeller av kolit har varit väsentliga för att öka kunskaperna om denna sjukdom. En populär modell Komplettera dricksvatten av möss med låg molekylvikt Dextran natriumsulfat (DSS), vilket resulterar i epitelial skador och en robust inflammatorisk reaktion i tjocktarmen varar flera dagar

Protocol

Del 1: Skador-Repair Kolit Modell

  1. Kroppsvikt bör erhållas för varje mus innan du börjar DSS behandling.
  2. Gör en 3% (w / v) dextransulfat lösning Natriumsalt i vatten och filtrera med ett 0,45 ìm cellulosaacetat filter.
  3. Byt ut dricksvatten i mus bur med 3% DSS lösning för fyra dagar. De möss bör inte ha tillgång till någon annan källa med vatten (dvs uteslutning tips placeras på automatiserade bevattningssystem).
  4. På dag fyra, byt DSS lösning med vatten i ytterligare tre dagar, vilket gör att en del kolon epiteliala återhämtning. Mössen ska vägas på dag fyra i syfte att kvantifiera systemiska konsekvenser av kolit. Viktminskning är vanligt med allvarliga skador.
  5. Dag 7, väga och offra möss. Möss kan avlivas genom inhalation överdos av isofluran eller andra institutionellt, godkända IACUC metoder.

Del 2: obduktion och skörd av Colon

  1. Exponera ventrala sidan av musen och säkra ben för att garantera obehindrad tillgång till buken. Blöt buken helt med 70% etanol.
  2. Ta tag i mittlinjen av buken med vävnad pincett och sträcker sig uppåt därmed camping huden. Använd spetsig sax incisionsfilm buken därmed utsätta buken innehåll. Utöka snittet till toppen av xyphoid processen på mittlinjen och sedan sträcka sig längs de underlägsna aspekten av costal marginalerna bilateralt.
  3. Identifiera tunntarmen, blindtarmen och tjocktarmen. Försiktigt dissekera / reta tjocktarmen från den omgivande tarmkäx. Transekt tjocktarmen djupt i bäckenet för att frigöra den distala tjocktarmen och ändtarmen. Transekt tjocktarmen vid colonocecal marginalen till fri den proximala kolon. Man måste vara försiktig under denna process inte skada kolon som rengör tjocktarmen blir problematiskt om perforering uppstår.
  4. Med hjälp av en 20G utfodring nål och 10 ml spruta, intuberas och spola kolon med iskalla PBS tills eluatet är helt fri från avföring.
  5. Vid denna punkt, om makroskopisk analys med en dissekera mikroskop önskas, kan kolon fastställas som "korvar". Om histologisk analys bara önskas än vidare till del 4 "Göra schweiziska Rolls för den histologiska analysen av akut kolit".

Del 3: Bearbetning som "Korv" för makroskopisk analys av hela tjocktarmen

  1. Det är viktigt att behålla rätt orientering i tjocktarmen, därför håller den distala änden av tjocktarmen närmast föraren. Klipp två bitar av icke-absorberbara suturer (SP116 1,5 metriska flätat silke), en ca 1 tum i längd, den andra 2 inches.
  2. Använd 2 tum bit sutur att knyta upp den distala änden så nära marginal som möjligt samtidigt bibehålla en bra tätning.
  3. Placera en 20G utfodring nål som innehåller 5 ml 10% buffrad formalin fosfat i proximala slutet av tjocktarmen. Löst binda resterande bit sutur omedelbart proximalt om glödlampan för utfodring nålen. Ta tag i tjocktarmen ordentligt på glödlampan för utfodring nål och ingjuta formalin fram till tjocktarmen är expanderat. Dra åt knut på suturen som du återkalla utfodring nålen, vilket lämnar den infunderade expanderade kolon som en "korv".
  4. Fyll en 15 ml koniska rör med 10% buffrad formalin fosfat och placera kolon i röret. Fix i 24 timmar.
  5. Häll bort formalin och ersätt med 70% EtOH för ytterligare 24 timmar. (Kolon kan lagras i 70% EtOH obegränsad tid vid rumstemperatur.)
  6. Ta bort tjocktarmen från det koniska röret och kapa strängarna på varje ände med en skalpell och var försiktig så du inte skadar tjocktarmen. Kom ihåg att den distala änden har längst snöre. Klipp längs från distalt proximala änden av tjocktarmen så att den bildar en lång plåt. Vid denna punkt kan tjocktarmen ses i dissekera mikroskop.

Del 4: Att göra schweiziska Rolls för den histologiska analysen av akut kolit

  1. Det är viktigt att behålla rätt orientering i tjocktarmen, därför håller den distala änden av tjocktarmen närmast föraren.
  2. Mät kolon längd. Det här måttet är en annan indikator på svårighetsgraden av skada. Kolit ökar ödem och förkortar den totala kolon längd.
  3. Använda spetsig sax, incisionsfilm longitudinellt från distalt proximala änden av tjocktarmen. Använd fin tippade pincett för att greppa kanten på snitt och öppna sidled arbeta dig distalt-> proximalt, vilket visar kolon som en platt ark.
  4. Rullande tjocktarmen kräver en pincett och en två räckte teknik. Ta tag i kanten på distal marginal med pincett. Gå vidare till sekventiellt rulla tjocktarmen genom att rotera och släppa pincett fortsätter till den proximala marginalen. Vilket genererar en spiral med en tredje dimension eller "Rulltårta".För att upprätthålla rullade formen, ta tag i rullen ordentligt med pincett och transekt den med en 27G1 / 2 st. Fäst nålen genom att böja nålen när det lämnar rullen.
  5. Placera rullen i en korrekt märkta vävnad kassetten och lägg i en burk med 10% buffrad formalin fosfat i rumstemperatur i 24 timmar för att säkerställa vävnad fixering.
  6. Häll bort formalin och ersätt med 70% EtOH för ytterligare 24 timmar. (Kolon kan lagras i 70% EtOH obegränsad tid vid rumstemperatur.)
  7. När provet har fastställts, kan det vara paraffininbäddade, sektioneras och monteras på objektglas för histologisk analys.

Del 5: representativa resultat

DSS modell för akut kolit kan forskaren få, korrigera och analysera ett kolon att modeller akut kolit. När Rulltårta kapas och monteras bör den utgöra en representativ bit av hela tjocktarmen om rullade på rätt sätt (Figur 1). Den monterade rullen kan färgas med H & E för att fastställa omfattningen av skador på tjocktarmen, från den distala (inuti) slut på den proximala (utanför) ände (figur 2). Immunohistokemi kan också utföras på Rulltårta att identifiera och kvantifiera inflammatoriska infiltrat. Om korven metoden har utförts korrekt, kommer den fasta tjocktarmen vara dilaterade och hela slemhinneepitel lätt kan manipuleras och visas under dissekera mikroskop (Figur 3).

Figur 1
Figur 1. Korrekt utförda "rulltårta". (A) kolon rullas från distalt proximala änden, transected med en nål och säkras genom att böja i slutet av nålen. Det är sedan placeras i en vävnad kassett för fixering. (B) H & E målat 5 ìm avsnitt av en rulltårta gjord av tjocktarmen som en mus som behandlats med DSS (d = distala kolon p = proximala colon).

Figur 2
Figur 2. DSS behandlas kolon visar tecken på akut kolit. Inflammation och crypt skador är tydligt i DSS-behandlade kolon jämfört med ett vatten som behandlats kontroll.

Figur 3
Figur 3. Ett exempel på en "korv". Korven infunderas med formalin och helt utökas. En liten vinkel kommer att vara närvarande sekundära till den naturliga krökning av tjocktarmen. Den öppnade korven ska ligga plant.

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Discussion

Detta protokoll kan modifieras för att modellera akut skada, skada, reparation, eller kronisk kolon processer skada.

Akut skada Ändring:

DSS behandling ad lib i 4-6 dagar följt av omedelbar offer

Skador-Reparation Ändring:

Skada med 4-6 dagars DSS behandling följt av återhämtning under 3-4 dagar och offer (som beskrivs i ovannämnda protokoll).

Kronisk Kolit Ändring:

Möss är placerade på tre fem-dagars cykler med 3% DSS med sexton dagars återhämtning mellan varje cykel. Möss offras efter den sista 16-dagars viloperiod.

Det finns flera frågor som forskare måste vara medvetna om med denna modell:

  1. Variabilitet i platsen för skadan: i våra händer, ser vi en övervägande distal skada mönster, med relativt sparsamt av den proximala kolon och blindtarmen. Andra har rapporterat mer proximalt dominans till den skada mönstret.
  2. Miljö-variabilitet. Det finns en betydande miljö-variabilitet. Detta är sannolikt delvis beroende på skillnader i tarmflorans, kost och andra miljöhänsyn.
  3. Denna modell är ineffektiv om hög molekylvikt DSS används (dvs. 500 tusen Da).
  4. Det finns stora påfrestningar variabilitet med denna modell 4.

Biokemiska analys av tjocktarmen kan utföras genom att ta prover från den proximala och distala marginaler. Beroende på graden av skada detta kan påverka din förmåga att generera en histologisk skada poäng. In vivo BrdU märkning för att mäta spridning kan uppnås genom intraperitoneal injektion på 16,7 mikrogram / kg BrdU 2 timmar före offra följt av α-BrdU IHC bearbetning.

Som ett alternativ till "korv", kan tjocktarmen fastställas helt platt. Line botten av en Tupperware behållare med Whatman papper och blöta pappret med 10% buffrad formalin fosfat. Dissekera tjocktarmen som beskrivs i "Del 2: obduktion och skörd av Colon". Återigen, håll den distala änden av tjocktarmen närmast föraren. Använda spetsig sax, incisionsfilm longitudinellt från distalt proximala änden av tjocktarmen. Med fin spets pincett grepp antingen kanten av snittet och öppna sidled arbeta, vilket visar kolon som en platt ark. Överför denna platta blad till pre-indränkt Whatman papper. Täck tillplattat kolon med en annan bit av Whatman och blöta med 10% buffrad formalin fosfat. Den Whatman ska vara helt täckt i formalin men inte så mycket att det lyfter tjocktarmen. Täta Tupperware och fastställa till 24 timmar. Ta bort tjocktarmen från Tupperware och placera den i 10 ml 70% etanol i en 15 ml koniska rör. (Kolon kan lagras i 70% EtOH obegränsad tid vid rumstemperatur.)

Det finns flera metoder för att kvantifiera kolon skada 5,6,7. En metod, till exempel, använder en flera parametrar skala med: inflammation, omfattning engagemang, förnyelse, krypta skador och procent inblandning 8.

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Disclosures

Försök på djur har utförts i enlighet med de riktlinjer och regler som respektive Vanderbilt IACUC.

Acknowledgements

Finansiering från NIH (1 K08 DK080221-01) och Vanderbilt institutionella fonder utveckling.

Materials

Name Company Catalog Number Comments
Dextran Sulfate Sodium Salt USB Corp., Affymetrix 9011-18-1 mol wt 40,000-50,000
10% Buffered Formalin Phosphate Fisher Scientific SF100-4
Isoflurane, USP Phoenix Pharmaceuticals, Inc. NDC 57319-474-06
TISSUE PATH Macrosette Cassettes Fisher Scientific 15182706
BD 10 ml Syringe BD Biosciences 309604
Ethyl Alcohol Pharmco-AAPER E190 Dilute to 70% with distilled water
20G Straight Feeding Needle VWR international 20068-612
27G1/2 PrecisionGlide Needle BD Biosciences 305109
Dissecting Scissors Sharp/Blunt Tip VWR international 82027-588
Waugh Forceps VWR international 82027-428
Non-absorbable Suture LOOK Surgical SP116
Whatman Blotting Paper VWR international 28298-020 Cut as needed
Nalgene Surfactant-Free Cellulose Acetate (SFCA) Filter Cole-Parmer EW-06731-2
Carbon fiber composites digital caliper Fisher Scientific 15-007-958

DOWNLOAD MATERIALS LIST

References

  1. Okayasu, I. A novel method in the induction of reliable experimental acute and chronic ulcerative colitis in mice. Gastroenterology. 98, (3), 694-702 (1990).
  2. Martinez, J. A. Deletion of Mtgr1 sensitizes the colonic epithelium to dextran sodium sulfate-induced colitis. Gastroenterology. 131, (2), 579-588 (2006).
  3. Moolenbeek, C., Ruitenberg, E. J. The "Swiss roll": a simple technique for histological studies of the rodent intestine. Lab Anim. 15, (1), 57-59 (1981).
  4. Mahler, M. Differential susceptibility of inbred mouse strains to dextran sulfate sodium-induced colitis. Am J Physiol. 274, G544-551 (1998).
  5. Aharoni, R. Immunomodulatory therapeutic effect of glatiramer acetate on several murine models of inflammatory bowel disease. J Pharmacol Exp Ther. 318, (1), 68-78 (2006).
  6. Hahm, K. B. Loss of transforming growth factor beta signalling in the intestine contributes to tissue injury in inflammatory bowel disease. Gut. 49, (2), 190-198 (2001).
  7. Green, B. T. Adrenergic modulation of Escherichia coli O157:H7 adherence to the colonic mucosa. Am J Physiol Gastrointest Liver Physiol. 287, (6), G1238-1246 (2004).
  8. Dieleman, L. A. Chronic experimental colitis induced by dextran sulphate sodium (DSS) is characterized by Th1 and Th2 cytokines. Clin Exp Immunol. 114, (3), 385-391 (1998).

Comments

55 Comments

  1. what kind of mice you used in the experiment? hao ole of the mice when you treatment with DSS

    Reply
    Posted by: echo r.
    March 18, 2010 - 8:43 PM
  2. We use both 1²9 and C57BL6 mice in this experiment but the protocol can be used in any background. The mice are at least 6 weeks old before we begin treatment with DSS.

    Reply
    Posted by: Caitlyn W.
    March 19, 2010 - 10:27 AM
  3. what kind of mice you used in the experiment? hao ole of the mice when you treatment with DSS

    Reply
    Posted by: echo r.
    March 18, 2010 - 9:36 PM
  4. what happens if we keep the colon more than ²4 hours in 10%formalin ?

    Reply
    Posted by: peyman m.
    May 23, 2010 - 6:17 AM
  5. Leaving them in the 10% formalin shouldn't be a problem. We have left them over the weekend. For counts, longer fixation shouldn't be problematic, but I would put them in 70% ethanol before the end of 3 days.

    Reply
    Posted by: Caitlyn W.
    May 23, 2010 - 6:38 PM
  6. should DSS be prepared daily? and why it need be filtered?&#²9²59;Is the coulor of the solution&#²9²59;(DSS solute in filter water ) yellow

    Reply
    Posted by: Anonymous
    June 15, 2010 - 8:50 AM
  7. The DSS dŒs not need to be prepared daily. You can fill a water bottle enough for the four days on DSS. It needs to be filtered because you will be handling it before filtering and it can harbor bacteria, etc. that you don't want to feed to your mice. Yes, the color of the solution is slightly yellow.

    Reply
    Posted by: Caitlyn W.
    June 15, 2010 - 11:52 AM
  8. should I store the solution in routine temperature or in the fridge?

    Reply
    Posted by: Anonymous
    June 16, 2010 - 2:50 AM
  9. It is stored at room temperature.

    Reply
    Posted by: Caitlyn W.
    June 16, 2010 - 12:54 PM
  10. should I change the solution containing dss daily ? dŒs it influence the result if i do not change the solution daily?

    Reply
    Posted by: Anonymous
    June 16, 2010 - 9:48 PM
  11. There is no need to change the solution daily. Just put enough DSS solution in the water bottle on day one to last for the duration of the first cycle of DSS.

    Reply
    Posted by: Anonymous
    June 17, 2010 - 12:02 PM
  12. In our animal facility ,autoclaved tap water is used for all the mice. can I prepare DSS solution in tap water and then filter , or only distilled water shuld be used.

    Reply
    Posted by: Anonymous
    June 17, 2010 - 12:57 AM
  13. You can prepare the solution in tap water and then filter.

    Reply
    Posted by: Anonymous
    June 17, 2010 - 12:03 PM
  14. Dear Drs Whittem, Williams and Williams:

    Hello, I am writing a protocol to my University's IACUC to perform DSS induction of colitis, and was hoping that you may be willing to answer some of my questions regarding the animals subjected to DSS. I will be using the acute injury and injury repair models mentioned in your article. Could you please provide additional details of the stress that the mice might experience throughout the course of the experiment? The signs stated on the tutorial included weight loss, bloody stools, and diarrhea. Do these mice have additional issues such as an inability to retrieve food from an overhead feeding dish/water bottle, anorexia, inability to groom, contractures (i.e. 'hunching' in response to pain) or additional signs that may be useful to note when submitting my protocol? Also, should I expect mortality during treatment? As recommended, I will use mice older than six weeks, but likely not several weeks older as I want to avoid age-related issues. Finally, would you recommend the use of any type of analgesic drug during the course of DSS-colitis experiments to minimize discomfort in these animals? If so, would you happen to know the effect the analgesic has on factors such as barrier function, healing, infection rate, overall survival etc.? As I do not yet have any personal experience with this model, any insight will be greatly appreciated. Thank you.

    Reply
    Posted by: Elle F.
    June 28, 2010 - 8:03 AM
  15. For the acute injury and injury repair models you should only see the stresses mentioned in the article, weight loss, bloody stool, and diarrhea. There are no additional issues and you should not expect mortality during treatment. I would not recommend the use of an analgesic drug because, for example, NSAIDs will injure the gut and they should not be needed. Good luck!

    Reply
    Posted by: Caitlyn W.
    June 28, 2010 - 10:52 AM
  16. How do you utilize the sausages? I assume you slit them lengthwise after fixation and then stain (with what?) and examine them enface in a dissecting scope. What can you learn from this type of examination? Do you have a preferred method for quantitation of the resultand data?

    Reply
    Posted by: John W.
    July 30, 2010 - 3:05 PM
  17. You are correct, the sausages are cut lengthwise and stained with methylene blue and analyzed for aberrant crypt foci and polyp burden. The data is collected as and quantified as numbers of ACF/colon or number or size of polyps per colon.

    Reply
    Posted by: Caitlyn W.
    July 30, 2010 - 3:19 PM
  18. Read the abstract,
    "Mice are euthanized at the conclusion ... and at necropsy ... and can be 'Swiss rolled" 3 to allow microscopic analysis of the entire colon or infused with formalin as "sausages" to allow macroscopic analysis. Tissue is then embedded in paraffin, sectioned, and stained for histologic review."

    "Swiss roll" is for histological analysis, while "sausage" is for macroscopic analysis of the ENTIRE colon.

    Reply
    Posted by: Anonymous
    December 6, 2010 - 11:21 AM
  19. is dextran a reliable model for colitis...?do i use the drug for treatment before or after colitis using dextran?

    Reply
    Posted by: Anonymous
    June 12, 2011 - 7:20 AM
  20. DSS is one of the better models for colitis. The papers found at the following sites as well as those that we cite in our paper give good information on the topic. http://www.ncbi.nlm.nih.gov/pubmed/9844047?dopt=Citation http://www.ncbi.nlm.nih.gov/pubmed/8881816?dopt=Citation http://www.ncbi.nlm.nih.gov/pubmed/14611673?dopt=Citation We have not performed treatments on mice on DSS. We mainly use it to analyze the response of knockout models to experimental colitis. Treatment times would depend on whether you would like to prevent or treat colitis.

    Reply
    Posted by: Caitlyn W.
    June 15, 2011 - 6:24 PM
  21. I'm working with this model, but using ²% DSS as described. We bought our chemicals from another company, but the molecular weight is the same. Can you tell me more about the bloody diarrhea. I have finished the DSS water, but did not notice any blood. There was a bit of an excessive amount of feces, but I did not see the mice hair (white) or bedding stained with dried blood. I've read in other papers that not all the mice have it, but dŒs the fact that I did not see any mean that something went wrong?

    Reply
    Posted by: Anonymous
    July 17, 2011 - 1:21 PM
  22. There can be considerable variability in the severity of colitis induced by DSS. This depends on manufacturer, lot #, environment, differences in enteric flora. We typically track weights, DAI which includes stool consistency, blood, etc, measure colon length and weight at necropsy and for us, we consider the histology observed from reviewing the H+E's as the gold standard for injury. I would recommend reviewing the histology with a GI pathologist.

    Reply
    Posted by: Anonymous
    July 20, 2011 - 3:00 PM
  23. I am submitting a protocol for DSS to the IACUC. They want to know how I "will deal with the diarrhea with occult blood". Is there anything that we can administer to the mice to make the IACUC happy? They want to know how long I will let these symptoms go on before removing the mouse from the study. Unfortunately, all of these things are facets of the disease.

    Reply
    Posted by: Anonymous
    August 18, 2011 - 10:09 AM
  24. Hello Jesse,
    We insure that IACUC knows that mice that drop below ²0% weight loss are sac'd. The amount of time that mice are on DSS results in diarrhea and blood for only 4-6 days and mice recover well. You should not have to worry about mice having symptoms for more than a week.

    Reply
    Posted by: Caitlyn W.
    August 18, 2011 - 12:00 PM
  25. Thank you for your response. Another question...if the mice are in distress(eg. bloody diarrhea) but we don't give them any analgesic, then the IACUC will make them go down as Category E by USDA standards. DŒs anyone else have experience with this? DŒs anyone else list them as Category E?

    Reply
    Posted by: Anonymous
    August 18, 2011 - 2:00 PM
  26. Ours are Category E

    Reply
    Posted by: Caitlyn W.
    August 18, 2011 - 6:19 PM
  27. The water to be used with the DSS should be autoclaved water?

    Reply
    Posted by: Lilian N.
    August 18, 2011 - 10:50 PM
  28. DŒs anybody do anything to combat dehydration in these mice?

    Reply
    Posted by: Anonymous
    August 19, 2011 - 10:56 AM
  29. Sucrose 6%.
    http://farncombe.mcmaster.ca/documents/Ghiaetal.GastrŒnterology²0091367²²80-²²88.pdf

    Reply
    Posted by: Lilian N.
    August 19, 2011 - 12:57 PM
  30. The water used with DSS dŒs not need to be autoclaved. You should filter it after making the DSS solution. We do IP saline injections.

    Reply
    Posted by: Caitlyn W.
    August 19, 2011 - 1:39 PM
  31. DSS will be degrade or not in water. How DSS solution will be filtered what is procedure for that.can u elaborate thanks

    Reply
    Posted by: Anonymous
    September 24, 2011 - 3:11 AM
  32. We have not officially tested whether DSS degrades in water over extended periods of time. We make fresh DSS solutions for each cycle of DSS that is administered and do not reuse the DSS that is remaining in the water bottles after each cycle. As mentioned in the protocol, we make a 3% (w/v) Dextran Sulfate Sodium Salt solution in water and filter with a 0.45 μm cellulose acetate filter (Nalgene Surfactant-Free Cellulose Acetate (SFCA) Filter, Cole-Parmer, catalog number: EW-06731-²). The directions for this filter come with it but it is simply a vacuum filtration system as pictured in the video (seen at 0:5², "Injury-Repair Colitis Model").

    Reply
    Posted by: Caitlyn W.
    September 25, 2011 - 3:50 PM
  33. Has anyone used buprenorphine in the DSS model?

    Reply
    Posted by: Anonymous
    September 27, 2011 - 1:29 PM
  34. Question: In this mouse model of DSS colitis, do we have to to remove the colon contents (stools) before histological analysis? How is this done? Thank you!

    Reply
    Posted by: Anonymous
    October 14, 2011 - 10:49 AM
  35. At 3:17 in the video the removal of stools is shown. The colon is flushed with PBS using a ²0G straight feeding needle.

    Reply
    Posted by: Caitlyn W.
    October 17, 2011 - 4:38 PM
  36. In some protocols, people sacrifice the mice 3 or 5 days after DSS treatment. What is the purpose of this? How is this different from killing the mice after 4 days of DSS treatment.

    Reply
    Posted by: Anonymous
    January 16, 2012 - 4:52 PM
  37. The number of days that you sac after DSS treatment will determine the amount of time the mice have to heal. we generally use 4 days because, in wild type mice, this is usually the best amount of time to see the beginnings of recovery while still noting damage in the mice of interest used in our lab. Basically, this is the point at which we see the most difference between the two groups. If you have a mouse that you expect to heal more quickly, 3 days may be suitable, and vice versa.

    Reply
    Posted by: Caitlyn W.
    January 18, 2012 - 1:21 PM
  38. Regarding the step 7 of part 4 in the protocol mentioned above, how do you remove a needle from a swiss-rolled tissue before going forward to paraffin-embedding? Just pull out? Do you have a specific way to do that?

    Reply
    Posted by: Anonymous
    January 17, 2012 - 6:05 PM
  39. Once the tissue is formalin fixed, the tissue will be stiff enough that you can either unbend or clip the needle and slide it out of the roll without altering the shape of the Swiss roll. Our tissue processing core knows how to handle our tissue, but if you need to do it yourself, insure that the tissue has been in 70% ethanol at least overnight and then use gloves and forceps to remove the needle from the roll.

    Reply
    Posted by: Caitlyn W.
    January 18, 2012 - 1:24 PM
  40. What are the strains of mice used for this model? And what is the health status of the animals?

    Reply
    Posted by: Anonymous
    February 10, 2012 - 11:16 AM
  41. We use C57Bl6 mice for our experiments. Our mice are healthy before treatment with DSS.

    Reply
    Posted by: Caitlyn W.
    December 13, 2012 - 1:04 PM
  42. Hello, I am writing a protocol to my University's IACUC to perform DSS induction of colitis.Should I put DSS in Hazardous material form?Is there any method to detect the DSS excretion in feces and urine?How can we deal with the waste from DSS (unused solution)?

    Thanks,

    Reply
    Posted by: Jinggang L.
    December 13, 2012 - 11:22 AM
  43. Our IACUC protocol dŒs not include DSS as a hazardous material and we pour waste DSS down the drain. As far as I know, there is not a method to detect DSS excretion in feces and urine.

    Reply
    Posted by: Caitlyn W.
    December 13, 2012 - 1:06 PM
  44. Hi
    In the materials you list Dextran Sulfate Sodium Salt USB Corp., Affymetrix 9011-18-1 mol wt 40,000-50,000.
    However when I check Affymetrix website, this catalog number belongs to a MW: 500,000 product (http://www.affymetrix.com/esearch/search.jsp?pd=131111&N=4²94967²93) . is this what you used or otherwise what is the correct catalog number. thanks!

    Reply
    Posted by: selcen o.
    May 6, 2013 - 10:50 AM
  45. I am so sorry for the confusion. The number listed in the protocol is the CAS# which is similar between the 500,000 and 50,000 M.W. DSS. The actual catalog number is 14489. This number should get you what you need (http://www.affymetrix.com/estore/browse/brand/usb/product.jsp?productId=130²61#1_1).

    Reply
    Posted by: Caitlyn W.
    May 6, 2013 - 12:00 PM
  46. Thanks a lot for the catalog number!
    Could you please explain the injection of saline to prevent dehydration? How often and how much should it be injected? I guess all mice should get the same amount at the same time, right?
    And when the mice get sick, should we put some food on the bottom of the cage, or are they usually capable of getting the food themselves? thanks again.

    Reply
    Posted by: selcen o.
    May 13, 2013 - 3:44 PM
  47. We usually only inject mice that are not looking healthy and we generally do this as needed. I will only inject a 1ml bolus on a single day. You can repeat this the next day, but this is really just a last resort. We sacrifice them once they reach ²0% weight loss so they don't have undue stress. When the mice get sick, I do tend to try and put food in the bottom of the cage in case the mice aren't able to reach up into the food distributor. If the mice are getting so sick that you need to inject with PBS or move the food down, you may want to try a smaller concentration of DSS (²% or 1%) or perform the treatment for a shorter time. We have had several genotypes that don't handle the DSS as well and decreasing the concentration or treatment time really helps them. There is also facility to facility variability dependent on mouse microbiomes so my mice may be less sensitive than your mice to DSS and even your WTs might have different responses than mine. In short, we often need to tweak the protocol so that we can get injury while insuring survival for the extent of the protocol that we need to perform.

    Reply
    Posted by: Caitlyn W.
    May 13, 2013 - 4:29 PM
  48. thanks a lot, Caityln, you are really helping a lot because we have no experience in that.

    Reply
    Posted by: selcen o.
    May 14, 2013 - 4:56 AM
  49. Inducing colorectal cancer in rats: I am in a predicament; instead of the DSS Mw 40,000, I have ordered DSS (Mr 5,000) by accident. Unfortunately I noticed this too late and have a very limited time. I have read that the Mw of DSS does play a role in inducing colitis, whereby 5 kDa could induce milder form of colitis. I plan to inject my rats with AOM (12 mg/Kg), expose the rats to 2 cycles of 1 week of 2% DSS following 2 weeks of water. These rats are quite old, 12 weeks to be exact. They are also Wistar rats so at this time they are prone to develop cancer easily. However, I would like to know is using 5 kDa DSS worth it? Could you please provide me with some advice?

    Reply
    Posted by: Lynn C.
    February 16, 2014 - 6:30 AM
  50. Unfortunately, I do not think that Mr 5,000 will work.

    Reply
    Posted by: Christopher W.
    February 19, 2014 - 9:21 AM
  51. can we use dss with m.w 500.000 for induce colitis?

    Reply
    Posted by: flora s.
    January 29, 2015 - 9:41 PM
  52. Is there a difference between female and male mice? Which would should i choose?

    Reply
    Posted by: stu a.
    February 17, 2015 - 11:42 AM
  53. We have not noticed a difference with gender; however, as the environment is a strong contributor to the severity of colitis, I would recommend test experiments with male and female WT mice in your facility.

    Reply
    Posted by: Christopher W.
    February 17, 2015 - 12:24 PM
  54. Would you also have an advice on how to minimize the variability between the cages? I was considering to mix treated and not treated animals in the same cage (our treatment is injected). However I am afraid that the groups will have cross effects on each other.
    Thank you

    Reply
    Posted by: stu a.
    February 17, 2015 - 2:25 PM
  55. Is it plausible to use 500,000 mw DSS? 1-2% for induction oftumours with AOM, I thank all advice.

    Reply
    Posted by: VERONICA M.
    May 14, 2015 - 5:04 PM

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