Name: performing and processing fna of anterior fat pad for amyloid
Uploaded: 2010-10-30T17:00:00
Duration: 9 min 41 s
Description: Watch this Scientific Journal Video about Performing and Processing FNA of Anterior Fat Pad for Amyloid at JoVE.com

Vi tackar Ms Bonnie Phetteplace, RN för assistans under video grafritning det FNAB förfarande demonstrationen.

Inledning Systemisk amyloidos är en mycket varierande sjukdom som refererar till extracellulära nedfallet av olika proteiner som tillsammans kallas amyloid. Deponering av dessa amyloidfibrerna med beta veckade konfiguration leder till olika kliniska presentationer beroende på de berörda organen. De mest kliniskt relevanta tecken på systemisk amyloidos noteras när det rör sig om kritiska organ som hjärta, lever och / eller njure. Historiskt skulle dessa involverade organ man ta biopsier ifrån för att påvisa förekomsten av amyloid. Dessa måttligt invasiva procedurer utförs en betydande risk, inklusive blödning. Fett pad ambition har sedan visat sig ge en tillförlitlig och icke-invasiv metod för detektion av amyloid i systemisk amyloidos 1. Detta förfarande är i huvudsak jämförbar med fettsugning av underhudsfett i främre bukväggen under lokalbedövning för att hämta fibroadipose vävnad för att utvärdera knappa amyloid insättningar i små blodkärlens väggar 2. Den mest frekvent används metoden för amyloid identifiering i vävnaden är fortfarande den karakteristiska äpple-gröna dubbelbrytning mönster ses när kongorött färgade snitt visualiseras i ett polariserat ljusmikroskop 3. När fettet pad aspiration utförs, kan kongorött fläckar göras antingen direkt kladdig diabilder eller cell förberedelser block av den aspirerade fettvävnaden. Men patienter i tidiga skeden av amyloidos har knappa amyloid insättningar, vilket kraftigt minskar känsligheten Kongo röda färgade snitt Cell Block 4,5. Ultrastrukturella utvärdering av fett pad aspirat genom elektronmikroskopi har bättre reproducerbarhet och bättre känslighet 4. Därför rekommenderas att lämna in allt fett pad aspirat för såväl förberedelserna av en cell block och för att utföra elektronmikroskopi 2. Fett pad strävan är en relativt låg kostnad och icke-invasiv metod för att erhålla vävnad för att diagnostisera systemisk amyloidos. Denna artikel beskriver fett förfarande pad aspiration tillsammans med information om provbearbetning att lämna prov för både Kongo röda fläckar och ultrastrukturella utvärdering av elektronmikroskopi. I denna video visar vi här reproducerbar och enkel procedur för att hämta optimala diagnostiskt material. 1. Utföra FNA av främre Fat Pad A. Anesthetization av det lokala området (Figur 1 &amp; 2) <ol><li> Använda spritkompresser eller huden rengöringsmedel föredras av en viss institution, rengör huden på nedre kvadranten delen av buken sidled med mittlinjen och under naveln (Figur 1). </li><li> Markera en romboid formad yta om 2 x 2 inches som visas i figur 1 med en märkpenna. </li><li> Aspirera ca 10 ml 1% lidokain med en 18G nål. Fäst en 25G 1 ½ tum nålen på sprutan. Avlägsna eventuella luftbubblor genom att knacka upprätt sprutan medan du trycker på kolven tills vätskan lämnas ut och inga luftbubblor är närvarande. </li><li> Bedöva längs gränser rombiska området redan markerats som visas i figur 2. Börja med att sätta in 25G nålen strax under huden vid punkt A och försiktigt in nålen subkutant till punkt X. Dra tillbaka kolven för att se till att du inte är inom ett fartyg. Sedan tryck långsamt in kolven för att infiltrera upp till 2,5 ml lidokain (ca ¼ av lidokain i 10 ml spruta) medan nålen dras ut till punkt A utan att låta nålen komma ut ur huden vid punkt A (figur 2A3). Med nålen fortfarande under huden ändra riktning mot punkten Y (Figur 2A4) och tryck nålen subkutant till punkt Y (Figur 2B1). Liksom tidigare bekräftar att nålen inte är i ett kärl och fördela cirka 2,5 mL av lidokain medan långsamt dra tillbaka nålen genom punkt A (figur 2b3 genom 2B4). Upprepa samma steg med början från punkt B och utlämning lidokain subkutan från punkt B till X och B till Y (figur 2C1 genom 2d4). Förhindra blödning från idioter A och B med fast tillämpning av steril mätare pjäs. </li><li> Nu kan du kontrollera att området är sövd genom att lätt vidröra huden inom bedövas rombiska med spets / hörnet av gasväv stycke, genom att jämföra med intilliggande unanesthetized hud. </li></ol> B. Utföra fett pad aspiration (Figur 3) (Tillämpning av lokalbedövning innan resultatet av fett pad aspiration kan kringgås, beroende på den regionala och individuella preferenser. Korrekt utförda FNAB förfarande för anteriora fett pad aspiration kan fyllas i på en prick. Men beroende på smärtgränsen för enskilda patientens , manövrering av 18G nålen fram och tillbaka in i subkutan fettvävnad är relativt distressjunga. Den anesthetization metod som beskrivs här uppnår effekten på bara två idioter med 25G nål och averts smärtan med förbättrad tolerans mot förfarandet.) <ol><li> Montera 18G 1 ½ tum nålen på en 10 ml spruta. Montera sprutan-nål montering i sprutan grepp (&quot;FNAB grepp / pistol&quot;) för korrekt tillämpning och frisättning av vakuum. </li><li> Sätt spetsen på nålen i underhudsfettet inom rengöras och bedövas romboid område (figur 3a). </li><li> Helt dra tillbaka kolven i sprutan med nålen in i den subkutana vävnaden för att generera vakuum (Figur 3b). </li><li> Bibehålla vakuum och manövrera nålen fram och tillbaka i olika riktningar in i underhudsfettet (Figur 3c genom 3i). Varje stroke bör vara så länge som möjligt med nålen vald utan att låta nålen komma ut ur huden. Maximal provtagning uppnås genom att byta riktning med varje slag (Figur 3i). Det är viktigt att hålla riktningen på nålen tangerar slemhinnorna och parallellt med hudens yta för att undvika punktering i bukhålan. </li><li> Den fibroadipose vävnaden ackumuleras i sprutan. När tillräckligt fibroadipose vävnad fragment (upp till 1 mL fragment rika blod blandas exemplar) hämtas, släpp vakuum helt och ta bort nålen (Figur 3k). </li><li> Har patienten eller den biträdande gäller fast tryck om ett område med förfarandet med ett block med gasbinda för att förhindra extravasation av blod. </li></ol> 2. Provbearbetning <ol><li> Placera minst 5-6 fragment av fibroadipose vävnad i glutaraldehyd lösning för elektronmikroskopi (Figur 4B). </li><li> Beroende på de institutionella protokollet, får några fläckar av fragmenten fibroadipose vävnad beredas genom att sprida dem mellan två bilder (Figur 4A). </li><li> Resterande material är tillåtna att koagulera i sprutan (det kan ta 5-7 minuter, beroende på koagulationstiden) (Figur 4C). </li><li> Aspirera 10% formalin i sprutan så att levrat fibroadipose vävnaden material lossnat från väggen i sprutan och fritt flytande (Figur 4C2). Avlägsna kolven från sprutan (Figur 4C3) och överför det levrat fibroadipose vävnaden i 10% formalin container från den öppna änden av sprutan mittemot munstycket änden (Figur 4C4). </li><li> Etikett behållarna lämpligt sätt och lämna glutaraldehyd för elektronmikroskop och formalin för H &amp; E avsnitt och kongorött fläcken för utvärdering inom polariserande mikroskopi. Om fläckar är förberedda kan de behandlas i enlighet med protokollet av det enskilda laboratoriet. </li></ol><img src="/files/ftp_upload/1747/1747fig1.jpg" alt="Figur 1" /> Figur 1. Område som ska bedövas för FNAB av främre fett pad. <img src="/files/ftp_upload/1747/1747fig2.jpg" alt="Figur 2" /> Figur 2. Anesthetization av det lokala området. <img src="/files/ftp_upload/1747/1747fig3.jpg" alt="Figur 3" /> Figur 3. Utföra FNAB av främre fett pad. <img src="/files/ftp_upload/1747/1747fig4.jpg" alt="Figur 4" /> Figur 4. Behandling av främre fett pad aspirera ska lämnas till laboratoriet för att upptäcka amyloid insättningar. 3. Representativa resultat <img src="/files/ftp_upload/1747/1747fig5.jpg" alt="Figur 5" /> Figur 5. Amyloid i väggen av små blodkärl i fragment fibroadipose vävnad. Var Fibroadipose vävnad formalinfixerade bearbetas, paraffininbäddade, färgas med kongorött, och granskas av polarisering ljusmikroskop. Amyloid i väggen av små blodkärl visar äppelgrön dubbelbrytning (vit pil). <img src="/files/ftp_upload/1747/1747fig6.jpg" alt="Figur 6" /> Figur 6. Tissue saknas amyloidos. Äpplet gröna dubbelbrytning är frånvarande i vävnader med en annan patient utan amyloidos. Blå dubbelbrytning (vit pil) av kollagen fibrer är oftast närvarande i nästan alla prov. <img src="/files/ftp_upload/1747/1747fig7.jpg" alt="Figur 7" /> Figur 7. Fibriller överensstämmer med amyloid i blodkärlsväggen. Elektron mikroskop av raka, icke förgrenade, slumpmässigt utspridda 8-10 fibriller nm diameter som bildas av amyloid i blodkärlsväggen.

<ol>
	<li>Westermark, P., Stenkvist, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+method+for+the+diagnosis+of+systemic+amyloidosis.">A new method for the diagnosis of systemic amyloidosis.</a> Arch Intern Med. 132, 522-523 (1973).</li><li>Devata, S., Hari, P., Markelova, N., Li, R., Shidham, V. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detecting+AL+Amyloid+in+Abdominal+fat+pad+aspirates:+Is+Congo+red+sufficient?.">Detecting AL Amyloid in Abdominal fat pad aspirates: Is Congo red sufficient?.</a> Amyloid. , .</li><li>Merlini, G., Bellotti, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+Mechanisms+of+Amyloidosis.">Molecular Mechanisms of Amyloidosis.</a> New Engl J Med. 349 (6), 583-596 (2003).</li><li>Shidham, V. B., Kumar, N., Cihlar, K., Varsegi, G., Markelova, N., Li, R., Hari, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fine+needle+aspiration+of+abdominal+fat+pad+for+diagnosis+of+early+amyloidosis:+How+can+the+clinical+role+of+the+test+be+improved?.">Fine needle aspiration of abdominal fat pad for diagnosis of early amyloidosis: How can the clinical role of the test be improved?.</a> Mod Pathol. 20, 1A-380A (2007).</li><li>Halloush, R., Lavrovskaya, E., Mody, D., Lager, D., Truong, L. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diagnosis+and+typing+of+systemic+amyloidosis:+The+role+of+abdominal+fat+pad+fine+needle+aspiration.">Diagnosis and typing of systemic amyloidosis: The role of abdominal fat pad fine needle aspiration.</a> Cytojournal. 6, 24-24 (2010).</li><li>Hazenberg, I. I., Rijswijk, C. V. a. n., H, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diagnostic+Accuracy+of+Subcutaneous+Abdominal+Fat+Tissue+Aspiration+for+Detecting+Systemic+Amyloidosis+and+Its+Utility+in+Clinical+Practice.">Diagnostic Accuracy of Subcutaneous Abdominal Fat Tissue Aspiration for Detecting Systemic Amyloidosis and Its Utility in Clinical Practice.</a> Arthritis Rheum. , 54-546 (2006).</li><li>Guy, C. D., Jones, C. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Abdominal+fat+pad+aspiration+biopsy+for+tissue+confirmation+of+systemic+amyloidosis:+specificity,+positive+predictive+value,+and+diagnostic+pitfalls.">Abdominal fat pad aspiration biopsy for tissue confirmation of systemic amyloidosis: specificity, positive predictive value, and diagnostic pitfalls.</a> Diagn Cytopathol. 24, 181-185 (2001).</li><li>Westermark, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diagnosing+Amyloidosis.">Diagnosing Amyloidosis.</a> Scand J Rheumatol Suppl. 24, 327-329 (1995).</li><li>Linke, R. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Highly+Sensitive+Diagnosis+of+Amyloid+and+Various+Amyloid+Syndromes+Using+Congo+Red+Fluorescence.">Highly Sensitive Diagnosis of Amyloid and Various Amyloid Syndromes Using Congo Red Fluorescence.</a> Virchow Arch. 436, 439-448 (2000).</li><li>Giorgadze, T. A., Shiina, N., Baloch, Z. W., Tomaszewski, J. E., Gupta, P. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+detection+of+amyloid+in+fat+pad+aspiration:+an+evaluation+of+Congo+red+stain+by+fluorescent+microscopy.">Improved detection of amyloid in fat pad aspiration: an evaluation of Congo red stain by fluorescent microscopy.</a> Diagn Cytopathol. 31, 300-306 (2004).</li><li>DeMay, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fine+Needle+Aspiration+Biopsy.">Fine Needle Aspiration Biopsy.</a> The Art &#38; Science of Cytopathology. , 465-46 (1996).</li><li>Arbustini, E., Vrga, L., Concardi, M., Pallandini, G., Obici, L., Merlini, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electron+and+Immuno-electron+Microscopy+of+Abdominal+Fat+Identifies+and+Characterizes+Amyloid+Fibrils+in+Suspected+Cardiac+Amyloidosis.">Electron and Immuno-electron Microscopy of Abdominal Fat Identifies and Characterizes Amyloid Fibrils in Suspected Cardiac Amyloidosis.</a> Amyloid. 9, 108-114 (2002).</li></ol>

Diagnos av amyloidos uppnås vanligen med en vävnad biopsi av de drabbade organ såsom njurar, lever och / eller hjärtat. Denna metod har hög diagnostisk avkastning, dock är invasiv och kan vara förenat med komplikationer, inklusive blödning 2. Ändtarm, tandkött och benbiopsier benmärg var att föredra för diagnos som relativt sett mindre invasiv strategi i 1960 6,7,8. Bukfett pad aspiration har rapporterats i 1973 som en säker, minimalt invasiva, enkelt och billigare förfarande för vävnaden diagnosen systemisk amyloidos 1. Men det är detaljerna i förfarandet och förhållningssätt till behandling av de hämtade provet inte konsekvent rapporteras. Denna video och artikeln beskrivs det förfarande steg för steg. Den metod för att upptäcka amyloid i fett pad aspiration, samma sätt är inte heller bra standardiserad med några studier som jämför och utvärderar olika angreppssätt 1,5,6,7,8. Utvärdering av främre fett pad aspirat med Kongo röda färgningen är mindre känslig med lägre interobserver reproducerbarhet särskilt i tidiga fall av amyloidos med knappa amyloid avlagringar 5. Immunohistokemi utföras på formalinfixerade fast paraffininbäddade Cell Block sektioner och kongorött fluorescens utförs på cytologi utstryk har rapporterats för att förbättra den diagnostiska sensitiviteten 9,10. Elektronmikroskopi förbättrar upptäckt och identifiering av knappa amyloidfibrerna i små väggar blodkärl i fibroadipose vävnaden i fett pad aspirat 4, 5. Baserat på denna information, beskriver denna artikel bearbetning av provet för att förbereda cell block (för utvärdering av kongorött färgade snitt cell block under polariserande mikroskop för diagnostik äppelgrön dubbelbrytning och även för immunhistokemi som anges) och för elektronmikroskopi. Men beroende på den strategi som valts för utvärdering av provet för amyloid, kan det bli föremål för cytologi smutskasta förberedelse, Cell Block förberedelse och bearbetning för elektronmikroskopi. Vissa laboratorier utför testerna på smetas förberedda från aspirerade fragment fibroadipose vävnad och färgas med kongorött att studera fluorescens 10. Generellt i en sen amyloidos kan ljusmikroskopi med kongorött polariserande mikroskopi vara tillräcklig, men i tidiga skeden av sjukdomen kongorött färgning med polariserande mikroskopi på delar cell block är mindre känslig och tillförlitlig för fett pad aspirat 4,5,11 . Andra speciella fläckar inklusive Thioflavin T kan användas med samma begränsningar. Andra metoder för att upptäcka amyloid inkludera utvärdering av aspirera utstryk för amyloid med kongorött polariserande mikroskopi och fluorescens 10. Enligt vår erfarenhet här metoden är mindre reproducerbar. Utvärdering av blodkärl i fett pad för amyloid genom elektronmikroskopi övervinner dessa begränsningar 4,11. Ytterligare karakterisering av amyloid har rapporterats av immunoelectron mikroskopi 12, dock kan dessa metoder inte är tillgängliga i stor utsträckning i en icke-tertiär vårdmiljö. Nålar som används för att utföra aspiration förfaranden kan kategoriseras enligt sin mäta storleken på böterna (21-25G), intermediära (18-20G), och stora (t.ex.-14G) 11. Anterior fett pad ambitioner har rapporterats utföras med variabel spårvidd från mellanliggande (18 till 20G) för fina (21 till 22 g) 2,10. Flesta av de stora skador är aspirerat med nålar finare mätare (t.ex. 25G) för att få bra cytologi fläckar tillsammans med ytterligare passerar med en bredare nål (t.ex. 18G) för att hämta ytterligare ett prov för cell block. Under främre fett pad aspiration, inte aspirera sammanhållande fibroadipose vävnaden inte bra med finare nålar. Cell Block förberedelse och elektronmikroskopi är viktiga för bedömning av små blodkärlens väggar i fragment fibroadipose vävnad i fett aspirat. Bredare spårvidd nålar (såsom 18G) rekommenderas att ge diagnostiska lämpligt material 2,10. Eftersom förfarandet är jämförbart med den konventionella FNAB, är fett pad strävan kallas FNAB trots bredare spårvidd nålar kan användas för att utföra den 5.

A correction was made to <a href="http://www.jove.com/index/details.stp?id=1747">Performing and Processing FNA of Anterior Fat Pad for Amyloid</a>. There was an error with an author's name. The author's last name had a typo and was corrected to:
Safwan S. Jaradeh
instead of:
Safwan S. Jardeh

Erratum: Performing and Processing FNA of Anterior Fat Pad for Amyloid

Spritsuddar gasbinda kuddar, märkpenna, 10 ml sprutor x2, 1% lidokain (lokalbedövningsmedel-Om lidokain används ensamt, det orsakar en initial sveda efter instillation. Detta kan förebyggas genom att använda en 1:1 blandning av 1% lidokain och 1% natrium-bi-karbonat), 18 gauge (G) 1 ½ tum nålar x2 (för att utföra FNAB), 25G 1 ½ tum nål (för att injicera lokalbedövningsmedel), FNA spruta hållare (&quot;pistol&quot;), bandage, injektionsflaska av glutaraldehyd (för elektronmikroskop) och biopsi behållare med 10% formalin.

performing and processing fna of anterior fat pad for amyloid

Historiskt sett var hjärta, lever och njure biopsier för att visa amyloid insättningar i amyloidos. Eftersom den kliniska presentationen av denna sjukdom är så varierande och icke-specifika, riskerna för dessa biopsier är för stora för det diagnostiska utbytet. Andra webbplatser som har en lägre biopsi risk, såsom hud eller tandkött, är också relativt invasiva och dyrt. Dessutom kan dessa biopsier inte alltid har tillräckligt amyloid insättningar för att fastställa diagnosen. Fett pad aspiration har visat god klinisk korrelation med låga kostnader och minimal morbiditet. Det finns dock inga standardiserade protokoll för att utföra detta förfarande eller behandling av det aspirerade provet, vilket leder till varierande och nonreproducible resultat. Den mest frekvent utnyttjade modalitet för detektion av amyloid i vävnaden är ett äpple-gröna dubbelbrytning på kongorött färgade snitt med ett polariserande mikroskop. Denna teknik kräver förberedelse cell block av aspirerade materialet. Tyvärr patienter i tidigt skede av amyloidos har minimala mängder amyloid vilket kraftigt minskar känsligheten Kongo röda färgade snitt cell block av fett pad aspirat. Därför bör ultrastrukturella utvärdering av fett pad aspirat med elektronmikroskopi användas, med tanke på ökad känslighet för amyloid upptäckt. Denna artikel visar en enkel och reproducerbar metod för genomförande främre fett pad strävan för detektion av amyloid använda både Kongo rödfärgning av sektioner Cell Block och elektronmikroskopi för ultrastrukturella identifiering.

Watch this Scientific Journal Video about Performing and Processing FNA of Anterior Fat Pad for Amyloid at JoVE.com

Performing and Processing FNA of Anterior Fat Pad for Amyloid

Fett pad ambition är att föredra, minimalt invasiva och låg kostnad förhållningssätt jämfört med andra metoder för att upptäcka amyloid för diagnostik av systemisk amyloidos. Denna video artikeln visar en processuella disposition för att utföra fett pad aspiration med lämplig behandling av provet för den optimala diagnostiska resultatet.

Utföra och behandling FNA av främre Fat Pad för Amyloid

Historically, heart, liver, and kidney biopsies were performed to demonstrate amyloid deposits in amyloidosis.  Since the clinical presentation of this ...

performing-and-processing-fna-of-anterior-fat-pad-for-amyloid

Research

JoVE Journal

Medicine

Purification and Aggregation of the Amyloid Precursor Protein Intracellular Domain

Amyloid precursor protein (APP) is a type I transmembrane protein associated with the pathogenesis of Alzheimer's disease (AD). APP is characterized by a large extracellular domain and a short cytosolic domain termed the APP intracellular domain (AICD). During maturation through the secretory pathway, APP can be cleaved by proteases termed &alpha;, &beta;, and &gamma;-secretases1. Sequential proteolytic cleavage of APP with &beta; and &gamma;-secretases leads to the production of a small proteolytic peptide, termed A&beta;, which is amyloidogenic and the core constituent of senile plaques. The AICD is also liberated from the membrane after secretase processing, and through interactions with Fe65 and Tip60, can translocate to the nucleus to participate in transcription regulation of multiple target genes2,3. Protein-protein interactions involving the AICD may affect trafficking, processing, and cellular functions of holo-APP and its C-terminal fragments. We have recently shown that AICD can aggregate in vitro, and this process is inhibited by the AD-implicated molecular chaperone ubiquilin-14. Consistent with these findings, the AICD has exposed hydrophobic domains and is intrinsically disordered in vitro5,6, however it obtains stable secondary structure when bound to Fe657. We have proposed that ubiquilin-1 prevents inappropriate inter- and intramolecular interactions of AICD, preventing aggregation in vitro and in intact cells4. While most studies focus on the role of APP in the pathogenesis of AD, the role of AICD in this process is not clear. Expression of AICD has been shown to induce apoptosis8, to modulate signaling pathways9, and to regulate calcium signaling10. Over-expression of AICD and Fe65 in a transgenic mouse model induces Alzheimer's like pathology11, and recently AICD has been detected in brain lysates by western blotting when using appropriate antigen retrieval techniques12. To facilitate structural, biochemical, and biophysical studies of the AICD, we have developed a procedure to produce recombinantly large amounts of highly pure AICD protein. We further describe a method for inducing the in vitro thermal aggregation of AICD and analysis by atomic force microscopy. The methods described are useful for biochemical, biophysical, and structural characterization of the AICD and the effects of molecular chaperones on AICD aggregation.

A method for large-scale purification of the APP intracellular domain (AICD) is described. We also describe methodology to induce in vitro AICD aggregation and visualization by atomic force microscopy. The methods described are useful for biochemical/structural characterization of the AICD and the effects of molecular chaperones on its aggregation.

Rening och aggregation av amyloidprekursorproteinet intracellulära domänen

Amyloid precursor protein (APP) is a type I transmembrane protein associated with the pathogenesis of Alzheimer's disease (AD). APP is characterized by a ...

Processing of Human Reduction Mammoplasty and Mastectomy Tissues for Cell Culture

Experimental examination of normal human mammary epithelial cell (HMEC) behavior, and how normal cells acquire abnormal properties, can be facilitated by in vitro culture systems that more accurately model in vivo biology. The use of human derived material for studying cellular differentiation, aging, senescence, and immortalization is particularly advantageous given the many significant molecular differences in these properties between human and commonly utilized rodent cells1-2. Mammary cells present a convenient model system because large quantities of normal and abnormal tissues are available due to the frequency of reduction mammoplasty and mastectomy surgeries.
The mammary gland consists of a complex admixture of many distinct cell types, e.g., epithelial, adipose, mesenchymal, endothelial. The epithelial cells are responsible for the differentiated mammary function of lactation, and are also the origin of the vast majority of human breast cancers. We have developed methods to process mammary gland surgical discard tissues into pure epithelial components as well as mesenchymal cells3. The processed material can be stored frozen indefinitely, or initiated into primary culture. Surgical discard material is transported to the laboratory and manually dissected to enrich for epithelial containing tissue. Subsequent digestion of the dissected tissue using collagenase and hyaluronidase strips stromal material from the epithelia at the basement membrane. The resulting small pieces of the epithelial tree (organoids) can be separated from the digested stroma by sequential filtration on membranes of fixed pore size. Depending upon pore size, fractions can be obtained consisting of larger ductal/alveolar pieces, smaller alveolar clusters, or stromal cells. We have observed superior growth when cultures are initiated as organoids rather than as dissociated single cells. Placement of organoids in culture using low-stress inducing media supports long-term growth of normal HMEC with markers of multiple lineage types (myoepithelial, luminal, progenitor)4-5. Sufficient numbers of cells can be obtained from one individual's tissue to allow extensive experimental examination using standardized cell batches, as well as interrogation using high throughput modalities.
Cultured HMEC have been employed in a wide variety of studies examining the normal processes governing growth, differentiation, aging, and senescence, and how these normal processes are altered during immortal and malignant transformation4-15,16. The effects of growth in the presence of extracellular matrix material, other cell types, and/or 3D culture can be compared with growth on plastic5,15. Cultured HMEC, starting with normal cells, provide an experimentally tractable system to examine factors that may propel or prevent human aging and carcinogenesis.

A method to process human mammary surgical discard material is described. Processed tissue, in the form of organoids, can be stored frozen indefinitely or placed in culture for long-term growth. This method enables experimental examination of normal human epithelial cell biology, and the effects of exogenous perturbations.

Behandling av mänskliga Minskning mammoplasty och vävnader mastektomi för cellodling

Experimental  examination of normal human mammary epithelial cell (HMEC) behavior, and how  normal cells acquire abnormal properties, can be facilitated ...

Orthotopic Injection of Breast Cancer Cells into the Mammary Fat Pad of Mice to Study Tumor Growth.

Breast cancer growth can be studied in mice using a plethora of models. Genetic manipulation of breast cancer cells may provide insights into the functions of proteins involved in oncogenic progression or help to discover new tumor suppressors. In addition, injecting cancer cells into mice with different genotypes might provide a better understanding of the importance of the stromal compartment. Many models may be useful to investigate certain aspects of disease progression but do not recapitulate the entire cancerous process. In contrast, breast cancer cells engraftment to the mammary fat pad of mice better recapitulates the location of the disease and presence of the proper stromal compartment and therefore better mimics human cancerous disease. In this article, we describe how to implant breast cancer cells into mice orthotopically and explain how to collect tissues to analyse the tumor milieu and metastasis to distant organs. Using this model, many aspects (growth, angiogenesis, and metastasis) of cancer can be investigated simply by providing a proper environment for tumor cells to grow.

Cancer is a complex disease that is influenced by the tissue surrounding the tumor as well as local pro- and anti-inflammatory mediators. Therefore, orthotropic injection models, rather than subcutaneous models may be useful to study cancer progression in a manner that better mimics human pathology.

Orthotopic Injektion av bröstcancerceller i juverfett Pad av möss att studera tumörtillväxt.

Breast cancer growth can be studied in mice using a plethora of models. Genetic manipulation of breast cancer cells may provide insights into the ...

Assessment of Viability of Human Fat Injection into Nude Mice with Micro-Computed Tomography

Lipotransfer is a vital tool in the surgeon&#8217;s armamentarium for the treatment of soft tissue deficits of throughout the body. Fat is the ideal soft tissue filler as it is readily available, easily obtained, inexpensive, and inherently biocompatible.1 However, despite its burgeoning popularity, fat grafting is hampered by unpredictable results and variable graft survival, with published retention rates ranging anywhere from 10-80%. 1-3
To facilitate investigations on fat grafting, we have therefore developed an animal model that allows for real-time analysis of injected fat volume retention. Briefly, a small cut is made in the scalp of a CD-1 nude mouse and 200-400 &#181;l&#160;of processed lipoaspirate is placed over the skull. The scalp is chosen as the recipient site because of its absence of native subcutaneous fat, and because of the excellent background contrast provided by the calvarium, which aids in the analysis process. Micro-computed tomography (micro-CT) is used to scan the graft at baseline and every two weeks thereafter. The CT images are reconstructed, and an imaging software is used to quantify graft volumes.
Traditionally, techniques to assess fat graft volume have necessitated euthanizing the study animal to provide just a single assessment of graft weight and volume by physical measurement ex vivo. Biochemical and histological comparisons have likewise required the study animal to be euthanized. This described imaging technique offers the advantage of visualizing and objectively quantifying volume at multiple time points after initial grafting without having to sacrifice the study animal. The technique is limited by the size of the graft able to be injected as larger grafts risk skin and fat necrosis. This method has utility for all studies evaluating fat graft viability and volume retention. It is particularly well-suited to providing a visual representation of fat grafts and following changes in volume over time.

Fat grafting is an essential technique for reconstructing soft tissue deficits. However, it remains an unpredictable procedure characterized by variable graft survival. Our goal was to devise a mouse model that utilizes a novel imaging method to compare volume retention between differing techniques of fat graft preparation and delivery.

Bedömning av livskraft Human Fat Injektion i nakna möss med Micro-datortomografi

Lipotransfer is a vital tool in the surgeon&#8217;s armamentarium for the treatment of soft tissue deficits of throughout the body. Fat is the ideal soft ...

Transplantation Into the Mouse Ovarian Fat Pad

Orthotopic transplantation assays in mice are invaluable for studies of cell regeneration and neoplastic transformation. Common approaches for orthotopic transplantation of ovarian surface and tubal epithelia include intraperitoneal and intrabursal administration of cells. The respective limitations of these methods include poorly defined location of injected cells and limited space volume. Furthermore, they are poorly suited for long-term structural preservation of transplanted organs. To address these challenges, we have developed an alternative approach, which is based on the introduction of cells and tissue fragments into the mouse fat pad. The mouse ovarian fat pad is located in the immediate vicinity of the ovary and uterine tube (aka oviduct, fallopian tube), and provides a familiar microenvironment for cells and tissues of these organs. In our approach fluorescence-labeled mouse and human cells, and fragments of the uterine tube are engrafted by using minimally traumatic dorsal incision surgery. Transplanted cells and their outgrowths are easily located in the ovarian fat pad for over 40 days. Long-term transplantation of the entire uterine tube allows correct preservation of all principle tissue components, and does not result in adverse side effects, such as fibrosis and inflammation. Our approach should be uniquely applicable for answering important biological questions such as differentiation, regenerative and neoplastic potential of specific cell populations. Furthermore, it should be suitable for studies of microenvironmental factors in normal development and cancer.

We describe an ovarian fad pad transplantation assay suitable for studies of normal and transformed epithelia of the female reproductive tract. The mouse fat pad allows transplantation of large tissue fragments, is easily accessible for surgery and imaging, and provides the most favorable native environment for tissues of M&#252;llerian origin.

Transplantation i musen äggstocksfettkudden

Orthotopic transplantation assays in mice are invaluable for studies of cell regeneration and neoplastic transformation. Common approaches for orthotopic ...

Confirmation of Myocardial Ischemia and Reperfusion Injury in Mice Using Surface Pad Electrocardiography

Many animal models have been established for the study of myocardial remodeling and heart failure due to its status as the number one cause of mortality worldwide. In humans, a pathologic occlusion forms in a coronary artery and reperfusion of that occluded artery is considered essential to maintain viability of the myocardium at risk. Although essential for myocardial recovery, reperfusion of the ischemic myocardium creates its own tissue injury. The physiologic response and healing of an ischemia/reperfusion injury is different from a chronic occlusion injury. Myocardial ischemia/reperfusion injury is gaining recognition as a clinically relevant model for myocardial infarction studies. For this reason, parallel animal models of ischemia/reperfusion are vital in advancing the knowledge base regarding myocardial injury. Typically, ischemia of the mouse heart after left anterior descending (LAD) coronary artery occlusion is confirmed by visible pallor of the myocardium below the occlusion (ligature). However, this offers only a subjective way of confirming correct or consistent ligature placement, as there are multiple major arteries that could cause pallor in different myocardial regions. A method of recording electrocardiographic changes to assess correct ligature placement and resultant ischemia as well as reperfusion, to supplement observed myocardial pallor, would help yield consistent infarct sizes in mouse models. In turn, this would help decrease the number of mice used. Additionally, electrocardiographic changes can continue to be recorded non-invasively in a time-dependent fashion after the surgery. This article will demonstrate a method of electrocardiographically confirming myocardial ischemia and reperfusion in real time.

During murine myocardial ischemia/reperfusion surgery, correct placement of the occluding ligature is typically confirmed by visible observation of myocardial pallor. Herein, a method of electrocardiographically confirming ischemia and reperfusion, to supplement observed myocardial pallor, is demonstrated in male C57Bl/6 mice.

Bekräftelse av ischemi och reperfusion skada i möss med användning av Surface Pad Elektrokardiografi

Many animal models have been established for the study of myocardial remodeling and heart failure due to its status as the number one cause of mortality ...

Stromal Vascular Fraction-enriched Fat Grafting for the Treatment of Symptomatic End-neuromata

The purpose of this study was to methodically illustrate and highlight the crucial steps of stromal vascular fraction (SVF)-enriched fat grafting as a novel treatment of symptomatic end-neuromata of peripheral sensory nerves, and in this study, specifically of the superficial branch of the radial nerve (SBRN). Despite a multitude of existing treatments, persistent postoperative pain and common pain relapse are still very common, independent of the procedure assessed. The neuroma is microsurgically excised accordingly to standardized protocol. Instead of the relocation of the regenerating nerve stump in neighboring anatomical structures, such as muscle or bone, a fat graft is applied perifocally and acts as a mechanical barrier. In order to reduce the fat resorption rate and boost the regenerative potential of the graft, the highly concentrated SVF is integrated in the grafting. The SVF is isolated from subcutaneous fat by enzymatic and mechanic separation of the lipoaspirate by a specific commercial isolation system. The SVF-enriched fat graft provides both a mechanical barrier and various biological effects at the cellular level, including improving angiogenesis, inflammation, and fibrosis. Both mechanical and biologic effects help to reduce the disorganized axonal outgrowth of the nerve stump during nerve regeneration and hence prevent the recurrence of painful end-neuromata.

The purpose of the study is to illustrate the technical procedure of stromal vascular fraction (SVF)-enriched fat grafting as a novel treatment of symptomatic end-neuromata. This technique provides advantages of both the mechanical barrier and the biological action at the cellular level by the processed and concentrated SVF.

Stromaceller vaskulär bråkdel-berikad fett ympning för behandling av symtomatisk slutet-neuromata

The purpose of this study was to methodically illustrate and highlight the crucial steps of stromal vascular fraction (SVF)-enriched fat grafting as a ...

Methodology for Sputum Induction and Laboratory Processing

The technique of sputum induction and processing is a recognized non-invasive method allowing the collection and analysis of cells from the airways, which is interesting in various respiratory diseases like asthma, chronic obstructive pulmonary disease (COPD), chronic cough, or idiopathic pulmonary fibrosis. This technique is well tolerated, safe and non-invasive, but is currently limited to research services and specialized centers in clinical practice because it is technically demanding, time-consuming, and requires trained staff. The success rate of sputum induction and analysis is about 80%.
Here, we describe the induction and laboratory processing of sputum samples. Sputum is induced by inhalation of hypertonic or isotonic saline with salbutamol. For the processing, we use the whole sputum technique. Dithiothreitol (DTT) is used to allow mucolysis of sputum samples. The primary aim of sputum processing is to obtain a differential cell count to study the cell types present in the airway lumen. Additional analyses may also be performed on sputum supernatant and sputum cells, which may allow further investigation into inflammatory processes and immune mechanisms. Examples include studying mediators in sputum supernatant and performing a large spectrum of analysis on sputum cells such as flow cytometry, genomics, or proteomics.
Finally, representative results of sputum analysis in healthy controls, asthmatics, and COPD patients are presented.

Here we describe the technique of sputum induction. This protocol also explains the sputum processing to perform a differential cell count and to collect sputum supernatant and cells for further analysis.

Metodik för Sputum induktion och laboratorium bearbetning

The technique of sputum induction and processing is a recognized non-invasive method allowing the collection and analysis of cells from the airways, which ...

Sampling Strategies and Processing of Biobank Tissue Samples from Porcine Biomedical Models

In translational medical research, porcine models have steadily become more popular. Considering the high value of individual animals, particularly of genetically modified pig models, and the often-limited number of available animals of these models, establishment of (biobank) collections of adequately processed tissue samples suited for a broad spectrum of subsequent analyses methods, including analyses not specified at the time point of sampling, represent meaningful approaches to take full advantage of the translational value of the model. With respect to the peculiarities of porcine anatomy, comprehensive guidelines have recently been established for standardized generation of representative, high-quality samples from different porcine organs and tissues. These guidelines are essential prerequisites for the reproducibility of results and their comparability between different studies and investigators. The recording of basic data, such as organ weights and volumes, the determination of the sampling locations and of the numbers of tissue samples to be generated, as well as their orientation, size, processing and trimming directions, are relevant factors determining the generalizability and usability of the specimen for molecular, qualitative, and quantitative morphological analyses. Here, an illustrative, practical, step-by-step demonstration of the most important techniques for generation of representative, multi-purpose biobank specimen from porcine tissues is presented. The methods described here include determination of organ/tissue volumes and densities, the application of a volume-weighted systematic random sampling procedure for parenchymal organs by point-counting, determination of the extent of tissue shrinkage related to histological embedding of samples, and generation of randomly oriented samples for quantitative stereological analyses, such as isotropic uniform random (IUR) sections generated by the &#34;Orientator&#34; and &#34;Isector&#34; methods, and vertical uniform random (VUR) sections.

The practical application and performance of methods for generation of representative tissue samples of porcine animal models for a broad spectrum of downstream analyses in biobank projects are demonstrated, including volumetry, systematic random sampling, and differential processing of tissue samples for qualitative and quantitative morphologic and molecular analyses types.

Strategier för provtagning och behandling av Biobank vävnadsprover från svin biomedicinska modeller

In translational medical research, porcine models have steadily become more popular. Considering the high value of individual animals, particularly of ...

A Training and Testing System for Performing Vascular Reconstruction In Vitro

Manual vascular reconstruction training is essential for a beginner surgeon. However, an optimal training system for vascular reconstruction in vitro has yet to be developed. In this study, we introduce an in vitro training and testing system using a magnetic anchoring technique with which a trainee can practice manual vascular reconstruction individually. Additionally, this system can also be used to test the quality of the reconstruction. The described system includes a vascular reconstruction training machine, magnetic tractors, and a magnetic suture puller. In this manuscript, we detail an end-to-end vein anastomosis using porcine right and left iliac veins. To identify the potential damage caused by a magnetic suture puller on the suture, we created three groups with six segments of 4-0 polypropylene sutures each: a control group with no intervention on the polypropylene suture, a group in which the polypropylene suture is manually pulled with sterile gloves 20x, and a magnetic puller group in which the magnetic puller pulled the polypropylene suture 20x. These groups were tested by light microscopy and breaking strength tests, and the effect of reconstruction was assessed. In the light microscopy test, the control group was less likely to be damaged (p &#60; 0.05) and the number of damaged points of the manual group and magnetic puller group were similar (p &#62; 0.05). The results of the breaking strength test were compared across groups and no significant difference was observed (p &#62; 0.05). The end-to-end anastomosis of the porcine iliac veins was successfully performed using this training system, and the reconstructed veins could undergo 2.0 kPa perfusion pressure. Using this training and testing system the trainee can practice manual vascular reconstruction in vitro individually with the aid of magnetic tractors and a magnetic suture puller, and the quality of the reconstruction can be tested.

Here we present a training and testing system where a trainee can complete manual vascular reconstruction in vitro individually using a magnetic anchoring technique. The system can also be used to test the quality of reconstruction.

Ett utbildnings-och test system för att utföra vaskulär rekonstruktion in vitro

Manual vascular reconstruction training is essential for a beginner surgeon. However, an optimal training system for vascular reconstruction in vitro has ...