Name: establishment of a clinic-based biorepository
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Dit werk werd ondersteund door fondsen van de Midwestern University College of Health Sciences Research Facilitation Grant, toegekend aan EEH, en het Midwestern University Office of Research en Gesponsorde Programma&#39;s Intramural Grant, toegekend aan KJL. Aanvullende ondersteuning is verleend door aangesloten laboratoria en geaffilieerde dermatologie. Wij bedanken Sarah Potekhen, Jamie Barto, Stefani Fawks, Cody Jording, Stacie Schimke, Heather Kissel en Ali Zaidi voor hun technische bijstand.

Alle experimenten op humane weefselmonsters werden goedgekeurd door Western IRB (WIRB Protocol # 20142461). Het enige criterium voor het verzamelen van weefsels voor deze procedures is een biopsie-bewezen diagnose van SCC. Er werden geen uitsluitingskriteria beschreven in het oorspronkelijke IRB-protocol, maar monsters van patiënten met een bekende overdraagbare overdraagbare ziekte werden niet gebruikt. Geïnformeerde toestemming werd verkregen voor het verzamelen van kutane weefsel, bloed en speeksel. Midwestern Inst...

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Naar de kennis van de auteur is dit protocol de eerste in zijn soort dat zich richt op de klinische aanbesteding van cutane weefselmonsters in zowel een kosteneffectieve als snelle aanpak. Patiënten die Mohs-microchirurgie ondergaan, worden meestal gepland tijdens specifieke tijdstippen, en de inzameling is beperkt tot deze perioden. Proefcollectie omvat inspanning van de medische assistent die betrokken is bij patiëntenzorg, de Mohs-histotechnoloog die de tumormonsters verwerkt en een aangewezen laboratoriumpersoneel...

Kanker- en biomarker onderzoek is gebaseerd op een aanbod van kwaliteit menselijk weefselmonsters, en de beperkte voorraad heeft onderzoek 1 , 2 gehinderd. Veel dermatologische studies zijn beperkt door de ontoereikende toevoer, de variabele kwaliteit en de kosten die verband houden met het gebruik van menselijk weefsel. De kosten voor het opzetten van een grote, toegewijde biobank zijn geschat op ongeveer twee miljoen dollar 3 , en deze kosten maken het gebruik van menselijk weefsel buiten bereik van veel onderzoekers. Bovendien bestaat het proces van het genereren en opslaan van onderzoeksmonsters het risico om klinische operaties te beïnvloeden en de patiëntenzorg te vertragen, indien niet zorgvuldig uitgevoerd. Een kosteneffectieve klinische biorepositorie is opgericht die zich toelegt op huidkankermonsters na aanbeveelde best practices en sample validatie 4 , 5 , 6 . <pClass = &quot;jove_content&quot;&gt; Dit protocol is ontwikkeld in een dermatologie kliniek die een groot aantal Mohs micrographische operaties uitvoert om plaveiselcellencarcinoom (SCC), basale celcarcinoom (BCC) en melanoma huidkanker te verwijderen. Vrijwillige donoren kunnen worden aangeworven uit deze patiëntenpopulatie. Het is belangrijk om de biorepositor op de plaats van verzameling vast te stellen om weefsel en bloed snel van toegestane patiënten vast te stellen zonder behandeling te vertragen. Het verzamelen van monsters uit dezelfde kliniek minimaliseert variaties in inzamelingstechnieken en minimaliseert variaties in de kwaliteit van monsters, wat problematisch kan zijn voor downstream toepassingen 7 , 8 . Het doel van de micrografische chirurgische techniek van Mohs is ervoor te zorgen dat alle kankerweefsels worden verwijderd, terwijl zoveel mogelijk gezond weefsel wordt behouden. De procedure omvat de geleidelijke verwijdering van dunne lagen tumorweefsel. Elke opeenvolgende laag is zijnTologisch onderzocht (na cryosectie van het tumorweefsel en het uitvoeren van H &amp; E-kleuring) door een dermatoloog om te bepalen of al het kankerweefsel is verwijderd. De excisie en het onderzoek van de volgende lagen weefsels wordt uitgevoerd terwijl de patiënt in het kantoor blijft. Deze techniek wordt beschouwd als de beste behandelingsoptie voor SCC 9 . Op dit punt is de wond afgesloten en, om de genezing en cosmetische verschijning te verbeteren, wordt aangrenzend normaal weefsel (ANT) vaak uitgesneden. Zo is deze chirurgische procedure om een ​​tumor te verwijderen ideaal geschikt voor het verzamelen van histologisch gekarakteriseerd weefsel voor toekomstige studies. De aanbestedingsprocedure voor het verkrijgen van tumorweefsel, aangrenzend normaal weefsel, speeksel en bloedmonsters is ontworpen om een ​​minimale invloed op de normale staffuncties te hebben ( Figuur 1 ). Medische assistenten verrichten de bloeddruk tijdens het voorbereiden van de patiënt voor de procedure. Na afloop van de Mohs procedure, de M<html> Ohs histotechnoloog bereidt aanvullende histologische dia&#39;s van het monster op en draagt ​​het weefsel over naar het biorepository. Kosten verbonden aan het opstellen van de biorepository zijn de aankoop van cryopreservation freezers, het creëren van bescheiden klinische laboratoriumruimte en de ontwikkeling van een inventarisatie programma. <img alt="Figuur 1" src="/files/ftp_upload/55583/55583fig1.jpg" /> Figuur 1: Opeenvolging van steekproefverzameling en verantwoordelijke medewerkers. Bij de patiënt inchecken en het bereiken van de toestemming van de patiënt verzamelt de medische assistent een buccale swab en voert hij een bloedtekening uit. De dermatoloog en medische assistent accijnzen dan de tumor en sluiten de wond, gedurende welke tijd SCC en ANT-monsters worden verzameld. Een toegewijde laboratoriumtechnicus verwerkt het bloed en deelt de SCC- en ANT-monsters voor weefselkweek, behoud en toegang tot de biorepository.Ftp_upload / 55583 / 55583fig1large.jpg &quot;target =&quot; _ blank &quot;&gt; Klik hier om een ​​grotere versie van deze figuur te bekijken. De verscheidenheid van verzamelde monsters zorgt voor een verscheidenheid aan experimentele benaderingen ( Figuur 2 ). Monsters die van de patiënt zijn verzameld zijn buccale swabs (speeksel kan ook worden verzameld indien nodig), heel bloed en uitgesneden weefsel. De buccale swabs en een monster van het gehele bloed worden opgeslagen, zonder verwerking, voor genotyping en weefsel matching. Geheel bloed wordt gescheiden in witte bloedcel (WBC) en plasmafracties voor toekomstige analyses. Na het verwerken van Mohs wordt de bevroren tumor direct in vloeibare stikstof geplaatst en overgebracht naar een -80 ° C vriezer. Vers, levensvatbaar tumorweefsel en ANT-monsters worden gekweekt onder toepassing van modificaties van eerdere technieken 10 , 11 en vervolgens cryopreserveerd. Tijdens het verzamelen wordt het nummer van elk steekproef opgenomen op een spreadsheet voorafgaand aan inschrijving<html> Het inventarisatieprogramma om de nauwkeurige verwerking te vergemakkelijken ( tabel 1 ). <img alt="Figuur 2" src="/files/ftp_upload/55583/55583fig2.jpg" /> Figuur 2: Overzicht van klinische biorepository-inzameling en verwerking van klinieken. Een buccale zwabber en bloedmonster worden verzameld van de patiënt en opgeslagen voor downstream genotyping en weefsel matching. Geheel bloed wordt verder verwerkt voor witte bloedcellen (WBC) isolatie en CTC analyse, evenals voor plasma-insameling en vloeibare biopsie analyse. Weefsel uitgesneden tijdens de Mohs-procedure wordt histologisch verwerkt voor diagnostische doeleinden, waarna de histologische dia&#39;s experimenteel kunnen worden gebruikt voor verdere immunohistochemische analyses. Met dien verstande dat het uitgesneden weefselmonster groot genoeg is, wordt een gedeelte van versweefsel verwijderd en gesneden voor eiwit- en RNA-isolatie en voor de oprichting van gekweekte cellijnen.55583 / 55583fig1large.jpg &quot;target =&quot; _ blank &quot;&gt; Klik hier om een ​​grotere versie van deze figuur te zien. <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td> Inzamelingsdatum: </td><td></td><td></td><td></td><td></td><td></td></tr><tr><td></td><td> Patiënt 1 </td><td> Patiënt 2 </td><td> Patiënt 3 </td><td> Patiënt 4 </td><td> Patiënt 5 </td></tr><tr><td> Voorletters en Geboortedatum </td><td></td><td></td><td></td><td></td><td></td></tr><tr><td> Oogkleur </td><td></td><td></td><td></td><td></td><td></td></tr><tr><td> Sample Type </td><td></td><td></td><td></td><td></td><td></td></tr><tr><td> Plaats van de steekproef </td><td></td><td></td><td></td><td></td><td></td></tr><tr><td> Speeksel </td><td></td><td></td><td></td><td></td><td></td></tr><tr><td> wGat bloed </td><td></td><td></td><td></td><td></td><td></td></tr><tr><td> Plasma </td><td></td><td></td><td></td><td></td><td></td></tr><tr><td> Tumor levensvatbaar </td><td></td><td></td><td></td><td></td><td></td></tr><tr><td> Weefsel Normaal Leefbaar </td><td></td><td></td><td></td><td></td><td></td></tr><tr><td> Tumor Mohs Liquid Stikstof </td><td></td><td></td><td></td><td></td><td></td></tr><tr><td> Weefsel Normaal vloeibaar stikstof </td><td></td><td></td><td></td><td></td><td></td></tr><tr><td> slides </td><td></td><td></td><td></td><td></td><td></td></tr></tbody></table> Tabel 1: Checklist om steekproeven te registreren. Gegevens die worden opgespoord en opgenomen met elk verzameld monster, omvatten patiënt initialen, geboortedatum en oogkleur (voor huidtypen), evenals de locatiOp het verwijderen van het monster. Het aantal speekselmonsters, bloedinzamelvolumes en het aantal levende en geconserveerde weefselmonsters verzameld worden ook opgenomen als referenties voor toewijzingen tot latere toepassingen. <a href="http://ecsource.jove.com/files/ftp_upload/55583/Table1.xlsx">Klik hier om dit bestand te downloaden.</a> Om de voorbeeldinzamelingsprocedures te valideren, is elk monstertype getest in downstream toepassingen. Door gebruik te maken van wijzigingen van eerdere technieken 12 , zijn tumor en ANT succesvol gebruikt in eiwit- en RNA-isolatie en kan deze mogelijk worden gebruikt voor DNA-isolatie. Bruikbare planten uit de weefselsecties zijn geëvalueerd door microscopie, terwijl opgeslagen histologische dia&#39;s zijn gebruikt voor immunohistochemie en immunofluorescentie. Door het hier beschreven protocol te volgen is het mogelijk om dit model uit te breiden naar andere dermatologische klinieken, andere tumortypen (zoals melanoom), En andere chirurgische specialiteiten en praktijken om menselijke weefselmonsters te leveren voor veelzijdig onderzoek naar menselijke kanker. Lichte wijzigingen van dit protocol zijn waarschijnlijk nodig voor andere praktijken, maar in principe is dit protocol van toepassing op elke chirurgische praktijk die routinematig de patiëntenmonsters verzamelt die tijdens de patiëntbehandeling zijn verzameld.

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			<td>Zeiss</td>
			<td>4300009901</td>
			<td></td>
		</tr>
		<tr>
			<td>Olympus IX51 phase contrast with DP72 camera</td>
			<td>Olympus</td>
			<td>IX511F3</td>
			<td></td>
		</tr>
	</tbody>
</table>

establishment of a clinic-based biorepository

De incidentie van huidkanker (bijvoorbeeld plaveiselcarcinoom, basaalcelcarcinoom en melanoom) is de afgelopen jaren toegenomen. Er wordt verwacht dat er sprake is van een parallelle vraag naar cutane tumormonsters voor biomedische onderzoeksstudies. De beschikbaarheid van weefsel is echter beperkt door de kosten van het opstellen van een biorepository en het ontbreken van protocollen die beschikbaar zijn voor het verkrijgen van klinische monsters die de klinische operaties niet interfereren. Er werd een protocol opgezet om kutane tumor en bijbehorende bloed- en speekselmonsters te verzamelen en te verwerken die minimale invloed hebben op routine klinische procedures op de datum van een Mohs-operatie. Tumormonsters worden verzameld en verwerkt van patiënten die hun eerste laag van Mohs-operatie ondergaan voor biopsie-bewezen kutane maligniteiten door de Mohs-histotechnoloog. Aangrenzend normaal weefsel wordt verzameld op het moment van chirurgische sluiting. Extra monsters die kunnen worden verzameld, zijn volledige bloed- en buccale swabs. Door gebruik te maken van weefselmonsters die normaal gesproken worden weggegooid, werd een biorepository gegenereerd die verschillende belangrijke voordelen biedt door in de kliniek versus de laboratoriumomgeving te zijn. Deze omvatten een breed scala van verzamelde monsters; Toegang tot ontdekte patiëntrecords, inclusief pathologieverslagen; En, voor de typische donor, toegang tot aanvullende monsters tijdens follow-upbezoeken.

Tumor en ANT zijn succesvol gebruikt bij het isoleren van eiwitten en getest door Western blotting. Zoals getoond in Figuur 3 kunnen cutane plaveiselcelcarcinoommarkers (Muc114, FHL-115 en p40 16 , 17 ) gedetecteerd worden in patiëntmonsters die zijn verzameld en opgeslagen in onze klinische biorepositor. <img alt="Figuur 3" src="/files/ftp_upload/5558...

Watch this Scientific Journal Video about Establishment of a Clinic-based Biorepository at JoVE.com

Establishment of a Clinic-based Biorepository

Kutane tumoren worden vaak weggegooid na Mohs micrografische chirurgie. Hier wordt een protocol beschreven dat klinisch ondersteunend personeel effectief kan verwerken en opslaan van huidkanker ( bv. Plaveiselcelcarcinoom, basale celcarcinoom en melanoom) monsters voor stroomafwaartse laboratoriumtoepassingen zonder in te grijpen bij klinische operaties.

Oprichting van een Biorepository op een kliniek

The incidence of skin cancer (e.g., squamous cell carcinoma, basal cell carcinoma, and melanoma) has been increasing over the past several years. It is ...

The overall goal of this protocol is to enable clinical support staff in a dermatology practice to effectively process and store cutaneous tumor samples for downstream laboratory applications, without interfering with routine clinical operations. This protocol was designed to enable medical assistants and Mohs histotechnicians to collect and store cutaneous tumor samples for future biomedical research projects, as part of their daily clinical operations. The main advantage of this protocol is that excised tissues that are normally discarded and destroyed are instead collected and preserved for research, using procedures, which minimally impact routine clinical operations.The variety of samples collected enables correlations between protein and RNA biomarkers in the tissue and plasma samples with patient outcome data. In principle, this protocol, once slightly modified is applicable to any surgical practice, that routinely discards patient samples, gathered in the course of patient treatment. Demonstrating the procedure will be Stefani Fawks, a Mohs histotechnician, and Dr.Mitchell Manway, a research fellow in our practice.To begin, use the tissue received from the Mohs surgeon and with a scalpel, excise a 10 to 50 milligram sample from the center of the apical side of the tissue. With tweezers, transfer the viable tissue to a labeled 1.5 milliliter tube, prefilled with culture medium. Make sure the tissue is entirely immersed in the medium.Place the sample in a refrigerator and store it at four degrees Celsius, for no longer than 24 hours before further processing. Harvest at least a 10 cubic millimeter tissue fragment from the apical side of the excised tissue, when the tumor sample is large enough, and such removal will not interfere with routine Mohs processing. Next, place the remaining unprocessed tumor sample on a cryostat tissue holder.Embed the tissue in an optimal cutting temperature compound, and freeze it within the holder to prepare histological slides. Using a cryostat, cut the tissue into seven micron thick sections, and layer up to three sections over a slide. After sectioning, fix the tissue by immersing the slides in acetone for one to two seconds.Then, allow the slides to air dry. Keeping the remaining sample in a cryostat chamber, pry the tissue from the cryo embedding medium, and transfer it to a labeled cryogenic tube. Immediately transfer the labeled vial into liquid nitrogen.Transfer of the tissue to a cryo vial after Mohs processing must be performed in the negative 20 degrees Celsius cryostat chamber. If performed properly and the tissue is kept frozen, this step will enable the successful extraction of RNA from post-Mohs tissue samples. Next, obtain adjacent normal tissue from the Mohs surgeon.Excise a sample from the apical side of the adjacent normal tissue. Then, using tweezers, transfer the excised sample to a labeled 1.5 milliliter tube filled with culture medium. Immerse the tissue in the same culture medium.Place it into an appropriate sample bag, and store at four degrees Celsius, until subsequent processing. Collect approximately 10 milliliters of venous blood into EDTA-coated collection tubes. Combine the blood samples in a sterile 15 milliliter centrifuge tube.Then, transfer approximately one milliliter of the blood sample into a 1.5 milliliter cryogenic tube, avoiding bubbles. Store this whole blood sample at minus 80 degrees Celsius, until subsequent genotyping and tissue matching. Next, centrifuge the remaining blood sample at 1000 times G for 30 minutes to obtain blood plasma.After spinning, collect and divide the plasma until one milliliter aliquots in 1.5 milliliter cryogenic tubes. And store the samples at minus 80 degrees Celsius for use during future liquid biopsy analyses. Enter the data from the checklist of patient samples, along with patient information and diagnosis, subtracted from the patient chart to the biorepository database.Finally, record the localization of the de-identified samples in the biorepository database, and move the samples to the research lab. Immediately after, transfer the tissue samples into 1.5 milliliter tubes, prefilled with appropriate extraction solutions, making sure the tissue is completely immersed. Store the samples at minus 80 degrees Celsius until further processing.Presented here are the images of representative Western blots of known squamous cell carcinoma markers, mucin one, FHL-one, and p40, obtained for adjacent normal tissue and a tumor sample. The quality of the RNA isolated from Mohs samples was first assessed using gel electrophoresis, followed by RNA integrity analysis. RNA integrity number values achieved for normal and tumor samples indicate that high quality and intact RNA was obtained.Cytometry profiles of the bands were plotted as fluorescence units versus nucleotide number for representative RNA samples from tumor and normal patient tissue. A squamous cell carcinoma tissue explant was imaged using phase contrast microscopy and shown to be a mixed keratinocyte and fibroblast culture, that might be subcultured or used for future cell line development. Finally, in the squamous cell carcinoma tissue sections, positive staining for the epithelial to mesenchymal transition marker was shown.Once mastered, this protocol will extend the standard work flow of the clinic staff to less than 30 minutes, if it is performed properly. The majority of the steps are performed by the existing clinical staff after patient care is completed. While attempting this protocol, it is important to maintain accurate record keeping, linking the samples with the patient's history and diagnosis.As this information is often needed for downstream applications. This technique will help pave the way for researchers in the field of dermatology for the development of liquid biopsies and biomarkers in squamous cell carcinomas and melanoma. After watching this video, you should have a good understanding of how to implement a clinic-based biorepository for cutaneous cancer samples, generated as part of daily practice.

establishment-of-a-clinic-based-biorepository

Research

JoVE Journal

Medicine

Minimally Invasive Establishment of Murine Orthotopic Bladder Xenografts

Orthotopic bladder cancer xenografts are the gold standard to study molecular cellular manipulations and new therapeutic agents&#160;in vivo. Suitable cell lines are inoculated either by intravesical instillation (model of nonmuscle invasive growth) or intramural injection into the bladder wall (model of invasive growth). Both procedures are complex and highly time-consuming. Additionally, the superficial model has its shortcomings due to the lack of cell lines that are tumorigenic following instillation. Intramural injection, on the other hand, is marred by the invasiveness of the procedure and the associated morbidity for the host mouse.
With these shortcomings in mind, we modified previous methods to develop a minimally invasive approach for creating orthotopic bladder cancer xenografts. Using ultrasound guidance we have successfully performed percutaneous inoculation of the bladder cancer cell lines UM-UC1, UM-UC3 and UM-UC13 into 50 athymic nude. We have been able to demonstrate that this approach is time efficient, precise and safe. With this technique, initially a space is created under the bladder mucosa with PBS, and tumor cells are then injected into this space in a second step. Tumor growth is monitored at regular intervals with bioluminescence imaging and ultrasound. The average tumor volumes increased steadily in in all but one of our 50 mice over the study period.
In our institution, this novel approach, which allows bladder cancer xenograft inoculation in a minimally-invasive, rapid and highly precise way, has replaced the traditional model.

The established technique to inoculate primary invasive orthotopic bladder cancer xenografts requires laparotomy and mobilization of the bladder. This procedure inflicts significant morbidity on the mice, is technically challenging and time-consuming. We therefore developed a high-precision, percutaneous approach utilizing ultrasound guidance.

Minimaal Invasieve Oprichting van Muriene Orthotope Blaas Xenografts

Orthotopic bladder cancer xenografts are the gold standard to study molecular cellular manipulations and new therapeutic agents&#160;in vivo. Suitable ...

Establishment of Human Epithelial Enteroids and Colonoids from Whole Tissue and Biopsy

The epithelium of the gastrointestinal tract is constantly renewed as it turns over. This process is triggered by the proliferation of intestinal stem cells (ISCs) and progeny that progressively migrate and differentiate toward the tip of the villi. These processes, essential for gastrointestinal homeostasis, have been extensively studied using multiple approaches. Ex vivo technologies, especially primary cell cultures have proven to be promising for understanding intestinal epithelial functions. A long-term primary culture system for mouse intestinal crypts has been established to generate 3-dimensional epithelial organoids. These epithelial structures contain crypt- and villus-like domains reminiscent of normal gut epithelium. Commonly, termed &#8220;enteroids&#8221; when derived from small intestine and &#8220;colonoids&#8221; when derived from colon, they are different from organoids that also contain mesenchyme tissue. Additionally, these enteroids/colonoids continuously produce all cell types found normally within the intestinal epithelium. This in vitro organ-like culture system is rapidly becoming the new gold standard for investigation of intestinal stem cell biology and epithelial cell physiology. This technology has been recently transferred to the study of human gut. The establishment of human derived epithelial enteroids and colonoids from small intestine and colon has been possible through the utilization of specific culture media that allow their growth and maintenance over time. Here, we describe a method to establish a small intestinal and colon crypt-derived system from human whole tissue or biopsies. We emphasize the culture modalities that are essential for the successful growth and maintenance of human enteroids and colonoids.

We describe a method to establish human enteroids from small intestinal crypts and colonoids from colon crypts collected from both surgical tissue and biopsies. In this methodological article, we present the culture modalities that are essential for the successful growth and maintenance of human enteroids and colonoids.

Oprichting van Human Epithelial Enteroids en Colonoids van Whole Tissue en Biopsie

The epithelium of the gastrointestinal tract is constantly renewed as it turns over. This process is triggered by the proliferation of intestinal stem ...

Establishment and Characterization of UTI and CAUTI in a Mouse Model

Urinary tract infections (UTI) are highly prevalent, a significant cause of morbidity and are increasingly resistant to treatment with antibiotics. Females are disproportionately afflicted by UTI: 50% of all women will have a UTI in their lifetime. Additionally, 20-40% of these women who have an initial UTI will suffer a recurrence with some suffering frequent recurrences with serious deterioration in the quality of life, pain and discomfort, disruption of daily activities, increased healthcare costs, and few treatment options other than long-term antibiotic prophylaxis. Uropathogenic Escherichia coli (UPEC) is the primary causative agent of community acquired UTI. Catheter-associated UTI (CAUTI) is the most common hospital acquired infection accounting for a million occurrences in the US annually and dramatic healthcare costs. While UPEC is also the primary cause of CAUTI, other causative agents are of increased significance including Enterococcus faecalis. Here we utilize two well-established mouse models that recapitulate many of the clinical characteristics of these human diseases. For UTI, a C3H/HeN model recapitulates many of the features of UPEC virulence observed in humans including host responses, IBC formation and filamentation. For CAUTI, a model using C57BL/6 mice, which retain catheter bladder implants, has been shown to be susceptible to E. faecalis bladder infection. These representative models are being used to gain striking new insights into the pathogenesis of UTI disease, which is leading to the development of novel therapeutics and management or prevention strategies.

The ability to model urinary tract infections (UTI) is crucial in order to be able to understand bacterial pathogenesis and spawn the development of novel therapeutics. This work&#8217;s goal is to demonstrate mouse models of experimental UTI and catheter associated UTI that recapitulate and predict findings seen in humans.

Oprichting en karakterisering van UTI en Cauti in een muismodel

Urinary tract infections (UTI) are highly prevalent, a significant cause of morbidity and are increasingly resistant to treatment with antibiotics. ...

Establishment of a Segmental Femoral Critical-size Defect Model in Mice Stabilized by Plate Osteosynthesis

The use of tissue-engineered bone constructs is an appealing strategy to overcome drawbacks of autografts for the treatment of massive bone defects. As a model organism, the mouse has already been widely used in bone-related research. Large diaphyseal bone defect models in mice, however, are sparse and often use bone fixation which fills the bone marrow cavity and does not provide optimal mechanical stability. The objectives of the current study were to develop a critical-size, segmental, femoral defect in nude mice. A 3.5-mm mid-diaphyseal femoral ostectomy (approximately 25% of the femur length) was performed using a dedicated jig, and was stabilized with an anterior located locking plate and 4 locking screws. The bone defect was subsequently either left empty or filled with a bone substitute (syngenic bone graft or coralline scaffold). Bone healing was monitored noninvasively using radiography and in vivo micro-computed-tomography and was subsequently assessed by ex vivo micro-computed-tomography and undecalcified histology after animal sacrifice, 10 weeks postoperatively. The recovery of all mice was excellent, a full-weight-bearing was observed within one day following the surgical procedure. Furthermore, stable bone fixation and consistent fixation of the implanted materials were achieved in all animals tested throughout the study. When the bone defects were left empty, non-union was consistently obtained. In contrast, when the bone defects were filled with syngenic bone grafts, bone union was always observed. When the bone defects were filled with coralline scaffolds, newly-formed bone was observed in the interface between bone resection edges and the scaffold, as well as within a short distance within the scaffold. 
The present model describes a reproducible critical-size femoral defect stabilized by plate osteosynthesis with low morbidity in mice. The new load-bearing segmental bone defect model could be useful for studying the underlying mechanisms in bone regeneration pertinent to orthopaedic applications.

Although mouse models are invaluable tools for bone tissue engineering, models of long bone defects are sparse. This need motivated development of the present protocol which uses a locking plate with four screws and a dedicated jig to perform and stabilize a reproducible, femoral, critical-size defect with low morbidity.

Oprichting van een Segmentale Femorale Critical-size Defect Model in Muizen gestabiliseerd door plaatosteosynthese

The use of tissue-engineered bone constructs is an appealing strategy to overcome drawbacks of autografts for the treatment of massive bone defects. As a ...

Establishment of a Severe Dry Eye Model Using Complete Dacryoadenectomy in Rabbits

Dry eye disease (DED) is a complex disease with multiple etiologies and variable symptoms, having ocular surface inflammation as its key pathophysiologic step. Despite advances in our understanding of DED, significant knowledge gaps remain. Advances are limited in part due to the lack of informative animal models. The authors recently reported on a method of DED induced by injecting all orbital lacrimal gland (LG) tissues with the lectin concanavalin A. Here, we report a novel model of aqueous-deficient DED based on the surgical resection of all orbital LG (dacryoadenectomy) tissues. Both methods use rabbits because of their similarity to human eyes in terms of the size and structure of the ocular surface. One week after removal of the nictitating membrane, the orbital superior LG was surgically removed under anesthesia, followed by removal of the palpebral superior LG, and finally removal of the inferior LG. Dacryoadenectomy induced severe DED, evidenced by a marked reduction in the tear break up time test and the Schirmer&#39;s tear test, and significantly increased tear osmolarity and rose bengal staining. Dacryoadenectomy-induced DED lasted at least eight weeks. There were no complications and animals tolerated the procedure well. The technique can be mastered relatively easily by those with adequate surgical experience and appreciation of the relevant rabbit anatomy. Since this model recapitulates the features of human aqueous-deficient DED, it is suitable for studies of ocular surface homeostasis, DED, and candidate therapeutics.

A novel approach is presented to induce chronic dry eye disease in rabbits by surgically removing all orbital lacrimal glands. This method, distinct from those previously reported, produces a stable, reproducible model of aqueous deficient dry eye well suited to study tear physiology and pathophysiology and ocular therapeutics.

Oprichting van een ernstig droog Oogmodel met volledige Dacryoadenectomie bij konijnen

Dry eye disease (DED) is a complex disease with multiple etiologies and variable symptoms, having ocular surface inflammation as its key pathophysiologic ...

Establishment and Evaluation of a Porcine Vein Graft Disease Model

Venous graft disease (VGD) is the leading cause of coronary artery bypass graft (CABG) failure. Large animal models of CABG-VGD are needed for the investigation of disease mechanisms and the development of therapeutic strategies. 
To perform the surgery, we enter the cardiac chamber through the third intercostal space and carefully dissect the internal mammary vein and immerse it in normal saline. The right main coronary artery is then treated for ischemia. The target vessel is incised, a shunt plug is placed, and the distal end of the graft vein is anastomosed. The ascending aorta is partially blocked, and the proximal end of the graft vein is anastomosed after perforation. The graft vein is checked for patency, and the proximal right coronary artery is ligated. 
CABG surgery is performed in minipigs to harvest the left internal mammary vein for its use as a vascular graft. Serum biochemical tests are used to evaluate the physiological status of the animals after surgery. Ultrasound examination shows that the proximal, middle, and distal end of the graft vessel are unobstructed. In the surgical model, turbulent blood flow in the graft is observed upon histological examination after the CABG surgery, and venous graft stenosis associated with intimal hyperplasia is observed in the graft. The study here provides detailed surgical procedures for the establishment of a repeatable CABG-induced VGD model.

In this protocol, novel pig vein bypass grafting was performed through a small incision in the left chest wall without cardiopulmonary bypass. A postoperative pathology study was done, which showed intimal thickening.

Vaststelling en evaluatie van een model voor varkensaderziekte

Venous graft disease (VGD) is the leading cause of coronary artery bypass graft (CABG) failure. Large animal models of CABG-VGD are needed for the ...

Establishment of a Simple and Effective Rat Model for Intraoperative Parathyroid Gland Imaging

Parathyroid gland (PG) identification is a critical unmet need in thyroidectomy. The identification of the PG is challenging in thyroid surgery as it is similar in color to the thyroid gland. The lack of effective animal models in preclinical research is a severe limitation for the development of PG identification techniques. This protocol allows for the establishment of a simple and effective rat model for PG identification. In this model, black iron oxide nanoparticles (IONPs) are injected locally in the thyroid gland and rapidly diffuse within the thyroid gland but not the PG. A negatively stained PG and a positively stained thyroid gland can be easily identified by the naked eye without requiring external microscopes. The position of the PG can be identified by increasing the color contrast between the thyroid gland and the PG, based on the color of the black IONPs. This rat model is low-cost and convenient for PG identification, and the IONPs are a novel PG contrast agent.

To date, the development of parathyroid gland (PG) identification methods is limited by the lack of animal models in preclinical research. Here, we establish a simple and effective rat model for intraoperative PG imaging and evaluate its effectiveness by using iron oxide nanoparticles as a novel PG contrast agent.

Oprichting van een eenvoudig en effectief rattenmodel voor intraoperatieve beeldvorming van bijschildklieren

Parathyroid gland (PG) identification is a critical unmet need in thyroidectomy. The identification of the PG is challenging in thyroid surgery as it is ...

Gemodificeerde arterioveneuze fistelchirurgie om het RV-volumeoverbelastingsmuismodel vast te stellen

Establishment and Confirmation of a Postnatal Right Ventricular Volume Overload Mouse Model

Right ventricular (RV) volume overload (VO) is common in children with congenital heart disease. In view of distinct developmental stages,the RV myocardium may respond differently to VO in children compared to adults. The present study aims to establish a postnatal RV VO model in mice using a modified abdominal arteriovenous fistula. To confirm the creation of VO and the following morphological and hemodynamic changes of the RV, abdominal ultrasound, echocardiography, and histochemical staining were performed for 3 months. As a result, the procedure in postnatal mice showed an acceptable survival and fistula success rate. In VO mice, the RV cavity was enlarged with a thickened free wall, and the stroke volume was increased by about 30%-40% within 2 months after surgery. Thereafter, the RV systolic pressure increased, corresponding pulmonary valve regurgitation was observed, and small pulmonary artery remodeling appeared. In conclusion, modified arteriovenous fistula (AVF) surgery is feasible to establish the RV VO model in postnatal mice. Considering the probability of fistula closure and elevated pulmonary artery resistance, abdominal ultrasound and echocardiography must be performed to confirm the model status before application.

Auteur in de schijnwerpers: Oprichting en bevestiging van een postnataal rechterventrikel volume-overbelastingsmuismodel  

This protocol presents the establishment and confirmation of a postnatal right ventricular volume overload (VO) model in mice with abdominal arteriovenous fistula (AVF), which can be applied to investigate how VO contributes to postnatal heart development.