Name: the superficial inferior epigastric artery axial flap to study ischemic preconditioning effects in a rat model
Uploaded: 2023-01-27T14:45:00
Description: Watch this Scientific Journal Video about The Superficial Inferior Epigastric Artery Axial Flap to Study Ischemic Preconditioning Effects in a Rat Model at JoVE.com

This work was funded by Massachusetts General Hospital (W.G.A) and Shriners Children&#39;s Boston (B.U, K.U, C.L.C). Y.B and I.F.v.R are funded by Shriners Hospitals for Children (Proposal ID: #970280 and #857829 respectively).

The Massachusetts General Hospital Institutional Animal Care and Use Committee approved the experimental protocol (IACUC- protocol #2022N000099).The authors followed the ARRIVE (Animal Research: Reporting In Vivo Experiments) guideline checklist for this work. All animals received humane care following the National Institute of Health Guide for the Care and Use of Laboratory Animals. A total of 12 male Lewis rats (250-350 g, 8-10 weeks old) were used for all experiments.
1. Anim...

<ol>
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E., Jennings, R. B., Reimer, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preconditioning+with+ischemia:+a+delay+of+lethal+cell+injury+in+ischemic+myocardium.">Preconditioning with ischemia: a delay of lethal cell injury in ischemic myocardium.</a> Circulation. 74 (5), 1124-1136 (1986).</li><li>Akcal, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Combination+of+ischemic+preconditioning+and+postconditioning+can+minimise+skin+flap+loss:+experimental+study.">Combination of ischemic preconditioning and postconditioning can minimise skin flap loss: experimental study.</a> Journal of Plastic Surgery and Hand Surgery. 50 (4), 233-238 (2016).</li><li>Ulker, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Does+ischemic+preconditioning+increase+flap+survival+by+ADORA2B+receptor+activation.">Does ischemic preconditioning increase flap survival by ADORA2B receptor activation.</a> Clinical Hemorheology and Microcirculation. 75 (2), 151-162 (2020).</li><li>Min, S. -. 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E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+rat+groin+flap+model+redesigned+for+evaluating+treatment+effects+on+ischemia-reperfusion+injury.">The rat groin flap model redesigned for evaluating treatment effects on ischemia-reperfusion injury.</a> Journal of Surgical Research. 222, 160-166 (2018).</li><li>M&#252;cke, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Indocyanine+green+videoangiography-assisted+prediction+of+flap+necrosis+in+the+rat+epigastric+flap+using+the+flow&#174;+800+tool.">Indocyanine green videoangiography-assisted prediction of flap necrosis in the rat epigastric flap using the flow&#174; 800 tool.</a> Microsurgery. 37 (3), 235-242 (2017).</li><li>Wang, D., Chen, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Indocyanine+green+angiography+for+continuously+monitoring+blood+flow+changes+and+predicting+perfusion+of+deep+inferior+epigastric+perforator+flap+in+rats.">Indocyanine green angiography for continuously monitoring blood flow changes and predicting perfusion of deep inferior epigastric perforator flap in rats.</a> Journal of Investigative Surgery. 34 (4), 393-400 (2021).</li><li>Berkane, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+to+secure+pedicled+flaps+using+perioperative+indocyanine+green+angiography:+a+prospective+study+about+10+cases.">How to secure pedicled flaps using perioperative indocyanine green angiography: a prospective study about 10 cases.</a> World Journal of Surgery and Surgical Research. 4 (1), 1319 (2021).</li><li>Alstrup, T., Christensen, B. O., Damsgaard, T. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ICG+angiography+in+immediate+and+delayed+autologous+breast+reconstructions:+peroperative+evaluation+and+postoperative+outcomes.">ICG angiography in immediate and delayed autologous breast reconstructions: peroperative evaluation and postoperative outcomes.</a> Journal of Plastic Surgery and Hand Surgery. 52 (5), 307-311 (2018).</li><li>K&#252;ntscher, M. V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ischemic+preconditioning+by+brief+extremity+ischemia+before+flap+ischemia+in+a+rat+model.">Ischemic preconditioning by brief extremity ischemia before flap ischemia in a rat model.</a> Plastic and Reconstructive Surgery. 109 (7), 2398-2404 (2002).</li><li>Liu, R. Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cost+analysis+of+indocyanine+green+fluorescence+angiography+for+prevention+of+anastomotic+leakage+in+colorectal+surgery.">Cost analysis of indocyanine green fluorescence angiography for prevention of anastomotic leakage in colorectal surgery.</a> Surgical Endoscopy. 36 (12), 9281-9287 (2022).</li><li>Cheng, M. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Devices+for+ischemic+preconditioning+of+the+pedicled+groin+flap.">Devices for ischemic preconditioning of the pedicled groin flap.</a> The Journal of Trauma. 48 (3), 552-557 (2000).</li><li>Xiao, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+innovative+and+economical+device+for+ischemic+preconditioning+of+the+forehead+flap+prior+to+pedicle+division:+a+comparative+study.">An innovative and economical device for ischemic preconditioning of the forehead flap prior to pedicle division: a comparative study.</a> Journal of Reconstructive Microsurgery. 38 (9), 703-710 (2022).</li><li>K&#252;ntscher, M. V., Hartmann, B., Germann, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Remote+ischemic+preconditioning+of+flaps:+a+review.">Remote ischemic preconditioning of flaps: a review.</a> Microsurgery. 25 (4), 346-352 (2005).</li></ol>

This article describes a reproducible fasciocutaneous flap model harvested in rats, allowing I.P. evaluation. This step-by-step surgical protocol gives research groups a reliable model to test different I.P. regimens. By preventing any vascularization other than the pedicle, this model allows for studying the flap's neovascularization from the wound bed and margin. This study performed the ligation on POD5, as previous studies have observed the autonomization of this flap in rats on POD5-711,...

Plastic and reconstructive surgery is constantly in evolution. The development of muscle, fasciocutaneous, and perforator flaps has made it possible to offer better-quality reconstructions while reducing morbidity. Combining this improved anatomical knowledge with enhanced technical skills, reconstructive surgeons can perform free flap transfers when defects are not close to any local solution. However, while perforator flap surgery is currently the most advanced technique in reconstructive surgery, the literature reports a 5% failure rate in free flap transfers1,2,3, and up to 20% for pedicled flap reconstruction4,5,6. Partial to total flap failure occurs when the flap&#39;s pedicle is compromised, therefore it is essential to continuously search for improvements to the current techniques. One of the methods to improve flap survival is to promote its neovascularization on the wound bed, thus allowing perfusion by a source other than the pedicle. Ischemic preconditioning (I.P.) has been initially described in a heart model7, demonstrating that an organ exposed to controlled ischemia survives to a higher degree after losing its primary blood supply by undergoing ischemia-induced neovascularization. Several authors have studied this cornerstone principle to optimize flap survival in preclinical and clinical models8,9,10.
The advantage of this technique over other methods to improve flap survival is its ease of implementation, consisting of clamp/declamp tests of the blood source. In the rat model, previous authors used the superficial inferior epigastric artery (SIEA) flap to study I.P. by clamping the main pedicle11,12,13. Nonetheless, several technical issues can be encountered with this model, and the literature lacks well-described protocols.
Therefore, this work aims to provide researchers with a detailed description of a rat SIEA flap procurement technique with an extended dissection of the femoral vessels to allow I.P. studies on an axial fasciocutaneous flap model. This model retains the integrity of the epigastric vessels and instead manipulates the femoral vessels, which are more resilient. We share our experience and tools to improve the study of this phenomenon and increase the replicability of this procedure.

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			<td>Lone Star elastic stays</td>
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			<td>Lone star Self-retaining retractor&#160;</td>
			<td>Cooper Surgical</td>
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			<td>Metronidazole tablets USP</td>
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			<td>Micro spring handle scissors&#160;</td>
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			<td>Microsurgical needle holder</td>
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			<td>Needle holder</td>
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			<td>Nosecone for Anesthesia&#160;</td>
			<td>World Precision Instruments</td>
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			<td>Pixel analysis software</td>
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			<td>Ragnell Scissors&#160;</td>
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			<td>Small animal ear tag</td>
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			<td>Style 1005-1</td>
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			<td>Peco Services Ltd</td>
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			<td>Dynarex Corporation</td>
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			<td>Sterile water for injection and irrigation&#160;</td>
			<td>Hospira</td>
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			<td>Surgical scrub &#8211; BD ChloraPrep Hi-Lite Orange 3 mL applicator with Sterile Solution</td>
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			<td>Webcol Alcohol prep pads&#160;</td>
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the superficial inferior epigastric artery axial flap to study ischemic preconditioning effects in a rat model

Fasciocutaneous flaps (FCF) have become the gold standard for complex defect reconstruction in plastic and reconstructive surgery. This muscle-sparing technique allows transferring vascularized tissues to cover any large defect. FCF can be used as pedicled flaps or as free flaps; however, in the literature, failure rates for pedicled FCF and free FCF are above 5%, leaving room for improvement for these techniques and further knowledge expansion in this area. Ischemic preconditioning (I.P.) has been widely studied, but the mechanisms and the optimization of the I.P. regimen are yet to be determined. This phenomenon is indeed poorly explored in plastic and reconstructive surgery. Here, a surgical model is presented to study the I.P. regimen in a rat axial fasciocutaneous flap model, describing how to safely and reliably assess the effects of I.P. on flap survival. This article describes the complete surgical procedure, including suggestions to improve the reliability of this model. The objective is to provide researchers with a reproducible and reliable model to test various ischemic preconditioning regimens and assess their effects on flap survivability.

All flaps were viable on POD5, showing good vascularization by the SIEA alone. Figure 5 shows the flap before and after IV fluorescein injection, showing a complete vascularization.
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/64980/64980fig05.jpg" /> 
Figure 5: Immediate intravenous fluorescein angiography (POD0). This assessment shows the flap&#39;s vascu...

Watch this Scientific Journal Video about The Superficial Inferior Epigastric Artery Axial Flap to Study Ischemic Preconditioning Effects in a Rat Model at JoVE.com

The Superficial Inferior Epigastric Artery Axial Flap to Study Ischemic Preconditioning Effects in a Rat Model

This protocol describes harvesting, suturing, and monitoring fasciocutaneous flaps in rats that allow for good visualization and manipulation of blood flow through the superficial inferior epigastric vessels by means of clamping and ligating the femoral vessels. This is critical for studies involving ischemic preconditioning.

Fasciocutaneous flaps (FCF) have become the gold standard for complex defect reconstruction in plastic and reconstructive surgery. This muscle-sparing ...

This surgical protocol provides a reliable model to study ischemic preconditioning regimen in fasciocutaneous flaps to improve the outcomes of these surgeries. This protocol is easy to learn, reproducible and reliable. The main difference with other protocols is the use of femoral vessels to provoke the ischemic preconditioning, avoiding harming small vessels.It can eventually lead to optimizing an ischemic preconditioning protocol for free and pedicled flap reconstruction to improve flap survivability and reduce the critical period of the flap vulnerability. This method can contribute to the field of reconstructive surgery and lends itself well to the study of ischemia reperfusion injuries. To begin, place the properly sedated animal in a supine position.Shave the abdomen from below the inguinal crease to above the level of the xiphoid process. Apply a hair removal cream on the shaved area and wait for five minutes before cleaning it. Using a sterile skin pen and a ruler, first mark the midline of the animal's abdomen and then mark the inguinal crease.Before making the incision, indicate the position of the desired flap on the animal's body by drawing an oval or a rectangle, having a vertical length of up to six centimeters and a horizontal width of about three centimeters extending cranially from the inguinal crease. Now draw five or six equidistant marks perpendicular to the flap limits. Then using a pair of Ragnell scissors, make a longitudinal incision of three to four centimeters on the inguinal crease.Be sure to pull skin upward to avoid damaging vessels. Expose and identify the femoral and epigastric vessels using a 4 jewelers microsurgical forceps by opening and closing the forceps to separate the fascia and to gain access to the vessels beneath the inguinal fat pad. Use the inguinal incision to start the flap incision using the Ragnell scissors.Pay attention to undermining the full thickness of the skin and the connective tissue over the abdominal muscle. As the tip of the flap is initially freed from the surrounding skin, continue the flap procurement by undermining from the distal to the proximal part using the Ragnell scissor tips to separate the flap from the muscle while cauterizing any perforator vessels and dermal plexus vessels around the flap. Once the flap is entirely harvested, aim to encapsulate the entirety of both branches of the superficial inferior epigastric artery or SIEA with the flap by gently lifting the flap upward to visualize the vessels.Next, separate the fat pads on the inferior aspect on both the medial and lateral sides of the flap. Use the bipolar cautery to cauterize the fat pads close to the border of the incision carefully without harming the superficial inferior epigastric pedicle. Before proceeding for the femoral vessel preparation after completion of the flap harvesting, inject the animal with a single dose of 17.5 international units of sodium heparin via the penile vein.To allow a better view of the vessels, place the animal within a lone star self-retaining retractor. Now working under a surgical microscope having a 40X magnification, use a couple of 4 microsurgical forceps to dissect the femoral vessels both proximally and distally until the emergence of the superficial epigastric vessels. On the distal femoral vessels, clean the fascia and gently free the nerve from the artery and vein.Using an 8-0 nylon suture, ligate the distal femoral vessels by circumventing the microsurgical needle holder under the artery and vein, clasping the suture and tying off these vessels right after the emergence of the epigastric vessels, leaving a one millimeter distance after the pedicle origin. On the proximal femoral vessels, repeat the same process of cleaning the connective tissue. However, separate the artery and vein from each other to allow efficient clamping.To induce intermittent ischemia, place microsurgical clamps separately on each proximal femoral artery and vein. When the ischemic injuries are completed, suture the flap to its original position lining the marks drawn preoperatively. Suture the flap using a running suture with 3-0 nylon at the inguinal crease medially, around the flap, and ending at the inguinal crease laterally.To verify the blood supply to the flap, inject 0.25 milliliters of 10%sterile fluorescein sodium into the the penile vein using the same technique and tools described for the previous injection. After three minutes, shine a long wave UV lamp at 366 nanometers to reveal the fluorescent areas corresponding to the perfused areas. After closure and verification, spread crushed metronidazole along the sutures to prevent auto-mutilation and spray liquid bandage on the same area.To remove the flap from the epigastric blood supply at the end of the ischemic preconditioning period, cauterize inferior to the fat pad along the inferior border of the flap. Immediate intravenous fluorescein angiography on postoperative day zero or POD0 showed complete vascularization of the flap by the SIEA alone. The tissue, including the whole flap paddle, was well perfused.The angiography of the negative control on POD5 and POD10 showed full viability of the flap paddle. All animals in this group where no ligation was performed on the feeding vessels remained healthy. For the positive control group, the absence of green fluorescence immediately after ligation showed no perfusion of the flap, proving the absence of neovascularization.This was confirmed on POD10 with observed necrosis of 85%of the skin paddle. Interestingly, the distal tip was viable and neovascularized. Statistical analysis of the flap surface viability on POD10 showed 99.5%survivability by the negative control group whereas the positive control group showed 11.25%survivability.An example experimental group meant to study ischemic preconditioning showed 13.67%survivability demonstrating that the particular ischemic preconditioning protocol was inefficient in this model. The two major steps are flap harvesting and vessel dissection under the microscope. The most important piece of advice is to have good-quality micro instruments.The next goal is to try different ischemic preconditioning protocols by varying the ischemia durations and rhythms to select the regimen allowing the best flap survivability following early flow disruption. This technique by providing a good model for ischemia fusion injuries can lead to new findings on ischemic preconditioning applied to modern reconstructive surgery and reconstructive transplant.

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Research

JoVE Journal

Immunology and Infection

Cutaneous Leishmaniasis in the Dorsal Skin of Hamsters: a Useful Model for the Screening of Antileishmanial Drugs

Traditionally, hamsters are experimentally inoculated in the snout or the footpad. However in these sites an ulcer not always occurs, measurement of lesion size is a hard procedure and animals show difficulty to eat, breathe and move because of the lesion. In order to optimize the hamster model for cutaneous leishmaniasis, young adult male and female golden hamsters (Mesocricetus auratus) were injected intradermally at the dorsal skin with 1 to 1.5 x l07 promastigotes of Leishmania species and progression of subsequent lesions were evaluated for up to 16 weeks post infection. The golden hamster was selected because it is considered the adequate bio-model to evaluate drugs against Leishmania as they are susceptible to infection by different species. Cutaneous infection of hamsters results in chronic but controlled lesions, and a clinical evolution with signs similar to those observed in humans. Therefore, the establishment of the extent of infection by measuring the size of the lesion according to the area of indurations and ulcers is feasible. This approach has proven its versatility and easy management during inoculation, follow up and characterization of typical lesions (ulcers), application of treatments through different ways and obtaining of clinical samples after different treatments. By using this method the quality of animal life regarding locomotion, search for food and water, play and social activities is also preserved.

Optimization of the experimental hamster model for cutaneous leishmaniasis by intradermal injection of Leishmania promastigotes at the dorsal skin. This approach is useful during inoculation, follow-up, characterization of lesions, application of treatments and obtaining of clinical samples. Locomotion, search for food and water, play and social activities are preserved.

Traditionally, hamsters are experimentally inoculated in the snout or the footpad. However in these sites an ulcer not always occurs, measurement of ...

An In vitro Model to Study Heterogeneity of Human Macrophage Differentiation and Polarization

Monocyte-derived macrophages represent an important cell type of the innate immune system. Mouse models studying macrophage biology suffer from the phenotypic and functional differences between murine and human monocyte-derived macrophages. Therefore, we here describe an in vitro model to generate and study primary human macrophages. Briefly, after density gradient centrifugation of peripheral blood drawn from a forearm vein, monocytes are isolated from peripheral blood mononuclear cells using negative magnetic bead isolation. These monocytes are then cultured for six days under specific conditions to induce different types of macrophage differentiation or polarization. The model is easy to use and circumvents the problems caused by species-specific differences between mouse and man. Furthermore, it is closer to the in vivo conditions than the use of immortalized cell lines. In conclusion, the model described here is suitable to study macrophage biology, identify disease mechanisms and novel therapeutic targets. Even though not fully replacing experiments with animals or human tissues obtained post mortem, the model described here allows identification and validation of disease mechanisms and therapeutic targets that may be highly relevant to various human diseases.

An In vitro Model to Study Heterogeneity of Human Macrophage Differentiation and Polarization

Monocyte-derived macrophages are important cells of the innate immune system. Here, we describe an easy to use in vitro model to generate these cells. Using gradient centrifugation, negative bead isolation and specific cell culture conditions, monocyte-derived macrophages can be generated for phenotypic and functional studies.

Monocyte-derived macrophages represent an important cell type of the innate immune system. Mouse models studying macrophage biology suffer from the ...

Non-Invasive Model of Neuropathogenic Escherichia coli Infection in the Neonatal Rat

Investigation of the interactions between animal host and bacterial pathogen is only meaningful if the infection model employed replicates the principal features of the natural infection. This protocol describes procedures for the establishment and evaluation of systemic infection due to neuropathogenic Escherichia coli K1 in the neonatal rat. Colonization of the gastrointestinal tract leads to dissemination of the pathogen along the gut-lymph-blood-brain course of infection and the model displays strong age dependency. A strain of E. coli O18:K1 with enhanced virulence for the neonatal rat produces exceptionally high rates of colonization, translocation to the blood compartment and invasion of the meninges following transit through the choroid plexus. As in the human host, penetration of the central nervous system is accompanied by local inflammation and an invariably lethal outcome. The model is of proven utility for studies of the mechanism of pathogenesis, for evaluation of therapeutic interventions and for assessment of bacterial virulence.

Non-Invasive Model of Neuropathogenic Escherichia coli Infection in the Neonatal Rat

Here, a procedure is described for the establishment of systemic infection in the neonatal rat with cultures of Escherichia coli K1. This non-invasive procedure permits colonization of the gastrointestinal tract, translocation of the pathogen to the systemic circulation, and invasion of the central nervous system at the choroid plexus.

Investigation of the interactions between animal host and bacterial pathogen is only meaningful if the infection model employed replicates the principal ...

In Vitro and In Vivo Model to Study Bacterial Adhesion to the Vessel Wall Under Flow Conditions

In order to cause endovascular infections and infective endocarditis, bacteria need to be able to adhere to the vessel wall while being exposed to the shear stress of flowing blood.
To identify the bacterial and host factors that contribute to vascular adhesion of microorganisms, appropriate models that study these interactions under physiological shear conditions are needed. Here, we describe an in vitro flow chamber model that allows to investigate bacterial adhesion to different components of the extracellular matrix or to endothelial cells, and an intravital microscopy model that was developed to directly visualize the initial adhesion of bacteria to the splanchnic circulation in vivo. These methods can be used to identify the bacterial and host factors required for the adhesion of bacteria under flow. We illustrate the relevance of shear stress and the role of von Willebrand factor for the adhesion of Staphylococcus aureus using both the in vitro and in vivo model.

In Vitro and In Vivo Model to Study Bacterial Adhesion to the Vessel Wall Under Flow Conditions

To study the interaction of bacteria with the blood vessels under shear stress, a flow chamber and an in vivo mesenteric intravital microscopy model are described that allow to dissect the bacterial and host factors contributing to vascular adhesion.

In order to cause endovascular infections and infective endocarditis, bacteria need to be able to adhere to the vessel wall while being exposed to the ...

Human Placental and Decidual Organ Cultures to Study Infections at the Maternal-fetal Interface

The placenta shows a large degree of interspecies anatomic variability. To best understand biology and pathophysiology of the human placenta, it is imperative to design experiments using human cells and tissues. An advantage of organ culture is maintenance of three-dimensional (3D) structural organization and extracellular matrix. The goal of the method described here is successful establishment of ex vivo human gestational tissue organ cultures and their healthy culture maintenance for 72-96 hr. The protocol details the immediate processing of research-consented, placental and decidual specimens fresh from the operating suite. These are abundant specimens that would otherwise be discarded. Detailed instructions on the sterile collection of these samples, including morphologic details on how to select appropriate tissues to establish 3D organ cultures, is provided. Placental villous and decidual tissues are microdissected into 2-3 mm3 pieces and placed separately on matrix-lined transwell filters and cultured for several days. Villous and decidual organ cultures are well suited for the study of human host-pathogen interaction. As compared to other model organisms, these human cultures are particularly advantageous to examine mechanism of infection for pathogens that demonstrate variable patterns of host specificity. As an example, we demonstrate infection of placental and decidual organ cultures with the clinically relevant, facultative intracellular bacterial pathogen Listeria monocytogenes.

A simple method to establish primary human placental (villous) and decidual organ cultures is described. Villous and decidual organ cultures are invaluable tools for studying pathogenesis at the human maternal-fetal interface. Infection with the facultative intracellular bacterium Listeria monocytogenes is demonstrated.

The placenta shows a large degree of interspecies anatomic variability. To best understand biology and pathophysiology of the human placenta, it is ...

Implementation of a Permeable Membrane Insert-based Infection System to Study the Effects of Secreted Bacterial Toxins on Mammalian Host Cells

Many bacterial pathogens secrete potent toxins to aid in the destruction of host tissue, to initiate signaling changes in host cells or to manipulate immune system responses during the course of infection. Though methods have been developed to successfully purify and produce many of these important virulence factors, there are still many bacterial toxins whose unique structure or extensive post-translational modifications make them difficult to purify and study in in vitro systems. Furthermore, even when pure toxin can be obtained, there are many challenges associated with studying the specific effects of a toxin under relevant physiological conditions. Most in vitro cell culture models designed to assess the effects of secreted bacterial toxins on host cells involve incubating host cells with a one-time dose of toxin. Such methods poorly approximate what host cells actually experience during an infection, where toxin is continually produced by bacterial cells and allowed to accumulate gradually during the course of infection. This protocol describes the design of a permeable membrane insert-based bacterial infection system to study the effects of Streptolysin S, a potent toxin produced by Group A Streptococcus, on human epithelial keratinocytes. This system more closely mimics the natural physiological environment during an infection than methods where pure toxin or bacterial supernatants are directly applied to host cells. Importantly, this method also eliminates the bias of host responses that are due to direct contact between the bacteria and host cells. This system has been utilized to effectively assess the effects of Streptolysin S (SLS) on host membrane integrity, cellular viability, and cellular signaling responses. This technique can be readily applied to the study of other secreted virulence factors on a variety of mammalian host cell types to investigate the specific role of a secreted bacterial factor during the course of infection.

Here, a method using a permeable membrane insert-based infection system to study the effects of Streptolysin S, a secreted toxin produced by Group A Streptococcus, on keratinocytes is described. This system can be readily applied to the study of other secreted bacterial proteins on various host cell types during infection.

Many bacterial pathogens secrete potent toxins to aid in the destruction of host tissue, to initiate signaling changes in host cells or to manipulate ...

Flow Cytometric Analysis of Particle-bound Bet v 1 Allergen in PM10

Flow cytometry is a method widely used to quantify suspended solids such as cells or bacteria in a size range from 0.5 to several tens of micrometers in diameter. In addition to a characterization of forward and sideward scatter properties, it enables the use of fluorescent labeled markers like antibodies to detect respective structures. Using indirect antibody staining, flow cytometry is employed here to quantify birch pollen allergen (precisely Bet v 1)-loaded particles of 0.5 to 10 &#181;m in diameter in inhalable particulate matter (PM10, particle size &#8804;10 &#181;m in diameter). PM10 particles may act as carriers of adsorbed allergens possibly transporting them to the lower respiratory tract, where they could trigger allergic reactions.
So far the allergen content of PM10 has been studied by means of enzyme linked immunosorbent assays (ELISAs) and scanning electron microscopy. ELISA measures the dissolved and not the particle-bound allergen. Compared to scanning electron microscopy, which can visualize allergen-loaded particles, flow cytometry may additionally quantify them. As allergen content of ambient air can deviate from birch pollen count, allergic symptoms might perhaps correlate better with allergen exposure than with pollen count. In conjunction with clinical data, the presented method offers the opportunity to test in future experiments whether allergic reactions to birch pollen antigens are associated with the Bet v 1 allergen content of PM10 particles &#62;0.5 &#181;m.

Here, we present a protocol to quantify allergen-loaded particles by flow cytometry. Ambient particulate matter particles may act as carriers of adsorbed allergens. We show here that flow cytometry, a method widely used to characterize suspended solids &#62;0.5 &#181;m in diameter, can be used to measure these allergen-loaded particles.

Flow cytometry is a method widely used to quantify suspended solids such as cells or bacteria in a size range from 0.5 to several tens of micrometers in ...

Visualizing the Effects of Sputum on Biofilm Development Using a Chambered Coverglass Model

Biofilms consist of groups of bacteria encased in a self-secreted matrix. They play an important role in industrial contamination as well as in the development and persistence of many health related infections. One of the most well described and studied biofilms in human disease occurs in chronic pulmonary infection of cystic fibrosis patients. When studying biofilms in the context of the host, many factors can impact biofilm formation and development. In order to identify how host factors may affect biofilm formation and development, we used a static chambered coverglass method to grow biofilms in the presence of host-derived factors in the form of sputum supernatants. Bacteria are seeded into chambers and exposed to sputum filtrates. Following 48 hr of growth, biofilms are stained with a commercial biofilm viability kit prior to confocal microscopy and analysis. Following image acquisition, biofilm properties can be assessed using different software platforms. This method allows us to visualize key properties of biofilm growth in presence of different substances including antibiotics.

This protocol describes the visualization of biofilm development following exposure to host-factors using a slide chamber model. This model allows for direct visualization of biofilm development as well as analysis of biofilm parameters using computer software programs.

Biofilms consist of groups of bacteria encased in a self-secreted matrix. They play an important role in industrial contamination as well as in the ...

Effects of Exposure of Formaldehyde to a Rat Model of Atopic Dermatitis Induced by Neonatal Capsaicin Treatment

Atopic dermatitis is chronically relapsing pruritic eczema and prevails around the world especially in developed countries. Complex interactions between genetic and environmental factors are known to play an important role in the pathophysiology of atopic dermatitis. However, we still lack a detailed picture of the pathogenesis of this disease. Thus, it is of importance to develop appropriate animal models for elucidating the progression of atopic dermatitis. Moreover, investigating the effect of environmental factors such as air pollutants on atopic dermatitis expands understanding of the disease. Here, we describe a method for inducing atopic dermatitis in rats with neonatal capsaicin treatment and a protocol for exposure of a constant concentration of formaldehyde to rats to reveal effects on the development of atopic dermatitis in infantile and adolescent periods. These protocols have been successfully applied to several experiments and can be used for other substances.

We describe here methods for neonatal capsaicin treatment to induce atopic dermatitis in rats and exposure of vaporized formaldehyde to investigate the effects of formaldehyde inhalation on atopic dermatitis.

Atopic dermatitis is chronically relapsing pruritic eczema and prevails around the world especially in developed countries. Complex interactions between ...

Stenosis of the Inferior Vena Cava: A Murine Model of Deep Vein Thrombosis

Deep vein thrombosis (DVT) and its devastating complication, pulmonary embolism, are a severe health problem with high mortality. Mechanisms of thrombus formation in veins remain obscure. Lack of mobility (e.g., after surgery or long-haul flights) is one of the main factors leading to DVT. The pathophysiological consequence of the lack of mobility is blood flow stagnation in venous valves. Here, a model is described that mimics such flow disturbance as a thrombosis-driving factor. In this model, partial flow restriction (stenosis) in the inferior vena cava (IVC) is created. Closure of about 90% of the IVC lumen for 48 h results in development of thrombi structurally similar to those in humans. The similarities are: i) most of the thrombus volume is red, i.e., consists of red blood cells and fibrin, ii) presence of a white part (lines of Zahn), iii) non-denuded endothelial monolayer, iv) elevated plasma D-Dimer levels, and v) possibility to prevent thrombosis by low molecular weight heparin. Limitations include variable size of thrombi and the fact that a certain percentage of wild-type mice (0 - 35%) may not produce a thrombus. In addition to visual observation and measurement, thrombi may be visualized by non-invasive technologies, such as ultrasonography, which allows for monitoring the dynamics of thrombus development. At shorter time points (1 - 6 h), intravital microscopy may be applied to directly observe events (e.g., recruitment of cells to the vessel wall) preceding thrombus formation. Use of this method by several teams around the world has made it possible to uncover basic mechanisms of DVT initiation and identify potential targets that might be beneficial for its prevention.

We describe here stenosis in the inferior vena cava as a murine model of deep vein thrombosis. This model recapitulates blood flow restriction, one of the major triggers of venous thrombosis in humans.

Deep vein thrombosis (DVT) and its devastating complication, pulmonary embolism, are a severe health problem with high mortality. Mechanisms of thrombus ...