eoe sub articles

EoE Sub Articles

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. Preparation of Bone Infection Models
<ol>
	<li>Keep male New Zealand white rabbits, aged 3 months, in individual cages, under air-controlled conditions (20 &#177; 1 &#176;C) and 12 h/12 h light-dark illumination cycles. Offer routine diet and tap water daily.</li>
	<li>Ensure that at the time of surgery that the rabbit weighs more than 3 kg.</li>
	<li>Anaesthetize rabbits by intraperitoneal injection with pentobarbital sodium (3 mg per 100 g of body weight). Make sure rabbits are fully anesthetized by a failure to respond to a paw pinch. Fix rabbits on the operating table during operation procedure. 
	NOTE:&#160;Make sure that the modeling procedure duration is less than 1 h.</li>
	<li>Shave the proximal tibia region using an electric shaver against the direction of hair growth. Disinfect the skin by applying a povidone-iodine solution.</li>
	<li>Mark the upper end of the tibia and the drilling hole position for injection with&#160;S. aureus&#160;(the distance to the upper end of the tibia is 1.5 cm) with pen and ruler. Make sure the drilling hole positions are in the middle of the tibial plateau horizontally (Figure 1A).</li>
	<li>Cut tibia skin using a No. 11 scalpel and make a 1 cm incision in the periosteum (Figure 1B, C). Punch a 2 mm diameter hole in the tibia using an electric bone drill unit (Figure 1D).</li>
	<li>Press the 2 mm diameter holes in the tibial plateau with a cylinder of bone wax of 2 mm diameter and 2 mm height (Figure 1E). Remove the spare bone wax along the horizontal plane of the tibial plateau (Figure 1F). Check that the 2 mm hole is full of bone wax (Figure 1G). 
	NOTE:&#160;Ensure that the holes are full of bone wax by checking the hole with or without blood overflow.</li>
	<li>Sew up the periosteum and skin with absorbable surgical suture in a vertical mattress suture to prevent the animal from chewing the sutures (Figure 1H).</li>
	<li>Inject 1 x 108&#160;CFU/mL of&#160;S. aureus&#160;solutions (30 &#181;L per 100 g of body weight) with a 1 mL asepsis injector (Figure 1I). Make sure the needles penetrate the bone wax and inject the&#160;S. aureus&#160;solution into bone marrow slowly.</li>
	<li>Keep the animal in warm and clean conditions to avoid heat loss after modelling. Monitor respiratory rate and heart rate. After waking up, house the rabbits in individual cages with free access to food and water.</li>
</ol>

<table><tbody><tr><td>absorbable surgical suture&amp;nbsp;</td><td>&amp;nbsp;Jinghuan</td><td>18S0604A</td><td></td></tr><tr><td>asepsis injector&amp;nbsp;</td><td>&amp;nbsp;Jinglong</td><td>20170501</td><td></td></tr><tr><td>bone wax</td><td>&amp;nbsp;ETHICON</td><td>&amp;nbsp;JH5CQLM</td><td></td></tr><tr><td>Electric bone drill unit&amp;nbsp;&amp;nbsp;</td><td>Bao Kang</td><td>BKZ-1</td><td></td></tr><tr><td>Electric shaver</td><td>&amp;nbsp;Codos</td><td>3800</td><td></td></tr><tr><td>New Zealand white rabbits&amp;nbsp;</td><td>Zhejiang Experimental Animal Center</td><td>&amp;nbsp;SCXK 2014-0047</td><td></td></tr><tr><td>No. 11 scalpel&amp;nbsp;</td><td>Yuanlikang&amp;nbsp;</td><td>20170604</td><td></td></tr><tr><td>pentobarbital sodium&amp;nbsp;&amp;nbsp;</td><td>Merk</td><td>2070124</td><td></td></tr><tr><td>S. aureus freeze drying powder&amp;nbsp;&amp;nbsp;</td><td>China General Microbiological Culture Collection Center</td><td>ATCC 6538</td><td></td></tr></tbody></table>

generating rabbit model of bone infection: a technique of inducing chronic infection by injecting bacteria directly in bone marrow

Source: Zhang, Y et al., <a href="https://www.jove.com/v/57294">Treatment with Vancomycin Loaded Calcium Sulphate and Autogenous Bone in an Improved Rabbit Model of Bone Infection.</a> J. Vis. Exp. (2019).
This video describes the technique of surgically inducing bacterial infection in the bone marrow of a rabbit by using bone wax to avoid the leakage of bacteria. This rabbit model can be used to study bone infection treatment and bone regeneration.

<img alt="Figure 1" src="/files/ftp_upload/20934/20934_Figure1.jpg" /> 
Figure 1. Surgical preparation of the bone infection model.&#160;(A) Shows the punching position on the tibia. The distance between the drilling hole position for injection with&#160;S. aureus&#160;to the upper end of tibia is 1.5 cm. (B) Incision made in the skin to expose the periosteum. (C) Shows incision made through the periosteum to expose the tibia. (D) Punch a 2-mm diameter hole in the tibia. (E) Fill the 2 mm diameter hole full of bone wax. (F) Cut off spare bone wax. (G) Show the bone wax filling the bone defect. (H) Sew up the periosteum and skin. (I) Inject with&#160;S. aureus&#160;solution.

Generating Rabbit Model of Bone Infection: A Technique of Inducing Chronic Infection by Injecting Bacteria Directly in Bone Marrow

Source: Zhang, Y et al., Treatment with Vancomycin Loaded Calcium Sulphate and Autogenous Bone in an Improved Rabbit Model of Bone Infection. J. Vis. Exp. ...

To generate a bacterial bone infection model, begin by placing an anesthetized rabbit in a lateral position. Remove the fur from the hindlimb to locate tibia - the bone connecting the knee to the ankle. Mark a point adjacent to the tibial plateau - the proximal end of tibia bone.
Incise the skin and periosteum - a tissue layer around the bone - exposing the tibia. Using the marked reference, make a cavity in the bone marrow. Insert a bone wax plug into the cavity. Suture the periosteum and the skin layers.
Next, take a syringe containing Staphylococcus aureus solution. Pierce the needle through the bone wax plug and inoculate the bacterial solution into the bone marrow. Eject the needle. Transfer the rabbit to a cage and monitor the spread of infection.
The bone wax plug prevents the leakage of the bacterial suspension, allowing the bacteria to proliferate in the bone marrow. These bacteria attract the leucocytes and inflammatory factors leading to osteomyelitis - an inflammation in the bone causing reduced blood supply.
These conditions eventually lead to the sequestrum formation - a necrotic area in the bone devoid of blood supply. Bacteria further infiltrate this area to escape the antibiotic treatment, resulting in a chronic infection.

generating-rabbit-model-bone-infection-technique-inducing-chronic

Research

JoVE Encyclopedia of Experiments

Biology

Large Animal Models

Leporine

1.1K Views. Source: Zhang, Y et al., Treatment with Vancomycin Loaded Calcium Sulphate and Autogenous Bone in an Improved Rabbit Model of Bone Infection. J. Vis. Exp. (2019). This video describes the technique of surgically inducing bacterial infection in the bone marrow of a rabbit by using bone wax to avoid the leakage of bacteria. This rabbit model can be used to study bone infection treatment and bone regeneration. 

Video: Generating Rabbit Model of Bone Infection: A Technique of Inducing Chronic Infection by Injecting Bacteria Directly in Bone Marrow - Concept

Combined In vivo Optical and &#181;CT Imaging to Monitor Infection, Inflammation, and Bone Anatomy in an Orthopaedic Implant Infection in Mice

Multimodality imaging has emerged as a common technological approach used in both preclinical and clinical research. Advanced techniques that combine in vivo optical and &#956;CT imaging allow the visualization of biological phenomena in an anatomical context. These imaging modalities may be especially useful to study conditions that impact bone. In particular, orthopaedic implant infections are an important problem in clinical orthopaedic surgery. These infections are difficult to treat because bacterial biofilms form on the foreign surgically implanted materials, leading to persistent inflammation, osteomyelitis and eventual osteolysis of the bone surrounding the implant, which ultimately results in implant loosening and failure. Here, a mouse model of an infected orthopaedic prosthetic implant was used that involved the surgical placement of a Kirschner-wire implant into an intramedullary canal in the femur in such a way that the end of the implant extended into the knee joint. In this model, LysEGFP mice, a mouse strain that has EGFP-fluorescent neutrophils, were employed in conjunction with a bioluminescent Staphylococcus aureus strain, which naturally emits light. The bacteria were inoculated into the knee joints of the mice prior to closing the surgical site. In vivo bioluminescent and fluorescent imaging was used to quantify the bacterial burden and neutrophil inflammatory response, respectively. In addition, &#956;CT imaging was performed on the same mice so that the 3D location of the bioluminescent and fluorescent optical signals could be co-registered with the anatomical &#956;CT images. To quantify the changes in the bone over time, the outer bone volume of the distal femurs were measured at specific time points using a semi-automated contour based segmentation process. Taken together, the combination of in vivo bioluminescent/fluorescent imaging with &#956;CT imaging may be especially useful for the noninvasive monitoring of the infection, inflammatory response and anatomical changes in bone over time.

Combined In vivo Optical and  &#181;CT Imaging to Monitor Infection, Inflammation, and Bone Anatomy in an Orthopaedic Implant Infection in Mice

Combined optical and &#956;CT imaging in a mouse model of orthopaedic implant infection, utilizing a bioluminescent engineered strain of Staphylococcus aureus, provided the capability to noninvasively and longitudinally monitor the dynamics of the bacterial infection, as well as the corresponding inflammatory response and anatomical changes in the bone.

Multimodality imaging has emerged as a common technological approach used in both preclinical and clinical research. Advanced techniques that combine in ...

JoVE Journal

Medicine

Surgical Angiogenesis in Porcine Tibial Allotransplantation: A New Large Animal Bone Vascularized Composite Allotransplantation Model

Segmental bone loss resulting from trauma, infection malignancy and congenital anomaly remains a major reconstructive challenge. Current therapeutic options have significant risk of failure and substantial morbidity.
Use of bone vascularized composite allotransplantation (VCA) would offer both a close match of resected bone size and shape and the healing and remodeling potential of living bone. At present, life-long drug immunosuppression (IS) is required. Organ toxicity, opportunistic infection and neoplasm risks are of concern to treat such non-lethal indications.
We have previously demonstrated that bone and joint VCA viability may be maintained in rats and rabbits without the need of long-term-immunosuppression by implantation of recipient derived vessels within the VCA. It generates an autogenous, neoangiogenic circulation with measurable flow and active bone remodeling, requiring only 2 weeks of IS. As small animals differ from man substantially in anatomy, bone physiology and immunology, we have developed a porcine bone VCA model to evaluate this technique before clinical application is undertaken. Miniature swine are currently widely used for allotransplantation research, given their immunologic, anatomic, physiologic and size similarities to man. Here, we describe a new porcine orthotopic tibial bone VCA model to test the role of autogenous surgical angiogenesis to maintain VCA viability.
The model reconstructs segmental tibial bone defects using size- and shape-matched allogeneic tibial bone segments, transplanted across a major swine leukocyte antigen (SLA) mismatch in Yucatan miniature swine. Nutrient vessel repair and implantation of recipient derived autogenous vessels into the medullary canal of allogeneic tibial bone segments is performed in combination with simultaneous short-term IS. This permits a neoangiogenic autogenous circulation to develop from the implanted tissue, maintaining flow through the allogeneic nutrient vessels for a short time. Once established, the new autogenous circulation maintains bone viability following cessation of drug therapy and subsequent nutrient vessel thrombosis.

Currently any kind of vascularized composite allotransplantation depends on long-term-immunosuppression, difficult to support for non-life-critical indications. We present a new porcine tibial VCA model that can be used to study bone VCA and demonstrate the use of surgical angiogenesis to maintain bone viability without the need of long-term immune-modulation.

Segmental bone loss resulting from trauma, infection malignancy and congenital anomaly remains a major reconstructive challenge. Current therapeutic ...

A Mouse Model to Assess Innate Immune Response to Staphylococcus aureus Infection

Staphylococcus aureus (S. aureus)&#160;infections, including methicillin resistant stains, are an enormous burden on the healthcare system. With incidence rates of S. aureus infection climbing annually, there is a demand for additional research in its&#160;pathogenicity. Animal models of infectious disease advance our understanding of the host-pathogen response and lead to the development of effective therapeutics. Neutrophils play a primary role in the innate immune response that controls S. aureus infections by forming an abscess to wall off the infection and facilitate bacterial clearance; the number of neutrophils that infiltrate an S. aureus skin infection often correlates with disease outcome. LysM-EGFP mice, which possess the enhanced green fluorescent protein (EGFP) inserted in the Lysozyme M (LysM) promoter region (expressed primarily by neutrophils), when used in conjunction with in vivo whole animal fluorescence imaging (FLI) provide&#160;a means of quantifying neutrophil emigration noninvasively and longitudinally into wounded skin. When combined with a bioluminescent S. aureus strain and sequential in vivo whole animal bioluminescent imaging (BLI), it is possible to longitudinally monitor both the neutrophil recruitment dynamics and in vivo bacterial burden at the site of infection in anesthetized mice from onset of infection to resolution or death. Mice are more resistant to a number of virulence factors produced by S. aureus that facilitate effective colonization and infection in humans. Immunodeficient mice provide a more sensitive animal model to examine persistent S. aureus infections and the ability of therapeutics to boost innate immune responses. Herein, we characterize responses in LysM-EGFP mice that have been bred to MyD88-deficient mice (LysM-EGFP&#215;MyD88-/- mice) along with wild-type LysM-EGFP mice to investigate S. aureus skin wound infection. Multispectral simultaneous detection enabled study of neutrophil recruitment dynamics by using in vivo FLI, bacterial burden by using in vivo BLI, and wound healing longitudinally and noninvasively over time.

A Mouse Model to Assess Innate Immune Response to Staphylococcus aureus Infection

An approach is described for real-time detection of the innate immune response to cutaneous wounding and Staphylococcus aureus infection of mice. By comparing LysM-EGFP mice (which possess fluorescent neutrophils) with a LysM-EGFP crossbred immunodeficient mouse strain, we advance our understanding of infection and the development of approaches to combat infection.

Staphylococcus aureus (S. aureus)&#160;infections, including methicillin resistant stains, are an enormous burden on the healthcare system. With incidence ...

Generating Rabbit Model of Bone Infection: A Technique of Inducing Chronic Infection by Injecting Bacteria Directly in Bone Marrow

Transcript

Generating Rabbit Model of Bone Infection: A Technique of Inducing Chronic Infection by Injecting Bacteria Directly in Bone Marrow

Combined In vivo Optical and µCT Imaging to Monitor Infection, Inflammation, and Bone Anatomy in an Orthopaedic Implant Infection in Mice

Surgical Angiogenesis in Porcine Tibial Allotransplantation: A New Large Animal Bone Vascularized Composite Allotransplantation Model

A Mouse Model to Assess Innate Immune Response to Staphylococcus aureus Infection