This work was supported by the NIH/NIDCR DE031431. We would like to thank the Translational Pathology Core Laboratory at UCLA for assistance with preparing the decalcified histological sections.

Procedures involving animal subjects have been approved by the Chancellor&#39;s Animal Research Committee of the University of California, Los Angeles (ARC protocol number 2002-125), and the Animal Research: Reporting In Vivo Experiments (ARRIVE)22. For this method, eighteen 3-week-old C57BL/6J male mice were used and underwent dental extractions, implant placement and peri-implantitis induction. All dental procedures were performed under 10&#215; microscopic magnification and carried out by trained and calibrated operators (Figure 1A).
1. Pre-extraction steps
<ol>
	<li>Perform the procedure in a surgical room complying with all biosafety and protection standards.</li>
	<li>Perform asepsis of all surfaces with triple friction using a 70% isopropyl alcohol solution.</li>
	<li>Anesthetize 3-week-old C57BL/6J male mice with 3% isoflurane. Check for the lack of response to paw withdrawing upon touch to ensure sufficient anesthetic depth is obtained.</li>
	<li>To avoid interfering with procedures in the mouth, use a nosecone to maintain isoflurane anesthesia.</li>
	<li>Have an auxiliary operator to stabilize the animal and maintain the mouth opening (Figure 1B).</li>
	<li>Apply ophthalmic lubricant, to prevent irritation in the eyes before starting the extraction.</li>
</ol>
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/66316/66316fig01.jpg" /> 
Figure 1: Operative adaptations: (A) Microscopic magnification. (B) Adapted inhalation anesthesia system and stabilization for mouth opening. <a href="https://www.jove.com/files/ftp_upload/66316/66316fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
2. Dental extraction
<ol>
	<li>For dental extractions, introduce a #5 dental explorer between the first and the second molar to start the elevation and luxation procedures by moving the instrument in the mesio-distal direction until the tooth moves in the socket (Figures 2A).</li>
	<li>Next, introduce the #5 dental explorer in the mesial site of the first molar. Do this by moving the instrument in the mesio-distal direction until the tooth moves in the socket.</li>
	<li>Following elevation, use the tip forceps and the suture tying forceps to remove the 1st molar.</li>
	<li>Next, introduce the dental explorer between the 2nd and 3rd molar to elevate and luxate the 2nd molar (Figure 2B).</li>
	<li>Then, use the tip forceps and/or the suture tying forceps to remove the tooth using the instruments to hold it and take out of the mouth. (Figure 2C).</li>
	<li>After dental extractions, ensure complete hemostasis achievement by using a sterile cotton tipped (Figure 2D) for 1 min (Figure 2E,F).</li>
	<li>Immediately after extraction, provide all animals with pain medication (Carprofen/Rimadyl 5 mg/kg) every 24 h. Administer the medication through subcutaneous injection.</li>
	<li>In addition, replace regular food with a soft diet. Administer the antibiotic (Amoxil 0.25 mg/mL) orally by incorporating the medication into the drinking water. Do this for four weeks after extractions.</li>
</ol>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/66316/66316fig02.jpg" /> 
Figure 2: Initial extraction sequence: (A, B) Maxillary region with 1st and 2nd molar teeth and use of dental explorer for elevation and luxation. (C) Use of the tip forceps and explorer for luxation and tooth removal. (D) Hemostasis. (E, F) Alveolar appearance after extractions. <a href="https://www.jove.com/files/ftp_upload/66316/66316fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
3. Implant placement
<ol>
	<li>Using a 15c blade, create a mesio-distal incision through the keratinized tissue in the area corresponding to the previously present teeth. Use the right maxillary molars as a spatial reference (Figure 3A).</li>
	<li>Raise the buccal and palatal full-thickness flaps by using a #5 dental explorer, ensuring complete flap elevation (Figure 3B).</li>
	<li>Perform the osteotomy using a 0.3 mm diameter carbide micro hand drill attached to a Pin Vise (hand drill) and activate it in a clockwise rotation. Create osteotomy sites with approximately 1 mm deep into the healed extraction sockets (Figure 3C).</li>
	<li>Place the custom-designed screw-shaped implants with a smooth machined surface (1.0 mm long and 0.5 mm in diameter) fabricated from 6AL4V titanium rods (Figure 3D), one per animal, by self-tapping in the region of the first and second maxillary left molars using a clockwise screwing motion (Figure 3E-I).</li>
	<li>Immediately after implant placement, administer pain medication (Carprofen/Rimadyl 5mg/kg every 24 h) through subcutaneous injection.</li>
	<li>Allow the implants to heal for four weeks, during which time administer antibiotics and feed as previously described.</li>
	<li>At the end of the healing period, ensure that the mucosal wound is completely closed and has a light pink appearance.</li>
</ol>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/66316/66316fig03.jpg" /> 
Figure 3: Implant placement sequence: (A) Incision using a 15c blade attached to the handle. (B) Full-thickness flaps using #5 dental explorer. (C) Osteotomy using a 0.3 mm carbide micro hand drill attached to a Pin Vise. (D) Titanium dental implant. (E, F) Implant support and implant holder. (G- I) Implant placement using a clockwise screwing motion. <a href="https://www.jove.com/files/ftp_upload/66316/66316fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
4. Peri-implantitis induction
<ol>
	<li>Place a silk ligature (6-0) around each fixture immediately apical to the implant head by contouring the surface of the implant and fixing it with a double knot. Keep the ligature for two weeks allowing the peri-implantitis development.</li>
	<li>Check the ligatures every two days to make sure they are still present. If missing, place a new ligature.</li>
</ol>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/66316/66316fig04.jpg" /> 
Figure 4: Ligature-induced peri-implantitis sequence. (A-D) Silk ligature (6.0) placed around the implant head. (E-G) Knot closure. (H) Ligature cut. (I) Final appearance. Clinical images obtained from live animals under sedation. <a href="https://www.jove.com/files/ftp_upload/66316/66316fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
5. Sacrifice
<ol>
	<li>After the peri-implantitis induction period (two weeks), sacrifice all animals with a high concentration of inhaled isoflurane, followed by a secondary method of cervical dislocation with a rod pressed into the base of the skull and quickly pulling the hind limbs, causing the separation of the cervical vertebrae from the skull.</li>
	<li>Separate the maxillae using sharp instruments, such as surgical scissors, that will separate the animal&#39;s entire jaw from the rest of the body.</li>
	<li>Photograph the maxillae using an optical microscope, fix it in 10% formalin for 24 h, then store in 70% ethanol.</li>
</ol>
6. Micro-computed tomography (&#181;CT )
<ol>
	<li>Scan the maxillae as previously described18,19 using micro-computed tomography (micro-CT) scanning.</li>
	<li>Turn on the scanner and the computer, then open the scan software, click on the radiation sign, and wait 15 minutes to preheat it.</li>
	<li>Open the equipment door by clicking on the door icon.</li>
	<li>Use a quarter-size rotary table. Place the maxilla on a 15ml conical tube. Fix the tube in the rotary table base. Place the rotary table in the equipment screw.</li>
	<li>Close the equipment door by clicking on the door icon.</li>
	<li>Turn on the X-Ray source.</li>
	<li>Click on the grab image icon and make sure the bone is on the field.</li>
	<li>Adjust a resolution of 10 &#181;m pixel size, X-ray energy of 55 KVp and 181 &#181;A, filter of AI 0.5 mm, rotation step 0.4, average frame 10. These settings will generate an estimated scanning time of 24 minutes.</li>
	<li>Start the acquisition.</li>
	<li>For reconstruction open the reconstruction software. Use beam hardening of 20%. Use ring artifact of 5%. Use a dynamic range of 0-0.13. Deselect label ON. Click on Preview. Choose the adequate view that is seen for the region of interest, and save it as TIFF.</li>
	<li>Orient the images obtained using software for viewing and analyzing data. Make the long axis of the implant head parallel to the sagittal and coronal axes and perpendicular to the axial axis.</li>
	<li>Save the sagittal image as a single image for linear bone analysis.</li>
	<li>Save transaxial image as a dataset for volumetric bone analysis.</li>
	<li>Perform linear bone analysis using the analyzing data software. Measure the distances in millimeters between the implant head and the alveolar bone in the sagittal and coronal planes, including mesial, distal, buccal, and palatal sites.</li>
	<li>Perform volumetric bone analysis using analyzing data software. Track bone loss around implants by drawing the region of interest in all slides corresponding to the bone loss.</li>
</ol>
7. Statistical analysis
<ol>
	<li>Measure linear and volumetric microtomographic bone loss and normalize these values by dividing each value by the average of the control group. Present an average for all groups, as mean &#177; standard error of the mean (SEM).</li>
	<li>Compare significance using a two-way analysis of variance (ANOVA) followed by a Tukey&#39;s test with a 95% confidence interval. (Prism 5). Apply significance levels as follows: p&#8804;0.01**, p&#8804;0.001****.</li>
</ol>

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The authors have nothing to disclose.

This protocol presents a descriptive report on surgical procedures for peri-implantitis induction utilizing a ligature model in mice. Working with mice has advantages, such as being cost-effective, the availability of an extensive genetic array given the many backgrounds23 among other aspects24,25. Over the years, several studies have successfully utilized mice in the medical and dental fields, including in peri-implantitis<sup class="xref...

Dental implants are increasingly prevalent as a desirable choice for replacing missing teeth1. The prevalence of dental implants in the US adult population is projected to increase up to 23% by 20262. Based on a market analysis report by Grand View Research (2022), the global market size of dental implants was projected to reach approximately US $4.6 billion in 2022. Furthermore, it is anticipated to exhibit a steady annual growth rate of around 10% until the year 20303. Unfortunately, the use of dental implants can lead to complications, such as peri-implantitis. Peri-implantitis has been defined as a biofilm-induced condition characterized by inflammation in the peri-implant mucosa and subsequent progressive loss of supporting bone4.
A systematic review found that the mean prevalence of peri-implantitis was 19.53% (95% Confidence interval [CI], 12.87 to 26.19%) at the patient level and 12.53% (95% CI 11.67 to 13.39%) at the implant-level5. Peri-implantitis represents a growing public health, due to an increase in implant failure and, consequently, substantial treatment costs6.
Understanding the pathogenesis of peri-implantitis is crucial to developing a systematic approach to prevent its onset and progression and maximize dental implants&#39; longevity in terms of aesthetics and function7,8. In this sense, using murine models in dental research has proven advantageous, given that mice share more than 95% of their genes with humans9,10, the number of available online genetic databases, and the ability to reproduce clinical scenarios11. All the described advantages allow the dissection of genetic mechanisms in different diseases12, accessible accommodation and management, and antibodies widely available as human panels, beyond the genetic modification availability (e.g., knockout and overexpression) for inflammatory tissue assessment and disease mapping13. Although advantageous, there are few publications addressing peri-implantitis in mice. This is due to methodological challenges, among others, including the difficulty in obtaining mini-implants or installing them.
To develop peri-implantitis in mice, many protocols have been described, such as ligature-induced peri-implantitis, bacteria-induced peri-implantitis14, Lipopolysaccharide (LPS)-induced peri-implantitis15, or the combination LPS + ligature-induced peri-implantitis16. Here, we will focus on the ligature model because it is the most widely accepted method to induce periodontitis17,18,19 and, more recently, peri-implantitis20,21. The ligature placed around the implants in a submucosal position stimulates plaque accumulation and, consequently, tissue inflamation. So, the development of this approach is based on the indication of a viable cost-benefit technique for pre-clinical investigations on peri-implant diseases. This study aims to describe an experimental model of ligature-induced peri-implantitis in mice and determine whether there is effectiveness in inducing this disease given the observed bone and tissue changes.
The overall goal of this article is to report the protocol applied to induce peri-implantitis in mice by ligature and to observe its effectiveness through tissue evaluation and bone loss around the implants.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>#5 dental explorer</td>
			<td>Hu-Friedy, Chicago, IL</td>
			<td>392-0911&#160;</td>
			<td>Dental luxation</td>
		</tr>
		<tr>
			<td>15c blade and surgical scalpel</td>
			<td>Henry Schein Inc., Melville, NY</td>
			<td>1126186</td>
			<td>Tissue incision</td>
		</tr>
		<tr>
			<td>6-0 silk ligatures</td>
			<td>Fisher Scientific, Hampton, NH</td>
			<td>NC9201232</td>
			<td>Ligature</td>
		</tr>
		<tr>
			<td>Amoxicillin 50&#956;g/mL</td>
			<td>Zoetis, San Diego, CA</td>
			<td>TS/DRUGS/57/2003</td>
			<td>Oral suspension</td>
		</tr>
		<tr>
			<td>Bacon Soft Diet</td>
			<td>Bio Serve&#174;, Frenchtown, NJ</td>
			<td>14-726-701</td>
			<td>-</td>
		</tr>
		<tr>
			<td>C57BL/6J male mice</td>
			<td>The Jackson Laboratories, Bar Harbor, ME, USA</td>
			<td>000664</td>
			<td>Age: 3-week-old</td>
		</tr>
		<tr>
			<td>CTAn software</td>
			<td>V.1.16 Bruker, Billerica, MA</td>
			<td>-</td>
			<td>Volumetric analysis</td>
		</tr>
		<tr>
			<td>Dolphin software</td>
			<td>Navantis, Toronto, CA</td>
			<td>-</td>
			<td>Linear bone analysis</td>
		</tr>
		<tr>
			<td>Implant carrier &#38; Tip</td>
			<td>D. P. Machining Inc., La Verne, CA</td>
			<td>Unique product&#160;</td>
			<td>Implant holder</td>
		</tr>
		<tr>
			<td>Implant support</td>
			<td>D. P. Machining Inc., La Verne, CA</td>
			<td>Unique product&#160;</td>
			<td>Implant capture</td>
		</tr>
		<tr>
			<td>Isoflurane&#160;</td>
			<td>Vet One, Boise, ID</td>
			<td>NDC13985-528-60</td>
			<td>Inhalational anesthetic</td>
		</tr>
		<tr>
			<td>Micro-CT scan 1172</td>
			<td>SkyScan, Kontich, Belgium</td>
			<td>-</td>
			<td>&#956;CT scans</td>
		</tr>
		<tr>
			<td>Nrecon Software</td>
			<td>Bruker Corporation, Billerica, MA</td>
			<td>-</td>
			<td>Images reconstruction</td>
		</tr>
		<tr>
			<td>&#216; 0.3mm - L 2.5mm Micro Drills&#160;</td>
			<td>Sphinx, Hoffman Estates, IL</td>
			<td>ART. 50699&#160;</td>
			<td>Osteotomy</td>
		</tr>
		<tr>
			<td>&#216; 0.5mm - L 1.0mm Titanium implants</td>
			<td>D. P. Machining Inc., La Verne, CA</td>
			<td>Unique product</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Ophthalmic lubricant</td>
			<td>Apexa, Ontario, CA</td>
			<td>NDC13985-600-03</td>
			<td>Artificial tears</td>
		</tr>
		<tr>
			<td>Pin Vise</td>
			<td>General Tools, Secaucus, NJ</td>
			<td>90</td>
			<td>Osteotomy</td>
		</tr>
		<tr>
			<td>Rimadyl 50mg/ml</td>
			<td>Zoetis, San Diego, CA</td>
			<td>4019449</td>
			<td>Anti-inflammatory</td>
		</tr>
		<tr>
			<td>Sterile cotton tipped</td>
			<td>Dynarex, Glendale, AZ</td>
			<td>4304-1</td>
			<td>Hemostasis</td>
		</tr>
		<tr>
			<td>Tip forceps</td>
			<td>Fine Science Tools, Foster City, CA</td>
			<td>11071-10</td>
			<td>Dental Extraction</td>
		</tr>
		<tr>
			<td>Tying forceps</td>
			<td>Fine Science Tools, Foster City, CA</td>
			<td>18025-10</td>
			<td>Ligature placement</td>
		</tr>
	</tbody>
</table>

experimental model of ligature-induced peri-implantitis in mice

Dental implants have a high success and survival rate. However, complications such as peri-implantitis (PI) are highly challenging to treat. PI is characterized by inflammation in the tissues around dental implants with progressive loss of supporting bone. To optimize dental implants' longevity in terms of health and functionality, it is crucial to understand the peri-implantitis pathophysiology. In this regard, using mouse models in research has proven clear benefits in recreating clinical circumstances. This study aimed to describe an experimental model of ligature-induced peri-implantitis in mice and determine whether there is effectiveness in inducing this disease, given the observed bone and tissue changes. The experimental peri-implantitis induction comprehends the following steps: teeth extraction, implant placement, and ligature-inducted PI. A sample of eighteen 3-week-old C57BL/6J male mice was divided into two groups, ligature (N=9) and control non-ligature (N=9). The evaluation of clinical, radiographical, and histological factors was performed. The ligature group showed significantly higher bone loss, increased soft tissue edema, and apical epithelial migration than the non-ligature group. It was concluded that this pre-clinical model can successfully induce peri-implantitis in mice.

For this method, eighteen 3-week-old C57BL/6J male mice were used and underwent dental extractions, implant placement and peri-implantitis induction. There were nine animals per group which was statistically significant, considering linear bone loss achieving 80% power, 15% standard deviation (&#963;) and 95% confidence interval (&#945; =0.05). Mice were fed a soft diet ad libitum during the experiment. Nine mice received a ligature (ligature-induced periimplantitis-experimental group), and nine mice did not rec...

Watch this Scientific Journal Video about Experimental Model of Ligature-Induced Peri-Implantitis in Mice at JoVE.com

Experimental Model of Ligature-Induced Peri-Implantitis in Mice

This is a report on an experimental model of ligature-induced peri-implantitis in mice. We describe all surgical steps, from pre- and post-operative management of the animals, extractions, implant placement, and ligature-induced peri-implantitis.

Dental implants have a high success and survival rate. However, complications such as peri-implantitis (PI) are highly challenging to treat. PI is ...

experimental-model-of-ligature-induced-peri-implantitis-in-mice

Research

JoVE Journal

Immunology and Infection

1.9K 조회수.  University of California. 이 기사의 전반적인 목표는 인찰에 의해 마우스의 임플란트 주위염을 유도하는 데 적용된 프로토콜을 보고하고 임플란트 주변의 조직 평가 및 뼈 손실을 통해 그 효과를 관찰하는 것입니다. 다음 절차는 모든 생물 안전 및 보호 표준을 준수하는 수술실에서 수행되었습니다. 모든 절차는 10X 현미경 배율로 수행되었으며 교육을 받고 보정된 작업자가 수행했습니다.모든 ?...

비디오: Experimental Model of Ligature-Induced Peri-Implantitis in Mice

Experimental Metastasis and CTL Adoptive Transfer Immunotherapy Mouse Model

Experimental metastasis mouse model is a simple and yet physiologically relevant metastasis model. The tumor cells are injected intravenously (i.v) into mouse tail veins and colonize in the lungs, thereby, resembling the last steps of tumor cell spontaneous metastasis: survival in the circulation, extravasation and colonization in the distal organs. From a therapeutic point of view, the experimental metastasis model is the simplest and ideal model since the target of therapies is often the end point of metastasis: established metastatic tumor in the distal organ. In this model, tumor cells are injected i.v into mouse tail veins and allowed to colonize and grow in the lungs. Tumor-specific CTLs are then injected i.v into the metastases-bearing mouse. The number and size of the lung metastases can be controlled by the number of tumor cells to be injected and the time of tumor growth. Therefore, various stages of metastasis, from minimal metastasis to extensive metastasis, can be modeled. Lung metastases are analyzed by inflation with ink, thus allowing easier visual observation and quantification.

An experimental lung metastasis and CTL immunotherapy mouse model for analysis of tumor cells-T cell interaction in vivo.

Experimental metastasis mouse model is a simple and yet physiologically relevant metastasis model. The tumor cells are injected intravenously (i.v) into ...

Induction of Experimental Autoimmune Hypophysitis in SJL Mice

Autoimmune hypophysitis can be reproduced experimentally by the injection of pituitary proteins mixed with an adjuvant into susceptible mice1. Mouse models allow us to study how diseases unfold, often providing a good replica of the same processes occurring in humans. For some autoimmune diseases, like type 1A diabetes, there are models (the NOD mouse) that spontaneously develop a disease similar to the human counterpart. For many other autoimmune diseases, however, the model needs to be induced experimentally. A common approach in this regard is to inject the mouse with a dominant antigen derived from the organ being studied. For example, investigators interested in autoimmune thyroiditis inject mice with thyroglobulin2, and those interested in myasthenia gravis inject them with the acetylcholine receptor3. If the autoantigen for a particular autoimmune disease is not known, investigators inject a crude protein extract from the organ targeted by the autoimmune reaction. For autoimmune hypophysitis, the pathogenic autoantigen(s) remain to be identified4, and thus a crude pituitary protein preparation is used. In this video article we demonstrate how to induce experimental autoimmune hypophysitis in SJL mice.

This video shows how to induce autoimmune hypophysitis in SJL mice and how to assess its severity by histopathology.

Autoimmune hypophysitis can be reproduced experimentally by the injection of pituitary proteins mixed with an adjuvant into susceptible mice1. Mouse ...

Protocols for Vaginal Inoculation and Sample Collection in the Experimental Mouse Model of Candida vaginitis

Vulvovaginal candidiasis (VVC), caused by Candida species, is a fungal infection of the lower female genital tract that affects approximately 75% of otherwise healthy women during their reproductive years18,32-34. Predisposing factors include antibiotic usage, uncontrolled diabetes and disturbance in reproductive hormone levels due to pregnancy, oral contraceptives or hormone replacement therapies33,34. Recurrent VVC (RVVC), defined as three or more episodes per year, affects a separate 5 to 8% of women with no predisposing factors33.
An experimental mouse model of VVC has been established and used to study the pathogenesis and mucosal host response to Candida3,4,11,16,17,19,21,25,37. This model has also been employed to test potential antifungal therapies in vivo13,24. The model requires that the animals be maintained in a state of pseudoestrus for optimal Candida colonization/infection6,14,23. Under such conditions, inoculated animals will have detectable vaginal fungal burden for weeks to months. Past studies show an extremely high parallel between the animal model and human infection relative to immunological and physiological properties3,16,21. Differences, however, include a lack of Candida as normal vaginal flora and a neutral vaginal pH in the mice.
Here, we demonstrate a series of key methods in the mouse vaginitis model that include vaginal inoculation, rapid collection of vaginal specimens, assessment of vaginal fungal burden, and tissue preparations for cellular extraction/isolation. This is followed by representative results for constituents of vaginal lavage fluid, fungal burden, and draining lymph node leukocyte yields. With the use of anesthetics, lavage samples can be collected at multiple time points on the same mice for longitudinal evaluation of infection/colonization. Furthermore, this model requires no immunosuppressive agents to initiate infection, allowing immunological studies under defined host conditions. Finally, the model and each technique introduced here could potentially give rise to use of the methodologies to examine other infectious diseases of the lower female genital tract (bacterial, parasitic, viral) and respective local or systemic host defenses.

Protocols for Vaginal Inoculation and Sample Collection in the Experimental Mouse Model of Candida vaginitis

Key techniques to be used in the evaluation of Candida vaginitis in an experimental animal model are described. The methods will allow rapid collection of vaginal specimens and lymphocytes from draining lumbar lymph nodes. These techniques could give rise to mouse models of other diseases in the female lower genital tract.

Vulvovaginal candidiasis (VVC), caused by Candida species, is a fungal infection of the lower female genital tract that affects approximately 75% of ...

Experimental Endocarditis Model of Methicillin Resistant Staphylococcus aureus (MRSA) in Rat

Endovascular infections, including endocarditis, are life-threatening infectious syndromes1-3. Staphylococcus aureus is the most common world-wide cause of such syndromes with unacceptably high morbidity and mortality even with appropriate antimicrobial agent treatments4-6. The increase in infections due to methicillin-resistant S. aureus (MRSA), the high rates of vancomycin clinical treatment failures and growing problems of linezolid and daptomycin resistance have all further complicated the management of patients with such infections, and led to high healthcare costs7, 8. In addition, it should be emphasized that most recent studies with antibiotic treatment outcomes have been based in clinical settings, and thus might well be influenced by host factors varying from patient-to-patient. Therefore, a relevant animal model of endovascular infection in which host factors are similar from animal-to-animal is more crucial to investigate microbial pathogenesis, as well as the efficacy of novel antimicrobial agents. Endocarditis in rat is a well-established experimental animal model that closely approximates human native valve endocarditis. This model has been used to examine the role of particular staphylococcal virulence factors and the efficacy of antibiotic treatment regimens for staphylococcal endocarditis. In this report, we describe the experimental endocarditis model due to MRSA that could be used to investigate bacterial pathogenesis and response to antibiotic treatment.

Experimental Endocarditis Model of Methicillin Resistant Staphylococcus aureus MRSA in Rat

Experimental rat endocarditis model due to methicillin-resistant S. aureus.

Endovascular infections, including endocarditis, are life-threatening infectious syndromes1-3. Staphylococcus aureus is the most common world-wide cause ...

Myelin Oligodendrocyte Glycoprotein (MOG35-55) Induced Experimental Autoimmune Encephalomyelitis (EAE) in C57BL/6 Mice

Multiple sclerosis is a chronic neuroinflammatory demyelinating disorder of the central nervous system with a strong neurodegenerative component. While the exact etiology of the disease is yet unclear, autoreactive T lymphocytes are thought to play a central role in its pathophysiology. MS therapy is only partially effective so far and research efforts continue to expand our knowledge on the pathophysiology of the disease and to develop novel treatment strategies. Experimental autoimmune encephalomyelitis (EAE) is the most common animal model for MS sharing many clinical and pathophysiological features. There is a broad diversity of EAE models which reflect different clinical, immunological and histological aspects of human MS. Actively-induced EAE in mice is the easiest inducible model with robust and replicable results. It is especially suited for investigating the effects of drugs or of particular genes by using transgenic mice challenged by autoimmune neuroinflammation. Therefore, mice are immunized with CNS homogenates or peptides of myelin proteins. Due to the low immunogenic potential of these peptides, strong adjuvants are used. EAE susceptibility and phenotype depends on the chosen antigen and rodent strain. C57BL/6 mice are the commonly used strain for transgenic mouse construction and respond among others to myelin oligodendrocyte glycoprotein (MOG). The immunogenic epitope MOG35-55 is suspended in complete Freund's adjuvant (CFA) prior to immunization and pertussis toxin is applied on the day of immunization and two days later. Mice develop a "classic" self-limited monophasic EAE with ascending flaccid paralysis within 9-14 days after immunization. Mice are evaluated daily using a clinical scoring system for 25-50 days. Special considerations for care taking of animals with EAE as well as potential applications and limitations of this model are discussed.

Myelin Oligodendrocyte Glycoprotein MOG35-55 Induced Experimental Autoimmune Encephalomyelitis EAE in C57BL/6 Mice

Experimental autoimmune encephalomyelitis (EAE) is an established animal model of multiple sclerosis. C57BL/6 mice are immunized with myelin oligodendrocyte glycoprotein (MOG) peptide 35-55 (MOG35-55), resulting in an ascending flaccid paralysis caused by autoreactive immune cells in the central nervous system. Protocols for disease induction and monitoring will be discussed.

Multiple sclerosis is a chronic neuroinflammatory demyelinating disorder of the central nervous system with a strong neurodegenerative component. While ...

Induction of Experimental Autoimmune Encephalomyelitis in Mice and Evaluation of the Disease-dependent Distribution of Immune Cells in Various Tissues

Multiple sclerosis is presumed to be an inflammatory autoimmune disease, which is characterized by lesion formation in the central nervous system (CNS) resulting in cognitive and motor impairment. Experimental autoimmune encephalomyelitis (EAE) is a useful animal model of MS, because it is also characterized by lesion formation in the CNS, motor impairment and is also driven by autoimmune and inflammatory reactions. One of the EAE models is induced with a peptide derived from the myelin oligodendrocyte protein (MOG)35-55 in mice. The EAE mice develop a progressive disease course. This course is divided into three phases: the preclinical phase (day 0 - 9), the disease onset (day 10 - 11) and the acute phase (day 12 - 14). MS and EAE are induced by autoreactive T cells that infiltrate the CNS. These T cells secrete chemokines and cytokines which lead to the recruitment of further immune cells. Therefore, the immune cell distribution in the spinal cord during the three disease phases was investigated. To highlight the time point of the disease at which the activation/proliferation/accumulation of T cells, B cells and monocytes starts, the immune cell distribution in lymph nodes, spleen and blood was also assessed. Furthermore, the levels of several cytokines (IL-1&#946;, IL-6, IL-23, TNF&#945;, IFN&#947;) in the three disease phases were determined, to gain insight into the inflammatory processes of the disease. In conclusion, the data provide an overview of the functional profile of immune cells during EAE pathology.

This manuscript describes the methods for induction and scoring of the experimental autoimmune encephalomyelitis (EAE) model, together with the assessment of immune cell distribution and mRNA cytokine levels in lymph nodes, spleen, blood and spinal cord using flow cytometry and quantitative PCR, respectively, at various disease phases.

Multiple sclerosis is presumed to be an inflammatory autoimmune disease, which is characterized by lesion formation in the central nervous system (CNS) ...

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 ...

Probiotic Studies in Neonatal Mice Using Gavage

Adult mouse models have been widely used to understand the mechanism behind disease progression in humans. The applicability of studies done in adult mouse models to neonatal diseases is limited. To better understand disease progression, host responses and long-term impact of interventions in neonates, a neonatal mouse model likely is a better fit. The sparse use of neonatal mouse models can in part be attributed to the technical difficulties of working with these small animals. A neonatal mouse model was developed to determine the effects of probiotic administration in early life and to specifically assess the ability to establish colonization in the newborn mouse intestinal tract. Specifically, to assess probiotic colonization in the neonatal mouse, Lactobacillus plantarum (LP) was delivered directly into the neonatal mouse gastrointestinal tract. To this end, LP was administered to mice by feeding through intra-esophageal (IE) gavage. A highly reproducible method was developed to standardize the process of IE gavage that allows an accurate administration of probiotic dosages while minimizing trauma, an aspect particularly important given the fragility of newborn mice. Limitations of this process include possibilities of esophageal irritation or damage and aspiration if gavaged incorrectly. This approach represents an improvement on current practices because IE gavage into the distal esophagus reduces the chances of aspiration. Following gavage, the colonization profile of the probiotic was traced using quantitative polymerase chain reaction (qPCR) of the extracted intestinal DNA with LP specific primers. Different litter settings and cage management techniques were used to assess the potential for colonization-spread. The protocol details the intricacies of IE neonatal mouse gavage and subsequent colonization quantification with LP.

This study details the process of gavaging precise amounts of probiotics to neonatal mice. The experimental set-up was optimized to include but is not limited to probiotic dosing, methods of administration, and quantification of bacteria in the intestines.

Adult mouse models have been widely used to understand the mechanism behind disease progression in humans. The applicability of studies done in adult ...

Robust Ligature-Induced Model of Murine Periodontitis for the Evaluation of Oral Neutrophils

The main advantages of studying the pathophysiology of periodontal disease utilizing murine models are the reduced cost of animals, array of genetically modified strains, the vast number of analyses that can be performed on harvested soft and hard tissues. However, many of these systems are subject to procedural criticisms. As an alternative, the ligature-induced model of periodontal disease, driven by the localized development and retention of a dysbiotic oral microbiome, can be employed, which is rapidly induced and relatively reliable. Unfortunately, the variants of ligature-induced murine periodontitis protocol are isolated to focal regions of the periodontium and subject to premature avulsion of the installed ligature. This minimizes the amount of tissue available for subsequent analyses and increases the number of animals required for study. This protocol describes the precise manipulations required to place extended molar ligatures with improved retention and usage of a novel rinse technique to recover oral neutrophils in mice with an alternative approach that mitigates the aforementioned technical challenges.

This article presents a protocol for establishing a ligature-induced model of murine periodontitis involving multiple maxillary molars, resulting in larger areas of the involved gingival tissue and bone for subsequent analysis as well as reduced animal usage. A technique to assess oral neutrophils in a manner analogous to human subjects is also described.

The main advantages of studying the pathophysiology of periodontal disease utilizing murine models are the reduced cost of animals, array of genetically ...

Experimental Autoimmune Uveitis: An Intraocular Inflammatory Mouse Model

Experimental Autoimmune Uveitis (EAU) is driven by immune cells responding to self-antigens. Many features of this non-infectious, intraocular inflammatory disease model recapitulate the clinical phenotype of posterior uveitis affecting humans. EAU has been used reliably to study the efficacy of novel inflammatory therapeutics, their mode of action and to further investigate the mechanisms that underpin disease progression of intraocular disorders. Here, we provide a detailed protocol on EAU induction in the C57BL/6J mouse - the most widely used model organism with susceptibility to this disease. Clinical assessment of disease severity and progression will be demonstrated using fundoscopy, histological examination and fluorescein angiography. The induction procedure involves subcutaneous&#160;injection of an emulsion containing a peptide (IRBP1-20) from the ocular protein interphotoreceptor retinoid binding protein (also known as retinol binding protein 3), Complete Freund&#39;s Adjuvant (CFA) and supplemented with killed Mycobacterium tuberculosis. Injection of this viscous emulsion on the back of the neck is followed by a single intraperitoneal injection of Bordetella pertussis toxin. At the onset of symptoms (day 12-14) and under general anesthesia, fundoscopic images are taken to assess disease progression through clinical examination. These data can be directly compared with those at later timepoints and peak disease (day 20-22) with differences analyzed. At the same time, this protocol allows the investigator to assess potential differences in vessel permeability and damage using fluorescein angiography. EAU can be induced in other mouse strains - both wildtype or genetically modified - and combined with novel therapies offering flexibility for studying drug efficacy and/or disease mechanisms.

In this report we present a protocol that allows the investigator to generate a mouse model of intraocular uveitis. More commonly referred to as experimental autoimmune uveitis (EAU), this established model captures many aspects of human disease. Herein, we will describe how to induce and monitor disease progression using several readouts.