This work was supported by Fundaci&#243; Universitat-Empresa de les Illes Balears (Proof of concept call 2020), by the Instituto de Salud Carlos III, Ministerio de Econom&#237;a y Competividad, co-funded by the ESF European Social Fund and the ERDF European Regional Development Fund (contract to M.M.B; FI18/00104) and by the Direcci&#243; General d&#39;Investigaci&#243;, Conselleria d&#39;Investigaci&#243;, Govern Balear (contract to M.M.F.C; FPI/040/2020). The authors thank Dr. Anna Tom&#225;s and Maria Tortosa for their help at the experimental surgery and platform of IdISBa. Finally, thanks to ADEMA School of Dentistry for the access to the CBCT scanner.

NOTE: The experimental protocol of the study was approved by the Ethical Committee of Animal Experimentation of the Balearic Islands Health Research Institute (CEEA-UIB; reference number 163/03/21).
1. Animal anesthesia and procedure preparation
<ol>
	<li>Sterilize all surgical instruments (aluminium mouth gags, dental explorer, diamond lance, surgical scissors, microsurgical pliers, a micro needle holder, a hollenback carver, a periosteal microsurgical elevator, and microsurgical scissors) (5 min at 135 &#176;C) before surgery.</li>
	<li>Prepare all the solutions needed for the procedure in sterile conditions, as described:
	<ol>
		<li>Prepare a mix of Ketamine (60 mg/mL) and Xylazine (8 mg/mL) by mixing 1.6 mL of Ketamine with 1 mL of Xylazine diluted in Phosphate Buffer Solution (PBS)/saline. Store the stock at 4 &#176;C.</li>
		<li>Dilute Atipamezole to a final concentration of 0.25 mg/mL in PBS/saline. Store the stock at 4 &#176;C.</li>
		<li>Dilute Buprenorphine to a final concentration of 0.03 mg/mL in PBS/saline. Store the stock at 4 &#176;C.</li>
		<li>Prepare 1 mL of Pg-LPS (1 mg/mL) in sterile saline. Store the stock at -20 &#176;C.</li>
	</ol>
	</li>
	<li>For the experiment, use female and male Wistar rats, aged 12 weeks and weighing 210-350 g at the time of surgery. Keep the animals housed in groups under the appropriate environment and constant conditions (20-24 &#176;C, 12 h per day light-dark cycles), with food and standard water, offered ad libitum.</li>
	<li>To induce anesthesia, weigh the rat and administer the mix of Ketamine/Xylazine at a concentration of 80/10 mg/kg intraperitoneally (IP), by using a 25 G sterile hypodermic needle and 1 mL syringe.</li>
	<li>After the rat is anesthetized, place the animal on its back on a heated surgical platform; during the procedure, cover the animal&#39;s body to prevent heat loss. 
	NOTE: Anesthesia depth is assessed by the loss of pedal reflex during the procedure and by monitoring vital signs. If needed, use a small nose cone to maintain anesthesia with 2% Isoflurane in 100% oxygen. Apply sterile ophthalmic ointment to both eyes after anesthesia induction to protect the corneas and prevent drying.&#160;</li>
	<li>During the procedures, administer 100% oxygen using a small nose cone, and monitor the pulse rate and oxygen saturation by pulse oximetry. 
	NOTE: If the oxygen saturation and pulse rate fall below 95% and 190 bpm, respectively, stop the procedure and place the animal in the lateral decubitus position until reaching normal values.</li>
	<li>Open the rat mouth using an aluminum mouth gag around the incisors (upper and lower), retracting the tongue with it and stabilizing the maxilla and mandible in an open, comfortable working position, enabling access to mandibular molars. 
	NOTE: If the animal needs to be placed in lateral decubitus, remove the mouth gag before changing its position to favor recovery. Once the animal is anesthetized, collect gingival crevicular fluid (GCF) before surgery (sample in basal conditions) (day 0).</li>
	<li>Collect GCF as described by the following steps:
	<ol>
		<li>Place the animal on its back on a surgical platform and stabilize the maxilla and mandible in an open position with aluminum mouth gags.</li>
		<li>Collect GCF using a total of four (two for each M1) absorbent paper point n&#186; 30 (0.03 cm diameter x 3 cm length), by inserting it into the gingival crevice (space between gingival epithelium and adjacent enamel) around the mesio-palatal of the M1 until slight resistance. Retain the paper point in the same position for a total of 30 s before immediate removal.</li>
		<li>After collection, transfer the paper point immediately into a plastic vial, and store at -80 &#176;C until assay performance.</li>
	</ol>
	</li>
</ol>
2. Retentive ligature technique and intragingival Pg -LPS injection
NOTE: The ligature model was created (day 0) by placing a sterile braided silk ligature (5/0) around the M1 bilaterally within the gingival sulcus using microsurgical instruments and securing it with the surgeon&#39;s knots on the palatal surface. The microsurgical instruments used were microsurgical pliers, a micro needle holder, a hollenback carver, a periosteal microsurgical elevator, and microsurgical scissors. Surgical loupes with an LED light source were also used (3.6x magnification).
<ol>
	<li>Position the distal tail of the suture on the palatal side of the dentition and insert the proximal segment between the contact of M1 and second maxillary molars (M2).</li>
	<li>Use the periosteal microsurgical elevator to insert the suture within the sulcus. Wrap the ligature around the buccal surface of the M1 very carefully, as the tissues at this level present a narrow zone of attached gingiva. On the palatal aspect, make sure the suture is tightened on both ends to ensure it is driven into the gingival sulcus. 
	NOTE: If resistance is observed when inserting the suture between the M1 and the M2, the contact may be opened slightly using a dental explorer and diamond lance-shaped bur.</li>
	<li>Tie the ends of the suture with a surgeon&#39;s knot and trim the tails as short as possible. Insert the knot in the sulcus. 
	NOTE: Tips of surgical instruments can cause oral trauma and bleeding. Prepare small segments of gauze or a cotton swab to remove blood from the oral cavity and apply pressure to stop any hemorrhage. Careful soft tissue management with a microsurgical approach minimizes surgical complications and leads to less tissue trauma.</li>
	<li>After ligature positioning, inject 40 &#181;L of Pg-LPS in sterile saline with a 25 G sterile hypodermic needle and a 1 mL syringe to the subgingival tissue (between the root or neck of a tooth and the gum margin) at the mesio-palatal side of the M1 bilaterally (day 0).</li>
</ol>
3. End of the procedure
<ol>
	<li>After ligation positioning and Pg-LPS application, release the rat from the surgical state and place it in a clean individual cage under a heat lamp.</li>
	<li>Inject the antagonist Atipamezole&#160;(0.5 mg/kg subcutaneously (SC))&#160;with a 25 G sterile hypodermic needle and a 1 mL syringe.</li>
	<li>For pain relief, inject 0.03 mg/kg Buprenorphine, SC.</li>
	<li>Monitor the animal&#39;s recovery until the effects of the operation are completely reversed. Individually house each rat in the appropriate environment under constant conditions (20-24 &#176;C, 12 h per day light-dark cycles). Offer food and deionized water ad libitum.</li>
	<li>During the first 2 days after the procedure, weigh the animals and inject Buprenorphine SC twice daily&#160;for pain relief. 
	NOTE: Buprenorphine can be administered before beginning the procedure to eliminate the windup effect.</li>
	<li>During the time of the experiment, monitor the animals by weight and general behavior assessment one or two times a week.</li>
</ol>
4. Post-procedure follow-up
NOTE: The induction of PD was maintained for 14 days to promote the accumulation of bacteria biofilm and consequent inflammation. The ligatures have to be examined and adjusted, and Pg-LPS is injected three times per week (day 2, day 4, day 6, day 8, day 10, and day 12).
<ol>
	<li>Examine and adjust the ligature (day 2, day 4, day 6, day 8, day 10, and day 12) as follows:
	<ol>
		<li>Anesthetize with 2% isoflurane in 100% oxygen using an anesthesia induction chamber.</li>
		<li>After the rat is anesthetized, place the animal on its back and use a small nose cone during the procedure with 1% isoflurane in 100% oxygen for maintenance of the animal anesthesia.</li>
		<li>Open the rat mouth using the aluminum mouth gag around the incisors (upper and lower), retracting the tongue with it and stabilizing the maxilla and mandible in an open, comfortable working position, enabling access to ligatures.</li>
		<li>Tighten the ligatures against the gingiva with the help of a periosteal microsurgical elevator, and make sure the suture of the ligatures is inserted, creating inflammation around the gingiva. 
		NOTE: It is possible that 7-10 days after surgery, ligatures are lost. If this happens, follow the protocol as explained for the injection of Pg-LPS (step 2.4).</li>
	</ol>
	</li>
	<li>After the ligature adjustment, bilaterally inject 40 &#181;L of Pg-LPS with a 25 G sterile hypodermic needle and a 1 mL syringe to the subgingival tissue at the mesio-palatal side of the M1 (day 2, day 4, day 6, day 8, day 10, and day 12).</li>
	<li>Remove the nose cone for anesthesia and place the rat in its cage. Monitor the animal&#39;s recovery until the effects of the anesthesia are completely reversed.</li>
</ol>
5. Animal sacrifice and analysis
NOTE: There are different options for evaluating the progression of PD. Here, the analysis described consists of an evaluation of pro-inflammatory cytokines at the gingival crevicular fluid (GCF), and an evaluation of the loss of alveolar bone.
<ol>
	<li>On day 14 of the study (day 14), sacrifice the animals with CO2&#160;in a carbon dioxide chamber. A&#160;displacement rate of 30% to 70% of the chamber volume/min is recommended for rodents. 
	NOTE: Lack of animal response to pedal reflex and absence of vital signs must be verified to confirm&#160;euthanasia.&#160;</li>
	<li>Collect GCF as described by the following steps: 
	NOTE: GCF is collected before PD induction (before surgery) (day 0) and after PD induction (after sacrifice) (day 14).
	<ol>
		<li>Place the animal on its back on a surgical platform, and stabilize the maxilla and mandible in an open position with aluminum mouth gags.</li>
		<li>Collect the GCF using adsorbent paper point n&#186; 30 (0.03 cm diameter x 3 cm length) by inserting it into the gingival crevice around the mesio-palatal of the M1 until slight resistance. Retain the paper point in the same position for a total of 30 s before immediate removal.</li>
		<li>After collection, transfer the paper point immediately into a plastic vial and store at -80 &#176;C until assay performance.</li>
	</ol>
	</li>
	<li>To evaluate proteins within the GCF, prepare the following solutions and follow the steps of the elution method as described: 
	NOTE: IL-1&#946; is evaluated on the GCF (day 14) using an immunoassay, according to the manufacturer&#39;s protocol.
	<ol>
		<li>Prepare the elution buffer fresh and keep it on ice throughout the entire extraction process to inhibit protease activity.</li>
		<li>Prepare all solutions needed for the elution buffer as described:
		<ol>
			<li>Prepare 1 mL of Aprotinin (1 mg/mL) in ultrapure water.</li>
			<li>Prepare 10 mL of phenylmethylsulfonylfluoride (PMSF) (200 mM) in methanol.</li>
			<li>Add 125 &#181;L of PMSF and 250 &#181;L of Aprotinin to 24.5 mL of PBS solution (pH = 7.4) to prepare the elution buffer.</li>
		</ol>
		</li>
		<li>Dissolve one commercial phosphatase inhibitor tablet with 10 mL of freshly prepared elution buffer for 10 min under agitation at 4 &#176;C. 
		NOTE: The duration of GCF elution is limited; use immediately for centrifugations after the addition of phosphatase inhibitor tablet within the first 30 min.</li>
		<li>After adding the phosphatase inhibitor, add 11 &#181;L of complete elution buffer directly onto the tube with the paper points.</li>
		<li>Centrifuge the tube at 452 x g for 5 min at 4 &#176;C.</li>
		<li>Transfer the eluted content to a new plastic vial. Repeat this process four additional times to yield a 50 &#181;L total volume.</li>
		<li>After the last centrifugation, add a total volume of 60 &#181;L directly onto the paper point, and centrifuge one last time at 452 x g for 5 min at 4 &#176;C.</li>
		<li>Use the required volume eluted for protein evaluation.</li>
	</ol>
	</li>
	<li>After euthanasia and GCF collection, with the help of surgical scissors, cut out the superior jaw of the rats. Try to peel off the mask of the rat and leave as little soft tissue as possible.</li>
	<li>Position the jaw in the microscope stage for visualization and take a picture at the desired magnification.</li>
	<li>Place the jaw directly in 4% paraformaldehyde (PFA) diluted in PBS. Refresh twice a week with new PFA for 12 days for a complete fixation. 
	CAUTION: Steps involving PFA should be performed in a fume hood following the Safety Data Sheet recommendations.</li>
	<li>After complete jaw fixation, evaluate bone loss using a cone-beam computed tomography (CBCT) scanner, as follows:
	<ol>
		<li>Open the PC.</li>
		<li>Open the CBCT analysis program.</li>
		<li>Select Scan Protocol &#62; Denture Scan Mode.</li>
		<li>Select Field of View (FOV) &#62; (11x8) HIRes (90 kV of voltage and 3 mA of current).</li>
		<li>Place the fixed upper maxilla of the rats in the gantry.</li>
		<li>Click Next.</li>
		<li>Click the X-ray firing button (press the X-ray remote control to make the emission and keep it pressed for the entire duration of the scan).</li>
		<li>If necessary, relocate the upper maxilla in the center of the frontal plane by pressing the control button, then click the X-ray firing button (press the X-ray remote control to make the emission and keep it pressed for the entire duration of the scan).</li>
		<li>Click Next.</li>
		<li>Click the X-ray firing button (press the X-ray remote control to make the emission and keep it pressed for the entire duration of the scan).</li>
		<li>If necessary, relocate the denture in the center of the sagittal plane by pressing the control button, then click the X-ray firing button (press the X-ray remote control to make the emission and keep it pressed for the entire duration of the scan).</li>
		<li>Click Next.</li>
		<li>Click the Start button (press the X-ray remote control to make the emission and keep it pressed for the entire duration of the exam). Wait until receiving a processing message, and then follow the instructions shown.</li>
		<li>A window view appears. Regulate the gray at 65% and click Apply.</li>
		<li>For saving the scan, click on File and save it in DICOM format. Then, click All Images and at the DICOM export selection window, select all the parameters shown (initial image, original axial, reformatted axial, multiplanar) and select predefined type.</li>
	</ol>
	</li>
	<li>Process the maxillary molars&#39; two-dimensional and sagittal representative images using an analysis program, as follows:
	<ol>
		<li>Open the software.</li>
		<li>Click File and Open, and then select the folder with images in DICOM format and click Open.</li>
		<li>Wait until the window &#34;Convert to 8 bit&#34; appears, select the range from 0 to 6,000, and click OK.</li>
		<li>Click Raw images.</li>
		<li>Define the top and bottom of the selection to limit the region of interest. Search for the first and last images where alveolar bone appears and select it with the top of selection and bottom of selection commands. 
		NOTE: Representative two-dimensional images of the alveolar bone of the molars could be exported, as in Figure 3A,B.</li>
		<li>For sagittal representative images, click on Toggle profile bar.</li>
		<li>Draw a sagittal line in the middle of the palate and click Reslice model. Determine parameters for delimiting the region of interest (slice spacing: 1; number of slices: 100) and click OK. Wait until the reslicing model ends. Finally, click save Resliced images. 
		NOTE: Representative sagittal imagines of the alveolar bone of the molars could be exported, as in Figure 3C,D.</li>
	</ol>
	</li>
</ol>

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The authors declare no conflict of interest.

This method describes the induction of PD in rats following a combined technique of Pg-LPS injections and ligature placement around the M1, revealing that significant changes in the periodontal tissues and alveolar bone could be induced in 14 days following this method.
During this procedure, attention to different critical steps must be provided. During animal anesthesia and procedure preparation, assessing the proper anesthesia during the surgical process is critical for its success...

Periodontitis (PD) is the sixth most prevalent public health condition worldwide, affecting approximately 11% of the total population, being an advanced, irreversible, and destructive form of periodontal disease1,2. PD is an inflammatory process that affects the gingival and periodontal tissues, which results in gingiva recession, apical migration of the junctional epithelium with pocket development, and the loss of alveolar bone3. Furthermore, PD is associated with several systemic diseases, including cardiovascular disease, obesity, diabetes, and rheumatoid arthritis, for which environmental and host-specific factors play a significant role4,5.
Hence, PD is a multifactorial disease primarily initiated by the accumulation of microbial plaque - resulting from dysbiosis of microbial communities - and by an exaggerated host immune response to periodontal pathogens, which leads to the breakdown of periodontal tissue4,6. Among several periodontal bacteria, the gram-negative anaerobic bacterium Porphyromonas gingivalis is one of the key pathogens in PD4. P. gingivalis contains a complex lipopolysaccharide (LPS) in its walls, a molecule known to induce polymorphonuclear leukocyte infiltration and vascular dilatation in inflamed periodontal tissues7. This results in the production of inflammatory mediators, such as interleukin 1 (IL-1), IL-6, and IL-8, tumor necrosis factor (TNF), or prostaglandins, with a subsequent osteoclast activation and bone resorption, leading to tissue destruction and ultimate tooth loss3.
Among the different advantages of animal models include the capacity to mimic cellular complexities as in humans, or to be more accurate than in vitro studies, which are carried out on plastic surfaces with limited cell types8. For modeling PD experimentally in vivo, different animal species have been used, like non-human primates, dogs, pigs, ferrets, rabbits, mice, and rats9. However, rats are the most extensively studied animal model for the pathogenesis of PD because they are inexpensive and easy to handle10. Their dental gingival tissue has similar structural features to human gingival tissue, with a shallow gingival sulcus and junctional epithelium attached to the tooth surface. Furthermore, as in humans, the junctional epithelium facilitates the passage of bacterial, foreign materials, and exudates from inflammatory cells 9.
One of the most reported experimental models of PD induction in rats is the placement of ligatures around the teeth, which is technically challenging but reliable10. The ligature placement facilitates dental plaque and bacterial accumulation, generating a dysbiosis in the gingival sulci, that causes periodontal tissue inflammation and destruction11. Loss of periodontal attachment and resorption of alveolar bone could occur in 7 days in this rat model8.
Another animal model for PD consists of the injection of LPS into the gingival tissue. As a result, osteoclastogenesis and bone loss are stimulated. The histopathological features of this model are similar to human-established PD, characterized by higher levels of proinflammatory cytokines, collagen degradation, and alveolar bone resorption6,8.
Thus, the aim of this study was to describe a simple rat model of experimental PD based on the techniques of P. gingivalis-LPS (Pg-LPS) injections, combined with ligature placement around the first maxillary molars (M1). This is a model with similar characteristics to those observed in human PD disease, which could be used in the study of disease progression mechanisms and future possible treatments.

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induction of periodontitis via a combination of ligature and lipopolysaccharide injection in a rat model

Periodontitis (PD) is a highly prevalent, chronic immune-inflammatory disease of the periodontium, that results in a loss of gingival soft tissue, periodontal ligament, cementum, and alveolar bone. In this study, a simple method of PD induction in rats is described. We provide detailed instructions for placement of the ligature model around the first maxillary molars (M1) and a combination of injections of lipopolysaccharide (LPS), derived from Porphyromonas gingivalis at the mesio-palatal side of the M1. The induction of periodontitis was maintained for 14 days, promoting the accumulation of bacteria biofilm and inflammation. To validate the animal model, IL-1&#946;, a key inflammatory mediator, was determined by an immunoassay in the gingival crevicular fluid (GCF), and alveolar bone loss was calculated using cone beam computed tomography (CBCT). This technique was effective in promoting gingiva recession, alveolar bone loss, and an increase in IL-1&#946; levels in the GCF at the end of the experimental procedure after 14 days. This method was effective in inducing PD, thus being able to be used in studies on disease progression mechanisms and future possible treatments.

A timeline of the experimental steps is presented in Figure 1. Figure 2A shows an image of the mandibula after surgical intervention, with ligature placement around the sulcus of the M1 at time 0 of the experiment. Figure 2B shows how, after 14 days of the procedure, the ligature around the M1 enters the gingival sulcus, causing inflammation of the gingiva and infiltrating accumulation.
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Watch this Scientific Journal Video about Induction of Periodontitis via a Combination of Ligature and Lipopolysaccharide Injection in a Rat Model at JoVE.com

Induction of Periodontitis via a Combination of Ligature and Lipopolysaccharide Injection in a Rat Model

In this study, a rat model of induction of periodontitis is presented via a combination of retentive ligature and repetitive injections of lipopolysaccharide derived from Porphyromonas gingivalis, over 14 days around the first maxillary molars. The ligation and LPS injection techniques were effective in inducing peridontitis, resulting in alveolar bone loss and inflammation.

Induction of Periodontitis via a Combination of Ligature and Lipopolysaccharide Injection in a Rat Model

Periodontitis (PD) is a highly prevalent, chronic immune-inflammatory disease of the periodontium, that results in a loss of gingival soft tissue, ...

induction-periodontitis-via-combination-ligature-lipopolysaccharide

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Immunology and Infection

3.4K Views.  University of the Balearic Islands (UIB). In this study, a rat model of induction of periodontitis is presented via a combination of retentive ligature and repetitive injections of lipopolysaccharide derived from Porphyromonas gingivalis, over 14 days around the first maxillary molars. The ligation and LPS injection techniques were effective in inducing peridontitis, resulting in alveolar bone loss and inflammation. 

Video: Induction of Periodontitis via a Combination of Ligature and Lipopolysaccharide Injection in a Rat Model

Preparation of Mouse Pituitary Immunogen for the Induction of Experimental Autoimmune Hypophysitis

Autoimmune hypophysitis is a chronic inflammation of the pituitary gland caused or accompanied by autoimmunity1. It has traditionally been considered a rare disease but reporting has increased markedly in recent years. Hypophysitis, in fact, develops not uncommonly as a "side effect" in cancer patients treated with antibodies that block inhibitory receptors expressed on T lymphocytes, such as CTLA-42 and PD-1 receptors. Autoimmune hypophysitis can be induced experimentally by injecting mice with pituitary proteins mixed with an adjuvant3. In this video article we demonstrate how to extract proteins from mouse pituitary glands and how to prepare them in a form suitable for inducing autoimmune hypophysitis in SJL mice.

Autoimmune hypophysitis can be reproduced in mice by injecting an extract of mouse pituitary proteins.

Autoimmune hypophysitis is a chronic inflammation of the pituitary gland caused or accompanied by autoimmunity1.  It has traditionally been considered a ...

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 ...

Induction of Graft-versus-host Disease and In Vivo T Cell Monitoring Using an MHC-matched Murine Model

Graft-versus-host disease (GVHD) is the limiting barrier to the broad use of bone marrow transplant as a curative therapy for a variety of hematological deficiencies. GVHD is caused by mature alloreactive T cells present in the bone marrow graft that are infused into the recipient and cause damage to host organs. However, in mice, T cells must be added to the bone marrow inoculum to cause GVHD. Although extensive work has been done to characterize T cell responses post transplant, bioluminescent imaging technology is a non-invasive method to monitor T cell trafficking patterns in vivo. 
Following lethal irradiation, recipient mice are transplanted with bone marrow cells and splenocytes from donor mice. T cell subsets from L2G85.B6 (transgenic mice that constitutively express luciferase) are included in the transplant. By only transplanting certain T cell subsets, one is able to track specific T cell subsets in vivo, and based on their location, develop hypotheses regarding the role of specific T cell subsets in promoting GVHD at various time points. At predetermined intervals post transplant, recipient mice are imaged using a Xenogen IVIS CCD camera. Light intensity can be quantified using Living Image software to generate a pseudo-color image based on photon intensity (red = high intensity, violet = low intensity). 
Between 4-7 days post transplant, recipient mice begin to show clinical signs of GVHD. Cooke et al.1 developed a scoring system to quantitate disease progression based on the recipient mice fur texture, skin integrity, activity, weight loss, and posture. Mice are scored daily, and euthanized when they become moribund. Recipient mice generally become moribund 20-30 days post transplant. 
Murine models are valuable tools for studying the immunology of GVHD. Selectively transplanting particular T cell subsets allows for careful identification of the roles each subset plays. Non-invasively tracking T cell responses in vivo adds another layer of value to murine GVHD models.

Induction of Graft-versus-host Disease and In Vivo T Cell Monitoring Using an MHC-matched Murine Model

Murine bone marrow transplantation is a widely used technique to study immunological mechanisms governing graft-versus-host disease in humans. The ability to monitor T cell trafficking patterns in vivo allows for detailed analysis of the development and perpetuation of T cell responses during graft-versus-host disease.

Graft-versus-host disease (GVHD) is the limiting barrier to the broad use of bone marrow transplant as a curative therapy for a variety of hematological ...

A Method for Generating Pulmonary Neutrophilia Using Aerosolized Lipopolysaccharide

Acute lung injury (ALI) is a severe disease characterized by alveolar neutrophilia, with limited treatment options and high mortality. Experimental models of ALI are key in enhancing our understanding of disease pathogenesis. Lipopolysaccharide (LPS) derived from gram positive bacteria induces neutrophilic inflammation in the airways and lung parenchyma of mice. Efficient pulmonary delivery of compounds such as LPS is, however, difficult to achieve. In the approach described here, pulmonary delivery in mice is achieved by challenge to aerosolized Pseudomonas aeruginosa LPS. Dissolved LPS was aerosolized by a nebulizer connected to compressed air. Mice were exposed to a continuous flow of LPS aerosol in a Plexiglas box for 10 min, followed by 2 min conditioning after the aerosol was discontinued. Tracheal intubation and subsequent bronchoalveolar lavage, followed by formalin perfusion was next performed, which allows for characterization of the sterile pulmonary inflammation. Aerosolized LPS generates a pulmonary inflammation characterized by alveolar neutrophilia, detected in bronchoalveolar lavage and by histological assessment. This technique can be set up at a small cost with few appliances, and requires minimal training and expertise. The exposure system can thus be routinely performed at any laboratory, with the potential to enhance our understanding of lung pathology.

We describe a method for inducing neutrophilic pulmonary inflammation by challenge to aerosolized lipopolysaccharide by nebulization, to model acute lung injury. In addition, basic surgical techniques for lung isolation, tracheal intubation and bronchoalveolar lavage are also described.

Acute lung injury (ALI) is a severe disease characterized by alveolar neutrophilia, with limited treatment options and high mortality. Experimental models ...

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 ...

Induction of Paralysis and Visual System Injury in Mice by T Cells Specific for Neuromyelitis Optica Autoantigen Aquaporin-4

While it is recognized that aquaporin-4 (AQP4)-specific T cells and antibodies participate in the pathogenesis of neuromyelitis optica (NMO), a human central nervous system (CNS) autoimmune demyelinating disease, creation of an AQP4-targeted model with both clinical and histologic manifestations of CNS autoimmunity has proven challenging. Immunization of wild-type (WT) mice with AQP4 peptides elicited T cell proliferation, although those T cells could not transfer disease to na&#239;ve recipient mice. Recently, two novel AQP4 T cell epitopes, peptide (p) 135-153 and p201-220, were identified when studying immune responses to AQP4 in AQP4-deficient (AQP4-/-) mice, suggesting T cell reactivity to these epitopes is normally controlled by thymic negative selection. AQP4-/- Th17 polarized T cells primed to either p135-153 or p201-220 induced paralysis in recipient WT mice, that was associated with predominantly leptomeningeal inflammation of the spinal cord and optic nerves. Inflammation surrounding optic nerves and involvement of the inner retinal layers (IRL) were manifested by changes in serial optical coherence tomography (OCT). Here, we illustrate the approaches used to create this new in vivo model of AQP4-targeted CNS autoimmunity (ATCA), which can now be employed to study mechanisms that permit development of pathogenic AQP4-specific T cells and how they may cooperate with B cells in NMO pathogenesis.

Here, we present a protocol to induce paralysis and opticospinal inflammation by transfer of aquaporin-4 (AQP4)-specific T cells from AQP4-/- mice into WT mice. In addition, we demonstrate how to use serial optical coherence tomography to monitor visual system dysfunction.

While it is recognized that aquaporin-4 (AQP4)-specific T cells and antibodies participate in the pathogenesis of neuromyelitis optica (NMO), a human ...

Two-photon Intravital Imaging of Leukocytes During the Immune Response in Lipopolysaccharide-treated Mouse Liver

Sepsis is a type of severe infection that can cause organ failure and tissue damage. Although the mortality and morbidity rates associated with sepsis are extremely high, no direct treatment or organ-related mechanism has been examined in detail in real time. The liver is the key organ that manages toxins and infections in the human body. Herein, we aimed to perform intravital imaging of mouse liver after induction of endotoxemia in order to track the motility of immune cells, such as neutrophils and liver capsular macrophages (LCMs). Accordingly, we designed a novel surgical method for exposure of the liver with minimally invasive surgery. Mice were intraperitoneally injected with lipopolysaccharide (LPS), a common endotoxin. Using our novel surgical approach for exposure and intravital imaging of the mouse liver, we found that neutrophil recruitment in LPS-treated LysM-green fluorescent protein (GFP) mouse liver was increased compared with that in phosphate-buffered saline-treated liver. After LPS treatment, the number of neutrophils increased significantly with time. Additionally, using CX3Cr1-GFP mice, we successfully visualized liver resident macrophages called LCMs. Therefore, to investigate the efficacy of new reagents to control immune mobility in vivo, determining the motility and morphology of neutrophils and LCMs in the liver may allow us to identify therapeutic effect in organ failure and tissue damage caused by leukocytes activation in sepsis.

We established a novel surgical protocol for two-photon imaging of live mice liver with minimal invasion. With this technique, we identified the detailed structure of the liver during lipopolysaccharide-induced endotoxemia. We anticipate that this method may be utilized to determine the effectiveness of various reagents treatment to hepatic leukocyte migration.

Sepsis is a type of severe infection that can cause organ failure and tissue damage. Although the mortality and morbidity rates associated with sepsis are ...

Deep Dermal Injection As a Model of Candida albicans Skin Infection for Histological Analyses

The skin is an extremely extended organ of the body and, due to this large surface, it is continuously exposed to microorganisms. Skin damage can easily lead to infections in the dermis which can, in turn, result in the dissemination of pathogens into the bloodstream. Understanding how the immune system fights infections at the very early stage and how the host can eliminate the pathogens is an important step to set the base for future therapeutic interventions. Here we describe a model of Candida albicans infection that can visualize the processes that occur early during an infection, including when the pathogen has passed the epithelial barrier, as well as the immune response elicited by the C. albicans invasion. We used this infection model to perform histological analyses that show the immune cells that infiltrate the skin as well as the presence and localization of the pathogen. Samples collected after the infection can be processed for RNA extraction.

Deep Dermal Injection As a Model of Candida albicans Skin Infection for Histological Analyses

Here we describe a protocol that allows histological and molecular analysis of skin samples after Candida albicans intradermal injection. This protocol maintains the structural integrity of the skin and allows for the localization of tissue-resident or newly recruited immune cells as well as the pathogen distribution.

The skin is an extremely extended organ of the body and, due to this large surface, it is continuously exposed to microorganisms. Skin damage can easily ...

Induction of Mouse Lung Injury by Endotracheal Injection of Bleomycin

Pulmonary fibrosis is a hallmark of several human lung diseases with a different etiology. Since current therapies are rather limited, mouse models continue to be an essential tool for developing new antifibrotic strategies. Here we provide an effective method to investigate in vivo antifibrotic activity of human mesenchymal stromal cells obtained from whole umbilical cord (hUC-MSC) in attenuating bleomycin-induced lung injury. C57BL/6 mice receive a single endotracheal injection of bleomycin (1.5 U/kg body weight) followed by a double infusion of hUC-MSC (2.5 x 105) into the tail vein, 24 h and 7 days after the bleomycin administration. Upon sacrifice at days 8, 14, or 21, inflammatory and fibrotic changes, collagen content, and hUC-MSC presence in explanted lung tissue are analyzed. The injection of bleomycin into the mouse&#160;trachea allows the direct targeting of the lungs, leading to extensive pulmonary inflammation and fibrosis. The systemic administration of a double dose of hUC-MSC results in the early blunting of the bleomycin-induced lung injury. Intravenously infused hUC-MSC are transiently engrafted into the mouse lungs, where they exert their anti-inflammatory and antifibrotic activity. In conclusion, this protocol has been successfully applied for the preclinical testing of hUC-MSC in an experimental mouse model of human pulmonary fibrosis. However, this technique can be easily extended both to study the effect of different endotracheally administered substances on the pathophysiology of the lungs and to validate new anti-inflammatory and antifibrotic systemic therapies.

Here we present an effective method to investigate the antifibrotic activity of intravenously infused human mesenchymal stromal cells obtained from the whole umbilical cord following the induction of lung injury by an endotracheal injection of bleomycin in C57BL/6 mice. This protocol can be easily extended to the preclinical testing of other therapeutics.

Pulmonary fibrosis is a hallmark of several human lung diseases with a different etiology. Since current therapies are rather limited, mouse models ...

Induction and Scoring of Graft-Versus-Host Disease in a Xenogeneic Murine Model and Quantification of Human T Cells in Mouse Tissues using Digital PCR

Acute graft-versus-host disease (GVHD) is a significant limitation for patients receiving hematopoietic stem cell transplant as therapy for hematological deficiencies and malignancies. Acute GVHD occurs when donor T cells recognize host tissues as a foreign antigen and mount an immune response to the host. Current treatments involve toxic immunosuppressive drugs that render patients susceptible to infection and recurrence. Thus, there is ongoing research to provide an acute GVHD therapy that can effectively target donor T cells and reduce side effects. Much of this pre-clinical work uses the xenogenic GVHD (xenoGVHD) murine model that allows for testing of immunosuppressive therapies on human cells rather than murine cells in an in vivo system. This protocol outlines how to induce xenoGVHD and how to blind and standardize clinical scoring to ensure consistent results. Additionally, this protocol describes how to use digital PCR to detect human T cells in mouse tissues, which can subsequently be used to quantify efficacy of tested therapies. The xenoGVHD model not only provides a model to test GVHD therapies but any therapy that can suppress human T cells, which could then be applied to many inflammatory diseases.

Here, we present a protocol to induce and score disease in a xenogeneic graft-versus-host disease (xenoGVHD) model. xenoGVHD provides an in vivo model to study immunosuppression of human T cells. Additionally, we describe how to detect human T cells in tissues with digital PCR as a tool to quantify immunosuppression.

Acute graft-versus-host disease (GVHD) is a significant limitation for patients receiving hematopoietic stem cell transplant as therapy for hematological ...