The authors thanks to Post Graduation Program in Medical Clinic of University of S&#227;o Paulo; Coordena&#231;&#227;o de Aperfei&#231;oamento de Pessoal de N&#237;vel Superior (CAPES) and University of S&#227;o Paulo Medical School (FMUSP).

This protocol was approved by the Ethics Committee for the use of animals of the USP Medicine School, Num. Project: 1343/2019-CEUA: FMUSP. For this protocol, C57BL/6 mice, aged 6 weeks, weighing 20 &#177; 2 g were used (n = 9/group).
1. Laparotomy
<ol>
	<li>Anesthetize animals with xylazine (10 mg/kg) and ketamine solution (80 mg/kg), subcutaneously (0.1 mL/10g of body weight) using a 1 mL syringe and 13x0.45mm needle 26G &#189;. Check for sufficient anesthesia depth by pinching the toe. Control body temperature using heated pads. Ensure that all surgical materials are sterile.</li>
	<li>Clean the abdominal area with 5% povidone-iodine solution and use a trimmer to remove hair between the chest and lower abdomen (approximately 2 cm2). Clean the surgical area with 70% alcohol.</li>
	<li>Immobilize the animal on the surgical board using surgical tape. Use scissors to cut 5 mm of the skin horizontally, on the upper part of the abdomen and 1 cm below the xiphoid process. Repeat the cut on the peritoneum. This will result in a laparotomy with minimal exposure of the cavity.</li>
</ol>
2. Locating and exposing the pancreas
<ol>
	<li>With the aid of a retractor, pull the liver towards the mouse&#39;s head, ~1 cm from the intestine.</li>
	<li>Locate the region of the pancreas that will be injected with sodium taurocholate (pancreas head). Locate the duodenum with reference to the liver- below the liver, on the right side (on the left as the mouse is viewed). The duodenum is the first part of the small intestine and is connected to the final portion of the stomach.</li>
	<li>With the aid of forceps, lift the liver towards the animal head, and gently pull the small intestine portion. Fix the two lateral ends of the small intestine with a 6-0 polypropylene suture to better view the distal portion of the common bile duct.</li>
</ol>
3. Severe acute pancreatitis induction
<ol>
	<li>Temporarily occlude the proximal common bile duct with a microvessel clip to prevent retrograde infusion from leaking into the liver. The common bile duct can be seen on the liver side of the duodenum and its junction with the duodenum will appear white. Expose the organ out of the abdominal cavity.</li>
	<li>Puncture the periampullary region (whitish part of the small intestine&#39;s wall) to access the common bile duct with a 0.4 mm needle connected to a 0.54 mm polyethylene tube.</li>
	<li>Make a temporary occlusion of the distal common bile duct with 8-0 suture to prevent the sodium taurocholate solution from leaking into the duodenum.</li>
	<li>Start the infusion pump and program a 2.5% sodium taurocholate solution (diluted in 0.9% saline) infusion at a constant speed of 10 &#181;L/10 g body weight for 3 min.</li>
	<li>After the infusion, remove the microvessel clip, the temporary 8-0 suture, and the injection needle from the bile pancreatic duct to reconstitute the physiological flow of the bile.</li>
	<li>At the end, suture the abdomen with 6-0 nonabsorbent monofilament polypropylene suture. The time between the laparotomy and the end suture should be a maximum of 30 min (see Figure 1).</li>
	<li>After the surgery, house the animals in polyethylene boxes lined with wood shavings and water and food ad libitum.</li>
	<li>Treat control mice in the same manner as the experimental mice but ensure that the infusate consists of saline only. Perform the surgical procedure and the infusion of saline solution (10 mL/min, for 3 min) in a control group (SHAM) to eliminate the inflammatory bias caused by surgery and cannulation.</li>
	<li>Use tramadol 12.5 mg/kg subcutaneously every 8 hours, starting after post-surgical recovery.</li>
</ol>
4. Methods for analysis
<ol>
	<li>At 12 h after AP induction, anesthetize the animals with xylazine (10&#160;mg/kg) and ketamine (80 mg/kg) to collect approximately 250 &#181;L of blood via the orbital plexus.
	<ol>
		<li>Gently hold the skin on the back, promoting a slight protrusion of the eyeball, and position it with the eye facing upwards.</li>
		<li>Instill a drop of eye ointment containing local anesthetic in the animal&#39;s eye.</li>
		<li>Position the end of the capillary tube in the medial corner of the eye and insert it gently under the eyeball, with an angle of ~30&#176;-45&#176;. Rotate the capillary tube until blood flow begins. Remember that it is not necessary to use force for the procedure.</li>
		<li>Once the collection is over, ensure homeostasis by keeping the eyelids closed by light compression with the gauze. Discard the capillary tube in the sharps container19.</li>
		<li>Centrifuge the serum (700 x g, 15 min) and stock the supernatant for amylase and IL-6 dosing (step 4.7 and 4.8).</li>
	</ol>
	</li>
	<li>Euthanize mice by CO2 asphyxiation.</li>
	<li>Use a 27 G needle to inject 4 mL of ice-cold 1x PBS into the peritoneal cavity. Tened the abdominal skin and ensure that the needle is pushed slowly in the peritoneum to not puncture any organs. After the injection, gently massage the peritoneum for 10 s to remove cells adhered to the peritoneum.</li>
	<li>Using scissors and tweezers, make a small cut (0.5 cm) on the inner skin and musculature to expose the abdominal cavity. Insert a bulb pipette in the peritoneum and collect the fluid. Be careful not to aspirate fatty tissue or other organs.</li>
	<li>Collect as much fluid as possible and deposit the collected cell suspension in tubes kept on ice. Discard the bulb pipette&#160;in the sharps container20. Centrifuge the peritoneal fluid (250 x g, 5 min) and stock the supernatant for IL-6 dosing (step 4.9).</li>
	<li>Collect the pancreas region adjacent to the duodenum (&#60;5mm).</li>
	<li>Process the pancreas by fixing in 10% formalin and incorporate it into paraffin.
	<ol>
		<li>Stain the slides with hematoxylin and eosin for histopathological analyzes under light microscopy. Use Schmidt&#39;s protocol21 (pancreatic edema, acinar cell, injury/necrosis, pancreatic inflammation) to assess the extent of AP.</li>
	</ol>
	</li>
	<li>Measure amylase (U/dL) using commercially available kits according to the manufacturer&#39;s recommendations.</li>
	<li>Measure IL-6 by Luminex assays using commercial kits according to the manufacturer&#39;s recommendations.</li>
	<li>Store the serum and peritoneal fluid supernatant obtained in the steps 4.1 and 4.4 in a freezer at -80 &#176;C if needed.</li>
</ol>

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The authors have nothing to disclose.

The method of inducing acute pancreatitis by retrograde sodium taurocholate infusion has already been shown in rats22,23,24. Three similar works, published in 2008, 2010 and 2015, served as a reference for the protocol3,14,15. In this work, we list all the critical steps for reproducing this method in C57BL/6 mice and some possibilities...

In humans, the presence of gallstones is the most common cause of pancreatitis due to the obstruction of the terminal portion of the choledochal, interrupting the flow of pancreatic secretions and causing an intense inflammatory process in the pancreas, with an increase in the concentration of digestive enzymes in the serum and inflammatory mediators1,2.
Two different theories have been proposed to explain the development of acute pancreatitis (AP). The &#34;common channel&#34; theory suggests that the stones present in the gallbladder obstruct the distal common bile duct system, allowing bile secretion to flow retrograde into the pancreatic duct. The second theory (the &#34;duct obstruction&#34; theory) suggests that the obstruction of the pancreatic duct by excess gallstones causes a blockage in the flow of pancreatic secretion to the duodenum, causing ductal hypertension3. Although the mechanisms that lead to acute biliary pancreatitis are not fully understood, the outcome is an intense inflammatory process. Digestive enzyme eruption and pancreas self-digestion lead to histopathological changes, an increase in inflammatory cytokines (IL-1&#946;, IL-6, TNF-&#945;) in ascitic fluid and serum, and an increase in acute phase proteins4,5,6.
Severe acute pancreatitis is a condition that deserves clinical attention due to the involvement of multiple organs and a high mortality risk. Animal models for the reproduction of acute pancreatitis (AP) are important as these explain the pathophysiological mechanisms of the disease and help in monitoring the evolution of inflammatory events, starting from the initial stages of the disease. This is usually not possible in the clinics2,7. In addition, access to pancreatic tissues is easy in preclinical studies, favoring the elucidation of changes linked to clinical conditions8 along with the possibility of working with isogenic species, eliminating undesirable variables, and mirroring clinical similarity with the outcomes observed in the human condition9.
Biliary and non-biliary models for the induction of acute pancreatitis in rats and mice species have been frequently studied in the scientific literature. Non-biliary methods of induction include administration of supramaximal stimulating doses of the cholecystokinin secretagogue or its analog cerulein10; administration of almost lethal doses of L-arginine; or administration of a choline-deficient diet supplemented with ethionine11. Although these methods are easy to reproduce and result in pancreatic inflammation, they do not replicate the mechanisms that in theory trigger AP (i.e., the reflux of bile secretion into the pancreatic duct). The technique that addresses the biliary model is based on the retrograde infusion of bile acids into the pancreatic duct and requires well-trained researchers to carry out this protocol. Several studies have been published using this method in rats (apparently for technical reasons since these experiments involve surgical procedures)12,13. However, the approach in mice may offer more interesting outcomes in the study of inflammation3,14,15. In this study, we will show a checklist of the steps to be followed for the reproduction of severe acute pancreatitis by infusion of sodium taurocholate in C57BL/6 anesthetized mice.
For works that involve the need for experiments with antibodies and analysis of the gene and protein expression, the use of mice is preferable because of the greater arsenal of materials for these animals and the possibility of working with isogenic and knockout species, among others that can be used relevant to studies16. Mice C57BL/6 is an inbred strain of mice originally developed for the study of antitumor activity and immunology. This strain is increasingly being preferred by researchers for being isogenic, allowing for a greater reproducibility of results, which may imply the use of a smaller number of animals in an experiment and less variability of results between the same group17,18.
Perides et al. (2010)14 published a protocol for AP induction in mice by sodium taurocholate infusion. Here we update this model using a higher sodium taurocholate concentration (2.5%) in C57BL/6 mice, with a defined volume and speed of infusion (Figure 1). The maximal level of severity is reached within 12 h of induction in mice. The elevation of the concentration of IL-6 both in the serum and in the peritoneal cavity is correlated with the progression of AP. With practice, the total estimated time from the induction of anesthesia to the completion of the infusion, is 25 min per animal. It is essential that a trained researcher conducts this experiment. To ensure that the solution is properly injected into the common bile duct, perform several pilot training sessions using methylene blue instead of sodium taurocholate.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.4 mm needle</td>
			<td>INTRAG MEDICAL TECH</td>
			<td>90183210</td>
			<td>30G</td>
		</tr>
		<tr>
			<td>&#160;0.54 mm polyethylene tube</td>
			<td>Tygon</td>
			<td>730010</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Styrofoam block</td>
			<td>-</td>
			<td>-</td>
			<td>-</td>
		</tr>
		<tr>
			<td>masking tape for mounting the mouse</td>
			<td>Missner</td>
			<td>1236</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Infusion pump scheduled to 10&#181;L / min.</td>
			<td>Havard aparatus-Peristaltic Pump Series</td>
			<td>MA1 55-7766</td>
			<td>&#160;Model 66 Small Peristaltic</td>
		</tr>
		<tr>
			<td>Scissors and forceps</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Antiseptic providine iodine</td>
			<td>Pfizer</td>
			<td>12086OR</td>
			<td>antisepsis</td>
		</tr>
		<tr>
			<td>70% ethanol</td>
			<td>SIGMA</td>
			<td>459836</td>
			<td>Mix 700 mL 100% ethanol with 300 mL dH2O</td>
		</tr>
		<tr>
			<td>Razor blade</td>
			<td>Lord</td>
			<td>bdk9a1ghk6</td>
			<td>For trichotomy</td>
		</tr>
		<tr>
			<td>Sodium taurocholate</td>
			<td>Sigma-Aldrich</td>
			<td>86339- 1G</td>
			<td>CAS NUMBER- 345909-26-4</td>
		</tr>
		<tr>
			<td>microvessel clip</td>
			<td>Medicon Surgical</td>
			<td>56.87.35</td>
			<td>Approximator, opening 4.0 mm, closing pressure 30 -&#160;40 g</td>
		</tr>
		<tr>
			<td>6-0 prolene</td>
			<td>Bioline</td>
			<td>5162</td>
			<td>Suture line</td>
		</tr>
		<tr>
			<td>Ketamin NP (cloridrato de dextrocetamina) 50mg/mL</td>
			<td>Crist&#225;lia</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Xilazine 2%</td>
			<td>Syntec</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile saline solution (0.9% (wt/vol) saline)</td>
			<td>Farmace</td>
			<td>105851</td>
			<td></td>
		</tr>
		<tr>
			<td>Methyl Blue</td>
			<td>Sigma-Aldrich Chemicals</td>
			<td>M5528</td>
			<td></td>
		</tr>
		<tr>
			<td>MILLIPLEX&#160;MAP&#160;Mouse Cytokine/Chemokine Magnetic Bead Panel - Immunology Multiplex Assay</td>
			<td>MERCK</td>
			<td>MCYTOMAG-70K</td>
			<td>Simultaneously analyze multiple cytokine and chemokine biomarkers with Bead-Based Multiplex Assays using the Luminex technology, in mouse serum, plasma and cell culture samples.</td>
		</tr>
		<tr>
			<td>Amylase Assay</td>
			<td>Labtest</td>
			<td>11</td>
			<td></td>
		</tr>
		<tr>
			<td>Desmarres retractor 13-mm 
			width</td>
			<td>ROBOZ</td>
			<td>RS-6672</td>
			<td></td>
		</tr>
	</tbody>
</table>

sodium taurocholate induced severe acute pancreatitis in c57bl/6 mice

Biliary acute pancreatitis induction by sodium taurocholate infusion has been widely used by the scientific community due to the representation of the human clinical condition and reproduction of inflammatory events corresponding to the onset of clinical biliary pancreatitis. The severity of pancreatic damage can be assessed by measuring the concentration, speed, and volume of the infused bile acid. This study provides an updated checklist of the materials and methods used in the protocol reproduction and shows the main results from this acute pancreatitis (AP) model. Most of the previous publications have limited themselves to reproducing this model in rats. We have applied this method in mice, which provides additional advantages (i.e., the availability of an arsenal of reagents and antibodies for these animals along with the possibility of working with genetically modified strains of mice) that may be relevant to the study. For acute pancreatitis induction in mice, we present a systematic protocol, with a defined dose of 2.5% sodium taurocholate at an infusion speed 10 &#181;L/min for 3 min in C57BL/6 mice that reaches its maximal level of severity within 12 h of induction and highlight results with outcomes that validate the method. With practice and technique, the total estimated time, from the induction of anesthesia to the completion of the infusion, is 25 min per animal.

Pancreatitis severity was scored between 0-3 according to the Schmidt&#39;s scale21 where zero corresponds to the absence, 1 corresponds to a mild presence (&#60;25%), 2 corresponds to a moderate presence (between 25 and 50%) and 3 corresponds to an intense presence (&#62; 50%) (Table 1). The measurements performed were plasma amylase activity, pancreatic edema, acinar cell, injury/necrosis, pancreatic inflammation (by histology analysis of H&#38;E...

Watch this Scientific Journal Video about Sodium Taurocholate Induced Severe Acute Pancreatitis in C57BL/6 Mice at JoVE.com

Sodium Taurocholate Induced Severe Acute Pancreatitis in C57BL/6 Mice

Animal models for severe acute pancreatitis enable the study of pathophysiological changes at the initial stage, facilitating observation of the evolution of inflammatory events. Here we provide a protocol for the induction of severe acute biliary pancreatitis by retrograde infusion of sodium taurocholate into the pancreatic duct of anesthetized C57BL/6 mice.

Biliary acute pancreatitis induction by sodium taurocholate infusion has been widely used by the scientific community due to the representation of the ...

sodium-taurocholate-induced-severe-acute-pancreatitis-in-c57bl6-mice

Research

JoVE Journal

Medicine

3.9K Views.  University of São Paulo Medical School. Animal models for severe acute pancreatitis enable the study of pathophysiological changes at the initial stage, facilitating observation of the evolution of inflammatory events. Here we provide a protocol for the induction of severe acute biliary pancreatitis by retrograde infusion of sodium taurocholate into the pancreatic duct of anesthetized C57BL/6 mice. 

Video: Sodium Taurocholate Induced Severe Acute Pancreatitis in C57BL/6 Mice

Expired CO2 Measurement in Intubated or Spontaneously Breathing Patients from the Emergency Department

Carbon dioxide (CO2) along with oxygen (O2) share the role of being the most important gases in the human body. The measuring of expired CO2 at the mouth has solicited growing clinical interest among physicians in the emergency department for various indications: (1) surveillance et monitoring of the intubated patient; (2) verification of the correct positioning of an endotracheal tube; (3) monitoring of a patient in cardiac arrest; (4) achieving normocapnia in intubated head trauma patients; (5) monitoring ventilation during procedural sedation. The video allows physicians to familiarize themselves with the use of capnography and the text offers a review of the theory and principals involved. In particular, the importance of CO2 for the organism, the relevance of measuring expired CO2, the differences between arterial and expired CO2, the material used in capnography with their artifacts and traps, will be reviewed. Since the main reluctance in the use of expired CO2 measurement is due to lack of correct knowledge concerning the physiopathology of CO2 by the physician, we hope that this explanation and the video sequences accompanying will help resolve this limitation.

Expired CO2 Measurement in Intubated or Spontaneously Breathing Patients from the Emergency Department

This video describes how to perform CO2 measurement in intubated as well as spontaneously breathing patients. The main clinical indications refer to emergency situations: (1) verifying adequate positioning of an endotracheal tube; (2) achieving normocapnia in trauma patients; (3) monitoring ventilation in the case of procedural sedation.

Carbon dioxide (CO2) along with oxygen (O2) share the role of being the most important gases in the human body. The measuring of expired CO2 at the mouth ...

Pressure Controlled Ventilation to Induce Acute Lung Injury in Mice

Murine models are extensively used to investigate acute injuries of different organs systems (1-34). Acute lung injury (ALI), which occurs with prolonged mechanical ventilation, contributes to morbidity and mortality of critical illness, and studies on novel genetic or pharmacological targets are areas of intense investigation (1-3, 5, 8, 26, 30, 33-36). ALI is defined by the acute onset of the disease, which leads to non-cardiac pulmonary edema and subsequent impairment of pulmonary gas exchange (36). We have developed a murine model of ALI by using a pressure-controlled ventilation to induce ventilator-induced lung injury (2). For this purpose, C57BL/6 mice are anesthetized and a tracheotomy is performed followed by induction of ALI via mechanical ventilation. Mice are ventilated in a pressure-controlled setting with an inspiratory peak pressure of 45 mbar over 1 - 3 hours. As outcome parameters, pulmonary edema (wet-to-dry ratio), bronchoalveolar fluid albumin content, bronchoalveolar fluid and pulmonary tissue myeloperoxidase content and pulmonary gas exchange are assessed (2). Using this technique we could show that it sufficiently induces acute lung inflammation and can distinguish between different treatment groups or genotypes (1-3, 5). Therefore this technique may be helpful for researchers who pursue molecular mechanisms involved in ALI using a genetic approach in mice with gene-targeted deletion.

A murine model for ventilator induced lung injury is an important tool to study an acute lung injury in vivo. Here, we report an easy applicable in situ model for acute lung injury using high-pressure mechanical ventilation to induce acute failure of the lung.

Murine models are extensively used to investigate acute injuries of different organs systems (1-34). Acute lung injury (ALI), which occurs with prolonged ...

Ischemia-reperfusion Model of Acute Kidney Injury and Post Injury Fibrosis in Mice

Ischemia-reperfusion induced acute kidney injury (IR-AKI) is widely used as a model of AKI in mice, but results are often quite variable with high, often unreported mortality rates that may confound analyses. Bilateral renal pedicle clamping is commonly used to induce IR-AKI, but differences between effective clamp pressures and/or renal responses to ischemia between kidneys often lead to more variable results. In addition, shorter clamp times are known to induce more variable tubular injury, and while mice undergoing bilateral injury with longer clamp times develop more consistent tubular injury, they often die within the first 3 days after injury due to severe renal insufficiency. To improve post-injury survival and obtain more consistent and predictable results, we have developed two models of unilateral ischemia-reperfusion injury followed by contralateral nephrectomy. Both surgeries are performed using a dorsal approach, reducing surgical stress resulting from ventral laparotomy, commonly used for mouse IR-AKI surgeries. For induction of moderate injury BALB/c mice undergo unilateral clamping of the renal pedicle for 26 min and also undergo simultaneous contralateral nephrectomy. Using this approach, 50-60% of mice develop moderate AKI 24 hr after injury but 90-100% of mice survive. To induce more severe AKI, BALB/c mice undergo renal pedicle clamping for 30 min followed by contralateral nephrectomy 8 days after injury. This allows functional assessment of renal recovery after injury with 90-100% survival. Early post-injury tubular damage as well as post injury fibrosis are highly consistent using this model.

We describe models of moderate and severe ischemia-reperfusion-induced kidney injury in which the mice undergo unilateral renal pedicle clamping followed by simultaneous or delayed contralateral nephrectomy, respectively. These models consistently give rise to renal dysfunction and post-injury fibrosis, but injury severity and survival are dependent on mouse background, age and surgical equipment.

Ischemia-reperfusion induced acute kidney injury (IR-AKI) is widely used as a model of AKI in mice, but results are often quite variable with high, often ...

Acute Brain Trauma in Mice Followed By Longitudinal Two-photon Imaging

Although acute brain trauma often results from head damage in different accidents and affects a substantial fraction of the population, there is no effective treatment for it yet. Limitations of currently used animal models impede understanding of the pathology mechanism. Multiphoton microscopy allows studying cells and tissues within intact animal brains longitudinally under physiological and pathological conditions. Here, we describe two models of acute brain injury studied by means of two-photon imaging of brain cell behavior under posttraumatic conditions. A selected brain region is injured with a sharp needle to produce a trauma of a controlled width and depth in the brain parenchyma. Our method uses stereotaxic prick with a syringe needle, which can be combined with simultaneous drug application. We propose that this method can be used as an advanced tool to study cellular mechanisms of pathophysiological consequences of acute trauma in mammalian brain in vivo. In this video, we combine acute brain injury with two preparations: cranial window and skull thinning. We also discuss advantages and limitations of both preparations for multisession imaging of brain regeneration after trauma.

Acute brain trauma is a severe injury that has no adequate treatment to date. Multiphoton microscopy allows studying longitudinally the process of acute brain trauma development and probing therapeutical strategies in rodents. Two models of acute brain trauma studied with in vivo two-photon imaging of brain are demonstrated in this protocol.

Although acute brain trauma often results from head damage in different accidents and affects a substantial fraction of the population, there is no ...

The bm12 Inducible Model of Systemic Lupus Erythematosus (SLE) in C57BL/6 Mice

Systemic lupus erythematosus (SLE) is an autoimmune disease with diverse clinical and immunological manifestations. Several spontaneous and inducible animal models mirror common components of human disease, including the bm12 transfer model. Upon transfer of bm12 splenocytes or purified CD4 T cells, C57BL/6 mice rapidly develop large frequencies of T follicular helper cells (Tfh), germinal center (GC) B cells, and plasma cells followed by high levels of circulating anti-nuclear antibodies. Since this model utilizes mice on a pure C57BL/6 background, researchers can quickly and easily study disease progression in transgenic or knockout mouse strains in a relatively short period of time. Here we describe protocols for the induction of the model and the quantitation Tfh, GC B cells, and plasma cells by multi-color flow cytometry. Importantly, these protocols can also be used to characterize disease in most mouse models of SLE and identify Tfh, GC B cells, and plasma cells in other disease models.

The bm12 Inducible Model of Systemic Lupus Erythematosus SLE in C57BL/6 Mice

The transfer of bm12 lymphocytes into a C57BL/6 recipient is an established model of systemic lupus erythematosus. Here we describe how to initiate disease using this model and how to characterize T follicular helper cells, germinal center B cells and plasma cells by flow cytometry.

Systemic lupus erythematosus (SLE) is an autoimmune disease with diverse clinical and immunological manifestations. Several spontaneous and inducible ...

Murine Distal Colostomy, A Novel Model of Diversion Colitis in C57BL/6 Mice

Diversion colitis (DC) is a frequent clinical condition occurring in patients with bowel segments excluded from the fecal stream as a result of a diverting enterostomy. The etiology of this disease remains ill-defined but appears to differ from that of classical inflammatory bowel diseases such as Crohn's disease and ulcerative colitis. Research aimed to decipher the pathophysiological mechanisms leading to the development of this disease has been severely hampered by the lack of an appropriate murine model. This protocol generates a murine model of DC that facilitates the study of the immune system's role and its interaction with the microbiome in the development of DC. In this model using C57BL/6 animals, distal parts of the colon are excluded from the fecal stream by creating a distal colostomy, triggering the development of mild to moderate inflammation in the excluded bowel segments and reproducing the hallmark lesions of human DC with a moderate systemic inflammatory response. In contrast to the rat model, a large number of genetically-modified murine models on the C57BL/6 background are available. The combination of these animals with our model allows the potential roles of individual cytokines, chemokines, or receptors of bioactive molecules (e.g., interleukin (IL)-17; IL-10, chemokine CXCL13, chemokine receptors CXCR5 and CCR7, and the sphingosine-1-phosphate receptor 4) to be assessed in the pathogenesis of DC. The availability of congenic mouse strains on the C57BL/6 background largely facilitates transfer experiments to establish the roles of distinct cell types involved in the etiology of DC. Finally, the model offers the opportunity to assess the influences of local interventions (e.g., modification of the local microbiome or local anti-inflammatory therapy) on mucosal immunity in affected and non-affected bowel segments and the on systemic immune homeostasis.

Murine distal colostomy provides a murine model for human diversion colitis, a predominantly lymphocytic colitis in colon segment excluded from the fecal stream.

Diversion colitis (DC) is a frequent clinical condition occurring in patients with bowel segments excluded from the fecal stream as a result of a ...

Real-time Pressure-volume Analysis of Acute Myocardial Infarction in Mice

Acute myocardial infarction can lead to acute heart failure and cardiogenic shock. The evaluation of hemodynamics is critical for the evaluation of any potential therapeutic approach directed against acute left ventricular (LV) dysfunction. Current imaging modalities (e.g., echocardiography and magnetic resonance imaging) have several limitations since data on LV pressure cannot directly be measured. LV catheterization in mice undergoing coronary artery occlusion could serve as a novel method for a real-time evaluation of LV function.
At the beginning of the procedure, mice were anesthetized followed by endotracheal intubation. For LV catheterization, the right carotid artery was exposed via middle-neck incision. The catheter was introduced and placed into the LV cavity. Left thoracotomy was conducted and the left main coronary artery (LCA) was ligated. To induce reperfusion, the suture was released after 45 min. Pressure-volume data was recorded at all times.
Ligation of the LCA caused a decrease in LV systolic function as evidenced by a 30% reduction in stroke volume, LV ejection fraction (EF), and cardiac output. Maximum dP/dt as a parameter for LV contractility was also significantly reduced and diastolic function was severely impaired (minimum dP/dt -40%). Reperfusion over a period of 20 min did not lead to a complete recovery of LV function.
Real-time pressure-volume analysis served as a valid procedure for monitoring cardiac function during acute myocardial infarction in mice. Maintaining stable anesthesia and a standardized surgical approach was crucial to ensure valid results. As the early phase of acute myocardial infarction is critical for morbidity and mortality, the delineated method could be beneficial for preclinical evaluation of new strategies for cardioprotection.

Acute myocardial infarction in mice induces acute but incompletely characterized changes in left ventricular (LV) function. LV catheterization in mice undergoing coronary artery occlusion serves as a novel method for a real-time evaluation of LV function.

Acute myocardial infarction can lead to acute heart failure and cardiogenic shock. The evaluation of hemodynamics is critical for the evaluation of any ...

Inducing Acute Lung Injury in Mice by Direct Intratracheal Lipopolysaccharide Instillation

Airway administration of lipopolysaccharide (LPS) is a common way to study pulmonary inflammation and acute lung injury (ALI) in small animal models. Various approaches have been described, such as the inhalation of aerosolized LPS as well as nasal or intratracheal instillation. The presented protocol describes a detailed step-by-step procedure to induce ALI in mice by direct intratracheal LPS instillation and perform FACS analysis of blood samples, bronchoalveolar lavage (BAL) fluid, and lung tissue. After intraperitoneal sedation, the trachea is exposed and LPS is administered via a 22 G venous catheter. A robust and reproducible inflammatory reaction with leukocyte invasion, upregulation of proinflammatory cytokines, and disruption of the alveolo-capillary barrier is induced within hours to days, depending on the LPS dosage used. Collection of blood samples, BAL fluid, and lung harvesting, as well as the processing for FACS analysis, are described in detail in the protocol. Although the use of the sterile LPS is not suitable to study pharmacologic interventions in infectious diseases, the described approach offers minimal invasiveness, simple handling, and good reproducibility to answer mechanistic immunological questions. Furthermore, dose titration as well as the use of alternative LPS preparations or mouse strains allow modulation of the clinical effects, which can exhibit different degrees of ALI severity or early vs. late onset of disease symptoms.

Presented here is a step-by-step procedure to induce acute lung injury in mice by direct intratracheal lipopolysaccharide instillation and to perform FACS analysis of blood samples, bronchoalveolar lavage fluid, and lung tissue. Minimal invasiveness, simple handling, good reproducibility, and titration of disease severity are advantages of this approach.

Airway administration of lipopolysaccharide (LPS) is a common way to study pulmonary inflammation and acute lung injury (ALI) in small animal models. ...

Acute Kidney Injury Model Induced by Cisplatin in Adult Zebrafish

Cisplatin is commonly used as chemotherapy. Although it has positive effects in cancer-treated individuals, cisplatin can easily accumulate in the kidney due to its low molecular weight. Such accumulation causes the death of tubular cells and can induce the development of Acute Kidney Injury (AKI), which is characterized by a quick decrease in kidney function, tissue damage, and immune cells infiltration. If administered in specific doses cisplatin can be a useful tool as an AKI inducer in animal models. The zebrafish has appeared as an interesting model to study renal function, kidney regeneration, and injury, as renal structures conserve functional similarities with mammals. Adult zebrafish injected with cisplatin shows decreased survival, kidney cell death, and increased inflammation markers after 24 h post-injection (hpi). In this model, immune cells infiltration and cell death can be assessed by flow cytometry and TUNEL assay. This protocol describes the procedures to induce AKI in adult zebrafish by intraperitoneal cisplatin injection and subsequently demonstrates how to collect the renal tissue for flow cytometry processing and cell death TUNEL assay. These techniques will be useful to understand the effects of cisplatin as a nephrotoxic agent and will contribute to the expansion of AKI models in adult zebrafish. This model can also be used to study kidney regeneration, in the search for compounds that treat or prevent kidney damage and to study inflammation in AKI. Moreover, the methods used in this protocol will improve the characterization of tissue damage and inflammation, which are therapeutic targets in kidney-associated comorbidities.

This protocol describes the procedures to induce Acute Kidney Injury (AKI) in adult zebrafish using cisplatin as a nephrotoxic agent. We detailed the steps to evaluate the reproducibility of the technique and two techniques to analyze inflammation and cell death in the renal tissue, flow cytometry and TUNEL, respectively.

Cisplatin is commonly used as chemotherapy. Although it has positive effects in cancer-treated individuals, cisplatin can easily accumulate in the kidney ...

Fertility Preservation in Patients with Severe Ovarian Dysfunction

Ovarian function progressively declines during aging and in some pathophysiological conditions including karyotype abnormality, autoimmune diseases, chemo- and radiation-therapies, as well as ovarian surgeries. In unmarried women with severe ovarian dysfunction, fertility preservation is important for future pregnancies. Although oocyte cryopreservation is an established method for fertility preservation, these patients could only preserve a limited number of oocytes even after ovarian hyperstimulation, leading to repeated stimulations to ensure sufficient oocytes to guarantee future pregnancy. To solve this issue, we have recently developed a drug-free in vitro activation (IVA) procedure, which enable us to stimulate early stages of ovarian follicles to develop to the preantral follicle stage. These preantral follicles can respond to the unique protocol of gonadotropin stimulation, resulting in increased number of retrieved oocytes per ovarian stimulation for cryopreservation. The drug-free IVA comprised from the surgical approach and ovarian stimulation. We removed a part of cortex from one or both ovaries from patients under laparoscopic surgery. The ovarian cortical tissues were cut into small cubes to disrupt the Hippo signaling pathway and stimulate the development of early stage follicles. These cubes were grafted orthotropically into remaining ovaries as well as beneath the serosa of both Fallopian tubes. We have already published the surgical procedure of the drug-free IVA and the protocol of subsequent ovarian stimulation, but herein we present the details of laboratory methods required for drug-free IVA.

We present details of laboratory procedures for drug-free in vitro activation (IVA) of ovarian follicles for patients with severe ovarian dysfunction. This method could increase the number of retrievable oocytes per ovarian hyperstimulation and benefit fertility preservation for those patients.

Ovarian function progressively declines during aging and in some pathophysiological conditions including karyotype abnormality, autoimmune diseases, ...