This work was supported by National Institutes of Health Grants R01 AI138203-01, AI109317-04, AI101973-01, and AI097532-01A1 to M. W. The University of North Dakota Core Facilities were supported by NIH grants (INBRE P20GM103442 and COBRE P20GM113123). This work was also supported by the Key Program of National Nature Science Foundation of China (81530063) to Jianxin Jiang. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. We thank Marvin Leier (Center for Rural Health, University of North Dakota) for making the video.

All methods described here were performed in accordance with the National Institute of Health Guide for Care and Use of Laboratory Animals and approved by the University of North Dakota Institutional Animal Care and Use Committee (IACUC).
1. Cecal ligation and puncture
NOTE: Female C57BL/6 mice (weight, 18-22 g; age, 6-8 weeks) are randomly divided into six groups: control group (Ctrl), infection group (SA for S. aureus), two sham groups, and two CLP groups. ...

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As the gold standard model for sepsis research, CLP has a combination of three insults, including tissue trauma caused by the laparotomy, necrosis due to ligation of the cecum, and infection as a result of microbial leakage that causes peritonitis with translocation of bacteria into blood8. Therefore, CLP mimics the complexity of human sepsis better than many other models. However, a major limitation of current CLP model is the inability to reflect the more prolonged phase of sepsis seen in patien...

Sepsis initiates a complex interplay of host pro-inflammatory and anti-inflammatory processes and is characterized by a hyperinflammatory response and subsequent immune dysfunction1,2. Sepsis represents a global health priority and causes a high number of deaths in intensive care units (ICUs)3. The incidence of sepsis is estimated to exceed 30 million cases worldwide per year, with mortality rates as high as 30% despite advances in ICU management4,5. In 2017, the World Health Organization adopted a resolution to improve the prevention, diagnosis, and management of this deadly disease5. However, recent studies have illustrated that death does not result from primary infection in severe septic patients but rather from secondary nosocomial infection (particularly pneumonia) that caused by immunosuppression6,7. Therefore, understanding the mechanisms of why septic patients develop secondary infection and discovering more effective treatments are urgently required. Herein, a dual model, also known as a double-hit model, to study the immunosuppressive phenomenon occurring in patients with protracted sepsis is described. 
As the gold standard experimental model in research on polymicrobial sepsis, cecal ligation and puncture (CLP) is a surgery characterized by cecum ligation and perforation, which contributes to polymicrobial peritonitis and sepsis8,9. The pathophysiological process and cytokine profiles, along with the kinetics and magnitude, are similar to clinical sepsis. The position of the ligation, needle size used for the puncture, and number of cecal punctures are major factors that impact the mortality following CLP.
The nosocomial pneumonia is the leading cause of mortality among critically ill patients with sepsis. The major type of organisms causing severe sepsis includes Staphylococcus aureus (20.5%), Pseudomonas species (19.9%), Enterobacteriacae (mainly E. coli, 16.0%), and fungi (19%). Meanwhile, recent studies have suggested an increasing incidence of gram-positive organisms, which are now almost as common as gram-negative infections3.
The method described in this protocol involves CLP, performed as the "first hit" to induce sublethal polymicrobial peritonitis, which manifests an immunosuppression condition. The procedure also involves subsequent intranasal instillation of S. aureus as the "second hit" to provide a clinically relevant research platform.

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			<td>Invitrogen</td>
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			<td style="width:201px;">NDC 0856-2013-01</td>
			<td>Amine hydrochloride injection</td>
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			<td>Molecular genetics, powder</td>
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			<td style="width:201px;">7100-1100</td>
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			<td style="width:191px;">DAVIS &#38; GECK</td>
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			<td style="width:191px;">General Supply</td>
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			<td style="width:191px;">General Supply</td>
			<td style="width:201px;">General Supply</td>
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			<td>ATCC</td>
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			<td style="width:191px;">General Supply</td>
			<td style="width:201px;">General Supply</td>
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			<td style="width:191px;">Alfa Aesar</td>
			<td style="width:201px;">7361-61-7</td>
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design of cecal ligation and puncture and intranasal infection dual model of sepsis-induced immunosuppression

Sepsis, a severe and complicated life-threatening infection, is characterized by an imbalance between pro- and anti-inflammatory responses in multiple organs. With the development of therapies, most patients survive the hyperinflammatory phase but progress to an immunosuppressive phase, which increases the emergence of secondary infections. Therefore, improved understanding of the pathogenesis underlying secondary hospital-acquired infections in the immunosuppressive phase during sepsis is of tremendous importance. Reported here is a model to test infectious outcomes by creating double-hit infections in mice. A standard surgical procedure is used to induce polymicrobial peritonitis by cecal ligation and puncture (CLP) and followed by intranasal infection of Staphylococcus aureus to simulate pneumonia occurring in immune suppression that is frequently seen in septic patients. This dual model can reflect the immunosuppressive state occurring in patients with protracted sepsis and susceptibility to secondary infection from nosocomial pneumonia. Hence, this model provides a simple experimental approach to investigate the pathophysiology of sepsis-induced secondary bacterial pneumonia, which may be used for discovering novel treatments for sepsis and its complications.

Depending on the experimental design and procedures, C57BL/6 mice were subjected to CLP, and after 3 days, they were administered bacteria intranasally (Figure 1). As shown in Figure 2, the mice began to die at ~12 h after induction of peritonitis. Two mice in the CLP+SA group and three mice in the CLP+NS group died before intranasal S. aureus instillation. No mortality was detected in uninfected non- or sham-operated mi...

Watch this Scientific Journal Video about Design of Cecal Ligation and Puncture and Intranasal Infection Dual Model of Sepsis-Induced Immunosuppression at JoVE.com

Design of Cecal Ligation and Puncture and Intranasal Infection Dual Model of Sepsis-Induced Immunosuppression

This protocol describes techniques to measure infectious outcomes underlying secondary hospital-acquired infections in the immunosuppressive condition, first by establishing cecal ligation/puncture mice then challenging them with intranasal infection to create a clinically relevant model of immunosuppression sepsis.

Sepsis, a severe and complicated life-threatening infection, is characterized by an imbalance between pro- and anti-inflammatory responses in multiple ...

Prepare the mouse for surgery. Shave the lower quadrants of the abdomen using a razor and disinfect the area with iodine. Place the mouse onto a clean work surface on its back with the head pointing away from the operator.Make a longitudinal skin incision about a half a centimeter long in the left lower abdomen. Be careful not to penetrate the peritoneal cavity. Use small scissors to extend the incision about one to two centimeters.Make an intramuscular incision to gain access into the peritoneal cavity. In most cases, the cecum is on the left side of the abdomen. Isolate the cecum.Using blunt, anatomical forceps, gently and carefully expose the cecum through the incision. Step four is the ligation. The position of the ligation on the cecum determines the severity level of sepsis.Ligating approximately 75%of the cecum induces severe sepsis. Ligating 50%of the cecum induces mid-level sepsis. Whereas ligating 25%or less of the cecum will result in minor sepsis.Ligate the cecum at the designated position. Make sure not to ligate the ileocecal valve. Step five is to puncture the cecum.Using a needle, perforate the cecum with a single through and through puncture mid-way between the ligation and the tip of the cecum. Make sure you do not puncture any blood vessels. To ensure that there was a complete puncture, extrude a small droplet of feces from both needle holes.Carefully replace the cecum into the abdominal cavity. Close the abdominal musculature by applying simple running sutures and close the skin. Clean the skin with iodine.Inject the mouse with a pre-warmed saline solution to resuscitate. Place the mouse back into the cage with unlimited access to food and water. After about three days from the cecal ligation and puncture or the CLP procedure, we subject the mouse to a second hit with an intranasal infection to stimulate pneumonia that often occurs in sepsis.Approach the neck of the mouse from behind, smoothly and gently. The thumb and index finger of the left hand form a U-shaped position. Hold the neck immediately behind the ears so the mouse head is completely restrained.Introduce the intranasal infection. Using a 100 microliter pipette, instill the staphylococcus aureus strain intranasally. Holding the mouse in an upright position, slowly drop two to three microliters of the bacterial solution into both nostrils of the mouse.Raise the mouse up and down with its head up and tail down to help the bacterial solution enter the trachea. Raise the mouse up quickly while lowering it gently and slowly. Repeat the instillation 15 times or until all the bacterial solution is instilled.The last step is to finish the procedure. Lay the mouse on the angled bedding at about a 35 degree angle to recover. Make sure the mouse is on its back with its head laying up.Watch the mouse to make sure it is completely recovered from the anesthesia and the procedure. After flattening the bedding, place the mouse back in the cage with unlimited access to food and water. Mice began to die at around 12 hours after induction of peritonitis.The mortality was much higher for mice with CLP before a pneumonia. Three days post-surgery, CLP mice had more mortality with an intranasal administration of staphylococcus aureus. The levels of serum pro-inflammatory cytokines significantly increased at 24 hours after bacterial instillation in sepsis mice compared to the mice that had CLP alone or the Sham operated mice with a second hit.However, double-hit mice exhibited significant decrease in BAL fluid cytokine levels compared to both SA and Sham surgery plus SA mice. Hence CLP impairs host defense and increases susceptibility to infection. This dual-hit model is a valuable tool to understand the molecular mechanisms for host-pathogen interaction, which may open the avenue to develop new, effective drugs and other therapeutics for superbug infections.

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Research

JoVE Journal

Immunology and Infection

9.7K visualizações.  Sichuan University. Prepare o rato para a cirurgia. Raspe os quadrantes inferiores do abdômen usando uma navalha e desinfete a área com iodo. Coloque o mouse sobre uma superfície de trabalho limpa em suas costas com a cabeça apontando para longe do operador.Faça uma incisão longitudinal da pele de cerca de meio centímetro de comprimento no abdômen inferior esquerdo. Tenha cuidado para não penetrar na cavidade peritoneal. Use uma tesoura pequena para estender a incisão de cerca de um a dois cent?...