This work was funded by NIH grant CA113734 awarded to F Yull and the Department of Veterans Affairs (TS Blackwell).

All animal experiments were approved by the Vanderbilt University Institutional Animal Care and Use Committee.
1. Preparing Transgenic Mice
<ol>
 <li>Female mice, positive for the NGL (NF-&kappa;B-GFP-Luciferase) reporter transgene (described previously1) are used for this procedure. When females are at least 6 weeks of age, mate them with an FVB wild type male. Harem breeding may be used (up to 4 females in a cage with 1 male). (Please note: FVB strain mice were used for the current study. Darker strain mice such as C57Bl6 may also be used, but these mice must be shaved prior to imaging).</li>
 <li>Starting at 15 days post-mating, check the females regularly for signs of pregnancy (large protruding girth). Each female must be separated once pregnant and the male removed from the breeding cage once the last female becomes pregnant.</li>
 <li>Watch the separated females and note the date of birth when a litter appears. A litter of at least 6 pups is required for adequate lactation. Eight days after the birth of the litter, proceed with the protocol (see Figure 1 for a timeline schematic).</li>
</ol>
2. Preparation for Injection
<ol>
 <li>Dissolve the E. coli LPS in PBS to a final concentration of 0.1 &mu;g/&mu;l. 50 &mu;l of this solution, or a total of 5 &mu;g, will be injected into the mammary gland. Prepare a second tube of PBS alone to be used for control injection.</li>
 <li>Set-up a dissecting scope and isoflurane anesthesia equipment. A nose-cone apparatus should be secured to the stage of the dissecting scope where the mouse will be placed. A good light source, whether from the scope itself or from extra spotlights should also be prepared.</li>
 <li>Once the set-up is complete, start the isoflurane machine and air flow.</li>
</ol>
3. Intraductal Injection of LPS
<ol>
 <li>Load the short needle syringe with 50 &mu;l of the LPS solution. Set this aside.</li>
 <li>Anesthetize the mouse to be injected by first placing the female into an isoflurane filled box and then once breathing is slowed, move her onto the stage and place her nose within the nose-cone. Use a toe pinch to confirm the mouse is fully anesthetized. Monitor the mouse's breathing throughout the procedure and adjust the isoflurane level accordingly. The cage containing the litter of pups that the female has been taken from should be carefully set aside until the dam can be returned after the procedure.</li>
 <li>Use a small drop of 70% ethanol to make the coat lay flat and to help visualize the nipple attached to the right #4 inguinal mammary gland on the mouse's lower abdomen.</li>
 <li>Focus the dissecting scope for an optimal view of the nipple. Carefully use a set of fine forceps to further draw out the nipple away from the skin, but do not puncture or injure the nipple in any way. The forceps should never be tightly clamped together when manipulating the tissue, but used with a light hold which requires the minimal amount of stress on the nipple.</li>
 <li>Use the forceps in the left hand to gently hold the nipple in place. Slowly bring the preloaded syringe into the field of view using the right hand. The syringe should be held so that no repositioning has to occur in order to inject the contents.</li>
 <li>Once the syringe is in view, aim carefully at the small ductal opening within the center of the nipple, and insert the syringe.</li>
 <li>Without repositioning the hand, inject the contents of the syringe and remove the needle from the nipple.</li>
 <li>Load a second syringe with 50 &mu;l of PBS for the control injection. Reposition the mouse on the stage and for the best view of the left #4 inguinal mammary gland nipple on the other side of the mouse's lower abdomen. Repeat steps 3.4 to 3.7 using the PBS-loaded syringe.</li>
 <li>Once both injections are complete, discontinue anesthesia and move the mouse to a recovery cage. Monitor the female to insure that she becomes mobile within 5 min. Allow the mouse to remain in the recovery cage for 1 hr. After 1 hr of recovery, place the female back into the cage with her litter of pups. Mice should be periodically monitored for 6 hr post injection before proceeding to imaging. In our experience, no adverse consequences were noted in either the female or the pups during the period between injection and imaging. However, if signs of distress are observed in the female (e.g. hunched posture, biting or guarding of the injection site, general lethargy) or the pups (no movement, bluish color of skin) the experiment should be discontinued.</li>
</ol>
4. Bioluminescent Imaging to Visualize NF-&kappa;B Activity
<ol>
 <li>Once mice have been transported to the imaging facility, begin the imaging procedure by opening the software and initializing the machine. Turn on the isoflurane machine and air flow, ensuring that the anesthesia is flowing to both the box and the imaging chamber.</li>
 <li>Place the mouse under isoflurane anesthesia in the clear anesthesia box. Once breathing has slowed, use a toe pinch to confirm the mouse is fully anesthetized. Then, inject 100 &mu;l of luciferin substrate into the mouse by retro-orbital injection5 using a sterile syringe.</li>
 <li>Immediately transfer the mouse to the imaging chamber and place her nose under the isoflurane nose-cone located there. The mouse should be supine. Secure each foot to the stage with tape.</li>
 <li>Input the appropriate settings to the IVIS software to take both a white light photographic image and an image of the bioluminescent activity. The exposure time should be 10 sec. Acquire the image.</li>
 <li>Once images have been acquired, allow the mouse to recover and then place her back in the cage with her pups and monitor periodically.</li>
 <li>The photographic and luminescent images are automatically overlaid and displayed on the screen, as in Figure 2. Analyze the acquired images by placing region of interest or "ROI" circles of equal size over each side of the mouse's lower abdomen (one over the PBS-injected gland and one over the LPS-injected gland). Set the read-out to photons. A number should be displayed indicating the luminescence detected within each ROI (see Figure 2B). Use this number in statistical analysis.</li>
 <li>To display the optimum image, further adjust the threshold of signal displayed. This will not alter the total luminescence read-out but will allow you to remove background signal and visualize the differences between the control and LPS-treated gland. Decrease the amount of luminescence shown until the PBS-injected gland is free of background signal. This should leave the increased signal in the LPS-treated gland still apparent in the image, as seen in Figure 2.</li>
 <li>Additional analysis, including protein and RNA assays, can be completed with mammary tissue collected from the female post-mortem6.</li>
</ol>
5. Representative Results
To obtain interpretable results from these methods, LPS (or PBS) must be infused only into the mammary ducts, leaving the adjacent tissue undisturbed. Injection into the subcutaneous tissue surrounding the nipple will result in inflammation due to injury rather than the simulated infection. Injection of trypan blue dye diluted in PBS is helpful when learning the technique. Figure 3A shows a successfully injected gland infused with 100 &mu;l of trypan blue in PBS. As shown, only the ductal tree should be filled with the injected solution, and there should be no bolus at the injection site. A "bolus" in this context is a build up of the injected liquid at the nipple or in the tissue surrounding the nipple without infusion into the ductal tree. This is shown in Figure 3B. Please note that the practice injections shown in Figure 3 were completed in a female at day 21 of lactation. During the height of lactation (day 8-10, the stage at which LPS is injected using the current protocol), milk is abundant and it is difficult to visualize the ducts and determine if the injection has been completed correctly. Therefore, later time points in lactation are better for practice injections.
Successful injection of LPS into the mammary ducts results in increased NF-&kappa;B activity within the gland. This should be evident in the reporter mouse upon IVIS imaging, and the LPS-injected gland should have substantially higher luciferase activity (measured by photon count) than the PBS-injected gland, as shown in Figure 2 (on average, the LPS gland reads 200% of the PBS control signal). A statistically significant increase in luminescence within the LPS-treated glands as compared to the contralateral control was achieved though the completion of a group of 4 mice 4. One additional study has also utilized intraductal mammary injection of either E. coli or LPS into NF-&kappa;B reporter transgenics. Similar to our own work, these authors saw a significant increase in reporter activity 6 hr after E. coli injection2.
Please note, due to constitutive NF-&kappa;B activity in the brain and other abdominal organs, the heads and abdomens of our reporter mice consistently show a signal when imaged, even at baseline prior to any manipulation. Thus, this signal should be ignored with regard to this experiment. An obvious signal should remain within the LPS-injected mammary gland after the image read-out is adjusted to minimize background luminescence in the PBS gland. Figure 2 shows images with the background reduced.
Unsuccessful injection of the gland will most likely result in damage to the nipple. This will cause pointed, skin-level inflammation that will be evident upon imaging. This type of wounding signal will be very strong and confined to the nipple area where the damage occurred, not emanating throughout the gland, as seen after a successful LPS injection into the ducts.
<img alt="Figure 1" src="/files/ftp_upload/4030/4030fig1.jpg" /> 
 Figure 1. Schematic of Procedure.
<img alt="Figure 2" src="/files/ftp_upload/4030/4030fig2.jpg" /> 
 Figure 2. Representative results from IVIS bioluminescent imaging. (A) Acquired image with background reduced, revealing increased luciferase activity in the LPS-injected gland. (B) Image from A with region of interest quantifications added. <a href="https://www.jove.com/files/ftp_upload/4030/4030fig2large.jpg" target="_blank"> Click here to view larger figure</a>.
<img alt="Figure 3" src="/files/ftp_upload/4030/4030fig3.jpg" /> 
 Figure 3. Practice injection: mammary gland injected with dye via intraductal injection. To test the accuracy of delivery via the nipple, a mouse at day 21 of lactation was injected with 100 &mu;l of trypan blue dye diluted in PBS. Following sacrifice, the gland was exposed for visualization. (A) successful injection and (B) bolus of dye at the nipple.

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No conflicts of interest declared.

Here we utilize a transgenic NF-&kappa;B reporter mouse and an optimized injection technique to model mastitis, inflammation of the breast tissue most commonly caused by bacterial infection. The most critical step in this procedure is the intraductal injection itself.
In practice, it is usually apparent whether the intraductal injection has been successful. The injection of the agents (LPS or PBS) should feel fluid and there should be little to no resistance as the syringe is emptied. The ...

<table border="1">
 <tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>Lipopolysaccharide from Escherichia coli (LPS)</td>
 <td>Sigma</td>
 <td>L 2880</td>
 <td>serotype 055:B5 </td>
 </tr>
 <tr>
 <td>PBS</td>
 <td>Mediatech, Inc.</td>
 <td>46-013-CM</td>
 <td></td>
 </tr>
 <tr>
 <td>31 Gauge insulin syringe</td>
 <td>Becton Dickinson</td>
 <td>328438</td>
 <td>Short needle</td>
 </tr>
 <tr>
 <td>Forceps</td>
 <td>World Precision Instruments </td>
 <td>15908 </td>
 <td></td>
 </tr>
 <tr>
 <td>Dissecting scope</td>
 <td>Leica </td>
 <td>M3Z</td>
 <td></td>
 </tr>
 <tr>
 <td>Terrell Isoflurane, USP</td>
 <td>MINRAD INC. for Rx Elite</td>
 <td>NDC 66794-011-25</td>
 <td></td>
 </tr>
 <tr>
 <td>IVIS 200 imaging system</td>
 <td>Caliper (formerly Xenogen)</td>
 <td>N/A</td>
 <td></td>
 </tr>
 <tr>
 <td>Syringe for luciferin injection</td>
 <td>Becton Dickinson</td>
 <td>309597</td>
 <td>1cc; 26G5/8 </td>
 </tr>
 <tr>
 <td>D-Luciferin Firefly, sodium salt monohydrate (Luciferin substrate)</td>
 <td>Biosynth Chemistry and Biology</td>
 <td>L-8240</td>
 <td>Dilute to 10 mg/ml</td>
 </tr>
 </tbody>
</table>

intraductal injection of lps as a mouse model of mastitis: signaling visualized via an nf-&kappa;b reporter transgenic

Animal models of human disease are necessary in order to rigorously study stages of disease progression and associated mechanisms, and ultimately, as pre-clinical models to test interventions. In these methods, we describe a technique in which lipopolysaccharide (LPS) is injected into the lactating mouse mammary gland via the nipple, effectively modeling mastitis, or inflammation, of the gland. This simulated infection results in increased nuclear factor kappa B (NF-&kappa;B) signaling, as visualized through bioluminescent imaging of an NF-&kappa;B luciferase reporter mouse1.
Our ultimate goal in developing these methods was to study the inflammation associated with mastitis in the lactating gland, which often includes redness, swelling, and immune cell infiltration2,3. Therefore, we were keenly aware that incision or any type of wounding of the skin, the nipple, or the gland in order to introduce the LPS could not be utilized in our methods since the approach would likely confound the read-out of inflammation. We also desired a straight-forward method that did not require specially made hand-drawn pipettes or the use of micromanipulators to hold these specialized tools in place. Thus, we determined to use a commercially available insulin syringe and to inject the agent into the mammary duct of an intact nipple. This method was successful and allowed us to study the inflammation associated with LPS injection without any additional effects overlaid by the process of injection. In addition, this method also utilized an NF-&kappa;B luciferase reporter transgenic mouse and bioluminescent imaging technology to visually and quantitatively show increased NF-&kappa;B signaling within the LPS-injected gland4.
These methods are of interest to researchers of many disciplines who wish to model disease within the lactating mammary gland, as ultimately, the technique described here could be utilized for injection of a number of substances, and is not limited to only LPS.

Watch this Scientific Journal Video about Intraductal Injection of LPS as a Mouse Model of Mastitis: Signaling Visualized via an NF-ÎºB Reporter Transgenic at JoVE.com

Intraductal Injection of LPS as a Mouse Model of Mastitis: Signaling Visualized via an NF-&amp;kappa;B Reporter Transgenic

Described here is a technique in which lipopolysaccharide is injected into the lactating mouse mammary gland via the nipple to simulate mastitis, a condition commonly caused by bacterial infection. Lipopolysaccharide injection results in increased nuclear factor kappa B (NF-&kappa;B) signaling, visualized through bioluminescent imaging of an NF-&kappa;B luciferase reporter mouse.

Intraductal Injection of LPS as a Mouse Model of Mastitis: Signaling Visualized via an NF-&kappa;B Reporter Transgenic

Animal models of human disease are necessary in order to rigorously study stages of disease progression and associated mechanisms, and ultimately, as ...

intraductal-injection-lps-as-mouse-model-mastitis-signaling

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JoVE Journal

Medicine

19.1K ビュー.  Vanderbilt University Medical Center. ここで説明する乳腺炎をシミュレートするために、ニップルを介して、授乳中のマウス乳腺に注入されるリポ多糖体で技術は、一般的に細菌感染によって引き起こされる症状である。増加した核因子κB（NF-κB）シグナル伝達におけるリポ多糖注射の結果は、NF-κBルシフェラーゼレポーターマウスの生物発光イメージングを通して可視化した。

ビデオ: Intraductal Injection of LPS as a Mouse Model of Mastitis: Signaling Visualized via an NF-&kappa;B Reporter Transgenic

Mouse Bladder Wall Injection

Mouse bladder wall injection is a useful technique to orthotopically study bladder phenomena, including stem cell, smooth muscle, and cancer biology. Before starting injections, the surgical area must be cleaned with soap and water and antiseptic solution. Surgical equipment must be sterilized before use and between each animal. Each mouse is placed under inhaled isoflurane anesthesia (2-5% for induction, 1-3% for maintenance) and its bladder exposed by making a midline abdominal incision with scissors. If the bladder is full, it is partially decompressed by gentle squeezing between two fingers. The cell suspension of interest is intramurally injected into the wall of the bladder dome using a 29 or 30 gauge needle and 1 cc or smaller syringe. The wound is then closed using wound clips and the mouse allowed to recover on a warming pad. Bladder wall injection is a delicate microsurgical technique that can be mastered with practice.

Mouse bladder wall injection is a useful approach to orthotopically study bladder stem cell and cancer biology. This delicate microsurgical method can be mastered with careful technique and practice.

Mouse bladder wall injection is a useful technique to orthotopically study bladder phenomena, including stem cell, smooth muscle, and cancer biology. ...

Initiation of Metastatic Breast Carcinoma by Targeting of the Ductal Epithelium with Adenovirus-Cre: A Novel Transgenic Mouse Model of Breast Cancer

Breast cancer is a heterogeneous disease involving complex cellular interactions between the developing tumor and immune system, eventually resulting in exponential tumor growth and metastasis to distal tissues and the collapse of anti-tumor immunity. Many useful animal models exist to study breast cancer, but none completely recapitulate the disease progression that occurs in humans. In order to gain a better understanding of the cellular interactions that result in the formation of latent metastasis and decreased survival, we have generated an inducible transgenic mouse model of YFP-expressing ductal carcinoma that develops after sexual maturity in immune-competent mice and is driven by consistent, endocrine-independent oncogene expression. Activation of YFP, ablation of p53, and expression of an oncogenic form of K-ras was achieved by the delivery of an adenovirus expressing Cre-recombinase into the mammary duct of sexually mature, virgin female mice. Tumors begin to appear 6 weeks after the initiation of oncogenic events. After tumors become apparent, they progress slowly for approximately two weeks before they begin to grow exponentially. After 7-8 weeks post-adenovirus injection, vasculature is observed connecting the tumor mass to distal lymph nodes, with eventual lymphovascular invasion of YFP+ tumor cells to the distal axillary lymph nodes. Infiltrating leukocyte populations are similar to those found in human breast carcinomas, including the presence of &#945;&#946; and &#947;&#948; T cells, macrophages and MDSCs. This unique model will facilitate the study of cellular and immunological mechanisms involved in latent metastasis and dormancy in addition to being useful for designing novel immunotherapeutic interventions to treat invasive breast cancer.

Activation of latent mutations with adenovirus-Cre into the mammary ductal system results in a clinically relevant metastatic breast cancer. Incorporation of a YFP promoter allows tracking of distal metastatic tumor cells. This model is useful to study latent metastasis, anti-tumor immunity, and for designing novel immunotherapies to treat breast cancer.

Breast cancer is a heterogeneous disease involving complex cellular interactions between the developing tumor and immune system, eventually resulting in ...

Excitotoxic Stimulation of Brain Microslices as an In vitro Model of Stroke

Examining molecular mechanisms involved in neuropathological conditions, such as ischemic stroke, can be difficult when using whole animal systems. As such, primary or &#39;neuronal-like&#39; cell culture systems are commonly utilized. While&#160;these systems are relatively easy to work with, and are useful model systems in which various functional outcomes (such as cell death) can be readily quantified, the examined outcomes and pathways in cultured immature neurons (such as excitotoxicity-mediated cell death pathways) are not necessarily the same as those observed in mature brain, or in intact tissue. Therefore, there is the need to develop models in which cellular mechanisms in mature neural tissue can be examined. We have developed an in vitro technique that can be used to investigate a variety of molecular pathways in intact nervous tissue. The technique described herein utilizes rat cortical tissue, but this technique can be adapted to use tissue from a variety of species (such as mouse, rabbit, guinea pig, and chicken) or brain regions (for example, hippocampus, striatum, etc.). Additionally, a variety of stimulations/treatments can be used (for example, excitotoxic, administration of inhibitors, etc.). In conclusion, the brain slice model described herein can be used to examine a variety of molecular mechanisms involved in excitotoxicity-mediated brain injury.

Excitotoxic Stimulation of Brain Microslices as an In vitro Model of Stroke

We have developed a brain slice model which can be used to examine molecular mechanisms involved in excitotoxicity-mediated brain injury.&#160;This technique generates viable mature brain tissue and reduces animal numbers required for experimentation, whilst keeping the neuronal circuitry, cellular interactions, and postsynaptic compartments partly intact.

Examining molecular mechanisms involved in neuropathological conditions, such as ischemic stroke, can be difficult when using whole animal systems. As ...

Development of an Alpha-synuclein Based Rat Model for Parkinson's Disease via Stereotactic Injection of a Recombinant Adeno-associated Viral Vector

In order to study the molecular pathways of Parkinson&#39;s disease (PD) and to develop novel therapeutic strategies, scientific investigators rely on animal models. The identification of PD-associated genes has led to the development of genetic PD models. Most transgenic &#945;-SYN mouse models develop gradual &#945;-SYN pathology but fail to display clear dopaminergic cell loss and dopamine-dependent behavioral deficits. This hurdle was overcome by direct targeting of the substantia nigra with viral vectors overexpressing PD-associated genes. Local gene delivery using viral vectors provides an attractive way to express transgenes in the central nervous system. Specific brain regions can be targeted (e.g. the substantia nigra), expression can be induced in the adult setting and high expression levels can be achieved. Further, different vector systems based on various viruses can be used. The protocol outlines all crucial steps to perform a viral vector injection in the substantia nigra of the rat to develop a viral vector-based alpha-synuclein animal model for Parkinson&#39;s disease.

Development of an Alpha-synuclein Based Rat Model for Parkinson&#039;s Disease via Stereotactic Injection of a Recombinant Adeno-associated Viral Vector

This manuscript describes how viral vector-mediated local gene delivery provides an attractive way to express transgenes in the central nervous system. The protocol outlines all crucial steps to perform a viral vector injection in the substantia nigra of the rat to develop a viral vector-based animal model for Parkinson's disease.

In order to study the molecular pathways of Parkinson&#39;s disease (PD) and to develop novel therapeutic strategies, scientific investigators rely on ...

Induction and Micro-CT Imaging of Cerebral Cavernous Malformations in Mouse Model

Mutations in the CCM1 (aka KRIT1), CCM2, or CCM3 (aka PDCD10) gene cause cerebral cavernous malformation (CCM) in humans. Mouse models of CCM disease have been established by tamoxifen induced deletion of Ccm genes in postnatal animals. These mouse models provide invaluable tools to investigate molecular mechanism and therapeutic approaches for CCM disease. An accurate and quantitative method to assess lesion burden and progression is essential to harness the full value of these animal models. Here, we demonstrate the induction of CCM disease in a mouse model and the use of the contrast enhanced X-ray micro computed tomography (micro-CT) method to measure CCM lesion burden in mouse brains. At postnatal day 1 (P1), we used 4-hydroxytamoxifen (4HT) to activate Cre recombinase activity from the Cdh5-CreErt2 transgene to cleave the floxed allele of Ccm2. CCM lesions in mouse brains were analyzed at P8. For micro-CT, iodine based Lugol&#39;s solution was used to enhance contrast in brain tissue. We have optimized the scan parameters and utilized a voxel dimension of 9.5 &#181;m, which lead to a minimum feature size of approximately 25 &#181;m. This resolution is sufficient to measure CCM lesion volume and number globally and accurately, and provide high-quality 3-D mapping of CCM lesions in mouse brains. This method enhances the value of the established mouse models to study the molecular basis and potential therapies for CCM and other cerebrovascular diseases.

This protocol demonstrates the induction of cerebral cavernous malformation disease in a mouse model and uses contrast enhanced micro computed tomography to measure lesion burden. This method enhances the value of established mouse models to study the molecular basis and potential therapies for cerebral cavernous malformation and other cerebrovascular diseases.

Mutations in the CCM1 (aka KRIT1), CCM2, or CCM3 (aka PDCD10) gene cause cerebral cavernous malformation (CCM) in humans. Mouse models of CCM disease have ...

Puncture-Induced Iris Neovascularization as a Mouse Model of Rubeosis Iridis

We describe a model of puncture-induced iris neovascularization as a general model for noninvasive evaluation of angiogenesis. The model is also relevant for targeting neovascular glaucoma, a sight-threatening complication of diabetic retinopathy. This method is based on the induction of iris vascular response by a series of self-sealing uveal punctures on BALB/c mice and takes advantage of the postpartum maturation of mouse ocular vasculature. Mouse pups undergo uveal punctures from postnatal day 12.5, when the pups naturally open their eyes, until postnatal day 24.5. Due to the transparency of the cornea, iris vasculature can be analyzed easily through time by noninvasive in vivo methods. Furthermore, the semitransparent iris of BALB/c mice can be flatmounted for detailed immunohistologic analysis with minimal non-specific background staining. In this model, angiogenesis is mainly driven by the inflammatory and plasminogen activating systems. The puncture-induced model is the first to induce iris neovascularization in small rodents, and has the advantage of allowing direct noninvasive in vivo analysis of the angiogenic process. Moreover, the model can be combined with angiogenic modulating substances, which highlights its potential in the study of angiogenesis with an in vivo perspective.

Iris neovascularization, a common complication of ischemic retinal disease, may lead to sight-threatening neovascular glaucoma. Here, we describe a murine protocol for inducing experimental iris neovascularization that may be used for noninvasive evaluation of angiogenesis-modulating substances.

We describe a model of puncture-induced iris neovascularization as a general model for noninvasive evaluation of angiogenesis. The model is also relevant ...

Oleic Acid-Injection in Pigs As a Model for Acute Respiratory Distress Syndrome

The acute respiratory distress syndrome is a relevant intensive care disease with an incidence ranging between 2.2% and 19% of intensive care unit patients. Despite treatment advances over the last decades, ARDS patients still suffer mortality rates between 35 and 40%. There is still a need for further research to improve the outcome of patients suffering from ARDS. One problem is that no single animal model can mimic the complex pathomechanism of the acute respiratory distress syndrome, but several models exist to study different parts of it. Oleic acid injection (OAI)-induced lung injury is a well-established model for studying ventilation strategies, lung mechanics and ventilation/perfusion distribution in animals. OAI leads to severely impaired gas exchange, deterioration of lung mechanics and disruption of the alveolo-capillary barrier. The disadvantage of this model is the controversial mechanistic relevance of this model and the necessity for central venous access, which is challenging especially in smaller animal models. In summary, OAI-induced lung injury leads to reproducible results in small and large animals and hence represents a well-suited model for studying ARDS. Nevertheless, further research is necessary to find a model that mimics all parts of ARDS and lacks the problems associated with the different models existing today.

In this article, we present a protocol to induce acute lung injury in pigs by central-venous injection of oleic acid. This is an established animal model for studying the acute respiratory distress syndrome (ARDS).

The acute respiratory distress syndrome is a relevant intensive care disease with an incidence ranging between 2.2% and 19% of intensive care unit ...

Isometric Contractility Measurement of the Mouse Mesenteric Artery Using Wire Myography

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and drugs. It is widely used in many studies on the physiological functions of vascular smooth muscle, the pathogenesis of vascular diseases such as hypertension, and the development of smooth muscle relaxant drugs. The mouse is a widely used model animal with a large pool of disease models and genetically modified strains. We introduced this method to measure the isometric contraction of mouse mesenteric artery in detail. A 1.4-mm segment of mouse mesenteric resistance artery was isolated and mounted on a myograph chamber by passing two steel wires through its lumen. After equilibration and normalization steps, the vessel segment was potentiated by a high-K+ solution twice prior to the contraction assay. As an example of the application of this method in drug development, we measured the relaxant effect of a novel natural substance, neoliensinine, isolated from a Chinese herb, embryos of the lotus seed (Nelumbo nucifera Gaertn.) on mouse mesenteric arteries. The vessel segments mounted on the myograph chamber were stimulated with a high-K+ solution. When the force tension reached a stable sustained phase, cumulative doses of neoliensinine were added to the chamber. We found that neoliensinine had a dose-dependent relaxant effect on smooth muscle contraction, thus suggesting that it bears potential activity against hypertension. In addition, as the vessel segment can survive at least 4 hours after mounting and maintain contractility induced by the high-K+ solution for many times, we suggest that the wire myograph system may be used for the time-consuming process of drug screening.

The wire myograph technique is used to investigate vascular smooth muscle functions and screen new drugs. We report a detailed protocol for measuring the isometric contractility of the mouse mesenteric artery and for screening new relaxants of vascular smooth muscle.

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and ...

Murine Precision-Cut Liver Slices as an Ex Vivo Model of Liver Biology

Understanding the mechanisms of liver injury, hepatic fibrosis, and cirrhosis that underlie chronic liver diseases (i.e., viral hepatitis, non-alcoholic fatty liver disease, metabolic liver disease, and liver cancer) requires experimental manipulation of animal models and in vitro cell cultures. Both techniques have limitations, such as the requirement of large numbers of animals for in vivo manipulation. However, in vitro cell cultures do not reproduce the structure and function of the multicellular hepatic environment. The use of precision-cut liver slices is a technique in which uniform slices of viable mouse liver are maintained in laboratory tissue culture for experimental manipulation. This technique occupies an experimental niche that exists between animal studies and in vitro cell culture methods. The presented protocol describes a straightforward and reliable method to isolate and culture precision-cut liver slices from mice. As an application of this technique, ex vivo liver slices are treated with bile acids to simulate cholestatic liver injury and ultimately assess the mechanisms of hepatic fibrogenesis.

This protocol provides a simple and reliable method for the production of viable precision-cut liver slices from mice. The ex vivo tissue samples can be maintained under laboratory tissue culture conditions for multiple days, providing a flexible model to examine liver pathobiology.

Understanding the mechanisms of liver injury, hepatic fibrosis, and cirrhosis that underlie chronic liver diseases (i.e., viral hepatitis, non-alcoholic ...

A Mouse Model for Chronic Pancreatitis via Bile Duct TNBS Infusion

Chronic pancreatitis (CP) is a complex disease involving pancreatic inflammation and fibrosis, glandular atrophy, abdominal pain and other symptoms. Several rodent models have been developed to study CP, of which the bile duct 2,4,6 -trinitrobenzene sulfonic acid (TNBS) infusion model replicates the features of neuropathic pain seen in CP. However, bile duct drug infusion in mice is technically challenging. This protocol demonstrates the procedure of bile duct TNBS infusion for generation of a CP mouse model. TNBS was infused into the pancreas through the ampulla of Vater in the duodenum. This protocol optimized drug volume, surgical techniques, and drug handling during the procedure. TNBS-treated mice showed features of CP as reflected by bodyweight and pancreas weight reductions, changes in pain-associated behaviors, and abnormal pancreatic morphology. With these improvements, mortality associated with TNBS injection was minimal. This procedure is not only critical in generating pancreatic disease models but is also useful in local pancreatic drug delivery.

Chronic pancreatitis (CP) is a disease characterized by inflammation and fibrosis of the pancreas, often associated with intractable abdominal pain. This article focuses on refining the technique to generate a mouse model of CP via bile duct infusion with 2,4,6 -trinitrobenzene sulfonic acid (TNBS).

Chronic pancreatitis (CP) is a complex disease involving pancreatic inflammation and fibrosis, glandular atrophy, abdominal pain and other symptoms. ...