We are grateful to the Preclinical Imaging Core at the NYU School of Medicine, with special thanks to Orlando Aristizabal and Youssef Zaim Wadghiri. The core is a shared resource partially supported by the Laura and Isaac Perlmutter Cancer Center Support Grant NIH/NCI 5P30CA016087 and NIBIB Biomedical Technology Resource Center Grant NIH P41 EB017183. This work was supported by the American Diabetes Association &#34;Pathway to Stop Diabetes&#34; to D.C. [grant number 1-16-ACE-08] and the NYU Applied Research Support Fund to P.R.

All methods described here have been approved by the Institutional Animal Care and Use Committee of New York University School of Medicine. All mice are housed behind a barrier and all personnel wear appropriate personal protective equipment.
1. Day 0: Preparation of Murine Model of Excisional Wound Healing
<ol>
	<li>Anesthetize diabetic (Leprdb/db) mice, aged 8&#8211;12 weeks, with inhalational 2% isoflurane. Confirm that each mouse has been properly anesthetized using the foot pad pinch test. Apply sterile ocular lubricant to each eye to prevent irritation from dryness.&#160;Researchers should follow their institutional veterinary staff&#8217;s guidelines when anesthetizing animals using isoflurane.</li>
	<li>Weigh the mice and record the body weight of each mouse.&#160;Confirm the diabetic status of animals by recording the blood glucose of each animal using a glucometer.&#160;</li>
	<li>Disinfect procedural workspace and anesthesia equipment. Remove dorsal hair of the mice using a hair trimmer, followed by application of hair removal lotion to wipe away excess hair. Use alcohol wipes to clean the exposed skin, twice, and allow to dry.</li>
	<li>Create two 10 mm full-thickness wounds extending through the panniculus carnosus using sterile 10 mm biopsy punches according to a well-established excisional wound healing technique7,8. Use sterile gloves for all survival surgery. Autoclave all surgical instruments in bags prior to surgery and open only in the surgical field.</li>
	<li>Splint the wounds open using a 0.5 mm thick silicone sheet with 10 mm circular cutouts and secure the stents in place using interrupted 4-0 silk sutures.</li>
	<li>Following surgery, remove animals from anesthesia and place on heating pad to facilitate proper recovery. Of note, if animal body temperature decreases during the procedure, the animal may be moved to the heating pad earlier to minimize body heat loss under anesthesia.&#160;Monitor the animals until they are awake and mobile.</li>
	<li>Once fully recovered, return animals to individual cages, containing food and water (ensure some food is on the floor of the cage for post-operative enrichment).&#160;Provide shredded paper towels as additional nesting material for 2 weeks. Do not house animals that have undergone surgery with other animals, to prevent adverse interactions and changes to wound healing status.</li>
	<li>For post-operative pain relief, inject mice subcutaneously with buprenorphine at 0.1 mg/kg of body weight twice a day, starting immediately following the procedure, for 3 days.</li>
</ol>
2. Day 1: Preparation of Keap1 siRNA
NOTE: Prepare all treatments inside a biosafety cabinet.
<ol>
	<li>Prepare siRNA dilution by combining 37.5 &#181;L of reduced serum medium with 12.5 &#181;L of 20 &#181;M Keap1 siRNA (siKeap1) (250 pmol) in a 1.5 mL microcentrifuge tube on ice.</li>
	<li>Prepare liposome dilution by combining 25 &#181;L of reduced serum medium with 25 &#181;L of liposome mix in a 1.5 mL microcentrifuge tube.</li>
	<li>Add 50 &#181;L of siRNA dilution to 50 &#181;L liposome dilution dropwise (1:1 volume), and gently mix.</li>
	<li>Incubate for 20 minutes at room temperature.</li>
	<li>Add 50 &#181;L of 2% methylcellulose gel in water and mix gently by pipetting up and down.</li>
	<li>Treat each animal with either a nonsense siNS (control) or siKeap1 (experimental). Apply the gel to the top of the wound. Wrap the animal&#8217;s torso with transparent film dressing to keep gel in place, keeping the limbs free to maintain mobility.</li>
</ol>
3. Day 3: Preparation of L-012 Solution
NOTE: Prepare all reagents in a biosafety cabinet.
<ol>
	<li>In a 1.5 mL microcentrifuge tube, prepare L-012 in 1X PBS at a concentration of 0.5 mg/100 mL.</li>
	<li>Manually vortex the microcentrifuge tube. The L-012 does not completely dissolve into the PBS, however it should be evenly suspended in the liquid. Of note, do not attempt to dissolve L-012 in water to avoid disturbing physiologic electrolyte balance following injection.</li>
	<li>Transfer the solution into a 1 mL syringe using a 27 G needle. 
	NOTE: Be sure to protect the L-012 solution from light.</li>
</ol>
4. Day 3: In Vivo Imaging of Diabetic Wounds
<ol>
	<li>Anesthetize mice with inhalational 2% isoflurane.&#160;Researchers should follow their institutional veterinary staff&#8217;s guidelines when anesthetizing animals using isoflurane. Confirm that each mouse has been properly anesthetized using the foot pad pinch test. Apply sterile ocular lubricant to each eye to prevent irritation from dryness.</li>
	<li>Gently remove the transparent film dressing from the mice without disturbing the wounds.</li>
	<li>Place the mice in the imaging chamber in their respective orders. To maintain proper O2 levels in the chamber, set the imaging system inflow and the induction chamber O2 levels to 1.0 L/min.</li>
	<li>Image the mice for bioluminescence and photograph at baseline before injection of L-012 compound.</li>
	<li>Wipe the abdomen with alcohol wipes and allow to dry. Perform an intraperitoneal injection of the L-012 solution at 5 mg per 200 g body weight using a 27-gauge needle. For example, a mouse weighing 20 g should receive 0.5 mg of L-012.</li>
	<li>Immediately following the L-012 injection, place the mice back in their respective locations in the imaging chamber. Image the mice over the course of 60 minutes, for 1 minute at 4 minute intervals. Define the 10-mm wound as the region of interest for determining level of ROS.</li>
	<li>Following surgery, remove animals from anesthesia and place on heating pad to facilitate proper recovery. Monitor the animals until they are awake and mobile.</li>
	<li>Once fully recovered, return animals to individual cages, maintaining the same post-operative enrichment environment previously described in 1.6.&#160;Do not house animals that have undergone surgery with other animals, to prevent adverse interactions and changes to wound healing status.</li>
</ol>

<ol><li>Nishinaka, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+new+sensitive+chemiluminescence+probe%2C+L-012%2C+for+measuring+the+production+of+superoxide+anion+by+cells%5BTitle%5D)+AND+%22Biochemical+and+Biophysical+Research+Communications%22%5BJournal%5D)">A new sensitive chemiluminescence probe, L-012, for measuring the production of superoxide anion by cells.</a> Biochemical and Biophysical Research Communications. 193 (2), 554-559 (1993).</li>
<li>Daiber, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Measurement+of+NAD%28P%29H+oxidase-derived+superoxide+with+the+luminol+analogue+L-012%5BTitle%5D)+AND+%22Free+Radical+Biology+and+Medicine%22%5BJournal%5D)">Measurement of NAD(P)H oxidase-derived superoxide with the luminol analogue L-012.</a> Free Radical Biology and Medicine. 36 (1), 101-111 (2004).</li>
<li>Imada, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Analysis+of+reactive+oxygen+species+generated+by+neutrophils+using+a+chemiluminescence+probe+L-012%5BTitle%5D)+AND+%22Analytical+Biochemistry%22%5BJournal%5D)">Analysis of reactive oxygen species generated by neutrophils using a chemiluminescence probe L-012.</a> Analytical Biochemistry. 271 (1), 53-58 (1999).</li>
<li>Sohn, H. Y., Gloe, T., Keller, M., Schoenafinger, K., Pohl, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Sensitive+superoxide+detection+in+vascular+cells+by+the+new+chemiluminescence+dye+L-012%5BTitle%5D)+AND+%22Journal+of+Vascular+Research%22%5BJournal%5D)">Sensitive superoxide detection in vascular cells by the new chemiluminescence dye L-012.</a> Journal of Vascular Research. 36 (6), 456-464 (1999).</li>
<li>Fuchs, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((In+vivo+Hypoxia+PET+Imaging+Quantifies+the+Severity+of+Arthritic+Joint+Inflammation+in+Line+with+Overexpression+of+Hypoxia-Inducible+Factor+and+Enhanced+Reactive+Oxygen+Species+Generation%5BTitle%5D)+AND+%22The+Journal+of+Nuclear+Medicine%22%5BJournal%5D)">In vivo Hypoxia PET Imaging Quantifies the Severity of Arthritic Joint Inflammation in Line with Overexpression of Hypoxia-Inducible Factor and Enhanced Reactive Oxygen Species Generation.</a> The Journal of Nuclear Medicine. 58 (5), 853-860 (2017).</li>
<li>Asghar, M. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((In+vivo+imaging+of+reactive+oxygen+and+nitrogen+species+in+murine+colitis%5BTitle%5D)+AND+%22Inflammatory+Bowel+Diseases%22%5BJournal%5D)">In vivo imaging of reactive oxygen and nitrogen species in murine colitis.</a> Inflammatory Bowel Diseases. 20 (8), 1435-1447 (2014).</li>
<li>Galiano, R. D., Michaels, J. t, Dobryansky, M., Levine, J. P., Gurtner, G. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Quantitative+and+reproducible+murine+model+of+excisional+wound+healing%5BTitle%5D)+AND+%22Wound+Repair+and+Regeneration%22%5BJournal%5D)">Quantitative and reproducible murine model of excisional wound healing.</a> Wound Repair and Regeneration. 12 (4), 485-492 (2004).</li>
<li>Soares, M. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Restoration+of+Nrf2+Signaling+Normalizes+the+Regenerative+Niche%5BTitle%5D)+AND+%22Diabetes%22%5BJournal%5D)">Restoration of Nrf2 Signaling Normalizes the Regenerative Niche.</a> Diabetes. 65 (3), 633-646 (2016).</li>
<li>Wang, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Imaging+ROS+signaling+in+cells+and+animals%5BTitle%5D)+AND+%22Journal+of+Molecular+Medicine%22%5BJournal%5D)">Imaging ROS signaling in cells and animals.</a> Journal of Molecular Medicine. 91 (8), 917-927 (2013).</li>
<li>Kielland, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((In+vivo+imaging+of+reactive+oxygen+and+nitrogen+species+in+inflammation+using+the+luminescent+probe+L-012%5BTitle%5D)+AND+%22Free+Radical+Biology+and+Medicine%22%5BJournal%5D)">In vivo imaging of reactive oxygen and nitrogen species in inflammation using the luminescent probe L-012.</a> Free Radical Biology and Medicine. 47 (6), 760-766 (2009).</li>
<li>Balke, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Visualizing+Oxidative+Cellular+Stress+Induced+by+Nanoparticles+in+the+Subcytotoxic+Range+Using+Fluorescence+Lifetime+Imaging%5BTitle%5D)+AND+%22Small%22%5BJournal%5D)">Visualizing Oxidative Cellular Stress Induced by Nanoparticles in the Subcytotoxic Range Using Fluorescence Lifetime Imaging.</a> Small. , (2018).</li>
<li>Zielonka, J., Lambeth, J. D., Kalyanaraman, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((On+the+use+of+L-012%2C+a+luminol-based+chemiluminescent+probe%2C+for+detecting+superoxide+and+identifying+inhibitors+of+NADPH+oxidase%3A+a+reevaluation%5BTitle%5D)+AND+%22Free+Radical+Biology+and+Medicine%22%5BJournal%5D)">On the use of L-012, a luminol-based chemiluminescent probe, for detecting superoxide and identifying inhibitors of NADPH oxidase: a reevaluation.</a> Free Radical Biology and Medicine. 65, 1310-1314 (2013).</li>
<li>Dikalov, S. I., Harrison, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Methods+for+detection+of+mitochondrial+and+cellular+reactive+oxygen+species%5BTitle%5D)+AND+%22Antioxidants+%26+Redox+Signalling%22%5BJournal%5D)">Methods for detection of mitochondrial and cellular reactive oxygen species.</a> Antioxidants & Redox Signalling. 20 (2), 372-382 (2014).</li>
<li>Rabbani, P. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Targeted+Nrf2+activation+therapy+with+RTA+408+enhances+regenerative+capacity+of+diabetic+wounds%5BTitle%5D)+AND+%22Diabetes+Research+and+Clinical+Practice%22%5BJournal%5D)">Targeted Nrf2 activation therapy with RTA 408 enhances regenerative capacity of diabetic wounds.</a> Diabetes Research and Clinical Practice. 139, 11-23 (2018).</li>
<li>Rabbani, P. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Novel+lipoproteoplex+delivers+Keap1+siRNA+based+gene+therapy+to+accelerate+diabetic+wound+healing%5BTitle%5D)+AND+%22Biomaterials%22%5BJournal%5D)">Novel lipoproteoplex delivers Keap1 siRNA based gene therapy to accelerate diabetic wound healing.</a> Biomaterials. 132, 1-15 (2017).</li>
</ol>

We have no disclosures to report.

Common techniques for measuring ROS have been limited by complex protocols requiring tissue extraction or similarly invasive techniques. In recent years, measurements of oxidative stress have been reported on the basis of innovative imaging modalities, thereby allowing for spatiotemporal assessments9,10,11. L-012 has several advantages as a chemiluminescent probe relative to luminol, lucigenin, and MCLA1<...

Oxygen metabolites generated through inflammatory processes contribute to various signaling cascades as well as destructive alteration of cellular components1. Utilizing sensitive and specific techniques to measure ROS is critical for studying inflammatory processes and characterizing the effects of oxidative stress. In vivo imaging is valuable because of its ability to provide dynamic spatial and temporal data in living tissue. L-012 is a synthetic chemiluminescent probe that is highly sensitive for superoxide anions and produces a higher light intensity than other fluorescent probes in cells, tissues, and whole blood1,2,3,4. It has been successfully employed for in vivo imaging in murine models to study several inflammatory diseases, including arthritis and colitis5,6. It has yet to be employed in an established cutaneous wound healing model. Measurement of ROS generated is equally relevant to assess the progression of wound healing under different conditions. The sensitivity and noninvasive nature of this method makes it a promising technique for studying wound healing across murine models.
Nrf2 is a major driver of the antioxidant response and a transcriptional factor with specificity for the antioxidant response element (ARE) common to the promoter regions of several antioxidant enzymes8. In the absence of oxidative stress, Nrf2 is sequestered in the cytoplasm by Keap1, which subsequently causes its ubiquitination and degradation. Imbalance of the Nrf2/Keap1 pathway has been implicated in inappropriate redox homeostasis and delayed wound healing in the setting of increased oxidative stress9. We have previously shown that suppression of Keap1 stimulates increased Nrf2 activity and promotes rescue of pathologic cutaneous wound healing in diabetic wounds9.
Here we describe a protocol that utilizes L-012-assisted bioluminescence imaging to measure ROS levels in an excisional cutaneous wound healing model, which is critical for highlighting the association between ROS and wound healing. This technique demonstrates real-time changes in oxidative burden within wounds and immediate periphery. Furthermore, this method allows for rapid assessment of interventions and mechanisms that affect redox handling. Here we use a model of Keap1 knockdown for the restoration of redox homeostasis to evaluate the applicability of our strategy. Because our technique is non-invasive and wounds are undisturbed, the same animal can be used for further confirmatory analyses on the basis of histology or cell lysates.

<table><tbody><tr><td>BKS.Cg-Dock7m+/+ Leprdb/J mice</td><td>Jackson Laboratories</td><td>000642</td><td></td></tr><tr><td>13 cm x 18 cm Silicone sheet (0.6 mm)</td><td>Sigma Aldrich&amp;nbsp;</td><td>665581</td><td></td></tr><tr><td>3M Tegaderm Transparent Film Dressings</td><td>3M</td><td>88-1626W</td><td></td></tr><tr><td>Lipofectamine 2000 Transfection Reagent</td><td>Life Technologies&amp;nbsp;</td><td>11668027</td><td></td></tr><tr><td>Keap1 Stealth siRNA</td><td>Thermofisher Scientific</td><td>1299001</td><td></td></tr><tr><td>Silencer negative control&amp;nbsp;</td><td>Thermofisher Scientific&amp;nbsp;</td><td>AM4635</td><td></td></tr><tr><td>Opti-MEM Reduced Serum</td><td>ThermoFisher Scientific</td><td>11058021</td><td></td></tr><tr><td>DPBS</td><td>ThermoFisher Scientific</td><td>14040133</td><td></td></tr><tr><td>Methyl-cellulose&amp;nbsp;</td><td>Sigma Aldrich</td><td>9004-67-5</td><td></td></tr><tr><td>L-012</td><td>Wako Chemicals</td><td>120-04891</td><td></td></tr><tr><td>IVIS Lumina III XR In Vivo Imaging System&amp;nbsp;</td><td>PerkinElmer</td><td></td><td></td></tr></tbody></table>

in vivo imaging of reactive oxygen species in a murine wound model

The generation of reactive oxygen species (ROS) is a hallmark of inflammatory processes, but in excess, oxidative stress is widely implicated in various pathologies such as cancer, atherosclerosis and diabetes. We have previously shown that dysfunction of the Nuclear factor (erythroid-derived 2)-like 2 (Nrf2)/ Kelch-like erythroid cell-derived protein 1 (Keap1) signaling pathway leads to extreme ROS imbalance during cutaneous wound healing in diabetes. Since ROS levels are an important indicator of progression of wound healing, specific and accurate quantification techniques are valuable. Several in vitro assays to measure ROS in cells and tissues have been described; however, they only provide a single cumulative measurement per sample. More recently, the development of protein-based indicators and imaging modalities have allowed for unique spatiotemporal analyses. L-012 (C13H8ClN4NaO2) is a luminol derivative that can be used for both in vivo and in vitro chemiluminescent detection of ROS generated by NAPDH oxidase. L-012 emits a stronger signal than other fluorescent probes and has been shown to be both sensitive and reliable for detecting ROS. The time lapse applicability of L-012-facilitated imaging provides valuable information about inflammatory processes while reducing the need for sacrifice and overall reducing the number of study animals. Here, we describe a protocol utilizing L-012-facilitated in vivo imaging to quantify oxidative stress in a model of excisional wound healing using diabetic mice with locally dysfunctional Nrf2/Keap1.

Three days after creating bilateral wounds according to an established excisional wound model (Figure 1A), diabetic mice are positioned in the imaging chamber. An initial photograph and a measure of bioluminescence are taken before injection of L-012 to account for background signal (Figure 1B). Following intraperitoneal injection with the L-012 solution, the mice are repositioned in the chamber and bioluminescence is visualized ...

Watch this Scientific Journal Video about In Vivo Imaging of Reactive Oxygen Species in a Murine Wound Model at JoVE.com

In Vivo Imaging of Reactive Oxygen Species in a Murine Wound Model

We describe a non-invasive in vivo imaging protocol that is streamlined and cost-effective, utilizing L-012, a chemiluminescent luminol-analog, to visualize and quantify reactive oxygen species (ROS) generated in a mouse excisional wound model.

In Vivo Imaging of Reactive Oxygen Species in a Murine Wound Model

The generation of reactive oxygen species (ROS) is a hallmark of inflammatory processes, but in excess, oxidative stress is widely implicated in various ...

in-vivo-imaging-of-reactive-oxygen-species-in-a-murine-wound-model

Research

JoVE Journal

Immunology and Infection

10.9K Views.  New York University School of Medicine. We describe a non-invasive in vivo imaging protocol that is streamlined and cost-effective, utilizing L-012, a chemiluminescent luminol-analog, to visualize and quantify reactive oxygen species (ROS) generated in a mouse excisional wound model.

Video: In Vivo Imaging of Reactive Oxygen Species in a Murine Wound Model

Bioluminescence Imaging of NADPH Oxidase Activity in Different Animal Models

NADPH oxidase is a critical enzyme that mediates antibacterial and antifungal host defense. In addition to its role in antimicrobial host defense, NADPH oxidase has critical signaling functions that modulate the inflammatory response 1. Thus, the development of a method to measure in "real-time" the kinetics of NADPH oxidase-derived ROS generation is expected to be a valuable research tool to understand mechanisms relevant to host defense, inflammation, and injury.
Chronic granulomatous disease (CGD) is an inherited disorder of the NADPH oxidase characterized by severe infections and excessive inflammation. Activation of the phagocyte NADPH oxidase requires translocation of its cytosolic subunits (p47phox, p67phox, and p40phox) and Rac to a membrane-bound flavocytochrome (composed of a gp91phox and p22phox heterodimer). Loss of function mutations in any of these NADPH oxidase components result in CGD. Similar to patients with CGD, gp91phox -deficient mice and p47phox-deficient mice have defective phagocyte NADPH oxidase activity and impaired host defense 2, 13. In addition to phagocytes, which contain the NADPH oxidase components described above, a variety of other cell types express different isoforms of NADPH oxidase.
Here, we describe a method to quantify ROS production in living mice and to delineate the contribution of NADPH oxidase to ROS generation in models of inflammation and injury. This method is based on ROS reacting with L-012 (an analogue of luminol) to emit luminescence that is recorded by a charge-coupled device (CCD). In the original description of the L-012 probe, L-012-dependent chemiluminescence was completely abolished by superoxide dismutase, indicating that the main ROS detected in this reaction was superoxide anion 14. Subsequent studies have shown that L-012 can detect other free radicals, including reactive nitrogen species 15, 16. Kielland et al. 16 showed that topical application of phorbol myristate acetate, a potent activator of NADPH oxidase, led to NADPH oxidase-dependent ROS generation that could be detected in mice using the luminescent probe L-012. In this model, they showed that L-012-dependent luminescence was abolished in p47phox-deficient mice.
We compared ROS generation in wildtype mice and NADPH oxidase-deficient p47phox-/- mice 2 in the following three models: 1) intratracheal administration of zymosan, a pro-inflammatory fungal cell wall-derived product that can activate NADPH oxidase; 2) cecal ligation and puncture (CLP), a model of intra-abdominal sepsis with secondary acute lung inflammation and injury; and 3) oral carbon tetrachloride (CCl4), a model of ROS-dependent hepatic injury. These models were specifically selected to evaluate NADPH oxidase-dependent ROS generation in the context of non-infectious inflammation, polymicrobial sepsis, and toxin-induced organ injury, respectively. Comparing bioluminescence in wildtype mice to p47phox-/- mice enables us to delineate the specific contribution of ROS generated by p47phox-containing NADPH oxidase to the bioluminescent signal in these models.
Bioluminescence imaging results that demonstrated increased ROS levels in wildtype mice compared to p47phox-/- mice indicated that NADPH oxidase is the major source of ROS generation in response to inflammatory stimuli. This method provides a minimally invasive approach for "real-time" monitoring of ROS generation during inflammation in vivo.

NADPH oxidase is the major source of reactive oxygen species (ROS) in phagocytes. Because of the ephemeral nature of ROS, it is difficult to measure and monitor ROS levels in living animals. A minimally invasive method for serial quantification of ROS in living mice is described.

NADPH oxidase is a critical enzyme that mediates antibacterial and antifungal host defense. In addition to its role in antimicrobial host defense, NADPH ...

Murine Model of Wound Healing

Wound healing and repair are the most complex biological processes that occur in human life. After injury, multiple biological pathways become activated. Impaired wound healing, which occurs in diabetic patients for example, can lead to severe unfavorable outcomes such as amputation. There is, therefore, an increasing impetus to develop novel agents that promote wound repair. The testing of these has been limited to large animal models such as swine, which are often impractical. Mice represent the ideal preclinical model, as they are economical and amenable to genetic manipulation, which allows for mechanistic investigation. However, wound healing in a mouse is fundamentally different to that of humans as it primarily occurs via contraction. Our murine model overcomes this by incorporating a splint around the wound. By splinting the wound, the repair process is then dependent on epithelialization, cellular proliferation and angiogenesis, which closely mirror the biological processes of human wound healing. Whilst requiring consistency and care, this murine model does not involve complicated surgical techniques and allows for the robust testing of promising agents that may, for example, promote angiogenesis or inhibit inflammation. Furthermore, each mouse acts as its own control as two wounds are prepared, enabling the application of both the test compound and the vehicle control on the same animal. In conclusion, we demonstrate a practical, easy-to-learn, and robust model of wound healing, which is comparable to that of humans.

A murine model of cutaneous wound healing that can be used to assess therapeutic compounds in physiological and pathophysiological settings.

Wound healing and repair are the most complex biological processes that occur in human life. After injury, multiple biological pathways become activated. ...

Medicine

Come to the Light Side: In Vivo Monitoring of Pseudomonas aeruginosa Biofilm Infections in Chronic Wounds in a Diabetic Hairless Murine Model

The presence of bacteria as structured biofilms in chronic wounds, especially in diabetic patients, is thought to prevent wound healing and resolution. Chronic mouse wounds models have been used to understand the underlying interactions between the microorganisms and the host. The models developed to date rely on the use of haired animals and terminal collection of wound tissue for determination of viable bacteria. While significant insight has been gained with these models, this experimental procedure requires a large number of animals and sampling is time consuming. We have developed a novel murine model that incorporates several optimal innovations to evaluate biofilm progression in chronic wounds: a) it utilizes hairless mice, eliminating the need for hair removal; b) applies pre-formed biofilms to the wounds allowing for the immediate evaluation of persistence and effect of these communities on host; c) monitors biofilm progression by quantifying light production by a genetically engineered bioluminescent strain of Pseudomonas aeruginosa, allowing real-time monitoring of the infection thus reducing the number of animals required per study. In this model, a single full-depth wound is produced on the back of STZ-induced diabetic hairless mice and inoculated with biofilms of the P. aeruginosa bioluminescent strain Xen 41. Light output from the wounds is recorded daily in an in vivo imaging system, allowing for in vivo and in situ rapid biofilm visualization and localization of biofilm bacteria within the wounds. This novel method is flexible as it can be used to study other microorganisms, including genetically engineered species and multi-species biofilms, and may be of special value in testing anti-biofilm strategies including antimicrobial occlusive dressings.

Come to the Light Side: In Vivo Monitoring of Pseudomonas aeruginosa Biofilm Infections in Chronic Wounds in a Diabetic Hairless Murine Model

Here we describe a novel diabetic murine model utilizing hairless mice for real-time, non-invasive, monitoring of biofilm wound infections of bioluminescent Pseudomonas aeruginosa. This method can be adapted to evaluate infection of other bacterial species and genetically modified microorganisms, including multi-species biofilms, and test the efficacy of antibiofilm strategies.

The presence of bacteria as structured biofilms in chronic wounds, especially in diabetic patients, is thought to prevent wound healing and resolution. ...

Inducing Ischemia-reperfusion Injury in the Mouse Ear Skin for Intravital Multiphoton Imaging of Immune Responses

Ischemia-reperfusion injury (IRI) occurs when there is transient hypoxia due to the obstruction of blood flow (ischemia) followed by a subsequent re-oxygenation of the tissues (reperfusion). In the skin, ischemia-reperfusion (IR) is the main contributing factor to the pathophysiology of pressure ulcers. While the cascade of events leading up to the inflammatory response has been well studied, the spatial and temporal responses of the different subsets of immune cells to an IR injury are not well understood. Existing models of IR using the clamping technique on the skin flank are highly invasive and unsuitable for studying immune responses to injury, while similar non-invasive magnet clamping studies in the skin flank are less-than-ideal for intravital imaging studies. In this protocol, we describe a robust model of non-invasive IR developed on mouse ear skin, where we aim to visualize in real-time the cellular response of immune cells after reperfusion via multiphoton intravital imaging (MP-IVM).

This protocol describes the induction of an ischemia-reperfusion (IR) model on mouse ear skin using magnet clamping. Using a custom-built intravital imaging model, we study in vivo inflammatory responses post-reperfusion. The rationale behind the development of this technique is to extend the understanding of how leukocytes respond to skin IR injury.

Ischemia-reperfusion injury (IRI) occurs when there is transient hypoxia due to the obstruction of blood flow (ischemia) followed by a subsequent ...

Visualization of Vascular Ca2+ Signaling Triggered by Paracrine Derived ROS

Oxidative stress has been implicated in a number of pathologic conditions including ischemia/reperfusion damage and sepsis. The concept of oxidative stress refers to the aberrant formation of ROS (reactive oxygen species), which include O2&bull;-, H2O2, and hydroxyl radicals. Reactive oxygen species influences a multitude of cellular processes including signal transduction, cell proliferation and cell death1-6. ROS have the potential to damage vascular and organ cells directly, and can initiate secondary chemical reactions and genetic alterations that ultimately result in an amplification of the initial ROS-mediated tissue damage. A key component of the amplification cascade that exacerbates irreversible tissue damage is the recruitment and activation of circulating inflammatory cells. During inflammation, inflammatory cells produce cytokines such as tumor necrosis factor-&alpha; (TNF&alpha;) and IL-1 that activate endothelial cells (EC) and epithelial cells and further augment the inflammatory response7. Vascular endothelial dysfunction is an established feature of acute inflammation. Macrophages contribute to endothelial dysfunction during inflammation by mechanisms that remain unclear. Activation of macrophages results in the extracellular release of O2&bull;- and various pro-inflammatory cytokines, which triggers pathologic signaling in adjacent cells8. NADPH oxidases are the major and primary source of ROS in most of the cell types. Recently, it is shown by us and others9,10 that ROS produced by NADPH oxidases induce the mitochondrial ROS production during many pathophysiological conditions. Hence measuring the mitochondrial ROS production is equally important in addition to measuring cytosolic ROS. Macrophages produce ROS by the flavoprotein enzyme NADPH oxidase which plays a primary role in inflammation. Once activated, phagocytic NADPH oxidase produces copious amounts of O2&bull;- that are important in the host defense mechanism11,12. Although paracrine-derived O2&bull;- plays an important role in the pathogenesis of vascular diseases, visualization of paracrine ROS-induced intracellular signaling including Ca2+ mobilization is still hypothesis. We have developed a model in which activated macrophages are used as a source of O2&bull;- to transduce a signal to adjacent endothelial cells. Using this model we demonstrate that macrophage-derived O2&bull;- lead to calcium signaling in adjacent endothelial cells.

Visualization of Vascular Ca2+ Signaling Triggered by Paracrine Derived ROS

An efficient method to gain insights into visualizing the paracrine-derived ROS induction of endothelial Ca2+ signaling is described. This method takes advantage of measuring paracrine derived ROS triggered Ca2+ mobilization in vascular endothelial cells in a co-culture model.

Oxidative stress has been implicated in a number of pathologic conditions including ischemia/reperfusion damage and sepsis. The concept of oxidative ...

Biology

A Mouse Model to Assess Innate Immune Response to Staphylococcus aureus Infection

Staphylococcus aureus (S. aureus)&#160;infections, including methicillin resistant stains, are an enormous burden on the healthcare system. With incidence rates of S. aureus infection climbing annually, there is a demand for additional research in its&#160;pathogenicity. Animal models of infectious disease advance our understanding of the host-pathogen response and lead to the development of effective therapeutics. Neutrophils play a primary role in the innate immune response that controls S. aureus infections by forming an abscess to wall off the infection and facilitate bacterial clearance; the number of neutrophils that infiltrate an S. aureus skin infection often correlates with disease outcome. LysM-EGFP mice, which possess the enhanced green fluorescent protein (EGFP) inserted in the Lysozyme M (LysM) promoter region (expressed primarily by neutrophils), when used in conjunction with in vivo whole animal fluorescence imaging (FLI) provide&#160;a means of quantifying neutrophil emigration noninvasively and longitudinally into wounded skin. When combined with a bioluminescent S. aureus strain and sequential in vivo whole animal bioluminescent imaging (BLI), it is possible to longitudinally monitor both the neutrophil recruitment dynamics and in vivo bacterial burden at the site of infection in anesthetized mice from onset of infection to resolution or death. Mice are more resistant to a number of virulence factors produced by S. aureus that facilitate effective colonization and infection in humans. Immunodeficient mice provide a more sensitive animal model to examine persistent S. aureus infections and the ability of therapeutics to boost innate immune responses. Herein, we characterize responses in LysM-EGFP mice that have been bred to MyD88-deficient mice (LysM-EGFP&#215;MyD88-/- mice) along with wild-type LysM-EGFP mice to investigate S. aureus skin wound infection. Multispectral simultaneous detection enabled study of neutrophil recruitment dynamics by using in vivo FLI, bacterial burden by using in vivo BLI, and wound healing longitudinally and noninvasively over time.

A Mouse Model to Assess Innate Immune Response to Staphylococcus aureus Infection

An approach is described for real-time detection of the innate immune response to cutaneous wounding and Staphylococcus aureus infection of mice. By comparing LysM-EGFP mice (which possess fluorescent neutrophils) with a LysM-EGFP crossbred immunodeficient mouse strain, we advance our understanding of infection and the development of approaches to combat infection.

Staphylococcus aureus (S. aureus)&#160;infections, including methicillin resistant stains, are an enormous burden on the healthcare system. With incidence ...

Stimulation of Stem Cell Niches and Tissue Regeneration in Mouse Skin by Switchable Protoporphyrin IX-Dependent Photogeneration of Reactive Oxygen Species In Situ

Here, we describe a protocol to induce switchable in vivo photogeneration of endogenous reactive oxygen species (ROS) in mouse skin. This transient production of ROS in situ efficiently activates cell proliferation in stem cell niches and stimulates tissue regeneration as strongly manifested through the acceleration of burn healing and hair follicle growth processes. The protocol is based on a regulatable photodynamic treatment that treats the tissue with precursors of the endogenous photosensitizer protoporphyrin IX and further irradiates the tissue with red light under tightly controlled physicochemical parameters. Overall, this protocol constitutes an interesting experimental tool to analyze ROS biology.

The aim of this protocol is to induce transient in vivo production of nonlethal levels of reactive oxygen species (ROS) in mouse skin, further promoting physiological responses in the tissue.

Here, we describe a protocol to induce switchable in vivo photogeneration of endogenous reactive oxygen species (ROS) in mouse skin. This transient ...

A Murine Model of a Burn Wound Reconstructed with an Allogeneic Skin Graft

Trivial superficial wounds heal without complications by primary intention. Deep wounds, such as full thickness burns, heal by secondary intention and require surgical debridement and skin grafting. Successful integration of the donor graft into a recipient wound bed depends on timely recruitment of immune cells, robust angiogenic response and new extracellular matrix formation. The development of novel therapeutic agents, which target some key processes involved in wound healing, are hindered by the lack of reliable preclinical models with optimized objective assessment of wound closure. Here, we describe an inexpensive and reproducible model of experimental full thickness burn wound reconstructed with an allogeneic skin graft. The wound is induced on the dorsum surface of anaesthetized inbred wild type mice from the BALB/C and SKH1-Hrhr backgrounds. The burn is produced using a brass template measuring 10 mm in diameter, which is preheated to 80 &#176;C and delivered at a constant pressure for 20 s. Burn eschar is excised 24 hours after the injury and replaced with a full thickness graft harvested from the tail of a genetically similar donor mouse. No specialized equipment is required for the procedure and surgical techniques are straightforward to follow. The method may be effortlessly implemented and reproduced in most research settings. Certain limitations are associated with the model. Due to technical difficulties, the harvest of thinner split thickness skin grafts is not possible. The surgical method we describe here allows for the reconstruction of burn wounds using full thickness skin grafts. It may be used to carry out preclinical therapeutic testing.

The aim of this study was to develop a murine model of burn wound healing. A thermal burn was induced on the dorsal skin of mice using a preheated brass template. Burned tissue was debrided and overlaid with a skin graft harvested from the tail of a genetically similar donor mouse.

Trivial superficial wounds heal without complications by primary intention. Deep wounds, such as full thickness burns, heal by secondary intention and ...

Analyzing Oxidative Stress in Murine Intestinal Organoids using Reactive Oxygen Species-Sensitive Fluorogenic Probe

Reactive oxygen species (ROS) play essential roles in intestinal homeostasis. ROS are natural by-products of cell metabolism. They are produced in response to infection or injury at the mucosal level as they are involved in antimicrobial responses and wound healing. They are also critical secondary messengers, regulating several pathways, including cell growth and differentiation. On the other hand, excessive ROS levels lead to oxidative stress, which can be deleterious for cells and favor intestinal diseases like chronic inflammation or cancer. This work provides a straightforward method to detect ROS in the intestinal murine organoids by live imaging and flow cytometry, using a commercially available fluorogenic probe. Here the protocol describes assaying the effect of compounds that modulate the redox balance in intestinal organoids and detect ROS levels in specific intestinal cell types, exemplified here by the analysis of the intestinal stem cells genetically labeled with GFP. This protocol may be used with other fluorescent probes.

The present protocol describes a method to detect reactive oxygen species (ROS) in the intestinal murine organoids using qualitative imaging and quantitative cytometry assays. This work can be potentially extended to other fluorescent probes to test the effect of selected compounds on ROS.

Reactive oxygen species (ROS) play essential roles in intestinal homeostasis. ROS are natural by-products of cell metabolism. They are produced in ...

A Murine Skin Wound Infection Model to Study the Immune Response against a Bacterial Pathogen

Source: Anderson, L. S., et al. <a href="https://app.jove.com/v/59015">A Mouse Model to Assess Innate Immune Response to Staphylococcus aureus Infection.</a> J. Vis. Exp. (2019).
This video demonstrates a technique to generate a murine model of skin wound infection. Upon infecting mice &#8212; engineered to express a fluorescent protein in neutrophils &#8212; with a genetically modified bioluminescent Staphylococcus aureus &#8212; a pathogenic bacteria. The bacterial burden at the infection site and the neutrophil recruitment dynamics can be studied using whole animal imaging.

In Vivo Imaging of Reactive Oxygen Species in a Murine Wound Model

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A Murine Model of a Burn Wound Reconstructed with an Allogeneic Skin Graft

Analyzing Oxidative Stress in Murine Intestinal Organoids using Reactive Oxygen Species-Sensitive Fluorogenic Probe

Model zakażenia rany skóry myszy do badania odpowiedzi immunologicznej na patogen bakteryjny