Name: in vivo imaging of reactive oxygen species in a murine wound model
Uploaded: 2018-11-17T13:00:00
Description: Watch this Scientific Journal Video about In Vivo Imaging of Reactive Oxygen Species in a Murine Wound Model at JoVE.com

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 p...

<ol>
	<li>Nishinaka, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+sensitive+chemiluminescence+probe,+L-012,+for+measuring+the+production+of+superoxide+anion+by+cells.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+NAD(P)H+oxidase-derived+superoxide+with+the+luminol+analogue+L-012.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+reactive+oxygen+species+generated+by+neutrophils+using+a+chemiluminescence+probe+L-012.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sensitive+superoxide+detection+in+vascular+cells+by+the+new+chemiluminescence+dye+L-012.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&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.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+imaging+of+reactive+oxygen+and+nitrogen+species+in+murine+colitis.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+and+reproducible+murine+model+of+excisional+wound+healing.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Restoration+of+Nrf2+Signaling+Normalizes+the+Regenerative+Niche.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+ROS+signaling+in+cells+and+animals.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+imaging+of+reactive+oxygen+and+nitrogen+species+in+inflammation+using+the+luminescent+probe+L-012.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualizing+Oxidative+Cellular+Stress+Induced+by+Nanoparticles+in+the+Subcytotoxic+Range+Using+Fluorescence+Lifetime+Imaging.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+the+use+of+L-012,+a+luminol-based+chemiluminescent+probe,+for+detecting+superoxide+and+identifying+inhibitors+of+NADPH+oxidase:+a+reevaluation.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+for+detection+of+mitochondrial+and+cellular+reactive+oxygen+species.">Methods for detection of mitochondrial and cellular reactive oxygen species.</a> Antioxidants &amp; Redox Signalling. 20 (2), 372-382 (2014).</li><li>Rabbani, P. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+Nrf2+activation+therapy+with+RTA+408+enhances+regenerative+capacity+of+diabetic+wounds.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+lipoproteoplex+delivers+Keap1+siRNA+based+gene+therapy+to+accelerate+diabetic+wound+healing.">Novel lipoproteoplex delivers Keap1 siRNA based gene therapy to accelerate diabetic wound healing.</a> Biomaterials. 132, 1-15 (2017).</li></ol>

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 border="0" cellpadding="0" cellspacing="0" width="618">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:265px;">BKS.Cg-Dock7m+/+ Leprdb/J mice</td>
			<td style="width:164px;">Jackson Laboratories</td>
			<td style="width:111px;">000642</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">13 cm x 18 cm Silicone sheet (0.6 mm)</td>
			<td>Sigma Aldrich&#160;</td>
			<td>665581</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">3M Tegaderm Transparent Film Dressings</td>
			<td>3M</td>
			<td>88-1626W</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Lipofectamine 2000 Transfection Reagent</td>
			<td>Life Technologies&#160;</td>
			<td>11668027</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Keap1 Stealth siRNA</td>
			<td>Thermofisher Scientific</td>
			<td>1299001</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Silencer negative control&#160;</td>
			<td>Thermofisher Scientific&#160;</td>
			<td>AM4635</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Opti-MEM Reduced Serum</td>
			<td>ThermoFisher Scientific</td>
			<td>11058021</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">DPBS</td>
			<td>ThermoFisher Scientific</td>
			<td>14040133</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Methyl-cellulose&#160;</td>
			<td>Sigma Aldrich</td>
			<td>9004-67-5</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">L-012</td>
			<td>Wako Chemicals</td>
			<td>120-04891</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">IVIS Lumina III XR In Vivo Imaging System&#160;</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 ...

Research

JoVE Journal

Immunology and Infection

In vivo Imaging of Transgenic Leishmania Parasites in a Live Host

In vivo Imaging of Transgenic Leishmania Parasites in a Live Host

Distinct species of Leishmania, a protozoan parasite of the family Trypanosomatidae, typically cause different human disease manifestations. The most ...

Non-invasive Imaging of Leukocyte Homing and Migration in vivo

Non-invasive Imaging of Leukocyte Homing and Migration in vivo

Two-photon (2P) microscopy is a high resolution imaging technique that has been broadly adapted by biologists.  The value of 2P microscopy is that it ...

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

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

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

Bioluminescent Bacterial Imaging In Vivo

Bioluminescent Bacterial Imaging In Vivo

This video describes the use of whole body bioluminesce imaging (BLI) for the study of bacterial trafficking in live mice, with an emphasis on the use of ...

Measurement of Tactile Allodynia in a Murine Model of Bacterial Prostatitis

Uropathogenic Escherichia coli (UPEC) are pathogens that play an important role in urinary tract infections and bacterial prostatitis1. We have recently ...

Total Protein Extraction and 2-D Gel Electrophoresis Methods for Burkholderia Species

Total Protein Extraction and 2-D Gel Electrophoresis Methods for Burkholderia Species

The investigation of  the intracellular protein levels of bacterial species is of importance to  understanding the pathogenic mechanisms of diseases ...

Femur Window Chamber Model for In Vivo Cell Tracking in the Murine Bone Marrow

Femur Window Chamber Model for In Vivo Cell Tracking in the Murine Bone Marrow

Bone marrow is a complex organ that contains various hematopoietic and non-hematopoietic cells. These cells are involved in many biological processes, ...

Murine Lymphocyte Labeling by 64Cu-Antibody Receptor Targeting for In Vivo Cell Trafficking by PET/CT

Murine Lymphocyte Labeling by 64Cu-Antibody Receptor Targeting for In Vivo  Cell Trafficking by PET/CT

This protocol illustrates the production of 64Cu and the chelator conjugation/radiolabeling of a monoclonal antibody (mAb) followed by murine lymphocyte ...

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

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. ...

Live Imaging of Chemokine Receptors in Zebrafish Neutrophils During Wound Responses

Leukocyte guidance by chemical gradients is essential for immune responses. Neutrophils are the first cells to be recruited to sites of tissue damage ...