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/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>

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 MCLA1,4. The compound is non-toxic, easily absorbed, and has much stronger bioluminescence as compared to luminol or similar probes12. Critically, L-012 can be safely administered multiple times in continuum for longitudinal analysis without any adverse effects or harm to animal models6. This strategy requires limited special training, and the equipment necessary is readily available in most research laboratories, making it a widely accessible protocol.
For optimal use, we suggest that reagents be properly up to date and protected from light to avoid degradation. Humanizing wounds through stenting allows for murine wounds to heal through migration and re-epithelialization, as opposed to by significant contraction. Sutures should be placed two thirds of the way through the stent perimeter to secure wound edges in place. Wrapping mice with transparent film dressing after gel application ensures that topical treatments remain in place. To prevent self-inflicted wounds in the mice, which may confound results, bitter-tasting spray may be applied to the periphery and suture knots to discourage biting. L-012 has limited solubility in PBS but will form a suspension by repetitive pipetting of the solution. These troubleshooting measures ensure consistency across different wounds, eliminating inter-user variability while maintaining animal comfort. Since this technique relies on the intraperitoneal injection of a solution into an animal model, there are several interfering factors inherent to the animals which cannot be controlled. For example, local blood flow and subsequent absorption and localization of L-012 to the areas of interest cannot be dictated. However, this variable can be mitigated through the use of an animal as its own internal control such that one wound can be treated with siNS and the other with the siKeap1.
We report on the utility of a strategy for the in vivo study of ROS in a cutaneous wound healing model. In our present study, treatment of wounds with Keap1 siRNA resulted in decreased oxidative burden which confirms our understanding of the significance of the Nrf2/Keap1 pathway for antioxidant handling. While this method allows for quantitative analysis, there are a number of important limitations. While L-012 is highly sensitive, it is not specific for ROS and has been shown to also react to reactive nitrogen species6. Further analysis with targeted probes and immunoassay-based methods complement this approach through enhanced specificity and subcellular localization of ROS correlates13. The proposed mechanism for L-012-based bioluminescence involves the oxidation of L-012 by molecular oxygen through the activity of peroxidase in combination with H2O212. This limits the use of this technique for studying antioxidant enzyme inhibitors that interact with peroxidase. More detailed analysis intended to specifically quantify ROS other than superoxide anion or reactive nitrogen species metabolites is not possible with the current strategy.
Given the high sensitivity of L-012 for detecting ROS broadly, it is readily adaptable for various cutaneous wound healing and other tissue models. We have demonstrated the expediency of this technique for assessing the redox implications of targeted therapeutics to diabetic wounds such as RTA 408 and the lipoproteoplex delivery of Keap1 siRNA14,15. Given the significant strengths of this protocol, there is immense future potential in applying similar strategies for studying ROS within deeper compartments through ex vivo approaches, focused dissections, and organoids.

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 in areas of the wound where ROS is detected (Figure 1C). After selecting the correct imaging and analysis settings as outlined in Figure 2, proceed with imaging. Bioluminescence is recorded for 1 minute at 5-minute intervals over the course of 60 minutes (Figure 3). This demonstrates the bioluminescence saturation of the region of interest over time. In our animal model, optimal imaging time to reach complete L-012 saturation was determined to be 50 min, after which no notable difference in bioluminescence was appreciated. This time will differ depending on the animal model utilized and should be independently determined and optimized for varying wound models depending on size, animal type, treatment, etc.
A region of interest (ROI) for the purpose of quantifying bioluminescence limited to the diabetic wound area is drawn on the overlay image (Figure 4). These ROIs are similarly defined for all wounds including before L-012 injection (Figure 4A), and after L-012 injection in nonsense siRNA-treated (Figure 4B) and Keap1 siRNA-treated mice (Figure 4C). Bioluminescence was calculated by dividing the total counts of light intensity by the area. Table 1 shows the calculations graphed in Figure 4D. The nonsense siRNA-treated diabetic wounds had 45,775 11,649 bioluminescence/cm2, while Keap1 suppression resulted in 19,405 5,939 bioluminescence/cm2 (mean SD). A Student&#39;s t-test demonstrated a significantly decreased bioluminescence (p=0.042) in the Keap1 siRNA-treated wound as compared to the nonsense siRNA-treated wound, correlating with lower oxidative burden with higher Nrf2 activity (Figure 4D). To confirm that the relative levels of ROS visualized by L-012 on siNS and siKeap1 correlate with other established means of measuring inflammation, we analyzed 10 day wound tissue sections. H&#38;E stains showed reduced cellular infiltration in siKeap1-treated wounds in contrast to siNS-treated ones, indicating reduced inflammatory morphology (Figure 5A). Immunoreactivity of F4/80, a protein macrophage marker, (red fluorescence) on wound tissue sections revealed reduced macrophages in siKeap1 treated wounds as compared to siNS-treated wounds (Figure 5B). This further demonstrates that the ROS levels visualized by L-012 are accurate and correlate with other established ROS measurement models. Critically, applying this technique did not necessitate sacrificing the animals for tissue sections as is required for H&#38;E and immunofluorescence staining.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58450/58450fig1.jpg" /> 
Figure 1: Experimental set-up for imaging. (A) Diabetic mice are wounded using an established excisional wound model. (B) A baseline overlay of photograph with bioluminescence pre-L-012 injection. (C) Following intraperitoneal injection with L-012, ROS is visualized. Luminescence scale for B and C. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58450/58450fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/58450/58450fig2.jpg" /> 
Figure 2: Image and analysis settings in in vitro imaging system program. (A) Select &#34;Imaging Wizard&#34; on the Acquisition Control Panel (red arrow). (B) &#34;Bioluminescence Imaging&#34; is the imaging mode selected. (C) &#34;Open filter&#34; is selected as the measurement technique. (D) Imaging subject selected is &#34;mouse&#34; and the &#34;time series study&#34; option is selected. The total number of segments and delay time between segments is input. (E) Select &#34;Acquire Sequence&#34; to proceed with imaging. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58450/58450fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/58450/58450fig3.jpg" /> 
Figure 3: Longitudinal studies of in vivo ROS imaging. Diabetic mice are imaged for 1 minute following intraperitoneal injection with L-012 at 5 minute intervals over the course of 60 min. Red circles: ROI. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58450/58450fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/58450/58450fig4.jpg" /> 
Figure 4: Quantifying bioluminescence. Photographs with bioluminescence overlay of diabetic wounds (A) prior to L-012 injection, (B) with wounds treated with siNS after L-012 injection, and (C) with wounds treated with siKeap1 after L-012 injection. (D) Quantification of average bioluminescence divided by surface area of the ROIs for siNS and siKeap1 treated wounds. Yellow circles: ROI. Data are represented as mean&#177;standard deviation; *p &#60; 0.05, n=4. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58450/58450fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/58450/58450fig5.jpg" /> 
Figure 5: ROS Correlations with immunohistochemical stains. (A) H&#38;E stains of diabetic wound tissue sections at 10 days showed reduced cellular infiltrate as evidence of reduced inflammatory morphology in the siKeap1 treated wounds as compared to the siNS-treated wounds. (B) F4/80 immunofluorescence (red) showed decreased number of macrophages in siKeap1 treated wound sections at 10 days as compared to their siNS-treated counterparts. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58450/58450fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>ROI</td>
			<td>si</td>
			<td>Total Counts</td>
			<td>Avg Counts</td>
			<td>Stdev Counts</td>
			<td>Area [cm&#178;]</td>
			<td>Total counts/area</td>
			<td>Average counts/area</td>
		</tr>
		<tr>
			<td>ROI 1</td>
			<td>NS</td>
			<td>9.38E+03</td>
			<td>1.14E+02</td>
			<td>3.85E+01</td>
			<td>2.48E-01</td>
			<td>3.78E+04</td>
			<td>4.58E+04</td>
		</tr>
		<tr>
			<td>ROI 2</td>
			<td>NS</td>
			<td>4.68E+03</td>
			<td>5.37E+01</td>
			<td>3.13E+01</td>
			<td>2.63E-01</td>
			<td>1.78E+04</td>
			<td rowspan="3"></td>
		</tr>
		<tr>
			<td>ROI 3</td>
			<td>NS</td>
			<td>1.72E+04</td>
			<td>2.18E+02</td>
			<td>1.54E+02</td>
			<td>2.39E-01</td>
			<td>7.20E+04</td>
		</tr>
		<tr>
			<td>ROI 4</td>
			<td>NS</td>
			<td>1.24E+04</td>
			<td>1.68E+02</td>
			<td>8.41E+01</td>
			<td>2.24E-01</td>
			<td>5.55E+04</td>
		</tr>
		<tr>
			<td>ROI 5</td>
			<td>Keap1</td>
			<td>3.21E+03</td>
			<td>3.41E+01</td>
			<td>2.76E+01</td>
			<td>2.84E-01</td>
			<td>1.13E+04</td>
			<td>1.94E+04</td>
		</tr>
		<tr>
			<td>ROI 6</td>
			<td>Keap1</td>
			<td>2.56E+03</td>
			<td>2.88E+01</td>
			<td>1.06E+01</td>
			<td>2.69E-01</td>
			<td>9.52E+03</td>
			<td rowspan="3"></td>
		</tr>
		<tr>
			<td>ROI 7</td>
			<td>Keap1</td>
			<td>4.92E+03</td>
			<td>6.48E+01</td>
			<td>5.03E+01</td>
			<td>2.30E-01</td>
			<td>2.14E+04</td>
		</tr>
		<tr>
			<td>ROI 8</td>
			<td>Keap1</td>
			<td>8.34E+03</td>
			<td>1.07E+02</td>
			<td>5.25E+01</td>
			<td>2.36E-01</td>
			<td>3.54E+04</td>
		</tr>
	</tbody>
</table>
Table 1: Quantifying bioluminescence for defined ROIs. Bioluminescence is measured for each ROI and standardized relative to surface area. Calculations for mean, standard deviation, and standard error are calculated accordingly. ROI 1, 2, 3, and 4 represent ROI from wounds of biological repeats of siNS treated diabetic wounds and ROI 5, 6, 7, and 8 represent ROIs from biological repeats of siKeap1 treated diabetic wounds.

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 ...

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Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

10.7K 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

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 common forms of disease are visceral leishmaniasis (VL) and cutaneous leishmaniasis (CL). Mouse models of leishmaniasis are widely used, but quantification of parasite burdens during murine disease requires mice to be euthanized at various times after infection. Parasite loads are then measured either by microscopy, limiting dilution assay, or qPCR amplification of parasite DNA. The in vivo imaging system (IVIS) has an integrated software package that allows the detection of a bioluminescent signal associated with cells in living organisms. Both to minimize animal usage and to follow infection longitudinally in individuals, in vivo models for imaging Leishmania spp. causing VL or CL were established. Parasites were engineered to express luciferase, and these were introduced into mice either intradermally or intravenously. Quantitative measurements of the luciferase driving bioluminescence of the transgenic Leishmania parasites within the mouse were made using IVIS. Individual mice can be imaged multiple times during longitudinal studies, allowing us to assess the inter-animal variation in the initial experimental parasite inocula, and to assess the multiplication of parasites in mouse tissues. Parasites are detected with high sensitivity in cutaneous locations. Although it is very likely that the signal (photons/second/parasite) is lower in deeper visceral organs than the skin, but quantitative comparisons of signals in superficial versus deep sites have not been done. It is possible that parasite numbers between body sites cannot be directly compared, although parasite loads in the same tissues can be compared between mice. Examples of one visceralizing species (L. infantum chagasi) and one species causing cutaneous leishmaniasis (L. mexicana) are shown. The IVIS procedure can be used for monitoring and analyzing small animal models of a wide variety of Leishmania species causing the different forms of human leishmaniasis.

In vivo Imaging of Transgenic Leishmania Parasites in a Live Host

An in vivo imaging system is used to generate quantitative measurements of murine infection with the Trypanosomatid protozoan Leishmania. This is a non-invasive and non-lethal method for detecting parasites expressing luciferase within many tissues throughout the course of chronic Leishmania spp. infection.

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

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 provides rich spatiotemporal information regarding cell behaviors within intact tissues and in live mice. Leukocyte recruitment plays a significant role in host defense against infection and when unchecked, can contribute to inflammatory and autoimmune diseases. Studying leukocyte recruitment in vivo is technically challenging since cells are moving rapidly within vessels located deep within light scattering tissues. To date, most intravital imaging studies require surgical preparation to expose the blood vessels and tissues. To avoid the tissue damage and inflammation induced by surgery itself, here, we describe a non-invasive single-cell imaging approach that can be used to study leukocyte trafficking in the mouse footpad and phalanges. We discuss the technical aspects of our 2P imaging preparation and walk the reader through a typical experiment from initial set up to execution and data collection.

Non-invasive Imaging of Leukocyte Homing and Migration in vivo

Here, we describe a non-invasive two-photon (2P) microscopy approach to study leukocyte homing in the mouse footpad. We discuss the technical aspects of our tissue imaging preparation and walk the reader through a typical experiment from initial set up to execution and data collection.

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 ...

Genotypic Inference of HIV-1 Tropism Using Population-based Sequencing of V3

Background: Prior to receiving a drug from CCR5-antagonist class in HIV therapy, a patient must undergo an HIV tropism test to confirm that his or her viral population uses the CCR5 coreceptor for cellular entry, and not an alternative coreceptor. One approach to tropism testing is to examine the sequence of the V3 region of the HIV envelope, which interacts with the coreceptor.
Methods: Viral RNA is extracted from blood plasma. The V3 region is amplified in triplicate with nested reverse transcriptase-PCR. The amplifications are then sequenced and analyzed using the software, RE_Call. Sequences are then submitted to a bioinformatic algorithm such as geno2pheno to infer viral tropism from the V3 region. Sequences are inferred to be non-R5 if their geno2pheno false positive rate falls below 5.75%. If any one of the three sequences from a sample is inferred to be non-R5, the patient is unlikely to respond to a CCR5-antagonist.

HIV tropism can be inferred from the V3 region of the viral envelope. V3 is PCR amplified in triplicate using nested RT-PCR, sequenced, and interpreted using bioinformatic software. Samples with with 1 or more sequence(s) with low g2P scores are classified as non-R5 virus.

Background: Prior to receiving a drug from CCR5-antagonist class in HIV therapy, a patient must undergo an HIV tropism test to confirm that his or her ...

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 deficiencies. GVHD is caused by mature alloreactive T cells present in the bone marrow graft that are infused into the recipient and cause damage to host organs. However, in mice, T cells must be added to the bone marrow inoculum to cause GVHD. Although extensive work has been done to characterize T cell responses post transplant, bioluminescent imaging technology is a non-invasive method to monitor T cell trafficking patterns in vivo. 
Following lethal irradiation, recipient mice are transplanted with bone marrow cells and splenocytes from donor mice. T cell subsets from L2G85.B6 (transgenic mice that constitutively express luciferase) are included in the transplant. By only transplanting certain T cell subsets, one is able to track specific T cell subsets in vivo, and based on their location, develop hypotheses regarding the role of specific T cell subsets in promoting GVHD at various time points. At predetermined intervals post transplant, recipient mice are imaged using a Xenogen IVIS CCD camera. Light intensity can be quantified using Living Image software to generate a pseudo-color image based on photon intensity (red = high intensity, violet = low intensity). 
Between 4-7 days post transplant, recipient mice begin to show clinical signs of GVHD. Cooke et al.1 developed a scoring system to quantitate disease progression based on the recipient mice fur texture, skin integrity, activity, weight loss, and posture. Mice are scored daily, and euthanized when they become moribund. Recipient mice generally become moribund 20-30 days post transplant. 
Murine models are valuable tools for studying the immunology of GVHD. Selectively transplanting particular T cell subsets allows for careful identification of the roles each subset plays. Non-invasively tracking T cell responses in vivo adds another layer of value to murine GVHD models.

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

Murine bone marrow transplantation is a widely used technique to study immunological mechanisms governing graft-versus-host disease in humans. The ability to monitor T cell trafficking patterns in vivo allows for detailed analysis of the development and perpetuation of T cell responses during graft-versus-host disease.

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 ...

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 caused by these organisms. Here we describe a procedure for protein extraction from Burkholderia species based on mechanical lysis using glass beads in the presence of ethylenediamine tetraacetic acid and phenylmethylsulfonyl fluoride in phosphate buffered saline. This method can be used for different Burkholderia species, for different growth conditions, and it is likely suitable for the use in proteomic studies of other bacteria. Following protein extraction, a two-dimensional (2-D) gel electrophoresis proteomic technique is described to study global changes in the proteomes of these organisms. This method consists of the separation of proteins according to their isoelectric point by isoelectric focusing in the first dimension, followed by separation on the basis of molecular weight by acrylamide gel electrophoresis in the second dimension. Visualization of separated proteins is carried out by silver staining.

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

Members of the Burkholderia genus are pathogens of clinical importance. We describe a method for total bacterial protein extraction, using mechanical disruption, and 2-D gel electrophoresis for subsequent proteomic analysis.

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

Bone marrow is a complex organ that contains various hematopoietic and non-hematopoietic cells. These cells are involved in many biological processes, including hematopoiesis, immune regulation and tumor regulation. Commonly used methods for understanding cellular actions in the bone marrow, such as histology and blood counts, provide static information rather than capturing the dynamic action of multiple cellular components in vivo. To complement the standard methods, a window chamber (WC)-based model was developed to enable serial in vivo imaging of cells and structures in the murine bone marrow. This protocol describes a surgical procedure for installing the WC in the femur, in order to facilitate long-term optical access to the femoral bone marrow. In particular, to demonstrate its experimental utility, this WC approach was used to image and track neutrophils within the vascular network of the femur, thereby providing a novel method to visualize and quantify immune cell trafficking and regulation in the bone marrow. This method can be applied to study various biological processes in the murine bone marrow, such as hematopoiesis, stem cell transplantation, and immune responses in pathological conditions, including cancer.

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

The protocol describes a novel murine femur window chamber model that can be used to track movement of cells in the femoral bone marrow in vivo. Intravital multiphoton fluorescence microscopy is used to image three components of the femoral bone marrow (vasculature, collagen matrix, and neutrophils) over time.

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

This protocol illustrates the production of 64Cu and the chelator conjugation/radiolabeling of a monoclonal antibody (mAb) followed by murine lymphocyte cell culture and 64Cu-antibody receptor targeting of the cells. In vitro evaluation of the radiolabel and non-invasive in vivo cell tracking in an animal model of an airway delayed-type hypersensitivity reaction (DTHR) by PET/CT are described.
In detail, the conjugation of a mAb with the chelator 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) is shown. Following the production of radioactive 64Cu, radiolabeling of the DOTA-conjugated mAb is described. Next, the expansion of chicken ovalbumin (cOVA)-specific CD4+ interferon (IFN)-&#947;-producing T helper cells (cOVA-TH1) and the subsequent radiolabeling of the cOVA-TH1 cells are depicted. Various in vitro techniques are presented to evaluate the effects of 64Cu-radiolabeling on the cells, such as the determination of cell viability by trypan blue exclusion, the staining for apoptosis with Annexin V for flow cytometry, and the assessment of functionality by IFN-&#947; enzyme-linked immunosorbent assay (ELISA). Furthermore, the determination of the radioactive uptake into the cells and the labeling stability are described in detail. This protocol further describes how to perform cell tracking studies in an animal model for an airway DTHR and, therefore, the induction of cOVA-induced acute airway DHTR in BALB/c mice is included. Finally, a robust PET/CT workflow including image acquisition, reconstruction, and analysis is presented.
The 64Cu-antibody receptor targeting approach with subsequent receptor internalization provides high specificity and stability, reduced cellular toxicity, and low efflux rates compared to common PET-tracers for cell labeling, e.g.64Cu-pyruvaldehyde bis(N4-methylthiosemicarbazone) (64Cu-PTSM). Finally, our approach enables non-invasive in vivo cell tracking by PET/CT with an optimal signal-to-background ratio for 48 h. This experimental approach can be transferred to different animal models and cell types with membrane-bound receptors that are internalized.

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

Following the preparation of a 64Cu-modified monoclonal antibody binding to a transgenic murine T cell receptor, T cells are radiolabeled in vivo, analyzed for viability, functionality, labeling stability and apoptosis, and adoptively transferred into mice with an airway delayed-type hypersensitivity reaction for non-invasive imaging by positron emission tomography/computed tomography (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

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. ...

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 where they execute crucial antimicrobial functions. Their trafficking to these loci is orchestrated by several inflammatory chemoattractants, including chemokines. At the molecular level, chemoattractant signaling is regulated by the intracellular trafficking of the corresponding receptors. However, it remains unclear how subcellular changes in chemokine receptors affect leukocyte migration dynamics at the cell and tissue level. Here we describe a methodology for live imaging and quantitative analysis of chemokine receptor dynamics in neutrophils during inflammatory responses to tissue damage. These tools have revealed that differential chemokine receptor trafficking in zebrafish neutrophils coordinates neutrophil clustering and dispersal at sites of tissue damage. This has implications for our understanding of how inflammatory responses are self-resolved. The described tools could be used to understand neutrophil migration patterns in a variety of physiological and pathological settings and the methodology could be expanded to other signaling receptors.

Here we describe protocols to perform live imaging and quantitative analysis of chemoattractant receptor dynamics in zebrafish neutrophils

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

Recurrent Escherichia coli Urinary Tract Infection Triggered by Gardnerella vaginalis Bladder Exposure in Mice

Recurrent urinary tract infections (rUTI) caused by uropathogenic Escherichia coli (UPEC) are common and costly. Previous articles describing models of UTI in male and female mice have illustrated the procedures for bacterial inoculation and enumeration in urine and tissues. During an initial bladder infection in C57BL/6 mice, UPEC establish latent reservoirs inside bladder epithelial cells that persist following clearance of UPEC bacteriuria. This model builds on these studies to examine rUTI caused by the emergence of UPEC from within latent bladder reservoirs. The urogenital bacterium Gardnerella vaginalis is used as the trigger of rUTI in this model because it is frequently present in the urogenital tracts of women, especially in the context of vaginal dysbiosis that has been associated with UTI. In addition, a method for in situ bladder fixation followed by scanning electron microscopy (SEM) analysis of bladder tissue is also described, with potential application to other studies involving the bladder.

Recurrent Escherichia coli Urinary Tract Infection Triggered by Gardnerella vaginalis Bladder Exposure in Mice

A mouse model of uropathogenic E. coli (UPEC) transurethral inoculation to establish latent intracellular bladder reservoirs and subsequent bladder exposure to G. vaginalis to induce recurrent UPEC UTI is demonstrated. Also demonstrated are the enumeration of bacteria, urine cytology, and in situ bladder fixation and processing for scanning electron microscopy.

Recurrent urinary tract infections (rUTI) caused by uropathogenic Escherichia coli (UPEC) are common and costly. Previous articles describing models of ...