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. Author manuscript; available in PMC: 2014 Nov 23.
Published in final edited form as: Mol Imaging. 2010 Feb;9(1):30–39.

Quantitative Assessment of Intestinal Injury Using a Novel In Vivo, Near-Infrared Imaging Technique

Todd W Costantini 1, Brian P Eliceiri 1, Carrie Y Peterson 1, William H Loomis 1, James G Putnam 1, Andrew Baird 1, Paul Wolf 1, Vishal Bansal 1, Raul Coimbra 1
PMCID: PMC4241240  NIHMSID: NIHMS359186  PMID: 20128996

Abstract

Intestinal injury owing to inflammation, severe trauma, and burn is a leading cause of morbidity and mortality. Currently, animal models employed to study the intestinal response to injury and inflammation depend on outdated methods of analysis. Given that these classic intestinal assays are lethal to the experimental animal, there is no ability to study the gut response to injury in the same animal over time. We postulated that by developing an in vivo assay to image intestinal injury using fluorescent dye, it could complement other expensive, time-consuming, and semiquantitative classic means of detecting intestinal injury. We describe a novel in vivo, noninvasive method to image intestinal injury using a charge-coupled device (CCD) camera that allows for serial visual and quantitative analysis of intestinal injury. Our results correlate with traditional, time–consuming, semiquantitative assays of intestinal injury, now allowing the noninvasive, nonlethal assessment of injury over time.

Although near-infrared imaging is increasingly being used to monitor diverse biologic conditions, it has never been applied to quantify the intestinal response to disease.1, 2 Regardless of its etiology, numerous medical conditions, including inflammatory bowel disease,3 necrotizing enterocolitis,4 sepsis,5 enteritis,6 liver disease,7 burn,8 and trauma,9 result in tight junction breakdown and increased intestinal epithelial permeability. Paracellular permeability and translocation of pathogens from within the gastrointestinal tract can result in an intestinal inflammatory response, characterized by proinflammatory cytokine synthesis. Intestinal barrier injury is an important inciting event that can result in the systemic inflammatory response, which is seen after severe injury.10

Translational research efforts studying the intestinal inflammatory response allow these clinical observations to be studied in the laboratory, where we rely on animal models to understand the gut response to injury. Current methods for studying intestinal injury include histology, immunohistochemistry, immunoblotting, and cytokine analysis. Intestinal permeability assays are important in determining intestinal barrier function. These assays study permeability by measuring the movement of tracers from the intestinal lumen into the systemic circulation. The classic methods of determining intestinal injury have been used for decades and continue to give researchers valuable information about intestinal function. These methods do have several limitations in that they require sacrifice of the animal prior to harvest of tissues and subsequent time-intensive methods of analysis. Given that these classic intestinal assays are lethal to the experimental animal, there is no ability to study the gut response to injury in the same animal over time.

Advances in imaging techniques and the evolution of near-infrared fluorescence have allowed for novel evaluations of the intestine following injury. Techniques such as magnetic resonance imaging and electron paramagnetic resonance spectroscopy have been used to monitor changes in the intestine following ischemia reperfusion.11, 12 Near-infrared spectroscopy is another method that has recently been used to study in vivo changes in gut pH and image the presence of cellular damage using models of intestinal injury.13, 14 Fluorescent proteins or near-infrared quantum dots can be bound to molecules of interest and tracked using fluorescence imaging techniques, serving as a biomarker based on the distribution of fluorescence signal.15, 16 As technology has progressed, the goal of developing an assay that allows for a relatively inexpensive, rapid screening of intestinal injury has become attainable.

The inability to easily, quickly, and effectively measure intestinal injury in animal models has limited our capacity to monitor the gut response to injury and the effects of candidate therapeutics. To this end, we postulated that by developing an in vivo assay to image intestinal injury using fluorescent dye, it could be an ideal alternative evaluation strategy with several advantages over other expensive, time-consuming, and semiquantitative classic means of detecting intestinal injury.

Materials and Methods

Burn Model

Male BALB/c mice weighing 20 to 24 g were purchased from Jackson Laboratory (Sacramento, CA). Animals were placed in a 12-hour light/dark cycle and provided with food and water ad libitum. Prior to the experiment, animals were anesthetized with inhaled isoflurane. The dorsal fur was removed using an electronic clipper. Animals were placed in a template estimating 30% total body surface area (TBSA). While under anesthesia, animals were subjected to a 7-second dorsal steam burn. Immediately following burn, animals were given a subcutaneous injection of 1.5 mL normal saline with buprenorphine in a nonburned area. Animals were returned to their cages to recover from anesthesia. Sham animals underwent general anesthesia, dorsal fur clipping, and injection of normal saline with buprenorphine but were not burned.

These experiments were approved by the University of California Animal Subjects Committee and are in accordance with guidelines established by the National Institutes for Health.

Intestinal Permeability Assay to 4 kDa FITC-Dextran

Animals (n ≥ 5 per group) were anesthetized with inhaled isoflurane and a midline laparotomy was performed. A 5 cm segment of distal ileum was isolated between silk ties. An intraluminal injection of 5 mg fluorescein isothiocyanate (FITC)-dextran (4 kDa, Sigma, St. Louis, MO) in 200 µL phosphate-buffered saline (PBS) was performed at various time points following burn. The abdominal wall was closed following administration of FITC-dextran. Thirty minutes following intraluminal injection, blood was obtained via cardiac puncture and placed in heparinized Eppendorf tubes on ice. Blood was centrifuged at 10,000 g for 10 minutes, and the plasma was removed. The plasma was then analyzed for the concentration of FITC-dextran using a SpectraMax M5 fluorescence spectrophotometer (Molecular Devices, Sunnyvale, CA). A standard curve was obtained by diluting serial concentrations of FITC-dextran in mouse serum.

Intestinal Permeability to Increasing Size FITC-Dextran

Animals (n ≥ 3 per group) were anesthetized 4 hours following burn injury. Laparotomy and isolation of the distal ileum were performed prior to intraluminal injection of 5 mg of either 20 or 70 kDa FITC-dextran (Sigma) in 200 µL PBS. Cardiac puncture was performed 30 minutes after intraluminal injection as previously described. Plasma was obtained for measurement in a SpectraMax M5 fluorescence spectrophotometer (Molecular Devices). A standard curve was obtained by diluting serial concentrations of each molecular-weight FITC-dextran in mouse plasma.

Histologic Evaluation

Animals (n = 3 per group) were sacrificed at several time points following burn for excision of the distal ileum for histologic evaluation. Specimens were stored in 10% PBS buffered formalin and embedded in paraffin blocks. Sections of distal ileum were cut 7 mm thick and placed onto glass slides. Specimens were stained with hematoxylin and eosin for imaging using an Olympus IX70 light microscope (Olympus, Center Valley, PA) at ×200 magnification. Images were obtained using Q-imaging software (Surrey, BC). Histologic gut injury was scored and reviewed by a pathologist blinded to the experimental groups. Three randomly selected fields from each specimen were graded based on a scoring system characterizing gut injury on a scale from 0 to 4: 0 = normal; no damage; 1 = mild; focal epithelial edema and necrosis; 2 = moderate; diffuse swelling or necrosis of the villi; 3 = severe; diffuse necrosis of the villi with evidence of neutrophil infiltration in the submucosa; 4 = major; widespread necrosis with massive neutrophil infiltration and hemorrhage as previously described.17

Fluorescence Imaging

Animals (n ≥ 3 per group) were anesthetized with inhaled isoflurane. A midline laparotomy was performed, and a 5 cm segment of distal ileum was isolated between silk ties. An injection of 5 mg FITC-dextran (4 kDa) or 1 µg Alexa Fluor 680 (3 kDa, Invitrogen, Carlsbad, CA) in 200 µL PBS was performed into the lumen of the isolated intestine. The abdominal wall was then closed with silk sutures. The animal was maintained under general anesthesia following the procedure. Thirty minutes following intraluminal injection of fluorescent tracer, the animal was placed in the Xenogen IVIS Lumina (Caliper LifeSciences, Hopkinton, MA) imaging system. Images were obtained with identical camera settings using Living Images 3.0 software (Caliper LifeSciences). Animal images were obtained using the Cy5.5 filter (Alexa Fluor 680) or the green fluorescent protein (GFP) filter (FITC) with 1-second exposure, f-stop of 1, from field of view C. Images of the excised intestinal segments were obtained using identical settings; however, they were imaged at field of view B. Quantification of fluorescence image was obtained by measuring the fluorescence within an equivalent region of interest for each animal using the Living Images software. Data are expressed as fluorescent intensity (arbitrary units) ± the standard error of the mean (SEM).

Gavage of Near-Infrared Tracer

With the animal under general anesthesia, a gavage was performed using PE-90 polyethylene tubing (Becton-Dickinson, Franklin Lakes, NJ). A 1 mL solution containing 5 µg of Alexa Fluor 680 was instilled into the stomach and small intestine (n = 4 animals per group). Serial images were obtained using the Xenogen IVIS Lumina imaging system with Living Images 3.0 software. To account for differences in the delivery of fluorescent dye into the gastrointestinal tract following gavage, data are represented as the decrease in fluorescent intensity seen from the same animal over the imaging time course. Relative fluorescent intensity was obtained by dividing the abdominal fluorescence obtained at each time point by the initial abdominal fluorescence obtained from that same animal immediately following gavage (time = 0).

Treatment with Pentoxifylline

To assess the ability of in vivo fluorescent imaging to detect changes in intestinal injury following therapeutic interventions, animals were treated with the antiinflammatory drug pentoxifylline (PTX; 1-[5-oxohexyl]–3,7-dimethylxanthine, Sigma). Immediately following 30% TBSA burn, animals underwent an intraperitoneal injection of 12.5 mg/kg PTX dissolved in normal saline (n = 6 animals) as previously described.8, 18 Animals were returned to their cage until 4 hours following burn injury, at which time they underwent gavage of Alexa 680 and serial imaging in the Xenogen IVIS Lumina.

Statistical Analysis

Data are expressed as the mean ± SEM. Statistical significance for gut injury scoring was performed using the Kruskal- Wallis test for nonparametric data with a post hoc Mann- Whitney test performed in pairwise fashion. The statistical significance among groups for the remainder of the experiments was determined using t -test or analysis of variance with Bonferroni correction where appropriate. Statistical analysis was performed using SPS S software v11.5 (SPSS, Chicago, IL). Statistical significance was defined as p < .05.

Results

Intestinal Permeability to FITC-Dextran

As molecular weight is known to affect paracellular permeability,19 we investigated intestinal permeability to 4, 20, and 70 kDa FITC-dextran at 4 hours following severe burn (Figure 1A). Whereas intestinal permeability to 4 kDa FITC-dextran was elevated, larger-size FITC-dextran (20, 70 kDa) showed decreased ability to leave the intestinal lumen and enter the systemic circulation. The kinetics of burn-induced intestinal permeability to 4 kDa FITC-dextran was also examined. Intestinal permeability to 4 kDa FITC-dextran peaks at 4 hours following severe burn and returns to baseline at 24 hours (Figure 1B).

Figure 1.

Figure 1

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Conventional methods of detecting burn-induced intestinal injury. A, Intestinal permeability to increasing molecular-weight FITC-dextran following severe burn injury. Intestinal permeability decreases with increasing molecular weight. B, Intestinal permeability to 4 kDa FITC-dextran at several time points following severe burn (n ≥ 5 animals per group). Intestinal permeability peaks 4 hours following burn. C, Hematoxylin and eosin–stained segments of intestine harvested at various time points following burn (n = 3 animals per group). Histologic gut injury characterized by blunting of villi noted at 4 hours with a normal intestinal architecture noted in animals 24 hours following injury. All sections imaged at ×200 magnification. Scale bar = 100 µm.

Histologic Intestinal Injury

Segments of distal ileum collected 4 hours following burn exhibited evidence of histologic gut injury as characterized by blunting of villi (Figure 1C). In contrast, sections of intestine from sham animals and animals at 2 and 24 hours following burn lacked histologic evidence of injury. In spite of increased permeability at 2 hours following burn, histologic evidence of injury lags behind the ability to detect changes in permeability. Histologic specimens were reviewed by a pathologist blinded to the experimental conditions and scored in three randomly selected regions on a scale from 0 (normal) to 4 (major; widespread necrosis with massive neutrophil infiltration). Maximal gut injury was seen at 6 hours following burn (2.67 ± 0.19). This was significantly higher than gut injury scores seen at 2 hours (1.33 ± 0.19, p < .05) and 24 hours (1.67 ± 0.33, p = .05) following injury.

Fluorescent Imaging of Intestinal Injury Using FITC-Dextran

A deep-cooled charge-coupled device (CCD)-based imaging system (Xenogen IVIS Lumina) was used to monitor changes in intestinal integrity following burn injury (Figure 2). Using FITC as a fluorescent dye for minimally invasive imaging led to a significant amount of quenching, demonstrated by disappearance of intestinal fluorescent signal when the abdominal wall is closed over the injected intestinal segment. The fact that the fluorescence signal from burned animals overcomes this quenching allows us to exploit this difference to detect intestinal injury in burned animals.

Figure 2.

Figure 2

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In vivo imaging of intraluminal FITC-dextran following severe burn. Image obtained of sham animals following intraluminal injection of FITC-dextran. Fluorescence signal is no longer seen after closure of the abdominal wall owing to quenching. The increased fluorescence signal after severe burn overcomes quenching of the abdominal wall, allowing imaging of intestinal injury. Images shown are from a representative mouse from each group (n ≥ 3 animals per group).

Near-Infrared Imaging of Intestinal Injury

To improve our image sensitivity and decrease the effects of quenching, we employed the near-infrared dye Alexa Fluor 680. The CCD camera was used to obtain images of near-infrared fluorescence using intraluminal Alexa 680 at several time points following severe burn (Figure 3A). Images indicate a plateau in fluorescence from animals at 4 to 6 hours following burn, with decreased fluorescence seen at later time points. These visual changes in fluorescence were confirmed by quantification of the fluorescent signal from all animals imaged at each time point (Figure 3C).

Figure 3.

Figure 3

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In vivo near-infrared imaging of intestinal injury following severe burn. A, CCD camera imaging of fluorescence 30 minutes after intraluminal injection of Alexa Fluor 680 following 30% total body surface area burn. B, Fluorescence imaging of intestinal specimens resected en bloc from animals at various time points following burn. C, Quantification of fluorescence from all animals imaged. Data are expressed as fluorescent intensity (arbitrary units) ± the standard error of the mean (SEM). D, Quantification of fluorescent signal from resected intestinal specimens. Images shown are from a representative mouse from each group (n ≥ 3 animals per group).

Intestinal segments were harvested en bloc to assess ex vivo fluorescence (Figure 3B). There is still evidence of quenching, and although it appears minimal, the quantification of the fluorescent signal is higher in the excised intestine (Figure 3D). The pattern of fluorescence imaging of the excised intestine across the time course is similar to the in vivo images, with increased fluorescence at 4 to 6 hours following burn. Noninjured animals that did not undergo injection of fluorescent dye were imaged to assess background fluorescence levels (data not shown). Some background fluorescence was noted within the intestinal lumen, likely from residual chow. The amount of background intestinal fluorescence was minimal and is no longer visualized after the adjustment of the standardized color bar settings used in these images.

Etiology of Increased Fluorescence in Animals with Intestinal Injury

We were then interested in determining the etiology of the changes in fluorescent signal between animals in each experimental group. We hypothesized that uninjured intestine was able to clear the fluorescent dye during the 30-minute assay while the injured intestine, perceivably owing to either decreased villous height or compromised perfusion, had decreased clearance of the near-infrared dye. Imaging was obtained immediately after intraluminal injection of dye to control for the effects of clearance. When comparing sham animals and animals 4 hours following severe burn, we found no difference in fluorescent signal between groups when imaging the abdomen or the resected intestine (Figure 4A) immediately following injection of the dye. Quantification of the fluorescence intensity from all animals imaged confirms no significant difference between abdominal fluorescence (Figure 4B) or intestinal fluorescence (Figure 4C) between sham and burned animals.

Figure 4.

Figure 4

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In vivo imaging obtained immediately following intraluminal injection of near-infrared tracer. A, Imaging obtained immediately after intraluminal injection of Alexa Fluor 680 showing equivalent fluorescent image between sham and burned animals. B and C, Quantification of fluorescence indicates no significant difference between sham and burn when the image is obtained immediately after intraluminal injection of near-infrared dye. These results suggest that differential imaging obtained between sham and burn animals is due to decreased clearance of the near-infrared tracer by the injured gut during the 30 minutes following intraluminal injection. Images shown are from a representative mouse from each group (n ≥ 3 animals per group).

Noninvasive Imaging of Intestinal Injury

To demonstrate that this method of imaging intestinal injury could be performed in a noninvasive fashion, animals were given a gavage of Alexa Fluor 680 prior to imaging. A time course of images was obtained in the same animal over the first 60 minutes after gavage of Alexa 680 to evaluate changes in fluorescence (Figure 5A). In sham animals, fluorescence decreases substantially over the 60-minute imaging sequence. Fluorescence decreases to a lesser degree over this time course in the burned animals, resulting in differential fluorescence between sham and burned animals.

Figure 5.

Figure 5

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In vivo, noninvasive imaging of burn-induced intestinal injury. Placement of intraluminal tracer accomplished noninvasively using gavage. A, Sequential imaging of the same animal over time following gavage of near-infrared tracer. Decreased clearance of the tracer in burned animals results in differential fluorescent imaging between sham and burn. B, Kinetics of fluorescent signal dissipation in sham and burned animals (n = 4 animals per group). Data are expressed as the mean relative fluorescent intensity ± SEM. The relative fluorescent intensity for each animal was obtained by dividing the fluorescence at each time point by the fluorescence measured immediately following gavage. The relative fluorescent intensity is significantly higher from the abdomen of animals 4 hours following burn compared with the sham animals when imaging at 20 and 30 minutes after gavage. * p = .01; ** p < .01 using t -test.

Next, we examined the differences in fluorescent signal dissipation over the imaging time course between animals 4 hours following burn and sham animals (Figure 5B). We measured the relative decrease in fluorescent signal from each animal by comparing the fluorescent intensity measured at each time point with the fluorescence measured immediately after gavage (time = 0). Fluorescent signal dissipates at a greater rate from the abdomen of the sham animal compared with the burned animal. We are able to exploit the different rates of signal decrease to obtain differential imaging and quantification of fluorescence between sham and burned animals. This difference becomes most evident at 20 and 30 minutes following gavage of near-infrared dye, suggesting that these are the ideal time points to use this imaging technique as a marker for intestinal injury.

Assessing Therapeutics Using Noninvasive Imaging of Intestinal Injury

The true utility of in vivo, near-infrared imaging of gut injury lies in its ability to monitor the effects of candidate therapeutics on intestinal injury. To assess the ability of this assay to detect modulation of gut injury following severe burn, we treated animals with the nonspecific phosphodiesterase inhibitor PTX immediately following injury. PTX has been shown to have significant antiinflammatory properties in models of sepsis,20 hemorrhagic shock,21 and pancreatitis.22 We recently studied the effects of PTX on intestinal barrier injury using this burn model, finding that a single dose of PTX immediately following burn decreases histologic gut injury, attenuates intestinal inflammation, and improves intestinal barrier integrity to 4 kDa FITC-dextran.8, 18

Burned animals treated with PTX underwent gavage of Alexa 680 at 4 hours following injury. Serial fluorescent imaging was then obtained at multiple time points following gavage, allowing for the comparison of changes in relative fluorescent intensity from the abdomen of all animals imaged (Figure 6). Compared to burn alone, abdominal fluorescence was significantly decreased in burned animals treated with PTX following gavage of Alexa 680 when quantifying fluorescence at the 30-minute imaging time point. There was no difference in relative fluorescent intensity when comparing sham and burned animals treated with PTX.

Figure 6.

Figure 6

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Treatment with an antiinflammatory drug decreases abdominal fluorescence following injury. Animals (n = 6) were treated with the phosphodiesterase inhibitor pentoxifylline (PTX; 12.5 mg/kg intraperitoneally) immediately after severe burn injury. Four hours following injury, animals were given a gavage of the near-infrared dye Alexa 680. In vivo, serial fluorescent imaging was obtained in the Xenogen IVIS Lumina. A, Representative images of animals from each experimental group obtained 30 minutes after gavage. B, Relative fluorescent intensity was calculated by dividing the fluorescent intensity at each time point by the fluorescent intensity measured immediately following gavage (t = 0). The mean relative fluorescent intensity ± SEM is shown for each group from images obtained at 20, 30, and 40 minutes following gavage of near-infrared dye. Relative fluorescent intensity is decreased from the abdomen of burned animals treated with PTX compared with animals subjected to burn alone. * p < .05 versus sham; ** p = .01 versus sham; † p = .05 versus burn; # p < .01 versus burn using analysis of variance.

Discussion

In this series of experiments, we demonstrated a novel method for the in vivo imaging of intestinal injury using near-infrared dye, which correlated with classic assays of intestinal injury including histology and intestinal permeability assays. We performed a 30% TBSA full-thickness steam burn on male BALB/c mice, an injury that is known to induce systemic inflammation, cause histologic gut injury, and increase intestinal permeability.18 We first validated our model by examining changes in intestinal permeability to increasing molecular-weight FITC-dextran. Our injury model resulted in increased permeability to 4 kDa FITC-dextran. Permeability decreased as the molecular weight of FITC-dextran increased, with minimal permeability to 20 and 70 kDa probes. The results of this assay focused our subsequent studies on 4 kDa molecular weight. We then investigated the kinetics of burn-induced intestinal permeability to 4 kDa FITC–dextran, demonstrating a time course of increased intestinal permeability at 4 hours postburn, returning to sham levels at 24 hours.

We then developed a technique for determining intestinal injury by imaging intraluminal fluorescent dye with a Xenogen IVIS Lumina. The Xenogen system uses a CCD camera to perform imaging of both fluorescence and luminescence. We initially imaged animals following injection of FITC-dextran; however, there was significant quenching of the fluorescent signal at this wavelength caused by the overlying abdominal wall. This necessitated our transition to imaging with near-infrared dye. At several time points following injury, animals underwent laparotomy and intraluminal injection of the near-infrared dye Alexa Fluor 680. Imaging was obtained 30 minutes following intraluminal injection. By performing imaging and quantification of the fluorescent intensity, we find a plateau in fluorescent signal at 4 to 6 hours following burn. This fluorescence decreases at later time points, suggesting either a decrease in intestinal injury or barrier recovery. The results of these images correlated with the classic assays (intestinal permeability to FITC-dextran, histology) that were performed and show maximal intestinal injury at 4 to 6 hours after injury, returning to baseline at 24 hours.

We then determined the explanation for our ability to measure increased fluorescence from the injured gut because each animal receives the same concentration of dye delivered to an equivalent length of gut. We hypothesized that the injured gut had decreased ability to clear the intraluminal dye during the 30-minute period between intraluminal injection and imaging. We postulated that this may be due to the decreased villous height that is known to exist in the injured intestine or decreased perfusion to the gut following injury. To test this hypothesis, we obtained imaging immediately following intraluminal injection of dye, leaving no time for clearance of dye in either group. These images showed both visually and quantitatively that there was no difference in fluorescence between the injured and the uninjured gut. These results supported the notion that the increased fluorescent signal seen at 4 and 6 hours following burn injury is due to the inability of the injured intestine to clear the intraluminal fluorescent marker over the 30 minutes of the assay. The differential ability to clear the fluorescent marker from the intestine results in the differential appearance of the injured and uninjured intestine. Therefore, this technique allows for in vivo imaging of intestinal injury using intraluminal placement of a near-infrared tracer.

Because the assessment of intestinal injury in a completely noninvasive fashion is clearly ideal, we developed a method to deliver the near-infrared dye using gavage rather than laparotomy. Animals were given a gavage of Alexa Fluor 680 to monitor gut barrier integrity. We compared the changes in fluorescent image between sham animals and animals 4 hours following severe burn and demonstrated our ability to adequately deliver and image the fluorescent dye with success similar to that using laparotomy followed by intraluminal injection. When the fluorescent image was followed serially in the same animal over time, there was increased fluorescent signal in animals 4 hours following burn compared with sham animals, which correlates with our previous results. The quantification of fluorescence from gavaged animals shows increased fluorescent intensity following burn. This demonstrates our ability to perform this assay in a non-invasive fashion, which will allow the assessment of intestinal injury in the same animal over minutes, hours, or days.

The true utility of this method for in vivo fluorescent imaging lies in its ability to quickly and easily assess the effects of therapeutics aimed at limiting intestinal injury following various insults. To demonstrate the ability of this assay to detect the attenuation of gut injury following burn, we treated a group of animals with the nonspecific phosphodiesterase inhibitor PTX immediately following burn insult. We chose PTX based on the extensive studies that have been conducted in our laboratory characterizing the antiinflammatory effects of PTX in various injury models.23–26 We recently published data demonstrating the protective effects of PTX on intestinal injury using this burn model. Specifically, we showed that treatment with PTX attenuates burn-induced intestinal permeability at the same 4-hour time point used in the gavage experiments performed in this study.8 We also demonstrated the ability of PTX to decrease histologic gut injury, modulate intestinal tight junction protein expression, and attenuate markers of intestinal inflammation, including activation of the nuclear factor κB signaling cascade.18

We demonstrated decreased fluorescent intensity from the abdomen of burned animals treated with PTX compared with sham animals using images obtained following gavage of near-infrared dye. Serial imaging once again suggests that imaging 30 minutes after gavage of near-infrared dye is the ideal time point for quantification of relative fluorescent intensity. This series of experiments confirms the ability of this assay to image and quantitatively measure modulation of intestinal injury following severe burn. The advantage of this assay over classic means of detecting intestinal injury clearly lies in the relative ease it takes to obtain these results using this real-time, in vivo technique.

Ideally, this imaging technique could be used in the future to measure gut barrier injury in large animal models. This current series of experiments uses the Xenogen IVIS Lumina imaging system to measure intestinal fluorescence, which limits the size of animal that can be placed in the imaging chamber. Technology certainly exists to perform imaging and measurement of fluorescence in a sensitive manner. Further studies would also be needed to understand the ideal volume of fluorescent dye that would need to be delivered to the gut as the size of the subject increases. In the future, performing in vivo studies of gut barrier injury in human subjects could certainly enhance our understanding of numerous pathologies altering gut barrier function. Clearly, studies will need to be undertaken to identify safe and effective means of delivering near-infrared fluorescent dye to the intestine of human subjects.

The ability to perform rapid, serial, in vivo assessments of intestinal injury using near-infrared fluorescent imaging could be ideal for use in burn research and have great utility in models of inflammatory bowel disease, intestinal ischemia, and other conditions causing significant gut injury. First, the results to date correlate well with other markers of intestinal injury using more classic means of evaluation, including intestinal permeability assays, microscopic evaluations, and changes in markers of serum and intestinal inflammation (tumor necrosis factor-α) measured in other studies we have published using this model.8 Second, near-infrared fluorescent imaging allows for a serial visual and quantitative evaluation of intestinal injury that can be used to evaluate intestinal injury in models of inflammation, shock, and inflammatory bowel disease. The ability to perform this in vivo assay in a noninvasive fashion allows for the imaging of the intestine over time in the same animal. Importantly, given that this imaging technique is not lethal, the number of animals required to complete these investigations could be reduced. This method of in vivo near-infrared imaging of intestinal injury complements classic assays of intestinal damage and may facilitate the monitoring of these important biologic changes and allow for the screening of candidate therapeutics with relative ease.

Footnotes

Financial disclosure of authors and reviewers: None reported.

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