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. 2013 Jun 17;14(8):736–741. doi: 10.4161/cbt.25094

Detection of DSS-induced gastrointestinal mucositis in mice by non-invasive optical near-infrared (NIR) imaging of cathepsin activity

Niklas K Finnberg 1,*, Yvette Liu 1, Wafik S El-Deiry 1
PMCID: PMC3841213  PMID: 23792573

Abstract

Approximately 1.4 million people of the US population suffer from Inflammatory Bowel Disease (IBD) of which the most common conditions are ulcerative colitis (UC) and Crohn disease (CD). Colonoscopy and small bowel follow through are considered the current gold standard in diagnosing IBD. However, improved imaging and increased diagnostic sensitivity could be beneficial. Optical molecular imaging has the potential to become a powerful and practical tool for early detection, image-guided biopsy, and surgery in diagnosing and treating patients with IBD. Here we used a well characterized chemical model to initiate experimental IBD in mice by feeding with dextran sulfate sodium (DSS) containing drinking water in an attempt to investigate the utility of non-invasive infrared (NIR) optical imaging in the detection gastrointestinal (GI) injury. We employed a “smart probe” (ProSense680) cleaved and fluorescently activated in the NIR-spectrum by various forms of secreted cathepsins. This probe has previously been shown to serve as a biomarker for the homing of inflammatory cells to injury. Our investigation suggests that NIR optical imaging can detect cathepsin-dependent probe cleavage non-invasively in animals with DSS-induced IBD. Increased tissue probe-retention and fluorescence was associated with increased infiltration of inflammatory cells, epithelial atrophy and sterilization of the mucosa. Furthermore, using NIR-imaging ex vivo we were able to document regional “hot spots” of inflammatory damage to the large intestine suggesting this method potentially could be coupled with colonoscopy investigation to aid in the sampling and the diagnostics of IBD.

Keywords: optical imaging, inflammatory bowel disease, Crohn disease, ulcerative colitis, near-infrared, cathepsin

Introduction

Approximately 1.4 million people in the US suffer from inflammatory bowel disease (IBD) where the most common conditions are ulcerative colitis (UC) and Crohn disease (CD).1 Although the underlying etiologies for the various forms of IBD remains largely unclear the diseases are characterized by chronic, uncontrolled inflammation of the intestinal mucosa, effecting any part of the gastro-intestinal tract with patchy or continuous inflammation. Regionally, involvement of the terminal ileum is common, but any site of the GI-tract may be affected and the inflammation of IBD may be transmural.2

IBD is believed to be the result of failure to downregulate the mucosal immune response to commensal bacteria and a significant role is played by the infiltration and activation of inflammatory cells.3 More specifically, the mucosa of IBD patients is commonly infiltrated by lymphocytes such as CD4+ T cells, leukocytes, and macrophages that form non-ceseating granulomas.4-6 Subsequently, diagnosis is based on the presence of tissue architectural abnormalities and/or the presentation of inflammatory cells in biopsies from the gastro-intestinal (GI) tract.

Colonoscopy and small bowel follow through are considered the current gold standard for diagnosing IBD. However, improvement and increased diagnostic sensitivity is necessary. As such, optical molecular imaging has the potential to become a powerful and practical tool for facilitating early detection, image guided biopsies, and surgery.7 Optical imaging instruments are typically low cost, portable, immune to electrical interference, and offer very good resolution that allows for additional spatial information about, e.g., the localization of inflammatory cells.8 Further valuable attributes of optical imaging is the lack of safety concern associated with (frequent) radiation exposures and information from clinically established imaging methods could be further augmented through the combination with optical imaging protocols.

The cathepsins are a family of lysosomal cysteine proteases, generally involved in cellular protein turnover, degradation, and tissue remodulation.9,10 In particular cathepsin B is highly expressed by and co-localizes closely with macrophages in experimental animal models for inflammatory disease and in inflammatory conditions of humans.11-14 Optical imaging using near-infrared (NIR) “smart” molecular probes, activatable through cathepsin-mediated cleavage, have been previously used for the detection of tumor-burden in animal colorectal cancer models.15-19 Here we used a well-defined and characterized chemical model to initiate IBD in mice by feeding with dextran sulfate sodium (DSS)-containing drinking water. This animal model of IBD recapitulates many important immunological and histopathological aspects of IBD in humans.20,21 We aimed to investigate the utility of cathepsin-activity as a biomarker, using non-invasive NIR optical imaging, for the detection of inflammatory injury to the GI tract. Our investigation suggests that non-invasive NIR optical imaging of a cathepsin-activatable probe (ProSense680) successfully identifies mice with DSS-induced IBD. On the microscopic level ProSense680-cleavage and NIR-fluorescence was associated with increased infiltration of inflammatory cells, epithelial atrophy and sterilization of the mucosa. Using this imaging method we were able to document regional “hot spots” of inflammatory damage in the large intestine of mice ex vivo suggesting this method has the potential to be coupled with colonoscopic investigation of the bowel as a part of diagnostic protocol for IBD.

Results

The presence of autofluorescence from the diet is a problem when performing optical imaging mice given that standard diet (SD) possess a high level of autofluorescence (Fig. 1A). Subsequently, mice were given an alfalfa-free imaging diet (ID) with low autofluorescence in the NIR-spectrum (750 nm<) two weeks prior to the initiation, during the DSS-treatment and prior to imaging. Mice were subjected to 2% DSS in the drinking water for 1 week and subsequently provided sterile drinking water without DSS for 1 week ad libitum. The body weight and body condition score was recorded during the course of the experiment. DSS-treatment is known to trigger an IBD-like inflammatory condition in C57BL6 mice that manifests as a reduction in body weight between day 6–14 following the initiation of treatment.22 Furthermore, female and male C57BL/6 mice were given DSS-containing drinking water concomitantly. Male mice are increasingly susceptible to DSS-induced IBD compared with female mice and develop more severe colonic damage and neutrophil influx23,24The precise molecular basis for this gender disparity observed in this model is unclear but was indeed confirmed in our model as well (Figs. 1B and 2A).

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Figure 1. Low fluorescence of imaging diet (ID) in the NIR wavelength and weight loss of mice subjected to a DSS-induced IBD. (A) Imaging diet (ID) provides better noise-to-signal compared to standard diet (SD) in the NIR wavelength of light compared to "green fluorescent" wavelength of light (515 nm). B/F-bright field. (B) Weight loss of female and male C57BL/6J mice subjected to DSS (2% w/v) in the drinking water. *P < 0.05, the Student t test.

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Figure 2. Whole-body non-invasive NIR optical imaging confirms DSS-induced IBD in mice injected with ProSense680. (A) Statistically significant differences in body weight between female and male C57BL/6J mice subjected DSS-induced IBD and/or non-invasive NIR-optical imaging (P < 0.0001, 2-way ANOVA). (B) NIR optical imaging (“non-invasive” and concomitant with necropsy) of female (f) and male (m) mice injected with ProSense680 reveals increased unmixed signals from male mice subjected to DSS (2% w/v) in the drinking water consistent with increased epithelial atrophy and inflammation in males compared to females as detected by H/E (C). Representative images are shown. (D) Ex vivo NIR imaging of “swiss-rolled” small intestines and colons from female and male mice with DSS-induced IBD. Duo, duodenum; jej, jejunum; ile,ileum; dc, descending colon; tc, transcending colon; ccc, cecum.

Mice were injected with ProSense680 and subsequently subjected to NIR optical imaging. Increased signal in the NIR-spectrum was detected from the bowel region of male (m) mice with DSS-induced IBD, evident by a clear reduction in body weight, but not female (f) mice (Fig. 2A and B). This was particularly obvious after spectral unmixing. There were no NIR-signal from the abdomen of mice not receiving DSS-treatment but injected with ProSense680 or mice receiving DSS-treatment and “sham” injected with PBS (data not shown). Euthanasia and necropsy confirmed that the NIR-signals observed were emitted from the small and large intestine of the mice (Fig. 2B). Histology of isolated small intestines from male and female mice revealed increased epithelial atrophy and inflammation was present in the gut of male mice, consistent with severe mucositis and poorer IBD-diagnosis compared with female mice (Fig. 2C). Ex vivo analysis of fresh small intestine and colon from female and male mice injected with ProSense680 showed increased signal from both the small intestine and colon of DSS-treated male mice (Fig. 2D). Thus it is possible, with sufficient sensitivity and specificity, to detect cathepsin-dependent probe activation through non-invasive optical NIR-imaging stemming from the GI-tract of mice with DSS-induced IBD.

Furthermore, we also aimed to investigate if NIR-imaging of ProSense680 signals could guide the sampling from regions of the colon with high IBD pathology score (increased inflammatory injury). Male mice with DSS-induced IBD, injected with ProSense680 were euthanized and their colon isolated. Stereomicroscopic NIR optical imaging of isolated colons revealed a heterogeneous distribution pattern of signal from the colon of the DSS-treated mice (Fig. 3). Areas with high (H) and low (L) NIR-signal was isolated from the rest of the colon by micro dissection and their histology evaluated by H/E-staining. Indeed we found that NIR “hot spot” areas; i.e., areas with high NIR-signal (H), showed a poorer pathology score compared “L” areas. Taken together this suggests that NIR-imaging of ProSense680 cleavage/cathepsin activity can guide biopsy sampling in animal models of IBD.

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Figure 3. NIR-imaging identifies inflammatory hot spots of the colon of male mice with DSS-induced IBD.The colon of male mice injected with ProSense680 was used for ex vivo optical NIR-imaging. Areas with high (H) and low (L) NIR-signal was micro-dissected from the surrounding tissue, fixed in paraformaldehyde and subjected for histology. H/E staining reveal that hot spots with high (H) NIR-signal show increased epithelial atrophy compared to areas with low (L) NIR-signal.

Discussion

Here we show that systemic injection of ProSense680, a cathepsin-activatable probe into mice with IBD allow for non-invasive detection of GI damage using NIR optical imaging. Male mice increasingly susceptible to DSS-induced IBD showed increased abdominal NIR-signal compared with resistant female mice (Fig. 2). Furthermore, ex vivo NIR optical imaging facilitated the detection of inflammatory NIR “hot spots” with a poorer histopathology compared with areas with low NIR-signal (Fig. 3). Subsequently, we believe that NIR optical imaging of cathepsin-activity has the potential to clinical translation aiding in the diagnosis of IBD or IBD-like conditions. NIR optical imaging is a non-invasive/no-contact imaging procedure that could be used concomitantly with colonoscopy or surgery with none of the safety concern linked to the radiation exposure of established imaging modalities. Furthermore, cathepsin-activated probes belong to the category of “smart probes” that in its inactive state is “silent” and allows for reduced background “noise” which is a common problem in the optical imaging field. Thus the development of future probes may allow for the molecular profiling of a particular disease condition and promote the convolution of enzymatically activated protein states as biomarkers for disease.

Diagnosis of IBD is often aided by imaging of the presence of architectural abnormalities such as abscessed and fistulas in the GI tract by CT.25 However, the presence of inflammatory cells without the presentation of architectural abnormalities is not readily made by CT. Subsequently, the developments of novel scintographic methods using e.g., 99mTc-HMPAO are ongoing.26 For some imaging modalities using radioactive tracers such as FDG-PET it is difficult to discriminate between uptake in the intestine and bone marrow in particular in the pelvic/perianal area. This may not pose a problem with NIR optical imaging using cathepsin-activatable probes since we did not experience any signal being emitted from the bone marrow in mice with DSS-induced IBD (data not shown).

Cathepsin B is a lysosomal cysteine protease involved in cellular protein turnover and degradation.9,10 Cathepsin B has been implicated in tumor progression and metastasis and high level of expression of cathepsin B has been shown to correlate inversely with patient survival.17 Previous data have shown that cathepsin is overexpressed in adenomas of the Apcmin/+ mice in comparison to normal mucosa.16 Cathepsin-activity is also known to be upregulated in inflammatory sites in other experimental animal models.27-29 Probing and NIR-imaging for activated cathepsin has successfully been performed for atherosclerosis,11 pulmonary inflammation,30 arthritis,31,32 and muscle dystrophy.33 Our findings suggest that during DSS-induced IBD in mice cathepsin expression is increasingly widespread in the GI sub-mucosa and epithelium (data not shown). Indeed, cathepsins are expressed in an presumably antigen processing manner in gastric epithelial cells for the presentation to CD4+ T cells34 as well as membrane-bound secretion from macrophages following bacterial stimuli.35 Thus it seems conceivable that induction of cathepsin expression in the context of IBD could be a result of Wnt-activation with subsequent tissue regeneration as a result of the damage to the mucosa and the concomitant infiltration of inflammatory cells. On the other hand, a potential limitation associated with optical imaging of activated cathepsins relates to that positive detection may only be allowed for during active disease (i.e., excretion of active cathepsins B) and not when the disease is quiescent or only mildly active.

As the majority of patients need a long-term follow-up it would be ideal to rely on a non-invasive technique with good compliance. For this purpose, ultrasound, CT, and magnetic resonance imaging (MRI) are more applied but also a lot of radiopharmaceuticals have been proposed for diagnostic and follow-up purposes. The majority of the IBD patients are in need for frequent follow-up with at least one colonoscopy per year should be offered according to national surveillance guidelines. Surveillance colonoscopy should be performed without disease activity and four tissue samples each 10 cm should be taken. It has been shown that dyes applied with a spraying catheter are of additional diagnostic values in the detection of IBD-related colorectal cancer with a factor of 3‒4 higher detection rate of intraepithelial neoplasia.36 Potentially, given the link between cathepsin excretion and colorectal cancer, optical imaging might also aid in detecting and directing colonoscopic biopsy sampling in the diagnosis of both inflammatory lesions and colonic neoplasms. Further experiments are required to address the potential of clinical translation of NIR-optical imaging of cathepsin-activity for the benefit of IBD-patients.

Material and Methods

Mice and treatments

Four- to six-week-old wild-type C57BL6/J (Jackson Laboratory) were housed in the animal facility of the animal research farm and central animal quarters of Penn State Hershey College of Medicine. All mice were housed in a controlled environment with regard to light, temperature and humidity. The animal care and treatment procedures were approved by the Institutional Animal Care and Use Committees of the Penn State College of Medicine. Mice were subjected to DSS in the drinking water as previously described.22 Briefly, female and male C57BL6/J mice were fed 2% (w/v) of DSS (mol. wt 40 kDa) in their drinking water for 7 d, followed by 2 weeks of consumption of water. The mice given DSS were divided into groups of female and male and imaged at day 13 and then sacrificed.

Histology and histochemistry

Following euthanasia the duodenum, jejunum, ileum, transverse colon and descending colon were harvested, subjected to fixation in 4% paraformaldehyde at 4 °C and further processed for histology. Embedded tissues was cut into 4-μm sections, mounted on glass slides, stained with hematoxylin and eosin (H&E) and subsequently analyzed by microscopy.

Non-invasive optical NIR imaging

Mice (n = 10) were given a alfalfa free diet (AIN-76A, Harland Tekland - Lab Animals Diet) with low autofluorescence in the near-infrared spectrum (650‒750 nm) 2 weeks prior to the initiation of the DSS-treatment. The activatable cathepsin B sensing probe ProSense680 (PerkinElmer) was injected IV (50 nmol/kg bw) into mice at day 14 following the initiation of DSS-treatment. Xylazine (30 mg/kg bw) and ketamine (200 mg/kg bw) anesthetized mice was imaged using the Maestro In vivo Imaging system (CRi systems) at 0, 2, and 24 h following injection and day 13 post initiation of the DSS-treatment. The GI tract was also isolated from mice injected with ProSense680 and investigated using a stereomicroscope coupled to a Nuance multi-spectral imaging system (GNIR Flex, CRi systems). Regions of interest in the GI-tract displaying high or absent signal in the unmixed NIR channel (650–750 nm) was isolated and fixed in 4% PFA as described under “Histology and histochemistry”. Image analysis was performed using Principal Component Analysis (PCA) coupled with unmixing and quantitation of NIR signal from the appropriate Region of Interest (ROI). The mean and SE of the pixel values was recorded and significance of differences among groups of animals and/or ROI within the GI tract was determined using the two-tailed Student t test (GraphPad software 5.0).

Statistical analysis

Analysis of statistically significant differences among treatment groups was determined using the Student t test in Excel (2003 Microsoft Office™) and the 2-way ANOVA test for statistical significance in GraphPad Prism 5.0.

Glossary

Abbreviations:

GI

gastro-intestinal

IBD

inflammatory bowel disease

UD

ulcerative colitis

CD

Crohn Disease

NIR

near-infrared

DSS

dextran sulphate sodium

Disclosure of Potential Conflicts of Interest

No potential conflict of interest was disclosed.

Footnotes

References

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