Characterizing intestinal strictures of Crohn’s disease in vivo by endoscopic photoacoustic imaging

: Crohn’s disease (CD) is one type of inflammatory bowel disease where both inflammation and fibrosis cause the thickening of the bowel wall and development of the strictures. Accurate assessment of the strictures is critical for the management of CD because the fibrotic strictures must be removed surgically. In this study, a prototype capsule-shaped acoustic resolution photoacoustic (PA) endoscope, which can perform mulitwavelength side-view scanning, was developed to characterize the intestinal strictures of CD. The imaging performance of the probe was tested in phantom experiments and a rabbit trinitrobenzene sulfonic acid (TNBS) model with acute (inflammatory only) or chronic (mixed fibrotic and inflammatory) colitis in vivo . The motion artifacts due to intestinal peristalsis and the respiratory motion of the animals were compensated to improve image qualities. Quantitative molecular component images derived from multi-wavelength PA measurements of normal, acute and chronic intestinal strictures demonstrated statistically significant differences among the three groups that were confirmed by histopathology. A longitudinal study demonstrated the capability of the system in monitoring the development of fibrosis. The results suggest that the proposed novel, capsule-shaped acoustic resolution PA endoscope can be used to characterize fibrostenotic disease in vivo .

endoscopic biopsies are limited to the superficial epithelial layer, and are unable to detect evidence of fibrosis [11]. Any evidence of inflammation is generally considered an indication for more corticosteroids [6], though this is futile in chronic fibromuscular strictures [12]. In addition, comprehensive evaluation of the bowel is inherently limited due to biopsy size.
Conventional imaging modalities including Ultrasound (US) imaging [13], Magnetic Resonance Imaging (MRI) [1,14], and Computed Tomography (CT) [4,5,15] can be applied to identify and locate the intestinal strictures, and measure bowel wall thickness. These modalities cannot assess intestinal strictures at molecular level, and cannot differentiate inflammatory from fibrotic strictures. This leaves a prognostic gap that is currently filled by repeated empiric courses of corticosteroids [6], which lead to significant adverse events and delays in timely treatment with surgery.
Photoacoustic (PA) imaging is an emerging non-radiative and non-ionizing technology combining optical spectroscopy and ultrasonography [16,17]. During a PA measurement, a pulsed or modulated laser illumination causes targeted molecules to vibrate, generating a tiny thermoexpansion within the targeted tissue. This thermoexpansion causes vibrations that propagate in the form of mechanical waves. The mechanical waves can be measured by US transducers for reconstruction of images representing the optical absorption properties within the illuminated tissue volume. PA imaging with sequential illumination at multiple wavelengths allows for the assessment of each individual molecular component with a tissue volume at ultrasonic resolution [18,19]. PA imaging systems are also capable of generating naturally co-registered ultrasonography images by using the pulse-echo mode of the US transducer [20][21][22]. In our previous work [23], we have validated the feasibility of characterizing the intestinal inflammation and fibrosis in CD by quantifying the hemoglobin and collagen content in ex vivo human and animal tissue using an acoustic resolution PA microscopy (ARPAM) system. The system has achieved PA imaging at the depth of 7 mm beneath stricture inner surface with a resolution of 50 µm. We have also demonstrated the feasibility of detecting intestinal inflammation and fibrosis in vivo in a trinitrobenzene sulfonic acid (TNBS) rat model [24].
Recently, several PA endoscopy systems have been developed [21,22,[25][26][27][28][29]. However, all of these studies focused on imaging the hemoglobin content which is only correlated to the inflammation. In addition, the circular scanning of these probes limited their capability of rapidly assessing a longitudinal segment of the intestine. The tomographic image reconstruction in a circular geometry also provides limited the resolution in deep tissue. In this study, a prototype capsule-shaped ARPAM probe with a longitudinal field of view was developed for characterizing both the inflammation and fibrosis in the intestinal strictures. The probe was compatible with clinical endoscopes and was preliminarily tested intra-rectally using TNBS rabbit models [30,31]. In a proof-of-concept longitudinal study in the chronic TNBS rabbit model, animals were scanned over 8 weeks to monitor disease progression from acute inflammation to chronic fibrosis. The quantitative imaging results were statistically analyzed and confirmed with the histopathology.

Development of the capsule-shaped ARPAM probe and peripheral systems
Figure 1(a) shows the schematic diagram of the prototype capsule-shaped ARPAM probe including the acquisition system. The probe consists of a capsule shell with a side-viewing window and a translation stage fabricated using a 3D printer with VisiJet M3 Crystal (ProJet 3500, 3D System, Rock Hill, SC, USA). As shown in Fig. 1(b), the movement of the translation stage was guided by two parallel tracks on the inner surface of the capsule shell. A spherically focused transducer (50 MHz central frequency, 80% bandwidth, 3.2 mm focal length, 2 mm aperture, LiNbO 3 , Medical Ultrasonic Transducer Resource, University of Southern California, CA, USA) and a multimode optical fiber (0.39 NA, 600 μm core, Thorlabs, Newton, NJ, USA) were fixed in the translation stage as shown in Fig. 1  ry motion of th and the tissu n. Such displa sponding to 3 nd white lines he reduced sig uare fitting w . Figure 3(

Longitudinal study of fibrotic progression in the chronic TNBS model using PA imaging
To test the utility of the PA probe for tracking disease development from acute inflammation to chronic fibrosis, a longitudinal study was performed with four animals using the chronic TNBS rabbit model. This longitudinal cohort received treatments identical to the chronic group in section 2.3.1, but were PA scanned at baseline, prior to each of the 3 TNBS treatments, and at the termination of the experiment at week 8, as illustrated in Fig. 8(a).

Histology
Proximal (untreated, i.e. normal) and distal (treated) colons of the euthanized rabbits were harvested and processed for histology. Hematoxylin and eosin (H&E) staining and Masson's trichrome staining were performed by the University of Michigan Cancer Center Histology and Immunoperoxidase Lab (Ann Arbor, MI, USA) and McClinchey Histology Lab (Stockbridge, MI, USA), respectively. Digital photomicrographs of proximal and distal colon sections were captured using an Olympus BX51 microscope at the University of Michigan Microscopy and Image Analysis Laboratory (Ann Arbor, MI, USA). To quantify the amount of collagen content, trichrome-stained slides were digitized at 20X ocular (~200x) magnification on a digital slide scanner (Leica Aperio AT2, Leica Biosystems, Buffalo Grove, IL). Annotated areas of lamina propria were digitally drawn and blue pixels were quantified using a commercial algorithm for color deconvolution (Leica Aperio Color Deconvolution, v.9.1, Leica Biosystems). Algorithm parameters were specifically optimized for the red and blue of the trichrome by selecting small areas with only the indicated colors and defining the RGB values for the stain, then using these values as the target stain input parameters. Threshold input parameters for weak, moderate, and strong positive staining were optimized by visual examination of a test image by a board-certified veterinary pathologist (KAE) in a blinded manner. Three-four random sections of lamina propria were quantified for each slide. Dense fibrosis was defined as the sum of percent strong and percent moderate staining per slide.

Statistics
The statistical analysis for differentiating the normal, acute and chronic conditions were performed using build-in ttest function in MATLAB (2017, Mathworks, Natick, MA, USA). The null hypothesis is that the three conditions cannot be differentiated using PA measurements. The correlation between the TNBS doses and the PA intensities were calculated using corrcoef function in MATLAB (2017, Mathworks, Natick, MA, USA). The null hypothesis is that the two values do not have correlation with each other.

Pathological analysis
The gross pathology and the histology photographs are shown in Fig. 6(a) and (b). In H&E stained sections of acute injury in Fig. 6(a) in the middle column, there is coagulation necrosis of the mucosa (bracket), inflammatory infiltrate (arrow), and hemorrhage (arrowhead). Chronic injury on the right column in Fig. 6(a) is characterized by widespread replacement of the mucosa by fibrous connective tissue (arrows) and increased density of fibrosis in the submucosa (arrowhead), not present in normal or acute sections. Statistical analysis of the fibrosis in the trichrome staining, as described in 2.3.5, has shown that the chronic animals has approximately two and half times collagen content compared to the normal and acute animals at the treated location [p = 0.032, Fig. 6(c)].  Figure 7(a) illustrates the representative PA molecular component images from normal, acute TNBS, and chronic TNBS rabbit colons, respectively. Hemoglobin tissue distribution was rendered in red and collagen distribution was rendered in green. As illustrated in Fig. 7(b), hemoglobin content significantly increased in both acute and chronic conditions compared to the normal condition. An approximately 2-fold hemoglobin signal intensity was observed in the acute condition compared to the normal condition (p = 0.0189). In the chronic condition, an approximately 3-fold collagen signal intensity was observed compared to the acute condition [p = 0.0002, Fig. 7(c)]. The quantitative PA results were consistent with the gross pathology and histological quantification.   [23], and used only 720 and 1310 nm targeting hemoglobin and collagen.

Quantitative characterization of inflammatory and fibrotic disease in the TNBS rabbit model
As illustrated in this study, the molecular components measured in the PA images in vivo are consistent with the gross pathology and the histopathology. The endoscopic PA imaging approach quantitatively differentiates inflammatory from fibrotic intestinal strictures, and objectively quantifies the development of fibrotic disease. In contrast to our transcutaneous PA imaging with resolution on the order of hundreds of microns [24], the endoscopic PA imaging approach provides an order higher resolution at tens of microns and, therefore, provides more detailed tissue pathology. Another advantage of the endoscopic approach over the transcutaneous one is that the endoscopic delivery of illumination avoids the optical energy attenuation through the skin and subcutaneous tissue.
This study demonstrates the feasibility of endoscopic PA imaging to characterize CD-like intestinal pathology in rabbit models. This is a dramatic improvement over current endoscopic technology, which can only measure inflammation in the surface epithelium, and can provide no data on fibromuscular damage in the deeper layers of the bowel wall. In future development, we will further miniaturize the capsule probe to be compatible with the instrument channel of a standard pediatric colonoscope. This miniaturized design will allow the withdrawal of the PA probe after prognostic assessment, and the insertion of conventional endoscopic instruments for interventional procedures such as balloon dilation without repositioning the colonoscope. The PA imaging speed can be potentially improved by using lasers with higher repetition rate, which will substantially mitigate the motion artifacts observed in Fig. 3. We will also integrate US imaging using a pulser-receiver for extracting structural information of the intestinal strictures, although extra scanning time will be introduced.

Conclusion
We have developed a novel, capsule-shaped ARPAM probe with axial resolution of 65 µm and lateral resolution of 90 µm to measure the molecular components characterizing inflammation and fibrosis in intestinal strictures. The in vivo discriminatory capability of the PA probe was validated in rabbit models of intestinal inflammation and fibrosis. In this proofof-concept longitudinal study in the rabbit TNBS models, PA imaging is feasible for monitoring the development of intestinal fibrosis over time, which could provide valuable information for clinical decision-making during CD management. Patients with strictures that are of the chronic fibromuscular phenotype could go to surgical resection in a timely fashion, avoiding months of intermittent intestinal obstruction, hospitalizations, and the side effects of futile empiric trials of corticosteroids. These results demonstrate that this PA endoscopic probe can be used to characterize fibrostenotic disease in vivo. Future development will be focused on the miniaturization and improvement of scanning speed of the probe.