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Abstract
We studied time-dependent changes in muscle optical properties during degeneration and regeneration using polarization-sensitive optical coherence tomography (PSOCT). Excised canine muscle transplants in a xenograft mouse model were imaged ex vivo from 3- to 112-day post-transplantation. PSOCT images were quantified to evaluate post-transplantation changes of three optical/structural properties: attenuation, birefringence and fiber alignment. The birefringence and fiber alignment decreased after transplantation until 20∼30-day and recovered thereafter. The attenuation coefficient showed a reversed trend over the same period of time. These results suggest that optical properties could be used for monitoring skeletal muscle degeneration and regeneration.
© 2020 Optical Society of America under the terms of the OSA Open Access Publishing Agreement
1. Introduction
Animal models of human diseases play key roles in improving our understanding of disease mechanisms and developing therapeutic strategies. Genetically engineered mouse models and xenograft mouse models are the two major types of animal models used in research. Xenograft mouse models are developed by transplanting tissues from other species to immunocompromised mice. In particular, grafting human tissues to an experimental animal host enables the direct study of human disease in human tissues. Due to such unique benefit, xenograft models are widely used in cancer research to study tumor progression and treatment response [1]. Xenograft mouse models have also been developed to investigate muscle regeneration and pathogenesis in muscular diseases [2–4]. Although neurovascular connections were not reconstructed during transplantation in these studies, the transplanted muscles were spontaneously vascularized, innervated, and successfully regenerated from muscle stem cells [2–4]. Experiments showed that regenerated xenograft muscles had similar contractility as the host muscle [3].
Conventional histology and immunohistology are standard laboratory techniques for evaluating tissue structural and compositional changes during muscle regeneration. These widely used methods are highly specific and sensitive. However, they are labor-intensive and often cannot evaluate the entire muscle sample. Standard clinical imaging modalities such as MRI [5] can evaluate the whole muscle, but lack sufficient resolution for imaging microscopic muscle damages in small animals. Therefore, a fast, high-resolution imaging technique that can quickly assess pathological changes of the entire muscle would be very useful to study muscle degeneration/regeneration in xenograft muscle models.
Optical coherence tomography (OCT) has shown potential for imaging damage-related morphological changes in skeletal muscles [6–8]. Klyen et al. studied the decay of OCT signals with depth and showed that a higher tissue attenuation was associated with necrotic muscle in mdx mice, a model of Duchenne muscular dystrophy [8]. Because skeletal muscles show strong optical birefringence, polarization-sensitive OCT (PSOCT) has also been used to image skeletal muscles [9–11]. Pasquesi et al. reported that muscle damage led to lower birefringence in the mdx mice [9]. By quantifying optical birefringence, Yang et al. demonstrated that low birefringence was a reliable marker for muscle necrosis [10]. Recently, Wang et al. visualized the 3D muscle fiber tractography using Jones matrix PSOCT and demonstrated the use of fiber disorganization to identify small damages in muscle [11].
In this study, we explored the possibility of using PSOCT to monitor structural and organizational changes during muscle degeneration and regeneration in a xenograft model in which a piece of canine muscle was grafted in an immune-deficient mouse. Three optical properties including optical attenuation, optical birefringence, and fiber alignment were extracted by quantifying PSOCT images. The results showed that all three parameters had significant temporal changes associated with muscle degeneration and regeneration.
2. Methods
2.1 Sample preparation
All animal experiments were approved by the institutional animal care and use committee at the University of Missouri and were in accordance with NIH guidelines. Muscle transplantation was performed as previously described [4]. The immune-deficient NSG host mice (NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ, Stock No: 005557) were generated in a specific pathogen free barrier facility using founders from The Jackson Laboratory (Bar Harbor, ME, USA). The donor muscle grafts were obtained from the biceps femoris muscle of a 14-month old male, mix-breed dog. Freshly dissected donor muscle blocks were stored in muscle graft media on ice. The samples were trimmed to about the size of a murine tibialis anterior (TA) muscle (∼9×4×2 mm3) prior to transplanting to 19 young adult NSG mice. Anesthesia was induced with 3-5% isoflurane and maintained by 1-2% isoflurane during the surgery. The proximal and distal tendinous attachments of the TA and extensor digitorum longus (EDL) muscles were identified and severed. These muscles were subsequently extracted and discarded. A suture loop was placed around the proximal and distal tendons of the adjacent peroneous longus (PL) muscle using a 4-0 silk suture. The donor graft was then transferred into the emptied TA compartment and sutured into place using the suture loops attached to the PL muscle. The incision was then closed using a combination of surgical glue and staples.
Animals were euthanized at 3, 7, 14, 28, 56, and 112 days post transplantation. Two to five animals were imaged at each time point. The graft muscles were dissected out and placed on filter paper soaked with PBS solution for optical imaging. After imaging, each sample was flash frozen in the optimal cutting temperature compound (Sakura Finetek Inc., Torrance, CA) in liquid nitrogen cooled isopentane and then stored at -80°C for histology. Tissue blocks were sectioned into 10 µm thick cryosections. Histology was evaluated by hematoxylin and eosin (HE) staining. Images were captured using a Nikon Eclipse E800 fluorescence microscope equipped with a Leica DFC7000 color camera.
2.2 Tissue imaging
A single-camera, 0.848 µm central wavelength, spectral domain Jones matrix based PSOCT system was used to image muscle samples [12]. The samples were scanned at a speed of 50k A-lines per second to construct an image volume of 1×6×6 mm3 (A×B×C). The imaging system had an axial resolution of 5.9 µm (in air) and lateral resolution of 11.8 µm.
In addition to conventional 3D intensity (I) images, the “cumulative” phase retardation (R) and fiber orientation (θC) images were constructed using the eigenvalues and eigenvectors from the measured Jones matrices [12]. Because these cumulative parameters (R and θC) do not represent the true sample birefringence properties at the local depth [13,14], images of local birefringence (Δn) and “local” optic axis (θL) were constructed using a Jones matrix based algorithm as described in detail previously [15]. The images were digitized into 16-bit values within [0, 25×10−4] for birefringence and [-90°, +90°] for optic axis. Once the local optic axis images were constructed, the tractography representation (T) of fiber orientation in an enface plane was obtained using the streamline functionality in MATLAB. To assess the fiber organization, images of “fiber alignment” (FA) were computed from the local optic axis images using circular statistics within a 3D moving window of 7×7×7 pixels [16]. The FA at each image pixel has a value between [0,1], where a higher value indicates a more organized fiber structure. In addition, images of local optical attenuation coefficient (µ) were calculated using intensity images following the algorithm described in detail previously [17]. The images of attenuation coefficient were digitized into 16-bit values within [0, 15 mm−1].
To quantify the overall optical properties (local birefringence, optical attenuation, and fiber organization) in a single muscle sample, the median values of birefringence, fiber alignment and attenuation coefficient were measured between 0.05-0.40 mm beneath the surface. All tissue regions close to locations of surgical suture were excluded in quantification.
3. Results and discussions
Figure 1 shows the PSOCT images of an example donor muscle before transplantation which was referred as the 0-day sample in this study.
Fig. 1. Image of a canine donor muscle before transplantation. (a) The cross-sectional images of the intensity (I), cumulative phase retardation (R), cumulative optic axis (θc), local birefringence (Δn), local optic axis (θL), attenuation coefficient (µ), and fiber alignment index (FA). (b) The corresponding enface images obtained at 200 µm beneath the tissue surface, along with the tractography (T) and cross-sectional HE histology result. The dotted line on the enface intensity image indicates the location of the cross-sectional images in panel (a). The red box indicated the region-of-interest (ROI) used for quantitative analysis in Fig. 2.
The cross-sectional intensity image (Fig. 1(a)) appeared to be quite homogeneous. Due to phase wrapping, the cumulative phase retardation (R) and optic axis (θC) images had banded appearance in muscle samples. The band thickness in the R image appeared to be consistent, suggesting a homogeneous distribution of birefringence. However, the phase wrapping in the θC image made it impossible to evaluate fiber orientation. In contrast, the constructed local birefringence image (Δn) and local optic axis image (θL) clearly revealed homogeneous distributions of optical birefringence and fiber orientation in the donor tissue. The quantitative attenuation (µ) and fiber alignment (FA) images suggested the same. The homogeneity was maintained throughout the entire imaging depth of 1-mm in the cross-sectional images.
The example enface images shown in Fig. 1(b) were extracted at the depth of 0.2 mm from the surface. The vertical dotted line on the enface intensity image indicates the location of the cross sections shown in Fig. 1(a). Similar to the cases in cross-sectional images (Fig. 1(a)), the variations shown in enface cumulative retardation (R) and optic axis (θc) images were largely artifacts due to phase wrapping; whereas the local birefringence (Δn) and axis (θL) images revealed homogeneous graft muscle fibers. The tractography (T) image provided a convenient visualization of muscle fiber orientations. Because the muscles were placed roughly perpendicular to the B-scan direction, a 0° orientation in optic axis was about aligned with the long axis of the graft muscle. The enface images of attenuation (µ) and fiber alignment (FA) also indicated a homogeneous muscle. The HE histology confirmed that the donor muscle had healthy muscle fibers indicated by clear boundaries and peripherally localized myonuclei.
Figure 2 shows the distributions of four imaging parameters obtained from the donor muscle before transplantation (the 0-day results shown in Fig. 1) obtained within the ROI shown in Fig. 1(b) from 0.05 to 0.4 mm beneath the surface. Quantitatively, the majority of the graft (87.7%) had a fiber alignment index of more than 0.9, confirming a highly aligned myofiber organization. About 64.3% of the graft had a birefringence within 3.0×10−4 and 8.0×10−4, whereas 54.0% of the graft had an optical attenuation coefficient between 1.5 mm−1 and 3.5 mm−1.
Fig. 2. Distributions of fiber orientation, fiber alignment, optical birefringence, and attenuation coefficient of the donor muscle measured before transplantation (0-day).
Figure 3 shows ex vivo imaging results of a sample excised on the 14th day post-transplantation. Some host tissues were also excised as part of the dissected muscle and imaged in this sample. In the enface images, the boundaries between the host and graft tissues were partially visible in the left side of the intensity images, but unclear in the right half. The boundaries became noticeably distinguishable in the four enface functional images of fiber orientation (θL), birefringence (Δn), attenuation (µ), and fiber alignment (FA). The host muscle (lower part of the tissue) showed homogeneous fiber orientation that resulted in higher fiber alignment. The host tissue also showed stronger birefringence and lower attenuation than the graft tissue.
Fig. 3. The PSOCT images of the intensity (I), birefringence (Δn), fiber orientation (θL), tractography (T), attenuation coefficient (µ), and fiber alignment index (FA) of the xenograft excised at 14-day post-transplantation. In the enface intensity image (extracted at the depth of 0.2 mm), the dashed circles indicate locations of surgical sutures (S). The white dotted line marks the estimated location of cross-sectional images for comparison with histology. The red and yellow boxes indicate ROIs used in quantitative analysis in Fig. 4. The red dashed line in the red box separates the ROI into two sub-regions. The small ROIs marked on cross-sectional PSOCT and histology images had the size of 200×200 µm2. All size bars indicated 1-mm.
As shown in the enface images, the orientation, fiber alignment, and birefringence in the graft varied significantly. The cross-sectional FA image suggested that some small regions of the graft at 0.4 ∼ 0.6 mm beneath the surface still had organized fibers. The top 0.4 mm of the graft had a significantly higher attenuation coefficient. The cross-sectional intensity image revealed a ∼0.2 mm thick dense tissue in superficial layer of the graft tissue which was also observed in the histology image. This layer appeared to have a relatively stronger birefringence, and thus its overall location inside the sample was easily identifiable in the enface birefringence image. In addition, fiber orientation of this structure appeared to be deviated from the long axis of the muscle as shown in the tractography image (T).
The HE histology confirmed the morphological difference between the host and graft observed in PSOCT. For the comparison, the 3D morphology of the PSOCT images were carefully examined to locate the cross-sectional image (Fig. 3) close to the location of histology sectioning. Overall, the host tissue showed clear muscle fibers, whereas the graft tissue appeared to be more heterogeneous. The dense layer on top of the graft was visible in the histology image, where connective tissue and myoblast cells lining up into myotubes can be identified. In most of the peripheral graft (for example ROI-1 in Fig. 3), histology showed damaged muscles with significant indications of necrosis and re-organization 14 days after transplantation. Interspersed were de novo centrally nucleated muscle fibers regenerated from donor stem cells. In the graft regions with relatively better fiber organization in PSOCT (ROI-2 in Fig. 3), histology also showed a higher frequency of organized residual muscle fibers. In addition, the histology suggested muscles in central graft (black dashed box in Fig. 3) still maintained better organization, which was not recognized in PSOCT due to limited imaging depth. This region contained necrotic muscle fibers that had not started to be degraded and re-organized at 14 days.
Figure 4 shows the distributions of fiber orientation, alignment, birefringence and attenuation in the 14-day graft and host obtained in the ROIs (marked in the enface “I” image in Fig. 3) from 0.05 to 0.40 mm beneath the surface. The 14-day graft had very different optical properties than the host muscle. Consistent with the image appearance in Fig. 3, the host tissue was homogeneous, highly aligned with the long axis of the muscle (0°), and had significantly higher birefringence and smaller attenuation than the graft. As a quantitative example, only 16.6% of the graft tissue had a greater than 0.8 alignment index in comparison to 74.6% in the host tissue. Overall, the birefringence in the 14-day host was higher than that of the 0-day graft, while its attenuation coefficient was drastically smaller than that of the 0-day graft. We speculated that the difference between 14-day host (Fig. 4) and 0-day graft (Fig. 2) may be species related. It may also be possible that the host muscle experienced some morphological and compositional changes triggered by transplantation.
Fig. 4. Distributions of fiber orientation, fiber alignment, optical birefringence, and attenuation coefficient of the graft and host tissues obtained from the 14-day sample in Fig. 3. The “sub-graft” distribution was obtained from the upper ROI (above the red dashed line shown in enface “I” image in Fig. 3).
In comparison to the 0-day graft (Fig. 2), the 14-day graft was more disorganized with a diverse angular distribution, although most fibers were still oriented near 0° (i.e. the long axis of the graft muscle). This broader orientation distribution resulted in poorer fiber alignment in the graft. The 14-day graft also had a weaker optical birefringence and higher attenuation coefficient than the 0-day graft. The dashed lines in Fig. 4 showed the distribution obtained in the top half of the ROI (above the red dashed line in the ROI shown in the enface “I” image in Fig. 3), which excluded the dense superficial tissue structure as described previously. As a result, the birefringence was greatly affected and shifted toward lower birefringence.
Figure 5 shows imaging results of a 56-day sample. Similar to the 14-day results, the host and graft were easily distinguishable in all functional images. The host also appeared darker in the intensity image. The differences between host and graft were most distinct in the birefringence and attenuation images. As in the case of the 14-day sample, the 56-day host tissue still had stronger birefringence and smaller attenuation coefficient than the graft. The cross-sectional images showed that the top 0.4 mm of the graft had much stronger attenuation than deeper tissues in most parts of the sample. However, in sharp contrast to the 14-day graft, the fiber alignment and orientation images revealed that many parts of the graft (excluding the areas with surgical suture) had organized fibers similar to the host muscle. The cross-sectional image indicated that organized fibers can be observed up to 0.6 mm in depth at this cross-sectional location. The histology image showed clear signs of surgical suture (marked in dashed circles) which helped to identify the cross-sectional location in the PSOCT images (dotted line in enface “I” image). To achieve a better match, the image volume was rotated 15° around the vertical axis in the enface plane (or the B-scan axis). Histology results clearly confirmed widespread muscle regeneration although heterogeneity can still be observed. For example, the ROI-2 showed more robust regeneration than ROI-1.
Fig. 5. The PSOCT images of the intensity (I), birefringence (Δn), fiber orientation (θL), tractography (T), attenuation coefficient (µ), and fiber alignment index (FA) of the xenograft excised at 56-day post transplantation. In the enface intensity image (extracted at the depth of 0.2 mm), the dotted line marks the estimated location of cross-sectional images for comparison with histology. The red and yellow boxes indicate ROIs used in quantitative analysis in Fig. 6. The dashed circles in the intensity images (enface and cross-section) and histology indicate locations of surgical sutures (S). The small ROIs marked on cross-sectional intensity and histology images had the size of 200×200 µm2. All size bars indicated 1-mm.
Figure 6 shows the distributions of fiber orientation, alignment, birefringence and attenuation coefficient in the graft and host of the 56-day sample shown in Fig. 5. The differences between host and graft as discussed above can be verified in these distributions. In this sample, the host muscle fibers were largely oriented ∼ -60° as visualized in the tractography (Fig. 5). Due to the broader distribution in orientation, 59.1% of the host had alignment index greater than 0.8, which was not as highly organized as in the 0-day graft and 14-day host. As a comparison, 44.2% of the graft at 56-day had a greater than 0.8 alignment index, which was significantly improved over the 16.6% value measured in the 14-day sample (Fig. 4). The graft birefringence was slightly higher at day 56 than at day 14 with median values of 5.2×10−4 vs. 4.6×10−4, respectively.
Fig. 6. Distributions of fiber orientation, fiber alignment, optical birefringence, and attenuation coefficient of the graft and host tissues obtained from the 56-day sample in Fig. 5.
To visualize the overall temporal changes in optical properties, Fig. 7 shows the birefringence, fiber alignment, and attenuation coefficient measured on the 3rd, 7th, 14th, 28th, 56th, and 112th day post-transplantation. Clear host tissues were imaged only in some samples excised at 14-day and later. Due to non-Gaussian distributions of these parameters (as shown in Figs. 4 and 6), median values of the corresponding parameter distributions were used to represent the overall measurements in the graft and host tissues at each time point. Each individual symbol in Fig. 7 represents the result from a different animal.
Fig. 7. Temporal changes of three optical properties: (a) fiber alignment FA, (b) birefringence Δn, and (c) attenuation coefficient µ, measured over a period of 112 days after transplantation. Each individual symbol represents data measured from a different animal. The dashed lines were plotted using the mean results obtained at each date point.
Overall, the median value of fiber alignment in grafts rapidly deteriorated after transplantation and reached a minimum of ∼0.5 at 28-day; it started to recover thereafter and eventually became >0.8 at 112-day, which was close to the level at pre-transplantation. The host tissues remained well organized during the same period. At 112-day, the alignment index was not statistically different between the host and graft (p=0.23, Student’s t-test). The birefringence followed a similar trend: it decreased after the transplantation, reached a minimum of 4.5×10−4 between 14- and 28-day, then started to increase and reached ∼6.5×10−4 at 112-day. In comparison, the birefringence in the host muscle remained at ∼ 8×10−4 during the entire study period. At 112-day, the graft birefringence values were similar to those measured before transplantation. However, the graft birefringence was consistently smaller than that of the host even at 112-day (p=0.01, Student’s t-test). The attenuation coefficient showed an opposite trend where it started to increase after transplantation until ∼ 28-day, then decreased to ∼ 3 mm−1 at 56-day, and remained at the same level afterwards till 112-day. In contrast to the stable alignment index and birefringence in host, attenuation coefficients in host tissue followed a similar temporal trend as the graft. However, attenuation in host was significantly smaller than graft during the entire testing period (p<0.001 at 112-day, Student’s t-test).
As described in the method section, tissues between 0.05 and 0.4 mm beneath the tissue surface were used in the quantification results (Fig. 7). Tissues above 0.05 mm were excluded in quantification to avoid the influence from the perimysium tissue wrapping around muscle fibers. Data calculated between 0.05 and 0.4 mm appeared to have the most consistent results, likely due to robust muscle regeneration and better signal-to-noise at smaller imaging depths. However, similar temporal trends were obtained in birefringence and alignment index even when the calculation depth was extended to 0.8 mm, while a similar temporal trend in attenuation coefficient was observed up to 0.5 mm in depth.
Among the three parameters, the attenuation coefficient showed the largest percentage increase of ∼57% from day 0 to day 28, in comparison to a ∼44% decrease in alignment index and a ∼22% decrease in birefringence over the same period. The observation of consistently different birefringence and attenuation coefficients between host and graft at 112-day was most likely the result of structural and compositional differences in muscle from different species (murine vs canine). The temporal changes in attenuation of the host tissue over the testing period suggested certain pathological changes may have occurred in host due to transplantation. Further studies with in-depth, quantitative histology analysis are necessary to elucidate the underlying mechanisms.
The decrease of graft birefringence and fiber alignment during the first 20∼30 days post transplantation was consistent with the destruction of pre-existing myofibers as observed in histology (Fig. 3). Our results (Fig. 5) showed that regenerated myofibers eventually became aligned with the original muscle orientation. However, the orientational characteristics of proliferating myoblasts and de novo myotubes during the early phase of regeneration were unclear as these small microstructures [18] cannot be fully resolved by our current system. A random distribution of organized microstructures may also lead to a reduced birefringence when the scale of such randomness is comparable to or smaller than the imaging pixel size [19]. A PSOCT system with improved resolution as recently reported [20] is needed to elucidate any fine morphological details of such micro myofiber structures. Nevertheless, all three imaging parameters showed a consistent transition at 20∼30 days post transplantation. This observation suggested that muscle regeneration started to outweigh any residual muscle degeneration in the graft muscle at this time point. As shown in previous studies, the specific duration of the degeneration and regeneration duration may vary depending on species used in the model [2–4]. For example, Nance et al. reported that transplanted mouse muscle became severely degenerated by 7-day and fully regenerated by 42-day post transplantation [4]. Zhang et al. reported that full regeneration of human muscle graft occurred at 90-day post transplantation [3]. Here, we demonstrate that PSOCT can monitor such processes. In practice, the ∼1-2 mm imaging depth in PSOCT may limit its applications to small animal tissues. As demonstrated in a recent study [21], implementing PSOCT using a fine needle optical probe can greatly extend the imaging depth, which may be a viable option for in vivo applications in a large muscle.
4. Summary
PSOCT was used to image the muscle degeneration and regeneration in a canine muscle xenograft model. Quantitative imaging parameters including orientation, birefringence, fiber alignment, and optical attenuation were measured in the graft and host tissues at multiple time points from 3- to 112-day post-transplantation. The results showed that the fiber alignment, birefringence, and attenuation demonstrate distinct and significant changes with time in grafts, following the expected changes in muscle degeneration and regeneration. Although these results require further confirmation with more samples, PSOCT showed promise as a useful tool to study muscle degeneration and regeneration.
Funding
National Institutes of Health (AR70517, NS90634); Jesse’s Journey-The Foundation for Cell and Gene Therapy; Ryan’s Quest; Michael’s Cause; Pietro’s Fight; Jackson Freel DMD Research Fund; Hope for Javier.
Acknowledgements
The authors thank Dr. Kathryn Wagner and Dr. Tracey Zhang for their guidance in developing the xenograft technique. We also thank Mr. Tanmoy Paul for his assistance in this project during the initial phase of data processing.
DD was supported by National Institutes of Health (NS90634, AR70517), Jesse’s Journey-The Foundation for Cell and Gene Therapy, Ryan’s Quest, Michael’s Cause, Pietro’s Fight, and Jackson Freel DMD Research Fund. MEN was supported by a pre-doctoral fellowship from Hope for Javier.
Disclosures
DD: Solid Biosciences (F, I, C, P, R, S), Edgewise Therapeutics (F). Other authors declare no conflicts of interest.
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