Muscular Pathology

We are investigating the use of optical coherence tomography (OCT) for examining muscle pathology, specifically the skeletal muscle tissue of mouse models of muscular dystrophy. This work is a collaborative effort with Professor Miranda GroundsSkeletal Muscle Research Group, and also Assoc/Prof Gavin Pinniger, both within the School of Anatomy, Physiology and Human Biology at The University of Western Australia.

Duchenne muscular dystrophy (DMD) is the most severe form of the human muscular dystrophies, characterised by the progressive degeneration of skeletal and cardiac muscle tissue. The disease is caused by a mutation of a recessive gene located on the X-chromosome that codes for the muscle membrane protein dystrophin, which is vital for preserving mechanical integrity of the muscle fibres (myofibres). Absence of a functional form of this protein results in a fragile membrane that is easily damaged, activating an inflammatory response that leads to myofibre death (necrosis). The disease occurs at a rate of ~1 in 3600 to 6000 male births worldwide, and proves fatal due to respiratory or cardiac failure.

Mice animal models are routinely used in pre-clinical studies aimed at understanding the cause and progression of the disease, and assessing the effectiveness of potential therapies for DMD. Histology is conventionally used to study the morphology of skeletal muscle, but the process is laborious and time consuming, and not amenable to examining large tissue volumes. Instead, we are exploring the use of OCT as a medical imaging alternative. OCT is a minimally-invasive imaging modality capable of acquiring three-dimensional (3D) scans of biological tissue with a resolution of 1 – 20 µm at depths up to 1 ~ 2 µm.

We have investigated OCT imaging of muscle pathology using two mouse models:

  • Whole muscle autograph (WMA) surgical model of skeletal muscle damage and inflammation [1]; and
  • Mdx mouse model for dystropathology in human DMD. [2]

We have performed 3D-imaging of large volumes of ex vivo mouse skeletal muscle tissue to identify features of interest, such as: regions of healthy and/or intact myofibres; damaged and/or necrotic myofibres; inflammation and infiltrating inflammatory cells; connective and adipose tissue; and areas of pathology. This has been achieved using the following techniques:

  1. Three-dimensional OCT (3D-OCT) imaging, using both time-domain [1], [2], [4] and spectral-domain [3], [5], [6] OCT systems;
  2. Needle probes for OCT for deep-tissue imaging [3]; and
  3. Parametric imaging of:
    1. Attenuation coefficient µt using 3D-OCT [4],
    2. Birefringence ∆n using polarisation-sensitive OCT (PS-OCT) [5], and
    3. Strain using optical coherence micro-elastography (OCME) [6].

Section 1. 1       3D-OCT imaging of muscle pathology

1) 1.1   Whole muscle autograft (WMA): Surgical model of muscle damage and inflammation [1]

We first investigated OCT imaging of muscle pathology, using the whole muscle autograft (WMA) [1]. The WMA is a surgical model of skeletal muscle damage, inflammation and regeneration, which has been used extensively in the research to help understand the basic biology of muscle. Specially, it has been used to understand the progression of pathology in skeletal muscle, the mechanisms of regenerating new tissue following this damage, and the effect of pharmaceutical interventions on these processes of necrosis, inflammation and regeneration of skeletal muscle tissue. En face OCT images obtained from the 3D-OCT datasets were matched with subsequent transverse hematoxylin and eosin (H&E)-stained histology, corresponding for depth and visually correlating structural features of interest. In the OCT images of the WMA, the damaged and inflamed graft tissue of the extensor digitorum (EDL) skeletal muscle could be readily distinguished from the healthy and intact host tibialis anterior (TA) muscle, meeting at a clearly defined boundary. Regions of tissue breakdown and inflammation could be identified in the graft tissue, and myofibres seen in the underlying host tissue. The features identified in the OCT images matched well with the corresponding H&E-histology, as shown in Figure 1.1.

applications_muscle-pathology_Fig1-1_WMA

Figure 1.1 En face OCT images [(a), (c), and (e)], and the corresponding H&E-histology [(b), (d), and (f)] of transverse sections of the autograft muscle sample at depths of (a) and (b) 100, (c) and (d) 250, and (e) and (f) 500 µm into the tissue. Surviving myofibres (S) are visible in the grafted tissue. The 2 × magnified view (insets) show the graft boundary.

2) 1.2   Mdx mouse: Genetic model of human Duchenne muscular dystrophy [2]

We next investigated OCT imaging of muscle pathology in the treadmill-exercised X-chromosome-linked muscular dystrophy (mdx) mouse model of DMD. Our results have shown that 3D-OCT is capable of imaging the large-scale structure and pathology of ex vivo muscle samples from the mdx. Necrotic lesions (areas containing necrotic myofibres with fragmented membranes and spilled cytoplasm, cellular debris, infiltrating inflammatory cells, connective and adipose tissue) occur in the skeletal muscles in response to the exercise and underlying disease. Location of these necrotic lesions is aided by the injection of the marked Evans blue dye (EBD) into the mice immediately after treadmill-exercise, but 24 hours prior to sampling. The EBD-positive degenerating myofibres can be macroscopically identified by their dark blue staining, which reveals the severity and spatial location of the muscle damage in the animal to help guide our 3D-OCT imaging. Necrotic lesions could be identified within these large volumes (mm3) by 3D-OCT based on the loss of intrinsic optical contrast, as seen in Figure 1.2.

applications_muscle-pathology_Fig1-2_3DOCT-mdx

Figure 1.2 OCT image sections from a 3D-OCT dataset of a dystrophic tibialis anterior skeletal muscle from the mdx mouse. (a) (y-z) OCT plane; (b) (x-z) OCT plane; (c) en face (x-y) OCT plane; (d) rendering of the sections with the axes marked corresponding to the section axes. White dashed lines indicate the sectioning planes. MF, myofibers (white arrowheads); DR, disrupted region (black arrowheads); CT, connective tissue (white arrows); SA, shadow artifact (black arrows). Scale bars 1 mm.

We validated OCT images by comparing them with co-located conventional histology using two staining techniques: hematoxylin and eosin (H&E) and fluorescent Evans blue dye (EBD), as seen in Figure 1.3 (c) and (d) respectively. The striation pattern observed in the en face OCT images correspond to areas of intact myofibres, which can clearly be seen in the co-located H&E-histology sections as centrally nucleated (CN) regenerated myofibres, and the source of the OCT appearance is attributed to the presence of the regular alignment of intact myofibres.

Regions of disrupted OCT signal are seen in the images as featureless areas corrupted by speckle that have lost the distinctive striation pattern, and are characterised by low signal and contrast within a relatively well-defined boundary. These disruption regions (DR) are attributed to necrotic lesions (NL) and the underlying change in the muscle tissue structure as a result of dystropathology, as seen in the corresponding H&E-stained histology sections Figure 1.3 (c) and F2 below. EBD-positive degenerating myofibers are also identified in the frozen histology sections by their red fluorescence, indicating regions containing permeable (leaky or necrotic) myofibres that are often associated with necrotic lesions.

applications_muscle-pathology_Fig1-3_3DOCT-mdx

Figure 1.3 Dystrophic tibialis anterior skeletal muscle tissue from a treadmill-exercised mdx mouse. (a) Photograph of the posterior surface showing Evans blue dye (EBD) accumulation (bright blue area, dashed outline). (b) En face (x-y) OCT image displaying the characteristic myofibre (MF) striation pattern and a large disruption region of that pattern in the OCT signal (white dashed outline). (c) Corresponding H&E-stained longitudinal histology section showing intact myofibres, dystropathology, and a large well-defined necrotic lesion, NL (black dashed outline). (d) EBD-fluorescence histology section showing a high accumulation of dye in a large area matching the previously identified necrotic lesion (white dashed outline) (Scale bars 1 mm). The 4 × magnified view (outsets) show regions of intact fibers (E1, F1, G1), the interior of the necrotic lesion (E2, F2, G2), and the lesion boundary (E3, F3, G3) (Scale bars 100 µm). IM, intact myofibers (white arrowheads); NM, necrotic myofibers (black arrowheads); CT, connective tissue (white arrows); IC, inflammatory cells (black arrows); CN, central nuclei (thin white arrows).

We have found that 3D-OCT is useful for the structural assessment of pathology and identification of lesions in skeletal muscle tissue from the mdx mouse model of DMD. It is able to image the structural features of interest from skeletal muscle tissue (myofibres, tendon, connective tissue, adipose tissue) based on the intrinsic differences in the scattering properties of these structures. It can identify muscle necrosis and lesions, which are seen in scans as regions of low signal, low contrast and a loss of the characteristic striation pattern of intact myofibres. We have also shown that EBD is a suitable in vivo marker for targeted 3D-OCT imaging of necrotic lesions and muscle pathology, as it does not interfere with the OCT imaging beam at 1300 nm.

Section 2. 2       Needle probe for deep tissue OCT imaging of muscle pathology [3]

We have also developed an extremely miniaturised OCT needle probe (outer diameter 310 μm) with a high sensitivity (108 dB) that has enabled us to image the structure and pathology of the mdx mouse deep within its skeletal muscle [3]. Details of this OCT needle probe are shown below in Figure 2.1.

applications_muscle-pathology_Fig2-1_Needle-OCT1

Figure 2.1 (a) Schematic of the ultrathin OCT needle probe used for deep-tissue imaging of skeletal muscle structure and pathology. (b) Microscope image of the angle-polished fibre probe before metallisation. (c) SEM image of the laser-drilled side opening. (d) Fully assembled needle probe showing the laser-drilled side opening. Red light from the aiming laser is visible

Using the miniaturised OCT needle probe, we were able to acquire three-dimensional volumetric images from ex vivo mouse tissue, examining both healthy mucle from wild-type mice and pathological muscle from dystrophic mdx mice. An example of this is shown below in Figure 2.2.

applications_muscle-pathology_Fig2-2_Needle-OCT2

Figure 2.2 Needle OCT imaging of normal, healthy mouse skeletal muscle tissue. (a) A rendered image of the volumetric 3D-OCT dataset at a depth of ~10mm, and three corresponding orthogonal cross-sections in (b) yellow dashed line, (c) red dashed line and (d) blue dashed line. 3D scale bar in (a) represents 500 μm within each plane. MF, myofibres (white arrowheads); C, connective tissue (white arrow); T, tendon (white arrow); N, needle tract (white arrow); B, birefringence artifacts (white double arrowheads).

Regions of intact myofibres were again seen as striations (Figure 2.3) whilst necrotic lesions were seen as a loss of these striations (Figure 2.4). We could also detect tendon and connective tissue deep within skeletal muscle tissue, based on their high backscattering. These observed structures were validated against co-registered hematoxylin and eosin (H&E) stained histology sections. This work has demonstrated the ability of using an OCT needle probe to visualise structure and pathology of at the microscopic level deep in skeletal muscle tissue in situ.

applications_muscle-pathology_Fig2-3_Needle-OCT3

Needle probe OCT imaging of normal skeletal muscle from a healthy wild-type mouse. (Left) OCT oblique slice taken from the 3D-OCT volumetric dataset. The striated appearance indicates the highly organised arrangement of the myofibres (MF, arrowheads). Several structures with higher signal intensity indicate tendon (T) and connective tissue (C). (Right) Corresponding H&E-stained histology section.

applications_muscle-pathology_Fig2-4_Needle-OCT4

Figure 2.4 Needle probe OCT imaging of skeletal muscle pathology in the dystrophic mdx mice. (Left) OCT oblique slice taken from the 3D-OCT volumetric dataset. The striated appearance indicates the highly organised arrangement of myofibres (MF, arrowheads). The structure with higher intensity indicates connective tissue (C). Muscle necrosis is visible as a region without striated appearance (Necrosis). (Right) Corresponding H&E-stained histology section.

Section 3. 3                Parametric OCT imaging of muscle pathology

Extending on our initial work of imaging skeletal muscle pathology using 3D-OCT, we have investigated three different techniques. These methods offer improved contrast of muscle pathology by extracting additional parameters from the raw 3D-OCT data.

1) 3.1   Attenuation coefficient µt using three-dimensional optical coherence tomography (3D-OCT) [4]

We assessed muscle pathology in ex vivo tissue samples from dystrophic mdx mice, by quantitative parametric imaging of the local total attenuation coefficient μt using three-dimensional optical coherence tomography [5]. An example of this is shown below Figure 3.1, where the parametric image of μt(x,y) improves the contrast between the muscle pathology (necrotic lesion) and the surrounding regions of intact myofibres.

applications_muscle-pathology_Fig3-1_3DOCT-mdx

Figure 3.1 Parametric imaging of skeletal muscle pathology using the attenuation coefficient µt. Images were obtained from two different regions: intact myofibres and a necrotic lesion; selected from within the same skeletal muscle tissue sample from a dystrophic mdx mouse. (a) – (c) Top row: Images of the entire muscle sample. Scale bars 1 mm. (d) – (f) Middle row (blue outline): Zoomed images from the region of intact myofibres. Scale bars 250 μm. (g) – (i) Bottom row (red outline): Zoomed images from the necrotic lesion containing necrotic myofibres and inflammatory cells. . Scale bars 250 μm.(a), (d) & (g) Co-registered H&E-stained histology sections indicating biological features of interest: (a) regions of intact myofibres (white asterisks) and necrotic lesions (black asterisks, outlined by black dashed-line), (d) intact myofibres (IM, white arrowheads), central nuclei (CN, thin black arrows), and inflammatory cells (IC, thick black arrows); (g) necrotic myofibres (NM, black arrowheads) and adipose cells (AC, doubled-headed white arrow). (b), (e) & (h) En face (x-y) OCT images at depth z = 350 μm from imaging glass-window / tissue interface, showing the characteristic OCT striation pattern (white @) of intact myofibres, and also disruption region (white hashtags, outlined by white dashed-line) of necrotic lesions. (c), (f) & (i) Parametric maps of attenuation coefficients μt(x,y). Points with μt < 0 are masked white. Regions of intact myofibres (black @) have µt values ranging 0.5 – 7.0 mm-1, giving µt = 3.7 ± 1.1 mm-1 (mean ± standard deviation) whilst regions within the necrotic lesion have µt values ranging 8.0 – 11.5 mm-1, giving μt = 10.1 ± 0.7 mm-1 (mean ± standard deviation).

 The resulting values of the local total attenuation coefficient μt (mean ± standard error) from necrotic lesions in the dystrophic skeletal muscle tissue of mdx mice were found to be are higher (9.6 ± 0.3 mm-1) than regions from the same tissue containing only necrotic myofibres (7.0 ± 0.6 mm-1), and significantly higher than values from intact myofibres, whether from an adjacent region within the same sample (4.8 ± 0.3 mm-1) or from healthy muscle tissue of wild-type mice (3.9 ± 0.2 mm-1) used as a control. These results are summarised in the histogram of Figure 3.2 below. Our results suggest that the attenuation coefficient could be used as a quantitative means to identify necrotic lesions and assess skeletal muscle tissue in mouse models of human Duchenne muscular dystrophy.

applications_muscle-pathology_Fig3-2_3DOCT-mdx2

Figure 3.2 Attenuation coefficients μt (mean ± standard error) of intact myofibres from the healthy muscles (n = 10) of wild-type mice, intact myofibers from the dystrophic muscles (n = 10) of mdx mice, necrotic myofibres (without the presence of inflammatory cells) from the dystrophic muscles (n = 2) of mdx mice, and necrotic lesions from the dystrophic muscles (n = 6) of mdx mice, calculated from all muscle samples. The attenuation coefficient μt of intact myofibres (whether from healthy wild-type or dystrophic mdx mice) is statistically significantly different from the attenuation coefficient μt of necrotic lesions from dystrophic mdx mice muscles.

2) 3.2   Birefringence ∆n using polarisation-sensitive optical coherence tomography (PS-OCT) [5]

Undamaged muscle tissue possesses high levels of optical birefringence due to its anisotropic ultrastructure, and this birefringence decreases when the tissue undergoes necrosis. Using polarisation-sensitive optical coherence tomography (PS-OCT) scans, we are able to quantify the birefringence in muscle tissue, generating an image indicative of the tissue ultrastructure, with areas of abnormally low birefringence indicating necrosis. The technique additionally gives a measure of the proportion (volume fraction) of necrotic tissue within the three-dimensional imaging field of view. Comparing the percentage necrosis assessed by this technique against the percentage necrosis obtained from manual assessment of histological sections, we found the difference between the two methods to be comparable to the interobserver variability of the histological assessment. This is the first published demonstration of PS-OCT to provide automated assessment of muscle necrosis [5].

applications_muscle-pathology_Fig3-3_PSOCT

Figure 3.3 Parametric birefringence ∆n(x,y) images of a triceps muscle from a dystrophic mdx mouse, illustrating the ability of PS-OCT to image skeletal muscle pathology. (A) Photograph of the whole muscle showing EBD staining. (B) Corresponding H&E-stained histology section from within the muscle sample. (C) En face OCT image (log-scaled intensity) of the whole muscle. (D) Parametric birefringence ∆n(x,y) image. Asterisk (*) in all images indicates the necrotic lesion (containing necrotic myofibres and inflammatory cells), which corresponds to the loss of the striation pattern in the en face OCT image (C) and the low birefringence regions within the parametric birefringence image (D).

3) 3.3   Mechanical strain mε using optical coherence micro-elastography (OCME) [6]

In muscle pathology, such as that seen in human DMD, impairment of skeletal muscle function is closely linked to changes in the mechanical properties of the muscle constituents. Optical coherence micro-elastography (OCME) uses optical coherence tomography (OCT) imaging of tissue under a quasi-static, compressive mechanical load to map variations in tissue mechanical properties on the micro-scale. We have conducted the first study of OCME on skeletal muscle tissue, in both healthy wild-type mice and dystrophic mdx mice exhibiting muscle pathology. Our results have shown that OCME can resolve the important features of muscle tissue, including myofibres, fascicles (connective tissue surrounding a bundle of many myofibres) and tendon, and can also detect necrotic lesions present in skeletal muscles of mdx mice, as shown below Figure 3.4. In many instances, OCME provides better or additional contrast complementary to that provided by OCT. Our results suggest that OCME could provide new understanding and opportunity for assessment of skeletal muscle pathology [6].

applications_muscle-pathology_Fig3-4_OCME

Figure 3.4 Parametric images of mechanical strain (mε) of a gastrocnemius muscle from a dystrophic mdx mouse. (a) H&E-stained histology section, (b) en face OCT image (log-scaled intensity) and (c) en face parametric micro-elastogram (milli-strain). (d), (f) and (h) Magnified insets (2 × 2 mm) of corresponding regions of intact myofibres. (e), (g) and (i) magnified insets of corresponding regions of a necrotic lesion. En face OCT images and micro-elastograms were taken 100 μm from the surface.

Section 4. Key techniques

OCT microscope-in-a-needle 

Parametric imaging of optical properties 

Polarization-sensitive OCT 

Optical coherence micro-elastography

Section 5. Key researchers

Key publications

  1. Blake R. Klyen, Julian J. Armstrong, Steven G. Adie, Hannah G. Radley, Miranda D. Grounds, David D. Sampson, “Three-dimensional optical coherence tomography of whole muscle autografts as a precursor to morphological assessment of muscular dystrophy in mice”, Journal of Biomedical Optics, 13(1), 011003 (2008).
  2. Blake R. Klyen, Thea Shavlakadze, Hannah G. Radley-Crabb, Miranda D. Grounds, David. D. Sampson, “Identification of muscle necrosis in the mdx mouse model of Duchenne muscular dystrophy using three-dimensional optical coherence tomography”, Journal of Biomedical Optics, 16(7), 076013 (2011).
  3. Xiaojie Yang, Dirk Lorenser, Robert A. McLaughlin, Rodney W. Kirk, Matthew Edmond, M. Cather Simpson, Miranda D. Grounds, and David D. Sampson, “Imaging deep skeletal muscle structure using a high-sensitivity ultrathin side-viewing optical coherence tomography needle probe”, Biomedical Optics Express, 5(1), pp. 136-148 (2014).
  4. Blake R. Klyen, Loretta Scolaro, Tea Shavlakadze, Miranda D. Grounds, and David D. Sampson, “Optical coherence tomography can assess skeletal muscle tissue from mouse models of muscular dystrophy by parametric imaging of the attenuation coefficient”, Biomedical Optics Express, 5(4), pp. 1217–1232 (2014).
  5. Xiaojie Yang, Lixin Chin, Blake R. Klyen, Tea Shavlakadze, Robert A. McLaughlin, Miranda D. Grounds, and David D. Sampson, “Quantitative assessment of muscle damage in the mdx mouse model of Duchenne muscular dystrophy using polarization-sensitive optical coherence tomography”, Journal of Applied Physiology, 115(9), pp. 1393-1401 (2013).
  6. Lixin Chin, Brendan F. Kennedy, Kelsey M. Kennedy, Philip Wijesinghe, Gavin J. Pinniger, Jessica R. Terrill, Robert A. McLaughlin and David D. Sampson, “Three-dimensional optical coherence micro-elastography of skeletal muscle tissue”, Biomedical Optics Express, 5(9), pp. 3090-3102, (2014).