Imaging Fourier-transform spectroscopy (IFTS) is a powerful method for biological hyperspectral analysis based on various imaging modalities, such as fluorescence or Raman. Since the measurements are taken in the Fourier space of the spectrum, it can also take advantage of compressed sensing strategies. IFTS has been readily implemented in high-throughput, high-content microscope systems based on wide-field imaging modalities. However, there are limitations in existing wide-field IFTS designs. Non-common-path approaches are less phase-stable. Alternatively, designs based on the common-path Sagnac interferometer are stable, but incompatible with high-throughput imaging. They require exhaustive sequential scanning over large interferometric path delays, making compressive strategic data acquisition impossible. In this paper, we present a novel phase-stable, near-common-path interferometer enabling high-throughput hyperspectral imaging based on strategic data acquisition. Our results suggest that this approach can improve throughput over those of many other wide-field spectral techniques by more than an order of magnitude without compromising phase stability.
Full text Figures and data Side by side Abstract Editor's evaluation Introduction Results Discussion Methods Appendix 1 Data availability References Decision letter Author response Article and author information Metrics Abstract The reasons for poor healing of pressure injuries are poorly understood. Vascular ulcers are worsened by extracellular release of hemoglobin, so we examined the impact of myoglobin (Mb) iron in murine muscle pressure injuries (mPI). Tests used Mb-knockout or treatment with deferoxamine iron chelator (DFO). Unlike acute injuries from cardiotoxin, mPI regenerated poorly with a lack of viable immune cells, persistence of dead tissue (necro-slough), and abnormal deposition of iron. However, Mb-knockout or DFO-treated mPI displayed a reversal of the pathology: decreased tissue death, decreased iron deposition, decrease in markers of oxidative damage, and higher numbers of intact immune cells. Subsequently, DFO treatment improved myofiber regeneration and morphology. We conclude that myoglobin iron contributes to tissue death in mPI. Remarkably, a large fraction of muscle death in untreated mPI occurred later than, and was preventable by, DFO treatment, even though treatment started 12 hr after pressure was removed. This demonstrates an opportunity for post-pressure prevention to salvage tissue viability. Editor's evaluation It is known that muscle pressure injury (mPI), tissue damage caused by sustained pressure, is difficult to be healed although the mechanism underlying has been poorly understood. In this study, the authors demonstrated a convincing evidence that myoglobin (Mb) released at the site of injury plays an important role in the size, severity, oxidative damage, and poor healing of mPI by causing the induction of immune cell (in particular phagocyte) death and delaying the clearance of dead tissues using Mb KO mice and iron chelation by deferoxamine. The authors' findings are valuable in developing a novel therapeutic option for mPI although further clinical corroboration of these findings would be of even greater value. https://doi.org/10.7554/eLife.85633.sa0 Decision letter eLife's review process Introduction Pressure injuries (also called pressure ulcers, bedsores, decubiti, or pressure sores) are tissue damage caused by sustained pressure. They are extremely painful (Girouard et al., 2008), costly to prevent and treat (Padula and Delarmente, 2019), and increase the risk of patient sepsis and death (Wassel et al., 2020). Tissue death can be caused by mechanical deformation, ischemia, or both. Ischemia is often studied as ischemia-reperfusion injury, but poor clearance of damage factors may have unappreciated importance (Gray et al., 2016). Pressure injuries often heal poorly (Mervis and Phillips, 2019), especially if they involve deeper layers such as muscle (Bouten et al., 2003; Preston et al., 2017), but the reasons are for poor quality and quantity of healing are not clear. Some cases can be explained by complications or comorbidities (infection, incontinence, poor circulation, hyperglycemia, chronic reinjury, advanced age), but pressure injuries can affect any immobile person (e.g., young adults with spinal cord injury). In this study, we ask whether some aspect of pressure-induced injury is intrinsically inhospitable to regeneration and in need of intervention. Chronic ulcers of veins or arteries (e.g., venous stasis ulcers, sickle cell ulcers) have high levels of extracellular hemoglobin (Hb) released in the wound. For example, many have deposits of hemosiderin. Extracellular Hb and its breakdown products (e.g., hemin, iron) create oxidative stress (Goldman et al., 1998; Reeder and Wilson, 2005) and other effects that are detrimental to regeneration. For example, Hb decreases nitric oxide for angiogenesis (Kato et al., 2017; Nader et al., 2020) and signals as a damage-associated molecular pattern (DAMP) to increase inflammation (Mendonça et al., 2016; Bozza and Jeney, 2020). Systems exist to detoxify Hb (Cherayil, 2011), but Hb is also an innate immune factor with evolutionarily conserved antimicrobial function. When extracellular Hb is activated by proteolytic cleavage, bacterial binding, or conformational change, it increases production of reactive oxygen species (ROS) Bogdan, 2007; this process has been called 'oxidative burst without phagocytes' (Bogdan, 2007; Jiang et al., 2007). Our earlier work used evolutionary conservation to identify ROS-producing fragments of Hb and crosstalk with tissue factor coagulation (Bahl et al., 2014; Bahl et al., 2011). Therefore, globin proteins have multiple functions that may be detrimental to chronic wounds (Tchanque-Fossuo et al., 2017). Myoglobin release into plasma or urine has been observed after muscle pressure injuries (mPI) in multiple studies including deep tissue injury (DTI) (Traa et al., 2019; Makhsous et al., 2010; Loerakker et al., 2012; Levine, 1993; Traa, 2019), but was studied as a readout of damage rather than a source of damage. We reason by analogy to hemoglobin that extracellular myoglobin might create a hostile wound environment. An extracellular environment oxidizes globins to a ferric (Fe3+) state, which can be further oxidized to ferryl (FeIV=O) globin in the presence of endogenous peroxides such as hydrogen peroxide (Reeder et al., 2008). Hydrogen peroxide is ubiquitous in contexts of cell stress, mitochondrial permeabilization, and cell death (Rojkind et al., 2002). Ferryl-Mb can oxidize macromolecules directly (Goldman et al., 1998; Kapralov et al., 2009; Plotnikov et al., 2009) and can form heme-to-protein crosslinks (Osawa and Williams, 1996). Most importantly, ferryl myoglobin can participate in a catalytic cycle of pseudo-peroxidase activity (redox cycling) (Boutaud and Roberts, 2011). In a tissue context, myoglobin can induce ferroptosis, which is a form of non-apoptotic cell death associated with iron and characterized by lipid peroxidation (Dixon et al., 2012). Dissociation of myoglobin into free heme or iron results in additional forms of toxicity, as described for hemoglobin. We hypothesize that mPI will have Mb-dependent pathologies, and that introducing Mb-knockout or iron chelation therapy will partially normalize the mPI pathologies. Deferoxamine (DFO), also known as desferrioxamine or desferoxamine, is an FDA-approved small-molecule iron chelator that improves iron overload syndromes (Velasquez and Wray, 2022; Karnon et al., 2012). DFO binds free iron and heme at a 1-to-1 ratio (Velasquez and Wray, 2022), scavenges free radicals (Reeder et al., 2008; Morel et al., 1992), reduces ferryl myoglobin to ferric myoglobin (Plotnikov et al., 2009; Vanek and Kozhli, 2022), inhibits crosslinking of heme to protein (Reeder and Wilson, 2005), and prevents the formation of pro-oxidant globin and heme species. DFO can function as an activator of Hif1α (Tchanque-Fossuo et al., 2017; Xiao et al., 2013), a tool for promoting angiogenesis (Duscher et al., 2015; Holden and Nair, 2019), an antioxidant (Sundin et al., 2000), or can join an anti-ischemic cocktail (Soloniuk et al., 1992). DFO appears in hundreds of studies of ischemic or inflammatory pathologies. In our study, subcutaneous DFO is used for testing the hypothesized role of myoglobin iron and as an anti-DAMP therapy for combating local iron overload. Assessing the contribution of myoglobin iron to pressure injury pathophysiology provides an opportunity to test several additional hypotheses about pressure injuries. First, our prior work in mathematical modeling (Jagannathan and Tucker-Kellogg, 2016) predicted that oxidative stress from myoglobin and other DAMPs could create secondary progression of pressure ulcers. Secondary progression means that otherwise viable tissue dies later from the environmental consequences of injury, rather than dying directly from the original injury. Pressure injuries are known to have gradual expansion of tissue death (Stadler et al., 2004), consistent with secondary progression, but blocking secondary progression has not been clinically recognized as a goal for intervention (European Pressure Ulcer Advisory Panel et al., 2019). Therefore, our studies are designed to test whether tissue margins can be saved from dying, if we initiate iron chelation therapy 12 hr after pressure has ended. Second, we hypothesize that iron chelation therapy, by improving the early stages of injury response, will lead to better muscle tissue architecture (better morphogenesis) in long-term regeneration, even after treatment has ended. This hypothesis will be tested by breeding inducible fluorescence into satellite cells (muscle stem cells bearing Pax7); this fluorescence causes newly regenerated muscle fibers light up against the dark background of pre-existing muscle. Third, establishing a pressure injury mouse model provides an opportunity to learn how much of the poor healing is independent of comorbidities and complications. To the best of our knowledge, pressure injuries have never been assessed for poor regeneration under aseptic conditions in young, healthy animals. We hypothesize that even under these ideal circumstances mPI will heal slowly and incompletely. Our fourth and final additional hypothesis is inspired by prior studies of blood-related conditions in which high levels of hemoglobin or heme/hemin could impair the survival, chemotaxis, and phagocytosis (Nader et al., 2020; Ferris and Harding, 2019; Sindrilaru et al., 2011; Ballart et al., 1986; Liu et al., 2019; Martins et al., 2016; Chen et al., 2009; Yefimova et al., 2002) of phagocytic cells. Given that pressure ulcers often have slough or eschar, we hypothesize that necrotic tissue will persist in the mPI wound bed, and that sterile mPI will have slough, despite the absence of bacterial biofilm. If correct, this would imply that slough by itself is not sufficient to indicate infection (or bacterial colonization) of a wound. Results Magnet-induced pressure injury causes delayed healing and failure of muscle regeneration To compare wound healing between acute and chronic wounds, we injured the dorsal skinfold of mice using either cardiotoxin (CTX) or pressure (Figure 1—figure supplement 1A–C) in healthy young adult mice under specific pathogen-free conditions. Both groups of mice received sham treatment (injected with 0.9% saline subcutaneously for 16 d or until mouse sacrifice, whichever was sooner). The normal uninjured mouse skinfold contains the following parallel layers: a thin epithelium (epidermis), a thicker layer of dermis, dermal white adipose tissue (dWAT), a muscle layer called the panniculus carnosus (PC), and a wavy layer of loose areolar tissue (Figure 1—figure supplement 1D). Despite comparable diameters of dead muscle between CTX and mPI at day 3, the wound diameters were vastly different at day 10 (Supplementary file 1a; p=0.578 at day 3 and p<0.0001 at day 10). In the CTX injury at day 3, many blood vessels were intact and carrying red blood cells, but in mPI at day 3, intact vasculature was not observed in the compressed region (Figure 1—figure supplement 2). Prior work showed that pressure affects blood and lymphatic vessels in many ways including potentially loss of flow (Gray et al., 2016; Kimura et al., 2020; Karahan et al., 2018). After CTX killed the panniculus muscle, substantial muscle regeneration occurred by 10 d, in which immature muscle fibers displayed central nuclei (Figure 1A). At 40 d after acute injury, muscle was completely regenerated and mature (evidenced by peripherally located nuclei, Figure 1C). In contrast, the pressure-injured wound bed remained filled with dead tissue at day 10 (Figure 1B and E–F; p<0.0001). Our pressure injuries showed no signs of infection and no epibole (Figure 1E). The dead epidermis, dermis, dWAT, and panniculus layers were pushed upward at day 7 ± 2 as slough (necroslough, per the nomenclature of Nasser, 2019) and remained at the surface, eventually becoming a dry eschar (Figure 1E). When the eschar dropped off (by day 15), the size of the epithelial opening was smaller than the eschar, meaning that re-epithelialization had occurred underneath the eschar. Re-epithelialization completed at day 21 ± 2. Despite successful closure of the epithelial layer, pressure injuries at 40 d had only partial regeneration of the panniculus carnosus layer (Figure 1D and G; p<0.0001). At 90 d after pressure injury, the dermis and epidermis had regenerated, but a hole remained in the panniculus muscle layer (Figure 1H), indicating a failure to regenerate (Nasir et al., 2022). Supplementary file 1b summarizes the timelines. We conclude that the mPI healed poorly. Figure 1 with 2 supplements see all Download asset Open asset Poor regeneration of muscle pressure injury (mPI), a model of chronic wound, compared with cardiotoxin (CTX), a model of acute injury. H&E-stained sections of saline-treated wound tissues (A, B) at day 10 post-injury and (C, D) day 40 post-injury. Red arrows point to the panniculus carnosus layer. '+' indicates where the panniculus layer should be. Scale bars: 50 µm. Uninjured control tissue, stained with H&E, is shown in Supplementary file 1C. (E) Full cross-section of H&E-stained mPI, including uninjured edges, at day 10 post-injury. Note the eschar attached to the wound surface. Scale bar: 500 µm. (F, G) Comparison of the regeneration scores for the panniculus layer between mPI and CTX injuries at days 10 and 40. (H) A multi-channel confocal image of the panniculus layer shows a round hole remains in the muscle, 90 d after mPI. Scale bar: 1000 µm. All injuries are saline-treated to permit comparison against later mPI treatments. All quantitative data are reported as means ± SD. n = 4–7. ****<0.0001 Statistical significance in (F) and (G) was computed by unpaired Student's t test. Compressed regions of pressure injury display an absence of viable immune cells To investigate why dead tissue remains at day 10, we studied tissue sections from day 3 post-injury. Both CTX-induced and pressure-induced injuries had comprehensive death of muscle tissue, as indicated by karyolysis (dissolution of nuclear components), karyorrhexis (fragmentation of the nucleus), and acidification (eosinification) in H&E staining of the panniculus carnosus (Figure 2B). CTX and pressure injuries had a difference in morphology: cells in pressure-injured tissues were flattened, and the thickness of the panniculus muscle layer was half of uninjured (Figure 2A–C; p<0.0001). Even more striking was the difference in immune cell numbers. The muscle layer of CTX wounds had sixfold higher levels of immune cell infiltrate than mPI (p<0.0001; Figure 2A–B and D). The panniculus layer of mPI was nearly devoid of intact immune cells. Some viable immune cells were found at the margins of the wound (at the boundary between injured and uninjured tissue), but not in the compressed region of mPI (Figure 2—figure supplement 1). The absence of immune cell infiltrate is noteworthy because iron-scavenging is performed by macrophages (Kristiansen et al., 2001; Soares and Hamza, 2016), and because free iron, when not adequately scavenged, can overstimulate the innate immune response (Bessman et al., 2020). Figure 2 with 2 supplements see all Download asset Open asset Early-stage pathologies in the injury-response of muscle pressure injury (mPI) (3 d after injury). (A, B) H&E-stained sections of saline-treated wound tissues, comparing magnet-induced mPI versus cardiotoxin (CTX) injury. Red arrows point to the panniculus carnosus (PC) layer. (C) Thickness of PC layer. (D) Histopathology scoring of immune infiltrate into the injured PC layer. (E, F) Immunostaining for citrullinated histone-3 (CitH3, a marker for extracellular traps, in red) in mPI versus CTX. DNA/nuclei were co-stained blue with DAPI. (G) Quantification of CitH3 staining. (H, I) Perls' Prussian blue iron staining (dark blue-gray), with nuclear fast red co-stain. (J) Quantification of Perls' staining. (K, L) Immunostaining for heme oxygenase-1 (HO-1, a marker of heme and iron) in mPI versus CTX. Note that the HO-1-positive signal in CTX injury is localized to the panniculus carnosus and is widespread across all layers in mPI. (M) Quantification of HO-1 staining. Scale bars: 50 µm. All quantitative data are reported as means ± SD. n = 4–7 mice. *<0.05, ****<0.0001. Statistical significance in (C, D), (G), (J), and (M) was computed by unpaired Student's t test. Another difference between mPI and CTX was in the level of citrullinated histone-3 (citH3), a marker of extracellular traps (ETs). ETs are formed when phagocytes citrullinate their histones and eject nuclear and/or mitochondrial DNA (and associated factors), which can trap and kill pathogens during infection. The process, ETosis, often kills the host cell. ETs have been observed in sickle cell ulcers (Chen et al., 2014). In day 3 wounds, levels of citH3 were tenfold higher in mPI than in CTX (Figure 2E–G; p=0.0280). Highest levels occurred near the muscle layer, such as the interface between the panniculus layer and the dWAT or dermis. This is consistent with the possibility that immune cells may have been present in the muscle layer of mPI before day 3 and then died of ETosis. Oxidative stress is a well-studied trigger of ETosis, and other stimuli include hemin and heme-activated platelets (Okubo et al., 2018; Ohbuchi et al., 2017). Free iron remains in wound tissues after pressure injury Iron deposition was very high in mPI, as measured by Perls' Prussian blue stain (Figure 2I), but iron was undetectable at the same time point after CTX injury (Figure 2H–J; p=0.0332). Prussian blue detects accumulation of ferric Fe3+, typically in the form of ferritin and hemosiderin, but it only shows high levels of iron, because it is unable 'to detect iron except in massive deposition' (Perl and Good, 1992; Liu et al., 2014). The blue speckles in Figure 2I are concentrated iron deposits in the extracellular matrix, and the blue ovals are iron-loaded immune cells (Kristiansen et al., 2001; Soares and Hamza, 2016). Levels of myoglobin and hemoglobin were also elevated in mPI compared to CTX injury (Figure 2—figure supplement 2; p=0.0091 for myoglobin and p=0.0474 for hemoglobin). Heme oxygenase-1 (HO-1) is an enzyme that performs heme degradation and serves as a marker of high heme or iron. HO-1 was expressed by mPI wound tissues, at similar levels to CTX-injured tissue (Figure 2K–M; p=0.998). However, HO-1 expression was localized to the panniculus layer after CTX injury, but it was widespread across all layers after mPI (Figure 2K–L). Levels of hemopexin and haptoglobin (iron detoxification factors) are shown in Figure 2—figure supplement 2. Myoglobin knockout mPI have less tissue death and greater immune infiltrate To measure the contribution of myoglobin iron to mPI pathogenesis, we developed myoglobin knockout mice (Mb−/−) via CRISPR deletion of the entire gene from the germline. Note that prior studies of adult Mb−/− mice found no obvious phenotype (Garry et al., 1998; Gödecke et al., 1999; Meeson et al., 2001). Mb−/− is often lethal to cardiac development during E9.5-E10.5, but some Mb−/− embryos survive to term (Meeson et al., 2001). Among our Mb−/− that were born, all developed with normal feeding, weight gain, grooming, social behavior, and life span. Deletion of Mb was confirmed by western blotting (Figure 3—figure supplement 1A), immunostaining (Figure 3—figure supplement 1B and C), and DNA gel electrophoresis (Figure 3—figure supplement 1D). With H&E staining, we detected no knockout-induced changes to the tissues of our injury model (skin, panniculus carnosus layer, or loose areolar tissue) other than increased capillary density (by 17%, p=0.0455; Figure 3—figure supplement 1E) and increased thickness of the dWAT layer in Mb−/− mice (by 43%, p=0.0232, Figure 3—figure supplement 1E). Total iron was not significantly decreased (p=0.0664, Figure 3—figure supplement 1F). We compared pressure ulcers in Mb−/− versus Mb+/+ mice (Figure 3—figure supplement 1G) using elderly 20-month-old animals. (The mPI in elderly were similar to mPI in young, except with milder increases in pressure-induced oxidative damage, Figure 3—figure supplement 2.) At day 3 post-injury, Mb+/+ mice had high levels of iron (Figure 3A), which appeared in the muscle, dWAT, and dermis, including both the extracellular space and in the small numbers of infiltrating immune cells. In contrast, Mb−/− mice had no detectable signal from Perls' stain in any layer of the wound (Figure 3B and C; p=0.0020). HO-1 was also decreased by 57% in Mb−/− mPI compared to Mb+/+ (Figure 3D–F; p=0.0438). Levels of immune cell infiltrate were 233% greater in Mb−/− compared to Mb+/+ (Figure 3G–I; p=0.0250). Figure 3 with 2 supplements see all Download asset Open asset Myoglobin knockout decreased iron deposits and tissue death after muscle pressure injury (mPI). (A, B) Perls' Prussian blue iron staining. (A) Note that iron deposits in the extracellular space and in immune cells of Mb+/+ wound tissue, and note that (B) Mb−/− tissues have no iron deposits in extracellular regions. (C) Quantification of Perls' staining. (D, E) Immunostaining for HO-1 in Mb+/+ versus Mb−/− tissues at day 3 after mPI. Note that HO-1 is elevated in all layers of Mb−/− except epidermis. (F) Quantification of HO-1 immunostaining. (G, H) H&E-stained sections of Mb+/+ versus Mb−/− mPI. Paraffin-embedded wound sections derived from elderly Mb-wildtype mice had poor cohesiveness (compared to elderly Mb-knockout or young) and exhibited greater cracking during identical sample handling. (I) Amount of immune infiltrate, quantified by histopathology scoring on a scale of 0–5, performed on day 3 sections. (J) External wound area in Mb+/+ and Mb−/− mice in the initial days following mPI from 12 mm magnets. Statistical analysis compared four wounds from two age- and sex-matched animals using a Student's t test for each day. Consistent with these results, Supplementary file 1C shows additional animals treated with different-sized magnets. (K) Tissue death in the PC muscle layer by histopathology scoring (3 indicates pervasive death). All quantitative data are reported as means ± SD. n = 3 mice. *<0.05, **<0.01. Statistical significance in (C), (F), and (I–K) was computed by unpaired Student's t test. The wound size was smaller in Mb−/− versus Mb+/+ (p=0.0297 at day 3, measured as external area, Figure 3J and Supplementary file 1c). Histopathology scoring in the center of the wound showed 50% decreased tissue death (p=0.0016, Figure 3G, H and K). Oxidative damage was lower in Mb-knockout wounds: DNA damage (8-hydroxy-2'-deoxyguanosine [8-OG]) was decreased by 87% (p=0.0485; Figure 4A–E), and lipid peroxidation (measured using BODIPY 581/591) was decreased by 61% (p=0.0298; Figure 4F–J). BODIPY 581/591 is a fatty acid analogue with specific sensitivity to oxidation (Drummen et al., 2002). When oxidized, its emission fluorescence shifts from 595 nm (red) to a maximal emission at 520 nm (green). The green fluorescence is what is shown in the figure. Similarly, Mb−/− had roughly 56% decrease in CitH3 (p=0.0127; Figure 4K–Q). These improvements in the wound microenvironment extended beyond the muscle layer because Mb+/+ wounds had high levels of BODIPY in the dWAT and dermis, and high levels of CitH3 throughout the wound. An additional measure of oxidative damage, 3-nitrotyrosine, showed the same pattern (56% decrease, p=0.0328; Figure 4—figure supplement 1). In summary, the wounds had less oxidative stress when myoglobin was absent. We also measured the impact of Mb on a monocyte/macrophage cell line in vitro. After pre-incubation with pro-inflammatory stimuli, RAW264.7 cells showed an increase in ROS and decrease in phagocytosis (measured as impaired efferocytosis or clearance of dead cells) in response to Mb treatment (Figure 4—figure supplement 2). A panel of cytokines, chemokines, and growth factors were measured in muscle homogenates of mPI from Mb−/− versus Mb+/+ at day 3 (Supplementary file 1d). There were no significant differences except the knockout wounds had lower levels of CXCL16 (a cytokine associated with lipid peroxidation Ma et al., 2018, Supplementary file 1d; p=0.0110) and higher levels of PAI-1 (also called Serpin E1, a protease inhibitor associated with TGFβ, Supplementary file 1d; p=0.0080). There were no significant differences in total protein between Mb−/− and Mb+/+. Figure 4 with 2 supplements see all Download asset Open asset Myoglobin knockout caused a more hospitable wound environment in muscle pressure injury (mPI). (A–D) Immunostaining of 8- oxaguanine (8-OG; in magenta) in Mb+/+ versus Mb−/− mPI. Nuclei were co-stained blue with DAPI. (B) and (D) are brightfield images of (A) and (C), respectively. (E) Quantification of 8-OG staining. (F–I) BODIPY staining (for lipid peroxidation) in Mb+/+ versus Mb−/−. (G) and (I) are brightfield images of (F) and (H), respectively. (J) Quantification of BODIPY staining. (K–P) Immunostaining for CitH3 (in red) in Mb+/+ versus Mb−/−. (L, O) DNA/nuclei were co-stained blue with DAPI. (M) and (P) are brightfield images of (K) and (N), respectively. (Q) Quantification of CitH3 staining. Scale bars: 50 µm. Blue dashed lines refer to mean fluorescence intensities for uninjured dorsal skinfolds. All quantitative data are reported as means ± SD. n = 3 mice. *<0.05. Statistical significance in (E), (J), and (Q) was computed by unpaired Student's t test. We next sought an orthogonal intervention to test the causal role of myoglobin iron. The FDA-approved iron chelation drug DFO was administered via injection under the dorsal skinfold of 5-month-old mice, starting the morning after pressure induction finished, and repeated twice daily for up 16 d (Figure 5A). DFO- or saline-treated tissues were analyzed at 3, 7, 10, 40, and 90 d (Supplementary file 1e). The same cohort of saline-treated mPI were compared against saline-treated CTX in Figures 1 and 2 (for days 3, 10, and 40 post-injury) and compared against DFO-treated mPI in Figures 5 and 6. Figure 5 with 2 supplements see all Download asset Open asset Deferoxamine (DFO) iron chelation therapy decreased iron deposits at day 3 after muscle pressure injury (mPI). (A) Experimental schedule shows confetti tamoxifen induction of fluorescence, mPI induction, treatment with DFO or saline, and tissue harvest. (B) Diameter of the dead region of the panniculus carnosus (PC) muscle in tissue sections from DFO- versus saline-treated mPI, 3 d post-injury. (C) Perls ' Prussian blue iron staining of DFO-treated wounds at the wound center. (D) Quantification of Perls ' staining, showing comparison against saline-treated mPI from Figure 2I. (E) Immunostaining of HO-1 in DFO-treated mPI. (F) Quantification of HO-1 staining, showing comparison against saline-treated mPI from Figure 2L. (G) H&E-stained sections of DFO-treated mPI at the wound center. (H) Histopathology scoring of immune infiltrate at all layers of the wound center at day 3, comparing DFO-treated versus saline-treated mPI, which appear in Figure 2B and (D). (I) Confirmation that injuries were properly created, according to death of PC tissue at the center of the wound (histopathology scoring where 3 indicates pervasive tissue death). (J) Skin wound area following mPI in 5-month-old Mb+/+ saline and DFO-treated mice, and in 20-month-old Mb+/+ and Mb-/- saline-treated mice. Scale bars: 50 µm. Blue dashed lines refer to histology scores and mean fluorescence intensities for uninjured dorsal skinfolds. All quantitative data are reported as means ± SD. n = 6–7 mice. *<0.05, **<0.01. Statistical significance in (B), (D), (F), and (H–J) was computed by paired Student's t test. Figure 6 with 3 supplements see all Download asset Open asset Deferoxamine (DFO) treatment improved the muscle pressure injury (mPI) microenvironment at early time point (day 3). (A–D) Immunostaining of 8-OG (for DNA damage) in saline-treated versus DFO-treated mPI. Nuclei were stained blue with DAPI. (B) and (D) are brightfield images of (A) and (C), respectively. (E) Quantification of 8-OG. (F–I) BODIPY staining (for lipid peroxidation) and brightfield in saline- versus DFO-treated mPI. (J) Quantification of BODIPY. (K) CitH3 immunostaining (L) with DNA/nuclear co-stain and (M) brightfield in DFO-treated mPI at day 3. (N) Quantification of CitH3 staining in DFO-treated versus the saline-treated mPI, which was analyzed in Figure 2F and G. Scale bars: 50 µm. Blue dashed lines refer to mean fluorescence intensities for uninjured dorsal skinfolds. All quantitative data are reported as means ± SD. n = 6 mice. *<0.05, **<0.01, ***<0.001. Statistical significance in (E), (J), and (N) was computed by paired Student's t test. Effects of iron chelation therapy on secondary progression of the wound Remarkably, DFO treatment caused a decrease in the amount of muscle tissue that died from the initial pressure injury, even though the pressure inductions were identical, and treatments did not begin until 12 hr after the last cycle of pressure. That is, intervention only started after deformation injury and reperfusion injury had already occurred. At day 3 of treatment, DFO-treated wounds had 35% smaller diameter of dead PC tissue (Figure 5B; p=0.0043). This observation of secondary progression confirms a prediction of our prior computational modeling (Jagannathan and Tucker-Kellogg, 2016). We also observed a decrease in apoptotic debris (measured as TUNEL staining, Figure 5—figure supplement 1; p=0.0105), which might reflect a decrease in apoptotic death and/or an increase in phagocytosis/efferocytosis. Eff
Primary images, data and quantification spreadsheets for "Myoglobin-derived iron causes wound enlargement and impaired regeneration in pressure injuries of muscle"
Primary images, data and quantification spreadsheets for "Myoglobin-derived iron causes wound enlargement and impaired regeneration in pressure injuries of muscle"
Summary Insufficient regeneration is implicated in muscle pathologies, but much remains unknown about the regenerative output of individual muscle stem cells, called satellite cells (SCs). Prior work showed that individual SCs contribute to regeneration of more than one muscle fiber (“fiber-crossing”) after full-muscle damage. We investigated whether fiber-crossing also occurred in peripheral regions of a localized muscle injury. To assess fiber-crossing with a minimum number of mice, we used lineage tracing with confetti fluorescence, and developed a novel stochastic modeling method to interpret the ambiguity of multi-color fluorescent lineage tags. Microscopy of the regenerated muscle showed that adjacent fibers often expressed the same-colored tags. Computational analysis concluded that the observed color patches would be extremely unlikely to occur by chance unless SCs contributed myonuclei to multiple adjacent fibers (26-33% of SCs contributing to at most 1-2 additional fibers). Interestingly, these results were similar across the different regions studied, suggesting that severe destruction is not required for fiber-crossing. Our method to assess fiber-crossing may be useful for future study of gene and cell therapies that use fiber-crossing to aid muscle regeneration.
Confetti fluorescence and other multi-color genetic labelling strategies are useful for observing stem cell regeneration and for other problems of cell lineage tracing. One difficulty of such strategies is segmenting the cell boundaries, which is a very different problem from segmenting color images from the real world. This paper addresses the difficulties and presents a superpixel-based framework for segmentation of regenerated muscle fibers in mice.We propose to integrate an edge detector into a superpixel algorithm and customize the method for multi-channel images. The enhanced superpixel method outperforms the original and another advanced superpixel algorithm in terms of both boundary recall and under-segmentation error. Our framework was applied to cross-section and lateral section images of regenerated muscle fibers from confetti-fluorescent mice. Compared with "ground-truth" segmentations, our framework yielded median Dice similarity coefficients of 0.92 and higher.Our segmentation framework is flexible and provides very good segmentations of multi-color muscle fibers. We anticipate our methods will be useful for segmenting a variety of tissues in confetti fluorecent mice and in mice with similar multi-color labels.
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