In vivo multiphoton microscopy detects longitudinal metabolic changes associated with delayed skin wound healing

Jones, Jake D.; Ramser, Hallie E.; Woessner, Alan E.; Quinn, Kyle P.

doi:10.1038/s42003-018-0206-4

Cited by 64 publications

(73 citation statements)

References 64 publications

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“…Lifetime measurements can distinguish between free and protein-bound forms of NAD(P)H and FAD, characterized by distinct molecular conformations that affect fluorescence quenching 49 . Previous studies have shown that metabolic autofluorescence imaging detects spatial and temporal changes in stromal cells across in vivo and 3D in vitro models 31,38,39,42,[50][51][52][53][54][55] . Thus, metabolic autofluorescence imaging of microscale 3D models was demonstrated to quantify metabolic activity and visualize macrophage heterogeneity within the 3D TME using primary human cancer, human cell lines, and mouse cell lines…”

Section: Introductionmentioning

confidence: 99%

Autofluorescence imaging of 3D tumor-macrophage microscale cultures resolves spatial and temporal dynamics of macrophage metabolism

Heaster

Humayun

et al. 2020

Preprint

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Macrophages within the tumor microenvironment (TME) exhibit a spectrum of pro-tumor and antitumor functions, yet it is unclear how the TME regulates this macrophage heterogeneity. Standard methods to measure macrophage heterogeneity require destructive processing, limiting spatiotemporal studies of function within the live, intact 3D TME. Here, we demonstrate twophoton autofluorescence imaging of NAD(P)H and FAD to non-destructively resolve spatiotemporal metabolic heterogeneity of individual macrophages within 3D microscale TME models. Fluorescence lifetimes and intensities of NAD(P)H and FAD were acquired at 24, 48, and 72 hours post-stimulation for mouse macrophages (RAW 264.7) stimulated with IFN-γ or IL-4 plus IL-13 in 2D culture, validating that autofluorescence measurements capture known metabolic phenotypes. To quantify metabolic dynamics of macrophages within the TME, mouse macrophages or human monocytes (RAW264.7 or THP-1) were cultured alone or with breast cancer cells (mouse PyVMT or primary human IDC) in 3D microfluidic platforms. Human monocytes and mouse macrophages in tumor co-cultures exhibited significantly different FAD mean lifetimes and greater migration than mono-cultures at 24, 48, and 72 hours post-seeding. In co-cultures with primary human cancer cells, actively-migrating monocyte-derived macrophages had greater redox ratios (NAD(P)H/FAD intensity) compared to passively-migrating monocytes at 24 and 48 hours post-seeding, reflecting metabolic heterogeneity in this subpopulation of monocytes. Genetic analyses further confirmed this metabolic heterogeneity. These results establish label-free autofluorescence imaging to quantify dynamic metabolism, polarization, and migration of macrophages at single-cell resolution within 3D microscale models. This combined culture and imaging system provides unique insights into spatiotemporal tumorimmune crosstalk within the 3D TME.

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Section: Introductionmentioning

confidence: 99%

Autofluorescence imaging of 3D tumor-macrophage microscale cultures resolves spatial and temporal dynamics of macrophage metabolism

Heaster

Humayun

et al. 2020

Preprint

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“…Measurement of intrinsic lifetime of NADH and FAD is emerging as a reliable technique for assessing cellular metabolism parameters in vitro 40 . NADH FLIM also enabled analysis of pharmacologically-induced alterations to mitochondrial metabolic processes from baseline cerebral metabolism in rat cerebral cortex 37 and metabolic changes at the front of wound re-epithelialization in diabetic and control mice 49 . This prompted us to apply protocol for measuring the spatial distribution of NADH in experimental models of tissue repair, as well as of cancer.…”

Section: Discussionmentioning

confidence: 99%

Evaluation of cell metabolic adaptation in wound and tumour by Fluorescence Lifetime Imaging Microscopy

Morone

Autilia

Schorn

et al. 2020

Sci Rep

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Acidic pH occurs in acute wounds progressing to healing as consequence of a cell metabolic adaptation in response to injury-induced tissue hypoperfusion. In tumours, high metabolic rate leads to acidosis affecting cancer progression. Acidic pH affects activities of remodelling cells in vitro. the pH measurement predicts healing in pathological wounds and success of surgical treatment of burns and chronic ulcers. However, current methods are limited to skin surface or based on detection of fluorescence intensity of specific sensitive probes that suffer of microenvironment factors. Herein, we ascertained relevance in vivo of cell metabolic adaptation in skin repair by interfering with anaerobic glycolysis. Moreover, a custom-designed skin imaging chamber, 2-Photon microscopy (2PM), fluorescence lifetime imaging (FLIM) and data mapping analyses were used to correlate maps of glycolytic activity in vivo as measurement of nADH intrinsic lifetime with areas of hypoxia and acidification in models of skin injury and cancer. The method was challenged by measuring the NADH profile by interfering with anaerobic glycolysis and oxidative phosphorylation in the mitochondrial respiratory chain. Therefore, intravital NADH FLIM represents a tool for investigating cell metabolic adaptation occurring in wounds, as well as the relationship between cell metabolism and cancer. In normal skin, dermis has an extracellular neutral-alkaline pH 1,2. After tissue damage, cells involved in tissue repair undergo a metabolic adaptation resulting from low oxygen tension towards a less energy-efficient process of anaerobic metabolism that leads to microenvironment acidosis 2,3. In vitro evidence suggests a beneficial effect of acidic pH in several processes implicated in wound healing, which involve cell adhesion 4,5 , migration and proliferation 6. In a previous report 7 , we described an unprecedented role of tissue acidification in setting PTX3, a key component of the humoral arm of innate immunity, in a tissue remodelling and repair mode. In wounds, reduced oxygen tension 8 and factors downstream of cell anaerobic glycolysis, such as lactate 9,10 , effectively stimulate immune and vascular endothelial cells to release factors that support angiogenesis. Subsequent neovascularization allows restoration of nutrient delivery and oxygen, and cells use oxidative metabolism for their longer-term functions contributing to restore the wound pH to values near to neutral 2,3. While an acidic pH occurs in the inflammatory phases of acute wounds that progress on healing, chronic and highly infected wounds are characterized by abundant recruitment of neutrophils and a non-acidic pH 11,12. Chronic non-healing wounds may occur secondarily to a high alkaline pH 11,13 , and studies report a relationship between wound pH and chronic wound healing 14-16. The effect of acidic pH in the wound bed has a clinical functional relevance on the healing of chronic wounds 11. In fact, a prolonged chemical acidification of the wound bed increases the healing rate in chron...

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“…All tissue samples, including the biopsied tissue at day 0, were frozen in OCT at −80°C. The frozen wound tissue were sectioned into 30 μm thick samples, transferred to glass slides, and stained with H&E …”

Section: Methodsmentioning

confidence: 99%

“…On either day 3, 5, or 10 post-wounding, approximately 1 cm 2 of skin surrounding the wound was excised from each mouse following euthanasia. All tissue samples, including the biopsied tissue at day 0, were frozen in OCT at −80 C. The frozen wound tissue were sectioned into 30 μm thick samples, transferred to glass slides, and stained with H&E. 13…”

Section: Sample Preparationmentioning

confidence: 99%

Characterizing differences in the collagen fiber organization of skin wounds using quantitative polarized light imaging

Woessner

McGee

Jones

et al. 2019

Wound Repair Regeneration

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Collagen fiber organization requires characterization in many biomedical applications, but it is difficult to objectively quantify in standard histology tissue sections. Quantitative polarized light imaging is a low‐cost technique that allows for rapid measurement of collagen fiber orientation and thickness. In this study, we utilize a quantitative polarized light imaging system to characterize fiber orientation and thickness from wound sections. Full thickness skin wound sections that were previously stained with hematoxylin and eosin were used to assess collagen fiber content and organization at different points during the wound healing process. Overall, wounds exhibited a measurable increase in collagen fiber thickness and a nonlinear change in fiber reorganization within the wound. Our study demonstrates that quantitative polarized light imaging is an inexpensive alternative or supplement to standard histology protocols, requiring no additional stains or dyes, and yields repeatable quantitative assessments of collagen organization.

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In vivo multiphoton microscopy detects longitudinal metabolic changes associated with delayed skin wound healing

Cited by 64 publications

References 64 publications

Autofluorescence imaging of 3D tumor-macrophage microscale cultures resolves spatial and temporal dynamics of macrophage metabolism

Autofluorescence imaging of 3D tumor-macrophage microscale cultures resolves spatial and temporal dynamics of macrophage metabolism

Evaluation of cell metabolic adaptation in wound and tumour by Fluorescence Lifetime Imaging Microscopy

Characterizing differences in the collagen fiber organization of skin wounds using quantitative polarized light imaging

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