Limbostomy: Longitudinal Intravital Microendoscopy in Murine Osteotomies

Stefanowski, Jonathan; Fiedler, Alexander; Köhler, M; Günther, Robert; Liublin, Wjatscheslaw; Tschaikner, Martin; Rauch, Ariana; Reismann, David; Matthys, Romano; Nützi, Reto; Bixel, M. Gabriele; Adams, Ralf H.; Niesner, Raluca; Duda, Georg N.; Hauser, Anja E.

doi:10.1002/cyto.a.23997

Cited by 10 publications

(7 citation statements)

References 56 publications

(102 reference statements)

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“…To give insight into the sequence of events contributing to these long-term processes spatio-temporal studies are of particular importance. Intravital two-photon microscopy (2PM) allowed us to analyze motility patterns of immune cells and their interactions with the environment, in murine marrow of flat bones 21 , of intact long bones of young or irradiated mice 10,11 , after surgical thinning of bone cortex in adult mice 5,15,60 , or by inserting micro-endoscopic lenses into the marrow cavity 24,61 . However, minimally invasive technologies for long bone imaging are still needed.…”

Section: Discussionmentioning

confidence: 99%

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In vivooptimized three-photon imaging of intact mouse tibia links plasma cell motility to functional states in the bone marrow

Rakhymzhan,

Fiedler,

Günther

et al. 2023

Preprint

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Intravital multi-photon imaging of the bone marrow is crucial to the study of cellular dynamics, communication with the microenvironment and functions, however, imaging of deep tissue areas is challenging and minimally invasive methods for deep-marrow imaging in intact long bones are needed. We developed a high pulse energy 1650 nm laser prototype, which permits to surpass >100 µm thick cortical bone and to perform three-photon microscopy (3PM) in more than 400 µm depth in the marrow cavity of intact mouse tibiain vivo. Its unique 3 and 4 MHz laser repetition rates allowed us to analyze motility patterns of rare cells over large fields of view deep within the unperturbed marrow. In this way, we found a bi-modal migratory behavior of marrow plasma cells. Besides, the analysis of third harmonics generation (THG) in the tibia identified this signal to be a label-free indicator of the abundance of cellular organelles, in particular the endoplasmic reticulum, reflecting protein biosynthesis capacity. We found that only one third of the plasma cells in the tibia marrow of adult mice have a strong THG signal and, thus, a high protein synthesis capacity, while the other two thirds of plasma cells display a low THG signal. Finally, we identified an inverse link between migratory behavior and THG signal strength in marrow plasma cells. As in these cells, the protein biosynthesis capacity indicated by a strong THG signal is mainly associated with antibody secretion, we could relate motility to functional states of plasma cellsin vivo. Our 3PM method retains the ability to connect cellular dynamics to protein biosynthesis capacity in various marrow cell types beyond plasma cells, as THG is a ubiquitous signal, opening new perspectives on understanding how tissue microenvironment impacts on cellular functions in the bone marrow.

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Section: Discussionmentioning

confidence: 99%

“…The CD19:tdRFP B lymphocyte fate mapping mice 11 , Prx1:tdRFP mesenchymal stromal cells fate mapping mice 77 and Cdh5:tdTomato x Histone:GFP (Cdh5:tdTom) fate mapping mice 61,78 (kind gift of Prof. R. Adams, Dr. M.G. Bixel) used for in vivo imaging were typically 12-24 weeks old females and males.…”

Section: Methodsmentioning

confidence: 99%

In vivooptimized three-photon imaging of intact mouse tibia links plasma cell motility to functional states in the bone marrow

Rakhymzhan,

Fiedler,

Günther

et al. 2023

Preprint

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“…We confirmed the compatibility of MarShie with the preservation of various fluorescent signal types. We used CX 3 CR1-GFP x Cdh5-tdTomato/histone-GFP reporter mice that express GFP in CX 3 CR1 + cells, spatially separated from histone-linked GFP in nuclei of the endothelial cells, and tdTomato (tdTom) in endothelial cell membranes via Cdh5linkage 26,27 . Fluorescent labeling for LSFM included (i) endogenous GFP, expressed in CX 3 CR1 + myeloid cells and histone-linked GFP in endothelial nuclei (ii) the red fluorescent protein tdTom driven by the Cadherin 5 (Cdh5) promoter (iii) a whole-mount stained signal using nanobodies against GFP, conjugated to Atto-647N, and (iv) intravenously injected antibodies against CD31, coupled to Alexa647 (Al647).…”

Section: Marshie Preserves Various Fluorescence Signals Throughout Th...mentioning

confidence: 99%

MarShie: a clearing protocol for 3D analysis of single cells throughout the bone marrow at subcellular resolution

Mertens,

Liebheit,

Ehl

et al. 2024

Nat Commun

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Analyzing immune cell interactions in the bone marrow is vital for understanding hematopoiesis and bone homeostasis. Three-dimensional analysis of the complete, intact bone marrow within the cortex of whole long bones remains a challenge, especially at subcellular resolution. We present a method that stabilizes the marrow and provides subcellular resolution of fluorescent signals throughout the murine femur, enabling identification and spatial characterization of hematopoietic and stromal cell subsets. By combining a pre-processing algorithm for stripe artifact removal with a machine-learning approach, we demonstrate reliable cell segmentation down to the deepest bone marrow regions. This reveals age-related changes in the marrow. It highlights the interaction between CX3CR1+ cells and the vascular system in homeostasis, in contrast to other myeloid cell types, and reveals their spatial characteristics after injury. The broad applicability of this method will contribute to a better understanding of bone marrow biology.

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“…During this healing cascade, immune cells play a key role [ 9 , 10 , 11 ]. They are among the first cells infiltrating a fracture zone after injury [ 12 , 13 ]. During the early healing phases, immune cells are responsible, among other aspects, for the clearance of cell debris (e.g., macrophages) and, by secreting signaling molecules, for the recruitment of all other cells necessary for the successful progression of healing [ 14 ].…”

Section: Introductionmentioning

confidence: 99%

Complex Spatio-Temporal Interplay of Distinct Immune and Bone Cell Subsets during Bone Fracture Healing

Schlundt,

Saß,

Bucher

et al. 2023

Cells

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Background: The healing of a bone injury is a highly complex process involving a multitude of different tissue and cell types, including immune cells, which play a major role in the initiation and progression of bone regeneration. Methods: We histologically analyzed the spatio-temporal occurrence of cells of the innate immune system (macrophages), the adaptive immune system (B and T lymphocytes), and bone cells (osteoblasts and osteoclasts) in the fracture area of a femoral osteotomy over the healing time. This study was performed in a bone osteotomy gap mouse model. We also investigated two key challenges of successful bone regeneration: hypoxia and revascularization. Results: Macrophages were present in and around the fracture gap throughout the entire healing period. The switch from initially pro-inflammatory M1 macrophages to the anti-inflammatory M2 phenotype coincided with the revascularization as well as the appearance of osteoblasts in the fracture area. This indicates that M2 macrophages are necessary for the restoration of vessels and that they also play an orchestrating role in osteoblastogenesis during bone healing. The presence of adaptive immune cells throughout the healing process emphasizes their essential role for regenerative processes that exceeds a mere pathogen defense. B and T cells co-localize consistently with bone cells throughout the healing process, consolidating their crucial role in guiding bone formation. These histological data provide, for the first time, comprehensive information about the complex interrelationships of the cellular network during the entire bone healing process in one standardized set up. With this, an overall picture of the spatio-temporal interplay of cellular key players in a bone healing scenario has been created. Conclusions: A spatio-temporal distribution of immune cells, bone cells, and factors driving bone healing at time points that are decisive for this process—especially during the initial steps of inflammation and revascularization, as well as the soft and hard callus phases—has been visualized. The results show that the bone healing cascade does not consist of five distinct, consecutive phases but is a rather complex interrelated and continuous process of events, especially at the onset of healing.

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Limbostomy: Longitudinal Intravital Microendoscopy in Murine Osteotomies

Cited by 10 publications

References 56 publications

In vivooptimized three-photon imaging of intact mouse tibia links plasma cell motility to functional states in the bone marrow

In vivooptimized three-photon imaging of intact mouse tibia links plasma cell motility to functional states in the bone marrow

MarShie: a clearing protocol for 3D analysis of single cells throughout the bone marrow at subcellular resolution

Complex Spatio-Temporal Interplay of Distinct Immune and Bone Cell Subsets during Bone Fracture Healing

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