Micro- and Macroscale Assessment of Posterior Cruciate Ligament Functionality Based on Advanced MRI Techniques

T2 mapping assesses tissue ultrastructure and composition, yet the association of imaging features and tissue functionality is oftentimes unclear. This study aimed to elucidate this association for the posterior cruciate ligament (PCL) across the micro- and macroscale and as a function of loading. Ten human cadaveric knee joints were imaged using a clinical 3.0T scanner and high-resolution morphologic and T2 mapping sequences. Emulating the posterior drawer test, the joints were imaged in the unloaded (δ0) and loaded (δ1) configurations. For the entire PCL, its subregions, and its osseous insertion sites, loading-induced changes were parameterized as summary statistics and texture variables, i.e., entropy, homogeneity, contrast, and variance. Histology confirmed structural integrity. Statistical analysis was based on parametric and non-parametric tests. Mean PCL length (37.8 ± 1.8 mm [δ0]; 44.0 ± 1.6 mm [δ1] [p < 0.01]), mean T2 (35.5 ± 2.0 ms [δ0]; 37.9 ± 1.3 ms [δ1] [p = 0.01]), and mean contrast values (4.0 ± 0.6 [δ0]; 4.9 ± 0.9 [δ1] [p = 0.01]) increased significantly under loading. Other texture features or ligamentous, osseous, and meniscal structures remained unaltered. Beyond providing normative T2 values across various scales and configurations, this study suggests that ligaments can be imaged morphologically and functionally based on joint loading and advanced MRI acquisition and post-processing techniques to assess ligament integrity and functionality in variable diagnostic contexts.

Preparation of large-scale digitization samples for automated electron microscopy of tissue and cell ultrastructure

Manual selection of targets in experimental or diagnostic samples by transmission electron microscopy (TEM), based on single overview and detail micrographs, has been time- consuming and susceptible to bias. Substantial information and throughput gain may now be achieved by automated acquisition of virtually all structures in a given EM section. Resulting datasets allow convenient pan-and-zoom examination of tissue ultrastructure with preserved microanatomical orientation. The technique is, however, critically sensitive to artifacts in sample preparation. We therefore established a methodology to prepare large-scale digitization samples (LDS) designed to acquire entire sections free of obscuring flaws. For evaluation, we highlight the supreme performance of scanning EM in transmission mode compared to other EM technology. The use of LDS will substantially facilitate access to EM data for a broad range of applications.

Visualizing cellular and tissue ultrastructure using Ten-fold Robust Expansion Microscopy (TREx)

ABSTRACTExpansion microscopy (ExM) is a powerful technique to overcome the diffraction limit of light microscopy that can be applied in both tissues and cells. In ExM, samples are embedded in a swellable polymer gel to physically expand the sample and isotropically increase resolution in x, y and z. The maximum resolution increase is limited by the expansion factor of the polymer gel, which is four-fold for the original ExM protocol. Variations on the original ExM method have been reported that allow for greater expansion factors, for example using iterative expansion, but at the cost of ease of adoption or versatility. Here, we systematically explore the ExM recipe space and present a novel method termed Ten-fold Robust Expansion Microscopy (TREx) that, like the original ExM method, requires no specialized equipment or procedures to carry out. We demonstrate that TREx gels expand ten-fold, can be handled easily, and can be applied to both thick tissue sections and cells enabling high-resolution subcellular imaging in a single expansion step. We show that applying TREx on antibody-stained samples can be combined with off-the-shelf small molecule stains for both total protein and membranes to provide ultrastructural context to subcellular protein localization.

Expression of a novel surfactant protein gene is associated with sites of extrapulmonary respiration in a lungless salamander

Numerous physiological and morphological adaptations were achieved during the transition to lungless respiration that accompanied evolutionary lung loss in plethodontid salamanders, including those that enable efficient gas exchange across extrapulmonary tissue. However, the molecular basis of these adaptations is unknown. Here, we show that lungless salamanders express in the larval integument and the adult buccopharynx—principal sites of respiratory gas exchange in these species—a novel paralogue of the gene surfactant-associated protein C ( SFTPC ), which is a critical component of pulmonary surfactant expressed exclusively in the lung in other vertebrates. The paralogous gene appears to be found only in salamanders, but, similar to SFTPC , in lunged salamanders it is expressed only in the lung. This heterotopic gene expression, combined with predictions from structural modelling and respiratory tissue ultrastructure, suggests that lungless salamanders may produce pulmonary surfactant-like secretions outside the lungs and that the novel paralogue of SFTPC might facilitate extrapulmonary respiration in the absence of lungs. Heterotopic expression of the SFTPC paralogue may have contributed to the remarkable evolutionary radiation of lungless salamanders, which account for more than two thirds of urodele species alive today.

A novel surfactant protein is associated with extrapulmonary respiration in lungless salamanders

Numerous physiological and morphological adaptations were achieved during the transition to lungless respiration following evolutionary lung loss in plethodontid salamanders, including those that enable efficient gas exchange across extrapulmonary tissue. However, the molecular basis of these adaptations is unknown. Here we show that lungless salamanders express in the skin and buccal cavity—the principal sites of respiratory gas exchange in these species—a novel paralog of the gene Surfactant-Associated Protein C (SFTPC), which is a critical component of pulmonary surfactant expressed exclusively in the lung in other vertebrates. The paralogous gene appears to be found only in salamanders, but, similar to SFTPC, in lunged salamanders it is expressed only in the lung. This heterotopic gene expression, combined with predictions from structural modeling and respiratory tissue ultrastructure, suggest that lungless salamanders produce pulmonary surfactant-like secretions outside the lungs and that the novel paralog of SFTPC might facilitate extrapulmonary respiration in the absence of lungs. Heterotopic expression of the SFTPC paralog may have contributed to the remarkable evolutionary radiation of lungless salamanders, which account for more than two thirds of urodele species alive today.