Cdk5-dependent rapid formation and stabilization of dendritic spines by corticotropin-releasing factor.

The neuropeptide corticotropin-releasing factor (CRF) exerts a pivotal role in modulating neuronal activity in the mammalian brain. The effects of CRF exhibit notable variations, depending on factors such as duration of exposure, concentration, and anatomical location. In the CA1 region of the hippocampus, the impact of CRF is dichotomous: chronic exposure to CRF impairs synapse formation and dendritic integrity, whereas brief exposure enhances synapse formation and plasticity. In the current study, we demonstrate long-term effects of acute CRF on the density and stability of mature mushroom spines ex vivo. We establish that both CRF receptors are present in this hippocampal region, and we pinpoint their precise subcellular localization within synapses by electron microscopy. Furthermore, both in vivo and ex vivo data collectively demonstrate that a transient surge of CRF in the CA1 activates the cyclin-dependent kinase 5 (Cdk5)-pathway. This activation leads to a notable augmentation in CRF-dependent spine formation. Overall, these data suggest that upon acute release of CRF in the CA1-SR synapse, both CRF-Rs can be activated and promote synaptic plasticity via activating different downstream signaling pathways, such as the Cdk5-pathway.

Challenges and advances in optical 3D mesoscale imaging.

Optical mesoscale imaging is a rapidly developing field that allows the visualisation of larger samples than is possible with standard light microscopy, and fills a gap between cell and organism resolution. It spans from advanced fluorescence imaging of micrometric cell clusters to centimetre-size complete organisms. However, with larger volume specimens, new problems arise. Imaging deeper into tissues at high resolution poses challenges ranging from optical distortions to shadowing from opaque structures. This manuscript discusses the latest developments in mesoscale imaging and highlights limitations, namely labelling, clearing, absorption, scattering, and also sample handling. We then focus on approaches that seek to turn mesoscale imaging into a more quantitative technique, analogous to quantitative tomography in medical imaging, highlighting a future role for digital and physical phantoms as well as artificial intelligence.

This review discusses the state of the art of an emerging field called mesoscale imaging. Mesoscale imaging refers to the trend towards imaging ever-larger samples that exceed the classic microscopy domain and is also referred to as 'mesoscopic imaging'. In optical imaging, this refers to objects between the microscopic and macroscopic scale that are imaged with subcellular resolution; in practice, this implies the imaging of objects from millimetre up to cm size with µm or nm resolution. As such, the mesoscopy field spans the boundary between classic 'biological' imaging and preclinical 'biomedical' imaging, typically utilising lower magnification objective lenses with a bigger field of view. We discuss the types of samples currently imaged with examples, and highlight how this type of imaging fills the gap between microscopic and macroscopic imaging, allowing further insight into the organisation of tissues in an organism. We also discuss the challenges of imaging such large samples, from sample handling to labelling and optical phenomena that stand in the way of quantitative imaging. Finally, we put the current state of the art into context within the neighbouring fields and outline future developments, such as the use of 'phantom' test samples and artificial intelligence for image analysis that will underpin the quality of mesoscale imaging.

A workflow for streamlined acquisition and correlation of serial regions of interest in array tomography.

BACKGROUND: Array tomography (AT) is a high-resolution imaging method to resolve fine details at the organelle level and has the advantage that it can provide 3D volumes to show the tissue context. AT can be carried out in a correlative way, combing light and electron microscopy (LM, EM) techniques. However, the correlation between modalities can be a challenge and delineating specific regions of interest in consecutive sections can be time-consuming. Integrated light and electron microscopes (iLEMs) offer the possibility to provide well-correlated images and may pose an ideal solution for correlative AT. Here, we report a workflow to automate navigation between regions of interest. RESULTS: We use a targeted approach that allows imaging specific tissue features, like organelles, cell processes, and nuclei at different scales to enable fast, directly correlated in situ AT using an integrated light and electron microscope (iLEM-AT). Our workflow is based on the detection of section boundaries on an initial transmitted light acquisition that serves as a reference space to compensate for changes in shape between sections, and we apply a stepwise refinement of localizations as the magnification increases from LM to EM. With minimal user interaction, this enables autonomous and speedy acquisition of regions containing cells and cellular organelles of interest correlated across different magnifications for LM and EM modalities, providing a more efficient way to obtain 3D images. We provide a proof of concept of our approach and the developed software tools using both Golgi neuronal impregnation staining and fluorescently labeled protein condensates in cells. CONCLUSIONS: Our method facilitates tracing and reconstructing cellular structures over multiple sections, is targeted at high resolution ILEMs, and can be integrated into existing devices, both commercial and custom-built systems.

Preservation of Fluorescence Signal and Imaging Optimization for Integrated Light and Electron Microscopy.

Life science research often needs to define where molecules are located within the complex environment of a cell or tissue. Genetically encoded fluorescent proteins and or fluorescence affinity-labeling are the go-to methods. Although recent fluorescent microscopy methods can provide localization of fluorescent molecules with relatively high resolution, an ultrastructural context is missing. This is solved by imaging a region of interest with correlative light and electron microscopy (CLEM). We have adopted a protocol that preserves both genetically-encoded and antibody-derived fluorescent signals in resin-embedded cell and tissue samples and provides high-resolution electron microscopy imaging of the same thin section. This method is particularly suitable for dedicated CLEM instruments that combine fluorescence and electron microscopy optics. In addition, we optimized scanning EM imaging parameters for samples of varying thicknesses. These protocols will enable rapid acquisition of CLEM information from samples and can be adapted for three-dimensional EM.

Influence of gap size, screw configuration, and nail materials in the stability of anterograde reamed intramedullary nail in femoral transverse fractures.

Femoral shaft fractures are among the most severe injuries of the skeleton. They are associated with high morbidity and mortality. The most appropriate treatment depending on the type of fracture and location level should be chosen. A finite element model of the femur has been developed, analyzing various types of fractures in the subtrochanteric and diaphyseal supracondylar area, with several gap sizes, being stabilized with a single combination of screws for the intramedullary nail. The mechanical strength of the nail against bending and compression efforts was studied comparing two materials for the nail: stainless steel and titanium alloy. Beside the finite elements (FE) simulations, a clinical follow-up was carried out, considering a sample of 55 patients, 24 males, and 31 females, with mean age of 52.5 years. Localizations of fractures were 22 in the right femur and 33 in the left one, respectively. A good agreement between clinical results and the simulated fractures in terms of gap size was found. Non-comminuted fractures have a mean consolidation time of 4.1 months, which coincides with the appropriate mobility at fracture site obtained in the FE simulations, whereas comminuted fractures have a higher mean consolidation period estimated in 7.1 months, corresponding to the excessive mobility at fracture site obtained by means of FE simulations. The obtained results between both nail materials (stainless steel and titanium alloy) show a higher mobility when using titanium nails, which produce a higher rate of strains at the fracture site, amplitude of micromotions and bigger global movements compared to stainless-steel nails. Steel nails provide stiffer osteosyntheses than the titanium nails. In conclusion, anterograde locked nail is particularly useful in the treatment of a wide range of supracondylar fractures with proximal extension into the femoral diaphysis.

Influence of screw combination and nail materials in the stability of anterograde reamed intramedullary nail in distal femoral fractures.

Intramedullary nailing (IM) is a technique universally accepted to treat femoral diaphyseal fractures. The treatment of fractures located in the distal third remains a controversial issue though. A finite element model of the femur has been developed, analyzing distal fractures with several gap sizes combined with different interlocking combinations of distal screws with one oblique screw proximally to stabilize the intramedullary nail. The mechanical strength of the nail against bending and compression efforts was also studied. Beside the FE simulations, a clinical follow-up of 15 patients, 6 males and 9 females, with mean age of 53.2 years was carried out. Localizations of fractures were 10 in the right femur and 5 in the left femur, respectively. A fairly good correspondence agreement between clinical results and the simulated fractures in terms of gap size was found. Non-comminuted fractures had a mean consolidation time of 20.5 weeks (4.8 months), a tendency corresponding well to the mobility obtained in the FE simulations; Comminuted fractures on the other hand exhibited a higher mean consolidation period of 22.2 weeks (5.2 months) secondary to the excessive mobility at fracture site obtained by means of FE simulations. The best stability at fracture site was found for the system with three distal screws and the system with two distal screws placed medial lateral. The highest leverage of distal screws was obtained maximizing the distance between them and choosing the coronal plane for their orientation. The results obtained with both nail materials (stainless steel and titanium alloy) show a higher mobility when using titanium nails. Steel nails provide stiffer osteosyntheses than the titanium nails. In conclusion, the best screw combination in terms of stability to produce fracture healing and the least difficulties during treatment is the one which had one oblique proximal screw with two distal lateral screw implanted in the coronal plane.

Finite element simulation and clinical follow-up of lumbar spine biomechanics with dynamic fixations.

Arthrodesis is a recommended treatment in advanced stages of degenerative disc disease. Despite dynamic fixations were designed to prevent abnormal motions with better physiological load transmission, improving lumbar pain and reducing stress on adjacent segments, contradictory results have been obtained. This study was designed to compare differences in the biomechanical behaviour between the healthy lumbar spine and the spine with DYNESYS and DIAM fixation, respectively, at L4-L5 level. Behaviour under flexion, extension, lateral bending and axial rotation are compared using healthy lumbar spine as reference. Three 3D finite element models of lumbar spine (healthy, DYNESYS and DIAM implemented, respectively) were developed, together a clinical follow-up of 58 patients operated on for degenerative disc disease. DYNESYS produced higher variations of motion with a maximum value for lateral bending, decreasing intradiscal pressure and facet joint forces at instrumented level, whereas screw insertion zones concentrated stress. DIAM increased movement during flexion, decreased it in another three movements, and produced stress concentration at the apophyses at instrumented level. Dynamic systems, used as single systems without vertebral fusion, could be a good alternative to degenerative disc disease for grade II and grade III of Pfirrmann.

Comparative Analysis of the Biomechanical Behaviour of Two Cementless Short Stems for Hip Replacement: Linea Anatomic and Minihip.

A comparative study between two stems (Linea Anatomic and Minihip) has been performed in order to analyse the differences in their biomechanical behaviour, concerning stem micromotions and load transmission between stem and bone. From the corresponding finite element models, a parametric study was carried out to quantify ranges of micromotions taking into account: friction coefficient in the stem-bone interface, press-fit and two types of gait cycle. Micromotions were evaluated for each stem at six different levels along repeated gait cycles. An initial and marked stem subsidence at the beginning of the simulation was observed, followed by an asymptotic decrease due to friction forces. Once migration occurs, a repeated reversible cyclic micromotion is developed and stabilized as gait cycle times are simulated. The general motion pattern exhibited higher amplitude of micromotion for Minihip compared to Linea stem. The load transmission mechanism was analyzed, identifying the main internal forces. The results show higher local forces for Minihip stem up to 80% greater than for Linea stem. The differences of design between Minihip and Linea conditioned different distributions of load, influencing the posterior stress-shielding. Consequently, short stems require high bone stock and quality should, being indicated for young patients with high bone quality.

Cementless hydroxyapatite coated hip prostheses.

More than twenty years ago, hydroxyapatite (HA), calcium phosphate ceramics, was introduced as a coating for cementless hip prostheses. The choice of this ceramic is due to its composition being similar to organic apatite bone crystals. This ceramic is biocompatible, bioactive, and osteoconductive. These qualities facilitate the primary stability and osseointegration of implants. Our surgical experience includes the implantation of more than 4,000 cementless hydroxyapatite coated hip prostheses since 1990. The models implanted are coated with HA in the acetabulum and in the metaphyseal area of the stem. The results corresponding to survival and stability of implants were very satisfactory in the long-term. From our experience, HA-coated hip implants are a reliable alternative which can achieve long term survival, provided that certain requirements are met: good design selection, sound choice of bearing surfaces based on patient life expectancy, meticulous surgical technique, and indications based on adequate bone quality.

Study of the polycarbonate-urethane/metal contact in different positions during gait cycle.

Nowadays, a growing number of young and more active patients receive hip replacement. More strenuous activities in such patients involve higher friction and wear rates, with friction on the bearing surface being crucial to ensure arthroplasty survival in the long term. Over the last years, the polycarbonate-urethane has offered a feasible alternative to conventional bearings. A finite element model of a healthy hip joint was developed and adjusted to three gait phases (heel strike, mid-stance, and toe-off), serving as a benchmark for the assessment of the results of joint replacement model. Three equivalent models were made with the polycarbonate-urethane Tribofit system implanted, one for each of the three gait phases, after reproducing a virtual surgery over the respective healthy models. Standard body-weight loads were considered: 230% body-weight toe-off, 275% body-weight mid-stance, and 350% body-weight heel strike. Contact pressures were obtained for the different models. When comparing the results corresponding to the healthy model to polycarbonate-urethane joint, contact areas are similar and so contact pressures are within a narrower value range. In conclusion, polycarbonate-urethane characteristics are similar to those of the joint cartilage. So, it is a favorable alternative to traditional bearing surfaces in total hip arthroplasty, especially in young patients.

Mid-term study of bone remodeling after femoral cemented stem implantation: comparison between DXA and finite element simulation.

This five-year prospective study was designed to investigate periprosthetic bone remodeling associated with two cemented stem models, ABG-II (Stryker) and VerSys (Zimmer), randomly implanted in patients older than 75 years. The sample consisted of 64 cases (32, ABG-II; 32, VerSys). Inclusion criterion was diagnosis of osteoarthritis recommended for cemented total hip arthroplasty. Besides clinical study, Finite Element (FE) simulation was used to analyze biomechanical changes caused by hip arthroplasty. Bone Mineral Density (BMD) measurements showed a progressive increase in bone mass throughout the entire follow-up period for both stems, well correlated with FE results except in Gruen zones 4, 5, 6 for ABG-II and in zones 4, 5 for VerSys, denoting that remodeling in those zones does not depend on mechanical factors but rather on biological or physiological ones.