Zahra Ayar

In the mammalian central nervous system (CNS), the ability to regenerate after injury drops sharply in the first weeks after birth, but how this change is reflected in cell morphology and organization is still not fully understood. Here we cultivated cortical cell cultures from the gray short-tailed opossum (Monodelphis domestica) and compared cells derived from early postnatal pups (postnatal day 3-6, "Pyoung") with those from older pups (postnatal day 16-18, "Pold"), which differ in their capacity to regenerate after injury. For each culture, we assessed marker-defined cellular composition using fluorescently labeled markers for neurons, glia, and immature/progenitor cells, and characterized their nanoscale-resolved three-dimensional morphology using label-free scanning ion conductance microscopy (SICM). Pyoung cultures exhibited active change in shape over time, contained a higher proportion of immature SOX2⁺ cells, and grew more neurites (TUJ1⁺) into the lesion site after injury. Moreover, Pold cultures showed more stable soma size (e.g., thickness, area, volume), a larger astroglial (SOX9⁺) population early in culture, and a glia-dominated lesion response. These early astroglia included more immature or reactive (vimentin⁺) astrocytes. SICM further revealed cellular bridges across the lesion, ridges left in the substrate by the injury, and aligned neurite outgrowth. Across all measurements, changes in molecular markers were associated with corresponding changes in cell morphology. This multimodal combination of fluorescence labeling and SICM therefore relates marker-defined cellular composition to physical organization across micro- and nanoscale features during and after postnatal in vitro development.

Biomolecular condensates are important regulators of cellular compartmentalization and biochemical processes. Understanding their material properties is critical to elucidate how they control molecular organization and dynamics within cells. However, quantitatively probing these properties remains challenging due to the wide range of length scales, concentrations, and timescales over which condensates operate, as well as the limited force ranges accessible to current nanoscale mechanical mapping methods. We explored the use of a non-contact 3D imaging tool Scanning Ion Conductance Microscopy (SICM) for stiffness measurements of liquid-liquid phase-separated biomolecular condensates. We focus on the Dhh1 protein, which is a regulator of cytoplasmic processing bodies (PBs) membrane-less cytoplasmic condensates that control the storage and degradation of untranslated mRNA. In our study, we investigate the properties of mCherry2- or His-mCherry2- tagged full-length Dhh1 and N- or C-terminus tail-deletion constructs, as well as the catalytically inactive mutant DQAD, under different pH and incubation times. We mapped both spatial and temporal changes in the material properties of the condensates, highlighting the capabilities of the instrument. We found that the removal of either of the two tails led to an increase in condensate stiffness upon shifting the pH from a stress-associated cellular environment (pH 6.5) to physiological conditions (pH 7.5). Additionally, the choice of protein tags led to vastly different results depending on the pH where mCherry2-Dhh1 exhibited a stiffening going from pH 6.0 to 6.5 while the double-tagged His-mCherry2 did not. Our measurements are verified and corroborated by established techniques such as optical tweezer-based fusion assays and fluorescence recovery after photobleaching (FRAP). Furthermore, we were able to track the same biomolecular condensate sample for up to 20 days getting insights on the ageing and evolution of the condensates. Overall, our study demonstrates the applicability of SICM for direct measurement of the material properties of biomolecular condensate.

Scanning Ion Conductance Microscopy (SICM) provides high‐resolution, nanoscale imaging of living cells, but it is generally limited by a slow scan rate, making it challenging to capture dynamic processes in real time. To tackle this challenge, an integrated data acquisition and computational framework is proposed that improves the temporal resolution of SICM by selectively skipping certain scan lines. A partial convolutional neural network (Partial‐CNN) model is developed and trained on SICM images and their corresponding masks to reconstruct the complete images from the undersampled data, ensuring the retention of structural integrity. This approach significantly reduces the image acquisition time (i.e., by 30–63%) without compromising quality, as validated through multiple quantitative metrics. Compared to conventional deep learning methods, the Partial‐CNN demonstrates higher accuracy in reconstructing fine details and maintaining consistent height maps across skipped regions. It is shown that this method provides an increased temporal resolution and retains image fidelity, making it suitable for real‐time dynamic SICM imaging and improving the smart scanning microscopy applications in time‐resolved biological imaging.

Scanning Ion Conductance Microscopy (SICM) provides high-resolution, nanoscale imaging of living cells, but it is generally limited by a slow scan rate, making it challenging to capture dynamic processes in real time. To tackle this challenge, we propose an integrated data acquisition and computational framework that improves the temporal resolution of SICM by selectively skipping certain scan lines. A partial convolutional neural network (Partial-CNN) model is developed and trained on SICM images and their corresponding masks to reconstruct the complete images from the under-sampled data, ensuring the retention of structural integrity. This approach significantly reduces the image acquisition time (i.e., by 30-60%) without compromising quality, as validated through multiple quantitative metrics. Compared to conventional deep learning methods, the Partial-CNN demonstrates higher accuracy in reconstructing fine details and maintaining consistent height maps across skipped regions. We show that this method provides an increased temporal resolution and retains image fidelity, making it suitable for real-time dynamic SICM imaging and improving the smart scanning microscopy applications in time-resolved biological imaging. Graphical

Time-lapse light microscopy combined with in vitro neuronal cultures has provided a significant contribution to the field of Developmental Neuroscience. The establishment of the neuronal polarity, i.e., formation of axons and dendrites, key structures responsible for inter-neuronal signaling, was described in 1988 by Dotti, Sullivan and Banker in a milestone paper that continues to be cited 30 years later. In the following decades, numerous fluorescently labeled tags and dyes were developed for live cell imaging, providing tremendous advancements in terms of resolution, acquisition speed and the ability to track specific cell structures. However, long-term recordings with fluorescence-based approaches remain challenging because of light-induced phototoxicity and/or interference of tags with cell physiology (e.g., perturbed cytoskeletal dynamics) resulting in compromised cell viability leading to cell death. Therefore, a label-free approach remains the most desirable method in long-term imaging of living neurons. In this paper we will focus on label-free high-resolution methods that can be successfully used over a prolonged period. We propose novel tools such as scanning ion conductance microscopy (SICM) or digital holography microscopy (DHM) that could provide new insights into live cell dynamics during neuronal development and regeneration after injury.

The loss of neurons is one of the most irreversible phenomena in traumatic injuries to the central nervous system (CNS). One of the recent approaches to replace the lost neurons is delivering stem cells to the lesion area and differentiating them using biodegradable scaffolds. Alginate has been known as an efficient hydrogel for this target. However, its bioactivity needs to be improved to promote cell adhesion/differentiation. In this study, alginate hydrogel was synthesized and optimized regarding mechanical properties of the CNS, and its bioactivity was improved using Schwann cell‐ and fibroblast‐derived extracellular matrix (ECM). The results of immunostaining showed higher differentiation efficacy on ECM‐modified hydrogels than controls. Although secreted protein content by fibroblast was 12.01 ± 2.09 folds higher than Schwann cells, no significant difference was observed between the differentiation potential of ECM‐modified hydrogels. Accordingly, both ECM‐modified hydrogels can be an appropriate scaffold to promote differentiation toward neuron‐like cells and, subsequently, nerve regeneration.

Hydrogels have been used as promising biomaterials for regeneration and control of pathophysiological events after traumatic spinal cord injuries (TSCI). However, no systematic comparison was conducted to show the effect of hydrogels on pathophysiological events. This study was designed to address this issue and evaluate the regenerative potential of hydrogels after TSCI. From 2857 records found in MEDLINE and EMBASE databases (April 23, 2021), 49 articles were included based on our inclusion/exclusion criteria. All studies discussing the effect of hydrogels on at least one of the main pathophysiological events after TSCI, including inflammation, axon growth, remyelination, glial scar formation, cavity size, and locomotor functional recovery were included. For statistical analysis, we used mean difference with 95% confidence intervals for locomotor functional recovery. The results showed that both natural and synthetic hydrogels could reduce the inflammatory response, hinder glial scar formation, and promote axon growth and vascularization. Also, the meta‐analysis of the BBB score showed that using the hydrogels can lead to locomotor functional recovery. It was found that hydrogels are more efficient when used in transection and hemisection injuries (SMD: 1.89; 95% CI: 1.26, 2.52;P < .00001) compared to other injury models. The pre‐formed implanted hydrogels (SMD: 1.79; 95% CI: 1.24, 2.34;P < .00001) found to be more effective compared to injection (SMD: 1.58; 95% CI: 0.64, 2.52;P = 0.0009). In conclusion, based on the available evidence, it was concluded that hydrogel composition as well as implantation method are dominant factors affecting tissue regeneration after TSCI and should be chosen according to the injury model in animal studies.

This study was designed to develop a drug delivery system based on poly(N‐isopropylacrylamide) (pNIPAM) hydrogel and a suitable solvent to enhance solubility and local release of curcumin. pNIPAM hydrogel was synthesized by radical polymerization. The chemical, mechanical and physical properties and biocompatibility of pNIPAM hydrogel were investigated as an implantable and rechargeable drug reservoir. Curcumin was loaded within pNIPAM hydrogel during swelling by using two different solvents; methanol, an organic solvent, and low molecular weight polyethylene glycol (PEG200), a polymeric solvent. The results of drug solubility showed that using PEG200 can increase curcumin solubility more than commonly used organic solvents such as methanol. Also, the release profile of drug‐loaded hydrogels demonstrated that PEG200 has a superior effect on the cumulative amount of released curcumin (33.163 ± 0.319 μg/ml) compared to methanol (8.765 ± 0.544 μg/ml) during 1 week. Based on our results, curcumin‐loaded hydrogels did not show any cytotoxicity, and pNIPAM/PEG combination represented an antibacterial effect within 12 hours. Accordingly, it can be concluded that pNIPAM hydrogel in combination with low molecular weight PEG200 could be used as an efficient drug delivery system to preserve and provide sustained release of curcumin as a hydrophobic drug.

This study was designed to determine the effective therapeutic parameters and evaluate the regenerative potential of low-level laser therapy (LLLT) after traumatic spinal cord injuries (TSCIs) in animal studies. The EMBASE and MEDLINE databases were searched on October 5, 2019, and followed with an update on January 2, 2021. All animal studies discussing the effect of LLLT on main pathophysiological events after TSCI, including inflammation, axon growth, remyelination, glial scar formation, cavity size, and locomotor recovery, were included. For statistical analysis, we used mean difference with 95% confidence intervals for locomotor recovery. In total, 19 articles were included based on our criteria. The results showed that regardless of laser type, laser beams with a wavelength between 600 and 850 nm significantly suppress inflammation and led inflammatory cells to M2 polarization and wound healing. Also, laser therapy using these wavelengths for more than 2 weeks significantly improved axon regeneration and remyelination. Improvement of locomotor recovery was more efficient using wavelengths less than 700 nm (SMD = 1.21; 95%CI: 0.09, 2.33; p = 0.03), lasers with energy densities less than 100 J/cm² (SMD = 1.72; 95%CI: 0.84, 2.59; p = 0.0001) and treatment duration between 1 and 2 weeks (SMD = 2.21; 95%CI: 1.24, 3.19; p < 0.00001). The LLLT showed promising potential to modulate pathophysiological events and recovery after TSCI, although there was heterogeneity in study design and reporting methods, which should be considered in future studies.