Microwave-assisted synthesis of TEMPO-labeled hydrogels traceable with MRI.

Polymer functionalization strategies have recently attracted considerable attention for several applications in biomaterials science. In particular, technological advancements in medical imaging have focused on the design of polymeric matrices to improve non-invasive approaches and diagnostic accuracy. In this scenario, the use of microwave irradiation of aqueous solutions containing appropriate combinations of polymers is gaining increasing interest in the synthesis of sterile hydrogels without using monomers, eliminating the need to remove unreacted species. In this study, we developed a method for the in situ fabrication of TEMPO-labeled hydrogels based on a one-pot microwave reaction that can then be tracked by magnetic resonance imaging (MRI) without using toxic compounds that could be hostile for the target tissue. Click chemistry was used to link TEMPO to the polymeric scaffold. In an in vivo model, the system was able to preserve its TEMPO paramagnetic activity up to 1 month after hydrogel injection, showing a clear detectable signal on T1-weighted MRI with a longitudinal relaxivity value of 0.29 mM s-1, comparable to a value of 0.31 mM s-1 characteristic of TEMPO application. The uncleavable conjugation between the contrast agent and the polymeric scaffold is a leading point to record these results: the use of TEMPO only physically entrapped in the polymeric scaffold did not show MRI traceability even after few hours. Moreover, the use of TEMPO-labeled hydrogels can also help to reduce the number of animals sacrificed being a longitudinal non-invasive technique.

Amyotrophic Lateral Sclerosis, a Multisystem Pathology: Insights into the Role of TNFα.

Amyotrophic lateral sclerosis (ALS) is considered a multifactorial, multisystem disease in which inflammation and the immune system play important roles in development and progression. The pleiotropic cytokine TNFα is one of the major players governing the inflammation in the central nervous system and peripheral districts such as the neuromuscular and immune system. Changes in TNFα levels are reported in blood, cerebrospinal fluid, and nerve tissues of ALS patients and animal models. However, whether they play a detrimental or protective role on the disease progression is still not clear. Our group and others have recently reported opposite involvements of TNFR1 and TNFR2 in motor neuron death. TNFR2 mediates TNFα toxic effects on these neurons presumably through the activation of MAP kinase-related pathways. On the other hand, TNFR2 regulates the function and proliferation of regulatory T cells (Treg) whose expression is inversely correlated with the disease progression rate in ALS patients. In addition, TNFα is considered a procachectic factor with a direct catabolic effect on skeletal muscles, causing wasting. We review and discuss the role of TNFα in ALS in the light of its multisystem nature.

Biomaterials and Cell Therapy Combination in Central Nervous System Treatments.

Current pharmacological and surgical therapies for the central nervous system (CNS) show a limited capacity to reduce the damage progression; that together with the intrinsic limited capability of the CNS to regenerate greatly reduces the hopes of recovery. Among all the therapies proposed, the tissue engineering strategies supplemented with therapeutic stem cells remain the most promising. Neural tissue engineering strategies are based on the development of devices presenting optimal physical, chemical, and mechanical properties which, once inserted in the injured site, can support therapeutic cells, limiting the effect of a hostile environment and supporting regenerative processes. Thus, this review focuses on the employment of hydrogel and nanofibrous scaffolds supplemented with stem cells as promising therapeutic tools for the central and peripheral nervous systems in preclinical and clinical applications.

Targeted therapy and deep learning insights into microglia modulation for spinal cord injury.

Spinal cord injury (SCI) is a devastating condition that can cause significant motor and sensory impairment. Microglia, the central nervous system's immune sentinels, are known to be promising therapeutic targets in both SCI and neurodegenerative diseases. The most effective way to deliver medications and control microglial inflammation is through nanovectors; however, because of the variability in microglial morphology and the lack of standardized techniques, it is still difficult to precisely measure their activation in preclinical models. This problem is especially important in SCI, where the intricacy of the glia response following traumatic events necessitates the use of a sophisticated method to automatically discern between various microglial cell activation states that vary over time and space as the secondary injury progresses. We address this issue by proposing a deep learning-based technique for quantifying microglial activation following drug-loaded nanovector treatment in a preclinical SCI model. Our method uses a convolutional neural network to segment and classify microglia based on morphological characteristics. Our approach's accuracy and efficiency are demonstrated through evaluation on a collection of histology pictures from injured and intact spinal cords. This robust computational technique has potential for analyzing microglial activation across various neuropathologies and demonstrating the usefulness of nanovectors in modifying microglia in SCI and other neurological disorders. It has the ability to speed development in this crucial sector by providing a standardized and objective way to compare therapeutic options.

Corrigendum to targeted therapy and deep learning insights into microglia modulation for spinal cord injury.

[This corrects the article DOI: 10.1016/j.mtbio.2024.101117.].

Synergistic Pharmacological Therapy to Modulate Glial Cells in Spinal Cord Injury.

Current treatments for modulating the glial-mediated inflammatory response after spinal cord injury (SCI) have limited ability to improve recovery. This is quite likely due to the lack of a selective therapeutic approach acting on microgliosis and astrocytosis, the glia components most involved after trauma, while maximizing efficacy and minimizing side effects. A new nanogel that can selectively release active compounds in microglial cells and astrocytes is developed and characterized. The degree of selectivity and subcellular distribution of the nanogel is evaluated by applying an innovative super-resolution microscopy technique, expansion microscopy. Two different administration schemes are then tested in a SCI mouse model: in an early phase, the nanogel loaded with Rolipram, an anti-inflammatory drug, achieves significant improvement in the animal's motor performance due to the increased recruitment of microglia and macrophages that are able to localize the lesion. Treatment in the late phase, however, gives opposite results, with worse motor recovery because of the widespread degeneration. These findings demonstrate that the nanovector can be selective and functional in the treatment of the glial component in different phases of SCI. They also open a new therapeutic scenario for tackling glia-mediated inflammation after neurodegenerative events in the central nervous system.

Structured-light-sheet imaging in an integrated optofluidic platform.

Heterogeneity investigation at the single-cell level reveals morphological and phenotypic characteristics in cell populations. In clinical research, heterogeneity has important implications in the correct detection and interpretation of prognostic markers and in the analysis of patient-derived material. Among single-cell analysis, imaging flow cytometry allows combining information retrieved by single cell images with the throughput of fluidic platforms. Nevertheless, these techniques might fail in a comprehensive heterogeneity evaluation because of limited image resolution and bidimensional analysis. Light sheet fluorescence microscopy opened new ways to study in 3D the complexity of cellular functionality in samples ranging from single-cells to micro-tissues, with remarkably fast acquisition and low photo-toxicity. In addition, structured illumination microscopy has been applied to single-cell studies enhancing the resolution of imaging beyond the conventional diffraction limit. The combination of these techniques in a microfluidic environment, which permits automatic sample delivery and translation, would allow exhaustive investigation of cellular heterogeneity with high throughput image acquisition at high resolution. Here we propose an integrated optofluidic platform capable of performing structured light sheet imaging flow cytometry (SLS-IFC). The system encompasses a multicolor directional coupler equipped with a thermo-optic phase shifter, cylindrical lenses and a microfluidic network to generate and shift a patterned light sheet within a microchannel. The absence of moving parts allows a stable alignment and an automated fluorescence signal acquisition during the sample flow. The platform enables 3D imaging of an entire cell in about 1 s with a resolution enhancement capable of revealing sub-cellular features and sub-diffraction limit details.

c-Jun N-terminal kinase regulates soluble Aß oligomers and cognitive impairment in AD mouse model.

Alzheimer disease (AD) is characterized by cognitive impairment that starts with memory loss to end in dementia. Loss of synapses and synaptic dysfunction are closely associated with cognitive impairment in AD patients. Biochemical and pathological evidence suggests that soluble Aß oligomers correlate with cognitive impairment. Here, we used the TgCRND8 AD mouse model to investigate the role of JNK in long term memory deficits. TgCRND8 mice were chronically treated with the cell-penetrating c-Jun N-terminal kinase inhibitor peptide (D-JNKI1). D-JNKI1, preventing JNK action, completely rescued memory impairments (behavioral studies) as well as the long term potentiation deficits of TgCRND8 mice. Moreover, D-JNKI1 inhibited APP phosphorylation in Thr-668 and reduced the amyloidogenic cleavage of APP and Aß oligomers in brain parenchyma of treated mice. In conclusion, by regulating key pathogenic mechanisms of AD, JNK might hold promise as innovative therapeutic target.

Specific inhibition of the JNK pathway promotes locomotor recovery and neuroprotection after mouse spinal cord injury.

Limiting the development of secondary damage represents one of the major goals of neuroprotective therapies after spinal cord injury. Here, we demonstrate that specific JNK inhibition via a single intraperitoneal injection of the cell permeable peptide D-JNKI1 6h after lesion improves locomotor recovery assessed by both the footprint and the BMS tests up to 4 months post-injury in mice. JNK inhibition prevents c-jun phosphorylation and caspase-3 cleavage, has neuroprotective effects and results in an increased sparing of white matter at the lesion site. Lastly, D-JNKI1 treated animals show a lower increase of erythrocyte extravasation and blood brain barrier permeability, thus indicating protection of the vascular system. In total, these results clearly point out JNK inhibition as a promising neuroprotective strategy for preventing the evolution of secondary damage after spinal cord injury.

Neuroprotective effects of toll-like receptor 4 antagonism in spinal cord cultures and in a mouse model of motor neuron degeneration.

Sustained inflammatory reactions are common pathological events associated with neuron loss in neurodegenerative diseases. Reported evidence suggests that Toll-like receptor 4 (TLR4) is a key player of neuroinflammation in several neurodegenerative diseases. However, the mechanisms by which TLR4 mediates neurotoxic signals remain poorly understood. We investigated the role of TLR4 in in vitro and in vivo settings of motor neuron degeneration. Using primary cultures from mouse spinal cords, we characterized both the proinflammatory and neurotoxic effects of TLR4 activation with lipopolysaccharide (activation of microglial cells, release of proinflammatory cytokines and motor neuron death) and the protective effects of a cyanobacteria-derived TLR4 antagonist (VB3323). With the use of TLR4-deficient cells, a critical role of the microglial component with functionally active TLR4 emerged in this setting. The in vivo experiments were carried out in a mouse model of spontaneous motor neuron degeneration, the wobbler mouse, where we preliminarily confirmed a protective effect of TLR4 antagonism. Compared with vehicle- and riluzole-treated mice, those chronically treated with VB3323 showed a decrease in microglial activation and morphological alterations of spinal cord neurons and a better performance in the paw abnormality and grip-strength tests. Taken together, our data add new understanding of the role of TLR4 in mediating neurotoxicity in the spinal cord and suggest that TLR4 antagonists could be considered in future studies as candidate protective agents for motor neurons in degenerative diseases.