Link Protein 1 Is Involved in the Activity-Dependent Modulation of Perineuronal Nets in the Spinal Cord.

One of the challenges of the mature nervous system is to maintain the stability of neural networks while providing a degree of plasticity to generate experience-dependent modifications. This plasticity-stability dynamism is regulated by perineuronal nets (PNNs) and is crucial for the proper functioning of the system. Previously, we found a relation between spinal PNNs reduction and maladaptive plasticity after spinal cord injury (SCI), which was attenuated by maintaining PNNs with activity-dependent therapies. Moreover, transgenic mice lacking the cartilage link protein 1 (Crtl1 KO mice) showed aberrant spinal PNNs and increased spinal plasticity. Therefore, the aim of this study is to evaluate the role of link protein 1 in the activity-dependent modulation of spinal PNNs surrounding motoneurons and its impact on the maladaptive plasticity observed following SCI. We first studied the activity-dependent modulation of spinal PNNs using a voluntary wheel-running protocol. This training protocol increased spinal PNNs in WT mice but did not modify PNN components in Crtl1 KO mice, suggesting that link protein 1 mediates the activity-dependent modulation of PNNs. Secondly, a thoracic SCI was performed, and functional outcomes were evaluated for 35 days. Interestingly, hyperreflexia and hyperalgesia found at the end of the experiment in WT-injured mice were already present at basal levels in Crtl1 KO mice and remained unchanged after the injury. These findings demonstrated that link protein 1 plays a dual role in the correct formation and in activity-dependent modulation of PNNs, turning it into an essential element for the proper function of PNN in spinal circuits.

Assessment of focused ultrasound stimulation to induce peripheral nerve activity and potential damage in vivo.

Introduction: Peripheral neuroprostheses are aimed to restore loss of sensory and motor functions by interfacing axons in the peripheral nerves. Most common interfaces in neuroprostheses are electrodes that establish electrical connection with peripheral axons. However, some challenges arise related to long-term functionality, durability, and body response. Recently, focused ultrasound stimulation (FUS) has emerged as a non-invasive approach to modulate the nervous system. However, it is controversial whether FUS can induce axon depolarization. Methods: We have assessed FUS applied in vivo to the rat peripheral nerve, with two objectives: first, to test whether FUS activates peripheral nerves under different stimulation conditions, and second, to evaluate if FUS inflicts damage to the nerve. FUS was delivered with three ultrasound transducers (Sonic Concept H115, H107, and H102) covering the largest set of parameters examined for FUS of peripheral nerves so far. Results: We did not obtain reliable evoked action potentials in either nerves or muscles, under any FUS condition applied, neither over the skin nor directly to the nerve exposed. Additional experiments ex vivo and in vivo on mice, confirmed this conclusion. When FUS stimulation was applied directly to the exposed sciatic nerve, neuromuscular function decreased significantly, and recovered one week later, except for FUS at 0.25 MHz. Histologically, degenerating nerve fibers were observed, with a tendency to be higher with the lower FUS frequency. Discussion: Past reports on the ability of ultrasound to stimulate the peripheral nerve are controversial. After testing a wide range of FUS conditions, we conclude that it is not a reliable and safe method for stimulating the peripheral nerve. Special consideration should be taken, especially when low-frequency FUS is applied, as it may lead to nerve damage.

Histone deacetylase inhibition by suberoylanilide hydroxamic acid during reperfusion promotes multifaceted brain and vascular protection in spontaneously hypertensive rats with transient ischaemic stroke.

Hypertension is the most prevalent modifiable risk factor for stroke and is associated with worse functional outcomes. Pharmacological inhibition of histone deacetylases by suberoylanilide hydroxamic acid (SAHA) modulates gene expression and has emerged as a promising therapeutic approach to reduce ischaemic brain injury. Here, we have tested the therapeutic potential of SAHA administered during reperfusion in adult male spontaneously hypertensive (SHR) rats subjected to transient middle cerebral artery occlusion (tMCAO; 90 min occlusion/24 h reperfusion). Animals received a single dose of SAHA (50 mg/kg) or vehicle i.p. at 1, 4, or 6 h after reperfusion onset. The time-course of brain histone H3 acetylation was studied. After tMCAO, drug brain penetrance and beneficial effects on behavioural outcomes, infarct volume, oedema, angiogenesis, blood-brain barrier integrity, cerebral artery oxidative stress and remodelling, and brain and vascular inflammation were evaluated. SAHA increased brain histone H3 acetylation from 1 to 6 h after injection, reaching the ischaemic brain administered during reperfusion. Treatment given at 4 h after reperfusion onset improved neurological score, reduced infarct volume and oedema, attenuated microglial activation, prevented exacerbated MCA angiogenic sprouting and blood-brain barrier breakdown, normalised MCA oxidative stress and remodelling, and modulated brain and cerebrovascular cytokine expression. Overall, we demonstrate that SAHA administered during early reperfusion exerts robust brain and vascular protection after tMCAO in hypertensive rats. These findings are aligned with previous research in ischaemic normotensive mice and help pave the way to optimise the design of clinical trials assessing the effectiveness and safety of SAHA in ischaemic stroke.

Beyond the limiting gap length: peripheral nerve regeneration through implantable nerve guidance conduits.

Peripheral nerve damage results in the loss of sensorimotor and autonomic functions, which is a significant burden to patients. Furthermore, nerve injuries greater than the limiting gap length require surgical repair. Although autografts are the preferred clinical choice, their usage is impeded by their limited availability, dimensional mismatch, and the sacrifice of another functional donor nerve. Accordingly, nerve guidance conduits, which are tubular scaffolds engineered to provide a biomimetic environment for nerve regeneration, have emerged as alternatives to autografts. Consequently, a few nerve guidance conduits have received clinical approval for the repair of short-mid nerve gaps but failed to regenerate limiting gap damage, which represents the bottleneck of this technology. Thus, it is still necessary to optimize the morphology and constituent materials of conduits. This review summarizes the recent advances in nerve conduit technology. Several manufacturing techniques and conduit designs are discussed, with emphasis on the structural improvement of simple hollow tubes, additive manufacturing techniques, and decellularized grafts. The main objective of this review is to provide a critical overview of nerve guidance conduit technology to support regeneration in long nerve defects, promote future developments, and speed up its clinical translation as a reliable alternative to autografts.

A mammalian-specific Alex3/Gαq protein complex regulates mitochondrial trafficking, dendritic complexity, and neuronal survival.

Mitochondrial dynamics and trafficking are essential to provide the energy required for neurotransmission and neural activity. We investigated how G protein-coupled receptors (GPCRs) and G proteins control mitochondrial dynamics and trafficking. The activation of Gαq inhibited mitochondrial trafficking in neurons through a mechanism that was independent of the canonical downstream PLCß pathway. Mitoproteome analysis revealed that Gαq interacted with the Eutherian-specific mitochondrial protein armadillo repeat-containing X-linked protein 3 (Alex3) and the Miro1/Trak2 complex, which acts as an adaptor for motor proteins involved in mitochondrial trafficking along dendrites and axons. By generating a CNS-specific Alex3 knockout mouse line, we demonstrated that Alex3 was required for the effects of Gαq on mitochondrial trafficking and dendritic growth in neurons. Alex3-deficient mice had altered amounts of ER stress response proteins, increased neuronal death, motor neuron loss, and severe motor deficits. These data revealed a mammalian-specific Alex3/Gαq mitochondrial complex, which enables control of mitochondrial trafficking and neuronal death by GPCRs.

ICA-27243 improves neuromuscular function and preserves motoneurons in the transgenic SOD1G93A mice.

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease characterized by the death of upper and lower motor neurons (MNs). Excessive neuronal excitability has been implicated in MN degeneration; thus, modulation of hyperexcitability appears as a promising therapeutic strategy. Potassium channels are attractive targets since they can be activated at subthreshold voltages and can regulate neuronal excitability. In this study, we assayed the effects of N-(6-Chloro-pyridin-3-yl)-3,4-difluorobenzamide compound, known as ICA-27243, as a potential treatment for ALS. ICA-27243 is a highly selective Kv7.2/7.3 opener used mainly in epilepsy models. In the in vitro model of spinal cord organotypic cultures (SCOCs) exposed to acute excitotoxicity, ICA-27243 prevented MN degeneration at a dose-of 10 â€‹µM. Administration of ICA-27243 to transgenic SOD1G93A ALS mice improved the decline of neuromuscular function, maintained locomotion and coordination in the rotarod, decreased spinal MN death and attenuated glial reactivity. In conclusion, we report here for the first time that ICA-27243 is an effective treatment for ALS, emphasizing the potential of targeting Kv channels to reduce neuronal hyperexcitability.

Nanoporous graphene-based thin-film microelectrodes for in vivo high-resolution neural recording and stimulation.

One of the critical factors determining the performance of neural interfaces is the electrode material used to establish electrical communication with the neural tissue, which needs to meet strict electrical, electrochemical, mechanical, biological and microfabrication compatibility requirements. This work presents a nanoporous graphene-based thin-film technology and its engineering to form flexible neural interfaces. The developed technology allows the fabrication of small microelectrodes (25 µm diameter) while achieving low impedance (∼25 kΩ) and high charge injection (3-5 mC cm-2). In vivo brain recording performance assessed in rodents reveals high-fidelity recordings (signal-to-noise ratio >10 dB for local field potentials), while stimulation performance assessed with an intrafascicular implant demonstrates low current thresholds (<100 µA) and high selectivity (>0.8) for activating subsets of axons within the rat sciatic nerve innervating tibialis anterior and plantar interosseous muscles. Furthermore, the tissue biocompatibility of the devices was validated by chronic epicortical (12 week) and intraneural (8 week) implantation. This work describes a graphene-based thin-film microelectrode technology and demonstrates its potential for high-precision and high-resolution neural interfacing.

Decellularized Graft for Repairing Severe Peripheral Nerve Injuries in Sheep.

BACKGROUND AND OBJECTIVES: Peripheral nerve injuries resulting in a nerve defect require surgical repair. The gold standard of autograft (AG) has several limitations, and therefore, new alternatives must be developed. The main objective of this study was to assess nerve regeneration through a long gap nerve injury (50 mm) in the peroneal nerve of sheep with a decellularized nerve allograft (DCA). METHODS: A 5-cm long nerve gap was made in the peroneal nerve of sheep and repaired using an AG or using a DCA. Functional tests were performed once a month and electrophysiology and echography evaluations at 6.5 and 9 months postsurgery. Nerve grafts were harvested at 9 months for immunohistochemical and morphological analyses. RESULTS: The decellularization protocol completely eliminated the cells while preserving the extracellular matrix of the nerve. No significant differences were observed in functional tests of locomotion and pain response. Reinnervation of the tibialis anterior muscles occurred in all animals, with some delay in the DCA group compared with the AG group. Histology showed a preserved fascicular structure in both AG and DCA; however, the number of axons distal to the nerve graft was higher in AG than in DCA. CONCLUSION: The decellularized graft assayed supported effective axonal regeneration when used to repair a 5-cm long gap in the sheep. As expected, a delay in functional recovery was observed compared with the AG because of the lack of Schwann cells.

Combining biophysical models and machine learning to optimize implant geometry and stimulation protocol for intraneural electrodes.

Objective.Peripheral nerve interfaces have the potential to restore sensory, motor, and visceral functions. In particular, intraneural interfaces allow targeting deep neural structures with high selectivity, even if their performance strongly depends upon the implantation procedure and the subject's anatomy. Currently, few alternatives exist for the determination of the target subject structural and functional anatomy, and statistical characterizations from cadaveric samples are limited because of their high cost. We propose an optimization workflow that can guide both the pre-surgical planning and the determination of maximally selective multisite stimulation protocols for implants consisting of several intraneural electrodes, and we characterize its performance in silico. We show that the availability of structural and functional information leads to very high performances and allows taking informed decisions on neuroprosthetic design.Approach.We employ hybrid models (HMs) of neuromodulation in conjunction with a machine learning-based surrogate model to determine fiber activation under electrical stimulation, and two steps of optimization through particle swarm optimization to optimize in silico implant geometry, implantation and stimulation protocols using morphological data from the human median nerve at a reduced computational cost.Main results.Our method allows establishing the optimal geometry of multi-electrode transverse intra-fascicular multichannel electrode implants, the optimal number of electrodes to implant, their optimal insertion, and a set of multipolar stimulation protocols that lead in silico to selective activation of all the muscles innervated by the human median nerve.Significance.We show how to use effectively HMs for optimizing personalized neuroprostheses for motor function restoration. We provide in-silico evidences about the potential of multipolar stimulation to increase greatly selectivity. We also show that the knowledge of structural and functional anatomies of the target subject leads to very high selectivity and motivate the development of methods for theirin vivocharacterization.

Massive Loss of Proprioceptive Ia Synapses in Rat Spinal Motoneurons after Nerve Crush Injuries in the Postnatal Period.

Peripheral nerve injuries (PNIs) induce the retraction from the ventral horn of the synaptic collaterals of Ia afferents injured in the nerve, effectively removing Ia synapses from α-motoneurons. The loss of Ia input impairs functional recovery and could explain, in part, better recovery after PNIs with better Ia synaptic preservation. Synaptic losses correlate with injury severity, speed, and efficiency of muscle reinnervation and requires ventral microglia activation. It is unknown whether this plasticity is age dependent. In neonates, axotomized motoneurons and sensory neurons undergo apoptosis, but after postnatal day 10 most survive. The goal of this study was to analyze vesicular glutamate transporter 1 (VGluT1)-labeled Ia synapses (which also include II afferents) after nerve crush in 10 day old rats, a PNI causing little Ia/II synapse loss in adult rats. We confirmed fast and efficient reinnervation of leg muscles; however, a massive number of VGluT1/Ia/II synapses were permanently lost. This synapse loss was similar to that after more severe nerve injuries involving full transection in adults. In adults, disappearance of ventrally directed Ia/II collaterals targeting α-motoneurons was associated with a prolonged microglia reaction and a CCR2 mechanism that included infiltration of CCR2 blood immune cells. By contrast, microgliosis after P10 injuries was fast, resolved in about a week, and there was no evidence of peripheral immune cell infiltration. We conclude that VGluT1/Ia/II synapse loss in young animals differs in mechanism, perhaps associated with higher microglia synaptic pruning activity at this age and results in larger losses after milder nerve injuries.

Repair of Long Peripheral Nerve Defects in Sheep: A Translational Model for Nerve Regeneration.

Despite advances in microsurgery, full functional recovery of severe peripheral nerve injuries is not commonly attained. The sheep appears as a good preclinical model since it presents nerves with similar characteristics to humans. In this study, we induced 5 or 7 cm resection in the peroneal nerve and repaired with an autograft. Functional evaluation was performed monthly. Electromyographic and ultrasound tests were performed at 6.5 and 9 months postoperation (mpo). No significant differences were found between groups with respect to functional tests, although slow improvements were seen from 5 mpo. Electrophysiological tests showed compound muscle action potentials (CMAP) of small amplitude at 6.5 mpo that increased at 9 mpo, although they were significantly lower than the contralateral side. Ultrasound tests showed significantly reduced size of tibialis anterior (TA) muscle at 6.5 mpo and partially recovered size at 9 mpo. Histological evaluation of the grafts showed good axonal regeneration in all except one sheep from autograft 7 cm (AG7) group, while distal to the graft there was a higher number of axons than in control nerves. The results indicate that sheep nerve repair is a useful model for investigating long-gap peripheral nerve injuries.

Are antihistamines still used during omalizumab treatment for chronic spontaneous urticaria?

Background: The guidelines for the treatment of chronic spontaneous urticaria (CSU) recommend adding omalizumab to the treatment of patients with uncontrolled disease despite four-fold doses of second-generation antihistamines (AH). On the contrary, some studies revealed that omalizumab was effective without concomitant AH and several authors suggest tapering off AH when CSU is controlled with omalizumab. Objectives: The aim of our study was to evaluate the use of AH during treatment with omalizumab in patients with CSU in real clinical practice. Materials & Methods: This was a multicentre cross-sectional and observational study conducted by the Catalan and Balearic Chronic Urticaria Network (XUrCB) based on a cohort of 298 CSU patients treated with omalizumab. Results: In total, 23.5% of our patients decided themselves to stop taking AH during omalizumab treatment. The ratio of patients with CSU without concomitant inducible urticaria and the percentage of patients with a good response to omalizumab (UAS7≤6 and/or UCT ≥12) were higher in those who stopped taking AH. Conclusion: More studies are required to identify the phenotypic characteristics of patients responding to omalizumab as monotherapy in order to avoid overtreating with AH. Our study suggests that patients with CSU without concomitant inducible urticaria and those who achieve a good response to omalizumab tend to be controlled by omalizumab without AH. In order to establish guidelines on how to stop AH, further evidenced-based studies are required.

Repair of Long Nerve Defects with a New Decellularized Nerve Graft in Rats and in Sheep.

Decellularized nerve allografts (DC) are an alternative to autografts (AG) for repairing severe peripheral nerve injuries. We have assessed a new DC provided by VERIGRAFT. The decellularization procedure completely removed cellularity while preserving the extracellular matrix. We first assessed the DC in a 15 mm gap in the sciatic nerve of rats, showing slightly delayed but effective regeneration. Then, we assayed the DC in a 70 mm gap in the peroneal nerve of sheep compared with AG. Evaluation of nerve regeneration and functional recovery was performed by clinical, electrophysiology and ultrasound tests. No significant differences were found in functional recovery between groups of sheep. Histology showed a preserved fascicular structure in the AG while in the DC grafts regenerated axons were grouped in small units. In conclusion, the DC was permissive for axonal regeneration and allowed to repair a 70 mm long gap in the sheep nerve.

Sensory Involvement in Amyotrophic Lateral Sclerosis.

Although amyotrophic lateral sclerosis (ALS) is pre-eminently a motor disease, the existence of non-motor manifestations, including sensory involvement, has been described in the last few years. Although from a clinical perspective, sensory symptoms are overshadowed by their motor manifestations, this does not mean that their pathological significance is not relevant. In this review, we have made an extensive description of the involvement of sensory and autonomic systems described to date in ALS, from clinical, neurophysiological, neuroimaging, neuropathological, functional, and molecular perspectives.

Multifaceted understanding of human nerve implants to design optimized electrodes for bioelectronics.

Bioelectronic medicine is a promising venue for treatment of disabilities using implantable neural interfaces. Peripheral neurostimulation of residual nerves recently enabled multiple functional benefits in amputees. Despite the preliminary promising impact on patients' life, the over-time stability of implants and the related nerve reactions are unclear. To unveil the mechanisms and inform the design of better nerve-electrode interfaces, we engaged a multifaceted approach, merging functional responses from patients, their histological data, and corresponding computational modelling. Neurostimulation evoked different selective sensation locations and qualities over-time, with respective perceptual thresholds, that showed different degree of time stabilities dependent from the stimulating active sites. The histological analysis after explant showed mild tissue reactions, while electromechanically active sites and substrates remained conserved. Computational models, based on patients' histology, revealed the direct influence of the simulated tissue reaction to change of thresholds and type of perceived sensations. Novel insights of electrode biocompatibility was observed compared to animals and the increase of thresholds could be predicted computationally. This multifaced framework suggest that future intraneural implants should have easier implantation and higher biocompatibility counteracting the sensations changes through AI-based stimulations and electrode coatings.

A transient inflammatory response contributes to oxaliplatin neurotoxicity in mice.

OBJECTIVES: Peripheral neuropathy is a relevant dose-limiting adverse event that can affect up to 90% of oncologic patients with colorectal cancer receiving oxaliplatin treatment. The severity of neurotoxicity often leads to dose reduction or even premature cessation of chemotherapy. Unfortunately, the limited knowledge about the molecular mechanisms related to oxaliplatin neurotoxicity leads to a lack of effective treatments to prevent the development of this clinical condition. In this context, the present work aimed to determine the exact molecular mechanisms involved in the development of oxaliplatin neurotoxicity in a murine model to try to find new therapeutical targets. METHODS: By single-cell RNA sequencing (scRNA-seq), we studied the transcriptomic profile of sensory neurons and satellite glial cells (SGC) of the Dorsal Root Ganglia (DRG) from a well-characterized mouse model of oxaliplatin neurotoxicity. RESULTS: Analysis of scRNA-seq data pointed to modulation of inflammatory processes in response to oxaliplatin treatment. In this line, we observed increased levels of NF-kB p65 protein, pro-inflammatory cytokines, and immune cell infiltration in DRGs and peripheral nerves of oxaliplatin-treated mice, which was accompanied by mechanical allodynia and decrease in sensory nerve amplitudes. INTERPRETATION: Our data show that, in addition to the well-described DNA damage, oxaliplatin neurotoxicity is related to an exacerbated pro-inflammatory response in DRG and peripheral nerves, and open new insights in the development of anti-inflammatory strategies as a treatment for preventing peripheral neuropathy induced by oxaliplatin.

A novel decellularized nerve graft for repairing peripheral nerve long gap injury in the rat.

Decellularized nerve allografts are an alternative to autograft for repairing severe nerve injuries, since they have higher availability and do not induce rejection. In this study, we have assessed the regenerative potential of a novel decellularization protocol for human and rat nerves for repairing nerve resections, compared to the gold standard autograft. A 15-mm gap in the sciatic nerve was repaired with decellularized rat allograft (DC-RA), decellularized human xenograft (DC-HX), or fresh autograft (AG). Electrophysiology tests were performed monthly to evaluate muscle reinnervation, whereas histological and immunohistochemical analyses of the grafts were evaluated at 4 months. A short-term study was also performed to compare the differences between the two decellularized grafts (DC-RA and DC-HX) in early phases of regeneration. The decellularization process eliminated cellularity while preserving the ECM and endoneurial tubules of both rat and human nerves. Higher amount of reinnervation was observed in the AG group compared to the DC-RA group, while only half of the animals of the DC-HX showed distal muscle reinnervation. The number of regenerating myelinated axons in the mid-graft was similar between AG and DC-RA and lower in DC-HX graft, but significantly lower in both DC grafts distally. At short term, fibroblasts repopulated the DC-RA graft, supporting regenerated axons, whereas an important fibrotic reaction was observed around DC-HX grafts. In conclusion, the decellularized allograft sustained regeneration through a long gap in the rat although at a slower rate compared to the ideal autograft, whereas regeneration was limited or even failed when using a decellularized xenograft.

Efficacy of Vafidemstat in Experimental Autoimmune Encephalomyelitis Highlights the KDM1A/RCOR1/HDAC Epigenetic Axis in Multiple Sclerosis.

Lysine specific demethylase 1 (LSD1; also known as KDM1A), is an epigenetic modulator that modifies the histone methylation status. KDM1A forms a part of protein complexes that regulate the expression of genes involved in the onset and progression of diseases such as cancer, central nervous system (CNS) disorders, viral infections, and others. Vafidemstat (ORY-2001) is a clinical stage inhibitor of KDM1A in development for the treatment of neurodegenerative and psychiatric diseases. However, the role of ORY-2001 targeting KDM1A in neuroinflammation remains to be explored. Here, we investigated the effect of ORY-2001 on immune-mediated and virus-induced encephalomyelitis, two experimental models of multiple sclerosis and neuronal damage. Oral administration of ORY-2001 ameliorated clinical signs, reduced lymphocyte egress and infiltration of immune cells into the spinal cord, and prevented demyelination. Interestingly, ORY-2001 was more effective and/or faster acting than a sphingosine 1-phosphate receptor antagonist in the effector phase of the disease and reduced the inflammatory gene expression signature characteristic ofEAE in the CNS of mice more potently. In addition, ORY-2001 induced gene expression changes concordant with a potential neuroprotective function in the brain and spinal cord and reduced neuronal glutamate excitotoxicity-derived damage in explants. These results pointed to ORY-2001 as a promising CNS epigenetic drug able to target neuroinflammatory and neurodegenerative diseases and provided preclinical support for the subsequent design of early-stage clinical trials.