Axonal Regrowth Research Articles

The Potential of Aloe Vera in Solution and in Blended Nanofibers Containing Poly (3-Hydroxybutyrate-Co-3-Hydroxyvalerate) as Substrates for Neurite Outgrowth.

This pilot study investigated the potential of aloe vera (AV) to promote neurite outgrowth in organotypic dorsal root ganglia (DRG) explants (n = 230) from neonatal rats (n = 15). Using this invitro model of acute axotomy, we assessed neurite outgrowth exceeding 1.5 times the explant diameter (viable explants) and measured the longest neurite length. These parameters were evaluated under control conditions and in cultures supplemented with commercial AV and four aligned scaffolds: poly-L-lactate (PLLA), polydioxanone (PDS), poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), and blended PHBV/AV. After 6 days of culture, explants were immunostained using neuron-specific marker Tuj1 and Schwann cell marker S100. Measurements were obtained with Image J software and analyzed using Jamovi 2.3. In control and AV dilution media, the study revealed radial tissue growth from the explant body with randomly oriented neurites, whereas in all scaffolds, bidirectional tissue growth occurred parallel to nanofibers. Binomial logistic regression analyses indicated that viable explants were more likely in the control group compared to PDS (p = 0.0042) and PHBV (p < 0.0001), with non-significant differences when compared to AV dilution, PLLA, and PHBV/AV. AV dilution showed a greater association with viable explants than PLLA (p = 0.0459), while non-significant difference was found between AV dilution and PHBV/AV. Additionally, the PHBV/AV scaffold predicted higher odds of viable explants than PLLA (p = 0.0479), PDS (p = 0.0001), and PHBV (p < 0.0001). Groups with similar probabilities of obtaining viable explants (control, AV dilution, and PHBV/AV) exhibited non-significant differences in their longest neurite lengths. In conclusion, control, AV dilution, and PHBV/AV yielded the highest probability of developing viable explants and the longest neurite lengths. This is the first study demonstrating the direct permissiveness of AV for axonal outgrowth. Furthermore, the blended PHBV/AV scaffold showed significant potential as a suitable scaffold for axonal regrowth and Schwann cell migration, ensuring controlled tissue formation for tissue engineering applications.

Dual Role of Ibuprofen and Indomethacin in Promoting Peripheral Nerve Regeneration In Vitro.

Peripheral nerve injuries (PNI) can result in significant losses of motor and sensory function. Although peripheral nerves have an innate capacity for regeneration, restoration of function after severe injury remains suboptimal. The gold standard for peripheral nerve regeneration (PNR) is autologous nerve transplantation, but this method is limited by the generation of an additional surgical site, donor-site morbidity, and neuroma formation at the site of harvest. Although targeted drug compounds have the potential to influence axonal growth, there are no drugs currently approved to treat PNI. Therefore, we propose to repurpose commonly used nonsteroidal anti-inflammatory drugs (NSAIDs) to enhance PNR, facilitating easier clinical translation. Additionally, calcium signaling plays a crucial role in neuronal connectivity and regeneration, but how specific drugs modulate this process remains unclear. We developed an in vitro hollow channel collagen gel platform that successfully supports neuronal network formation. This study evaluated the effects of commonly used NSAIDs, namely ibuprofen and indomethacin, in our in vitro model of axonal growth, regeneration, and calcium signaling as potential treatments for PNI. Our results demonstrate enhanced axonal growth and regrowth with both ibuprofen and indomethacin, suggesting a positive influence on PNR. Further, these drugs showed enhanced calcium signaling dynamics, which we posit is a crucial aspect for nerve repair. Taken together, these findings highlight the potential of ibuprofen and indomethacin to be used as treatment options for PNI, given their dual capability to promote axonal growth and enhance calcium signaling.

Synaptotagmin 4 Supports Spontaneous Axon Sprouting after Spinal Cord Injury.

Injuries to the central nervous system (CNS) can cause severe neurological deficits. Axonal regrowth is a fundamental process for the reconstruction of compensatory neuronal networks after injury; however, it is extremely limited in the adult mammalian CNS. In this study, we conducted a loss-of-function genetic screen in cortical neurons, combined with a Web resource-based phenotypic screen, and identified synaptotagmin 4 (Syt4) as a novel regulator of axon elongation. Silencing Syt4 in primary cultured cortical neurons inhibits neurite elongation, with changes in gene expression involved in signaling pathways related to neuronal development. In a spinal cord injury model, inhibition of Syt4 expression in cortical neurons prevented axonal sprouting of the corticospinal tract, as well as neurological recovery after injury. These results provide a novel therapeutic approach to CNS injury by modulating Syt4 function.

Dnajc3 (HSP40) Modulates Axon Regeneration in the Mouse Optic Nerve.

The present study is designed to identify the genes modulating optic nerve regeneration in the mouse. Using the BXD mouse strains as a genetic mapping panel, we examined differential responses to axon regeneration in order to map genomic loci modulating axonal regeneration. To study regeneration in the optic nerve, Pten was knocked down in the retinal ganglion cells using adeno-associated virus (AAV) delivery of an shRNA, followed by the induction of a mild inflammatory response by an intravitreal injection of Zymosan with CPT-cAMP. The axons of the retinal ganglion cells were damaged by optic nerve crush (ONC). Following a 12-day survival period, regenerating axons were labeled by Cholera Toxin B. Two days later, the regenerating axons within the optic nerve were examined to determine the number of regenerating axons and the distance traveled down the optic nerve. An integral genomic map was made using the regenerative response. Candidate genes were tested by knocking down expression using shRNA or by overexpressing the gene in AAV vectors. The analysis revealed a considerable amount of differential axonal regeneration across all 33 BXD strains, demonstrated by the number of axons regenerating and the length of the regenerating axons. Some strains (BXD99, BXD90, and BXD29) demonstrated significant axonal regeneration; while other strains (BXD13, BXD18, and BXD34) had very little axon regrowth. Within the regenerative data, there was a 4-fold increase in distance regenerated and a 7.5-fold difference in the number of regenerating axons. These data were used to map a quantitative trait locus modulating axonal regeneration to Chromosome 14 (115 to 119 Mb). Within this locus were 16 annotated genes. Subsequent testing revealed that one candidate gene, Dnajc3 , modulates axonal regeneration. Knocking down of Dnajc3 led to a decreased regeneration response in the high regenerative strains (BXD90), while overexpression of Dnajc3 resulted in an increased regeneration response in C57BL/6J and a low regenerative strain (BXD34). In this study, Dnajc3 (encodes Heat Shock Protein 40, HSP40, a molecular chaperone) was identified as a modulator of axon regeneration in mice. This is the first report defining the role of Dnajc3 (HSP40) in axon regeneration.

Local glycolysis supports injury-induced axonal regeneration.

Successful axonal regeneration following injury requires the effective allocation of energy. How axons withstand the initial disruption in mitochondrial energy production caused by the injury and subsequently initiate regrowth is poorly understood. Transcriptomic data showed increased expression of glycolytic genes after optic nerve crush in retinal ganglion cells with the co-deletion of Pten and Socs3. Using retinal cultures in a multicompartment microfluidic device, we observed increased regrowth and enhanced mitochondrial trafficking in the axons of Pten and Socs3 co-deleted neurons. While wild-type axons relied on mitochondrial metabolism, after injury, in the absence of Pten and Socs3, energy production was supported by local glycolysis. Specific inhibition of lactate production hindered injury survival and the initiation of regrowth while slowing down glycolysis upstream impaired regrowth initiation, axonal elongation, and energy production. Together, these observations reveal that glycolytic ATP, combined with sustained mitochondrial transport, is essential for injury-induced axonal regrowth, providing new insights into the metabolic underpinnings of axonal regeneration.

Schwann cell-secreted frizzled-related protein 1 dictates neuroinflammation and peripheral nerve degeneration after neurotrauma.

Neurotrauma in limbs can induce sustained neuroinflammation, resulting in persistent disruption of nerve tissue architecture and retardation of axon regrowth. Despite macrophage-mediated inflammation promoting the removal of necrotic neural components and stimulating neo-vessel ingrowth, detrimental shifts in macrophage phenotype exacerbate nerve degeneration. Herein, we find that peripheral nerve injuries (PNIs) result in abundant secreted frizzled-related protein 1 (sFRP1) expression, particularly by Schwann cells (SCs). Heat shock protein 90 (HSP90) in macrophages recognizes sFRP1 and triggers a dysregulated secretion of inflammatory mediators. Single-cell atlas of human injured peripheral nerves reveals the appearance of sFRP1-expressing SCs with mesenchymal traits and macrophages with a proinflammatory genetic profile. Deletion of either SC-specific sFRP1 or macrophage-specific HSP90 alleviates neuroinflammation and prevents the progression of nerve degeneration. Together, our findings implicate the response of macrophages to SC-derived sFRP1 in exacerbating nerve damage following PNIs.

Inhibition of KIF5b-mediated Nav1.8 transport by ropivacaine contributes to axonal regeneration following sciatic nerve injury in rats

Peripheral nerve injury (PNI), typically caused by traumatic accidents or medical events, is currently one of the most common diseases that leads to limb disability. After PNI, tetrodotoxin-resistant voltage-gated sodium channel Nav1.8 is upregulated at the lesion site. Our earlier study suggested that ropivacaine promotes axon regrowth by regulating Nav1.8-mediated macrophage signaling. Nevertheless, the mechanism of ropivacaine in regulation of Nav1.8 expression remains incompletely understood. Kinesin family 5b (KIF5b) was reported to mediate the Nav1.8 axonal transport from dorsal root ganglia (DRGs) to lesion site. Herein, we investigated whether ropivacaine promotes axon regeneration through inhibition of KIF5b-mediated Nav1.8 transport. Reduced levels of KIF5b and Nav1.8 in DRGs coincide with their increase at the lesion site. Nav1.8 mRNA was significantly increased at the lesion site but not in DRGs. Surprisingly, ropivacaine reversed the alterations of Nav1.8 and KIF5b protein expression without affecting Nav1.8 mRNA level. Due to KIF5b overexpression in DRGs, Nav1.8 protein level was significantly decreased in DRGs and increased at the lesion site. We also found KIF5b overexpression significantly impaired behavioral functions, reduced the recovery index of compound muscle action potential (CMAP) amplitude, inhibited axonal regrowth, slowed M1 macrophage infiltration and shift to M2 phenotype, and delayed myelin debris clearance. Notably, all aforementioned results caused by KIF5b overexpression were alleviated by ropivacaine. Furthermore, we demonstrated that inhibition of Nav1.8 transport by A-803467 produced mitigating effects on the impairment of regenerative capacity induced by KIF5b overexpression similar to ropivacaine. These results suggest that ropivacaine promotes axonal regeneration at least partially by inhibiting KIF5b-mediated Nav1.8 forward transport.

Repeated intrathecal injections of peripheral nerve-derived stem cell spheroids improve outcomes in a rat model of traumatic brain injury

BackgroundTraumatic brain injury (TBI) is a major cause of disability and mortality worldwide. However, existing treatments still face numerous clinical challenges. Building on our prior research showing peripheral nerve-derived stem cell (PNSC) spheroids with Schwann cell-like phenotypes can secrete neurotrophic factors to aid in neural tissue regeneration, we hypothesized that repeated intrathecal injections of PNSC spheroids would improve the delivery of neurotrophic factors, thereby facilitating the restoration of neurological function and brain tissue repair post-TBI.MethodsWe generated PNSC spheroids from human peripheral nerve tissue using suspension culture techniques. These spheroids were characterized using flow cytometry, immunofluorescence, and reverse-transcription polymerase chain reaction. The conditioned media were evaluated in SH-SY5Y and RAW264.7 cell lines to assess their effects on neurogenesis and inflammation. To simulate TBI, we established a controlled cortical impact (CCI) model in rats. The animals were administered intrathecal injections of PNSC spheroids on three occasions, with each injection spaced at a 3-day interval. Recovery of sensory and motor function was assessed using the modified neurological severity score (mNSS) and rotarod tests, while histological (hematoxylin and eosin, Luxol fast blue staining) and T2-weighted magnetic resonance imaging analyses, alongside immunofluorescence, were conducted to evaluate the recovery of neural structures and pathophysiology.ResultsPNSC spheroids expressed high levels of Schwann cell markers and neurotrophic factors, such as neurotrophin-3 and Ephrin B3. Their conditioned medium was found to promote neurite outgrowth, reduce reactive oxygen species-mediated cell death and inflammation, and influence M1-M2 macrophage polarization. In the CCI rat model, rats receiving repeated triple intrathecal injections of PNSC spheroids showed significant improvements in sensory and motor function, with considerable neural tissue recovery in damaged areas. Notably, this treatment promoted nerve regeneration, axon regrowth, and remyelination. It also reduced glial scar formation and inflammation, while encouraging angiogenesis.ConclusionOur findings suggest that repeated intrathecal injections of PNSC spheroids can significantly enhance neural recovery after TBI. This effect is mediated by the diverse neurotrophic factors secreted by PNSC spheroids. Thus, the strategy of combining therapeutic cell delivery with multiple intrathecal injections holds promise as a novel clinical treatment for TBI recovery.

Nuclear Magnetic Resonance Treatment Induces ßNGF Release from Schwann Cells and Enhances the Neurite Growth of Dorsal Root Ganglion Neurons In Vitro.

Peripheral nerve regeneration depends on close interaction between neurons and Schwann cells (SCs). After nerve injury, SCs produce growth factors and cytokines that are crucial for axon re-growth. Previous studies revealed the supernatant of SCs exposed to nuclear magnetic resonance therapy (NMRT) treatment to increase survival and neurite formation of rat dorsal root ganglion (DRG) neurons in vitro. The aim of this study was to identify factors involved in transferring the observed NMRT-induced effects to SCs and consequently to DRG neurons. Conditioned media of NMRT-treated (CM NMRT) and untreated SCs (CM CTRL) were tested by beta-nerve growth factor (ßNGF) ELISA and multiplex cytokine panels to profile secreted factors. The expression of nociceptive transient receptor potential vanilloid 1 (TRPV1) channels was assessed and the intracellular calcium response in DRG neurons to high-potassium solution, capsaicin or adenosine triphosphate was measured mimicking noxious stimuli. NMRT induced the secretion of ßNGF and pro-regenerative-signaling factors. Blocking antibody experiments confirmed ßNGF as the main factor responsible for neurotrophic/neuritogenic effects of CM NMRT. The TRPV1 expression or sensitivity to specific stimuli was not altered, whereas the viability of cultured DRG neurons was increased. Positive effects of CM NMRT supernatant on DRG neurons are primarily mediated by increased ßNGF levels.

Axonal Regeneration after Spinal Cord Injury: Molecular Mechanisms, Regulatory Pathways, and Novel Strategies.

Axonal regeneration in the spinal cord after traumatic injuries presents a challenge for researchers, primarily due to the nature of adult neurons and the inhibitory environment that obstructs neuronal regrowth. Here, we review current knowledge of the intricate network of molecular and cellular mechanisms that hinder axonal regeneration, with a focus on myelin-associated inhibitors (MAIs) and other inhibitory guidance molecules, as well as the pivotal pathways implicated in both inhibiting and facilitating axonal regrowth, such as PKA/AMP, PI3K/Akt/mTOR, and Trk, alongside the regulatory roles of neurotrophins and axonal guidance cues. We also examine current insights into gene therapy, tissue engineering, and pharmacological interventions that show promise in overcoming barriers to axonal regrowth.

Axon guidance during mouse central nervous system regeneration is required for specific brain innervation.

Reconstructing functional neuronal circuits is one major challenge of central nervous system repair. Through activation of pro-growth signaling pathways, some neurons achieve long-distance axon regrowth. Yet, functional reconnection has hardly been obtained, as these regenerating axons fail to resume their initial trajectory and reinnervate their proper target. Axon guidance is considered to be active only during development. Here, using the mouse visual system, we show that axon guidance is still active in the adult brain in regenerative conditions. We highlight that regenerating retinal ganglion cell axons avoid one of their primary targets, the suprachiasmatic nucleus (SCN), due to Slit/Robo repulsive signaling. Together with promoting regeneration, silencing Slit/Robo invivo enables regenerating axons to enter the SCN and form active synapses. The newly formed circuit is associated with neuronal activation and functional recovery. Our results provide evidence that axon guidance mechanisms are required to reconnect regenerating axons to specific brain nuclei.

TFEB/3 Govern Repair Schwann Cell Generation and Function Following Peripheral Nerve Injury.

TFEB and TFE3 (TFEB/3), key regulators of lysosomal biogenesis and autophagy, play diverse roles depending on cell type. This study highlights a hitherto unrecognized role of TFEB/3 crucial for peripheral nerve repair. Specifically, they promote the generation of progenitor-like repair Schwann cells after axonal injury. In Schwann cell-specific TFEB/3 double knock-out mice of either sex, the TFEB/3 loss disrupts the transcriptomic reprogramming that is essential for the formation of repair Schwann cells. Consequently, mutant mice fail to populate the injured nerve with repair Schwann cells and exhibit defects in axon regrowth, target reinnervation, and functional recovery. TFEB/3 deficiency inhibits the expression of injury-responsive repair Schwann cell genes, despite the continued expression of c-jun, a previously identified regulator of repair Schwann cell function. TFEB/3 binding motifs are enriched in the enhancer regions of injury-responsive genes, suggesting their role in repair gene activation. Autophagy-dependent myelin breakdown is not impaired despite TFEB/3 deficiency. These findings underscore a unique role of TFEB/3 in adult Schwann cells that is required for proper peripheral nerve regeneration.

Electrostimulation via a 3D-printed, biomimetic, neurotrophic, electroconductive scaffold for the promotion of axonal regrowth after spinal cord injury

Spinal cord injury (SCI) is a devastating neurotrauma, affecting 250,000 to 500,000 people annually, and typically results in paralysis. Electrostimulation can promote neuronal growth, but the formation of a lesion cavity post-SCI inhibits regrowth, limiting its efficacy. Bridging the lesion with a structured, electroactive substrate to direct electrostimulation to growing neurites could support and drive neuronal regrowth through the lesion to enable functional recovery but to date, no such platform exists. This study describes the development of an electroconductive (15 ± 5 S/m), 3D-printed scaffold, comprising a polypyrrole/polycaprolactone framework filled with biomimetic & neurotrophic extracellular matrix. 3D printing allowed inclusion of channels in the scaffold designed to mimic the size of human corticospinal tracts to direct electrostimulation to growing neurons. Scaffolds exhibited excellent biocompatibility with both neurons and human primary astrocytes and maintained electrical and biofunctionality when scaled to match the size of human corticospinal tracts. When neurons were cultured for 7 days on the scaffolds under continuous electrostimulation (200 mV/mm, 12 Hz), significantly longer neurites were observed on electrically stimulated electroconductive scaffolds. These results demonstrate that electrostimulation applied via an anatomically-mimetic, 3D-printed electroconductive scaffold drives neurite outgrowth and represents a promising approach for treatment of spinal cord injury.

AAV-Mediated Expression of miR-17 Enhances Neurite and Axon Regeneration In Vitro.

Neurodegenerative disorders, including traumatic injuries to the central nervous system (CNS) and neurodegenerative diseases, are characterized by early axonal damage, which does not regenerate in the adult mammalian CNS, leading to permanent neurological deficits. One of the primary causes of the loss of regenerative ability is thought to be a developmental decline in neurons' intrinsic capability for axon growth. Different molecules are involved in the developmental loss of the ability for axon regeneration, including many transcription factors. However, the function of microRNAs (miRNAs), which are also modulators of gene expression, in axon re-growth is still unclear. Among the various miRNAs recently identified with roles in the CNS, miR-17, which is highly expressed during early development, emerges as a promising target to promote axon regeneration. Here, we used adeno-associated viral (AAV) vectors to overexpress miR-17 (AAV.miR-17) in primary cortical neurons and evaluate its effects on neurite and axon regeneration in vitro. Although AAV.miR-17 had no significant effect on neurite outgrowth and arborization, it significantly enhances neurite regeneration after scratch lesion and axon regeneration after axotomy of neurons cultured in microfluidic chambers. Target prediction and functional annotation analyses suggest that miR-17 regulates gene expression associated with autophagy and cell metabolism. Our findings suggest that miR-17 promotes regenerative response and thus could mitigate neurodegenerative effects.

Schwann cell JUN expression worsens motor performance in an amyotrophic lateral sclerosis mouse model.

Amyotrophic lateral sclerosis is a devastating neurodegenerative disease characterized by motor neuron death and distal axonopathy. Despite its clinical severity and profound impact in the patients and their families, many questions about its pathogenesis remain still unclear, including the role of Schwann cells and axon-glial signaling in disease progression. Upon axonal injury, upregulation of JUN transcription factor promotes Schwann cell reprogramming into a repair phenotype that favors axon regrowth and neuronal survival. To study the potential role of repair Schwann cells on motoneuron survival in amyotrophic lateral sclerosis, we generated a mouse line that over-expresses JUN in the Schwann cells of the SOD1G93A mutant, a mouse model of this disease. Then, we explored disease progression by evaluating survival, motor performance and histology of peripheral nerves and spinal cord of these mice. We found that Schwann cell JUN overexpression does not prevent axon degeneration neither motor neuron death in the SOD1G93A mice. Instead, it induces a partial demyelination of medium and large size axons, worsening motor performance and resulting in more aggressive disease phenotype.

Hybrid construction of tissue-engineered nerve graft using skin derived precursors induced neurons and Schwann cells to enhance peripheral neuroregeneration

Peripheral nerve injury is a major challenge in clinical treatment due to the limited intrinsic capacity for nerve regeneration. Tissue engineering approaches offer promising solutions by providing biomimetic scaffolds and cell sources to promote nerve regeneration. In the present work, we investigated the potential role of skin-derived progenitors (SKPs), which are induced into neurons and Schwann cells (SCs), and their extracellular matrix in tissue-engineered nerve grafts (TENGs) to enhance peripheral neuroregeneration. SKPs were induced to differentiate into neurons and SCs in vitro and incorporated into nerve grafts composed of a biocompatible scaffold including chitosan neural conduit and silk fibroin filaments. In vivo experiments using a rat model of peripheral nerve injury showed that TENGs significantly enhanced nerve regeneration compared to the scaffold control group, catching up with the autograft group. Histological analysis showed improved axonal regrowth, myelination and functional recovery in animals treated with these TENGs. In addition, immunohistochemical staining confirmed the presence of induced neurons and SCs within the regenerated nerve tissue. Our results suggest that SKP-induced neurons and SCs in tissue-engineered nerve grafts have great potential for promoting peripheral nerve regeneration and represent a promising approach for clinical translation in the treatment of peripheral nerve injury. Further optimization and characterization of these engineered constructs is warranted to improve their clinical applicability and efficacy.

Epigenetic Cross-Talk Between Sirt1 and Dnmt1 Promotes Axonal Regeneration After Spinal Cord Injury in Zebrafish.

Though spinal cord injury (SCI) causes irreversible sensory and motor impairments in human, adult zebrafish retain the potent regenerative capacity by injury-induced proliferation of central nervous system (CNS)-resident progenitor cells to develop new functional neurons at the lesion site. The hallmark of SCI in zebrafish lies in a series of changes in the epigenetic landscape, specifically DNA methylation and histone modifications. Decoding the post-SCI epigenetic modifications is therefore critical for the development of therapeutic remedies that boost SCI recovery process. Here, we have studied on Sirtuin1 (Sirt1), a non-classical histone deacetylase that potentially plays a critical role in neural progenitor cells (NPC) proliferation and axonal regrowth following SCI in zebrafish. We investigated the role of Sirt1 in NPC proliferation and axonal regrowth in response to injury in the regenerating spinal cord and found that Sirt1 is involved in the induction of NPC proliferation along with glial bridging during spinal cord regeneration. We also demonstrate that Sirt1 plays a pivotal role in regulating the HIPPO pathway through deacetylation-mediated inactivation of Dnmt1 and subsequent hypomethylation of yap1 promoter, leading to the induction of ctgfa expression, which drives the NPC proliferation and axonal regrowth to complete the regenerative process. In conclusion, our study reveals a novel cross-talk between two important epigenetic effectors, Sirt1 and Dnmt1, in the context of spinal cord regeneration, establishing a previously undisclosed relation between Sirt1 and Yap1 which provides a deeper understanding of the underlying mechanisms governing injury-induced NPC proliferation and axonal regrowth. Therefore, we have identified Sirt1 as a novel, major epigenetic regulator of spinal cord regeneration by modulating the HIPPO pathway in zebrafish.

Generation of an Armcx1 Conditional Knockout Mouse.

Armadillo repeat-containing X-linked protein-1 (Armcx1) is a poorly characterized transmembrane protein that regulates mitochondrial transport in neurons. Its overexpression has been shown to induce neurite outgrowth in embryonic neurons and to promote retinal ganglion cell (RGC) survival and axonal regrowth in a mouse optic nerve crush model. In order to evaluate the functions of endogenous Armcx1 in vivo, we have created a conditional Armcx1 knockout mouse line in which the entire coding region of the Armcx1 gene is flanked by loxP sites. This Armcx1fl line was crossed with mouse strains in which Cre recombinase expression is driven by the promoters for β-actin and Six3, in order to achieve deletion of Armcx1 globally and in retinal neurons, respectively. Having confirmed deletion of the gene, we proceeded to characterize the abundance and morphology of RGCs in Armcx1 knockout mice aged to 15 months. Under normal physiological conditions, no evidence of aberrant retinal or optic nerve development or RGC degeneration was observed in these mice. The Armcx1fl mouse should be valuable for future studies investigating mitochondrial morphology and transport in the absence of Armcx1 and in determining the susceptibility of Armcx1-deficient neurons to degeneration in the setting of additional heritable or environmental stressors.

Assessment of corneal nerve regeneration after axotomy in a compartmentalized microfluidic chip model with automated 3D high resolution live-imaging.

Damage to the corneal nerves can result in discomfort and chronic pain, profoundly impacting the quality of life of patients. Development of novel in vitro method is crucial to better understand corneal nerve regeneration and to find new treatments for the patients. Existing in vitro models often overlook the physiology of primary sensory neurons, for which the soma is separated from the nerve endings. To overcome this limitation, our novel model combines a compartmentalized microfluidic culture of trigeminal ganglion neurons from adult mice with live-imaging and automated 3D image analysis offering robust way to assess axonal regrowth after axotomy. Physical axotomy performed by a two-second aspiration led to a reproducible 70% axonal loss and altered the phenotype of the neurons, increasing the number of substance P-positive neurons 72 h post-axotomy. To validate our new model, we investigated axonal regeneration after exposure to pharmacological compounds. We selected various targets known to enhance or inhibit axonal regrowth and analyzed their basal expression in trigeminal ganglion cells by scRNAseq. NGF/GDNF, insulin, and Dooku-1 (Piezo1 antagonist) enhanced regrowth by 81, 74 and 157%, respectively, while Yoda-1 (Piezo1 agonist) had no effect. Furthermore, SARM1-IN-2 (Sarm1 inhibitor) inhibited axonal regrowth, leading to only 6% regrowth after 72 h of exposure (versus 34% regrowth without any compound). Combining compartmentalized trigeminal neuronal culture with advanced imaging and analysis allowed a thorough evaluation of the extent of the axotomy and subsequent axonal regrowth. This innovative approach holds great promise for advancing our understanding of corneal nerve injuries and regeneration and ultimately improving the quality of life for patients suffering from sensory abnormalities, and related conditions.

Fabrication of ECM protein coated hollow collagen channels to study peripheral nerve regeneration

Peripheral nerve injury is a prevalent clinical problem that often leads to lifelong disability and reduced quality of life. Although peripheral nerves can regenerate, recovery after severe injury is slow and incomplete. The current gold standard treatment, autologous nerve transplantation, has limitations including donor site morbidity and poor functional outcomes, highlighting the need for improved repair strategies. We developed a reproducible in vitro hollow channel collagen gel construct to investigate peripheral nerve regeneration (PNR) by exploring the influence of key extracellular matrix (ECM) proteins on axonal growth and regeneration. Channels were coated with ECM proteins: collagen IV, laminin, or fibronectin and seeded with dorsal root ganglia (DRG) collected from E16 rat embryos to compare the ability of the ECM proteins to enhance axonal growth. Robust axonal extension and Schwann cell (SC) infiltration were observed in fibronectin-coated channels, suggesting its superiority over other ECM proteins. Differential effects of ECM proteins on axons and SCs indicated direct growth stimulation beyond SC-mediated guidance. In vitro laceration injury modeling further confirmed fibronectin’s superior pro-regenerative effects, showcasing its potential in enhancing axonal regrowth post-injury. Advancing in vitro modeling that closely replicates native microenvironments will accelerate progress in overcoming the limitations of current nerve repair approaches.