Nerve Regeneration Research Articles

Influencing factors and repair advancements in rodent models of peripheral nerve regeneration.

Peripheral nerve injuries lead to severe functional impairments, with rodent models essential for studying regeneration. This review examines key factors affecting outcomes. Age-related declines, like reduced nerve fiber density and impaired axonal transport of vesicles, hinder recovery. Hormonal differences influence regeneration, with BDNF/trkB critical for testosterone and nerve growth factor for estrogen signaling pathways. Species and strain selection impact outcomes, with C57BL/6 mice and Sprague-Dawley rats exhibiting varying regenerative capacities. Injury models - crush for early regeneration, chronic constriction for neuropathic pain, stretch for traumatic elongation and transection for severe lacerations - provide insights into clinically relevant scenarios. Repair techniques, such as nerve grafts and conduits, show that autografts are the gold standard for gaps over 3cm, with success influenced by graft type and diameter. Time course analysis highlights crucial early degeneration and regeneration phases within the first month, with functional recovery stabilizing by three to six months. Early intervention optimizes regeneration by reducing scar tissue formation, while later interventions focus on remyelination. Understanding these factors is vital for designing robust preclinical studies and translating research into effective clinical treatments for peripheral nerve injuries.

Immunomodulation by the combination of statin and matrix-bound nanovesicle enhances optic nerve regeneration

Modulating inflammation is critical to enhance nerve regeneration after injury. However, clinically applicable regenerative therapies that modulate inflammation have not yet been established. Here, we demonstrate synergistic effects of the combination of an HMG-CoA reductase inhibitor, statin/fluvastatin and critical components of the extracellular matrix, Matrix-Bound Nanovesicles (MBV) to enhance axon regeneration and neuroprotection after mouse optic nerve injury. Mechanistically, co-intravitreal injections of fluvastatin and MBV robustly promote infiltration of monocytes and neutrophils, which lead to RGC protection and axon regeneration. Furthermore, monocyte infiltration is triggered by elevated expression of CCL2, a chemokine, in the superficial layer of the retina after treatment with a combination of fluvastatin and MBV or IL-33, a cytokine contained within MBV. Finally, this therapy can be further combined with AAV-based gene therapy blocking anti-regenerative pathways in RGCs to extend regenerated axons. These data highlight novel molecular insights into the development of immunomodulatory regenerative therapy.

Nanozyme Hydrogels Promote Nerve Regeneration in Spinal Cord Injury by Reducing Oxidative Stress.

Inhibiting secondary cell death and promoting neuronal regeneration are critical for nerve repair after spinal cord injury (SCI). The excessive accumulation of reactive oxygen species (ROS) after SCI causes cell death and induces apoptosis. These reactions further increase the level of ROS production, leading to a vicious cycle of spinal cord tissue damage. Therefore, intervention targeting ROS is a potential therapeutic approach to improve the recovery of locomotor function after SCI. In this study, we designed and synthesized a nanozyme hydrogel delivery system loaded with multiple drugs, LA/Me/Se NPs-h. LA/Me/Se NPs-h exhibited a satisfactory size distribution and excellent stability, enhancing the bioavailability of therapeutic drugs. Moreover, we explored the antioxidant and protective effects of LA/Me/Se NPs-h against oxidative stress-induced cell damage caused by ROS production after SCI in vitro. In the mice SCI model, the Basso mouse scale and gait analysis showed that LA/Me/Se NPs-h significantly promoted the recovery of locomotor function after SCI. The histological and immunofluorescence results of the injury site revealed that LA/Me/Se NPs-h upregulated the expression of GFAP, NF-200, and superoxide dismutase in spinal cord lesion, reduced caspase-3 expression, improved spinal cord continuity, reduced lesion cavity, and inhibited the axonal demyelination. Consequently, LA/Me/Se NPs-h increased the activity of antioxidant enzymes and reduced neuronal apoptosis by reducing oxidative stress and ultimately promoted nerve regeneration. Taken together, this study demonstrated promising nanozyme hydrogels and provided an effective therapeutic strategy for SCI and other ROS-related diseases.

Low-Energy extracorporeal shockwave therapy improves locomotor functions, tissue regeneration and modulating the inflammation induced FGF1 and FGF2 signaling to protect damaged tissue in spinal cord injury of rat model: An experimental animal study.

Spinal cord injury (SCI) is a debilitating condition that results in severe motor function impairments. Current therapeutic options remain limited, underscoring the need for novel treatments. Extracorporeal shockwave therapy (ESWT) has emerged as a promising noninvasive approach for treating musculoskeletal disorders and nerve regeneration. This study explored the effects of low-energy ESWT on locomotor function, tissue regeneration, inflammation, and mitochondrial function in a rat SCI model. Experiments were performed using locomotor function assays, CatWalk gait analysis, histopathological examination, immunohistochemical and immunofluorescence staining. The findings demonstrated that low-energy ESWT had a dose-dependent effect, with three treatment sessions (ESWT3) showing superior outcomes compared to a single session. ESWT3 significantly improved motor functions (run patterns, run average speed, and maximum variation, as well as the Basso, Beattie, and Bresnahan (BBB) score) and promoted tissue regeneration while reducing inflammation. ESWT3 significantly decreased levels of IL-1β, IL6 and macrophages (CD68) while increasing leucocyte (CD45) infiltration. Additionally, ESWT3 upregulated NueN and mitofusin 2 (MFN2), suggesting enhanced neuronal health and mitochondrial function. Moreover, ESWT3 modulated the expression of fibroblast growth factor 1 (FGF1), FGF2, their receptor FGFR1 and phosphorylation of ERK, aiding tissue repair and regeneration in SCI. This study highlights the potential of low-energy ESWT as an effective noninvasive treatment for SCI, demonstrating significant improvements in motor recovery, tissue regeneration, anti-inflammatory effects, and mitochondrial protection. These findings provide valuable insights into the mechanisms of ESWT and its therapeutic application for SCI recovery.

Long-term nerve regeneration in diabetic keratopathy mediated by a novel NGF delivery system.

Diabetic keratopathy (DK) is a common chronic metabolic disorder that causes ocular surface complications. Among various therapeutic approaches, local delivery of nerve growth factor (NGF) remains the most effective treatment for DK. However, achieving a sustained therapeutic effect with NGF and the frequent drug delivery burden remain challenging during clinical practice. Here, we developed a novel adeno-associated virus (AAV)-based NGF delivery system that achieved one-year-long-lasting effects by a single injection. We refined the corneal stromal injection technique, resulting in reduced corneal edema and improved AAV distribution homogeneity. AAV serotype AAV.rh10 exhibited high tropism and specificity to corneal nerves. A dose of 2×109 vector genomes (vg) was determined to achieve efficient Ngf gene expression without inducing corneal immune responses. Moreover, NGF protein was highly expressed in trigeminal ganglion (TG) through a retrograde transport mechanism, indicating the capacity for repairing corneal nerve damage both at the root and corneal nerve endings. In a mouse DK model, a single injection of AAV-Ngf into the corneal stroma led to marked corneal nerve regeneration for over 5 months. Together, we provide a novel therapeutic paradigm for long-term effective treatment of DK and this therapeutic approach is superior to current DK therapies.

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.

Multilevel analysis of the central-peripheral-target organ pathway: contributing to recovery after peripheral nerve injury.

Peripheral nerve injury is a common neurological condition that often leads to severe functional limitations and disabilities. Research on the pathogenesis of peripheral nerve injury has focused on pathological changes at individual injury sites, neglecting multilevel pathological analysis of the overall nervous system and target organs. This has led to restrictions on current therapeutic approaches. In this paper, we first summarize the potential mechanisms of peripheral nerve injury from a holistic perspective, covering the central nervous system, peripheral nervous system, and target organs. After peripheral nerve injury, the cortical plasticity of the brain is altered due to damage to and regeneration of peripheral nerves; changes such as neuronal apoptosis and axonal demyelination occur in the spinal cord. The nerve will undergo axonal regeneration, activation of Schwann cells, inflammatory response, and vascular system regeneration at the injury site. Corresponding damage to target organs can occur, including skeletal muscle atrophy and sensory receptor disruption. We then provide a brief review of the research advances in therapeutic approaches to peripheral nerve injury. The main current treatments are conducted passively and include physical factor rehabilitation, pharmacological treatments, cell-based therapies, and physical exercise. However, most treatments only partially address the problem and cannot complete the systematic recovery of the entire central nervous system-peripheral nervous system-target organ pathway. Therefore, we should further explore multilevel treatment options that produce effective, long-lasting results, perhaps requiring a combination of passive (traditional) and active (novel) treatment methods to stimulate rehabilitation at the central-peripheral-target organ levels to achieve better functional recovery.

Nervonic acid as novel therapeutics initiates both neurogenesis and angiogenesis for comprehensive wound repair and healing

Rapid tissue reconstruction in acute and chronic injuries are challengeable, the inefficient repair mainly due to the difficulty in simultaneous promoting the regeneration of peripheral nerves and vascular, which are closely related. Main clinical medication strategy of tissue repair depends on different cytokines to achieve nerves, blood vessels or granulation tissue regeneration, respectively. However, their effect is still limited to single aspect with biorisk exists upon long-time use. Herein, for the first time, we have demonstrated that NA isolated from Malania oleifera has potential to simultaneously promote both neurogenesis and angiogenesis in vitro and in vivo. First, NA was identified by NMR and FTIR structural characterization analysis. In a model of oxidative stress in neural cells induced by hydrogen peroxide, the cells viability of RSC96 and PC12 were protected from oxidative stress injury by NA. Similarly, based on the rat wound healing model, effective blood vessel formation and wound healing can be observed in tissue staining under NA treatment. In addition, according to the identification of nerve and vascular related markers in the wound tissue, the mechanism of NA promoting nerve regeneration lies in the upregulation of the secretion NGF, NF-200 and S100 protein, and NA treatment was also able to up-regulate VEGF and CD31 to directly promote angiogenesis during wound healing. This study provides an important candidate drug molecules for acute or chronic wound healing and nerve vascular synchronous regeneration.

Advances in therapies using mesenchymal stem cells and their exosomes for treatment of peripheral nerve injury: state of the art and future perspectives.

"Peripheral nerve injury" refers to damage or trauma affecting nerves outside the brain and spinal cord. Peripheral nerve injury results in movements or sensation impairments, and represents a serious public health problem. Although severed peripheral nerves have been effectively joined and various therapies have been offered, recovery of sensory or motor functions remains limited, and efficacious therapies for complete repair of a nerve injury remain elusive. The emerging field of mesenchymal stem cells and their exosome-based therapies hold promise for enhancing nerve regeneration and function. Mesenchymal stem cells, as large living cells responsive to the environment, secrete various factors and exosomes. The latter are nano-sized extracellular vesicles containing bioactive molecules such as proteins, microRNA, and messenger RNA derived from parent mesenchymal stem cells. Exosomes have pivotal roles in cell-to-cell communication and nervous tissue function, offering solutions to changes associated with cell-based therapies. Despite ongoing investigations, mesenchymal stem cells and mesenchymal stem cell-derived exosome-based therapies are in the exploratory stage. A comprehensive review of the latest preclinical experiments and clinical trials is essential for deep understanding of therapeutic strategies and for facilitating clinical translation. This review initially explores current investigations of mesenchymal stem cells and mesenchymal stem cell-derived exosomes in peripheral nerve injury, exploring the underlying mechanisms. Subsequently, it provides an overview of the current status of mesenchymal stem cell and exosome-based therapies in clinical trials, followed by a comparative analysis of therapies utilizing mesenchymal stem cells and exosomes. Finally, the review addresses the limitations and challenges associated with use of mesenchymal stem cell-derived exosomes, offering potential solutions and guiding future directions.

In Vitro and In Vivo Methods for Studying Retinal Ganglion Cell Survival and Optic Nerve Regeneration.

Glaucoma is marked by a progressive degeneration of the optic nerve and delayed loss of retinal ganglion cells (RGCs), the projection neurons of the eye. Because RGCs are not replaced and because surviving RGCs cannot regenerate their axons, the visual loss in glaucoma is largely irreversible. Here we describe methods to evaluate treatments that may be beneficial for treating glaucoma using in vitro cell culture models (immunopanning to isolate neonatal RGCs, dissociated mature retinal neurons, retinal explants) and in vivo models that test potential treatments or investigate underlying molecular mechanisms in an intact system. Potentially, the use of these models can help investigators continue to improve treatments to preserve RGCs and restore visual function in patients with glaucoma.

Combination of nusinersen and rehabilitation using hybrid assistive limb for type 3 spinal muscular atrophy

AbstractAn antisense oligonucleotide drug nusinersen can improve motor function of spinal muscular atrophy (SMA) patients. The Hybrid Assistive Limb (HAL) is a wearable robot assisting the wearers' movements in real time. Gait rehabilitation using HAL is useful for SMA. However, the efficacy of combined treatment has never been reported before. We have investigated the efficacy of combining both therapies. A 21‐year‐old man, genetically diagnosed with type 3 SMA, was treated by combining nusinersen and five sessions of gait rehabilitation using HAL over 2 years. Hammersmith Functional Motor Scale‐Expanded (HFMSE) score improved by 12 points after treatment. Electrophysiological parameters of the tibial nerves improved. Muscle volume also increased by 8% at 39 months. The combined therapy resulted in a good clinical outcome. This may be due to promotion of nerve regeneration and recovery of muscle volume by a synergy of nusinersen and motor learning correctly guided by HAL.

Outcome of End to Side Nerve Modified Double Oberlin Procedure and End to End Dorsal Approach Spinal Accessory to Suprascapular Nerve in Partial Postganglionic BPI: A Case Report

Introduction: Brachial plexus injury (BPI) is a devastating peripheral nerve injury that mostly affecting young men, often due to motor vehicle accidents. BPIs can be classified into preganglionic and postganglionic lesions, with postganglionic injuries allowing potential nerve regeneration. The Oberlin method, aimed at reinnervating the biceps, and the Mackinnon technique, targeting both biceps and brachialis muscles, have shown promising outcomes in BPI management. Case report: A 45-year-old man presented with right hand weakness and occasional tingling in the upper arm and fingers two months after a motorcycle accident. Motor examination revealed muscle strength of 0/5 in the right trapezius, rhomboids, serratus anterior (positive winging scapula), pectoralis minor and major, latissimus dorsi, deltoid, biceps, and elbow flexion. Wrist and finger functions remained intact. Diagnosed with post-ganglionic BPI, the patient underwent two nerve transfer surgeries: the Mackinnon technique, transferring the spinal accessory nerve to the suprascapular nerve to reinnervate shoulder muscles, and the Double Oberlin procedure, transferring fascicles from the median and ulnar nerves to the musculocutaneous nerve for reinnervation of the biceps and brachialis muscles. Post-surgery, the patient engaged in an aggressive rehabilitation program focusing on strength, range of motion, and sensory re-education. Over nine months, the patient showed significant improvement in elbow flexion (MRC grade 3), wrist extension (MRC grade 5), hand grip strength (MRC grade 3) with improved sensation across the affected limb. He returned to work and daily activities with mild residual weakness and sensory deficits. Discussion: This case illustrates the effectiveness of the Mackinnon and Double Oberlin nerve transfer techniques in restoring function in a patient with total post-ganglionic BPI. The Mackinnon technique enabled shoulder muscle reinnervation, essential for abduction and stabilization, while the Double Oberlin procedure enhanced elbow flexion by reinnervating both the biceps and brachialis muscles. The combined surgical and intensive rehabilitation approach facilitated significant functional recovery. Conclusion: The successful application of the Double Oberlin and Mackinnon techniques in this case demonstrates their potential in managing total post-ganglionic BPI. Early surgical intervention and consistent rehabilitation are crucial for optimizing recovery outcomes. This case highlights the importance of a multidisciplinary approach in treating complex peripheral nerve injuries. Keywords: Double Oberlin, Mackinnon, Brachial Plexus Injury

Toll-Like Receptor 4 (TLR4) Promotes DRG Regeneration and Repair after Sciatic Nerve Injury via the ERK-NF-kB Pathway.

Previously, we found that the expression of Toll-like receptor 4 (TLR4) is altered after sciatic nerve injury, and its differential expression plays a key role in recovery. However, the mechanisms by which TLR4 affects neuronal function in the dorsal root ganglion (DRG) have not been completely evaluated. The objective is to determine TLR4 expression in DRG tissues after sciatic neural injury and exploring the effects of TLR4 knockdown and overexpression in the DRG on neuronal function and nerve regeneration in rats in vivo and in vitro. We established a model of nerve injury and utilized molecular biology and cell biology experiments to explore the molecular mechanisms by which TLR4 in the DRG affects sciatic nerve restoration and regeneration after injury. Verified the localization of TLR4 in DRG neurons. Investigated pathways that related to apoptosis or nerve regeneration by which TLR4 regulates the function of DRG neurons. TLR4 expression was upregulated in the DRG tissues of rats after sciatic nerve injury. TLR4 overexpression promoted axon regeneration and inhibited apoptosis in DRG neurons. TLR4 promoted the regeneration of axons and the recovery of motor and sensory functions in the sciatic nerve after injury in vivo, and the data showed that TLR4 may regulate the function of DRG neurons and promote nerve repair and regeneration through the ERK and NF-κB signaling pathways in vivo and ex vivo. The study suggests that TLR4 may regulate the function of DRG neurons and promote nerve regeneration by affecting the ERK and NF-κB signaling pathways.

Impact of Platelet-Rich Plasma on Sciatic Nerve Injury in Rats: HSP 70 Expression and Histological Changes in the Anterior Horn of the Spinal Cord

Peripheral nerve injuries are a significant and growing cause of disability worldwide, as reported by the World Health Organization (WHO). These injuries initiate complex cellular and molecular responses, including the activation of protective pathways that upregulate proteins such as Heat Shock Protein 70 (HSP 70), which plays a crucial role in neuroprotection and the inhibition of neuronal apoptosis. This study investigates the effects of Platelet-Rich Plasma (PRP) on peripheral nerve regeneration, focusing on neuron density, Nissl body presence, and HSP 70 expression as key indicators of recovery. The study utilized 36 rat spinal cord samples, divided into two main groups based on the evaluation time points, day 7 and day 42 post-injury. Each time point group was further subdivided into three: control, sciatica injury, and sciatica injury treated with PRP. Results demonstrated that PRP treatment significantly enhances peripheral nerve regeneration. This was evidenced by increased neuron density, improved Nissl body formation, and a modulation of HSP 70 expression levels, leading to accelerated recovery of motor function, particularly noticeable by day 7 post-injury. These findings suggest that PRP could be a promising therapeutic option for enhancing nerve regeneration and functional recovery after peripheral nerve injuries.

Electrospun PCL/Alginate/Nanoclay Nerve Conduit with Olfactory Ectomesenchymal Stem Cells for Nerve Regeneration.

Biocompatible and biodegradable nerve growth conduits (NGCs) provide a promising alternative to conventional nerve grafting for peripheral nerve regeneration. Incorporating nanoclay (NC) has been shown to increase the hydrophilicity and flexibility of polymeric scaffolds. In the present study, poly caprolactone-alginate (PCL-ALG) conduits with varying percentages of NC (0.1%, 0.2%, and 0.5%) were fabricated using the electrospinning technique. The conduit containing 0.5% NC showed a greater increase in elongation (33%) and porosity, reaching 95% with the lowest contact angle (10°). For in vitro, human olfactory ectomesenchymal stem cells (OE-MSCs) were used as a favorable choice for neuronal differentiation owing to the origin from the neural crest. The viability and proliferation of OE-MSCs were maintained after 5 days on scaffolds with 0.5% NC, as confirmed by the MTT assay, cell adhesion analysis, and live/dead staining. Furthermore, the impact of 0.5% PCL-ALG-NC on the paracrine activity of OE-MSCs was studied for a period of 7 days. Our results indicated that human OE-MSCs, when cocultured with PC12 cells on NGC, have the capability to release nerve growth factor levels of up to 1392.83 pg/mL. In summary, the electrospun PCL-ALG conduit containing an optimal NC dosage (0.5%) and seeded with human OE-MSCs shows promising outcomes as NGC scaffold for peripheral nerve regeneration.

Macrophages’ Functions in the Central and Peripheral Nervous Regeneration

Macrophages’ Functions in the Central and Peripheral Nervous Regeneration

Marine bioactives: a new frontier in neurosurgical therapeutics.

Bioactives derived from marine sources are opening up new therapeutic opportunities for neurosurgery. These include tissue regeneration, neuroprotection, and infection control. These molecules derived from extreme marine conditions have generated new modes of action, for example, selective inhibition of voltage-gated ion channels and influencing pathways of neuroinflammation. Representative examples include chitosan for nerve regeneration, conotoxins for pain modulation, and fucoidan for neuroprotection. Despite therapeutic promise, major challenges remain: those of sustainable sourcing and regulatory validation. This letter points up the potential of marine bioactives to change neurosurgical care and challenges the brain communities to work together across disciplines in order to make that happen.

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.

Impact of Neurostimulation, Immunomodulation, Topical Medication Application, and Surgical Reconstruction on Corneal Nerve Function and Regeneration.

The corneal epithelium, supplied by thousands of nerve endings, plays a substantial role in absorbing and distributing nutrients along the ocular surface. Many studies have explored the influence of various modalities in regulating tear production to manage corneal disorders and dry eye disease. These findings have highlighted the advantages of enhancing corneal nerve function and regeneration through neurostimulation, neural signaling, immunomodulation, topical medication application, and surgical reconstruction. The purpose of this narrative review article was to provide an overview of the current state of knowledge on this topic based on a PubMed database literature search for relevant animal and human studies investigating the modification of the trigeminal pathway to restore corneal nerve function and improve overall ocular health. Further investigation into this area of research is important to help guide new therapeutic targets for the prevention and development of treatments of corneal degeneration.

Design and construction of a GAP-43 reporter system for potential identification of effective therapeutics for peripheral nerve regeneration

Background: Peripheral nerve injury (PNI) is a condition that can result in muscle paralysis and sensory disturbances. Electrical stimulation and/or the application of exogenous neurotrophic factors and cytokines are effective at enhancing nerve regeneration and is mediated via the expression of regeneration associated genes (RAGs) such as the growth associated protein GAP-43. Therapeutic upregulation of GAP-43 has potential use as a treatment for improving recovery from PNI. Few studies have investigated the potential of increasing GAP-43 for PNI therapeutic purposes and current methods for measuring GAP-43 expression are limited. Aims and Objectives: The broader aim of this work was to construct a motor neuron-like cell model with a GAP-43 reporter system. Such a model would have potential use in screening for novel therapeutics that upregulate GAP-43 and in the optimisation of electrical stimulation treatment in combination with these therapies. The key aim of the work was to design and construct a Cas9 expressing plasmid bearing a gRNA that targets GAP-43 cleavage and a donor plasmid bearing a reporter GFP or Neo cassette flanked with 5’ and 3’ GAP-43 homology arms (HAs) to facilitate the insertion of the reporter immediately 3’ of the GAP-43 promoter via CRISPR/Cas9 homology directed repair (HDR). Such an insertion would enable quantitative measurement of endogenous expression of the GAP-43 gene. Methods and Results: To guide the Cas9 nuclease to the target location, GAP-43 gRNA oligomers were designed and cloned downstream of the U6 promoter in the Cas9 expression plasmid px330, which also expresses the Cas9 gene and the cloned gRNA when transfected into cells. For CRISPR/Cas9 HDR, the 5’ and 3’ GAP-43 HAs were amplified from mouse genomic DNA and cloned into the donor plasmid using Gibson Assembly so that they flanked the reporter cassette. Results: This work successfully constructed the Cas9 gRNA expressing plasmid to target cleavage of the GAP-43 gene in mouse cell lines and has provided the complete design and construction foundation for generation of the reporter system for the endogenous GAP-43 gene in mice.