Transcriptome Analyses Reveal Systematic Molecular Pathology After Optic Nerve Crush

The function of glial cells in axonal regeneration after injury has been the subject of controversy in recent years. Thus, deeper insight into glial cells is urgently needed. Many studies on glial cells have elucidated the mechanisms of a certain gene or cell type in axon regeneration. However, studies that manipulate a single variable may overlook other changes. Here, we performed a series of comprehensive transcriptome analyses of the optic nerve head over a period of 90 days after optic nerve crush (ONC), showing systematic molecular changes in the optic nerve head (ONH). Furthermore, using weighted gene coexpression network analysis (WGCNA), we established gene module programs corresponding to various pathological events at different times post-ONC and found hub genes that may be potential therapeutic targets. In addition, we analyzed the changes in different glial cells based on their subtype markers. We revealed that the transition trend of different glial cells depended on the time course, which provides clues for modulating glial function in further research.

Citrus Naringenin Increases Neuron Survival in Optic Nerve Crush Injury Model by Inhibiting JNK-JUN Pathway

Traumatic nerve injury activates cell stress pathways, resulting in neuronal death and loss of vital neural functions. To date, there are no available neuroprotectants for the treatment of traumatic neural injuries. Here, we studied three important flavanones of citrus components, in vitro and in vivo, to reveal their roles in inhibiting the JNK (c-Jun N-terminal kinase)-JUN pathway and their neuroprotective effects in the optic nerve crush injury model, a kind of traumatic nerve injury in the central nervous system. Results showed that both neural injury in vivo and cell stress in vitro activated the JNK-JUN pathway and increased JUN phosphorylation. We also demonstrated that naringenin treatment completely inhibited stress-induced JUN phosphorylation in cultured cells, whereas nobiletin and hesperidin only partially inhibited JUN phosphorylation. Neuroprotection studies in optic nerve crush injury mouse models revealed that naringenin treatment increased the survival of retinal ganglion cells after traumatic optic nerve injury, while the other two components had no neuroprotective effect. The neuroprotection effect of naringenin was due to the inhibition of JUN phosphorylation in crush-injured retinal ganglion cells. Therefore, the citrus component naringenin provides neuroprotection through the inhibition of the JNK-JUN pathway by inhibiting JUN phosphorylation, indicating the potential application of citrus chemical components in the clinical therapy of traumatic optic nerve injuries.

Polydopamine nanoparticles attenuate retina ganglion cell degeneration and restore visual function after optic nerve injury

Background Oxidative stress contributes to retina ganglion cells (RGCs) loss in variety of ocular diseases, including ocular trauma, ocular vein occlusion, and glaucoma. Scavenging the excessed reactive oxygen species (ROS) in retinal neurovascular unit could be beneficial to RGCs survival. In this study, a polydopamine (PDA)-based nanoplatform is developed to protect RGCs. Results The PDA nanoparticles efficiently eliminate multi-types of ROS, protect endothelia and neuronal cells from oxidative damage, and inhibit microglia activation in retinas. In an optic nerve crush (ONC) model, single intravitreal injection of PDA nanoparticles could significantly attenuate RGCs loss via eliminating ROS in retinas, reducing the inflammatory response and maintaining barrier function of retinal vascular endothelia. Comparative transcriptome analysis of the retina implied that PDA nanoparticles improve RGCs survival probably by altering the expression of genes involved in inflammation and ROS production. Importantly, as a versatile drug carrier, PDA nanoparticles could deliver brimonidine (a neuroprotection drug) to synergistically attenuate RGCs loss and promote axon regeneration, thus restore visual function. Conclusions The PDA nanoparticle-based therapeutic nanoplatform displayed excellent performance in ROS elimination, providing a promising probability for treating retinal degeneration diseases. Graphical Abstract

CD82 protects against glaucomatous axonal transport deficits via mTORC1 activation in mice

Glaucoma is a leading cause of irreversible blindness worldwide and is characterized by progressive optic nerve degeneration and retinal ganglion cell loss. Axonal transport deficits have been demonstrated to be the earliest crucial pathophysiological changes underlying axonal degeneration in glaucoma. Here, we explored the role of the tetraspanin superfamily member CD82 in an acute ocular hypertension model. We found a transient downregulation of CD82 after acute IOP elevation, with parallel emergence of axonal transport deficits. The overexpression of CD82 with an AAV2/9 vector in the mouse retina improved optic nerve axonal transport and ameliorated subsequent axon degeneration. Moreover, the CD82 overexpression stimulated optic nerve regeneration and restored vision in a mouse optic nerve crush model. CD82 exerted a protective effect through the upregulation of TRAF2, which is an E3 ubiquitin ligase, and activated mTORC1 through K63-linked ubiquitylation and intracellular repositioning of Raptor. Therefore, our study offers deeper insight into the tetraspanin superfamily and demonstrates a potential neuroprotective strategy in glaucoma treatment.

Calcium Homeostasis in Parvalbumin DRG Neurons is Altered After Sciatic Nerve Crush and Sciatic Nerve Transection Injuries

Reflex abnormalities mediated by proprioceptive sensory neurons after peripheral nerve injury (PNI) can limit functional improvement, leaving patients with disability that affects their quality of life. We examined post-injury calcium transients in a subpopulation of DRG neurons consisting primarily of proprioceptors to determine whether alterations in calcium homeostasis are present in proprioceptors, as has been documented in other DRG neurons after PNI. Using transgenic mice, we restricted expression of the calcium indicator GCaMP6s to DRG neurons containing parvalbumin (PV). Mice of both sexes were randomly assigned to sham, sciatic nerve crush, or sciatic nerve transection and resuture conditions. Calcium transients were recorded from ex-vivo preparations of animals at one of three post-surgery time points: 1-3 days, 7-11 days, and after 60 days of recovery. Results demonstrated that the post-PNI calcium transients of PV DRG neurons are significantly different than sham. Abnormalities were not present during the acute response to injury (1-3 days), but transients were significantly different than sham at the recovery stage where axon regeneration is thought to be underway (7-11 days). During late-stage recovery (60 days post-injury), disturbances in the decay time course of calcium transients in transection animals persisted, whereas parameters of transients from crush animals returned to normal. These findings identify a deficit in calcium homeostasis in proprioceptive neurons, which may contribute to the failure to fully recover proprioceptive reflexes after PNI. Significant differences in the calcium transients of crush versus transection animals after reinnervation illustrate calcium homeostasis alterations are distinctive to injury type.

Effect of unacylated ghrelin on peripheral nerve regeneration

Ghrelin is a circulating peptide hormone released by enteroendocrine cells of the gastrointestinal tract as two forms, acylated and unacylated. Acylated ghrelin (AG) binds to the growth hormone secretagogue receptor 1a (GHSR1a), thus stimulating food intake, growth hormone release, and gastrointestinal motility. Conversely, unacylated GHR (UnAG), through binding to a yet unidentified receptor, protects the skeletal muscle from atrophy, stimulates muscle regeneration, and protects cardiomyocytes from ischemic damage. Recently, interest about ghrelin has raised also among neuroscientists because of its effect on the nervous system, especially the stimulation of neurogenesis in spinal cord, brain stem, and hippocampus. However, few information is still available about its effectiveness on peripheral nerve regeneration. To partially fill this gap, the aim of this study was to assess the effect of UnAG on peripheral nerve regeneration after median nerve crush injury and after nerve transection immediately repaired by means of an end-to-end suture. To this end, we exploited FVB1 Myh6/Ghrl transgenic mice in which overexpression of the ghrelin gene (Ghrl) results in selective up-regulation of circulating UnAG levels, but not of AG. Regeneration was assessed by both functional evaluation (grasping test) and morphometrical analysis of regenerated myelinated axons. Results obtained lead to conclude that UnAG could have a role in development of peripheral nerves and during more severe lesions.

Interactions Among Nerve Regeneration, Angiogenesis, and the Immune Response Immediately After Sciatic Nerve Crush Injury in Sprague-Dawley Rats

To preliminarily explore the primary changes in the expression of genes involved in peripheral nerve processes, namely, regeneration, angiogenesis, and the immune response, and to identify important molecular therapeutic targets, 45 Sprague-Dawley (SD) rats were randomly divided into a control group and an injury group. In the injury group, tissue samples were collected at 4 and 7 days after the injury for next-generation sequencing (NGS) analysis combined with gene ontology (GO) analysis, Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis and Venn diagram construction to identify the differentially expressed mRNAs (DEmRNAs) associated with regeneration, angiogenesis, and the immune response of the nerve. The expression of genes in the distal and proximal ends of the injured nerve after injury was analyzed by qRT-PCR. NGS revealed that compared with the control group, the injury group had 4020 DEmRNAs 4 days after injury and 3278 DEmRNAs 7 days after injury. A bioinformatics analysis showed that C-C chemokine receptor type 5 (CCR5), Thy1 cell surface antigen (Thy1), Notch homolog 1 (Notch1), and semaphorin 4A (Sema4A) were all associated with regeneration, angiogenesis, and the immune response of the nerve at both 4 and 7 days after injury, but qPCR revealed no significant difference in the expression of Thy1 (P = 0.29) or Sema4A (P = 0.82) in the proximal end, whereas a significant difference was observed in CCR5 and Notch1 (P < 0.05). The trend in the Notch1 change was basically consistent with the RNA-seq result after injury, which implied its indispensable role during endothelial cell proliferation and migration, macrophage recruitment, and neurotrophic factor secretion.

Polydopamine Nanoparticles Attenuate Retina Ganglion Cell Degeneration and Restore Visual Function After Optic Nerve Injury

Background: Oxidative stress contributes to retina ganglion cells (RGCs) loss in variety of ocular diseases, including ocular trauma, ocular vein occlusion, and glaucoma. Scavenging the excessed reactive oxygen species (ROS) in retinal neurovascular unit could be beneficial to RGCs survival. In this study, a polydopamine (PDA)-based nanoplatform is developed to protect RGCs. Results: The PDA nanoparticles efficiently eliminate multi-types of ROS, protect endothelia and neuronal cells from oxidative damage, and inhibit microglia activation in retinas. In an optic nerve crush (ONC) model, single intravitreal injection of PDA nanoparticles could significantly attenuate RGCs loss via eliminating ROS in retinas, reducing the inflammatory response and maintaining barrier function of retinal vascular endothelia. Comparative transcriptome analysis of the retina implied that PDA nanoparticles improve RGCs survival probably by altering the expression of genes involved in inflammation and ROS production. Importantly, as a versatile drug carrier, PDA nanoparticles could deliver brimonidine (a neuroprotection drug) to synergistically attenuate RGCs loss and promote axon regeneration, thus restore visual function. Conclusions: the PDA nanoparticle-based therapeutic nanoplatform displayed excellent performance in ROS elimination, providing a promising probability for treating retinal degeneration diseases.