Cytoprotective Small Compound M109S Attenuated Retinal Ganglion Cell Degeneration Induced by Optic Nerve Crush in Mice.

BAX plays an essential role in retinal ganglion cell (RGC) death induced by optic nerve injury. Recently, we developed M109S, an orally bioactive and cytoprotective small compound (CPSC) that inhibits BAX-mediated cell death. We examined whether M109S can protect RGC from optic nerve crush (ONC)-induced apoptosis. M109S was administered starting 5 h after ONC for 7 days. M109S was orally administered in two groups (5 mg/kg twice a day or 7.5 mg/kg once a day). The retina was stained with anti-BRN3A and cleaved Caspase-3 (active Caspase-3) that are the markers of RGC and apoptotic cells, respectively. ONC decreased the number of BRN3A-positive RGC and increased the number of active Caspase-3-expressing apoptotic cells. In ONC-treated retina, there were cells that were double stained with anti-BRN3A and ant-cleaved Caspase-3, indicating that apoptosis in BRN3A-positive RGCs occurred. M109S inhibited the decrease of BRN3A-positive cells whereas it inhibited the increase of active Caspase-3-positive cells in the retina of ONC-treated mice, suggesting that M109S inhibited apoptosis in RGCs. M109S did not induce detectable histological damage to the lungs or kidneys in mice, suggesting that M109S did not show toxicities in the lung or kidneys when the therapeutic dose was used. The present study suggests that M109S is effective in rescuing damaged RGCs. Since M109S is an orally bioactive small compound, M109S may become the basis for a portable patient-friendly medicine that can be used to prevent blindness by rescuing damaged optic nerve cells from death.

CD38 Deficiency Protects Mouse Retinal Ganglion Cells Through Activating the NAD+/Sirt1 Pathway in Ischemia-Reperfusion and Optic Nerve Crush Models.

Purpose: The purpose of this study was to investigate the protective effect of CD38 deletion on retinal ganglion cells (RGCs) in a mouse retinal ischemia/reperfusion (I/R) model and an optic nerve crush (ONC) model, and to elucidate the underlying molecular mechanisms. Methods: Retinal I/R and ONC models were constructed in mice. PCR was used to identify the deletion of CD38 gene in mice, hematoxylin and eosin (H&E) staining was used to evaluate the changes in retinal morphology, and electroretinogram (ERG) was used to evaluate the changes in retinal function. The survival of RGCs and activation of retinal macroglia were evaluated by immunofluorescence staining. The expression of Sirt1, CD38, Ac-p65, Ac-p53, TNF-α, IL-1ß, and Caspase3 proteins in the retina was further evaluated by protein imprinting. Results: In retinal I/R and ONC models, CD38 deficiency reduced the loss of RGCs and activation of macroglia and protected the retinal function. CD38 deficiency increased the concentration of NAD+, reduced the degree of acetylation of NF-κB p65 and p53, and reduced expression of the downstream inflammatory cytokines TNFα, IL-1ß, and apoptotic protein Caspase3 in the retina in the ONC model. Intraperitoneal injection of the Sirt1 inhibitor EX-527 partially counteracted the effects of CD38 deficiency, suggesting that CD38 deficiency acts at least in part through the NAD+/Sirt1 pathway. Conclusions: CD38 plays an important role in the pathogenesis of retinal I/R and ONC injury. CD38 deletion protects RGCs by attenuating inflammatory responses and apoptosis through the NAD+/Sirt1 pathway.

Enriching new transplantable RGC-like cells from retinal organoids for RGC replacement therapy.

Optic neuropathies, such as glaucoma, are due to progressive retinal ganglion cells (RGCs) degeneration, result in irreversible vision loss. The promising RGCs replacement therapy for restoring vision are impeded by insufficient RGC-like cells sources. The present work was enriched one new type RGC-like cells using two surface markers CD184 and CD171 from human induced pluripotent stem cells (hiPSCs) by FACS sorting firstly. These new kind cells have well proliferation ability and possessed passage tolerance in vitro 2D or 3D spheroids culture, which kept expressing Pax6, Brn3b and ßIII-Tubulin and so on. The transplanted CD184+CD171+ RGC-like cells could survive and integrate into the normal and optic nerve crush (ONC) mice retina, especially they were more inclined to across the optic nerve head and extend to the damaged optic nerve. These data support the feasible application for cell replacement therapy in RGC degenerative diseases, as well as help to develop new commercial cells sorting reagents and establish good manufacturing practice (GMP) grade RGC-like donor cells for further clinical application.

A Comparative Investigation of Axon-Blood Vessel Growth Interaction in the Regenerating Sciatic and Optic Nerves in Adult Mice.

The vascular and the nervous systems share similarities in addition to their complex role in providing oxygen and nutrients to all cells. Both are highly branched networks that frequently grow close to one another during development. Vascular patterning and neural wiring share families of guidance cues and receptors. Most recently, this relationship has been investigated in terms of peripheral nervous system (PNS) regeneration, where nerves and blood vessels often run in parallel so endothelial cells guide the formation of the Büngner bands which support axonal regeneration. Here, we characterized the vascular response in regenerative models of the central and peripheral nervous system. After sciatic nerve crush, followed by axon regeneration, there was a significant increase in the blood vessel density 7 days after injury. In addition, the optic nerve crush model was used to evaluate intrinsic regenerative potential activated with a combined treatment that stimulated retinal ganglion cells (RGCs) regrowth. We observed that a 2-fold change in the total number of blood vessels occurred 7 days after optic nerve crush compared to the uncrushed nerve. The difference increased up to a 2.7-fold change 2 weeks after the crush. Interestingly, we did not observe differences in the total number of blood vessels 2 weeks after crush, compared to animals that had received combined treatment for regeneration and controls. Therefore, the vascular characterization showed that the increase in vascular density was not related to the efficiency of both peripheral and central axonal regeneration.

The Pulsed Nd:YAG Laser Therapy Enhanced Nerve Regeneration via Apoptosis Inhibition in a Rat Crushed Sciatic Nerve Model.

The study was aimed to validate the efficacy of the pulsed Nd:YAG laser on nerve regeneration in a rat sciatic nerve crushed model. 54 Wistar rats were randomly assigned into three groups: shame control, crush control, and laser treated group. For the laser treated group, the pulsed Nd:YAG laser (10 Hz) with 350 mJ per pulse in energy density and 50 J/cm2 in fluence was applied extracorporeally at the lesion site for 12 min to daily deliver 500 J immediately and consecutive 9 days following the crush injury. At week 1, the apoptosis-related activities in the injured nerve were examined (n = 8/each group). The sciatic functional index (SFI) was measured preoperatively and weekly until 4 weeks after the index procedure. The injured nerve and the innervated gastrocnemius muscle histology were assessed at week 4 (n = 10/each group). At week 1, the laser group showed the significant less TUNEL-positive ratio (P < 0.05), and the lower expression of cleaved caspase3/procaspase-3 and beclin-2/beclin-2-associated protein X ratios compared with the crush control. Furthermore, the laser group revealed significantly better SFI since week 1 and throughout the study (P < 0.05, all) compared with the crush control. At week 4, the laser group showed significantly higher axon density, lower myelin g-ratio, and the corresponding higher glycogen expression (P < 0.05, all) in the gastrocnemius muscle compared with those in the crush control. The pulsed Nd:YAG might enhance the injured nerve regeneration via apoptosis inhibition.

Calbindin 2-specific deletion of arginase 2 preserves visual function after optic nerve crush.

We previously found that global deletion of the mitochondrial enzyme arginase 2 (A2) limits optic nerve crush (ONC)-induced neuronal death. Herein, we examined the cell-specific role of A2 in this pathology by studies using wild type (WT), neuronal-specific calbindin 2 A2 KO (Calb2cre/+ A2 f/f), myeloid-specific A2 KO (LysMcre/+ A2f/f), endothelial-specific A2 KO (Cdh5cre/+ A2f/f), and floxed controls. We also examined the impact of A2 overexpression on mitochondrial function in retinal neuronal R28 cells. Immunolabeling showed increased A2 expression in ganglion cell layer (GCL) neurons of WT mice within 6 h-post injury and inner retinal neurons after 7 days. Calb2 A2 KO mice showed improved neuronal survival, decreased TUNEL-positive neurons, and improved retinal function compared to floxed littermates. Neuronal loss was unchanged by A2 deletion in myeloid or endothelial cells. We also found increased expression of neurotrophins (BDNF, FGF2) and improved survival signaling (pAKT, pERK1/2) in Calb2 A2 KO retinas within 24-hour post-ONC along with suppression of inflammatory mediators (IL1ß, TNFα, IL6, and iNOS) and apoptotic markers (cleavage of caspase3 and PARP). ONC increased GFAP and Iba1 immunostaining in floxed controls, and Calb2 A2 KO dampened this effect. Overexpression of A2 in R28 cells increased Drp1 expression, and decreased mitochondrial respiration, whereas ABH-induced inhibition of A2 decreased Drp1 expression and improved mitochondrial respiration. Finally, A2 overexpression or excitotoxic treatment with glutamate significantly impaired mitochondrial function in R28 cells as shown by significant reductions in basal respiration, maximal respiration, and ATP production. Further, glutamate treatment of A2 overexpressing cells did not induce further deterioration in their mitochondrial function, indicating that A2 overexpression or glutamate insult induce comparable alterations in mitochondrial function. Our data indicate that neuronal A2 expression is neurotoxic after injury, and A2 deletion in Calb2 expressing neurons limits ONC-induced retinal neurodegeneration and improves visual function.

Regional Gene Expression in the Retina, Optic Nerve Head, and Optic Nerve of Mice with Optic Nerve Crush and Experimental Glaucoma.

A major risk factor for glaucomatous optic neuropathy is the level of intraocular pressure (IOP), which can lead to retinal ganglion cell axon injury and cell death. The optic nerve has a rostral unmyelinated portion at the optic nerve head followed by a caudal myelinated region. The unmyelinated region is differentially susceptible to IOP-induced damage in rodent models and human glaucoma. While several studies have analyzed gene expression changes in the mouse optic nerve following optic nerve injury, few were designed to consider the regional gene expression differences that exist between these distinct areas. We performed bulk RNA-sequencing on the retina and separately micro-dissected unmyelinated and myelinated optic nerve regions from naïve C57BL/6 mice, mice after optic nerve crush, and mice with microbead-induced experimental glaucoma (total = 36). Gene expression patterns in the naïve unmyelinated optic nerve showed significant enrichment of the Wnt, Hippo, PI3K-Akt, and transforming growth factor ß pathways, as well as extracellular matrix-receptor and cell membrane signaling pathways, compared to the myelinated optic nerve and retina. Gene expression changes induced by both injuries were more extensive in the myelinated optic nerve than the unmyelinated region, and greater after nerve crush than glaucoma. Changes present three and fourteen days after injury largely subsided by six weeks. Gene markers of reactive astrocytes did not consistently differ between injury states. Overall, the transcriptomic phenotype of the mouse unmyelinated optic nerve was significantly different from immediately adjacent tissues, likely dominated by expression in astrocytes, whose junctional complexes are inherently important in responding to IOP elevation.

Neuroinflammation and gliosis in the injured and contralateral retinas after unilateral optic nerve crush.

The main purpose of this study is to analyze the effects of unilateral optic nerve crush in the gene expression of pro- and anti-inflammatory mediators, and gliosis markers in injured and contralateral retinas. Retinas from intact, unilaterally optic nerve injured or sham-operated C57BL/6J mice were analyzed 1, 3, 9 and 30 days after the surgery (n = 5/group and time point) and the relative expression of TGF-ß1, IL-1ß, TNF-α, Iba1, AQP4, GFAP, MHCII, and TSPO was analyzed in injured and contralateral using qPCR. The results indicated that compared with intact retinas, sham-operated animals showed an early (day 1) upregulation of IL-1ß, TNF-α and TSPO and a late (day 30) upregulation of TNF-α. In sham-contralateral retinas, TNF-α and TSPO mRNA expression were upregulated and day 30 while GFAP, Iba1, AQP4 and MHCII downregulated at day 9. Compared with sham-operated animals, in retinas affected by optic nerve crush GFAP and TSPO upregulated at day 1 and TNF-α, Iba1, AQP4 and MHCII at day 3. In the crushed-contralateral retinas, TGF-ß1, TNF-α, Iba1 and MHCII were upregulated at day 1. TSPO was upregulated up to day 30 whereas TGF-ß1 and Iba1 downregulated after day 9. In conclusion, both sham surgery and optic nerve crush changed the profile of inflammatory and gliosis markers in the injured and contralateral retinas, changes that were more pronounced for optic nerve crush when compared to sham.

Establishment of a facial nerve trunk crush injury model and evaluation of facial nerve self-healing in rats.

INTRODUCTION/AIMS: To facilitate further investigation into the mechanisms of facial nerve regeneration, a simple and reliable model of facial nerve crush injury is essential. Nevertheless, the establishment of such models lacks standardization and repeatability, while the healing capacity of the nerve is often overlooked, potentially affecting future studies. METHODS: We made facial nerve trunk crush injury models with different pressing times and detected the changes from the distal nerves to the motoneurons via behavior analysis, electrophysiological test, and histomorphometry analysis. RESULTS: It revealed a particular capacity for self-healing following facial nerve crush damage because there was almost no facial motoneuron apoptosis in the MC group during the observation period, and rats in MC group had total facial paralysis in behavioral tests following surgery and varying degrees of recovery 28 days postoperatively with no treatments. As the pressing time increased, the latency, wave amplitude, nerve fiber damage degree, nerve axon ratio, myelin thickness, electroneurograph (ENoG) value, ultrastructural damage, abnormal morphological changes, and the buccal muscle atrophy of each MC group gradually increased or got worse during the observation period. However, after 28 postoperative days, only the ENoG values of the M10min and M12min groups were beyond 90%, indicating no self-healing. DISCUSSION: It suggests that a stable model of peripheral facial palsy may be created by applying a 12.5 cm mosquito clamped to the facial nerve trunk for at least 10 min, which laid the foundation for the subsequent research to objectively evaluate facial nerve regeneration.

Optic Nerve Crush Injury in Rodents to Study Retinal Ganglion Cell Neuroprotection and Regeneration.

Optic nerve crush injury is a useful model for studying the response of central nervous system neurons (CNS) to injury. A particular focus of this model has been to elucidate therapeutic factors in promoting neuroprotection and axon regeneration after injury. Here we describe a step-by-step protocol in accessing the optic nerve and creating a crush injury. This can be used to create a reproducible model to study the response of retinal ganglion cells (RGC), the main projection neurons of the eye, to injury.

AxonQuantifier: A semi-automated program for quantifying axonal density from whole-mounted optic nerves.

BACKGROUND: Here, we present a semi-automated method for quantifying retinal ganglion cell (RGC) axon density at different distances from the optic nerve crush site using longitudinal, confocal microscopy images taken from whole-mounted optic nerves. This method employs the algorithm AxonQuantifier which operates on the freely available program, ImageJ. NEW METHOD: To validate this method, seven adult male Long Evans rats underwent optic nerve crush injury followed by in vivo treatment with electric fields of varying strengths for 30 days to produce optic nerves with a wide range of axon densities distal to the optic nerve crush site. Prior to euthanasia, RGC axons were labelled with intravitreal injections of cholera toxin B conjugated to Alexa Fluor 647. After dissection, optic nerves underwent tissue clearing, were whole-mounted, and imaged longitudinally using confocal microscopy. COMPARISON WITH EXISTING METHODS: Five masked raters quantified RGC axon density at 250, 500, 750, 1000, 1250, 1500, 1750, and 2000 µm distances past the optic nerve crush site for the seven optic nerves manually and using AxonQuantifier. Agreement between these methods was assessed using Bland-Altman plots and linear regression. Inter-rater agreement was assessed using the intra-class coefficient. RESULTS: Semi-automated quantification of RGC axon density demonstrated improved inter-rater agreement and reduced bias values as compared to manual quantification, while also increasing time efficiency 4-fold. Relative to manual quantification, AxonQuantifier tended to underestimate axon density. CONCLUSIONS: AxonQuantifier is a reliable and efficient method for quantifying axon density from whole mount optic nerves.

Long-term tactile hypersensitivity after nerve crush injury in mice is characterized by the persistence of intact sensory axons.

ABSTRACT: Traumatic peripheral nerve injuries are at high risk of neuropathic pain for which novel effective therapies are urgently needed. Preclinical models of neuropathic pain typically involve irreversible ligation and/or nerve transection (neurotmesis). However, translation of findings to the clinic has so far been unsuccessful, raising questions on injury model validity and clinically relevance. Traumatic nerve injuries seen in the clinic commonly result in axonotmesis (ie, crush), yet the neuropathic phenotype of "painful" nerve crush injuries remains poorly understood. We report the neuropathology and sensory symptoms of a focal nerve crush injury using custom-modified hemostats resulting in either complete ("full") or incomplete ("partial") axonotmesis in adult mice. Assays of thermal and mechanically evoked pain-like behavior were paralleled by transmission electron microscopy, immunohistochemistry, and anatomical tracing of the peripheral nerve. In both crush models, motor function was equally affected early after injury; by contrast, partial crush of the nerve resulted in the early return of pinprick sensitivity, followed by a transient thermal and chronic tactile hypersensitivity of the affected hind paw, which was not observed after a full crush injury. The partially crushed nerve was characterized by the sparing of small-diameter myelinated axons and intraepidermal nerve fibers, fewer dorsal root ganglia expressing the injury marker activating transcription factor 3, and lower serum levels of neurofilament light chain. By day 30, axons showed signs of reduced myelin thickness. In summary, the escape of small-diameter axons from Wallerian degeneration is likely a determinant of chronic pain pathophysiology distinct from the general response to complete nerve injury.

Effect of growth differentiation factor 11 expression after peripheral nerve injury in Sprague-Dawley rats.

OBJECTIVES: We attempt to investigate the expression pattern of GDF11 in the sciatic nerves after injury. METHODS: Thirty-six healthy male Sprague Dawley (SD) rats were divided into three groups at random and were labelled as: day 1, day 4, and day 7 post-surgery. The sciatic nerve crush model was established on the left-hind limb, while the right limb was untreated, and served as the control. Nerve samples were collected at post-injury day 1, day 4 and day 7. Nerve samples collected from the proximal and distal stump of the injury site underwent immunofluorescence staining with GDF11, NF200 and CD31. GDF11 mRNA expression was analyzed by qRT-PCR. CCK-8 assay, after si-GDF11 transfection in Schwann cells (RSC96) was applied to verify its effect in cell proliferation rate. RESULTS: GDF11 was abundantly expressed in axons stained with NF200 and Schwann cells stained with S100. However, no GDF11 expression was observed in vascular endothelial tissues stained with CD31. From day 4 onwards, the level of GDF11 showed an increasing trend, up to a twofold level at day 7 after injury. Proliferation rate of RSC96 cells showed a significant decrease after the down-regulation of GDF11 by siRNAs compared to the control group. CONCLUSIONS: GDF11 may play a role in the proliferation of Schwann cell during nerve regeneration process.

A small-molecule PI3Kα activator for cardioprotection and neuroregeneration.

Harnessing the potential beneficial effects of kinase signalling through the generation of direct kinase activators remains an underexplored area of drug development1-5. This also applies to the PI3K signalling pathway, which has been extensively targeted by inhibitors for conditions with PI3K overactivation, such as cancer and immune dysregulation. Here we report the discovery of UCL-TRO-1938 (referred to as 1938 hereon), a small-molecule activator of the PI3Kα isoform, a crucial effector of growth factor signalling. 1938 allosterically activates PI3Kα through a distinct mechanism by enhancing multiple steps of the PI3Kα catalytic cycle and causes both local and global conformational changes in the PI3Kα structure. This compound is selective for PI3Kα over other PI3K isoforms and multiple protein and lipid kinases. It transiently activates PI3K signalling in all rodent and human cells tested, resulting in cellular responses such as proliferation and neurite outgrowth. In rodent models, acute treatment with 1938 provides cardioprotection from ischaemia-reperfusion injury and, after local administration, enhances nerve regeneration following nerve crush. This study identifies a chemical tool to directly probe the PI3Kα signalling pathway and a new approach to modulate PI3K activity, widening the therapeutic potential of targeting these enzymes through short-term activation for tissue protection and regeneration. Our findings illustrate the potential of activating kinases for therapeutic benefit, a currently largely untapped area of drug development.

Park7 protects retinal ganglion cells and promotes functional preservation after optic nerve crush via regulation of the Nrf2 signaling pathway.

PURPOSE: We aim to investigate the effect of Park7 on mice RGC survival and function following optic nerve crush (ONC), and to explore its potential mechanism. METHODS: Wild-type male C57BL/6J mice were subjected to optic nerve crush. Six weeks before ONC, mice received rAAV-shRNA (Park7)-EGFP or rAAV-EGFP intravitreally. Western blotting was used to detect Park7 levels. RGC survival was measured using immunofluorescence. Retinal cell apoptosis was detected using terminal deoxynucleotidyl transferase nick-end-labelling. An electroretinogram (ERG) and the optomotor response (OMR) were used to assess RGC function. Kelch-like ECH-associated protein 1 (Keap1), nuclear factor erythroid 2-related factor (Nrf2), and heme oxygenase 1 (HO-1) levels were assessed using western blotting. RESULTS: ONC injury increased the relative expression of Park7 significantly and decreased RGC survival, the amplitude of the photopic negative response (PhNR), and OMR. Intravitreal injection of rAAV-shRNA(Park7)-EGFP downregulated Park7 expression and was clearly demonstrated by the green fluorescence protein in many retinal layers. Moreover, Park7 downregulation aggravated the decrease in RGC survival and amplitude of PhNR as well as the visual acuity after ONC. However, inhibition of Park7 significantly increased Keap1 levels, decreased the total and nuclear Nrf2 levels, and reduced HO-1 levels. CONCLUSIONS: Park7 downregulation enhanced RGC injury and decreased retinal electrophysiological response and OMR after ONC in mice via the Keap1-Nrf2-HO-1 signaling pathway. Park7 may have neuroprotective effects and could represent a novel way to treat optic neuropathy.

Microglial CD11b Knockout Contributes to Axonal Debris Clearance and Axonal Degradation Attenuation via IGF-1 After Acute Optic Nerve Injury.

Purpose: Microglial clearance of axonal debris is an essential response for management of traumatic optic neuropathy. Inadequate removal of axonal debris leads to increased inflammation and axonal degeneration after traumatic optic neuropathy. The present study investigated the role of CD11b (Itgam) in axonal debris clearance and axonal degeneration. Methods: Western blot and immunofluorescence were used to detect CD11b expression in the mouse optic nerve crush (ONC) model. Bioinformatics analysis predicted the possible role of CD11b. Cholera toxin subunit B (CTB) and zymosan were used to assay phagocytosis by microglia in vivo and in vitro, respectively. CTB was also used to label functionally intact axons after ONC. Results: CD11b is abundantly expressed after ONC and participates in phagocytosis. Microglia from Itgam-/- mice exhibited more significant phagocytosis of axonal debris than wild-type microglia. In vitro experiments confirmed that the CD11b gene defect in M2 microglia leads to increased insulin-like growth factor-1 secretion and thus promotes phagocytosis. Lastly, following ONC, Itgam-/- mice exhibited elevated expression of neurofilament heavy peptide and Tuj1, along with more intact CTB-labeled axons when compared with wild-type mice. Moreover, the inhibition of insulin-like growth factor-1 decreased CTB labeling in Itgam-/- mice after injury. Conclusions: CD11b limits microglial phagocytosis of axonal debris in traumatic optic neuropathy, as demonstrated by increased phagocytosis with CD11b knockout. The inhibition of CD11b activity may be a novel approach to promote central nerve repair.

Ligand-Induced Activation of GPR110 (ADGRF1) to Improve Visual Function Impaired by Optic Nerve Injury.

It is extremely difficult to achieve functional recovery after axonal injury in the adult central nervous system. The activation of G-protein coupled receptor 110 (GPR110, ADGRF1) has been shown to stimulate neurite extension in developing neurons and after axonal injury in adult mice. Here, we demonstrate that GPR110 activation partially restores visual function impaired by optic nerve injury in adult mice. Intravitreal injection of GPR110 ligands, synaptamide and its stable analogue dimethylsynaptamide (A8) after optic nerve crush significantly reduced axonal degeneration and improved axonal integrity and visual function in wild-type but not gpr110 knockout mice. The retina obtained from the injured mice treated with GPR110 ligands also showed a significant reduction in the crush-induced loss of retinal ganglion cells. Our data suggest that targeting GPR110 may be a viable strategy for functional recovery after optic nerve injury.

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.

Effect of Azithromycin on Sciatic Nerve Injury in the Wistar Rats.

After a severe peripheral nerve injury, complete functional recovery is rare. Modulating the inflammatory response could be an effective way to enhance peripheral nerve regeneration. The present study aimed to determine the effect of azithromycin on functional recovery following sciatic nerve crush in Wistar rats. 40 male Wistar rats were used in four groups, including: the negative control, sham, and two groups of azithromycin (15 and 150 mg/kg/day) (n = 10).The rats' right sciatic nerve was crushed using a non-serrated clamp. In experimental groups, animals were treated with azithromycin (15 and 150 mg/kg/day) for 7 days. Then, sensory-motor functions were evaluated over eight weeks. Real-time PCR was used to measure the expression of NGF and BDNF genes. At the end of the 4th week, the sensory recovery accelerated in the azithromycin-treated rats so that the reaction times in the groups treated with 15 mg/kg and 150 mg/kg doses of azithromycin reached 5.14 s and 6.61 s, respectively, which were significantly lower than the 12 s in the negative control group (P < 0.05).Eventually, the mean SFI values in the negative control and both azithromycin-treated groups recovered to preoperative levels in the 8th week, with no significant difference between the sciatic lesion groups. Findings showed a seven-day course of azithromycin administered immediately after a sciatic nerve crush could accelerate regeneration and improve motor and sensory function recovery compared to negative controls. These significant effects were observed in both the azithromycin 15 mg/kg and the azithromycin 150 mg/kg treatment groups. Azithromycin treatment upregulated the expression of NGF and BDNF genes in crushed sciatic nerve. Our findings suggest that a seven-day treatment of azithromycin after a sciatic nerve injury could accelerate the regeneration process and improve functional recovery.

The spatial-temporal reactive changes of compressed optic nerve in a clinically relevant rabbit model of persistent compressive optic nerve axonopathy.

The optic nerve (ON) can get compressed in different diseases. However, the pathological and functional changes occurring in the compressed ON over time under constant compression are still unclear. In the present study, we implanted an artificial tube around the optic nerve of a rabbit to primarily create a clinically relevant persistent compressive optic nerve axonopathy (PCOA). Due to the protuberance on the inner ring of the tube, steady and persistent compressions were maintained. In this model, we investigated the thickness of ganglion cell complex (GCC), retinal ganglion cell (RGC) density, axon density of optic nerve, flash visual evoked potential (FVEP), and anterograde axonal transport at various times in four different groups viz. the no comp, 1/2 comp, 3/4 comp, and crush groups. The GCC thickness, RGC density, and axon density of ON were hierarchically and significantly decreased in 1/2 comp, 3/4 comp, and crush groups. Compared to no comp eyes, the P2 amplitude ratio of FVEP was significantly decreased in 3/4 comp but not in 1/2 comp eyes. Only a portion of the optic nerve lost the ability of anterograde axonal transport in the 1/2 comp group. However, it was evident at 2-wpo and more prominent at 4-wpo in 3/4 comp eyes. This study reveals that the compression only induces the homolateral ON axons impairment and the proportion of the affected axons maintains the same for mild compression for at least three months. Furthermore, an underlying threshold effect highlights that mild compression does not require urgent surgery, while the severe compression warrants immediate surgical intervention.