Satellite glial cell manipulation prior to axotomy enhances developing dorsal root ganglion central branch regrowth into the spinal cord.

The central and peripheral nervous systems (CNS and PNS, respectively) exhibit remarkable diversity in the capacity to regenerate following neuronal injury with PNS injuries being much more likely to regenerate than those that occur in the CNS. Glial responses to damage greatly influence the likelihood of regeneration by either promoting or inhibiting axonal regrowth over time. However, despite our understanding of how some glial lineages participate in nerve degeneration and regeneration, less is known about the contributions of peripheral satellite glial cells (SGC) to regeneration failure following central axon branch injury of dorsal root ganglia (DRG) sensory neurons. Here, using in vivo, time-lapse imaging in larval zebrafish coupled with laser axotomy, we investigate the role of SGCs in axonal regeneration. In our studies we show that SGCs respond to injury by relocating their nuclei to the injury site during the same period that DRG neurons produce new central branch neurites. Laser ablation of SGCs prior to axon injury results in more neurite growth attempts and ultimately a higher rate of successful central axon regrowth, implicating SGCs as inhibitors of regeneration. We also demonstrate that this SGC response is mediated in part by ErbB signaling, as chemical inhibition of this receptor results in reduced SGC motility and enhanced central axon regrowth. These findings provide new insights into SGC-neuron interactions under injury conditions and how these interactions influence nervous system repair.

A fluid-walled microfluidic platform for human neuron microcircuits and directed axotomy.

In our brains, different neurons make appropriate connections; however, there remain few in vitro models of such circuits. We use an open microfluidic approach to build and study neuronal circuits in vitro in ways that fit easily into existing bio-medical workflows. Dumbbell-shaped circuits are built in minutes in standard Petri dishes; the aqueous phase is confined by fluid walls - interfaces between cell-growth medium and an immiscible fluorocarbon, FC40. Conditions are established that ensure post-mitotic neurons derived from human induced pluripotent stem cells (iPSCs) plated in one chamber of a dumbbell remain where deposited. After seeding cortical neurons on one side, axons grow through the connecting conduit to ramify amongst striatal neurons on the other - an arrangement mimicking unidirectional cortico-striatal connectivity. We also develop a moderate-throughput non-contact axotomy assay. Cortical axons in conduits are severed by a media jet; then, brain-derived neurotrophic factor and striatal neurons in distal chambers promote axon regeneration. As additional conduits and chambers are easily added, this opens up the possibility of mimicking complex neuronal networks, and screening drugs for their effects on connectivity.

Neuron-specific RNA-sequencing reveals different responses in peripheral neurons after nerve injury.

Peripheral neurons are heterogeneous and functionally diverse, but all share the capability to switch to a pro-regenerative state after nerve injury. Despite the assumption that the injury response is similar among neuronal subtypes, functional recovery may differ. Understanding the distinct intrinsic regenerative properties between neurons may help to improve the quality of regeneration, prioritizing the growth of axon subpopulations to their targets. Here, we present a comparative analysis of regeneration across four key peripheral neuron populations: motoneurons, proprioceptors, cutaneous mechanoreceptors, and nociceptors. Using Cre/Ai9 mice that allow fluorescent labeling of neuronal subtypes, we found that nociceptors showed the greater regeneration after a sciatic crush, followed by motoneurons, mechanoreceptors, and, finally, proprioceptors. By breeding these Cre mice with Ribotag mice, we isolated specific translatomes and defined the regenerative response of these neuronal subtypes after axotomy. Only 20% of the regulated genes were common, revealing a diverse response to injury among neurons, which was also supported by the differential influence of neurotrophins among neuron subtypes. Among differentially regulated genes, we proposed MED12 as a specific regulator of the regeneration of proprioceptors. Altogether, we demonstrate that the intrinsic regenerative capacity differs between peripheral neuron subtypes, opening the door to selectively modulate these responses.

Functional Recovery Associated with Dendrite Regeneration in PVD Neuron of Caenorhabditis elegans.

PVD neuron of Caenorhabditis elegans is a highly polarized cell with well-defined axonal, and dendritic compartments. PVD neuron operates in multiple sensory modalities including the control of both nociceptive touch sensation and body posture. Although both the axon and dendrites of this neuron show a regeneration response following laser-assisted injury, it is rather unclear how the behavior associated with this neuron is affected by the loss of these structures. It is also unclear whether neurite regrowth would lead to functional restoration in these neurons. Upon axotomy, using a femtosecond laser, we saw that harsh touch response was specifically affected leaving the body posture unperturbed. Subsequently, recovery in the touch response is highly correlated to the axon regrowth, which was dependent on DLK-1/MLK-1 MAP Kinase. Dendrotomy of both major and minor primary dendrites affected the wavelength and amplitude of sinusoidal movement without any apparent effect on harsh touch response. We further correlated the recovery in posture behavior to the type of dendrite regeneration events. We found that dendrite regeneration through the fusion and reconnection between the proximal and distal branches of the injured dendrite corresponded to improved recovery in posture. Our data revealed that the axons and dendrites of PVD neurons regulate the nociception and proprioception in worms, respectively. It also revealed that dendrite and axon regeneration lead to the restoration of these differential sensory modalities.

In Vivo Imaging of Secondary Neurodegeneration Associated With Phosphatidylserine Externalization Along Axotomized Axons.

Purpose: Axonal degeneration in acute and chronic disorders is well-characterized, comprising retrograde (proximal) and Wallerian (distal) degeneration, but the mechanism of propagation remains less understood. Methods: Laser injury with a diode-pumped solid-state 532 nm laser was used to axotomize retinal ganglion cell axons. We used confocal in vivo imaging to demonstrate that phosphatidylserine externalization is a biomarker of early axonal degeneration after selective intraretinal axotomy. Results: Quantitative dynamic analysis revealed that the rate of axonal degeneration was fastest within 40 minutes, then decreased exponentially afterwards. Axonal degeneration was constrained within the same axotomized axonal bundles. Remarkably, axon degeneration arising from the site of injury induced a secondary degeneration of distal normal axons. Conclusions: Axonal degeneration in vivo is a progressive process associated with phosphatidylserine externalization, which can propagate not only along the axon but to adjacent uninjured axons. This finding has implications for acute and chronic neurodegenerative disorders associated with axonal injury.

Preparation of Viable Human Neurites for Neurobiological and Neurodegeneration Studies.

Few models allow the study of neurite damage in the human central nervous system. We used here dopaminergic LUHMES neurons to establish a culture system that allows for (i) the observation of highly enriched neurites, (ii) the preparation of the neurite fraction for biochemical studies, and (iii) the measurement of neurite markers and metabolites after axotomy. LUHMES-based spheroids, plated in culture dishes, extended neurites of several thousand µm length, while all somata remained aggregated. These cultures allowed an easy microscopic observation of live or fixed neurites. Neurite-only cultures (NOC) were produced by cutting out the still-aggregated somata. The potential application of such cultures was exemplified by determinations of their protein and RNA contents. For instance, the mitochondrial TOM20 protein was highly abundant, while nuclear histone H3 was absent. Similarly, mitochondrial-encoded RNAs were found at relatively high levels, while the mRNA for a histone or the neuronal nuclear marker NeuN (RBFOX3) were relatively depleted in NOC. Another potential use of NOC is the study of neurite degeneration. For this purpose, an algorithm to quantify neurite integrity was developed. Using this tool, we found that the addition of nicotinamide drastically reduced neurite degeneration. Also, the chelation of Ca2+ in NOC delayed the degeneration, while inhibitors of calpains had no effect. Thus, NOC proved to be suitable for biochemical analysis and for studying degeneration processes after a defined cut injury.

Restoration of injured motoneurons reduces microglial proliferation in the adult rat facial nucleus.

In the axotomized facial nucleus (axotFN), the levels of choline acetyltransferase, vesicular acetylcholine transporter, and gamma amino butyric acid A receptor α1 are decreased, after which the microglia begin to proliferate around injured motoneuron cell bodies. We conjectured that an injury signal released from the injured motoneurons triggers the microglial proliferation in the axotFN. However, it is unclear whether the level of microglial proliferation is dependent on the degree of motoneuronal insult. In this study, we investigated the relationship between the extents of motoneuronal injury and microglial proliferation in a rat axotFN model. Administration of glial cell line-derived neurotrophic factor, N-acetyl L-cysteine, or salubrinal at the transection site ameliorated the increase in c-Jun and the reductions in levels of phosphorylated cAMP response element binding protein (p-CREB) and functional molecules in the injured motoneurons. Concurrently, the levels of the microglial marker ionized calcium-binding adapter molecule 1 and of macrophage colony-stimulating factor (cFms), proliferating cell nuclear antigen, and p-p38/p38 were significantly downregulated in microglia. These results demonstrate that the recovery of motoneuron function resulted in the reduction in microglial proliferation. We conclude that the degree of neuronal injury regulates the levels of microglial proliferation in the axotFN.

Exogenous Hsp70 exerts neuroprotective effects in peripheral nerve rupture model.

Hsp70 is the main molecular chaperone responsible for cellular proteostasis under normal conditions and for restoring the conformation or utilization of proteins damaged by stress. Increased expression of endogenous Hsp70 or administration of exogenous Hsp70 is known to exert neuroprotective effects in models of many neurodegenerative diseases. In this study, we have investigated the effect of exogenous Hsp70 on recovery from peripheral nerve injury in a model of sciatic nerve transection in rats. It was shown that recombinant Hsp70 after being added to the conduit connecting the ends of the nerve at the site of its extended severance, migrates along the nerve into the spinal ganglion and is retained there at least three days. In animals with the addition of recombinant Hsp70 to the conduit, a decrease in apoptosis in the spinal ganglion cells after nerve rupture, an increase in the level of PTEN-induced kinase 1 (PINK1), an increase in markers of nerve tissue regeneration and a decrease in functional deficit were observed compared to control animals. The obtained data indicate the possibility of using recombinant Hsp70 preparations to accelerate the recovery of patients after neurotrauma.

The Role of Hydrogen Sulfide in the Localization and Expression of p53 and Cell Death in the Nervous Tissue in Traumatic Brain Injury and Axotomy.

Traumatic brain injury (TBI) is one of the leading causes of disability and death worldwide. It is characterized by various molecular-cellular events, with the main ones being apoptosis and damage to axons. To date, there are no clinically effective neuroprotective drugs. In this study, we examined the role of hydrogen sulfide (H2S) in the localization and expression of the key pro-apoptotic protein p53, as well as cell death in the nervous tissue in TBI and axotomy. We used a fast donor (sodium sulphide, Na2S) H2S and a classic inhibitor (aminooxyacetic acid, AOAA) of cystathionine ß-synthase (CBS), which is a key enzyme in H2S synthesis. These studies were carried out on three models of neurotrauma in vertebrates and invertebrates. As a result, it was found that Na2S exhibits a pronounced neuroprotective effect that reduces the number of TUNEL-positive neurons and glial cells in TBI and apoptotic glia in axotomy. This effect could be realized through the Na2S-dependent decrease in the level of p53 in the cells of the nervous tissue of vertebrates and invertebrates, which we observed in our study. We also observed the opposite effect when using AOAA, which indicates the important role of CBS in the regulation of p53 expression and death of neurons and glial cells in TBI and axotomy.

Laser-Induced Axotomy of Human iPSC-Derived and Murine Primary Neurons Decreases Somatic Tau and AT8 Tau Phosphorylation: A Single-Cell Approach to Study Effects of Acute Axonal Damage.

The microtubule-associated protein Tau is highly enriched in axons of brain neurons where it regulates axonal outgrowth, plasticity, and transport. Efficient axonal Tau sorting is critical since somatodendritic Tau missorting is a major hallmark of Alzheimer's disease and other tauopathies. However, the molecular mechanisms of axonal Tau sorting are still not fully understood. In this study, we aimed to unravel to which extent anterograde protein transport contributes to axonal Tau sorting. We developed a laser-based axotomy approach with single-cell resolution and combined it with spinning disk confocal microscopy enabling multi live-cell monitoring. We cultivated human iPSC-derived cortical neurons and mouse primary forebrain neurons in specialized chambers allowing reliable post-fixation identification and Tau analysis. Using this approach, we achieved high post-axotomy survival rates and observed axonal regrowth in a subset of neurons. When we assessed somatic missorting and phosphorylation levels of endogenous human or murine Tau at different time points after axotomy, we surprisingly did not observe somatic Tau accumulation or hyperphosphorylation, regardless of their regrowing activity, consistent for both models. These results indicate that impairment of anterograde transit of Tau protein and acute axonal damage may not play a role for the development of somatic Tau pathology. In sum, we developed a laser-based axotomy model suitable for studying the impact of different Tau sorting mechanisms in a highly controllable and reproducible setting, and we provide evidence that acute axon loss does not induce somatic Tau accumulation and AT8 Tau phosphorylation. UV laser-induced axotomy of human iPSC-derived and mouse primary neurons results in decreased somatic levels of endogenous Tau and AT8 Tau phosphorylation.

Interleukin (IL)-1 Receptor Signaling Is Required for Complete Taste Bud Regeneration and the Recovery of Neural Taste Responses following Axotomy.

Experimental or traumatic nerve injury causes the degeneration of associated taste buds. Unlike most sensory systems, the sectioned nerve and associated taste buds can then regenerate, restoring neural responses to tastants. It was previously unknown whether injury-induced immune factors mediate this process. The proinflammatory cytokines, interleukin (IL)-1α and IL-1ß, and their requisite receptor are strongly expressed by anterior taste buds innervated by the chorda tympani nerve. We tested taste bud regeneration and functional recovery in mice lacking the IL-1 receptor. After axotomy, the chorda tympani nerve regenerated but was initially unresponsive to tastants in both WT and Il1r KO mice. In the absence of Il1r signaling, however, neural taste responses remained minimal even >8 weeks after injury in both male and female mice, whereas normal taste function recovered by 3 weeks in WT mice. Failed recovery was because of a 57.8% decrease in regenerated taste buds in Il1r KO compared with WT axotomized mice. Il1a gene expression was chronically dysregulated, and the subset of regenerated taste buds were reinnervated more slowly and never reached full volume as progenitor cell proliferation lagged in KO mice. Il1r signaling is thus required for complete taste bud regeneration and the recovery of normal taste transmission, likely by impairing taste progenitor cell proliferation. This is the first identification of a cytokine response that promotes taste recovery. The remarkable plasticity of the taste system makes it ideal for identifying injury-induced mechanisms mediating successful regeneration and recovery.SIGNIFICANCE STATEMENT Taste plays a critical role in nutrition and quality of life. The adult taste system is highly plastic and able to regenerate following the disappearance of most taste buds after experimental nerve injury. Several growth factors needed for taste bud regeneration have been identified, but we demonstrate the first cytokine pathway required for the recovery of taste function. In the absence of IL-1 cytokine signaling, taste bud regeneration is incomplete, preventing the transmission of taste activity to the brain. These results open a new direction in revealing injury-specific mechanisms that could be harnessed to promote the recovery of taste perception after trauma or disease.

Epigenomic Profiling of Dorsal Root Ganglia upon Regenerative and Non-regenerative Axonal Injury.

RNA sequencing (RNA-seq), chromatin immunoprecipitation sequencing (ChIP-seq), and assay for transposase-accessible chromatin sequencing (ATAC-seq) are genome-wide techniques that provide information relative to gene expression, chromatin binding sites, and chromatin accessibility, respectively. Here we describe RNA-seq, H3K9ac, H3K27ac and H3K27me3 ChIP-seq, and ATAC-seq in dorsal root ganglia (DRG) after sciatic nerve or dorsal column axotomy, to characterize the transcriptional and epigenetic signatures of DRG upon regenerative vs non-regenerative axonal lesion.

Live Imaging of Axonal Dynamics After Laser Axotomy of Peripheral Neurons in Zebrafish.

Axon severing results in diverse outcomes, including successful regeneration and reestablishment of function, failure to regenerate, or neuronal cell death. Experimentally injuring an axon makes it possible to study degeneration of the distal stump that was detached from the cell body and document the successive steps of regeneration. Precise injury reduces damage to the environment surrounding an axon, and thereby the involvement of extrinsic processes, such as scarring or inflammation, enabling researchers to isolate the role that intrinsic factors play in regeneration. Several methods have been used to sever axons, each with advantages and disadvantages. This chapter describes using a laser on a two-photon microscope to cut individual axons of touch-sensing neurons in zebrafish larvae, and live confocal imaging to monitor its regeneration, a method that provides exceptional resolution.

Drosophila Laser Axotomy Injury Model to Investigate RNA Repair and Splicing in Axon Regeneration.

The limited axon regeneration capacity of mature neurons often leads to insufficient functional recovery after damage to the central nervous system (CNS). To promote CNS nerve repair, there is an urgent need to understand the regeneration machinery in order to develop effective clinical therapies. To this aim, we developed a Drosophila sensory neuron injury model and the accompanying behavioral assay to examine axon regeneration competence and functional recovery after injury in the peripheral and central nervous systems. Specifically, we used a two-photon laser to induce axotomy and performed live imaging to assess axon regeneration, combined with the analysis of the thermonociceptive behavior as a readout of functional recovery. Using this model, we found that the RNA 3'-terminal phosphate cyclase (Rtca), which acts as a regulator for RNA repair and splicing, responds to injury-induced cellular stress and impedes axon regeneration after axon breakage. Here we describe how we utilize our Drosophila model to assess the role of Rtca during neuroregeneration.

Glycolytic System in Axons Supplement Decreased ATP Levels after Axotomy of the Peripheral Nerve.

Wallerian degeneration (WD) occurs in the early stages of numerous neurologic disorders, and clarifying WD pathology is crucial for the advancement of neurologic therapies. ATP is acknowledged as one of the key pathologic substances in WD. The ATP-related pathologic pathways that regulate WD have been defined. The elevation of ATP levels in axon contributes to delay WD and protects axons. However, ATP is necessary for the active processes to proceed WD, given that WD is stringently managed by auto-destruction programs. But little is known about the bioenergetics during WD. In this study, we made sciatic nerve transection models for GO-ATeam2 knock-in rats and mice. We presented the spatiotemporal ATP distribution in the injured axons with in vivo ATP imaging systems, and investigated the metabolic source of ATP in the distal nerve stump. A gradual decrease in ATP levels was observed before the progression of WD. In addition, the glycolytic system and monocarboxylate transporters (MCTs) were activated in Schwann cells following axotomy. Interestingly, in axons, we found the activation of glycolytic system and the inactivation of the tricarboxylic acid (TCA) cycle. Glycolytic inhibitors, 2-deoxyglucose (2-DG) and MCT inhibitors, a-cyano-4-hydroxycinnamic acid (4-CIN) decreased ATP and enhanced WD progression, whereas mitochondrial pyruvate carrier (MPC) inhibitors (MSDC-0160) did not change. Finally, ethyl pyruvate (EP) increased ATP levels and delayed WD. Together, our findings suggest that glycolytic system, both in Schwann cells and axons, is the main source of maintaining ATP levels in the distal nerve stump.

Multifocal electroretinography increases following experimental glaucoma in nonhuman primates with retinal ganglion cell axotomy.

PURPOSE: To determine whether short-latency changes in multifocal electroretinography (mfERG) observed in experimental glaucoma (EG) are secondary solely to retinal ganglion cell (RGC) loss or whether there is a separate contribution from elevated intraocular pressure (IOP). METHODS: Prior to operative procedures, a series of baseline mfERGs were recorded from six rhesus macaques using a 241-element unstretched stimulus. Animals then underwent hemiretinal endodiathermy axotomy (HEA) by placing burns along the inferior 180° of the optic nerve margin in the right eye (OD). mfERG recordings were obtained in each animal at regular intervals following for 3-4 months to allow stabilization of the HEA effects. Laser trabecular meshwork destruction (LTD) to elevate IOP was then performed; first-order kernel (K1) waveform root-mean-square (RMS) amplitudes for the short-latency segment of the mfERG wave (9-35 ms) were computed for two 7-hexagon groupings-the first located within the superior (non-axotomized) macula and the second within the inferior (axotomized) macula. Immunohistochemistry for glial fibrillary acidic protein (GFAP) was done. RESULTS: By 3 months post HEA, there was marked thinning of the inferior nerve fiber layer as measured by optical coherence tomography. Compared with baseline, no statistically significant changes in 9-35 ms K1 RMS amplitudes were evident in either the axotomized or non-axotomized portions of the macula. Following LTD, mean IOP in HEA eyes rose to 46 ± 9 compared with 20 ± 2 mmHg (SD) in the fellow control eyes. In the HEA + EG eyes, statistically significant increases in K1 RMS amplitude were present in both the axotomized inferior and non-axotomized superior portions of the OD retinas. No changes in K1 RMS amplitude were found in the fellow control eyes from baseline to HEA epoch, but there was a smaller increase from baseline to HEA + EG. Upregulation of GFAP in the Müller cells was evident in both non-axotomized and axotomized retina in eyes with elevated IOP. CONCLUSIONS: The RMS amplitudes of the short-latency mfERG K1 waveforms are not altered following axotomy but undergo marked increases following elevated IOP. This suggests that the increase in mfERG amplitude was not solely a result of RGC loss and may reflect photoreceptor and bipolar cell dysfunction and/or changes in Müller cells.

Transcutaneous and Direct Electrical Stimulation of Mouse Sciatic Nerve Accelerates Functional Recovery After Nerve Transection and Immediate Repair.

BACKGROUND: Electrical stimulation can accelerate peripheral nerve regeneration after injury and repair. Clinically, direct electrical stimulation (DES) may involve longer operating times, increasing risks of perioperative complications. Transcutaneous electrical stimulation (TCES) is a noninvasive alternative. In this study, we investigate how transcutaneous and DES compare for accelerating functional nerve recovery in a mouse sciatic nerve model. METHODS: Twenty-eight mice were divided into sham (n = 4), axotomy (n = 8), DES (n = 8), and TCES (n = 8) groups. After sciatic nerve transection and repair, the proximal nerve was subjected to DES or TCES at 20 Hz for 1 hour. Sciatic functional index was measured before the injury, and at weeks 1, 2, 4, 6, 8, 10, and 12 by walking-track analysis. Electrophysiological measures were taken at week 12. RESULTS: Kinematic studies showed significant improvement from the 8th week to the 12th week for both electrical stimulation groups compared with the axotomy group (P < 0.05), with no difference between the electrical stimulation groups. At the 12th week, both DES and TCES groups had significantly faster average conduction velocity than the axotomy group. CONCLUSIONS: Functional recovery was significantly better from 8 weeks onward in mice receiving either DES or TCES stimulation when compared with axotomy and repair alone. Transcutaneous electrical stimulation is a minimally invasive alternative treatment for accelerating functional recovery after peripheral nerve injury.

Comparative Analysis of Retinal Organotypic Cultures and In Vivo Axotomized Retinas.

Retinal organotypic cultures (ROCs) are used as an in vivo surrogate to study retinal ganglion cell (RGC) loss and neuroprotection. In vivo, the gold standard to study RGC degeneration and neuroprotection is optic nerve lesion. We propose here to compare the course of RGC death and glial activation between both models. The left optic nerve of C57BL/6 male mice was crushed, and retinas analyzed from 1 to 9 days after the injury. ROCs were analyzed at the same time points. As a control, intact retinas were used. Retinas were studied anatomically to assess RGC survival, microglial, and macroglial activation. Macroglial and microglial cells showed different morphological activation between models and were activated earlier in ROCs. Furthermore, microglial cell density in the ganglion cell layer was always lower in ROCs than in vivo. RGC loss after axotomy and in vitro followed the same trend up to 5 days. Thereafter, there was an abrupt decrease in viable RGCs in ROCs. However, RGC somas were still immuno-identified by several molecular markers. ROCs are useful for proof-of-concept studies on neuroprotection, but long-term experiments should be carried out in vivo. Importantly, the differential glial activation observed between models and the concomitant death of photoreceptors that occurs in vitro may alter the efficacy of RGC neuroprotective therapies when tested in in vivo models of optic nerve injury.

Axotomy results in an increase in Thy-1 protein in the 35-day-old rat supraoptic nucleus.

We demonstrated previously that the hypothalamic supraoptic nucleus (SON) undergoes an axonal sprouting response following a unilateral lesion of the hypothalamo-neurohypophysial tract in a 35-day-old rat to repopulate the partially denervated neural lobe (NL). However, no sprouting occurs following the same injury in a 125-day-old rat. We previously reported a significant increase in Thy-1 protein in the SON of a 125-day-old rat compared to a 35-day-old rat in the absence of injury. Thy-1 is a cell surface glycoprotein shown to inhibit axonal outgrowth following injury; however, we did not look at axotomy's effect on Thy-1 in the SON. Therefore, we sought to determine the integrin ligands that bind Thy-1 in the SON and how axotomy impacts Thy-1. Like what others have shown, the co-immunoprecipitation analysis demonstrated that Thy-1 interacts with αvß3 and αvß5 integrin dimers in the SON. We used western blot analysis to examine protein levels of Thy-1 and integrin subunits following injury in the 35- and 125-day-old rat SON and NL. Our results demonstrated that Thy-1 protein levels increase in the lesion SON in a 35-day-old rat. The quantitative dual-fluorescent analysis showed that the increase in Thy-1 in the lesion SON occurred in astrocytes. There was no change in Thy-1 or integrin protein levels following injury in the 125-day-old following injury. Furthermore, the axotomy significantly decreased Thy-1 protein levels in the NL of both 35- and 125-day-old rats. These results provide evidence that Thy-1 protein levels are injury dependent in the magnocellular neurosecretory system.

Facial nerve axotomy induces morphological changes in hippocampal pyramidal neurons.

Facial nerve injury in rats have been widely used to study functional and structural changes that occur in the injured motoneurons and other central nervous system structures related with sensorimotor processing. A decrease in long-term potentiation of hippocampal CA3-to-CA1 commissural synapse has recently been reported related to this peripheral injury. Additionally, it has been found increased corticosterone plasmatic levels, impairment in spatial memory consolidation, and hippocampal microglial activation in animals with facial nerve axotomy. In this work, we analyzed the neuronal morphology of hippocampal CA1 and CA3 pyramidal neurons in animals with either reversible or irreversible facial nerve injury. For this purpose, brain tissues of injured animals sacrificed at different postlesion times, were stained with the Golgi-Cox method and compared with control brains. It was found that both reversible and irreversible facial nerve injury-induced significant decreases in dendritic tree complexity, dendritic length, branch points, and spine density of hippocampal neurons. However, such changes' timing varied according to hippocampal area (CA1 vs. CA3), dendritic area (apical vs. basal), and lesion type (reversible vs. irreversible). In general, the observed changes were transient when animals had the possibility of motor recovery (reversible injury), but perdurable if the recovery from the lesion was impeded (irreversible injury). CA1 apical and CA3 basal dendritic tree morphology were more sensible to irreversible injury. It is concluded that facial nerve injury induced significant changes in hippocampal CA1 and CA3 pyramidal neurons morphology, which could be related to LTP impairments and microglial activation in the hippocampal formation, previously described.