ABSTRACT
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.
Subject(s)
Axons , Wallerian Degeneration , Animals , Rats , Mice , Axotomy , Axons/metabolism , Wallerian Degeneration/metabolism , Sciatic Nerve/metabolism , Adenosine Triphosphate/metabolism , Nerve Regeneration/physiologyABSTRACT
Achyranthes bidentata polypeptide k (ABPPk), a powerful active component from a traditional Chinese medicinal herb-Achyranthes bidentata Bl., has exhibited promising neuroprotective activity due to its multiple-targeting capability. However, the effect of ABPPk on the survival, growth and axonal regeneration of spinal cord motor neurons remains unclear. Here, a modified method, which is more optimized for embryonic cells in ambient carbon dioxide levels, was used for acquisition of rat embryonic spinal cord motor neurons with high survival and purity. ABPPk concentration-dependently enhanced the neuronal viability and promoted the neurite outgrowth. Co-culture of motor neurons and skeletal myocytes model indicated that ABPPk enhanced the neuromuscular junction development and maturation. A microfluidic axotomy model was further established for the axonal disconnection, and ABPPk significantly accelerated the axonal regeneration of motor neurons. Furthermore, we demonstrated that the upregulation of three neurofilament protein subunits in motor neurons might be relevant to the mechanisms of the growth-promoting effect of ABPPk. Our findings provide an experimental and theoretical basis for the development of ABPPk as a potential application in the development of treatment strategy for nerve injury diseases.
Subject(s)
Achyranthes , Axons/drug effects , Motor Neurons/drug effects , Muscle Fibers, Skeletal/drug effects , Nerve Regeneration/drug effects , Neuromuscular Junction/drug effects , Neuronal Outgrowth/drug effects , Plant Extracts/pharmacology , Animals , Axotomy , Cell Survival/drug effects , Coculture Techniques , GAP-43 Protein/drug effects , GAP-43 Protein/metabolism , In Vitro Techniques , Neurofilament Proteins/drug effects , Neurofilament Proteins/metabolism , Peptides/pharmacology , Peripheral Nerve Injuries , Primary Cell Culture , Rats , Rats, Sprague-Dawley , Spinal Cord/cytologyABSTRACT
Following facial nerve axotomy, nerve function is not fully restored even after reconstruction. This may be attributed to axon degeneration/neuronal death and sustained neuroinflammation. CD38 is an enzyme that catalyses the hydrolysis of nicotinamide adenine dinucleotide (NAD+) and is a candidate molecule for regulating neurodegeneration and neuroinflammation. In this study, we analyzed the effect of CD38 deletion and NAD+ supplementation on neuronal death and glial activation in the facial nucleus in the brain stem, and on axon degeneration and immune cell infiltration in the distal portion of the facial nerve after axotomy in mice. Compared with wild-type mice, CD38 knockout (KO) mice showed reduced microglial activation in the facial nucleus, whereas the levels of neuronal death were not significantly different. In contrast, the axon degeneration and demyelination were delayed, and macrophage accumulation was reduced in the facial nerve of CD38 KO mice after axotomy. Supplementation of NAD+ with nicotinamide riboside slowed the axon degeneration and demyelination, although it did not alter the level of macrophage infiltration after axotomy. These results suggest that CD38 deletion and supplementation of NAD+ may protect transected axon cell-autonomously after facial nerve axotomy.
Subject(s)
ADP-ribosyl Cyclase 1/metabolism , Axons/physiology , Axotomy/methods , Facial Nerve Diseases/metabolism , Facial Nerve/pathology , NAD/metabolism , ADP-ribosyl Cyclase 1/genetics , Animals , Cell Count , Cells, Cultured , Dietary Supplements , Disease Models, Animal , Facial Nerve Diseases/genetics , Facial Nerve Diseases/therapy , Humans , Mice , Mice, Inbred ICR , Mice, Knockout , Nerve DegenerationABSTRACT
Neuronal injury leads to rapid, programmed disintegration of axons distal to the site of lesion. Much like other forms of axon degeneration (e.g. developmental pruning, toxic insult from neurodegenerative disorder), Wallerian degeneration associated with injury is preceded by spheroid formation along axons. The mechanisms by which injury leads to formation of spheroids and whether these spheroids have a functional role in degeneration remain elusive. Here, using neonatal mouse primary sympathetic neurons, we investigate the roles of players previously implicated in the progression of Wallerian degeneration in injury-induced spheroid formation. We find that intra-axonal calcium flux is accompanied by actin-Rho dependent growth of calcium rich axonal spheroids that eventually rupture, releasing material to the extracellular space prior to catastrophic axon degeneration. Importantly, after injury, Sarm1-/- and DR6-/-, but not Wlds (excess NAD+) neurons, are capable of forming spheroids that eventually rupture, releasing their contents to the extracellular space to promote degeneration. Supplementation of exogenous NAD+ or expressing WLDs suppresses Rho-dependent spheroid formation and degeneration in response to injury. Moreover, injured or trophically deprived Sarm1-/- and DR6-/-, but not Wlds neurons, are resistant to degeneration induced by conditioned media collected from wild-type axons after spheroid rupture. Taken together, these findings place Rho-actin and NAD+ upstream of spheroid formation and may suggest that other mediators of degeneration, such as DR6 and SARM1, mediate post-spheroid rupture events that lead to catastrophic axon disassembly.
Subject(s)
Armadillo Domain Proteins/physiology , Cytoskeletal Proteins/physiology , Nerve Tissue Proteins/physiology , Neurodegenerative Diseases/pathology , Neurons/pathology , Receptors, Tumor Necrosis Factor/physiology , Spheroids, Cellular/pathology , Wallerian Degeneration/physiopathology , Animals , Axons/pathology , Axotomy , Calcium/metabolism , Female , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Neurodegenerative Diseases/etiology , Neurodegenerative Diseases/metabolism , Neurons/metabolismABSTRACT
A network of intersecting molecular pathways interacts to initiate and execute axon destruction. Maximum protection against axon degeneration likely requires more than manipulation of a single target. Here, we describe the process of designing a high-throughput arrayed screening assay for the identification of key factors responsible for axon destruction and/or protection. First, we go over some existing screens in the literature, then discuss the planning, tracking, analysis, and statistics around such a screening experiment. Prioritization of perturbations may allow laboratories to cost-effectively explore the process of screening. We also present the pairing of a combinatorial drug screen with a machine learning algorithm, predicting how to best modulate neurodegenerative and neuroprotective components.
Subject(s)
Axons/physiology , High-Throughput Screening Assays/methods , Nerve Degeneration/physiopathology , Animals , Axotomy , CRISPR-Cas Systems , Combinatorial Chemistry Techniques , Computer Simulation , Drug Evaluation, Preclinical/methods , Finches/embryology , High-Throughput Screening Assays/instrumentation , Image Processing, Computer-Assisted , Phenotype , Quality Control , RNA Interference , Retinal Ganglion Cells/cytology , Sensitivity and Specificity , Support Vector MachineABSTRACT
ETHNOPHARMACOLOGICAL RELEVANCE: Buyang huanwu decoction (BYHWD) is a classic recipe in traditional Chinese medicine (TCM) to supplement Qi and activate blood. It has been used to recover the neural function after the injury of central nervous system for hundreds of years in China. AIM OF THE STUDY: This study investigated whether Buyang huanwu decoction (BYHWD) combined with bone marrow mesenchymal stem cells (BMSCs) transplantation had synergistic effect on neuroprotection of red nucleus neurons after spinal cord injury (SCI). MATERIALS AND METHODS: Rubrospinal tract (RST) transection model was established and BMSCs were collected. The forelimb locomotor function was recorded using inclined plate test and spontaneous vertical exploration. cAMP level in red nucleus was detected with Enzyme-linked immunosorbent assay (ELISA). Morphology and number of red nucleus neurons was observed using Nissl's staining. Expression of cAMP-response element binding protein (CREB), ras homolog gene family member A (RhoA) and nerve growth factor (NGF) in red nucleus was detected using immunohistochemistry, qRT-PCR and Western-blotting. RESULTS: The combination of BYHWD and BMSCs transplantation could improve the forelimb locomotor function significantly and give the red nucleus somas a better protection. Meanwhile, cAMP level, CREB and NGF increased, while RhoA decreased remarkably in the BYHWD+BMSCs group. CONCLUSIONS: BYHWD combined with BMSCs transplantation had synergistic effect on neuroprotection of red nucleus neurons after SCI; the mechanism may be related to up-regulating cAMP level, activating the cAMP/CREB/RhoA signaling pathway, and promoting expression of NGF.
Subject(s)
Drugs, Chinese Herbal/therapeutic use , Mesenchymal Stem Cells , Neurons/physiology , Red Nucleus/physiology , Spinal Cord Injuries/therapy , Animals , Axotomy , Male , Mesenchymal Stem Cell Transplantation , Phytotherapy , Rats, Sprague-Dawley , Spinal Cord Injuries/physiopathologyABSTRACT
Immature peripheral nervous system damage, such as the transection of a peripheral nerve, results in the extensive degeneration of motoneurons and dorsal root ganglia (DRG) sensory neurons, mostly due to apoptotic events. We have previously shown that cannabidiol (CBD), the most abundant non-psychotropic molecule present in the Cannabis sativa plant, exhibits neuroprotective action when administered daily at a dose of 15â¯mg/kg. This study shows that use of the fluorinated synthetic version of CBD (4'-fluoro-cannabidiol, HUF-101) significantly improves neuronal survival by 2-fold compared to that achieved with traditional CBD at one-third the dose. Furthermore, we show that HUF-101 administration significantly upregulates anti-apoptotic genes and blocks the expression of pro-apoptotic nuclear factors. Two-day-old Wistar rats were subjected to unilateral sectioning of the sciatic nerve and treated daily with HUF-101 (1, 2.5, 5â¯mg/kg/day, i.p.) or a vehicle solution for five days. The results were evaluated by Nissl staining, immunohistochemistry, and qRT-PCR. Neuronal counting revealed a 47% rescue of spinal motoneurons and a 79% rescue of DRG neurons (HUF-101, 5â¯mg/kg). Survival was associated with complete depletion of p53 and a 60-fold elevation in BCL2-like 1 gene expression. Additionally, peroxisome proliferator-activated receptor gamma (PPAR-gamma) gene expression was downregulated by 80%. Neuronal preservation was coupled with a high preservation of synaptic coverage and a reduction in astroglial and microglial reactions that were evaluated in nearby spinal motoneurons present in the ventral horn of the lumbar intumescence. Overall, these data strongly indicate that HUF-101 exerts potent neuroprotective effects that are related to anti-apoptotic protection and the reduction of glial reactivity.
Subject(s)
Cannabidiol/analogs & derivatives , Gliosis/drug therapy , Neuroprotective Agents/therapeutic use , Sciatic Nerve/surgery , Animals , Apoptosis Regulatory Proteins/biosynthesis , Axotomy , Cannabidiol/pharmacology , Cannabidiol/therapeutic use , Cell Survival/drug effects , Dose-Response Relationship, Drug , Down-Regulation/drug effects , Ganglia, Spinal/drug effects , Motor Neurons/drug effects , Neuroprotective Agents/pharmacology , PPAR gamma/biosynthesis , Rats , Tumor Suppressor Protein p53/metabolism , Up-Regulation/drug effects , bcl-X ProteinABSTRACT
Injury of CNS nerve tracts remodels circuitry through dendritic spine loss and hyper-excitability, thus influencing recovery. Due to the complexity of the CNS, a mechanistic understanding of injury-induced synaptic remodeling remains unclear. Using microfluidic chambers to separate and injure distal axons, we show that axotomy causes retrograde dendritic spine loss at directly injured pyramidal neurons followed by retrograde presynaptic hyper-excitability. These remodeling events require activity at the site of injury, axon-to-soma signaling, and transcription. Similarly, directly injured corticospinal neurons in vivo also exhibit a specific increase in spiking following axon injury. Axotomy-induced hyper-excitability of cultured neurons coincides with elimination of inhibitory inputs onto injured neurons, including those formed onto dendritic spines. Netrin-1 downregulation occurs following axon injury and exogenous netrin-1 applied after injury normalizes spine density, presynaptic excitability, and inhibitory inputs at injured neurons. Our findings show that intrinsic signaling within damaged neurons regulates synaptic remodeling and involves netrin-1 signaling.Spinal cord injury can induce synaptic reorganization and remodeling in the brain. Here the authors study how severed distal axons signal back to the cell body to induce hyperexcitability, loss of inhibition and enhanced presynaptic release through netrin-1.
Subject(s)
Dendritic Spines/physiology , Netrin-1/metabolism , Neuronal Plasticity , Pyramidal Cells/physiology , Synapses/physiology , Animals , Axotomy , Embryo, Mammalian , Gene Expression , Glutamic Acid/metabolism , Microfluidic Analytical Techniques , Motor Cortex/physiopathology , Primary Cell Culture , Rats, Sprague-Dawley , Spinal Cord Injuries/physiopathologyABSTRACT
SETTING: Interventional procedures directed toward sources of pain in the axial and appendicular musculoskeletal system are performed with increasing frequency. Despite the presence of evidence-based guidelines for such procedures, there are wide variations in practice. Case reports of serious complications such as spinal cord infarction or infection from spine injections lack appropriate context and create a misleading view of the risks of appropriately performed interventional pain procedures. OBJECTIVE: To evaluate adverse event rate for interventional spine procedures performed at three academic interventional spine practices. METHODS: Quality assurance databases at three academic interventional pain management practices that utilize evidence-based guidelines [1] were interrogated for immediate complications from interventional pain procedures. Review of the electronic medical record verified or refuted the occurrence of a complication. Same-day emergency department transfers or visits were also identified by a records search. RESULTS: Immediate complication data were available for 26,061 consecutive procedures. A radiology practice performed 19,170 epidural steroid (primarily transforaminal), facet, sacroiliac, and trigger point injections (2006-2013). A physiatry practice performed 6,190 spine interventions (2004-2009). A second physiatry practice performed 701 spine procedures (2009-2010). There were no major complications (permanent neurologic deficit or clinically significant bleeding [e.g., epidural hematoma]) with any procedure. Overall complication rate was 1.9% (493/26,061). Vasovagal reactions were the most frequent event (1.1%). Nineteen patients (<0.1%) were transferred to emergency departments for: allergic reactions, chest pain, symptomatic hypertension, and a vasovagal reaction. CONCLUSION: This study demonstrates that interventional pain procedures are safely performed with extremely low immediate adverse event rates when evidence-based guidelines are observed.
Subject(s)
Back Pain/therapy , Catheter Ablation/adverse effects , Injections, Epidural/adverse effects , Nerve Block/adverse effects , Pain Management/adverse effects , Adrenal Cortex Hormones/administration & dosage , Adult , Aged , Axotomy/adverse effects , Axotomy/methods , Female , Humans , Injections, Intra-Articular , Male , Middle Aged , Pain Management/methods , Retrospective StudiesABSTRACT
Microglial activation plays a crucial role in the pathological processes of various retinal and optic nerve diseases. TNF-α is a pro-inflammatory cytokine that is rapidly upregulated and promotes retinal ganglion cells (RGCs) death after optic nerve injury. However, the cellular source of TNF-α after optic nerve injury remains unclear. Thus, we aimed to determine the changes of retinal microglial activation in a rat model of optic nerve transection (ONT) after transcorneal electrical stimulation (TES). Furthermore, we assessed TNF-α expression after ONT and evaluated the effects of TES on TNF-α production. Rats were divided into 2 control groups receiving a sham surgery procedure, 2 ONT+Sham TES groups, and 2 ONT+TES groups. The rats were sacrificed on day 7 or 14 after ONT. RGCs were retrogradely labelled by Fluorogold (FG) 7 days before ONT, one TES group and corresponding controls were stimulated on day 0, 4, and the second were stimulated on day 0, 4, 7, 10. Whole-mount immunohistofluorescence, quantification of RGCs and microglia, and western blot analysis were performed on day 7 and 14 after ONT. TES significantly increased RGCs survival on day 7 and 14 after ONT, which was accompanied by reduced microglia on day 7, but not 14. TNF-α was co-localized with ameboid microglia and significantly increased on day 7 and 14 after ONT. TES significantly reduced TNF-α production on day 7 and 14 after ONT. Our study demonstrated that TES promotes RGCs survival after ONT accompanied by reduced microglial activation and microglia-derived TNF-α production.
Subject(s)
Retinal Ganglion Cells/drug effects , Retinal Ganglion Cells/physiology , Animals , Axotomy/methods , Cell Count , Cell Survival/physiology , Cornea , Electric Stimulation , Electric Stimulation Therapy/methods , Male , Microglia/metabolism , Optic Nerve/physiology , Optic Nerve Injuries/metabolism , Rats , Rats, Sprague-Dawley , Retina/metabolism , Retinal Ganglion Cells/metabolism , Tumor Necrosis Factor-alpha/drug effects , Up-RegulationABSTRACT
Activity and disuse of synapses are thought to influence progression of several neurodegenerative diseases in which synaptic degeneration is an early sign. Here we tested whether stimulation or disuse renders neuromuscular synapses more or less vulnerable to degeneration, using axotomy as a robust trigger. We took advantage of the slow synaptic degeneration phenotype of axotomized neuromuscular junctions in flexor digitorum brevis (FDB) and deep lumbrical (DL) muscles of Wallerian degeneration-Slow (Wld(S)) mutant mice. First, we maintained ex vivo FDB and DL nerve-muscle explants at 32°C for up to 48 h. About 90% of fibers from Wld(S) mice remained innervated, compared with about 36% in wild-type muscles at the 24-h checkpoint. Periodic high-frequency nerve stimulation (100 Hz: 1s/100s) reduced synaptic protection in Wld(S) preparations by about 50%. This effect was abolished in reduced Ca(2+) solutions. Next, we assayed FDB and DL innervation after 7 days of complete tetrodotoxin (TTX)-block of sciatic nerve conduction in vivo, followed by tibial nerve axotomy. Five days later, only about 9% of motor endplates remained innervated in the paralyzed muscles, compared with about 50% in 5 day-axotomized muscles from saline-control-treated Wld(S) mice with no conditioning nerve block. Finally, we gave mice access to running wheels for up to 4 weeks prior to axotomy. Surprisingly, exercising Wld(S) mice ad libitum for 4 weeks increased about twofold the amount of subsequent axotomy-induced synaptic degeneration. Together, the data suggest that vulnerability of mature neuromuscular synapses to axotomy, a potent neurodegenerative trigger, may be enhanced bimodally, either by disuse or by hyperactivity.
Subject(s)
Neuromuscular Junction/physiopathology , Wallerian Degeneration/physiopathology , Animals , Axotomy , Calcium/metabolism , Electric Stimulation Therapy , Female , Male , Mice, Inbred C57BL , Mice, Transgenic , Muscle, Skeletal/pathology , Muscle, Skeletal/physiopathology , Nerve Tissue Proteins/genetics , Nerve Tissue Proteins/metabolism , Neuromuscular Junction/pathology , Running/physiology , Sciatic Nerve/drug effects , Sciatic Nerve/physiopathology , Sodium Channel Blockers/pharmacology , Tetrodotoxin/pharmacology , Tibial Nerve/injuries , Tibial Nerve/physiopathology , Tissue Culture Techniques , Wallerian Degeneration/pathology , Wallerian Degeneration/prevention & controlABSTRACT
In 2014, a phase II randomised, double blind clinical trial assessing the efficacy of cholecalciferol (vitamin D3) in patients with a cervical trauma will be set up. This trial stems from previous studies showing that vitamin D supplementation improves functional recovery in rat models of peripheral or central nerve injury. In a first series of experiments, we used a rat model of peripheral nerve trauma to demonstrate the therapeutic efficiency of vitamin D. We first demonstrated that ergocalciferol (vitamin D2) increases the number and the diameter of newly formed axons and improves the response of metabosensitive fibers from tibialis muscle, in a model of transected peroneal nerve. Then, we compared vitamin D2 and vitamin D3 and observed that the latter is more efficient. At the dose of 500 IU/kg/day, vitamin D3 induces a dramatic functional recovery. We also demonstrated that vitamin D3 increases the number of preserved or newly formed axons in the proximal end, the mean axon diameter in the distal end, neurite myelination in both the distal and proximal ends as well as the expression of genes involved in axogenesis and myelination. In parallel, we assessed the therapeutic role of vitamin D on the central nervous system. In a first study, using a rat model of spinal cord compression at the T10 thoracic level, we delivered vitamin D3 (cholecalciferol) orally at the dose of 50 IU/kg/day or 200 IU/kg/day. When compared to control animals, vitamin D-treated rats displayed, three months after injury, a significant improvement of ventilatory frequency and a reduction of H reflex indicating functional improvements at three months post-injury. In a second study, we used a rat model of cervical hemisection (C2) with a higher dose of oral vitamin D3 (500 IU/kg/day) delivered weekly, during 12 weeks. We observed an improved locomotor recovery, a reduced spasticity and a significantly higher rate of axons crossing the lesion site in treated animals. However, it must be pointed out that the functional improvement is reduced when vitamin D is provided one week after the trauma.
Subject(s)
Spinal Cord Injuries/drug therapy , Vitamin D/therapeutic use , Animals , Axons/drug effects , Axotomy , Cervical Vertebrae , Cholecalciferol/administration & dosage , Cholecalciferol/therapeutic use , Demyelinating Diseases/drug therapy , Drug Evaluation, Preclinical , Ergocalciferols/administration & dosage , Ergocalciferols/therapeutic use , Gene Expression Regulation , Humans , Muscle, Skeletal/innervation , Nerve Fibers, Myelinated/drug effects , Nerve Regeneration/drug effects , Neuroprotective Agents/administration & dosage , Neuroprotective Agents/therapeutic use , Peroneal Nerve/injuries , Rats , Vitamin D/administration & dosage , Vitamin D/physiologyABSTRACT
Local effect of acetyl salicylic acid (ASA) on peripheral nerve regeneration was studied using a rat sciatic nerve transection model. Forty-five male healthy White Wistar rats were divided into three experimental groups (n = 15), randomly: Sham-operation (SHAM), control (SIL), and ASA-treated (SIL/ASA) groups. In SHAM group after anesthesia left sciatic nerve was exposed through a gluteal muscle incision and after homeostasis the muscle was sutured. In SIL group the left sciatic nerve was exposed the same way and transected proximal to tibio-peroneal bifurcation leaving a 10-mm gap. Proximal and distal stumps were each inserted into a silicone tube and filled with 10 µl phosphate buffered solution. In SIL/ASA group defect was bridged using a silicone tube filled with 10 µl acetyl salisylic acid (0.1 mg/ml). Each group was subdivided into three subgroups of five animals each and were studied 4, 8, and 12 weeks after surgery. Data were analyzed statistically by factorial analysis of variance (ANOVA) and the Bonferroni test for pair-wise comparisons. Functional study confirmed faster and better recovery of regenerated axons in SIL/ASA than in SIL group (p < 0.05). Gastrocnemius muscle mass in SIL/ASA was significantly more than in SIL group. Morphometric indices of regenerated fibers showed that the number and diameter of the myelinated fibers in SIL/ASA were significantly higher than in control group. In immuohistochemistry, location of reactions to S-100 in SIL/ASA was clearly more positive than in SIL group. Response to local treatment of ASA demonstrates that it influences and improves functional recovery of peripheral nerve regeneration.
Subject(s)
Aspirin/therapeutic use , Nerve Regeneration/drug effects , Peripheral Nerve Injuries/drug therapy , Sciatic Nerve/injuries , Administration, Topical , Animals , Aspirin/administration & dosage , Axotomy , Drug Evaluation, Preclinical , Guided Tissue Regeneration/instrumentation , Guided Tissue Regeneration/methods , Male , Microsurgery , Muscle, Skeletal/innervation , Muscular Atrophy/prevention & control , Nerve Fibers, Myelinated/drug effects , Nerve Fibers, Myelinated/ultrastructure , Peripheral Nerve Injuries/pathology , Postoperative Complications/prevention & control , Random Allocation , Rats , Rats, Wistar , Recovery of Function , Sciatic Nerve/surgery , Sciatic Nerve/ultrastructure , Tissue Scaffolds , WalkingABSTRACT
Recovery of mimic function after facial nerve transection is poor: the successful regrowth of axotomized motoneurons to their targets is compromised by (1) poor axonal navigation and excessive collateral branching, (2) abnormal exchange of nerve impulses between adjacent regrowing axons, and (3) insufficient synaptic input to facial motoneurons. As a result, axotomized motoneurons get hyperexcitable and unable to discharge. Since improvement of growth cone navigation and reduction of the ephaptic cross talk between axons turn out be very difficult, we concentrated our efforts on the third detrimental component and proposed that an intensification of the trigeminal input to axotomized electrophysiologically silent facial motoneurons might improve specificity of reinnervation. To test our hypothesis we compared behavioral, electrophysiological, and morphological parameters after single reconstructive surgery on the facial nerve (or its buccal branch) with those obtained after identical facial nerve surgery but combined with direct or indirect stimulation of the ipsilateral infraorbital (ION) nerve. We found that in all cases, trigeminal stimulation was beneficial for the outcome by improving the quality of target reinnervation and recovery of vibrissa! motor performance.
Subject(s)
Electric Stimulation Therapy/methods , Facial Nerve Injuries/physiopathology , Facial Nerve Injuries/therapy , Nerve Regeneration/physiology , Recovery of Function/physiology , Trigeminal Nerve/physiology , Afferent Pathways/anatomy & histology , Afferent Pathways/physiology , Animals , Axotomy/methods , Disease Models, Animal , Facial Muscles/innervation , Facial Nerve/cytology , Facial Nerve/physiology , Female , Growth Cones/physiology , Growth Cones/ultrastructure , Motor Neurons/cytology , Motor Neurons/physiology , Rats , Rats, Wistar , Trigeminal Nerve/anatomy & histology , Vibrissae/innervationABSTRACT
Laser killing of cell nuclei has long been a powerful means of examining the roles of individual cells in C. elegans. Advances in genetics, laser technology, and imaging have further expanded the capabilities and usefulness of laser surgery. Here, we review the implementation and application of currently used methods for target edoptical disruption in C. elegans.
Subject(s)
Caenorhabditis elegans/physiology , Larva/physiology , Laser Therapy/methods , Microsurgery/methods , Neurons/physiology , Animals , Axotomy , Caenorhabditis elegans/cytology , Caenorhabditis elegans/radiation effects , Cell Lineage , Cell Nucleus/radiation effects , Cell Nucleus/ultrastructure , Fluorescent Dyes , Green Fluorescent Proteins , Larva/cytology , Larva/radiation effects , Lasers , Low-Level Light Therapy , Microfluidics , Neurons/radiation effects , Photosensitizing AgentsABSTRACT
Severing the axons of retinal ganglion cells (RGC) by crushing the optic nerve (ONC) causes the majority of RGC to degenerate and die, primarily by apoptosis. We showed recently that after ONC in adult rats, caspase-2 activation occurred specifically in RGC while no localisation of caspase-3 was observed in ganglion cells but in cells of the inner nuclear layer. We further showed that inhibition of caspase-2 using a single injection of stably modified siRNA to caspase-2 protected almost all RGC from death at 7 days, offering significant protection for up to 1 month after ONC. In the present study, we confirmed that cleaved caspase-2 was localised and activated in RGC (and occasional neurons in the inner nuclear layer), while TUNEL⺠RGC were also observed after ONC. We then investigated if suppression of caspase-2 using serial intravitreal injections of the pharmacological inhibitor z-VDVAD-fmk (z-VDVAD) protected RGC from death for 15 days after ONC. Treatment of eyes with z-VDVAD suppressed cleaved caspase-2 activation by >85% at 3-4 days after ONC. Increasing concentrations of z-VDVAD protected greater numbers of RGC from death at 15 days after ONC, up to a maximum of 60% using 4000 ng/ml of z-VDVAD, compared to PBS treated controls. The 15-day treatment with 4000 ng/ml of z-VDVAD after ONC suppressed levels of cleaved caspase-2 but no significant changes in levels of cleaved caspase-3, -6, -7 or -8 were detected. Although suppression of caspase-2 protected 60% of RGC from death, RGC axon regeneration was not promoted. These results suggest that caspase-2 specifically mediates death of RGC after ONC and that suppression of caspase-2 may be a useful therapeutic strategy to enhance RGC survival not only after axotomy but also in diseases where RGC death occurs such as glaucoma and optic neuritis.
Subject(s)
Apoptosis/drug effects , Caspase 2/metabolism , Caspase Inhibitors/pharmacology , Cytoprotection/drug effects , Oligopeptides/pharmacology , Retinal Ganglion Cells/drug effects , Animals , Axotomy , Caspase Inhibitors/administration & dosage , Drug Evaluation, Preclinical , Female , Intravitreal Injections , Oligopeptides/administration & dosage , Optic Nerve/cytology , Optic Nerve/drug effects , Optic Nerve/physiology , Optic Nerve/surgery , Rats , Rats, Sprague-Dawley , Retinal Ganglion Cells/physiologyABSTRACT
Some antibiotics are suggested to exert neuroprotective effects via regulation of glial responses. Attenuation of microglial activation by minocycline prevents neuronal death in a variety of experimental models for neurological diseases, such as cerebral ischemia, Parkinson's and Huntington's disease. Ceftriaxone delays loss of neurons in genetic animal models of amyotrophic lateral sclerosis through upregulation of astrocytic glutamate transporter expression (GLT-1). However, it remains largely unknown whether these antibiotics are able to protect neurons in axotomy models for progressive motor neuron diseases. Recent studies have shown that the axotomized motoneurons of the adult rat can survive, whereas those of the adult mouse undergo neuronal degeneration. We thus examined the possible effects of ceftriaxone and minocycline on neuronal loss and glial reactions in the mouse hypoglossal nucleus after axotomy. The survival rate of lesioned motoneurons at 28 days after axotomy (D28) was significantly improved by ceftriaxone and minocycline treatment. There were no significant differences in the cellular densities of astrocytes between ceftriaxone-treated and saline-treated animals. Ceftriaxone administration increased the expression of GLT-1 in the hypoglossal nucleus, while it suppressed the reactive increase of glial fibrillary acidic protein (GFAP) expression to control level. The cellular densities of microglia at D28 were significantly lower in minocycline-treated mice than in saline-treated mice. The time course analysis showed that immediate increase in microglia at D3 and D7 was not suppressed by minocycline. The present observations show that minocycline and ceftriaxone promote survival of lesioned motoneurons in the mouse hypoglossal nucleus, and also suggest that alterations in glial responses might be involved in neuroprotective actions of antibiotics.
Subject(s)
Astrocytes/drug effects , Axotomy/adverse effects , Ceftriaxone/therapeutic use , Hypoglossal Nerve Injuries/drug therapy , Microglia/drug effects , Minocycline/therapeutic use , Neurons/drug effects , Neuroprotective Agents/therapeutic use , Animals , Ceftriaxone/pharmacology , Cell Survival , Disease Models, Animal , Drug Evaluation, Preclinical , Drug Interactions , Glial Fibrillary Acidic Protein , Hypoglossal Nerve Injuries/pathology , Male , Mice , Mice, Inbred C57BL , Minocycline/pharmacology , Motor Neuron Disease , Nerve Tissue Proteins/biosynthesis , Nerve Tissue Proteins/genetics , Neuroprotective Agents/pharmacologyABSTRACT
BACKGROUND: The adult medicinal leech central nervous system (CNS) is capable of regenerating specific synaptic circuitry after a mechanical lesion, displaying evidence of anatomical repair within a few days and functional recovery within a few weeks. In the present work, spatiotemporal changes in molecular distributions during this phenomenon are explored. Moreover, the hypothesis that neural regeneration involves some molecular factors initially employed during embryonic neural development is tested. RESULTS: Imaging mass spectrometry coupled to peptidomic and lipidomic methodologies allowed the selection of molecules whose spatiotemporal pattern of expression was of potential interest. The identification of peptides was aided by comparing MS/MS spectra obtained for the peptidome extracted from embryonic and adult tissues to leech transcriptome and genome databases. Through the parallel use of a classical lipidomic approach and secondary ion mass spectrometry, specific lipids, including cannabinoids, gangliosides and several other types, were detected in adult ganglia following mechanical damage to connected nerves. These observations motivated a search for possible effects of cannabinoids on neurite outgrowth. Exposing nervous tissues to Transient Receptor Potential Vanilloid (TRPV) receptor agonists resulted in enhanced neurite outgrowth from a cut nerve, while exposure to antagonists blocked such outgrowth. CONCLUSION: The experiments on the regenerating adult leech CNS reported here provide direct evidence of increased titers of proteins that are thought to play important roles in early stages of neural development. Our data further suggest that endocannabinoids also play key roles in CNS regeneration, mediated through the activation of leech TRPVs, as a thorough search of leech genome databases failed to reveal any leech orthologs of the mammalian cannabinoid receptors but revealed putative TRPVs. In sum, our observations identify a number of lipids and proteins that may contribute to different aspects of the complex phenomenon of leech nerve regeneration, establishing an important base for future functional assays.
Subject(s)
Hirudo medicinalis/metabolism , Lipid Metabolism , Nerve Regeneration/physiology , Nervous System/metabolism , Peptides/metabolism , Amino Acid Sequence , Animals , Axotomy , Cannabinoids/metabolism , Chromatography, High Pressure Liquid , Cluster Analysis , Embryo, Nonmammalian/metabolism , Ganglia, Invertebrate/metabolism , Ganglia, Invertebrate/pathology , Hirudo medicinalis/embryology , Molecular Sequence Data , Nervous System/pathology , Peptides/chemistry , Phylogeny , Proteome/metabolism , Receptors, Cannabinoid/genetics , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Spinal Cord/metabolism , Spinal Cord/pathology , Stress, Mechanical , TRPV Cation Channels/metabolism , Time FactorsABSTRACT
Poor functional recovery found after peripheral nerve injury has been attributed to the misdirection of regenerating axons to reinnervate functionally inappropriate muscles. We applied brief electrical stimulation (ES) to the common fibular (CF) but not the tibial (Tib) nerve just prior to transection and repair of the entire rat sciatic nerve, to attempt to influence the misdirection of its regenerating axons. The specificity with which regenerating axons reinnervated appropriate targets was evaluated physiologically using compound muscle action potentials (M responses) evoked from stimulation of the two nerve branches above the injury site. Functional recovery was assayed using the timing of electromyography (EMG) activity recorded from the tibialis anterior (TA) and soleus (Sol) muscles during treadmill locomotion and kinematic analysis of hindlimb locomotor movements. Selective ES of the CF nerve resulted in restored M-responses at earlier times than in unstimulated controls in both TA and Sol muscles. Stimulated CF axons reinnervated inappropriate targets to a greater extent than unstimulated Tib axons. During locomotion, functional antagonist muscles, TA and Sol, were coactivated both in stimulated rats and in unstimulated but injured rats. Hindlimb kinematics in stimulated rats were comparable to untreated rats, but significantly different from intact controls. Selective ES promotes enhanced axon regeneration but does so with decreased fidelity of muscle reinnervation. Functional recovery is neither improved nor degraded, suggesting that compensatory changes in the outputs of the spinal circuits driving locomotion may occur irrespective of the extent of misdirection of regenerating axons in the periphery.
Subject(s)
Axons/physiology , Electric Stimulation Therapy , Nerve Regeneration/physiology , Recovery of Function/physiology , Sciatic Nerve/injuries , Animals , Axotomy , Electromyography , Evoked Potentials, Motor , Female , Muscles/innervation , Rats , Rats, Sprague-Dawley , Sciatic Nerve/physiologyABSTRACT
Spinal cord injury (SCI) is a serious condition often affecting young and healthy individuals around the world. Electro-acupuncture (EA) has been proven to contribute towards neurologic and functional recoveries in SCI, but the underlying mechanism remains largely unknown especially regarding neural specific proteins involved in the development of EA. The protein expression profile of spinal cord in both SCI and EA treatment models was analyzed by using two-dimensional electrophoresis-based proteomics. Using a MALDI-TOF/TOF MS and subsequent protein database searching, we identified changes in 15 proteins in the spinal cord following Governor Vessel (GV) EA treatment on SCI. These proteins are involved in inflammation, cell adhesion and migration, signal transduction and apoptosis processes. We selected 2 proteins (ANXA5 and CRMP2) beneficial to neuronal survival and axonal regeneration, and further identified these protein changes using Western blot analysis. Subsequently, Nissl staining and immunofluorescence double labeling approaches were used to explore possible role of the two neural specific proteins in the process of GV-EA treatment on SCI. Our results suggest that ANXA5 and CRMP2 may be neural specific proteins in the process of GV-EA treatment on SCI. This work might contribute to the better understanding of the mechanism involved in EA treatment on SCI at protein levels and provide a new therapeutic strategy for SCI.