Sex differences in the effects of high-fat diet on mouse sciatic nerves

ABSTRACTDysregulated complement activation drives peripheral inflammatory neuropathies by promoting immune attacks that exacerbate inflammation and tissue damage. Beyond immune functions, complement signaling may influence neuronal activity. To explore the role of C3a receptor (C3aR) and C5a receptor 1 (C5aR1) in peripheral nerve pathology, we examined their localization in mouse sciatic and human sural nerves and assessed their impact on nerve conduction. Immunofluorescence identified C3aR and C5aR1 in mice and human nerves. qPCR and western blot confirmed receptor expression in mouse sciatic nerves. Ex vivo electrophysiology assessed neural responses in control and treated nerves exposed to C3aR and C5aR1 agonist or agonist + antagonists. C3aR localized to the glial paranodal region of large‐myelinated fibers, while C5aR1 is primarily in small unmyelinated fibers. C3aR activation enhanced large‐fiber responses, reduced the refractory period, and increased excitability. The C3aR antagonist prevented these effects. In contrast, C5aR1 activation had minimal impact on conduction. These findings highlight distinct roles of C3aR and C5aR1 in peripheral nerves and suggest that Schwann cell C3aR regulates neuronal excitability. Targeting these pathways may help modulate nerve activity and inflammation in conditions like Guillain–Barré syndrome and diabetic neuropathy.

The localization of voltage-sensitive sodium channels was investigated in mouse, rat and rabbit sciatic nerves using iodinated alpha- and beta-Scorpion toxins (ScTx) as specific probes. Saturable specific binding for a beta-ScTx was detected in mouse sciatic nerve homogenates (Kd = 90 pM, binding site capacity = 90 fmol mg-1 protein). LM autoradiographic studies demonstrated that the two types of ScTx stained the Ranvier nodes of the myelinated fibres, and also showed a clear but weaker labelling of the unmyelinated Remak bundles. In the sciatic nerve, which is widely considered as a model 'myelinated nerve', the nodal membrane represented only a small fraction of the total axonal membranes (0.2% and 0.05% for mouse and rabbit sciatic nerves respectively). Therefore, despite their high channel density, nodal membranes contribute only a small proportion of the total labelling by beta-ScTx (15% and 2.3% for mouse and rabbit sciatic nerves respectively), with the major contribution to labelling arising from unmyelinated axons. The distribution of specific binding sites for a beta-Scorpion toxin was then analysed in cross-sections of rabbit sciatic nerve at the EM level. The quantitative analysis of autoradiograms involved three methods, the 50% probability circle method, and two cross-fire analyses using either systematically distributed hypothetical sources or hypothetical sources only located on the plasma membranes of axons and of Schwann cells associated with unmyelinated Remak bundles. No specific beta-Scorpion toxin binding sites were detected at the plasma membrane of Schwann cells from either myelinated fibres or unmyelinated bundles, or at the internodal surface of myelinated axons. Sites were only detected at the surface of unmyelinated axons and at nodal axolemma. Their density in unmyelinated axons was found to be in the range of 1-6 per micron2 of plasma membrane surface area by combining quantitative EM autoradiography and stereological measurements.

Schmidt-Lanterman incisures (SLIs) are a specific feature of myelinated nerve fibers in the peripheral nervous system (PNS). In this study, we report localization of a signal transduction protein, Src, in the SLIs of mouse sciatic nerves, and its phosphorylation states in Y527 and Y418 (P527 and P418, respectively) under normal conditions or deletion of a membrane skeletal protein, 4.1G. In adult mouse sciatic nerves, Src was immunolocalized in SLIs as a cone-shape, as well as in paranodes and some areas of structures reminiscent of Cajal bands. By immunostaining in normal nerves, P527-Src was strongly detected in SLIs, whereas P418-Src was much weaker. Developmentally, P418-Src was detected in SLIs of early postnatal mouse sciatic nerves. The staining patterns for P527 and P418 in normal adult nerve fibers were opposite to those in primary culture Schwann cells and a Schwannoma cell line, RT4-D6P2T. In 4.1G-deficient nerve fibers, which had neither 4.1G nor the membrane protein palmitoylated 6 (MPP6) in SLIs, the P418-Src immunoreactivity in SLIs was clearly detected at a stronger level than that in the wild type. An immunoprecipitation study revealed Src interaction with MPP6. These findings indicate that the Src-MPP6-4.1G protein complex in SLIs has a role in signal transduction in the PNS.

Congenital Hypomyelination (CH) IS the most severe demyelinating form of Hereditary Motor and Sensory Neuropathies and manifests at birth in human. Some subtypes of CH are due to dominant mutations in the gene coding for PO glycoprotein, which functions as a homophilic adhesion protein, responsible for compaction of opposing myelin lamellae. By homologous recombination in ES cells, we have generated a mouse containing a nonsense mutation in the intracellular portion of PO (Q215X) that, in the heterozygous state, is associated with CH neuropathy in humans. This mutation is predicted to encode a truncated PO protein, lacking part of the cytoplasmic domain. Expression analysis demonstrated that Q215X heterozygous mice produce both wildtype PO and a smaller, truncated PO glycoprotein; furthermore, the levels of mRNA and protein produced by the mutated allele are less abundant, relative to the wildtype. We demonstrated then that this reduction in mRNA and protein levels could be paIiially explained by inefficient transcription of the mutated allele, due to the presence of a LoxP site with intron 5. Behavioral analysis of the Q215X1+ mice revealed reduced motor performance at 11 days after birth. Morphological analysis performed on sciatic nerves of mice between PI and P 14 revealed defects in the process of axonal sorting by Schwann cells, with the presence of bundles of mixed large and small calibre axons surrounded, but not ensheathed, by single Schwann cells. These morphogical defects are rescued after the second week of life: sciatic nerves from adult mice, in fact, show only mild hypomyelination, which is much less severe than the morphology reported in patients. From these preliminary data, we conclude that the Q215X mutation results in a truncated PO protein; since the phenotype of Q215X1+ mice and PO +1- mice differs, Q215X probably produces a gain of function. Finally, we studied in vivo the intracellular location of the truncated protein, in order to clarify aspects of the pathogenetic mechanism of the Q215X PO mutation: we found that the mutated protein is not properly trafficked within Schwann cells, being partially retained in the ER compartment. The phenotype we have observed in the mutant mice presents similarities to other hypomyelinating mice that carry mutations in different genes, that are all involved in the laminin pathway: this suggests that mutant PO may interfere with laminin signaling, required for the correct timing of axonal sorting by Schwann cells. Thus, from these data we conclude that the Q215X mouse is a partial model of Congenital Hypomyelination, less severely affected, if compared to human patients. This difference in disease severity could be partially explained by the inefficient transcription of the Q215X allele. Thus, we believe that the mechanism of this hypomyelination is likely to be related to the mechanism of the more severe neuropathy in human. This mouse will be useful to reveal the pathogenesis of the mutation.

The present study was designed to investigate the effect of ramipril (angiotensin-converting enzyme inhibitor) on the chronic constriction injury of sciatic nerve induced neuropathic pain in mice. The neuropathic pain was induced by four loose ligations of the right sciatic nerve in mice. The battery of behavioral tests, i.e. plantar, pin prick, tail flick, tail pinch, rota rod tests, were performed to assess the degree of thermal and mechanical hyperalgesia in ipsilateral paw and tail, and motor in-coordination activity respectively. In addition, the biochemical tests, i.e. total protein, thiobarbituric acid reactive substances and reduced glutathione, were also performed in sciatic nerve tissue samples. The administration of ramipril (2 and 4 mg/kg, p.o.) significantly attenuated chronic constriction injury-induced rise in peripheral as well as central pain sensitivity (thermal and mechanical) along with impairment of motor in-coordination activity. Further, it also produces ameliorative effects on chronic constriction injury-induced rise in thiobarbituric acid reactive substances and decrease in glutathione levels when compared with a normal control group. It may be concluded that angiotensin-converting enzyme inhibitor may be a potential new target for the management of neuropathic pain.

Further evidence on a role of chemokines in injury‐related pain hypersensitivity

It is well-known that denervation of motor nerves induces atrophy and decreases contractile force of the skeletal muscle. However, it is not completely understood how denervation alters calcium handling in the skeletal muscle. We investigated the effect of denervation on the expression and function of proteins involved in calcium handling. Two weeks after denervation of the right sciatic nerve in mice, we observed a significant decrease in mass and cross-sectional area of the ipsilateral tibialis anterior (TA) and flexor digitorum brevis (FDB) muscles. Also, we observed a significant decrease in the specific tetanus contractile force in the ipsilateral TA muscle. Calcium imaging of the ipsilateral FDB showed that the peak twitch and tetanus calcium concentrations were significantly decreased due to a decrease in calcium content of the sarcoplasmic reticulum (SR). Denervation reduced the maximum rate of sarcoplasmic/endoplasmic calcium ATPase (SERCA) activity. Interestingly, the amount of phospholamban (PLN), but not its transcripts, was increased in the ipsilateral vs. contralateral side after denervation, suggesting that denervation impairs constitutive regulation of PLN. Immunohistochemical analysis revealed increased PLN in all major fiber types in TA with IIx fibers showing a threefold higher expression than the contralateral side. These results suggest that the abnormal increase in PLN in the ipsilateral fast-twitch fibers may be involved in decreased SERCA activity, SR calcium content, peak calcium transients, and contractile forces of denervated muscles.

The inhibition of the nitric oxide–cGMP–PKG–JNK signaling pathway avoids the development of tolerance to the local antiallodynic effects produced by morphine during neuropathic pain

To investigate the effect of sevoflurane on neuropathic pain induced by chronic constriction injury (CCI) of sciatic nerve in mice, and to elucidate its mechanism by animal experiments. Thirty-two C57BL/6 mice were randomly divided into four groups: Sham group, Model group, Control group and Sevoflurane group. First, a mouse model of neuropathic pain was established. Then, the mice in each group were killed on Day 14 after operation to harvest the enlarged lumbosacral spinal cord. In contrast with the Model group, the Sevoflurane group displayed a significantly increased paw withdrawal mechanical threshold (PWMT) and significantly prolonged paw withdrawal thermal latency (PWTL) from Day 5 after operation. The morphological changes of lumbosacral spinal cord were observed by hematoxylin-eosin (HE) staining and transmission electron microscopy. Pathological results showed that sevoflurane reduced nuclear pyknosis in lumbosacral spinal cord tissue, with a large number of mitochondrial crista disappearance and mitochondrial swelling. The results of Western blotting showed that sevoflurane significantly decreased the protein expressions of phosphorylated phospholipase Cγ (p-PLCγ), phosphorylated calcium/calmodulin-dependent protein kinase II (p-CaMKII) and phosphorylated inositol 1,4,5-triphosphate receptor (p-IP3R), and reduced the protein expressions of endoplasmic reticulum (ER) stress proteins glucose-regulated protein 78 (GRP78) and GRP94, oxidative stress-related proteins P22 and P47 and inflammatory factors nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3), interleukin-1 β (IL-1β), and tumor necrosis factor-α (TNF-α). Sevoflurane inhibits neuropathic pain by maintaining ER stress and oxidative stress homeostasis through inhibiting the activation of the PLCγ/CaMKII/IP3R signaling pathway.

Translational readthrough-inducing agents have been developed for the treatment of nonsense mutations in hereditary diseases. The clinical effectiveness of readthrough agents has been reported, although newly developed agents are still desired because of their toxicities or limited clinical effectiveness. Recently, novel negamycin-derived readthrough agents without antimicrobial activity have been developed. Our aim was to evaluate the activities of these readthrough agents by monitoring the production of large myelin protein zero (L-MPZ), the programmed translational readthrough isoform of myelin protein zero (P0, MPZ) mRNA, and to clarify the influence of these agents on the sciatic nerve in vivo. First, we examined the readthrough activities of novel negamycin-derived agents using cell-free and cell culture systems using plasmids encoding human MPZ (hP0) cDNA. Three of the negamycin derivatives, TCP-112, TCP-169, and TCP-1109, suppressed the canonical stop codon to induce readthrough. Direct injection of TCP-1109, which showed higher readthrough activity for Mpz in mouse sciatic nerves, exhibited a 1.3-fold increase in the L-MPZ/P0 ratio compared to that with the vehicle control on western blotting. The nerve conduction velocity and beam walk test showed abnormalities in the classical readthrough agent G418-treated group, but not in the TCP-1109-treated group. Immunofluorescence analysis showed that TCP-1109 caused less damage to the sciatic nerve than G418. In the semi-thin sections, a lower g-ratio and more tomacula-like structures were observed in TCP-1109-treated nerves. Thus, the present results indicate that negamycin-derived readthrough agents enhance programmed translational readthrough, and the management of readthrough activities using canonical stop codons may be important.

The aim of this study was to define morphological and neurophysiological features of an age-related neuropathy in mice and to explore possible underlying pathogenetic mechanisms. Teased-fibre analysis of sciatic nerves in aged animals demonstrated a high incidence of demyelination. Electron microscopic analysis of sciatic nerve also revealed axonal atrophy. Both forms of pathology preferentially effect large myelinated fibres. The morphological evidence suggests that demyelination may have resulted from two processes; a primary event and a late consequence of axonal shrinkage. A high-affinity ouabain-binding form of (Na+,K+)ATPase was found in both mice sciatic nerve and spinal roots. A progressive and ultimately severe reduction of enzyme concentration with age was demonstrated in sciatic nerve and dorsal root. Since no change with age was seen in fast (anterograde or retrograde) axoplasmic flow, reduced peripheral nerve (Na+,K+)ATPase is not due to impaired enzyme translocation. Motor nerve conduction velocity decreased significantly with age, while minimum F-wave latency and somatosensory evoked potential latency increased. Impaired conduction velocities in aged animals may be attributed to the interaction of many factors including demyelination, remyelination, a disproportionate loss of large myelinated fibres, axonal atrophy, nerve regeneration and reduced peripheral nerve (Na+,K+)ATPase. It is concluded that the neuropathy in senescent mice is not species specific and is less severe, even in long-lived mice species, compared with that seen in the rat.

Nerve repair is crucial for restoring function following axonal injury. However, mature neurons in the central nervous system (CNS) have a limited ability to regenerate their axons after injury due to diminished intrinsic growth capacity and a hostile environment. In contrast, neurons in the peripheral nervous system (PNS) exhibit robust regenerative potential. Among them, adult dorsal root ganglion (DRG) neurons are particularly well-known for their ability to regenerate effectively following peripheral nerve injuries, making them an ideal model for studying the cellular and molecular mechanisms underlying nerve repair. Here, a modified microsuture technique, termed "loop-before-transection," is described for generating a sciatic nerve transection and resuture model in mice, followed by analysis of axon regeneration. Behavioral assessments and muscle reinnervation are also demonstrated to evaluate functional recovery. After sciatic nerve transection and resuture, animals initially exhibit behavioral deficits, but full recovery is typically observed by 30 days post-injury. Applying drugs that promote axonal fusion at the injury site significantly accelerates functional recovery. This model system offers a powerful tool for investigating the mechanisms governing mammalian nerve repair in vivo.

BTBR ob/ob mice as a novel diabetic neuropathy model: Neurological characterization and gene expression analyses

Source: Li, Q., et al. Investigating Mammalian Axon Regeneration: In Vivo Electroporation of Adult Mouse Dorsal Root Ganglion. J. Vis. Exp. (2018)This video demonstrates the process of axonal regeneration in a mouse sciatic nerve following a crush injury. The isolated nerve is fixed and prepared for imaging, with fluorescent proteins expressed in neurons to visualize axon growth. Using an epifluorescence microscope, axon regeneration is traced from the injury site to assess the extent of regrowth.

Muscular dystrophy with laminin deficiency decreases the content of butyrylcholinesterase tetramers in sciatic nerves of Lama2dy mice