Effect of peripheral blood mononuclear cells on ischemia-reperfusion injury of sciatic nerve of adult male albino rat: histological, immunohistochemical, and ultrastructural study.

Ischemia/reperfusion (I/R) injury of sciatic nerve is a serious condition that results in nerve fiber degeneration, and reperfusion causes oxidative injury. Peripheral blood mononuclear cells (PBMNCs) have neuroregenerative power. This study was carried out to evaluate the potential ameliorative effect of PBMNCs on changes induced by I/R injury of the sciatic nerve. Fifty adult male albino rats were divided into donor and experimental groups that were subdivided into four groups: group I (control group), group II received 50 µL PBNMCs once intravenously via the tail vein, group III rubber tourniquet was placed around their Rt hind limb root for 2 hours to cause ischemia, group IV was subjected to limb ischemia as group III, then they were injected with 50 ul PBMNCs as group II before reperfusion. I/R injury showed disorganization of nerve fascicles with wide spaces in between nerve fibers. The mean area of collagen fibers, iNOS immunoexpression, and number of GFAP-positive Schwann cells of myelinated fibers showed a highly significant increase, while a highly significant reduction in the G-ratio and neurofilament immunoexpression was observed. Myelin splitting, invagination, evagination, and myelin figures were detected. PBMNC-treated group showed a marked improvement that was confirmed by histological, immunohistochemical, and ultrastructural findings.

Deletion of the Gene Encoding the NMDA Receptor GluN1 Subunit in Schwann Cells Causes Ultrastructural Changes in Remak Bundles and Hypersensitivity in Pain Processing.

Abnormalities in interactions between sensory neurons and Schwann cells (SCs) may result in heightened pain processing and chronic pain states. We previously reported that SCs express the NMDA receptor (NMDA-R), which activates cell signaling in response to glutamate and specific protein ligands, such as tissue-type plasminogen activator. Herein, we genetically targeted grin1 encoding the essential GluN1 NMDA-R subunit, conditionally in SCs, to create a novel mouse model in which SCs are NMDA-R-deficient (GluN1- mice). These mice demonstrated increased sensitivity to light touch, pinprick, and thermal hyperalgesia in the absence of injury, without associated changes in motor function. Ultrastructural analysis of adult sciatic nerve in GluN1- mice revealed increases in the density of Aδ fibers and Remak bundles and a decrease in the density of Aß fibers, without altered g-ratios. Abnormalities in adult Remak bundle ultrastructure were also present including aberrant C-fiber ensheathment, distances between axons, and increased poly-axonal pockets. Developmental and post radial sorting defects contributed to altered nerve fiber densities in adult. Uninjured sciatic nerves in GluN1- mice did not demonstrate an increase in neuroinflammatory infiltrates. Transcriptome profiling of dorsal root ganglia (DRGs) revealed 138 differentially regulated genes in GluN1- mice. One third of the regulated genes are known to be involved in pain processing, including sprr1a, npy, fgf3, atf3, and cckbr, which were significantly increased. The intraepidermal nerve fiber density (IENFD) was significantly decreased in the skin of GluN1- mice. Collectively, these findings demonstrate that SC NMDA-R is essential for normal PNS development and for preventing development of pain states.SIGNIFICANCE STATEMENT Chronic unremitting pain is a prevalent medical condition; however, the molecular mechanisms that underlie heightened pain processing remain incompletely understood. Emerging data suggest that abnormalities in Schwann cells (SCs) may cause neuropathic pain. We established a novel mouse model for small fiber neuropathy (SFN) in which grin1, the gene that encodes the NMDA receptor (NMDA-R) GluN1 subunit, is deleted in SCs. These mice demonstrate hypersensitivity in pain processing in the absence of nerve injury. Changes in the density of intraepidermal small fibers, the ultrastructure of Remak bundles, and the transcriptome of dorsal root ganglia (DRGs) provide possible explanations for the increase in pain processing. Our results support the hypothesis that abnormalities in communication between sensory nerve fibers and SCs may result in pain states.

Protective Effects of PACAP in a Rat Model of Diabetic Neuropathy.

Pituitary adenylate cyclase-activating peptide (PACAP) is a neuropeptide with a widespread occurrence and diverse effects. PACAP has well-documented neuro- and cytoprotective effects, proven in numerous studies. Among others, PACAP is protective in models of diabetes-associated diseases, such as diabetic nephropathy and retinopathy. As the neuropeptide has strong neurotrophic and neuroprotective actions, we aimed at investigating the effects of PACAP in a rat model of streptozotocin-induced diabetic neuropathy, another common complication of diabetes. Rats were treated with PACAP1-38 every second day for 8 weeks starting simultaneously with the streptozotocin injection. Nerve fiber morphology was examined with electron microscopy, chronic neuronal activation in pain processing centers was studied with FosB immunohistochemistry, and functionality was assessed by determining the mechanical nociceptive threshold. PACAP treatment did not alter body weight or blood glucose levels during the 8-week observation period. However, PACAP attenuated the mechanical hyperalgesia, compared to vehicle-treated diabetic animals, and it markedly reduced the morphological signs characteristic for neuropathy: axon-myelin separation, mitochondrial fission, unmyelinated fiber atrophy, and basement membrane thickening of endoneurial vessels. Furthermore, PACAP attenuated the increase in FosB immunoreactivity in the dorsal spinal horn and periaqueductal grey matter. Our results show that PACAP is a promising therapeutic agent in diabetes-associated complications, including diabetic neuropathy.

Laminin 211 inhibits protein kinase A in Schwann cells to modulate neuregulin 1 type III-driven myelination.

Myelin is required for proper nervous system function. Schwann cells in developing nerves depend on extrinsic signals from the axon and from the extracellular matrix to first sort and ensheathe a single axon and then myelinate it. Neuregulin 1 type III (Nrg1III) and laminin α2ß1γ1 (Lm211) are the key axonal and matrix signals, respectively, but how their signaling is integrated and if each molecule controls both axonal sorting and myelination is unclear. Here, we use a series of epistasis experiments to show that Lm211 modulates neuregulin signaling to ensure the correct timing and amount of myelination. Lm211 can inhibit Nrg1III by limiting protein kinase A (PKA) activation, which is required to initiate myelination. We provide evidence that excessive PKA activation amplifies promyelinating signals downstream of neuregulin, including direct activation of the neuregulin receptor ErbB2 and its effector Grb2-Associated Binder-1 (Gab1), thereby elevating the expression of the key transcription factors Oct6 and early growth response protein 2 (Egr2). The inhibitory effect of Lm211 is seen only in fibers of small caliber. These data may explain why hereditary neuropathies associated with decreased laminin function are characterized by focally thick and redundant myelin.

Neural Selective Cryoneurolysis with Ice Slurry Injection in a Rat Model.

BACKGROUND: Postoperative pain caused by trauma to nerves and tissue around the surgical site is a major problem. Perioperative steps to reduce postoperative pain include local anesthetics and opioids, the latter of which are addictive and have contributed to the opioid epidemic. Cryoneurolysis is a nonopioid and long-lasting treatment for reducing postoperative pain. However, current methods of cryoneurolysis are invasive, technically demanding, and are not tissue-selective. This project aims to determine whether ice slurry can be used as a novel, injectable, drug-free, and tissue-selective method of cryoneurolysis and resulting analgesia. METHODS: The authors developed an injectable and selective method of cryoneurolysis using biocompatible ice slurry, using rat sciatic nerve to investigate the effect of slurry injection on the structure and function of the nerve. Sixty-two naïve, male Sprague-Dawley rats were used in this study. Advanced Coherent anti-Stokes Raman Scattering microscopy, light, and fluorescent microscopy imaging were used at baseline and at various time points after treatment for evaluation and quantification of myelin sheath and axon structural integrity. Validated motor and sensory testing were used for evaluating the sciatic nerve function in response to ice slurry treatment. RESULTS: Ice slurry injection can selectively target the rat sciatic nerve. Being injectable, it can infiltrate around the nerve. The authors demonstrate that a single injection is safe and selective for reversibly disrupting the myelin sheaths and axon density, with complete structural recovery by day 112. This leads to decreased nocifensive function for up to 60 days, with complete recovery by day 112. There was up to median [interquartile range]: 68% [60 to 94%] reduction in mechanical pain response after treatment. CONCLUSIONS: Ice slurry injection selectively targets the rat sciatic nerve, causing no damage to surrounding tissue. Injection of ice slurry around the rat sciatic nerve induced decreased nociceptive response from the baseline through neural selective cryoneurolysis.

Histological and ultrastructural study of AflatoxinB1 induced neurotoxicity in Sciatic nerve of adult male Albino rats.

Aflatoxins are mycotoxins produced by Aspergillus spp. which is a common contaminant of food items such as corn, spices, rice, nuts, and flour. Aflatoxin contamination of foods is a worldwide problem. Chronic aflatoxin exposure is found to be associated with Sciatic nerve damage. In vivo study was carried out to evaluate the toxic effect of aflatoxin B1 (AFB1) on the Sciatic nerve. Twenty-one adult male rats were included and divided equally into 3 groups (7 rats each): Group I (control group), group II (olive oil group) and group III: (AflatoxinB1 group). The rats received AFB1 (250 µg/kg B.W./day) orally by gastric tube 5 days/week for 4 weeks. Sciatic nerve specimens were prepared, and semi-thin sections were stained with Toluidine blue, examined by light microscope and photographed. Ultrathin sections (50-80 nm) from selected areas of the trimmed blocks were made, examined and photographed by transmission electron microscopy (JEOL-JSM-1011) in King Saud University Electron Microscopy Unit. The findings indicate that the administration of AFB1 to rats' results in degeneration in the sciatic nerve in the form of Wallerian degeneration in the myelin sheath. Macrophages appear to engulf the degenerated myelin and neutrophils.

Reorganization of Destabilized Nodes of Ranvier in ßIV Spectrin Mutants Uncovers Critical Timelines for Nodal Restoration and Prevention of Motor Paresis.

Disorganization of nodes of Ranvier is associated with motor and sensory dysfunctions. Mechanisms that allow nodal recovery during pathological processes remain poorly understood. A highly enriched nodal cytoskeletal protein ßIV spectrin anchors and stabilizes the nodal complex to actin cytoskeleton. Loss of murine ßIV spectrin allows the initial nodal organization, but causes gradual nodal destabilization. Mutations in human ßIV spectrin cause auditory neuropathy and impairment in motor coordination. Similar phenotypes are caused by nodal disruption due to demyelination. Here we report on the precise timelines of nodal disorganization and reorganization by following disassembly and reassembly of key nodal proteins in ßIV spectrin mice of both sexes before and after ßIV spectrin re-expression at specifically chosen developmental time points. We show that the timeline of nodal restoration has different outcomes in the PNS and CNS with respect to nodal reassembly and functional restoration. In the PNS, restoration of nodes occurs within 1 month regardless of the time of ßIV spectrin re-expression. In contrast, the CNS nodal reorganization and functional restoration occurs within a critical time window; after that, nodal reorganization diminishes, leading to less efficient motor recovery. We demonstrate that timely restoration of nodes can improve both the functional properties and the ultrastructure of myelinated fibers affected by long-term nodal disorganization. Our studies, which indicate a critical timeline for nodal restoration together with overall motor performance and prolonged life span, further support the idea that nodal restoration is more beneficial if initiated before any axonal damage, which is critically relevant to demyelinating disorders.SIGNIFICANCE STATEMENT Nodes of Ranvier are integral to efficient and rapid signal transmission along myelinated fibers. Various demyelinating disorders are characterized by destabilization of the nodal molecular complex, accompanied by severe reduction in nerve conduction and the onset of motor and sensory dysfunctions. This study is the first to report in vivo reassembly of destabilized nodes with sequential improvement in overall motor performance. Our study reveals that nodal restoration is achievable before any axonal damage, and that long-term nodal destabilization causes irreversible axonal structural changes that prevent functional restoration. Our studies provide significant insights into timely restoration of nodal domains as a potential therapeutic approach in treatment of demyelinating disorders.

SIL1 deficiency causes degenerative changes of peripheral nerves and neuromuscular junctions in fish, mice and human.

BACKGROUND: Marinesco-Sjögren Syndrome (MSS) is a rare neuromuscular condition caused by recessive mutations in the SIL1 gene resulting in the absence of functional SIL1 protein, a co-chaperone for the major ER chaperone, BiP. As BiP is decisive for proper protein processing, loss of SIL1 results in the accumulation of misshaped proteins. This accumulation likely damages and destroys cells in vulnerable tissues, leading to congenital cataracts, cerebellar ataxia, vacuolar myopathy and other MSS phenotypes. Whether the peripheral nervous system (PNS) is affected in MSS has not been conclusively shown. METHODS: To study PNS vulnerability in MSS, intramuscular nerves fibres from MSS patients and from SIL1-deficient mice (woozy) as well as sciatic nerves and neuromuscular junctions (NMJ) from these mice have been investigated via transmission electron microscopic and immunofluorescence studies accompanied by transcript studies and unbiased proteomic profiling. In addition, PNS and NMJ integrity were analyzed via immunofluorescence studies in an MSS-zebrafish model which has been generated for that purpose. RESULTS: Electron microscopy revealed morphological changes indicative of impaired autophagy and mitochondrial maintenance in distal axons and in Schwann cells. Moreover, changes of the morphology of NMJs as well as of transcripts encoding proteins important for NMJ function were detected in woozy mice. These findings were in line with a grossly abnormal structure of NMJs in SIL1-deficient zebrafish embryos. Proteome profiling of sciatic nerve specimens from woozy mice revealed altered levels of proteins implicated in neuronal maintenance suggesting the activation of compensatory mechanisms. CONCLUSION: Taken together, our combined data expand the spectrum of tissues affected by SIL1-loss and suggest that impaired neuromuscular transmission might be part of MSS pathophysiology.

SCO2 mutations cause early-onset axonal Charcot-Marie-Tooth disease associated with cellular copper deficiency.

Recessive mutations in the mitochondrial copper-binding protein SCO2, cytochrome c oxidase (COX) assembly protein, have been reported in several cases with fatal infantile cardioencephalomyopathy with COX deficiency. Significantly expanding the known phenotypic spectrum, we identified compound heterozygous variants in SCO2 in two unrelated patients with axonal polyneuropathy, also known as Charcot-Marie-Tooth disease type 4. Different from previously described cases, our patients developed predominantly motor neuropathy, they survived infancy, and they have not yet developed the cardiomyopathy that causes death in early infancy in reported patients. Both of our patients harbour missense mutations near the conserved copper-binding motif (CXXXC), including the common pathogenic variant E140K and a novel change D135G. In addition, each patient carries a second mutation located at the same loop region, resulting in compound heterozygote changes E140K/P169T and D135G/R171Q. Patient fibroblasts showed reduced levels of SCO2, decreased copper levels and COX deficiency. Given that another Charcot-Marie-Tooth disease gene, ATP7A, is a known copper transporter, our findings further underline the relevance of copper metabolism in Charcot-Marie-Tooth disease.

Metformin Enhances Functional Recovery of Peripheral Nerve in Rats with Sciatic Nerve Crush Injury.

BACKGROUND The aim of this study was to explore the effect of metformin by inducing autophagy for enhancing functional recovery of peripheral nerve in rats with sciatic nerve crush injury. MATERIAL AND METHODS Autophagy was determined by electron microscopy, immunofluorescence, and Western blot analysis. Motor function recovery was studied by the footprint intensity method. Axonal growth and regeneration were detected through Western blot while axonal remyelination was analysed through immunocytochemistry. Sensory and functional recovery were assessed by reflexive motor function analysis. RESULTS The present study deciphered the role of autophagy induction by metformin in motor functions and peripheral nerve regeneration following sciatic nerve crush injury in rats. The process was detected by measuring autophagosomes and the expression of microtubule-associated protein 1A/1B-light chain 3 upon metformin treatment of sciatic nerve crush-injured rats. Neurobehavioral recovery by metformin was tested by CatWalk gait analysis, and we quantified expression of myelin basic protein MBP and neurofilament NF200 at the damage sight by immunoblotting. In metformin-treated injured rats, autophagy was upregulated, by which the number of dead cells was decreased. Motor function was also recovered after metformin treatment, which was accompanied by upregulation of MBP and NF200 through autophagy induction. Surprisingly, the motor regenerative capability was reduced by treatment with 3-methyl adenine (an autophagy inhibitor) in nerve-injured rats. CONCLUSIONS Our study revealed that pharmacological induction of autophagy has an important and active role in the regeneration of nerve and motor function regain.

Toxic effect of acetamiprid on Rana ridibunda sciatic nerve (electrophysiological and histopathological potential).

In this study, the effects of a neonicotinoid insecticide acetamiprid on the sciatic nerve of Rana ridibunda were investigated by using electrophysiological and histological methods. A total of 35 preparations of sciatic nerve isolated from 35 frogs (Nervus ischiadicus) were used in the experiments. Experiments were designed as four different dose groups (n = 8 per group). Acetamiprid solutions of 1 (group 1), 10 (group 2), 100 (group 3), and 1000 µM (group 4) were applied to the nerves in dose groups. In each group, action potentials were recorded before application of acetamiprid which served as control data. The extracellular action potentials were recorded for each group of 30th, 60th, 90th and 120th min of application time. Action potential amplitude and area were measured from recordings. Histological evaluation was performed by transmission electron microscopy. In electrophysiological examination, all doses in which acetamiprid applied have shown the effect from the 30th min and suppressed the sciatic nerve action potential. Acetamiprid significantly reduced the amplitude at the rate of 78-96% and the area at the rate of 79-98% (p < 0.05). In electron microscopic examination, the control nerves were in normal appearance. Disorganization, irregularity, dense ovoid body formation, fragmentation of the myelin sheath, and loss on some axoplasm of the nerves in the dose group have been observed. Our findings showed that acetamiprid can cause neuropathic changes in sciatic nerve at all applied doses. These results indicate that acetamiprid as other insecticides can have harmful effects on non-target organisms.

Translocator protein agonist Ro5-4864 alleviates neuropathic pain and promotes remyelination in the sciatic nerve.

Our previous study reported the translocator protein to play a critical role in neuropathic pain and the possible mechanisms in the spinal cord. However, its mechanism in the peripheral nervous system is poorly understood. This study was undertaken to explore the distribution of translocator protein in the dorsal root ganglion and the possible mechanisms in peripheral nervous system in a rat model of spared nerve injury. Our results showed that translocator protein was activated in dorsal root ganglion after spared nerve injury. The translocator protein signals were primarily colocalized with neurons in dorsal root ganglion. A single intrathecal (i.t.) injection of translocator protein agonist (7-chloro-5-4-chlorophenyl)-1,3-dihydro-1-methyl-2-H-1,4-benzodiaze-pine-2) (Ro5-4864) exerted remarkable analgesic effect compared with the spared nerve injury group ( P < 0.01). After i.t. administration of 2 µg Ro5-4864 on day 3, the expression of translocator protein in ipsilateral dorsal root ganglion was significantly increased on day 7( P < 0.01) but decreased on day 14 ( P < 0.05) compared with the same point in time in the control group. The duration of translocator protein activation in dorsal root ganglion was remarkably shortened. Ro5-4864 also inhibited the activation of phospho-extracellular signal-regulated kinase 1(p-ERK1) ( P < 0.01), p-ERK2 (D7: P < 0.01, D14: P < 0.05), and brain-derived neurotrophic factor ( P < 0.05) in dorsal root ganglion. Meanwhile, i.t. administration of 2 µg Ro5-4864 on day 3 further accelerated the expression of myelin protein zero(P0) and peripheral myelin protein 22 (PMP22). Our results suggested Ro5-4864 could alleviate neuropathic pain and attenuate p-ERK and brain-derived neurotrophic factor activation in dorsal root ganglion. Furthermore, Ro5-4864 stimulated the expression of myelin regeneration proteins which may also be an important factor against neuropathic pain development. Translocator protein may present a novel target for the treatment of neuropathic pain both in the central and peripheral nervous systems.

Bone marrow-derived mesenchymal stem/stromal cells reverse the sensorial diabetic neuropathy via modulation of spinal neuroinflammatory cascades.

BACKGROUND: Diabetic neuropathy (DN) is a frequent and debilitating manifestation of diabetes mellitus, to which there are no effective therapeutic approaches. Mesenchymal stem/stromal cells (MSC) have a great potential for the treatment of this syndrome, possibly through regenerative actions on peripheral nerves. Here, we evaluated the therapeutic effects of MSC on spinal neuroinflammation, as well as on ultrastructural aspects of the peripheral nerve in DN-associated sensorial dysfunction. METHODS: C57Bl/6 mice were treated with bone marrow-derived MSC (1 × 106), conditioned medium from MSC cultures (CM-MSC) or vehicle by endovenous route following the onset of streptozotocin (STZ)-induced diabetes. Paw mechanical and thermal nociceptive thresholds were evaluated by using von Frey filaments and Hargreaves test, respectively. Morphological and morphometric analysis of the sciatic nerve was performed by light microscopy and transmission electron microscopy. Mediators and markers of neuroinflammation in the spinal cord were measured by radioimmunoassay, real-time PCR, and immunofluorescence analyses. RESULTS: Diabetic mice presented behavioral signs of sensory neuropathy, mechanical allodynia, and heat hypoalgesia, which were completely reversed by a single administration of MSC or CM-MSC. The ultrastructural analysis of the sciatic nerve showed that diabetic mice exhibited morphological and morphometric alterations, considered hallmarks of DN, such as degenerative changes in axons and myelin sheath, and reduced area and density of unmyelinated fibers. In MSC-treated mice, these structural alterations were markedly less commonly observed and/or less pronounced. Moreover, MSC transplantation inhibited multiple parameters of spinal neuroinflammation found in diabetic mice, causing the reduction of activated astrocytes and microglia, oxidative stress signals, galectin-3, IL-1ß, and TNF-α production. Conversely, MSC increased the levels of anti-inflammatory cytokines, IL-10, and TGF-ß. CONCLUSIONS: The present study described the modulatory effects of MSC on spinal cord neuroinflammation in diabetic mice, suggesting new mechanisms by which MSC can improve DN.

Neonatal Hypoxia Results in Peripheral Nerve Abnormalities.

Although the adverse effects of neonatal hypoxia associated with premature birth on the central nervous system are well known, the contribution of hypoxic damage to the peripheral nervous system (PNS) has not been addressed. We demonstrate that neonatal hypoxia results in hypomyelination and delayed axonal sorting in mice leading to electrophysiological and motor deficits that persist into adulthood. These findings support a potential role for PNS hypoxic damage in the motor impairment that results from premature birth and suggest that therapies designed to protect the PNS may provide clinical benefit.

Anti-VEGF treatment improves neurological function and augments radiation response in NF2 schwannoma model.

Hearing loss is the main limitation of radiation therapy for vestibular schwannoma (VS), and identifying treatment options that minimize hearing loss are urgently needed. Treatment with bevacizumab is associated with tumor control and hearing improvement in neurofibromatosis type 2 (NF2) patients; however, its effect is not durable and its mechanism of action on nerve function is unknown. We modeled the effect anti-VEGF therapy on neurological function in the sciatic nerve model and found that it improves neurological function by alleviating tumor edema, which may further improve results by decreasing muscle atrophy and increasing nerve regeneration. Using a cranial window model, we showed that anti-VEGF treatment may achieve these effects via normalizing the tumor vasculature, improving vessel perfusion, and delivery of oxygenation. It is known that oxygen is a potent radiosensitizer; therefore, we further demonstrated that combining anti-VEGF with radiation therapy can achieve a better tumor control and help lower the radiation dose and, thus, minimize radiation-related neurological toxicity. Our results provide compelling rationale for testing combined therapy in human VS.

The leptin receptor mutation of the obese Zucker rat causes sciatic nerve demyelination with a centripetal pattern defect.

Young male Zucker rats with a leptin receptor mutation are obese, have a non-insulin-dependent diabetes mellitus (NIDDM), and other endocrinopathies. Tibial branches of the sciatic nerve reveal a progressive demyelination that progresses out of the Schwann cells (SCs) where electron-contrast deposits are accumulated while the minor lines or intermembranous SC contacts display exaggerated spacings. Cajal bands contain diversely contrasted vesicles adjacent to the abaxonal myelin layer with blemishes; they appear dispatched centripetally out of many narrow electron densities, regularly spaced around the myelin annulus. These anomalies widen and yield into sectors across the stacked myelin layers. Throughout the worse degradations, the adaxonal membrane remains along the axonal neuroplasm. This peripheral neuropathy with irresponsive leptin cannot modulate hypothalamic-pituitary-adrenal axis and SC neurosteroids, thus exacerbates NIDDM condition. Additionally, the ultrastructure of the progressive myelin alterations may have unraveled a peculiar, centripetal mode of trafficking maintenance of the peripheral nervous system myelin, while some adhesive glycoproteins remain between myelin layers, somewhat hindering the axon mutilation. Heading title: Peripheral neuropathy and myelin.

[Effects of ShenFu Injection on Peripheral Neurotoxicity of Paclitaxel].

OBJECTIVE: To investigate the effects and the underlying mechanisms of ShenFu Injection on paclitaxel-induced peripheral neuropathy. METHODS: Twenty-eight adult male Wister rats were randomized into 4 groups (n=7) : control group, paclitaxel group, paclitaxel combined with low or high dose of ShenFu Injection groups. Rats were intraperitoneally injected with paclitaxel 8 mg/kg every 4 d for a total of 4 doses except control group. From Day 1 of the experiment (injection),low dose (4 mL/kg) and high dose (8 mL/kg) of Shenfu Injection were intraperitoneally injected daily in the combination groups for a total of 21 d respectively,while normal saline (NS) was injected in control group in the same way instead. Mechanical withdraw threshold (MWT) and thermal withdraw latency (TWL) of rats' hind paw were measured before (0 d) and after the first injection (6 d,14 d). The level of nerve growth factor (NGF) in the serum was measured at 22 d before the euthanasia,and the ultrastructure of the sciatic nerve was observed with transmission electron microscope. RESULTS: The MWT and TWL of 14 d in paclitaxel group significantly increased compared with those of 0 d and control group ( P<0.05). The combination of paclitaxel with ShenFu Injection,especially the high dose ( P<0.05),significantly reduced the MWT and TWL when compared to paclitaxel group at 14 d. Compared with simultaneous control group,there was no remarkably increased MWT and TWL in the low and high dose of ShenFu Injection (P>0.05) . Compared with control group,the serum NGF level significantly decreased ( P<0.05) in paclitaxel group,while the serum NGF level in low and high dose of ShenFu Injection groups were higher than paclitaxel group,particularly in the high dose group ( P<0.05). When compared to control group,the sciatic nerve fiber structure in the paclitaxel group was generally damaged,including myelin sheath swelling,fragmentation and vacuolization,endoplasmic reticulum swelling and matrix structure disorder in Schwann cells. The structural damages were mitigated in the low dose and high dose groups,especially the latter one,when compared to the paclitaxel group. CONCLUSION: Shenfu Injection can reduce the peripheral neurotoxicity of paclitaxel by promoting the expression of NGF in serum.

Akt Regulates Axon Wrapping and Myelin Sheath Thickness in the PNS.

The signaling pathways that regulate myelination in the PNS remain poorly understood. Phosphatidylinositol-4,5-bisphosphate 3-kinase 1A, activated in Schwann cells by neuregulin and the extracellular matrix, has an essential role in the early events of myelination. Akt/PKB, a key effector of phosphatidylinositol-4,5-bisphosphate 3-kinase 1A, was previously implicated in CNS, but not PNS myelination. Here we demonstrate that Akt plays a crucial role in axon ensheathment and in the regulation of myelin sheath thickness in the PNS. Pharmacological inhibition of Akt in DRG neuron-Schwann cell cocultures dramatically decreased MBP and P0 levels and myelin sheath formation without affecting expression of Krox20/Egr2, a key transcriptional regulator of myelination. Conversely, expression of an activated form of Akt in purified Schwann cells increased expression of myelin proteins, but not Krox20/Egr2, and the levels of activated Rac1. Transgenic mice expressing a membrane-targeted, activated form of Akt under control of the 2',3'-cyclic nucleotide 3'-phosphodiesterase promoter, exhibited thicker PNS and CNS myelin sheaths, and PNS myelin abnormalities, such as tomacula and myelin infoldings/outfoldings, centered around the paranodes and Schmidt Lanterman incisures. These effects were corrected by rapamycin treatmentin vivo Importantly, Akt activity in the transgenic mice did not induce myelination of nonmyelinating Schwann cells in the sympathetic trunk or Remak fibers of the dorsal roots, although, in those structures, they wrapped membranes redundantly around axons. Together, our data indicate that Akt is crucial for PNS myelination driving axonal wrapping by unmyelinated and myelinated Schwann cells and enhancing myelin protein synthesis in myelinating Schwann cells. SIGNIFICANCE STATEMENT: Although the role of the key serine/threonine kinase Akt in promoting CNS myelination has been demonstrated, its role in the PNS has not been established and remains uncertain. This work reveals that Akt controls several key steps of the PNS myelination. First, its activity promotes membrane production and axonal wrapping independent of a transcriptional effect. In myelinated axons, it also enhances myelin thickness through the mTOR pathway. Finally, sustained Akt activation in Schwann cells leads to hypermyelination/dysmyelination, mimicking some features present in neuropathies, such as hereditary neuropathy with liability to pressure palsies or demyelinating forms of Charcot-Marie-Tooth disease. Together, these data demonstrate the role of Akt in regulatory mechanisms underlying axonal wrapping and myelination in the PNS.

Schwann Cell O-GlcNAc Glycosylation Is Required for Myelin Maintenance and Axon Integrity.

UNLABELLED: Schwann cells (SCs), ensheathing glia of the peripheral nervous system, support axonal survival and function. Abnormalities in SC metabolism affect their ability to provide this support and maintain axon integrity. To further interrogate this metabolic influence on axon-glial interactions, we generated OGT-SCKO mice with SC-specific deletion of the metabolic/nutrient sensing protein O-GlcNAc transferase that mediates the O-linked addition of N-acetylglucosamine (GlcNAc) moieties to Ser and Thr residues. The OGT-SCKO mice develop tomaculous demyelinating neuropathy characterized by focal thickenings of the myelin sheath (tomacula), progressive demyelination, axonal loss, and motor and sensory nerve dysfunction. Proteomic analysis identified more than 100 O-GlcNAcylated proteins in rat sciatic nerve, including Periaxin (PRX), a myelin protein whose mutation causes inherited neuropathy in humans. PRX lacking O-GlcNAcylation is mislocalized within the myelin sheath of these mutant animals. Furthermore, phenotypes of OGT-SCKO and Prx-deficient mice are very similar, suggesting that metabolic control of PRX O-GlcNAcylation is crucial for myelin maintenance and axonal integrity. SIGNIFICANCE STATEMENT: The nutrient sensing protein O-GlcNAc transferase (OGT) mediates post-translational O-linked N-acetylglucosamine (GlcNAc) modification. Here we find that OGT functions in Schwann cells (SCs) to maintain normal myelin and prevent axonal loss. SC-specific deletion of OGT (OGT-SCKO mice) causes a tomaculous demyelinating neuropathy accompanied with progressive axon degeneration and motor and sensory nerve dysfunction. We also found Periaxin (PRX), a myelin protein whose mutation causes inherited neuropathy in humans, is O-GlcNAcylated. Importantly, phenotypes of OGT-SCKO and Prx mutant mice are very similar, implying that compromised PRX function contributes to the neuropathy of OGT-SCKO mice. This study will be useful in understanding how SC metabolism contributes to PNS function and in developing new strategies for treating peripheral neuropathy by targeting SC function.

Resolvin D1 Programs Inflammation Resolution by Increasing TGF-ß Expression Induced by Dying Cell Clearance in Experimental Autoimmune Neuritis.

UNLABELLED: Experimental autoimmune neuritis (EAN) is the animal model of human acute inflammatory demyelinating polyradiculoneuropathies (AIDP), an auto-immune inflammatory demyelination disease of the peripheral nervous system (PNS) and the world's leading cause of acute autoimmune neuromuscular paralysis. EAN and AIDP are characterized by self-limitation with spontaneous recovery; however, endogenous pathways that regulate inflammation resolution in EAN and AIDP remain elusive. A pathway of endogenous mediators, especially resolvins and clearance of apoptotic cells, may be involved. Here, we determined that resolvin D1 (RvD1), its synthetic enzyme, and its receptor were greatly increased in PNS during the recovery stage of EAN. Both endogenous and exogenous RvD1 increased regulatory T (Treg) cell and anti-inflammatory macrophage counts in PNS, enhanced inflammation resolution, and promoted disease recovery in EAN rats. Moreover, RvD1 upregulated the transforming growth factor-ß (TGF-ß) level and pharmacologic inhibition of TGF-ß signaling suppressed RvD1-induced Treg cell counts, but not anti-inflammatory macrophage counts, and RvD1-improved inflammation resolution and disease recovery in EAN rats. Mechanistically, the RvD1-enhanced macrophage phagocytosis of apoptotic T cells leading to reduced apoptotic T-cell accumulation in PNS induced TGF-ß production and caused Treg cells to promote inflammation resolution and disease recovery in EAN. Therefore, these data highlight the crucial role of RvD1 as an important pro-resolving molecule in EAN and suggest its potential as a therapeutic target in human neuropathies. SIGNIFICANCE STATEMENT: Experimental autoimmune neuritis (EAN) is the animal model of human acute inflammatory demyelinating polyradiculoneuropathies, an auto-immune inflammatory demyelination disease of the peripheral nervous system (PNS) and the world's leading cause of acute autoimmune neuromuscular paralysis. Here, we demonstrated that resolvin D1 (RvD1) promoted macrophage phagocytosis of apoptotic T cells in PNS, thereby upregulating transforming growth factor-ß by macrophages, increased local Treg cell counts, and finally promoted inflammation resolution and disease recovery in EAN. These data highlight the crucial role of RvD1 as an important pro-resolving molecule in EAN and suggest that it has potential as a therapeutic target in human neuritis.