[Thinking of the treatment for lumbar spinal stenosis with high-dense silven needle therapy based on the theory of six-meridian differentiation].

The paper introduces the thinking of the diagnosis and treatment with high-dense silver needle therapy for lumbar spinal stenosis (LSS) based on the theory of six-meridian differentiation. According to the severity of LSS and the depth of illness location, LSS is differentiated as six syndromes/patterns, including taiyang disorder, yangming disorder, shaoyang disorder, shaoyin disorder, jueyin disorder and taiyin disorder. The high-dense silver needle therapy is used. The main points include the bilateral Jiaji points (EX-B 2) from L1 to L5 and the acupoints of the bladder meridian of foot-taiyang (1.5 cun lateral to each side of L1 to L5); and the supplementary points are selected from the affected meridians. According to the disorders of six meridians, the length of moxa stick is adjusted in warm acupuncture, targeting the tender sites of soft tissue damage. In order to obtain the satisfactory effects, the appropriate physical exercise is applicable rather than absolutely limiting the movement of affected vertebrae during the treatment.

Chorioamnionitis accelerates granule cell and oligodendrocyte maturation in the cerebellum of preterm nonhuman primates.

BACKGROUND: Preterm birth is often associated with chorioamnionitis and leads to increased risk of neurodevelopmental disorders, such as autism. Preterm birth can lead to cerebellar underdevelopment, but the mechanisms of disrupted cerebellar development in preterm infants are not well understood. The cerebellum is consistently affected in people with autism spectrum disorders, showing reduction of Purkinje cells, decreased cerebellar grey matter, and altered connectivity. METHODS: Preterm rhesus macaque fetuses were exposed to intra-amniotic LPS (1 mg, E. coli O55:B5) at 127 days (80%) gestation and delivered by c-section 5 days after injections. Maternal and fetal plasma were sampled for cytokine measurements. Chorio-decidua was analyzed for immune cell populations by flow cytometry. Fetal cerebellum was sampled for histology and molecular analysis by single-nuclei RNA-sequencing (snRNA-seq) on a 10× chromium platform. snRNA-seq data were analyzed for differences in cell populations, cell-type specific gene expression, and inferred cellular communications. RESULTS: We leveraged snRNA-seq of the cerebellum in a clinically relevant rhesus macaque model of chorioamnionitis and preterm birth, to show that chorioamnionitis leads to Purkinje cell loss and disrupted maturation of granule cells and oligodendrocytes in the fetal cerebellum at late gestation. Purkinje cell loss is accompanied by decreased sonic hedgehog signaling from Purkinje cells to granule cells, which show an accelerated maturation, and to oligodendrocytes, which show accelerated maturation from pre-oligodendrocytes into myelinating oligodendrocytes. CONCLUSION: These findings suggest a role of chorioamnionitis on disrupted cerebellar maturation associated with preterm birth and on the pathogenesis of neurodevelopmental disorders among preterm infants.

c-Myc Drives inflammation of the maternal-fetal interface, and neonatal lung remodeling induced by intra-amniotic inflammation.

Background: Intra-amniotic inflammation (IAI) is associated with increased risk of preterm birth and bronchopulmonary dysplasia (BPD), but the mechanisms by which IAI leads to preterm birth and BPD are poorly understood, and there are no effective therapies for preterm birth and BPD. The transcription factor c-Myc regulates various biological processes like cell growth, apoptosis, and inflammation. We hypothesized that c-Myc modulates inflammation at the maternal-fetal interface, and neonatal lung remodeling. The objectives of our study were 1) to determine the kinetics of c-Myc in the placenta, fetal membranes and neonatal lungs exposed to IAI, and 2) to determine the role of c-Myc in modulating inflammation at the maternal-fetal interface, and neonatal lung remodeling induced by IAI. Methods: Pregnant Sprague-Dawley rats were randomized into three groups: 1) Intra-amniotic saline injections only (control), 2) Intra-amniotic lipopolysaccharide (LPS) injections only, and 3) Intra-amniotic LPS injections with c-Myc inhibitor 10058-F4. c-Myc expression, markers of inflammation, angiogenesis, immunohistochemistry, and transcriptomic analyses were performed on placenta and fetal membranes, and neonatal lungs to determine kinetics of c-Myc expression in response to IAI, and effects of prenatal systemic c-Myc inhibition on lung remodeling at postnatal day 14. Results: c-Myc was upregulated in the placenta, fetal membranes, and neonatal lungs exposed to IAI. IAI caused neutrophil infiltration and neutrophil extracellular trap (NET) formation in the placenta and fetal membranes, and neonatal lung remodeling with pulmonary hypertension consistent with a BPD phenotype. Prenatal inhibition of c-Myc with 10058-F4 in IAI decreased neutrophil infiltration and NET formation, and improved neonatal lung remodeling induced by LPS, with improved alveolarization, increased angiogenesis, and decreased pulmonary vascular remodeling. Discussion: In a rat model of IAI, c-Myc regulates neutrophil recruitment and NET formation in the placenta and fetal membranes. c-Myc also participates in neonatal lung remodeling induced by IAI. Further studies are needed to investigate c-Myc as a potential therapeutic target for IAI and IAI-associated BPD.

Derivation of iPSC line UMi029-A bearing a hearing-loss associated variant in the SMPX gene.

Hereditary hearing loss (HL) is the most common sensory disorder with multiple potential modes of inheritance, such as X-linked. Multiple loci have been associated with X-linked HL, including variants in the Small Muscle Protein X-Linked (SMPX) gene responsible for deafness, X-linked 4 (DFNX4) (OMIM 300066). Here we describe the derivation of an induced pluripotent stem cell (iPSC) line from an individual bearing a novel splice variant (c.133-1 G > A) that leads to a frameshift creating a premature stop codon (p.(Gly45Val*36)) in SMPX[1].

Characterization of UMi028-A-1 stem cell line that contains a CRISPR/Cas9 induced hearing loss-associated variant (V60L (c.178G > T)) in the P2RX2 gene.

UMi028-A-1 hiPSC line contains a CRISPR/Cas9-induced heterozygous, hearing loss-associated variant (V60L (GTA > TTA)) in the Purinergic Receptor P2X2 (P2RX2) gene. This line, derived from an unaffected male iPSC line, has been successfully characterized for its cellular and genetic properties. The c.178G > T variant in P2RX2 is associated with non-syndromic, dominant, progressive hearing loss. Once differentiated into inner ear cell types, UMi028-A-1 will serve as a resource for understanding the molecular mechanisms underlying hearing loss and serve as a potential platform for testing therapeutic approaches to restore inner ear function.

Human carbonic anhydrase-8 AAV8 gene therapy inhibits nerve growth factor signaling producing prolonged analgesia and anti-hyperalgesia in mice.

Carbonic anhydrase-8 (Car8; murine gene symbol) is an allosteric inhibitor of inositol trisphosphate receptor-1 (ITPR1), which regulates neuronal intracellular calcium release. We previously reported that wild-type Car8 overexpression corrects the baseline allodynia and hyperalgesia associated with calcium dysregulation in the waddle (wdl) mouse due to a 19 bp deletion in exon 8 of the Car8 gene. In this report, we provide preliminary evidence that overexpression of the human wild-type ortholog of Car8 (CA8WT), but not the reported CA8 S100P loss-of-function mutation (CA8MT), inhibits nerve growth factor (NGF)-induced phosphorylation of ITPR1, TrkA (NGF high-affinity receptor), and ITPR1-mediated cytoplasmic free calcium release in vitro. In addition, we show that gene transfer using AAV8-V5-CA8WT viral particles via sciatic nerve injection demonstrates retrograde transport to dorsal root ganglia (DRG) producing prolonged V5-CA8WT expression, pITPR1 and pTrkA inhibition, and profound analgesia and anti-hyperalgesia in male C57BL/6J mice. AAV8-V5-CA8WT-mediated overexpression prevented and treated allodynia and hyperalgesia associated with chronic neuropathic pain produced by the spinal nerve ligation (SNL) model. These AAV8-V5-CA8 data provide a proof-of-concept for precision medicine through targeted gene therapy of NGF-responsive somatosensory neurons as a long-acting local analgesic able to prevent and treat chronic neuropathic pain through regulating TrkA signaling, ITPR1 activation, and intracellular free calcium release by ITPR1.

[Exploration of the teaching model of acupuncture in the foreign fair].

The teaching experience is getting enriched during the past 40-year practice in Beijing International Acupuncture Training Center. The new teaching model is explored constantly and enable to adapt to the development and demand in the new era. In the paper, the necessity and feasibility of the reform of acupuncture teaching model are explained in five aspects, named developing teaching materials and promoting traditional culture, brea-king routine sequence and striving to the combination of the dynamic and the static, enhancing clinical case practice and improving practice ability, adjusting teaching time and emphasizing clinical teaching, reforming checking me-thod and increasing practice skills. An acupuncture teaching model in foreign fair is proposed in terms of individual and flexible teaching and practice emphasis.

Marked potentiation of cell swelling by cytokines in ammonia-sensitized cultured astrocytes.

BACKGROUND: Brain edema leading to high intracranial pressure is a lethal complication of acute liver failure (ALF), which is believed to be cytotoxic due to swelling of astrocytes. In addition to the traditional view that elevated levels of blood and brain ammonia are involved in the mechanism of brain edema in ALF, emerging evidence suggests that inflammatory cytokines also contribute to this process. We earlier reported that treatment of astrocyte cultures with a pathophysiological concentration of ammonia (5 mM NH4Cl) resulted in the activation of nuclear factor-kappaB (NF-κB) and that inhibition of such activation diminished astrocyte swelling, suggesting a key role of NF-κB in the mechanism of ammonia-induced astrocyte swelling. Since cytokines are also well-known to activate NF-κB, this study examined for additive/synergistic effects of ammonia and cytokines in the activation of NF-κB and their role in astrocyte swelling. METHODS: Primary cultures of astrocytes were treated with ammonia and cytokines (TNF-α, IL-1, IL-6, IFN-γ, each at 10 ng/ml), individually or in combination, and cell volume was determined by the [3H]-O-methylglucose equilibration method. The effect of ammonia and cytokines on the activation of NF-κB was determined by immunoblots. RESULTS: Cell swelling was increased by ammonia (43%) and by cytokines (37%) at 24 h. Simultaneous co-treatment with cytokines and ammonia showed no additional swelling. By contrast, cultures pretreated with ammonia for 24 h and then exposed to cytokines for an additional 24 h, showed a marked increase in astrocyte swelling (129%). Treatment of cultures with ammonia or cytokines alone also activated NF-κB (80-130%), while co-treatment had no additive effect. However, in cultures pre-treated with ammonia for 24 h, cytokines induced a marked activation of NF-κB (428%). BAY 11-7082, an inhibitor of NF-κB, completely blocked the astrocyte swelling in cultures pre-treated with ammonia and followed by the addition of a mixture of cytokines. CONCLUSION: Our results indicate that ammonia and a mixture of cytokines each cause astrocyte swelling but when these agents are added simultaneously, no additive effects were found. On the other hand, when cells were initially treated with ammonia and 24 h later given a mixture of cytokines, a marked potentiation in cell swelling and NF-κB activation occurred. These data suggest that the potentiation in cell swelling is a consequence of the initial activation of NF-κB by ammonia. These findings provide a likely mechanism for the exacerbation of brain edema in patients with ALF in the setting of sepsis/inflammation.

Brain aquaporin-4 in experimental acute liver failure.

Intracranial hypertension caused by brain edema and associated astrocyte swelling is a potentially lethal complication of acute liver failure (ALF). Mechanisms of edema formation are not well understood, but elevated levels of blood and brain ammonia and its by-product glutamine have been implicated in this process. Since aquaporin-4 (AQP4) has been implicated in brain edema in other conditions, we examined its role in a rat model of ALF induced by the hepatotoxin thioacetamide. Rats with ALF showed increased AQP4 protein in the plasma membrane (PM). Total tissue levels of AQP4 protein and mRNA levels were not altered, indicating that increased AQP4 is not transcriptionally mediated but likely reflects a more stable anchoring of AQP4 to the PM and/or interference with its degradation. An increase inAQP4 immunoreactivity in thePM was observed in perivascular astrocytes in ALF. Rats with ALF also showed increased levels of α-syntrophin, a protein involved in anchoringAQP4 to perivascular astrocytic end-feet. Increased AQP4 andα-syntrophin levels were inhibited by L-histidine, an inhibitor of glutamine transport into mitochondria, suggesting a role for glutamine in the increase of PM levels of AQP4. These results indicate that increased AQP4 PM levels in perivascular astrocytic end-feet are likely critical to the development of brain edema in ALF.

Aquaporin-4 in manganese-treated cultured astrocytes.

Manganese in excess is neurotoxic and causes CNS injury resembling that of Parkinson's disease. In brain, astrocytes predominantly take up and accumulate manganese and are thus vulnerable to its toxicity. Manganese was shown to induce cell swelling in cultured astrocytes, and oxidative/nitrosative stress (ONS) mediates such swelling. As aquaporin-4 (AQP4) is important in the mechanism of astrocyte swelling, we examined the effect of manganese on AQP4 protein levels in cultured astrocytes. Treatment of cultures with manganese increased AQP4 protein in the plasma membrane (PM), whereas total cellular AQP4 protein and mRNA levels were unchanged, suggesting that increased AQP4 levels is due to its increased stability and/or increased trafficking to the PM and not to its neosynthesis. AQP4 gene silencing by small interfering ribonucleic acid resulted in a marked reduction in astrocyte swelling by manganese. Antioxidants, as well as an inhibitor of nitric oxide synthase, diminished the increase in AQP4 protein expression, suggesting a role of ONS in the mechanism of AQP4 increase. As ONS is known to activate mitogen-activated protein kinases (MAPKs) and MAPK activation has been implicated in astrocyte swelling, we examined the effect of manganese on the activation of MAPKs and found an increased phosphorylation of extracellular signal-regulated kinase (ERK)1/2, C-Jun amino-terminal kinase (JNK)1/2/3, and p38-MAPK. Inhibitors of ERK1/2 and p38-MAPK (but not of JNK) blocked (40-60%) the manganese-induced increase in AQP4 protein content and astrocyte swelling, suggesting the involvement of these kinases in the increased AQP4 content. Inhibition of oxidative stress or MAPKs may represent potential strategies for counteracting AQP4-related neurological complications associated with manganese toxicity.

Brain edema in acute liver failure: inhibition by L-histidine.

Brain edema and the associated increase in intracranial pressure are potentially lethal complications of acute liver failure (ALF). Astrocyte swelling (cytotoxic edema) represents a significant component of the brain edema in ALF, and elevated blood and brain ammonia levels have been strongly implicated in its formation. We earlier showed in cultured astrocytes that oxidative stress (OS) and the mitochondrial permeability transition (mPT) play major roles in the mechanism of ammonia-induced astrocyte swelling. Glutamine, a byproduct of ammonia metabolism, has also been shown to induce OS, the mPT, and astrocyte swelling. Such effects of glutamine were suggested to be mediated by its hydrolysis in mitochondria, potentially yielding high levels of ammonia in this organelle and leading to OS and the mPT. L-histidine, an inhibitor of mitochondrial glutamine transport, was recently shown to mitigate OS, mPT, and cell swelling in cultured astrocytes treated with ammonia. The present study examined whether L-histidine similarly abolishes OS, the mPT, and brain edema in a rat model of ALF. Treatment of rats with thioacetamide caused a significant degree of brain edema, which was associated with induction of OS and the mPT. These changes were completely abolished by L-histidine, supporting a key role of mitochondrial glutamine transport and hydrolysis in the mechanism of the brain edema associated with ALF.

Calcium in the mechanism of ammonia-induced astrocyte swelling.

Brain edema, due largely to astrocyte swelling, is an important clinical problem in patients with acute liver failure. While mechanisms underlying astrocyte swelling in this condition are not fully understood, ammonia and associated oxidative/nitrosative stress appear to be involved. Mechanisms responsible for the increase in reactive oxygen/nitrogen species (RONS) and their role in ammonia-induced astrocyte swelling, however, are poorly understood. Recent studies have demonstrated a transient increase in intracellular Ca2+ in cultured astrocytes exposed to ammonia. As Ca2+ is a known inducer of RONS, we investigated potential mechanisms by which Ca2+ may be responsible for the production of RONS and cell swelling in cultured astrocytes after treatment with ammonia. Exposure of cultured astrocytes to ammonia (5 mM) increased the formation of free radicals, including nitric oxide, and such increase was significantly diminished by treatment with the Ca2+ chelator 1,2-bis-(o-aminophenoxy)-ethane-N,N,-N',N'-tetraacetic acid tetraacetoxy-methyl ester (BAPTA). We then examined the activity of Ca2+-dependent enzymes that are known to generate RONS and found that ammonia significantly increased the activities of NADPH oxidase (NOX), constitutive nitric oxide synthase (cNOS), and phospholipase A2 (PLA2) and such increases in activity were significantly diminished by BAPTA. Pre-treatment of cultures with 7-nitroindazole, apocyanin, and quinacrine, respective inhibitors of cNOS, NOX, and PLA2, all significantly diminished RONS production. Additionally, treatment of cultures with BAPTA or with inhibitors of cNOS, NOX, and PLA2 reduced ammonia-induced astrocyte swelling. These studies suggest that the ammonia-induced rise in intracellular Ca2+ activates free radical producing enzymes that ultimately contribute to the mechanism of astrocyte swelling.