Zonisamide Enhances Neurite Elongation of Primary Motor Neurons and Facilitates Peripheral Nerve Regeneration In Vitro and in a Mouse Model

No clinically applicable drug is currently available to enhance neurite elongation after nerve injury. To identify a clinically applicable drug, we screened pre-approved drugs for neurite elongation in the motor neuron-like NSC34 cells. We found that zonisamide, an anti-epileptic and anti-Parkinson’s disease drug, promoted neurite elongation in cultured primary motor neurons and NSC34 cells in a concentration-dependent manner. The neurite-scratch assay revealed that zonisamide enhanced neurite regeneration. Zonisamide was also protective against oxidative stress-induced cell death of primary motor neurons. Zonisamide induced mRNA expression of nerve growth factors (BDNF, NGF, and neurotrophin-4/5), and their receptors (tropomyosin receptor kinase A and B). In a mouse model of sciatic nerve autograft, intragastric administration of zonisamide for 1 week increased the size of axons distal to the transected site 3.9-fold. Zonisamide also improved the sciatic function index, a marker for motor function of hindlimbs after sciatic nerve autograft, from 6 weeks after surgery. At 8 weeks after surgery, zonisamide was protective against denervation-induced muscle degeneration in tibialis anterior, and increased gene expression of Chrne, Colq, and Rapsn, which are specifically expressed at the neuromuscular junction. We propose that zonisamide is a potential therapeutic agent for peripheral nerve injuries as well as for neuropathies due to other etiologies.     

周围神经组织液对大鼠EPI-NCSCs体外分化的影响

为了探讨周围神经组织环境因素对EPI-NCSCs（epidermalneuralcreststemcells）分化的影响,该文用坐骨神经组织提取液为环境因素,对体外培养的EPI—NCSCs进行分化诱导。以正常培养的EPI—NCSCs为对照组,细胞培养液成分为：DMEM／F12、10％FBS、2％B27、20ng／mLbFGF。在细胞培养液中添加1％周围神经组织液作为实验组。MTT结果显示。周围神经组织液对EPI-NCSCs的活性和增殖无影响;对照组和实验组EPI-NCSCs都呈多种形态。RT-PCR分析显示,在对照组和实验组中EPI．NCSCs均表达GFAP、S-100、β-IIITubulin（Tujl）,但在实验组中,这3种蛋白的表达量均高于对照组。免疫组化分析显示,在对照组中,EPI—NCSCs同时表现了雪旺细胞和早期神经元的鉴定特征;在实验组中,EPI—NCSCs没有神经元的鉴定特征,只表达雪旺氏细胞的鉴定特征。这表明,周围神经组织液可以诱导体外培养的EPI-NCSCs向雪旺氏细胞分化。     

Dorsal Root Ganglia Neurons and Differentiated Adipose-derived Stem Cells: An In Vitro Co-culture Model to Study Peripheral Nerve Regeneration

Dorsal root ganglia (DRG) neurons, located in the intervertebral foramina of the spinal column, can be used to create an in vitro system facilitating the study of nerve regeneration and myelination. The glial cells of the peripheral nervous system, Schwann cells (SC), are key facilitators of these processes; it is therefore crucial that the interactions of these cellular components are studied together. Direct contact between DRG neurons and glial cells provides additional stimuli sensed by specific membrane receptors, further improving the neuronal response. SC release growth factors and proteins in the culture medium, which enhance neuron survival and stimulate neurite sprouting and extension. However, SC require long proliferation time to be used for tissue engineering applications and the sacrifice of an healthy nerve for their sourcing. Adipose-derived stem cells (ASC) differentiated into SC phenotype are a valid alternative to SC for the set-up of a co-culture model with DRG neurons to study nerve regeneration. The present work presents a detailed and reproducible step-by-step protocol to harvest both DRG neurons and ASC from adult rats; to differentiate ASC towards a SC phenotype; and combines the two cell types in a direct co-culture system to investigate the interplay between neurons and SC in the peripheral nervous system. This tool has great potential in the optimization of tissue-engineered constructs for peripheral nerve repair.     

In Vitro Retina as an Experimental Model of the Central Nervous System

Abstract Methods are described for isolating adult rabbit retina and maintaining it in a medium designed to resemble CSF. Morphologic, metabolic, and electrophysiologic measurements obtained on the in vitro retinas showed that they remained in a nearly physiological state for at least 8 h, and even after 2 days in vitro they still exhibited a high level of metabolic activity and electrical responsiveness to light. Physiological activity was modified by photic stimulation, and data are presented to document changes in metabolism in response to the changes in function. The isolated retina appears to offer a number of unusual advantages for studying relationships between function and metabolism in organized mammalian central nervous tissue     

Lentiviral Vector-Mediated Gradients of GDNF in the Injured Peripheral Nerve: Effects on Nerve Coil Formation,Schwann Cell Maturation and Myelination

Although the peripheral nerve is capable of regeneration, only a small minority of patients regain normal function after surgical reconstruction of a major peripheral nerve lesion, resulting in a severe and lasting negative impact on the quality of life. Glial cell-line derived neurotrophic factor (GDNF) has potent survival- and outgrowth-promoting effects on motoneurons, but locally elevated levels of GDNF cause trapping of regenerating axons and the formation of nerve coils. This phenomenon has been called the “candy store” effect. In this study we created gradients of GDNF in the sciatic nerve after a ventral root avulsion. This approach also allowed us to study the effect of increasing concentrations of GDNF on Schwann cell proliferation and morphology in the injured peripheral nerve. We demonstrate that lentiviral vectors can be used to create a 4 cm long GDNF gradient in the intact and lesioned rat sciatic nerve. Nerve coils were formed throughout the gradient and the number and size of the nerve coils increased with increasing GDNF levels in the nerve. In the nerve coils, Schwann cell density is increased, their morphology is disrupted and myelination of axons is severely impaired. The total number of regenerated and surviving motoneurons is not enhanced after the distal application of a GDNF gradient, but increased sprouting does result in higher number of motor axon in the distal segment of the sciatic nerve. These results show that lentiviral vector mediated overexpression of GDNF exerts multiple effects on both Schwann cells and axons and that nerve coil formation already occurs at relatively low concentrations of exogenous GDNF. Controlled expression of GDNF, by using a viral vector with regulatable GDNF expression, may be required to avoid motor axon trapping and to prevent the effects on Schwann cell proliferation and myelination.     

The Transcription Factor TFCP2L1 is Associated with Myelination via miR708-5p Regulation in the Peripheral Nerve System

Neurochemical Research - MicroRNAs (miRNAs) have been implicated in nerve injury and demyelination; however, their functions in peripheral nerves remain unclear. To determine the potential...     

In Vitro Model using Mouse Hepatocytes for Study of Alcohol Stress

In this study, the effects of ethanol and allyl alcohol on primary mouse hepatocytes were investigated. No cytotoxicity was observed by ethanol treatments, but more toxicity to cells was found in the response to allyl alcohol treatment. The expression of cytochrome P450 2E1 (CYP2E1), phase I enzyme was examined in response to ethanol and allyl alcohol. Both xenobiotics induced CYP2E1 up to 1.5～5 fold at the protein level. The effects of insulin on CYP2E1 expression were also measured. Insulin, which has been regarded as an essential hormone for primary hepatocytes, was shown to decrease the level of CYP2E1 protein, and did not affect cell viability. These results on CYP2E1 induction demonstrate that primary mouse hepatocytes, when using ethanol and allyl alcohol as substrates and in insulin-free medium, provide a suitable system for the studies of the role of CYP2E1 in xenobiotic metabolism and toxicity.     

In Vitro System for Production of Mouse Mammary Tumor Virus

An in vitro system for production, purification, and concentration of mouse mammary tumor virus is described. Monolayer cultures of C(3)H mouse mammary tumor cells propagated at 34 C in roller bottles in the presence of dexamethasone, a glucocorticoid hormone, release B-type particles which possess ribonucleic acid and a ribonucleic acid-dependent deoxyribonucleic acid polymerase. One thousandfold concentration by ultracentrifugation with subsequent gradient fractionation yielded > 7 x 10(10) particles per ml in the 1.16- to 1.18-g/ml region. Mouse mammary tumor virus produced in this system was free of detectable C-type virus.     

Involvement of TRPM2 in Peripheral Nerve Injury-Induced Infiltration of Peripheral Immune Cells into the Spinal Cord in Mouse Neuropathic Pain Model

Recent evidence suggests that transient receptor potential melastatin 2 (TRPM2) expressed in immune cells plays an important role in immune and inflammatory responses. We recently reported that TRPM2 expressed in macrophages and spinal microglia contributes to the pathogenesis of inflammatory and neuropathic pain aggravating peripheral and central pronociceptive inflammatory responses in mice. To further elucidate the contribution of TRPM2 expressed by peripheral immune cells to neuropathic pain, we examined the development of peripheral nerve injury-induced neuropathic pain and the infiltration of immune cells (particularly macrophages) into the injured nerve and spinal cord by using bone marrow (BM) chimeric mice by crossing wildtype (WT) and TRPM2-knockout (TRPM2-KO) mice. Four types of BM chimeric mice were prepared, in which irradiated WT or TRPM2-KO recipient mice were transplanted with either WT-or TRPM2-KO donor mouse-derived green fluorescence protein-positive (GFP⁺) BM cells (TRPM2^BM+/Rec+, TRPM2^BM–/Rec+, TRPM2^BM+/Rec–, and TRPM2^BM–/Rec– mice). Mechanical allodynia induced by partial sciatic nerve ligation observed in TRPM2^BM+/Rec+ mice was attenuated in TRPM2^BM–/Rec+, TRPM2^BM+/Rec–, and TRPM2^BM–/Rec– mice. The numbers of GFP⁺ BM-derived cells and Iba1/GFP double-positive macrophages in the injured sciatic nerve did not differ among chimeric mice 14 days after the nerve injury. In the spinal cord, the number of GFP⁺ BM-derived cells, particularly GFP/Iba1 double-positive macrophages, was significantly decreased in the three TRPM2-KO chimeric mouse groups compared with TRPM2^BM+/Rec+ mice. However, the numbers of GFP^–/Iba1⁺ resident microglia did not differ among chimeric mice. These results suggest that TRPM2 plays an important role in the infiltration of peripheral immune cells, particularly macrophages, into the spinal cord, rather than the infiltration of peripheral immune cells into the injured nerves and activation of spinal-resident microglia. The spinal infiltration of macrophages mediated by TRPM2 may contribute to the pathogenesis of neuropathic pain.     

低温保存的小鼠卵母细胞的体外授精研究

采用75分钟和150分钟两种精卵作用时间,对经6℃低温处理2小时和6小时的小鼠卵母细胞进行体外受精。精卵作用75分钟后,经6℃处理的卵子无一受精,而对照组的受精率为30.7％。作用150分钟后,低温处理2小时、6小时和对照组的受精率分别为62.0％,36.55％和76.0％。并对试验所出现的现象作了讨论。     

Comparative Cardiac Toxicity of Anthracyclines In Vitro and In Vivo in the Mouse

Purpose

The antineoplastic efficacy of anthracyclines is limited by their cardiac toxicity. In this study, we evaluated the toxicity of doxorubicin, non-pegylated liposomal-delivered doxorubicin, and epirubicin in HL-1 adult cardiomyocytes in culture as well as in the mouse in vivo.

Methods

The cardiomyocytes were incubated with the three anthracyclines (1 µM) to assess reactive oxygen generation, DNA damage and apoptotic cell death. CF-1 mice (10/group) received doxorubicin, epirubicin or non-pegylated liposomal-doxorubicin (10 mg/kg) and cardiac function was monitored by Doppler echocardiography to measure left ventricular ejection fraction (LVEF), heart rate (HR) and cardiac output (CO) both prior to and 10 days after drug treatment.

Results

In HL-1 cells, non-pegylated liposomal-doxorubicin generated significantly less reactive oxygen species (ROS), as well as less DNA damage and apoptosis activation when compared with doxorubicin and epirubicin. Cultured breast tumor cells showed similar sensitivity to the three anthracyclines. In the healthy mouse, non-pegylated liposomal doxorubicin showed a minimal and non-significant decrease in LVEF with no change in HR or CO, compared to doxorubicin and epirubicin.

Conclusion

This study provides evidence for reduced cardiac toxicity of non-pegylated-liposomal doxorubicin characterized by attenuation of ROS generation, DNA damage and apoptosis in comparison to epirubicin and doxorubicin.     

Effect of pH on an In Vitro Model of Gastric Microbiota in Enteral Nutrition Patients

Patients with dysphagia due to oropharyngeal disease or cerebrovascular accident require long-term nutritional support via enteral feeding, which often results in microbial overgrowth in the upper gastrointestinal (GI) tract. Gastric acid is the primary innate defense mechanism in the stomach and has been assumed to provide an effective barrier to microbial colonization at pH values of <4. To evaluate the efficacy of gastric acid as a barrier to overgrowth, the microbiota of gastric and duodenal aspirates was assessed by culturing methods. Additionally, a fermentor-based model incorporating enteral nutrition tubing of the gastric microbiota of enteral nutrition (EN) patients was constructed to assess the effect of pH on the microbiota. Results showed that gastric acidity had a relatively small effect on the numbers of microorganisms recovered from intestinal aspirates but did influence microbiota composition. Similarly, at pH 3 in the fermentor, a complex microbiota developed in the planktonic phase and in biofilms. The effect of pH on microbiota composition was similar in aspirates and in the fermentors. Candidas and lactobacilli were aciduric, while recoveries of Escherichia coli and Klebsiella pneumoniae decreased as pH was reduced, although both were still present in significant numbers at pH 3. Only Staphylococcus aureus and Bifidobacterium adolescentis persisted at higher pH values both in vitro and in vivo. Lactate and acetate were the main organic acids detected in both aspirates and fermentors. These data show that the simulator used in this investigation was capable of modeling the effects of environmental influences on the upper GI microbiota of EN patients and that gastric pH of <4 is not sufficient to prevent microbial overgrowth in these individuals.     

F11-Mediated Inhibition of RhoA Signalling Enhances the Spread of Vaccinia Virus In Vitro and In Vivo in an Intranasal Mouse Model of Infection

The cortical actin cytoskeleton beneath the plasma membrane represents a physical barrier that vaccinia virus has to overcome during its exit from an infected cell. Previous observations using overexpression and pharmacological approaches suggest that vaccinia enhances its release by modulating the cortical actin cytoskeleton by inhibiting RhoA signalling using the viral protein F11. We have now examined the role of F11 and its ability to interact with RhoA to inhibit its downstream signalling in the spread of vaccinia infection both in vitro and in vivo. Live cell imaging over 48 hours reveals that loss of F11 or its ability to bind RhoA dramatically reduces the rate of cell-to-cell spread of the virus in a cell monolayer. Cells infected with the ΔF11L virus also maintained their cell-to-cell contacts, and did not undergo virus-induced motility as observed during wild-type infections. The ΔF11L virus is also attenuated in intranasal mouse models of infection, as it is impaired in its ability to spread from the initial sites of infection to the lungs and spleen. Loss of the ability of F11 to bind RhoA also reduces viral spread in vivo. Our results clearly establish that viral-mediated inibition of RhoA signalling can enhance the spread of infection not only in cell monolayers, but also in vivo.     

Electrophysiological Properties of Motor Neurons in a Mouse Model of Severe Spinal Muscular Atrophy: In Vitro versus In Vivo Development

We examined the electrophysiological activity of motor neurons from the mouse model of severe spinal muscular atrophy (SMA) using two different methods: whole cell patch clamp of neurons cultured from day 13 embryos; and multi-electrode recording of ventral horns in spinal cord slices from pups on post-natal days 5 and 6. We used the MED64 multi-electrode array to record electrophysiological activity from motor neurons in slices from the lumbar spinal cord of SMA pups and their unaffected littermates. Recording simultaneously from up to 32 sites across the ventral horn, we observed a significant decrease in the number of active neurons in 5–6 day-old SMA pups compared to littermates. Ventral horn activity in control pups is significantly activated by serotonin and depressed by GABA, while these agents had much less effect on SMA slices. In contrast to the large differences observed in spinal cord, neurons cultured from SMA embryos for up to 21 days showed no significant differences in electrophysiological activity compared to littermates. No differences were observed in membrane potential, frequency of spiking and synaptic activity in cells from SMA embryos compared to controls. In addition, we observed no difference in cell survival between cells from SMA embryos and their unaffected littermates. Our results represent the first report on the electrophysiology of SMN-deficient motor neurons, and suggest that motor neuron development in vitro follows a different path than in vivo development, a path in which loss of SMN expression has little effect on motor neuron function and survival.     

Chronic Nerve Growth Factor Exposure Increases Apoptosis in a Model of In Vitro Induced Conjunctival Myofibroblasts

In the conjunctiva, repeated or prolonged exposure to injury leads to tissue remodeling and fibrosis associated with dryness, lost of corneal transparency and defect of ocular function. At the site of injury, fibroblasts (FB) migrate and differentiate into myofibroblasts (myoFB), contributing to the healing process together with other cell types, cytokines and growth factors. While the physiological deletion of MyoFB is necessary to successfully end the healing process, myoFB prolonged survival characterizes the pathological process of fibrosis. The reason for myoFB persistence is poorly understood. Nerve Growth Factor (NGF), often increased in inflamed stromal conjunctiva, may represent an important molecule both in many inflammatory processes characterized by tissue remodeling and in promoting wound-healing and well-balanced repair in humans. NGF effects are mediated by the specific expression of the NGF neurotrophic tyrosine kinase receptor type 1 (trkA^NGFR) and/or the pan-neurotrophin glycoprotein receptor (p75^NTR). Therefore, a conjunctival myoFB model (TGFβ1-induced myoFB) was developed and characterized for cell viability/proliferation as well as αSMA, p75^NTR and trkA^NGFR expression. MyoFB were exposed to acute and chronic NGF treatment and examined for their p75^NTR/trkA^NGFR, αSMA/TGFβ1 expression, and apoptosis. Both NGF treatments significantly increased the expression of p75^NTR, associated with a deregulation of both αSMA/TGFβ1 genes. Acute and chronic NGF exposures induced apoptosis in p75^NTR expressing myoFB, an effect counteracted by the specific trkA^NGFR and/or p75^NTR inhibitors. Focused single p75^NTR and double trkA^NGFR/p75^NTR knocking-down experiments highlighted the role of p75^NTR in NGF-induced apoptosis. Our current data indicate that NGF is able to trigger in vitro myoFB apoptosis, mainly via p75^NTR. The trkA^NGFR/p75^NTR ratio in favor of p75^NTR characterizes this process. Due to the lack of effective pharmacological agents for balanced tissue repairs, these new findings suggest that NGF might be a suitable therapeutic tool in conditions with impaired tissue healing.     

Inhibition of EGFR-AKT Axis Results in the Suppression of Ovarian Tumors In Vitro and in Preclinical Mouse Model

Ovarian cancer is the leading cause of cancer related deaths in women. Genetic alterations including overexpression of EGFR play a crucial role in ovarian carcinogenesis. Here we evaluated the effect of phenethyl isothiocyanate (PEITC) in ovarian tumor cells in vitro and in vivo. Oral administration of 12 μmol PEITC resulted in drastically suppressing ovarian tumor growth in a preclinical mouse model. Our in vitro studies demonstrated that PEITC suppress the growth of SKOV-3, OVCAR-3 and TOV-21G human ovarian cancer cells by inducing apoptosis in a concentration-dependent manner. Growth inhibitory effects of PEITC were mediated by inhibition of EGFR and AKT, which are known to be overexpressed in ovarian tumors. PEITC treatment caused significant down regulation of constitutive protein levels as well as phosphorylation of EGFR at Tyr1068 in various ovarian cancer cells. In addition, PEITC treatment drastically reduced the phosphorylation of AKT which is downstream to EGFR and disrupted mTOR signaling. PEITC treatment also inhibited the kinase activity of AKT as observed by the down regulation of p-GSK in OVCAR-3 and TOV-21G cells. AKT overexpression or TGF treatment blocked PEITC induced apoptosis in ovarian cancer cells. These results suggest that PEITC targets EGFR/AKT pathway in our model. In conclusion, our study suggests that PEITC could be used alone or in combination with other therapeutic agents to treat ovarian cancer.