Detection of single mRNAs in individual cells of the auditory system.
Cochlea; Immunohistochemistry; Inner hair cell; Outer hair cell; Single-molecule fluorescence in situ hybridization; Spiral ganglion neuron
Gene expression analysis is essential for understanding the rich repertoire of cellular functions. With the development of sensitive molecular tools such as single-cell RNA sequencing, extensive gene expression data can be obtained and analyzed from various tissues. Single-molecule fluorescence in situ hybridization (smFISH) has emerged as a powerful complementary tool for single-cell genomics studies because of its ability to map and quantify the spatial distributions of single mRNAs at the subcellular level in their native tissue. Here, we present a detailed method to study the copy numbers and spatial localizations of single mRNAs in the cochlea and inferior colliculus. First, we demonstrate that smFISH can be performed successfully in adult cochlear tissue after decalcification. Second, we show that the smFISH signals can be detected with high specificity. Third, we adapt an automated transcript analysis pipeline to quantify and identify single mRNAs in a cell-specific manner. Lastly, we show that our method can be used to study possible correlations between transcriptional and translational activities of single genes. Thus, we have developed a detailed smFISH protocol that can be used to study the expression of single mRNAs in specific cell types of the peripheral and central auditory systems.
Salehi Pezhman; Nelson Charlie N; Chen Yingying; Lei Debin; Crish Samuel D; Nelson Jovitha; Zuo Hongyan; Bao Jianxin
Hearing research
2018
2018-09
Article information provided for research and reference use only. All rights are retained by the journal listed under publisher and/or the creator(s).
<a href="http://doi.org/10.1016/j.heares.2018.07.008" target="_blank" rel="noreferrer noopener">10.1016/j.heares.2018.07.008</a>
Otoprotective Effects of Stephania tetrandra S. Moore Herb Isolate against Acoustic Trauma.
calcium channel; hair cells; noise-induced hearing loss; spiral ganglion neurons; Stephania tetrandra; synapse; Tetrandrine
Noise is the most common occupational and environmental hazard, and noise-induced hearing loss (NIHL) is the second most common form of sensorineural hearing deficit. Although therapeutics that target the free-radical pathway have shown promise, none of these compounds is currently approved against NIHL by the United States Food and Drug Administration. The present study has demonstrated that tetrandrine (TET), a traditional Chinese medicinal alkaloid and the main chemical isolate of the Stephania tetrandra S. Moore herb, significantly attenuated NIHL in CBA/CaJ mice. TET is known to exert antihypertensive and antiarrhythmic effects through the blocking of calcium channels. Whole-cell patch-clamp recording from adult spiral ganglion neurons showed that TET blocked the transient Ca(2+) current in a dose-dependent manner and the half-blocking concentration was 0.6 + 0.1 muM. Consistent with previous findings that modulations of calcium-based signaling pathways have both prophylactic and therapeutic effects against neural trauma, NIHL was significantly diminished by TET administration. Importantly, TET has a long-lasting protective effect after noise exposure (48 weeks) in comparison to 2 weeks after noise exposure. The otoprotective effects of TET were achieved mainly by preventing outer hair cell damage and synapse loss between inner hair cells and spiral ganglion neurons. Thus, our data indicate that TET has great potential in the prevention and treatment of NIHL.
Yu Yan; Hu Bing; Bao Jianxin; Mulvany Jessica; Bielefeld Eric; Harrison Ryan T; Neton Sarah A; Thirumala Partha; Chen Yingying; Lei Debin; Qiu Ziyu; Zheng Qingyin; Ren Jihao; Perez-Flores Maria Cristina; Yamoah Ebenezer N; Salehi Pezhman
Journal of the Association for Research in Otolaryngology : JARO
2018
2018-12
Article information provided for research and reference use only. All rights are retained by the journal listed under publisher and/or the creator(s).
<a href="http://doi.org/10.1007/s10162-018-00690-3" target="_blank" rel="noreferrer noopener">10.1007/s10162-018-00690-3</a>
Demethyleneberberine alleviates inflammatory bowel disease in mice through regulating NF-kappaB signaling and T-helper cell homeostasis.
Animals; Anti-Inflammatory Agents/*pharmacology/therapeutic use; Berberine/*analogs & derivatives/pharmacology/therapeutic use; Colitis; Colon/drug effects/immunology/pathology; Cytokines/immunology; Dextran Sulfate; DMB; Female; Helper-Inducer/*drug effects/immunology; Homeostasis/drug effects; Immunoglobulin G/blood; Inbred C57BL; Inflammatory Bowel Diseases/blood/chemically induced/drug therapy/*immunology; Mice; NF-kappa B/*antagonists & inhibitors/immunology; NF-kappaB; RAW 264.7 Cells; Reactive Oxygen Species/metabolism; Signal Transduction/drug effects; Spleen/cytology; T-Lymphocytes; Th cell
OBJECTIVE: The activation of NF-kappaB signaling and unbalance of T-helper (Th) cells have been reported to play a key role in the pathogenesis of colitis. Cortex Phellodendri Chinensis (CPC) is commonly used to treat inflammation and diarrhea. Demethyleneberberine (DMB), a component of CPC, was reported to treat alcoholic liver disease as a novel natural mitochondria-targeted antioxidant in our previous study. In this study, we investigated whether DMB could protect against dextran sulfate sodium (DSS)-induced inflammatory colitis in mice by regulation of NF-kappaB pathway and Th cells homeostatis. METHODS: Inflammatory colitis mice were induced by 3% DSS, and DMB were orally administered on the doses of 150 and 300 mg/kg. In vitro, DMB (10, 20, 40 muM) and N-acetyl cysteine (NAC, 5 mM) were co-cultured with RAW264.7 for 2 h prior to lipopolysaccharide (LPS) stimulation, and splenocytes from the mice were cultured ex vivo for 48 h for immune response test. RESULTS: In vivo, DMB significantly alleviated the weight loss and diminished myeloperoxidase (MPO) activity, while significantly reduced the production of pro-inflammatory cytokines, such as interleukin (IL)-6 and tumor necrosis factor-alpha (TNF-alpha), and inhibited the activation of
Chen Yingying; Li Rui-Yan; Shi Mei-Jing; Zhao Ya-Xing; Yan Yan; Xu Xin-Xin; Zhang Miao; Zhao Xiao-Tong; Zhang Yu-Bin
Inflammation research : official journal of the European Histamine Research Society ... [et al.]
2017
2017-02
Article information provided for research and reference use only. All rights are retained by the journal listed under publisher and/or the creator(s).
<a href="http://doi.org/10.1007/s00011-016-1005-3" target="_blank" rel="noreferrer noopener">10.1007/s00011-016-1005-3</a>