\nResearch Proposal\n<\/p>\n
\nDiscuss the role of connexin in deafness\n<\/p>\n
\n\u00a0<\/strong>\n<\/p>\n \nIntroduction: <\/strong>\n<\/p>\n \nGap junctions (GJ) in the mammalian cochlear consist of Cx26 and 30 (Liu et al, 2016) coded for by the genes GJB2 and 6 respectively, on chromosome 13q. The GJs allow the transfer of\u00a0 ions and small molecules up to ~1.5 kDa (metabolites and potential signalling molecules) down their concentration gradient (Alberts, 2008) due to the large pore size ~10\u201315 \u00c5 (Jagger and Forge, 2015). Fluid homeostasis and intercellular cell signalling are enabled through the connection (Nickel and Forge, 2008). Researchers identified that Cx26\/30 are involved in cochlear function and hearing but their exact role and contribution to cochlear physiology remains unknown.\n<\/p>\n \n \nFigure 1:<\/strong> Highlights that the GJs are formed by two hemichannels composed of six connexin subunits spanning across adjacent cells. These GJs connect the cytoplasms of adjacent cells as seen in the bottom image (Image modified from Verheule and Kaese, 2013)\n<\/p>\n \nThe GJs have many roles but one proposed role that hasn\u2019t been investigated as much is their role in nutrient and energy supply. The cochlear is supplied by the labyrinthine artery alone (Nakashima et al., 2003). Hoang Dinh et al., 2009 showed that glucose from the cardiovascular circulation reaches cochlear supporting cells through GJs. Furthermore the organ of corti\u2019s sensory epithelium is an avascular organ where microcirculation to hair cells (HCs) and supporting cells is lacking (____). This is important as Cx-mutation linked defects are usually seen in regions with poor microcirculation or when metabolite transportation is demanding which explains why the cochlear is a target. Glucose is the major energy source within cellular metabolic activities, required for development of the cochlear.\n<\/p>\n \nResearchers have proposed that deafness seen in loss-of-function Cx mutants was due to reduced transportation of energetic metabolites across the GJs in the organ of corti where microcirculation is poor (chang et al., 2008).\n<\/p>\n \nHypotheses<\/strong>:\n<\/p>\n \n\u00a0<\/strong>\n<\/p>\n \nAim<\/strong>: Understanding how metabolite transfer is effected in Cx26 mutants and how this decreased transfer of glucose disrupts hearing and cochlear development. This will be done by monitoring glucose movement throughout the cochlear comparing the WT and Cx26 KO mice to see if transfer\u2019s impaired. Then to take this further, checking whether decreased glucose availability is linked to an increase in ROS which promotes cell death, as previous reports have suggested there may be a link here (___)\n<\/p>\n \nAll procedures are performed at three key postnatal stages including P4.2, as previous investigations have concluded that Cx26 is vital for early on development and if mutated prior to P4.2 hearing loss is seen (Chen et al., 2014). P6 where Cx30 begins to take over and is suggested to compensate for Cx26 deficiency, and P12 when the onset of hearing begins.\n<\/p>\n \nMethods:<\/strong>\n<\/p>\n \nProducing Cx26 KO mouse<\/strong>\n<\/p>\n \nHere Cx26loxP\/loxP\u00a0mice are crossed with the\u00a0Gt(ROSA)26Sortm1(Cre\/Esr1)Nat\/J mouse strain (Chen et al., 2014).\u00a0\n<\/p>\n \nThis has a tamoxifen-inducible Cre-mediated recombination system determined by the endogenous mouse Gt(ROSA)26Sor promoter.\n<\/p>\n \n4-hydroxytamoxifen (4-HTMX) is added via after birth for 3 days via intraperitoneal injection to delete Cx26 hence activating cre-expression (0.5 mg\/10g body weight).\n<\/p>\n \nControl:\n<\/p>\n \n–\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0 The WT and mutant mice will come from the same litter and multiple litters will be used to ensure result reliability.\n<\/p>\n \n–\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0 WT is exposed to 4-HTMX, to confirm 4-HTMX isn\u2019t causing abnormal cochlear morphology or hearing loss.\n<\/p>\n \nImmunofluorescent staining<\/strong>\n<\/p>\n \nThis method is performed on the cochlear cells. 4% paraformaldehyde is added to fix the cells alongside PBS (0.1M) for half an hour. Then 0.1% triton is used to permeabalise the sample for another half an hour. Blockage is then achieved by adding 10% goat serum for an hour.\n<\/p>\n \nFirstly the tissue\u2019s incubated with a primary antibody (monoclonal mouse anti-Cx26 antibodies) within that blocking solution overnight at a low temperature (4 degrees). PBS is used to wash the sample, repeated three times.\u00a0 Then incubate with a secondary Alexa Fluor\u00ae 488 and 568 conjugated antibodies (Chen et al., 2014), again with the blockage solution for another hour. Finally the sample is washed out placed in a fluorescence mounting medium and observed either via a confocal or fluorescence microscope.\n<\/p>\n \nFluorescent dye diffusion assay <\/strong>\n<\/p>\n \nThis method checks whether GJs are assembling correctly in mutants. Membrane impermeable florescent dye, Luciferase yellow (LY), gets injected by a pipette into the cytoplasm of a single cell located in the cochlear (e.g. Claudius cells) through the whole-cell patch-clamp mode.\n<\/p>\n \nIntercellular dye diffusion patterns are recorded by a cooled digital CCD camera every 5 minutes for 30 minutes, comparing the distance the dye\u2019s moved. AxioVision 3.1 software controls image attainment. The cells presenting florescence are counted under a microscope to see how far the dye has travelled hence It\u2019s important that the cell borders are clearly recognised.\u00a0 Under room temperature.\n<\/p>\n \nControl\n<\/p>\n \n–\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0 The dye\u2019s injected into a marginal cell which don\u2019t contain GJs to certify the dye\u2019s GJs selective and not moving via alternative means\n<\/p>\n \n\u00a0<\/strong>\n<\/p>\n \nUse of 2-NBDG to mimic glucose transfer<\/strong>\n<\/p>\n \nThis technique show how much and how easily glucose travels in the WT and mutant. Mice are anesthetized via xylazine and ketamine injections (i.p., 10 mg\/ kg and i.p., 100 mg\/kg respectively). Targeting the mice\u2019s cardiovascular system the hearts left ventricle is penetrated with a shielded wing needle to add the solutions (table 1). A peristaltic pump works to control the speed of perfusion. Animals are divided into four experimental groups which allow the confirmation of the results\n<\/p>\n \nMouse model used\n<\/p>\n<\/td>\n \nWhat was required\n<\/p>\n<\/td>\n<\/tr>\n \n1.WT mice with 2-NBDG\n<\/p>\n<\/td>\n \n3.8 mM\u00a0 of 2-NBDG (in HBSS) is added for 10 minutes\n<\/p>\n<\/td>\n<\/tr>\n \n2. WT mice with octanol and 2-NBDG\n<\/p>\n<\/td>\n \n2 mM of octanol (in HBSS) added for 10 minutes\n<\/p>\n \n3.8 mM of 2-NBDG plus 2 mM of octanol (in PBS) is added for another 10 minutes\u00a0\n<\/p>\n<\/td>\n<\/tr>\n \n3. WT mice with 2-NBDG and glucose\n<\/p>\n<\/td>\n \n3.8 mM of 2-NBDG\n<\/p>\n \n0.5 M of glucose (in PBS)\n<\/p>\n \nAdded for 10 minutes\n<\/p>\n<\/td>\n<\/tr>\n \n4. Cx26 KO mice and 2-NBDG\n<\/p>\n<\/td>\n \n3.8 mM of 2-NBDG (in HBSS) added for 10 minutes\n<\/p>\n<\/td>\n<\/tr>\n<\/table>\n \nTable 1:<\/strong> outlining what is used in each 4 mouse models to look at movement of 2-NBDG allowing a comparison between the WT and mutant as well as utilising controls. 2-NBDG concentration impersonates the concentration of glucose in the plasma and cochlea. 2mM of octonol is used during preparation to prevent loaded 2-NBDG escaping from the supporting cells as the GJ channel sin the WT are unaffected so it would easily pass through them. Therefore model 3 check the dye isn\u2019t moving via other means. 1-3 are controls to check the experiment will work whilst 4 is being tested with the mutant.\n<\/p>\n \nWhen 2-NBDG is seen in the SV we know the perfusion was successfully. The samples mentioned above are relocated to perfusion chambers to allow observation through an inverted fluorescent microscope. Cooled-CCD camera works to obtain images whilst ImageJ software measures the florescence emitted.\n<\/p>\n \n\u00a0<\/strong>\n<\/p>\n \nMeasuring intracellular ROS level<\/strong>\n<\/p>\n \nFluorescent indicator dye, CM-H2DCFDA, monitors intracellular ROS levels. This compound remains non-florescent until its acetate group is removed by the enzyme esterase or when living cell experience oxidation occurs. After 70 minutes within the supporting cells the intracellular fluorescent intensity of H2DCFDA(10mM) should stabilise, indicating that the intracellular dye is loaded. After 80 minutes images are taken using an AxioCam digital camera alongside AxioVision 4.6 software. This will be compared between the WT and Cx26 KO. Here we can also observe any signs of cell death having occurred by the change in ROS levels.\n<\/p>\n \nThe ROS produced in the supporting cells will correlate with the intensity of florescence, which is measured by ImageJ software and averages are taken.\n<\/p>\n \nStatistical methods<\/strong>\n<\/p>\n \nMultiple comparison 2 way ANOVA\n<\/p>\n \nResults:<\/strong>\n<\/p>\n \nMethod<\/strong>\n<\/p>\n<\/td>\n \nResults for WT<\/strong>\n<\/p>\n<\/td>\n \nResults for Cx26 KO<\/strong>\n<\/p>\n<\/td>\n \nReason for use?<\/strong>\n<\/p>\n<\/td>\n<\/tr>\n \nImmunofluorescent staining <\/strong>\n<\/p>\n<\/td>\n \nNormal florescent staining for Cx26\n<\/p>\n<\/td>\n \nNo florescent staining to be seen regarding Cx26 as its been KO\n<\/p>\n<\/td>\n \nUsed to check for the presence of Cx26 hence the success of the KO\n<\/p>\n<\/td>\n<\/tr>\n \nFluorescent dye diffusion assay <\/strong>\n<\/p>\n<\/td>\n \nThe dye injected should reach a large diameter of cells faster\n<\/p>\n<\/td>\n \nThe dye will be localised around the place it was injected and it shouldn\u2019t have moved far as the GJs haven\u2019t formed properly hence no movement should be seen\n<\/p>\n \nWhen looking at P6\/12 movement should still be seen but not as effectively as the WT\n<\/p>\n<\/td>\n \nThis informs us on whether the GJs have correctly assembled\n<\/p>\n<\/td>\n<\/tr>\n \nUse of 2-NBDG<\/strong>\n<\/p>\n<\/td>\n \nWT mice show 2NBDG reaching longer distances faster\n<\/p>\n<\/td>\n \nKO shows 2-NBDG reaching significantly shorter distance slower. This may vary being a little more for the P6\/12 mice models but same principle applies\n<\/p>\n<\/td>\n \nThis is used to get an clear picture of how much glucose is making its way into the cochlear\n<\/p>\n<\/td>\n<\/tr>\n \nMeasuring intracellular ROS levels<\/strong>\n<\/p>\n<\/td>\n \nThe levels should be relatively low hence florescence should be very minimal.\n<\/p>\n \nCell death here will be at normal levels and the cochlear should be developing as normal\n<\/p>\n<\/td>\n \nWe see increased ROS levels hence the sample appears highly florescent.\n<\/p>\n \nAdditionally we should also observe using for example a confocal microscope cell death to be seen in the cochlear\n<\/p>\n<\/td>\n \nShows the levels of ROS present a compound associated with apoptosis\n<\/p>\n<\/td>\n<\/tr>\n<\/table>\n \nTable 2: <\/strong>This tablesummarises the results that are expected to be seen in terms of the following experiments within both the WT and Cx26 KO but reasons why are explained below.<\/p>\n \n(Alberts, 2008)\n<\/p>\n \nResearch has suggested the Intercellular coupling GJs provide plays a role in allowing local glucose and other metabolites transfers (Albert, ____). Immunofluorescent staining informs us on Cx26-KO success. If successful the antibodies targeting Cx26 can\u2019t bind hence no florescence is seen.\n<\/p>\n \nTo identify Cx26s role in metabolic coupling within the cochlear supporting cells dye diffusion is performed using LY known to pass selectively through GJs. LY is used in supporting cells like Claudius cells to determine if metabolite transfer is altered when the GJs are incorrectly assembled within Cx26-KO. For Cx26-KOs, I\u2019d expect to see reduced LY transfer due to these poorly assembled channels. Whilst for the WT LY reaches a large range of cells as GJ assembly is unaltered.\n<\/p>\n \nTo identify differences in glucose transfer from the blood vessels to the inner ear cells via GJs, 2-NBDG a florescent dye that mimics glucose movement is used and florescence is measured and quantified.\u00a0 For the WT I\u2019d expect fast diffusion of 2-NBDG across the cochlear supporting cells, reaching a significantly wider distance than the mutant channels. If there is still movement of glucose in the mutant this may be down to co-assembly of Cxs in cochlear GJs hence Cx30 may compensate for the Cx26-KO as these GJs can also assemble as homomeric Cx30 channels (Figure 1). But it\u2019s not until beyond P5 when Cx30 is dominant hence why we\u2019re using stages P6 and P12.\u00a0 Therefore we can investigate whether complete eradication of GJ-mediated intercellular biochemical coupling is a likely outcome.\n<\/p>\n \n\u00a0<\/strong>\n<\/p>\n \nGlucose is vital in many processes throughout the organism, if absent cells would begin to undergo apoptosis and development wouldn\u2019t occur successfully. Using a range of stages provides a better understanding and possibly highlights the time when glucose demand is at a peak hence disturbances in glucose transfer have drastic outcome (deafness). It\u2019s suggested that glucose deficiency decreases ATP availability whilst increasing ROS generation in supporting cells, through Cx30 null mice models (Zhu et\u00a0al., 2015a). Increased ROS increases mitochondrial permeability releasing cytochrome-C and other pro-apoptotic factors into the cytosol (Mammano and Bortolozzi, 2018). Proposing that this ROS rise causes cell death and cochlear dysfunction.\n<\/p>\n \nMy last experiment works to prove whether there is a correlation between decreased glucose availability and ROS increase therefore cell death in the cochlear meaning sound waves can\u2019t be converted into electrical signals for the brain to process. This proposed cell death phenotype will be compared under the microscope between the WT and mutants, giving a clear visual answer to this theory.\n<\/p>\n \nConclusion: <\/strong>\n<\/p>\n \nThis proposal is an important in identifying connexins role in deafness. Mutations in connexin26 are responsible for non-syndromic childhood deafness within humans (Jagger and Forge, 2014).\n<\/p>\n \nMy research, if everything goes as expected would display that reduced glucose delivery worsens ATP availability and causes ROS accumulation in supporting cells and consequently cytochrome C release hence cell death and cochlear dysfunction. I believe this would apply more to earlier stages rather than later ones due to that idea of redundancy between connexins26\/30. This would disrupt other pathways as we know ATP is involved in K+<\/sup> recycling and Ca2+<\/sup> wave propagation. Previous experiments have proved that intracellular conductance remains unaltered in these mutants hence the proposed pathways possibly aren\u2019t the issue but rather glucose deficiency is. This would need to be explored after the completion of this investigation.\n<\/p>\n \nReferences:<\/strong>\n<\/p>\n \n Study Bay Coursework Assignment Writing Help Research Proposal Discuss the role of connexin in deafness \u00a0 Introduction: Gap junctions (GJ) in the mammalian cochlear consist\u2026<\/p>\n","protected":false},"author":3,"featured_media":0,"comment_status":"open","ping_status":"open","sticky":false,"template":"","format":"standard","meta":{"footnotes":""},"categories":[8092,8093,7625,7823,8094,7826,8078],"tags":[7828,8095,450,594,8100,574,8098,8097,8096,8099],"class_list":["post-86549","post","type-post","status-publish","format-standard","hentry","category-affordable-dissertation-writing-service","category-are-dissertation-writing-services-legal","category-assignment-help-uk","category-best-dissertation-writing-service","category-dissertation-writing-service-australia","category-dissertation-writing-services-in-usa","category-professional-dissertation-writing-service","tag-best-dissertation-writing-service-review","tag-best-phd-thesis-writing-services","tag-dissertation-writing-services","tag-dissertation-writing-services-near-me","tag-essay-writer-usa","tag-free-dissertation-writing-services","tag-my-assignment-services","tag-phd-thesis-writing-assistance","tag-phd-writer","tag-research-paper-writing-service-australia"],"_links":{"self":[{"href":"https:\/\/www.studyproessays.com\/help\/wp-json\/wp\/v2\/posts\/86549","targetHints":{"allow":["GET"]}}],"collection":[{"href":"https:\/\/www.studyproessays.com\/help\/wp-json\/wp\/v2\/posts"}],"about":[{"href":"https:\/\/www.studyproessays.com\/help\/wp-json\/wp\/v2\/types\/post"}],"author":[{"embeddable":true,"href":"https:\/\/www.studyproessays.com\/help\/wp-json\/wp\/v2\/users\/3"}],"replies":[{"embeddable":true,"href":"https:\/\/www.studyproessays.com\/help\/wp-json\/wp\/v2\/comments?post=86549"}],"version-history":[{"count":0,"href":"https:\/\/www.studyproessays.com\/help\/wp-json\/wp\/v2\/posts\/86549\/revisions"}],"wp:attachment":[{"href":"https:\/\/www.studyproessays.com\/help\/wp-json\/wp\/v2\/media?parent=86549"}],"wp:term":[{"taxonomy":"category","embeddable":true,"href":"https:\/\/www.studyproessays.com\/help\/wp-json\/wp\/v2\/categories?post=86549"},{"taxonomy":"post_tag","embeddable":true,"href":"https:\/\/www.studyproessays.com\/help\/wp-json\/wp\/v2\/tags?post=86549"}],"curies":[{"name":"wp","href":"https:\/\/api.w.org\/{rel}","templated":true}]}}![\"\"](\"images\/0458011.001.png\")
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