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Staging Of Neurofibrillary Pathology: Alzheimer Disease

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Staging Of Neurofibrillary Pathology: Alzheimer Disease Question: Discuss about the Report on Staging Of Neurofibrillary Pathology Related To Alzheimer Disease With The Help Of Paraffin Sections And Immunocy to chemistry?   Answer: Aim And Justification The aim of the research is to amend the staging procedure of AD-related neurofibrillary pathology described in the year 1991 by highlighting immunoreactions for visualizing hyperphosphorylated tau. Adaptation of the tissue selection would be taken up and processed to the requirements of the regular diagnostic laboratory. Staging of the AD-related neurofibrillary pathology in six different stages is the main aim. The focus would be on the plexuses formed of both pretangle and tangle material. The difference is that paraffin sections would be used that would be immunostained for hyperphosphorylated tau and processed on an automated basis . For undertaking a proper assessment of Alzheimer’s disease related neurofibrillary pathology, there is particularly need of any method permitting a adequate distinction between early, intermediate and late stages (Gonzalez-Lima 2013). A main element of Alzheimer’s disease is the deposition of tau protein in a slow process, phosphorylated in nature, within specific neuronal types in particular areas (Lock 2013). The staging of Alzheimer’s disease related neurofibrillary pathology first came into light in the first half of the 1990s. Thick sections were used that were unconventional (100 micrometer). The staging had the use of an advanced silver technique. It reflected the progression of the disease that had the basis of the topographic expansion of the lesions occurring within the body (Thal et al. 2013). There is a requirement to set up new procedures for better meeting the demands cropping up in routine laboratories researching on Alzheimer’s disease (Morris et al. 2016). The aim of the research hereby proposed is to revise this procedure by adapting tissue selectioning. In the research to be undertaken, thin paraffin sections would be used up (about 5–15 micrometer). Strong immunoreactions (AT8) for hyperphosphorylated tau protein having the ability to processed on an automated basis would be used.  The justification for taking up this topic for the research proposal is that there is an anticipation that the revised method, as put forward by the new research, would demonstrate a more suitable staging procedure’s application.  Background Information Alzheimer’s disease accounts for almost 70% cases of dementia (de la Monte and Tong 2013). It is a significant chronic neurodegenerative disease starting slowly and getting worse over time. The most commonly seen early symptoms are short-term memory loss, problems with language, mood swings, disorientation, loss of motivation and issues with behaviour. Gradually the functioning of the body is lost (Ramanathan 2013).  The cause for such disease is not understood to the full extent. The disease progress has relation with tangles and plaques in the brain. Examination of the brain is the way to make a definite diagnosis of the disease. No treatment can reverse the progression of the disease (RavdinandKatzen 2013). A key element of the pathological processes of Alzheimer’s disease is the development of lesions that grow intraneuronally at the vulnerable sites of the brain. The main composition of the lesions is hyperphosphorylated tau protein including pretangled material that is the neurofibrillary tangles (NFT) in cell bodies, neuritic plaques (NP) and neutrophil threads (NT) (Hyman et al. 2012). The Alzheimer’s disease pathological process takes a lot of time to be established and in this time period, the lesions have a tendency to develop depending on a predictable sequence. A staging system that was introduced in 1991 for the intraneuronal lesions differentiated early, intermediate and late phases of the disease process. It was for patients who were symptomatic and non-symptomatic. The staging system was considered in the NIH-Reagan criteria for the neuropathological diagnosis of the disease. The procedure was based on the assessment of 100 micrometer sections that was undertaken by the silveriodate technique. The first section had the anterior parahippocampal gyrus, hippocampal formation at uncus level and sections of the adjoining occipito-temporal gyrus. The other part took into consideration sections of the parastriate area, striate area and peristriate region. Distinct dissimilarity in the neurofibrillary lesion’s topographical distribution pattern gave a chance to the observer for assigning an autopsy case to one out of the six stages (Lasagna-Reeves et al. 2012). However, there are certain problems with such system in relation to regular diagnostic purpose. The main reason is that it requires unusually thick sections that need to be cut from blocks included in an unconventional medium. Moreover, the method needs free floating sections to be stained by experienced assistant in the laboratory by a non-automated silver technique. These factors clearly demonstrate the limitation of the feasibility of the process for routine diagnosis. There is a need of a uniform staging procedure that would be easy and cost-effective. This issue would be addressed by the proposed methodology that would make possible the proper utilisation of the staging procedure that can be undertaken in a more efficient manner than previous.  The goal for such research is the same and that is to stage the Alzheimer’s disease related neurofibrillary pathology in six stages. The limelight for the proposed research would be on the plexus arising from tangle and pretangle material. Immunocytochemistry is the only method of detecting the pretangle material and the revised staging procedure considers this aspect However, the paraffin section immunostained for the tau protein would be processed on automated basis.   Methods And Experimental Design Materials Required Brains taken for autopsy (This would need ethical approval. The local Standard Operating Procedures (SOPs) along with legal  regulations, ethical  guide-lines are  to   be   considered   for the research  specimen) 4% aqueous solution of HCHO (Formaldehyde) Monoclonal antibody AT8 (Phospho-PHF-tau pSer202+Thr205 Antibody) Biotinylated antibody (anti-mouse IgG) Tenside solution De-ionised water Methodology Of Staging Of Neurofibrillary Pathology Related To Alzheimer Disease The procedure that would be followed is outlined hereby. Brains taken for autopsy would be fixed by immersing them in 10% formalin. This is 4% aqueous solution of HCHO. The time allotted would be one week or it may be longer even. The meninges would be partially removed for uncovering the collateral sulcus, Calarine fissure and rhinal sulcus. Immunostained paraffined sections taken from three blocks having conventional size fitting normal tissue cassettes would be needed. On an alternative basis, the tissue on one side of the cut may be used for paraffin implanting into thin sections. The first block would include anteromedial sections of the temporal lobe with the mid-uncal or amygdala level having the cut and taking into account anterior sections of both the occipito-temporal gyrus and the parahippocampalgyrus. The second section would include sections of the medial and superior temporal gyri and would be taken from the similar slice as the previous block. The third block would be halfway between the connection of the parieto-occipital sulcus and the occipital pole. The cut would be oriented perpendicular to the calcarine. The mounted paraffin would be of 5-15 micrometer in thickness. These would be de-waxed followed by re-hydration. The monoclonal antibody AT8 is a commercially available antibody having strong immunoreactivity for hyperphosphorylated tau protein (Rosseels et al. 2014). This antibody does not have the feature of cross-reacting with normal tau epitopes nor does it need special pre-treatments (Castillo-Carranza et al. 2014). When performed on paraffin sections, these immunoreactions allow counter-staining for other structures too. The sections would be incubated at 4oC for 40 hours with the AT8 antibody. The ratio for the sample and the antibody would be maintained would be 1:2,000. The next step would be to process it with a second biotinylated antibody. The antibody for this case is anti-mouse antibody. The reactions would then be visualised by the use of Reactions are visible with the ABC-complex (Vectastain) and 3,3-diaminobenzidine (Sigma). Long duration of brain tissue fixation in the solution of formaldehyde have the potential to cause metachromatic precipitations (Senturk et al. 2014). The elements of the material react to some extent with silver methods and immunoreactions. The precipitations woud be removed by tenside solution ( the ratio would be 1 unit volume Tween 20 and 9 unit volumes de-ionized water) or pyridine at 80oC 30 minutes. The sections would then be rinsed in a thorough manner in tap water and then shifted to de-ionised water. Gantt Chart:   Week 1  Week 2 Week 3 Week 4  Week 5 Research on the topic ·                   Experiment carried out according to the protocol   ·           ·               Establishment of results       ·             Presentation of reaserch         ·           Expected Results And Impact            Alzheimer’s disease progression in the patients is an ongoing process and it is never static (Wimo et al. 2013). The staging protocol of 1990s had undergone a series of permutations but no particular changes were taken up (Montine et al. 2012). The scientific procedure proposed in this research would be a changed description of the early staging process that have the potentiality to be performed on sections of the paraffin after immunostaining with AT8 antibody and processing on an automated basis. This would fulfill the requirements of the regular laboratory. Ease as well as consistency are two features of the staging system and these are to be undertaken as the key features and would contribute to draw a strong comparison between the results of the laboratory. The procedure would be a reliable one and also have a reproducible classification of the Alzheimer’s disease.   References Castillo-Carranza, D., Sengupta, U., Guerrero-Munoz, M., Lasagna-Reeves, C., Gerson, J., Singh, G., Estes, D., Barrett, A., Dineley, K., Jackson, G. and Kayed, R. 2014. Passive Immunization with Tau Oligomer Monoclonal Antibody Reverses Tauopathy Phenotypes without Affecting Hyperphosphorylated Neurofibrillary Tangles. Journal of Neuroscience, 34(12), pp.4260-4272. de la Monte, S. M., and Tong, M. 2013. Insulin resistance and metabolic failure underlie Alzheimer disease. Metabolic Syndrome and Neurological Disorders, 1-30. Gonzalez-Lima, F. (Ed.). 2013. Cytochrome oxidase in neuronal metabolism and Alzheimer’s disease. Springer Science & Business Media. Hyman, B., Phelps, C., Beach, T., Bigio, E., Cairns, N., Carrillo, M., Dickson, D., Duyckaerts, C., Frosch, M., Masliah, E., Mirra, S., Nelson, P., Schneider, J., Thal, D., Thies, B., Trojanowski, J., Vinters, H. and Montine, T. 2012. National Institute on Aging–Alzheimer’s Association guidelines for the neuropathologic assessment of Alzheimer’s disease. Alzheimer’s & Dementia, 8(1), pp.1-13. Lasagna-Reeves, C. A., Castillo-Carranza, D. L., Sengupta, U., Sarmiento, J., Troncoso, J., Jackson, G. R., andKayed, R. 2012. Identification of oligomers at early stages of tau aggregation in Alzheimer’s disease. The FASEB Journal, 26(5), pp.1946-1959. Lock, M. 2013. The Alzheimer conundrum: Entanglements of dementia and aging. Princeton University Press. Montine, T.J., Phelps, C.H., Beach, T.G., Bigio, E.H., Cairns, N.J., Dickson, D.W., Duyckaerts, C., Frosch, M.P., Masliah, E., Mirra, S.S. and Nelson, P.T., 2012. National Institute on Aging–Alzheimer’s Association guidelines for the neuropathologic assessment of Alzheimer’s disease: a practical approach. Acta neuropathologica, 123(1), pp.1-1. Morris, E., Chalkidou, A., Hammers, A., Peacock, J., Summers, J., andKeevil, S. 2016. Diagnostic accuracy of 18F amyloid PET tracers for the diagnosis of Alzheimer’s disease: a systematic review and meta-analysis.European journal of nuclear medicine and molecular imaging, 43(2), pp.374-385. Ramanathan, V. 2013. Alzheimer discourse: Some sociolinguistic dimensions. Routledge. Ravdin, L. D., andKatzen, H. L. 2013. Handbook on the Neuropsychology of Aging and Dementia. New York, NY, USA:: Springer. Rosseels, J., Van den Brande, J., Violet, M., Jacobs, D., Grognet, P., Lopez, J., Huvent, I., Caldara, M., Swinnen, E., Papegaey, A., Caillierez, R., Buée-Scherrer, V., Engelborghs, S., Lippens, G., Colin, M., Buée, L., Galas, M., Vanmechelen, E. and Winderickx, J. 2014. Tau Monoclonal Antibody Generation Based on Humanized Yeast Models. Journal of Biological Chemistry, 290(7), pp.4059-4074. Senturk, G. E., andCanillioglu, Y. E. 2014. Which histochemical staining technique should I choose for biological specimens. Microscopy: advances in scientific research and education. Formatex Research Center, Badajoz, Spain, pp.769-775. Thal, D. R., von Arnim, C., Griffin, W. S. T., Yamaguchi, H., Mrak, R. E.,Attems, J., andUpadhaya, A. R. 2013. Pathology of clinical and preclinical Alzheimer’s disease. European archives of psychiatry and clinical neuroscience, 263(2), pp.137-145. Wimo, A., Jönsson, L., Bond, J., Prince, M., Winblad, B. and International, A.D., 2013. The worldwide economic impact of dementia 2010. Alzheimer’s & Dementia, 9(1), pp.1-11.

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