A neurodegenerative disease is where the neurons of some parts of the brain die out with time, leading to degradation of the functions of the nervous system. They are often incurable, occur due to combination of genetic and environmental factors, and are more likely to be diagnosed in old age. Examples of such diseases include Alzheimer’s disease, Parkinson’s disease, and motor neuron disease (Physiopedia, 2021). Out of these, Alzheimer’s disease commonly affects the cerebral cortex while others such as Parkinson’s affect deeper regions of the brain (Braak et.al, 1996). To understand temporal patterns of neuronal loss, we use mice at different stages of the disease and a variety of histology techniques. To understand neuron morphology changes, we use stains for specific cell types of the neurons.
Symptoms of Alzheimer’s
Common symptoms of Alzheimer’s include forgetfulness, increasing cognitive decline with time, inability to perform daily tasks, and behavioral symptoms such as decreased independence and decline in social skills, impairing the affected person’s ability to live independently. These symptoms persist and become worse with old age. Associated morphological changes in the neurons of the brain include tangles and plaque formation caused by Amyloid beta (A-beta) and tau proteins causing neuronal death, and microglial cells that damage the synaptic connections between neurons (Braak et.al, 1996).
Use of mouse models
Mouse models have long been considered for studying Alzheimer’s (Elder et al., 2010). In case of Alzheimer’s, getting human tissue for study of disease progression is difficult since it is typically diagnosed late and often the tissue is available only after the subject is deceased. Using mouse models can be useful for several reasons. Mice have a life span of only 18 months, allowing one to observe brain degeneration as the disease progresses. They are small, easy to handle and can be grown relatively easily to produce a homogenous population. They have mammalian genes similar to humans, and exhibit similar behavior traits as humans, such as loss of social interactions and hyperactivity. One can study transgenic mice whose genes are altered to have Alzheimer-like symptoms, by inducing them to express proteins linked to Alzheimer’s such as Tau, APP and Amyloid beta. This can help us understand how the disease affects human brains, as well as investigate treatments to alleviate the symptoms (Hafezparast et al., 2002).
However, there are also some limitations to using mouse models for Alzheimer’s (Demetrius, 2005). One limitation is the possibility of different strains of mice having different genes or mutations, that may cause mice in the same experiment to behave differently or abnormally. This can be mitigated by ensuring that all the mice used in the study are from the same strain. Also, since the root cause of Alzheimer’s is still not well known, using mouse models is only as useful as reproducing some well-known symptoms of Alzheimer’s and investigating the cure for those symptoms, rather than root causes. Mouse strains relevant for study of the progression of Alzheimer’s include C57BL/6, FVB/N, and SJL/J (Qosa & Kaddoumi, 2016).
Investigating temporal patterns of neuronal loss
Using a mouse model to investigate the time patterns of neuronal loss in areas of the brain affected by Alzheimer’s can be done by using a combination of neuroimaging techniques (in vivo) and histological techniques (in vitro). In both cases, we observe the brain structure and function in transgenic mice at different progressive stages of Alzheimer’s, say from a 3-month-old brain to the lifespan of the mouse. One can also have a control group of mice with healthy brains, to compare with the structure of the cortex in brains having Alzheimer’s.
One advantage of using neuroimaging is that we can observe the brain scans of the same mice at regular time intervals to observe disease progression, and there would be no genetic variability, compared to histological techniques where multiple mice at different stages are needed to get the brain slices. Imaging techniques that have been used in mouse models of Alzheimer’s include resting state fMRI, which has good spatial resolution (Shah et al., 2013), EEG, which has good temporal resolution (Vogler et al., 2017), and Computed Tomography (Stenzel et al., 2019).
Histological techniques involve taking slices of the mouse brains at different stages of Alzheimer’s, staining them appropriately and observing under the light microscope or electron microscope to understand how neuronal loss progressively happens in different areas of the cortex. It involves the following stages: fixing the brain tissues using paraffin wax or Cryo techniques, sectioning into slices using microtomes or cryostats, staining with dyes or immunostaining, and finally analysis under the microscope. Fig. 1 shows the different steps of histological analysis.
Table 1 shows some of the common dyes used to stain different cell types in the neuron. Multiple dyes can also be combined for histological staining of different neuronal cell types. For example, one can use dyes which stain neuron nuclei, such as Hematoxylin, along with Eosin for the cell bodies.
Immunohistochemistry or IHC can also be used to identify different cell types in neurons and understand how much brain loss is occurring. This involves labelling neurons by staining them with monoclonal and polyclonal antibodies (called immunostaining), that bind to and are used as markers for various proteins or antigens, corresponding to the various neuronal cell types. Both primary antibodies, that bind to the protein, and secondary antibodies, that bind to the primary antibodies, can be used. After the immunostaining is complete, they are observed under a light microscope. Common antibodies that can perform such labelling include MAP2 for dendrites, Tau-1 for axons, GFAP for Glia and PSD95 for synapses (Carter and Shieh, 2015). A study involving immunohistochemistry to study the progression for Alzheimer’s using transgenic 3xTg-AD mice at ages varying from 2 to 26 months was performed by Mastrangelo and Bowers (2008), where the APP Y188 antibody was used to recognize the hAPP protein.
Immunofluorescence techniques, a variant of IHC, can also be used after the mouse cortex slices have been hardened, sectioned, and stained. Here the antigen proteins in different cell types are immunostained by incubating with antibodies to label the proteins linked to Alzheimer’s, as before. The advantage with IHC is that they are visualized with antibodies linked to fluorochromes in different colours for the different proteins, enabling easier identification under the microscope. Solé-Domènech et al.(2013) performed a study where p-FTAA fluorescent dyes were used for the staining.
A study involving quantification of neuronal degeneration due to Alzheimer’s using mouse models and histological techniques was conducted by Yamaguchi et al. (2013). Staining steps involved Nissl staining using Cresyl violet, immunostaining using primary antibodies such as anti-MAP-2 and secondary antibodies such as BA-9200, and immunofluorescence staining using fluorescent dyes such as Alexa Fluor, and Fluoro-Jade B staining. Finally, the quantification of the amount of the neuronal loss was done using BIOQUANT software and optical fractionator and observing the stained brain sections under the microscope.
Investigating changes in the morphology of neurons
To observe changes in the morphology of the individual neurons in Alzheimer’s disease, we need to identify differences to the structure of individual neurons in brain tissue, when appropriately stained and observed under the microscope. This can be done using stains that label different cell types within the neurons, as well as those that label the whole neurons. Combinations of stains can also be used to clearly visualize different cell types that are stained differently, for example staining the cell body red using Eosin and the nuclei blue using Hematoxylin.
Golgi staining is a method that stains the complete structure of individual neurons including soma, dendrites and axons. Staining of brain slices is performed using silver nitrate and other chemicals, and randomly stains a few neurons black, allowing them to be observed clearly under the microscope. By using Golgi staining and comparing healthy brain tissue with Alzheimer’s affected tissue both taken from the mouse cortex, one can observe if the structure of the dendrites or other parts of the individual neurons have been damaged. Baloyannis (2013) surveyed different ways in which Golgi staining could be used to study damage to dendrites in Alzheimer’s. Golgi-cox technique, a variant of the Golgi technique, was used to determine damage to dendritic spines in Alzheimer’s in a mouse model by Kartalou et al. (2020).
Immunohistochemistry techniques also be used to observe the morphology of individual neurons using antibodies to label antigens that bind to specific cell types of the neurons, such as dendrites, axons or Glia. One study (Fix et al., 1996) used antibody stains including primary polyclonal rabbit anti-cow GFAP antibody and a goat anti-rabbit secondary antibody to detect the antigens in Glial cells. Another study by Pisa et al. (2015) studied fungal infections in mouse brains with Alzheimer’s, using donkey anti-rabbit IgG primary antibodies, anti-tau antibodies and donkey anti-mouse IgG secondary antibodies.
Conclusion
In this article, we discussed various techniques to investigate temporal patterns of neuronal loss due to Alzheimer’s as well as changes in morphology of individual neurons, including Golgi staining that stains the entire structure of neurons, as well as immunohistochemistry techniques using primary and secondary antibodies that stain specific cell types such as dendrites or Glia.
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