Some of the analysis methods are manual while others have varying levels of automation.
Use of Campenot chambers or more modern microfluidic device technology facilitates the examination of axons by physically separating somata from axonal projections in primary neuron cultures. Alternatively, neuronal phenotype markers, structural proteins, or axon-specific markers can be useful to identify the process of axon degeneration. One of the simplest forms of imaging degenerating axons in cultures is to use phase contrast and manually score or count the extent of degenerating and intact axons. Ī number of methods are used to identify and quantify degenerating axons in cultured neurons. The length of axons can be measured in both 3D tissue sections using the spaceballs stereology probe. Stereological quantification is the archetypical method by which degenerated neurons are quantified because of its efficiency in counting an unbiased sample of neurons for an accurate population estimation.
The use of stereology to quantify degenerating axons would provide benefit to the field to standardize the approach to axonal quantification. This method provides versatility as a relatively rapid and useful analysis of different axon projections in tissue however, this method does not utilize random, unbiased, systematic sampling (i.e., stereology).
Using this approach, axon degeneration is determined by manually counting all axons that remain intact while crossing both parallel lines. A recently described method quantifies axons crossing through a single 30μm thick tissue section using two parallel sampling lines. Unfortunately, this approach does not provide information regarding morphological characteristics of the axons. Axonal loss is often quantified using relatively straightforward image analysis techniques, such as optical density measurements of regional staining intensity for either a phenotype-specific marker or an axonal marker. These morphological changes in the axons can be used to study the process and extent of axonal degeneration both in vivo and in vitro.ĭespite the rapidly growing interest in studying axonal degeneration, a somewhat limited number of methods exist for quantifying axon degeneration in tissue sections. Demyelination of axons can lead to a similar process of fragmentation and axon death. The factors of demyelination associated with traumatic injury and autoimmune disease have significant effects on the health and structure of the axon. Other factors independent from the neuron itself can affect the health of the axon. Eventually, the axon debris is degraded beyond detection. As degeneration progresses, the axons become physically fragmented, breaking apart at the thinned segments, leaving only the spheroids. Spheroids are readily apparent features of axons that are undergoing degeneration. Next, segmentation begins, creating thinned segments and larger rounded segments, known as spheroids. First, axons become dystrophic, as they swell along their length. Īxons tend to undergo a relatively consistent pattern of progressive physical changes during the process of degeneration. Interestingly, the molecular events that characterize injury-induced axonal degeneration are distinct from those involved in apoptosis, but the precise mechanisms involved in axonal degeneration in many common neurodegenerative diseases remain unknown. Axon degeneration appears to precede cell loss in several of these diseases and can proceed in either a retrograde (“dying-back”) or anterograde direction from the site of insult or injury. There has long been an appreciation that axons are particularly vulnerable to degeneration and that neurons with long projections are often those most affected in neurodegenerative diseases, but the majority of pathological studies focus on the loss of neuronal cell bodies. Similarly, the axons of neurons affected in AD show signs of degeneration early in the disease. For example, the dopaminergic projections from the substantia nigra to the striatum are severely affected early during disease and degeneration of these projections precedes overt loss of cell bodies. Parkinson’s disease (PD), Alzheimer’s disease (AD), amyotrophic lateral sclerosis and traumatic brain injury) and an important component of the neural disconnection that occurs in these diseases. Axonal degeneration is a common feature in several neurodegenerative diseases (e.g.
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