Central Nervous System

Analysis of how astrocytes in thick tissues could improve knowledge about ALS

Sean J. Miller, Jeffrey D. Rothstein, Johns Hopkins University School of Medicine

The star shaped glial cells known as astrocytes are the most abundant cell type in the central nervous system (CNS), but scientists don’t yet fully understand the extent of their functions. Sean J. Miller and Jeffrey D. Rothstein from the Johns Hopkins University School of Medicine are using an optical clearing technique and Imaris to reconstruct and quantify different astrocyte populations to better understand their role in health and disease.

A) 3d-rendering of a single astrocyte at super resolution shows overlap with other types of local cells, B) automated morphological reconstruction shows minor and major processes with detailed contextual resolution. Reprinted from Miller SJ et al. PLoS ONE 11(8): e0160391.

In healthy people, astrocytes are essential for maintenance and continued survival of neurons. However, dysfunctional astrocytes are implicated in diseases such as epilepsy and ALS. One major challenge to studying the local and regional biology of astrocytes has been the inability to image them in thick tissue with high resolution microscopy. Additionally, the large size of mice astrocytes can make it hard to observe their entire cellular structure.

Miller and Rothstein developed a modified form of the CLARITY optical clearing technique and combined it with high-resolution microscopy and Imaris image analysis to obtain detailed information about astrocytes in very thick tissue sections. Their technique can be used to better understand the biological processes involved in ALS and to examine astrocyte properties after an experimental treatment, for example.

Automated analysis

The new approach provides automated cell counting in very thick tissue sections, which can be helpful in evaluating astrocyte proliferation, death and subtypes. It also allows the targeting of astrocyte-interacting cells to study potentially altered interactions between cell types in the same tissue. The researchers also incorporated Imaris Spots Detection to quantify fluorescence puncta, which can be used to determine altered expression levels for biological targets of interest. Finally, using Imaris for 3D reconstruction of the astrocytes allows the identification of areas of astrogliosis — a well-established marker of CNS insult — based on astrocyte hypertrophy and molecular profile.

The researchers demonstrated their new approach by using it to quantify and create 3D reconstructions of thousands of cells. Using Imaris, they counted and sorted thousands of astrocytes into their subpopulations in the cortex and also reconstructed these cellular populations to identify areas of physical connection and to quantify their overall volume.

“In particular, the Spots detection application was extremely useful in quantifying the overall populations of different astrocytes throughout the cortex,” said Miller. “We also used the Surfaces reconstruction application when evaluating the different morphologies of astrocytes in our thick tissue sections. Both of these features of Imaris were essential to our study.”

The researchers are applying these new tools to study ALS by using them to locate areas of inflammation by finding astrocyte populations that become hypertrophied (swollen and enlarged). “We have used Imaris extensively to evaluate not only the astrocyte populations, but also the neurons in which the astrocytes interact with in their local environment,” said Miller. “We are also using Imaris Filament Tracer to reconstruct neuronal dendrites and evaluate expression of different synaptic markers in both health and disease.”

Research Paper: Miller SJ, Rothstein JD (2016) Astroglia in Thick Tissue with Super Resolution and Cellular Reconstruction. PLoS ONE 11(8): e0160391.

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