generating-analyzing-high-parameter-histology-images-with-histoflow

Generating and Analyzing High-Parameter Histology Images with Histoflow Cytometry

The usage of histology to investigate immune cell diversity in tissue sections such as those derived from the central nervous system (CNS) is critically ...

University of Calgary

The mesencephalic locomotor region (MLR) is an important integrative area for the initiation and modulation of locomotion. Recently it has been realized that dopamine (DA) projections from the substantia nigra pars compacta project to the MLR. Here we explore DA projections from an area of the medial zona incerta (ZI) known for its role in motor control onto the MLR. We provide evidence that dopaminergic (DAergic) A13 neurons have connectivity to the cuneiform nucleus (CnF) and pedunculopontine tegmental nucleus (PPTg) of the MLR. No ascending connectivity to the dorsolateral striatum was observed. On the other hand, DAergic A13 projections to the medullary reticular formation (MRF) and the lumbar spinal cord were sparse. A small number of non-DAergic neurons within the medial ZI projected to the lumbar spinal cord. We then characterized the DA A13 cells and report that these cells differ from canonical DA neurons since they lack the Dopamine Transporter (DAT). The lack of DAT expression, and possibly the lack of a dopamine reuptake mechanism, points to a longer time of action compared to typical dopamine neurons. Collectively our data suggest a parallel descending DAergic pathway from the A13 neurons of the medial ZI to the MLR, which we expect is important for modulating movement.

Parallel descending dopaminergic connectivity of A13 cells to the brainstem locomotor centers.

The objective of this study was to test whether regenerating motor axons from a donor nerve can travel in a retrograde fashion using sensory branches to successfully reinnervate a motor nerve end organ.

Successful retrograde regeneration using a sensory branch for motor nerve transfer.

Immunofluorescence histology is commonly used to study immune cells in tissues where the number of fluorescence parameters is normally limited to four or less. This makes it impossible to interrogate multiple subsets of immune cells in tissue with the same precision as flow cytometry. The latter, however, dissociates tissues and loses spatial information. To bridge the gap between these technologies, we developed a workflow to expand the number of fluorescence parameters that can be imaged on widely available microscopes. We instituted a method for identifying single cells in tissue and exporting the data for flow cytometry-based analysis. This histoflow cytometry technique successfully separates spectrally overlapping dyes and identifies similar numbers of cells in tissue sections as manual cell counts. Populations identified through flow cytometry-like gating strategies are mapped to the original tissue to spatially localize gated subsets. We applied histoflow cytometry to immune cells in the spinal cords of mice with experimental autoimmune encephalomyelitis. We ascertained that B cells, T cells, neutrophils, and phagocytes differed in their frequencies in CNS immune cell infiltrates and were increased relative to healthy controls. Spatial analysis determined that B cells and T cells/phagocytes preferentially localized to CNS barriers and parenchyma, respectively. By spatially mapping these immune cells, we inferred their preferred interacting partners within immune cell clusters. Overall, we demonstrate the ease and utility of histoflow cytometry, which expands the number of fluorescent channels used in conventional immunofluorescence and enables quantitative cytometry and spatial localization of histological analyses.

David A. Elliott

Hotchkiss Brain Institute

University of Calgary

Generating and Analyzing High-Parameter Histology Images with Histoflow Cytometry