After After
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10142 - Toluidine Blue, Neuroscience

INTRODUCTION
This protocol can be used for evaluation of nervous tissue. Nerves consist of sensory, motor, and autonomic nerve axons. Particularly those of larger diameter, are surrounded by myelin. This APP can quantify axon cross-sectional area, myelin thickness, and the G-ratio (axon major axis length / myelin major axis length), which is relevant for the study of neurodegenerative diseases and a variety of neuropathies.

The protocol automatically detects cross sections of nerves. Each nerve section is evaluated for large, small, and degenerated nerve fibers. The small and large nerve fibers are partitioned into axon and myelin and important parameters, as major axis length, are exported per axon.

KEYWORDS
Neuroscience, Toluidine Blue, Nerve, Axons, Myelin, G-ratio, Major Axis Length

METHODS
Initially, myelin bands are enhanced using a local linear filter on the intensity of the toluidine stain. Thin and thick myelin bands are differentiated by using two different filter sizes; a large filter for thick myelin bands and a smaller filter for thin myelin bands. An initial threshold classification is used, followed by post processing steps to clean up false detections of nerve fibers. Lastly, different ROIs are generated for small and large nerve fibers, to enable per object outputs for each individual nerve fiber.

QUANTITATIVE OUTPUT VARIABLES
The output variables obtained from this protocol are:

  • # Small: number of small axon fibers
  • # Large: number of large axon fibers
  • # Degenerate: number of degenerate axon fibers
  • # Total: total number of axon fibers (#Small + #Large + #Degenerate)
  • Tissue Area: The ROI area
  • Nerve Density: (#Total / Tissue Area)
  • Degenerate Area obj: Area of degenerated axon fibers per object
  • Large Axon Area obj: Area of large axons1
  • Large Myelin Area obj: Area of large axon myelin per object1
  • Small Axon Area obj: Area of small axon per object2
  • Small Myelin Area obj: Area of small axon myelin per object2
  • Large Axon Major Axis Length obj: Major axis length of large axons per object1
  • Large Myelin Major Axis Length obj: Major axis length of large axon myelin per object1

1Per object calculations based on all large axon fibers
2Per object calculations based on all small axon fibers

AUXILIARY APPs
APP: "01 Nerve Detection"
The auxiliary APP '01 Nerve Detection " is used for automatic detection of nerve- components.

WORKFLOW
Step 1: Load and run the APP “01 Nerve Detection” for nerve identification or manually outline ROIs
Step 2: Load and run the APP “02 Nerve Quantification” for quantification of nerve fibers (axon+myelin)

STAINING PROTOCOL
Nerves were fixed in 2.5% glutaraldehyde, placed in 1% osmium tetroxide, dehydrated through a graded series of ethanol and propylene oxide, cut at 0.5 µm, and stained with toluidine blue.

ADDITIONAL INFORMATION
The APP utilizes the Visiopharm EngineTM and Viewer software modules, where EngineTM offers an execution platform to expand processing capability and speed of image analysis. The Viewer allows a fast review together with several types of image adjustment properties e.g. outlining of regions, annotations and direct measures of distance, curve length, radius, etc.
By adding the AuthorTM module the APP can be customized to fit other purposes. AuthorTM offers a comprehensive and dedicated set of tools for creating new fit-for-purpose analysis APPs, and no programming experience is required.

REFERENCES
USERS
This APP was developed for Professor Osvaldo Delbono, Wake Forest School of Medicine.

RUO
Figure 1
Figure 1
Macroscopic view of toluidine blue stained nervous tissue.
Figure 2
Figure 2
Automatically detected outline of nerves.
Figure 3
Figure 3
Nervous tissue. Zoom in on Figure 1.
Figure 4
Figure 4
Initial classification of nervous tissue in figure 3.
Figure 5
Figure 5
Post processing enables the detection of small (light green) and large (green) axons for the nerve fibers fully surrounded by myelin.
Figure 6
Figure 6
Final identification of the nervous tissue seen in Figure 1. Axons with myelin are detected: Large axon (green), large myelin (red), small axon (light green), small myelin (orange), and degenerate fiber (black).
Figure 7
Figure 7
Regions used for per object calculations. Large nerve fibers (yellow outline) and small nerve fibers (pink outline).