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Figure 1: Subbasal nerve plexus and stromal nerves in the cornea of a thy1-YFP mouse. Shown is (A) an overview, (B) a section, and (C) quantification of single subbasal nerves. Scale bar 50 um

If current trends continue, it is predicted that by the year 2035, one in 10 adults will suffer from diabetes mellitus, a disease that is becoming an ever more pressing medical challenge.

One of the key factors underlying this metabolic disorder is the impaired function of the insulin-producing β-cells of the pancreas, with a subsequent drop in insulin secretion leading to abnormally elevated blood sugar levels and resulting in a variety of physiological complications.

While type 1 diabetes stems from the autoimmune destruction of β-cells, type 2 diabetes is characterized by both β-cell failure and insulin resistance in tissues around the body. Understanding the mechanisms and implications of this complex disorder is therefore highly important—and Simone Baltrusch, a professor of medical biochemistry and molecular biology at the University of Rostock (Germany), has found confocal laser scanning microscopy to be an ideal tool for this research endeavor.

Compared to standard widefield microscopy, confocal laser scanning microscopy delivers a sharp image derived only from a defined focal plane. Baltrusch’s group uses the technique to study not only the molecular level, but also for understanding wider aspects of the disease in relation to the affected organelles and cells.

The diabetic mitochondrial network
As knowledge of biological systems progresses, it is becoming increasingly clear that what were once considered static components are instead highly interconnected networks. This is even true of the mitochondria.

“Textbook diagrams of mitochondria within the cell often show separated organelles with maybe 10 to 20 per cell, but this is not actually the case,” Baltrusch explained. “This is a dynamic network undergoing a continuous process of fusion and fission—it’s changing all the time.”

Interestingly, a correlation has been observed between type 2 diabetes and an increase in mitochondrial mutations. Carrying mitochondrial mutations and a wild type nucleus, conplastic mouse models are ideal for understanding this relationship.

“We want to know what happens when such mutations accumulate over time,” said Baltrusch. “Does the mitochondrial network structure or function change? Studying mice carrying such mutations, we can see what happens compared to the wild type strain.”

The increased mitochondrial fragmentation observed within diabetes means it becomes problematic to get enough energy for cells, and has been implicated in insulin resistance of skeletal muscle. On the other hand, very elongated mitochondria are also problematic. Interestingly, studying images from different networks, Baltrusch’s group has found this is very much dependent on cell type and physiological conditions.

“The mitochondrial network adapts to what is needed, how much energy is available and where this comes from—fat or carbohydrate. Each affects the network, and we’ve really learned a lot,” Baltrusch said.

Toward non-invasive detection
While understanding the underlying mechanisms of diabetes is vital for developing new treatments and advancing our knowledge of biological systems, it is equally valuable to look at the impact diabetes has on quality of life.

A second project utilizing confocal microscopy works toward the development of a new technique for early detection of diabetic neuropathy, or neuronal damage. When hyperglycaemia (an elevated blood sugar level) persists over five years or longer, physiological damage can be quite expansive—and neuropathy is the most common long-term complication of diabetes. It arises when glycation of blood proteins gives rise to what are known as advanced glycation end products (AGEs), which bind receptors on the surface of neurones and trigger damaging processes, such as apoptosis.

Early diagnosis is therefore crucial for disease management, and yet current techniques are non-quantifiable, depending on the patient’s verbal response to a sensory trigger applied to the foot. A more quantitative technique is needed, and while analyzing the neuronal network from a skin biopsy can identify any fibers lost, this is painful and wound healing is also a problem for diabetics. A collaborative project between Baltrusch’s group and Oliver Stachs’s group from the Department of Ophthalmology looks at analyzing neuronal networks in the cornea of the eye.

Working with diabetic mouse models, with a focus on a type of diabetes not triggered by autoimmunity, ß-cells of the pancreatic islet cells were specifically destroyed using the compound streptozotocin. Samples were investigated using antibody staining for insulin, and visualized using confocal microscopy. The shape was comparable to a living pancreatic islet isolated by collagenase digestion of the mouse pancreas.

Using a transgenic mouse with nerve fibers labeled with yellow fluorescent protein (the thy1-YFP mouse), corneal nerves were visualized ex vivo. Figure 1 shows the larger stromal nerve and the very thin nerves of the subbasal nerve plexus. However, the cellular architecture of the cornea is complex. The nerve plexus is close to the Bowman’s layer, which arises from the penetrating A delta fibers, and has subbasal orientation.

“The subbasal nerves are very sensitive to damage, and you see a loss of these in people and animals with diabetes,” explained Baltrusch.

Comparing old and new techniques, skin samples from the mice are also analyzed, and corneal nerves are found to be much more susceptible, owing to the presence of more receptors for AGEs.

“It is crucial to find the right layer of the cornea and analyze the correct nerves, but being so thin this can be difficult,” said the professor.  
Confocal microscopy is ideally suited to this application, as it allows selection of the correct z-position and facilitates analysis with 3-D imaging.

The bigger picture
One question in this study is how many pictures are required to get a good representation of corneal neurone health. In addition to nerve density, nerve fiber length is also an indicator of neuronal health, and this requires a large field of view. Acquiring a larger field of view requires a microscope with an automatic image stitching function.

Figure 2 shows an acquisition of 36 images with 25 z-slices generated overnight using the FluoView FV10i (Olympus), while imaging one quarter of the cornea, which takes three days. Throughout these extended experiments, the machine runs autonomously, with the autofocus routine ensuring the scan field remains in focus between multiple images.

For quantifying nerve fiber density and the length of individual nerves, image stitching yields a much more insightful representation of the cornea. While the whole cornea would amount to 160 GB of data, the stitched image shown in Figure 2—taken from less than a quarter of the cornea—constitutes approximately 6 GB.
In addition to facilitating the detection of neuropathy, these investigations have opened other avenues in research.

“We are interested in which areas are damaged first, and when mice are treated with insulin, what comes back first? What is really interesting is that you can see (in Figure 2B) these dots in the nerves, and we know that these beaded nerve fibers branch, and thus are important for new nerves,” said Baltrusch.

Figure 2: Stitched image of a part of the thy1-YFP mouse cornea. (A) Multi area time laps viewer: 3-D maximum projection of 36 single images with 25 z-layers each. The image in the marked white square is shown in 3-D maximum projection (B) and as 3-D volume view. (C) Image stitching helps to visualize subbasal corneal nerves at large (red square in A and D).
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