Study shows 3D structure of damaged nerves in diabetic neuropathy
The use of three-dimensional imaging of human nerve tissue at the sub-cellular scale may help researchers better understand the pathophysiology of diabetic neuropathy, one of the most common complications of type 1 and type 2 diabetes.
In a study published in Scientific Reports, researchers from Lund University and Skåne University Hospital, Linköping University, and the European Synchrotron Radiation Facility in Copenhagen, demonstrated the feasibility of using X-ray phase contrast holographic nanotomography to enable 3D imaging of nerves at high resolution, while covering a relatively large tissue volume. The technology offered researchers a glimpse of subcomponents of human peripheral nerves in biopsies from adults with and without diabetes — many of which have not been previously described in literature.
Healio spoke with Lars B. Dahlin MD, PhD, professor and senior consultant in hand surgery in the department of translational medicine at Lund University and Skåne University Hospital in Malmö, Sweden, about how biopsies from a carpal tunnel study led to 3D imaging of nerves, the use of X-ray phase contrast holographic nanotomography to identify regenerative nerve clusters in an individual with type 1 diabetes, and how the research could lead to a better understanding of the pathogenesis of diabetic neuropathy.
What led you and your colleagues to take on this study?
Dahlin: Diabetic neuropathy is a serious complication of diabetes, affecting not only the lower extremities, but also the upper extremities. Neuropathy, for several reasons, more frequently affects the lower extremities, and complications, such as a nerve compression syndrome, cause disability. The most common nerve compression syndrome is carpal tunnel syndrome — compression of the median nerve at wrist level. As part of a previous prospective study that evaluated the outcome of surgical release of the median nerve, we also took a nerve biopsy from an uncompressed nerve at the same level as the median nerve, the terminal branch of the posterior interosseous nerve. We examined these nerve biopsies with conventional morphological techniques, only allowing two-dimensional imaging, but then I realized that we could use the synchrotron light technique to get 3D images of the nerve.
How does X-ray phase contrast holographic nanotomography works?
Dahlin: Basically, it is a gigantic X-ray method, with the tomography technique enabling multiple images and then the segmentation technique enabling 3D images, performed with very high resolution at the nano-level. If you compare synchrotron light with the X-ray equipment used in a hospital, the synchrotron source is about a hundred billion times more intense. It is like a microscope, but with X-ray light that has a much shorter wavelength than regular light. This, in turn, allows you to study soft tissue at the cellular level without making incisions.
Can you describe the study design?
Dahlin: We decided to look at nerves from healthy adults and from individuals with type 1 and type 2 diabetes, who were also included in the above mentioned prospective clinical study focusing on surgical treatment of carpal tunnel syndrome.
Our primary intention was to look at the blood vessels in the nerve. However, due to extreme contrast from the nerve fibers, we noticed that we could get an amazing view of the nerve fibers in 3D — particularly, the small nerve fibers that had degenerated due to the neuropathy, but then tried to regrow. These are known as regenerative clusters.
What surprised you during this work?
Dahlin: Interestingly, we were able to show the detailed 3D structure of the regenerative clusters in the patient with type 1 diabetes in contrast to the normal appearance of the myelinated wave-formed nerve fibers in the fascicle found in the healthy individual. The visualization of these clusters has never been shown previously. Furthermore, we could also see in detail a degenerating nerve fiber, where the fiber tried to regrow again. The latter finding was a completely novel observation in a nerve affected by neuropathy. This is a whole new way of studying nerves. The 3D visualization of pathological alterations at the cellular level in diabetic neuropathy is not possible with conventional imaging techniques, including micro-CT, for describing 3D structure. I was surprised that we could look at the nerve fibers, also including other morphological details, such as the node of Ranvier and myelin incisures.
How can this technique help future diabetic neuropathy research?
Dahlin: This study has demonstrated the feasibility of using X-ray holographic nanotomography to visualize the cellular and subcellular structures of single human peripheral nerves. Quantitative 3D structural data, which can be related to conventional two-dimensional images, is important to further understand the pathophysiology and the detailed cellular networks of peripheral nerves in diabetic
Even if a variety of other techniques are available to visualize nerve fibers, the strength with the synchrotron technique is that we can demonstrate the detailed structure of myelinated healthy and regenerating axons over longer distances, both longitudinally and cross-sectionally, with a resolution in the sub-micron range and with the potential, in the future, to statistically analyze morphometry in a larger number of samples.
With this technique, we can understand, in detail, how nerve fibers react during degeneration in diabetic neuropathy, and how the nerve fibers try to regrow as a compensation mechanism. This will contribute, together with other complimentary techniques, to a better understanding of the development of neuropathy in diabetes. We are now working on a larger, follow-up study where we hope to be able to further identify more nerve fibers. The study will investigate how the thickness of the nerve fibers varies, as well as the extent to which regenerative clusters occur.