University of Nebraska-Lincoln College of EngineeringOnline: Summer 2011
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  UNL-UNMC collaboration investigagtes and innovates against peripheral arterial disease  


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Peripheral arterial disease affects an estimated five percent of Americans older than age 55. People with PAD have blockages, generated by atherosclerosis, in arteries supplying blood to their legs. When arteries in the legs clog up, blood flow is reduced—a condition called ischemia; over time, lower blood flow damages every structure in the patient’s leg including muscles, nerves and skin.

The University of Nebraska Medical Center has two leaders in PAD work: George Casale, Ph.D. and Iraklis Pipinos, M.D. Now, a team led by Jeyamkondan Subbiah of UNL’s Department of Biological Systems Engineering adds its expertise to their collaboration on PAD morphometrics. The bio-engineers are developing mathematical equations based on multivariate statistics, to process multiple factors in the image characteristics of muscle fibers (myofibers) and yield new diagnostic tools for modeling that helps categorize PAD instances according to the degree of ischemic damage.

PAD presents in different stages, said Subbiah, an associate professor. In the first stage the patient has blockages in his arteries but they produce minimal or no symptoms. The second and by far most common stage is called claudication and affects the patient’s ability to walk. Patients with claudication cannot walk normally and have to stop after very short distances due to pain and limited movement of the muscles in their legs. The third and the most severe stage of PAD is known as critical limb ischemia, characterized by severe pain in the patient’s foot or tissue loss in the form of non-healing ulcers or gangrene. A patient in this stage frequently ends up losing his affected leg to amputation.

Casale and Pipinos have been exploring the pathways that connect blockages in the arteries to the damage in the legs, with a particular focus on damage to the muscle tissue responsible for the function of the legs. Subbiah said Pipinos, a vascular surgeon, specializes in the diagnosis and treatment of patients with PAD, and Casale is the first investigator to work on imaging of muscle fiber (myofiber) biochemistry in PAD using quantitative fluorescence microscopy. Subbiah’s team augments their approaches with sophisticated image-processing algorithms that analyze myofibers with a high level of accuracy in assessing PAD.

The team aims to more efficiently detect PAD in people’s muscles, making it easier for doctors to address the condition before it needs surgery. “Fiber by fiber,” Subbiah said, “we can objectively and specifically measure the morphology, or size and shape, of PAD-affected muscle fibers.” It’s an exciting type of collaboration, Pipinos said, with different expertise coming together and looking at a problem from different angles, with the objective of helping patients.

Subbiah, whose hyperspectral image analysis work is also highly regarded by the beef industry for predicting meat tenderness, said he values his team applying its capabilities to improve a serious health issue. He predicted that his team’s analysis work could serve in developing a computer program that mimics an expert surgeon’s diagnostic knowledge, helping rural areas access high-level expertise regarding this disease.


The bio-engineers use tissue samples obtained from 56 of Pipinos’ patients and images acquired in Casale’s quantitative fluorescence microscopy lab, to quantify the extent and type of muscle damage. Pipinos, who treats patients at Omaha’s Veterans Administration Medical Center, described his PAD patients—most recently, a 60-year-old Vietnam veteran who agreed to provide a sample via a leg muscle biopsy—as another key part of the research to advance knowledge in fighting the disease. “These veterans are still serving their country,” Pipinos said.

The work progresses at Subbiah's UNL East Campus lab, where doctoral student Govindarajan Konda Naganathan (“KN”) helps with identifying and separating individual fibers in the images. Then Kim Cluff, another doctoral student, works on the analysis and development of models to characterize PAD severity. Cluff has achieved overall accuracy of 82.1 percent with his model in classifying the samples into the disease’s three categories.

Cluff said he is excited about the results because “nobody’s gone in and objectively measured the morphometrics in such detail and used advanced multivariate statistics for characterizing the disease before.” Further stages of this UNL-UNMC research look to incorporate additional objective factors—oxidative damage, mitochondrial dysfunction, auto-fluorescence signals and molecular fingerprinting using Raman and Fourier-Transform Infrared Spectroscopy—to improve diagnostic accuracy. Subbiah said: “We are looking for patterns, good and bad, to aid prediction and prevention” of advanced PAD occurrences.”

Subbiah’s team augments the approaches with sophisticated image-processing algorithms that analyze myofibers with a high level of accuracy in assessing PAD.

The initial UNL findings, presented by UNMC postdoctoral research fellow Dimitrios Miserlis at an October 2010 meeting of the Midwest Vascular Surgical Society, showed promise for developing and refining objective criteria to determine the severity of the muscle damage produced by PAD. Miserlis said with UNL’s morphometrics work, each category of damage can be targeted for biochemical analysis, yielding precisely determined biochemical signatures of how PAD begins and progresses.

This is important because “biochemical signatures may be used to direct and assess stage-specific therapeutic interventions,” Miserlis told the gathering. Since PAD primarily affects senior citizens, the benefits of surgery to widen or bypass the clogged arteries should outweigh the risks of surgery, but Subbiah noted that sometimes the surgery carries higher risks than benefits. For instance, if the muscle has already been severely damaged, it may not respond to the renewed blood flow. Subbiah compared this outcome to a farmer widening an irrigation channel, when the plants intended to receive the water might already be too far wilted. He added that the imaging and image analysis tool developed to characterize muscle damage would be useful for surgeons to provide earlier, individualized therapy for patients.

This work should substantially improve therapy at all levels by this mathematical way to identify and map a disease’s progression, Subbiah said, for better decisions and more personalized medicine—because individual medical treatment is not “one size fits all.”

- Carole Wilbeck


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