BELLINGHAM, Washington, January 25, 2023 — As the popularity of fluorescent surgery grows, so does the need to design and develop fluorophores that can distinguish target tissue from other tissues and subsequently guide surgical steps. The current list of FDA-approved fluorophores for clinical use is limited to three: indocyanine green, fluorescein, and methylene blue.
Although these agents have several clinical applications, they are not targeted, which limits their specificity. Many candidate fluorophores appear to be effective in animal models, but their clinical application requires extensive testing and significant financial investment.
To enable more precise screening and testing of fluorophores, researchers at Dartmouth-Hitchcock Medical Center and Dartmouth College partnered with the Oregon Health and Science University to create a first-of-its-kind perfused model of an amputated human limb. The model allowed the collection of human data for preclinical testing, evaluation and selection of leading fluorescent agents for clinical trials. In addition, the model has the potential to be used to study peripheral pathologies in a controlled environment.
In a study by colleagues, the researchers tested the fluorescence intensity values and tissue specificity of the preclinical fluorophore targeting neural tissue, as well as the ability of the model to be used to select the lead fluorescent agent in the future. The team reported the results of one patient from their original 10-patient pilot study.
Tissue examination began shortly after the amputation to avoid tissue destruction due to lack of oxygen. Saline was perfused through the dominant artery followed by a standard dose of nerve-specific fluorophore. Ten minutes of perfusion with the fluorophore was followed by a 20-minute wash with saline alone. At this time, the limb was drained by gravity and the collected perfusate was recirculated back into the circuit to simulate circulation.
After 30 minutes of perfusion, neural tissue was imaged in situ and ex vivo using commercial open and closed field fluorescent imaging systems. The use of saline in combination with a cardiac perfusion pump allowed researchers to simulate the osmotic and vascular pressure of the physiological system in situ during limb perfusion.
Perfusion of the amputated limb with saline and a fluorescent agent (eg, a fluorophore) administered through the dominant artery. Saline and target fluorophore were delivered through the artery, the limb was drained by gravity, and the perfusate was recirculated back into the circuit. Courtesy of Bateman et al., doi: 10.1117/1.JBO.28.8.082802. The fluorophore showed excellent signal to background ratio (SBR), indicating a strong signal from neural tissue compared to background noise. In situ open field imaging demonstrated an SBR of 4.7 when comparing nerve to adjacent muscle tissue. Closed field imaging demonstrated an SBR of 3.8 when comparing nerve to adipose tissue and an SBR of 4.8 when comparing nerve to muscle.
The results showed that the nerve-specific fluorophore can achieve the optical performance required for target tissue isolation.
“We are impressed with the SBR observed with this fluorophore and believe that it will perform excellently in the clinical setting as well,” said Professor Eric Henderson. “By seeing these contrast values, we are confident that the perfusion model is adequately delivering the fluorophore to the target tissue.”
The team believes that the human limb model can be used to research and select other fluorescent agents in the future. Preclinical testing of fluorophores using this approach can help determine tissue toxicity, fluorophore clearance time, and formation of harmful metabolites, in addition to evaluating target fluorophores for use in clinical trials and surgery.
In addition to improving the accuracy and safety of fluorescent agents, researcher Logan M. Bateman said the study also represents a move to lower development costs and minimize potential harm to patients. In addition, the team sees the platform being used to study peripheral diseases and pathological features in tissues under controlled conditions, as well as to study changes caused by tumor growth.
The study was published in the Journal of Biomedical Optics (www.doi.org/10.1117/1.JBO.28.8.082802).