As the popularity of fluorescence-guided surgery grows, so does the demand to design and develop fluorophores, which serves to distinguish the 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. Though these agents have several clinical applications, they are untargeted, which limits their specificity. Many candidate fluorophores appear effective in animal models — but their clinical translation necessitates rigorous testing and significant financial investment. To perform more accurate selection and testing of fluorophores, researchers at Dartmouth-Hitchcock Medical Center and Dartmouth College have partnered with Oregon Health & Science University to create a first-in-kind, perfused, amputated human limb model. The model allowed for the collection of human data for the preclinical testing, evaluation, and selection of lead fluorescent agents for clinical trials. In addition, the model can potentially be used to study peripheral pathologies in a controlled environment. In the collaborators’ study, the researchers tested the fluorescence intensity values and tissue specificity of a preclinical, nerve tissue-targeted fluorophore, as well as the capacity of the model to be used for lead fluorescent agent selection in the future. The team reported on results from a single patient out of its initial 10-patient proof-of-concept pilot study. Tissue examination began soon after amputation to avoid tissue breakdown due to a lack of oxygen. Saline was perfused through a dominant artery, and then a standard dose of the nerve-specific fluorophore was administered. Ten minutes of perfusion with the fluorophore were followed by a 20-minute washout with just saline. During this time, the limb was gravity-drained and the collected perfusate was recycled back into the circuit to mimic blood circulation. After 30 minutes of perfusion, the nerve tissue was imaged in situ and ex vivo with both open-field and closed-field commercial fluorescence imaging systems. The use of normal saline in combination with a cardiac perfusion pump allowed the researchers to mimic the osmotic and vascular pressure of an in situ physiologic system during perfusion of the limb. Perfusion of an amputated limb with saline and a fluorescent agent (i.e., fluorophore) administered via a dominant artery. The saline and targeted fluorophore entered via the artery, the limb was drained through gravity, and the perfusate recycled back into the circuit. Courtesy of Bateman et al., doi: 10.1117/1.JBO.28.8.082802. The fluorophore demonstrated an excellent signal-to-background ratio (SBR), indicating a strong signal from the nerve tissue relative to the background noise. In situ, open-field imaging demonstrated an SBR of 4.7 when the nerve was compared with adjacent muscle tissue. Closed-field imaging demonstrated an SBR of 3.8 when the nerve was compared with adipose tissue and an SBR of 4.8 when the nerve was compared with muscle. The results showed that the nerve-specific fluorophore could achieve the optical performance necessary to highlight the target tissue. “We are impressed with the SBR seen using this fluorophore and believe that it will perform excellently clinically 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 could be used to investigate and select other fluorescent agents in the future. Preclinical testing of fluorophores using this approach could help identify tissue toxicity, clearance time of fluorophores, and the production of harmful metabolites, in addition to evaluating targeted fluorophores for use in clinical trials and surgery. Beyond enhancing the accuracy and safety of fluorescent agents, the research also represents a step toward reducing development costs and minimizing potential harm to patients, researcher Logan M. Bateman said. Additionally, the team sees the platform being used to study peripheral diseases and pathological features in tissues under controlled conditions and to investigate changes caused by tumor growth. The research was published in Journal of Biomedical Optics (www.doi.org/10.1117/1.JBO.28.8.082802).