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Defining the Zone of Acute Peripheral Nerve Injury using Fluorescence Lifetime Imaging in a Crush Injury Sheep Model
Dattesh R Dave, MD, MSc
1; Alba Alfonso Garcia, Phd
2; Lisanne Kraft, M.sc.
2; Laura Marcu, Phd
2; Clifford T Pereira, M.D.
31UC Davis Plastic Surgery, Sacramento, CA; 2University of California, Davis, Davis, CA; 3University of California Davis, Sacramento, CA
Hypothesis: Current technologies to define the zone of acute peripheral nerve injury intraoperatively are limited by surgical experience, time, cumbersome electrodiagnostic equipment and interpreter reliability. In this pilot study, we evaluate a novel, real-time label-free optical technique for intraoperative nerve injury imaging. We hypothesize that fluorescence lifetime imaging (FLIm) will detect a difference between the time-resolved fluorescence signatures for acute crush injuries versus uninjured segments of sheep peripheral nerves, corresponding to histology.
Methods: Label-free FLIm uses ultraviolet laser pulses to excite endogenous tissue fluorophores and detect their fluorescent decay over time – generating real time tissue specific signatures. Based on sample size need, a crush injury was produced in 8 peripheral nerves of 2 sheep. A handheld FLIm instrument captured the time-resolved fluorescence signatures of injured and uninjured nerve segments across 3 spectral emission channels (390/40 nm, 470/28 nm, and 540/50 nm). Two-sample T-test evaluated average FLIm parameters (i.e., lifetime and intensity ratios) for injured and uninjured nerve segments. We harnessed linear discriminant analysis (LDA) to differentiate between crushed and uninjured nerve segments. Nerve samples were collected for H&E staining and evaluated for perineural thinning, perineural edema, and nuclear atypia.
Results: Sampling produced a total of 23,692 point- measurements from 8 peripheral nerves of two sheep. Histology confirmed the zone of injury via perineural thinning, edema, and nuclear atypia. Average lifetime at 470 nm and 540 nm were statistically different between crushed and uninjured sheep nerve segments (p<.01, 95% CI 1.9-4.4). The LDA analysis differentiated between crushed and uninjured areas of 8 nerve segments with 92% sensitivity, 85% specificity, and 88% accuracy.
Summary:
-In this pilot study, FLIm significantly detected differing average lifetime values for crushed versus uninjured sheep peripheral nerves with high sensitivity, specificity, and accuracy.
-FLIm findings corresponded to histologic analysis.
-Future studies will evaluate FLIm's ability to detect more subtle acute and chronic nerve injuries.
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