Objective  To explore the appropriate imaging dose-time combination （ADTC）of indocyanine green（ICG）for peripheral nerve imaging in rats using near-infrared fluorescence imaging（NIRF）technology, and to analyze the structural fidelity and resolution efficiency of neuroimaging under this ADTC strategy. Analyzing the structural fidelity and resolution performance of nerve imaging under this ADTC strategy, and evaluating the biological toxicity of the approach.

Methods  In in-vivo imaging experiments of rats, different ICG dosing concentrations（0, 30, 40, 50, 60, 70 mg/kg）and different observation time points post-administration（0.5, 10, 15, 20, 25, 30 h）were designed. Under corresponding conditions, various types of peripheral nerves（sciatic nerve, brachial plexus nerve, median nerve）were dissected and exposed in rats, and fluorescence images were captured and collected using near-infrared fluorescence imaging（NIRF） technology. The signal-to-background ratio（SBR）was calculated to screen for ADTC. In the fluorescence images corresponding to the ADTC strategy, all images with SBR ≥ 1.5 were selected. The full width at half maximum（FWHM）method was applied to measure the nerve diameter in the fluorescence images and compared with the nerve diameter measured in the white light images to assess structural restoration, and the correlation between SBR and nerve diameter was investigated. The vagus nerve was used as the observation object to validate the rationality of the ADTC strategy.The appropriate imaging concentration was screened for potential biological toxicity by comparing the levels of aspartate aminotransferase （AST）, alanine aminotransferase （ALT）, and creatinine （CREA） in different dose groups after 24 hours, combined with analysis of HE-stained liver and kidney tissue results.

Results  The overall SBR level at the 60 mg/kg ICG dose was superior to other dose groups (all P < 0.05). Further observation at multiple time points at the 60 mg/kg dose revealed that the SBR value at 20 hours post-imaging was superior to other time groups (all P < 0.05). The optimal dose-time combination for ADTC was thus determined to be 60 mg/kg-20 h. Image analysis of ADTC-treated samples revealed no statistically significant differences in nerve diameter between fluorescent and white-light images for sciatic nerve, brachial plexus, and median nerve (all P > 0.05). Linear regression analysis showed that SBR decreased with increasing nerve diameter across all peripheral nerve types (all P < 0.05). The vagus nerve exhibited an SBR ≥ 1.5 under the ADTC regimen, with no statistically significant difference in nerve diameter between dual-field measurements (P > 0.05). Compared to the control group, only the 70 mg/kg dose showed elevated CREA levels (P < 0.05). Hematoxylin and eosin (HE) staining revealed pathological changes in the liver and kidneys exclusively in the 70 mg/kg dose group.

Conclusion  Under NIRF technology, ICG-guided rat peripheral nerve imaging ADTC was 60 mg/kg-20 h, and this ADTC strategy showed good applicability in a variety of peripheral nerves. ICG-enhanced nerve had good structural fidelity and higher resolution efficacy for finer nerve structures.ADTC strategy exhibits no biological toxicity within 24 hours.