Nerve-targeted probes for fluorescence-guided intraoperative imaging

Abstract

A fundamental goal of many surgeries is nerve preservation, as inadvertent injury can lead to patient morbidity including numbness, pain, localized paralysis and incontinence. Nerve identification during surgery relies on multiple parameters including anatomy, texture, color and relationship to surrounding structures using white light illumination. We propose that fluorescent labeling of nerves can enhance the contrast between nerves and adjacent tissue during surgery which may lead to improved outcomes. Methods: Nerve binding peptide sequences including HNP401 were identified by phage display using selective binding to dissected nerve tissue. Peptide dye conjugates including FAM-HNP401 and structural variants were synthesized and screened for nerve binding after topical application on fresh rodent and human tissue and in-vivo after systemic IV administration into both mice and rats. Nerve to muscle contrast was quantified by measuring fluorescent intensity after topical or systemic administration of peptide dye conjugate. Results: Peptide dye conjugate FAM-HNP401 showed selective binding to human sural nerve with 10.9x fluorescence signal intensity (1374.44 ± 425.96) compared to a previously identified peptide FAM-NP41 (126.17 ± 61.03). FAM-HNP401 showed nerve-to-muscle contrast of 3.03 ± 0.57. FAM-HNP401 binds and highlight multiple human peripheral nerves including lower leg sural, upper arm medial antebrachial as well as autonomic nerves isolated from human prostate. Conclusion: Phage display has identified a novel peptide that selectively binds to ex-vivo human nerves and in-vivo using rodent models. FAM-HNP401 or an optimized variant could be translated for use in a clinical setting for intraoperative identification of human nerves to improve visualization and potentially decrease the incidence of intra-surgical nerve injury.

Keywords: fluorescence imaging; human nerve; nerve targeting; translational.

Figure 1

In-vivo fluorescent labelling of autonomic nerves in rodents. (A) Low magnification fluorescence image showing bladder, vas deferens and urethra running through the prostate with adjacent autonomic nerve labeled with FAM-NP41 in mice. Higher magnification white light reflectance image (B) and corresponding fluorescence grayscale image (C) of the autonomic nerve running adjacent to the urethra. Quantitation of autonomic nerve detection by fluorescence compared to white light detection in mice (D). Nerve-to-muscle contrast for reflectance/fluorescence were plotted for individual nerve branches. Values to the right of the line indicate that there is improved visualization with fluorescence compared to reflected light. Images (E-G) are analogous to (A-C) except that they highlight FAM-NP41-dependent labeling of autonomic nerve in rat prostate versus mouse, with white light imaging showing non-visible nerve (F). (H) FAM-NP41-labeled prostate nerve is also detectable using a clinical grade Zeiss Pentero Surgical Microscope.

Figure 2

Comparison of FAM-HNP401 and FAM-NP41 in binding and labelling of human sural nerve. Topical application of 100 μM of FAM-HNP401 on 10 μm sections of unfixed human sural nerve tissue (A) and human temporalis muscle tissue (E) kept adjacent on the same glass slide and imaged on a confocal microscope with 488 nm laser excitation. For comparison, FAM-NP41 was applied to a sequential section of human nerve (B) and muscle (F) under identical conditions as for (A, E). H&E staining of the nerve (C) and muscle (G). (D) Signal intensity of perineurium of nerve tissue treated with FAM-HNP401 (n=4) compared with FAM-NP41 (n=4). (H) Nerve-to-muscle contrast of peptides applied topically to human tissue sections (n=4).

Figure 3

In vivo imaging of nerve-binding peptides in mice and rats with pharmacokinetics. In-vivo fluorescence images of sciatic nerves from 6-month-old SKH1 mice that had been previously intravenously injected with 450 nmol of FAM-HNP401 (~48.4 mg/kg) (A) or FAM-NP41 (~39 mg/kg) (B). (C) The fluorescence intensity of the sciatic nerve measured and quantitated in Image J showed a 2.3-fold increase for FAM-HNP401 compared to FAM-NP41. (D) Nerve-to-muscle contrast for the two peptides were comparable at 5.79 ± 0.81 for FAM-HNP401 and 6.63 ± 1.63 for FAM-NP41. (E) In vivo fluorescence image of rat sciatic nerve 5 h after intravenous injection of 2 µmol of FAM-HNP401 (~54 mg/kg). Rat prostate nerve imaged with a real-time custom surgical imaging system (F) and Lumar small animal microscope (G) 5 h after intravenous injection of 2 µmol of FAM-HNP401. (H) The blood clearance curve shows FAM signal obtained from equal volume blood draws taken from five SKH1-Elite male mice. Each mouse was injected intravenously with 100 nmol (~11 mg/kg) of FAM-HNP401 prior to blood collection at 1 min, 10 min, 20 min, 30 min, 1 h and 2 h time points.

Figure 4

HNP401 binds to fresh viable nerve from the prostate gland and median anti-brachial cutaneous human nerve. Fluorescence imaging after topical application of 100 µM FAM-HNP401 or FAM-NP41 on 10 µm sections on cryosectioning tape of nerve within human prostate gland, (A-B) or from median anti-brachial cutaneous human nerve (E-F). Nerves were imaged immediately after sectioning and application of peptide using confocal microscopy. Immunohistochemistry analysis with dual labelling for neurofilament antibody SMI312 (red) and DAPI-stained nuclei (blue) (C, G) and corresponding H&E staining (D, H) of a fixed section of nerve on glass slides.

Figure 5

Comparison of truncated sequences to determine binding efficiency. Representative fluorescence images of unfixed human sural nerve that were treated topically with 100 µMof FAM-labelled N-2 (A), N-4 (B), N-6 (C), N-8 (D), C-2 (E), C-4 (F), C-6 (G), C-8 (H) or HNP401 (I). Due to poor solubility, C-6 had a final concentration of ^a73 µM and C-8 had a final concentration of ^b80.6 µM for topical tests. (J) Comparison of signal intensity of peptides normalized to FAM-HNP401 were made to test for improved binding. (K) Normalized sural nerve to temporalis muscle contrast was determined for FAM-HNP401 and FAM-HNP401-N-2 (Student t-test, unpaired, one-tail, p=0.011).