Intraoperative visualization of nerves using a myelin protein-zero specific fluorescent tracer

Background Surgically induced nerve damage is a common but debilitating side effect in oncological surgery. With the aim to use fluorescence guidance to enable nerve-sparing interventions in future surgery, a fluorescent tracer was developed that specifically targets myelin protein zero (P0). Results Truncated homotypic P0 protein-based peptide sequences were C-terminally functionalized with the far-red cyanine dye Cy5. The lead compound Cy5-P0101–125 was selected after initial solubility, (photo)physical and in vitro evaluation (including P0-blocking experiments). Cy5-P0101–125 (KD = 105 ± 17 nM) allowed in vitro and ex vivo P0-related staining. Furthermore, Cy5-P0101–125 enabled in vivo fluorescence imaging of the Sciatic nerve in mice after local intravenous (i.v.) administration and showed compatibility with a clinical fluorescence laparoscope during evaluation in a porcine model undergoing robot-assisted surgery. Biodistribution data revealed that i.v. administered [111In]In-DTPA-P0101–125 does not enter the central nervous system (CNS). Conclusion P0101–125 has proven to be a potent nerve-specific agent that is able to target P0/myelin under in vitro, ex vivo, and in vivo conditions without posing a threat for CNS-related toxicity. Supplementary Information The online version contains supplementary material available at 10.1186/s13550-021-00792-9.


Background
Surgery, and science therein, has come a long way since Mr. Gunning and Lord Thurlow, respectively, stated to each other in 1796: "there is no more science in surgery", in reply "than there is in butchery" [1]. Contradictory to these statements, the rapid translation of innovative minimally invasive surgical technologies has initiated the concept of "precision surgery" (2)(3)(4)(5). This concept is mostly driven by engineering efforts in the form of medical devices such as endoscopic cameras, refined instruments, and robotic manipulators that enable modern surgeons to intervene in the human body in ways that were previously not thought possible [3,4,6]. Imaging provides an alternative to impact surgical accuracy; the application of minimally invasive procedures is strengthened by the ability to map areas of disease in the context of healthy anatomy (so-called surgical roadmaps) using non-invasive preoperative imaging modalities such as MRI or PET/CT [7][8][9]. Unfortunately, intraoperative detection of preoperatively identified lesions/structures can be challenging. For instance, increased distancing between the surgeon and the patient limits the surgeon's sensory experience in the form of palpation (e.g. when a surgical robot is used). This shortcoming can at least in part be compensated through the use of interventional molecular imaging. To date, this imaging sub-discipline has predominantly focused on intraoperative detection of cancerous lesions using either radio-or fluorescence guidance [10,11]. Here the main applications have included complex anatomies such as the head-and-neck or pelvic area where image guidance is exploited for both detection of nodal metastases and primary tumour margins [12][13][14][15][16]. However, in these same anatomies accidental surgical damage to nerves can yield debilitating side effects such as loss of sensory feeling or speech, incontinence and/or erectile dysfunction. This occurrence of surgically induced nerve damage is not uncommon: despite the fact that more than 70% of prostate cancer patients receive nerve-sparing surgery, it is accepted that 30% of patients suffer from loss of erectile function at 1 year post-surgery [17]. Here, it should be noted that the extent of the damage may be hard to predict [18]. In addition, 10-15% of patients suffer from urinary incontinence [19]. In head and neck cancer patients, nerve anatomies are complex [20] and recurrent laryngeal nerve injury and mandibular nerve injury is seen in 14% of patients undergoing, respectively, thyroid surgery or neck dissection [21,22]. Permanent paralysis is seen in 4-7% of patients [22].
Nerve-specific fluorescence imaging has been poised as a means to allow high-resolution nerve identification in real time [23]. For this application, a number of different targeting strategies have been evaluated, ranging from neuronal tracing to targeting intracellular expressed proteins in myelin sheets such as myelin basic protein [23][24][25][26][27]. In some cases, the nerve-specific target is unknown [27][28][29][30]. When pursuing conventional receptor-targeted molecular imaging strategies, extracellularly expressed targets are generally sought after. In that sense, myelin protein zero (MPZ, or P0), a 124-amino-acid-residueslarge homotypic protein that makes up 80% of the protein content in peripheral myelin (Fig. 1A, [31]), and that is located on the outer membrane of the Schwann cells that form the myelin sheath, would most certainly be a target that is worth exploring. Uniquely, P0 is specific for the peripheral nervous system (PNS) and is not expressed in the central nervous system (CNS). In the CNS myelin sheath formation is facilitated by the adhesive properties of myelin proteolipid protein (PLP; [32]).
Building on the truncation of the extracellular portion of P0 (Fig. 1B) that was previously proposed by Makowska et al. [33] and the homotypic binding properties of P0 a b Fig. 1 Myelin protein zero as a target for nerve imaging. A schematic overview of the localization of myelin protein zero (P0) in the peripheral nervous system with (I) the location of the nervus ischiadicus (encircled in grey), (II) myelinated axon within this nerve, (III) the myelin sheath encapsulating the axon, (IV) densely packed myelin within the myelin sheath, (V) homotypic binding of P0 within the myelin sheath (location P0 on outer membrane and between layers annotated in black) and (VI) the crystal structure of the extracellular portion of P0 (P0 ex ). B Peptides P0 1-25 , P0 21-45 , P0 41-65 , P0 61-85 , P0 81-105 , P0 95-120 and P0 101-125 derived from the crystal structure of P0 ex with the specific section of the amino acid sequence included in the peptide highlighted in blue and the location of Cy5 functionalization represented by a red-light bulb (e.g. intrinsic binding between P0 and P0), fluorescently labelled nerve-specific synthetic P0-derived peptides were extrapolated from the crystal structure of P0 ( Fig. 1B; peptides in blue). After initial solubility, (photo) physical and in vitro evaluation (affinity and microscopic localization) a lead compound that showed the most ideal properties was selected. This fluorescent tracer was further scrutinized in three-dimensional (3D) dorsal root ganglion (DRG) cell cultures, on ex vivo nerve specimens and in vivo in mice (macroscopic localization, nerve/ myelin-specificity, biodistribution). In vivo nerve visualization in a porcine model was performed as a proof of principle for real-time nerve visualization in a robotassisted surgery setting.

General chemistry
All chemicals were obtained from commercial sources and used without further purification. DMF was dried over 4 Å molecular sieves. High-pressure liquid chromatography (HPLC) was performed on a Waters HPLC system (Waters Chromatography B.V., Etten-Leur, The Netherlands) using a 1525EF pump and a 2489 UV detector. For preparative HPLC, a Maisch ReproSil-Pur 120 C18-AQ 10 μM (250 mm × 20 mm) column (Dr. Maisch HPLC GmbH, Ammerbuch-Entringen, Germany) was used at a flow rate of 12 mL/min. For analytical HPLC, a Maisch ReproSil-Pur C18-AQ 5 μM (250 mm × 4.6 mm) column was used with a gradient of 0.1% trifluoroacetic acid (

Detailed analysis of the selected lead compound Cy5-P0 101-125
Cy5-P0 101-125 was selected as lead compound and subjected to more detailed chemical analysis. Methods and results for assessment of the (photo)physical properties of Cy5-P0 101-125 (i.e. serum protein binding and LogP o/w , chemical stability, stability at different temperatures and the molar extinction coefficient and relative quantum yield and brightness) are described in Additional file 1: chemical properties.
In line with their use by Whitney et al. [27], transgenic B6.Cg-Tg(Thy1-YFP)-16Jrs/J (THY-1 YFP) mice were obtained from JAX (the Jackson Laboratory) and were used for ex vivo and in vivo nerve staining and in vivo biodistribution studies (8-15 weeks old). THY-1 YFP mice express spectral variants of GFP (yellow fluorescent protein-YFP; ex 488, em 520) at high levels in motor and sensory neurons. The fluorescent signal in the nerves was used as an internal control for the staining (pattern) of the developed imaging agents. Balb/c nude mice were used as non-fluorescent control.

Flow cytometry
Analysis of the binding affinity (K D ) of Cy5-P0 1-25 , Cy5-P0 41-65 , Cy5-P0 61-85 , Cy5-P0 81-105 , Cy5-P0 95-120 and Cy5-P0 101-125 for P0 was performed using P0-expressing RT4 D6P2T cells and a previously described flow cytometric method [36]. For saturation, binding experiments were performed for each of the fluorescent peptides in a concentration range of 0-2000 nM. All measurements were taken in triplicate, and experiments were repeated at least three times per tracer. Fluorescence was measured using a FACSCanto II flow cytometry device (BD Biosciences) in the APC-A channel. The normalized geometric means were fitted with equations in the GraphPad Prism 5 software. A fluorescence-linked immunoabsorbent assay (FLISA) that was used to confirm the specificity of Cy5-P0 101-125 for P0 is described in Additional file 1: Fluorescence-linked immunoabsorbent assay.

Fluorescence microscopy of cells
Cells were trypsinized and seeded onto 35-mm culture dishes that contained a glass insert (MatTek co) on the day prior to the imaging experiment.
In vitro and ex vivo fluorescence confocal images were acquired using a Leica SP8 WL at sequential settings and 10 × or 63 × magnification. Image analysis was performed using Leica Confocal Software (Leica Microsystems). For blocking studies, quantification of the fluorescence signal intensity (N = 10 for blocked and non-blocked) in the obtained images was performed using ImageJ according to previously described methods [37,38]. Statistical evaluation was performed based on a Student's t test.

More detailed ex vivo and in vivo studies with lead compound Cy5-P0 101-125 Culture and imaging of 3D dorsal root ganglion (DRG) explant cultures from THY-1 YFP mouse embryos
For evaluation of the staining pattern of P0, 3D DRG explant cultures were used (all N = 3 per peptide or control staining). Description of the methods used for ex vivo culture and imaging of 3D DRG explant cultures from THY-1 YFP mouse embryos are provided in Additional file 1: 3D culture of DRG explants. Cy5-P0 Ab-H60 , Cy5-P0 ex as well as Cy5-NP-41 were used as controls.

Ex vivo tissue of mice
Fluorescence immunohistochemistry was performed on fresh frozen samples of the nervus ischiadicus that were embedded in Tissue-Tek and cut into 5 µM frozen sections. Cryo-sections were fixed in pre-cooled acetone (VWR Chemicals, 67-64-1) for 10 min and dried on air for 1 h and washed with 1 × phosphate-buffered saline (PBS) (Life Technologies, 10010-015) to remove Tissue-Tek. Slides were incubated for one hour at room temperature with 1 µM of Cy5-P0 101-125 . Sections were rinsed and dehydrated using ethanol and mounted with ProLong Gold Antifade Mountant with DAPI (Fisher, P-36931). Images were obtained using a fluorescence confocal microscope. Standard antibody-based immunohistochemistry was used as control; for details, see Additional file 1: Immunohistochemistry.
For direct ex vivo assessment of freshly excised, nontreated tissue non-fixed sections of the nervus ischiadicus (mouse; N = 3) were incubated in 1.5-mL vials (Eppendorf, Falcon) containing 1 µM Cy5-P0 101-125 for 1 h. Confocal imaging was performed after washing with PBS. Non-incubated sections of the nerve were used as control.

In vivo assessment in mice
For evaluation of in vivo staining in mice, Cy5-P0 101-125 was administered (20 μL; 5 nmol) either intravenously in the v. femoralis or directly into the nerve sheath (intraneural) of THY-1 TFP mice (N = 3 per injection method) or Balb/c nude mice. Injection was performed under general anaesthetics (hypnorm/dormicum/H 2 O solution (1:1:2; 5 µL/g) via intraperitoneal injection). After placement of the mouse in the microscope stand, images were collected prior and during the dissection of the Nervus Ischiadicus. Staining was evaluated at 1 h after injection; N = 3. Mice were killed via cervical dislocation before the start of the imaging session. Animals that received no tracer or that received an intravenous (v. femoralis) injection of Cy5-NP-41 were used as control. In vivo fluorescence confocal microscopy was performed using a Zeiss 710 NLO upright confocal microscope. For collection and evaluation of the in vivo images, ZEN 2011 software was used. Furthermore, the nervus ischiadicus (in vivo; mouse model) and the Pudendal Nerve (excised after in vivo imaging; porcine model) were imaged using a Dino-lite handheld digital fluorescence microscope (AM4115T-DFRW for Cy5 imaging; Dino-lite Digital Microscope; λ ex 620 nm, λ em 650 nm). In-house developed image-processing software [39] that allowed colour coding of the fluorescence signal for improved visualization and distinction of intensity differences was used to depict the nerve-tobackground ratio (NBR; ratio between relative fluorescence units in the tumour and surrounding tissue). The provided pseudo-coloured fluorescence overlay was accompanied by an intensity-based scalebar representing the NBR (fluorescence signal intensity differences represented via a colour spectrum). Confirmation of the TBR values was obtained using ImageJ software by dividing the fluorescent signal intensity in the tumour by the fluorescent signal intensity in background tissue.

Biodistribution of [ 111 In]In-DTPA-P0 101-125 in mice
Synthesis and radiolabelling of DTPA-P0 101-125 with 111 In is described in Additional file 1: synthesis of control compounds. For quantitative assessment of the biodistribution of [ 111 In]In-DTPA-P0 101-125 , 10 MBq of the labelled tracer was injected intravenously (tail vein). The percentage of the injected activity per gram of tissue (%IA/g) was assessed at 2 h post-injection as previously described [40,41]. Excretion was defined as: (MBq present in animal at 24 h post-tracer administration/injected activity) * 100%.

In vivo assessment in a porcine model
To evaluate whether Cy5-P0 101-125 (100 μg, 25.6 nmol) was compatible with a real-life surgical setting, its use evaluated was in a porcine model undergoing robotassisted surgery using a da Vinci Si or Xi system (Intuitive). Pigs (N = 3) were injected directly in the Pudendal nerve (intraneural administration). Using a prototype and Cy5 dedicated KARL STORZ fluorescence laparoscope [42] introduced through the assistant trocar, a similar set-up was initially applied in the clinical setting [43,44]; fluorescence imaging of the nerve and surrounding tissues was performed at 1 h after tracer administration. Animals were maintained under Isoflurane anaesthesia for the complete duration of the surgical training and subsequent nerve imaging experiments and were euthanized before awakening from the anaesthesia. After resection, fluorescence microscopy images were made of the fresh nerve to confirm staining. Image processing was performed using in-house custom-developed software as described above. Table 1 Fluorescently labelled P0 peptides, sequences and outcome synthesis a Italic alanine residue replacing the cysteine from native P0, bolded and underlined residues were implemented via the above-mentioned pseudoproline method [34], underlined cysteines were non-native residues added to the C-terminus

Peptide
Amino acid sequence Negative/positive charges

More detailed studies with lead compound Cy5-P0 101-125 Chemical analysis
Chemical analysis of Cy5-P0 101-125 revealed that this tracer was 99% stable after incubation in serum for 24 h at 37 °C and > 99% stable at temperatures > 0 °C for at least 4 h (Additional file 1: Figure SI2). Additional chemical-and photophysical features of Cy5-P0 101-125 are presented in Additional file 1: Table SI2.

Imaging of 3D dorsal root ganglion (DRG) explant cultures from THY-1 YFP mouse embryos
3D cultures based on DRG explants obtained from THY-1 YFP mouse embryos provided an intermediate step between in vitro and in vivo evaluation (Fig. 3A, B). These 3D cultures contained a centre ganglion (*) and axonal outgrowths (white arrow) and are known as wellestablished neuronal cultures for drug discovery for neuronal neuropathies [46]. Incubation with Cy5-P0 101-125 resulted in a spotted staining pattern of cells residing along the course of the developed axonal outgrowths as well as in the DRG explant itself (Fig. 3AII and AIII). Again, staining with Cy5-P0 ex (Fig. 3AIII and BIII) confirmed the findings.

Ex vivo assessment of murine nerve tissue
Immunohistochemical assessment of concurrent freshfrozen sections of the nervus ischiadicus of THY1-YFP mice revealed a clear overlap between the location of P0 between staining obtained after incubation with Cy5-P0 101-125 (fluorescence immunohistochemistry; Fig. 3C) and an anti-P0 antibody (standard immunohistochemistry; insert Fig. 3C).
More detailed microscopic assessment of viable (nonfrozen, non-pretreated) samples of the nervus ischiadicus that were incubated ex vivo with Cy5-P0 101-125 revealed an identical wavy staining pattern ( Fig. 4AI; in red). Here, the intrinsic YFP signal within the axons of the THY-1 YFP mice provided an extra confirmation that staining of Cy5-P0 101-125 co-localized with the myelin sheath surrounding the axons.

In vivo assessment in mice
To reduce the dose and staining beyond the area of interest, in vivo administration intraneural (Fig. 4AII) Fig. 4AIII and B) administration were applied. Both tracer administration routes provided identical results compared to ex vivo incubation with Cy5-P0 101-125 . Similar to assessment in vitro (Fig. 2), the non-P0-specific peptide CY5-NP41 (Fig. 4AIV)  nervus ischiadicus based on the emitted fluorescence signal (Fig. 4B). Macroscopic in vivo assessment of the nervous ischiadicus (whitelight image; Fig. 5C) resulted in a mean NBR of 6.0 ± 2.2 after administration of Cy5-P0 101-125 (Fig. 4CIII). Comparable results in non-YFP mice ( Fig. 5CIV; mean NBR: 3.7 ± 1.0) help exclude the possibility of spectral overlap between YFP and Cy5.

In vivo assessment in a porcine model
The compatibility of Cy5-P0 101-125 with clinical grade imaging modalities applied during robot-assisted minimally invasive surgery was evaluated in a porcine model (Fig. 6). Here, the use of a dedicated Cy5 fluorescence laparoscope (KARL STORZ; [42]) allowed in vivo visualization of the pudendal nerve (Fig. 6B, C; white arrow). Image processing based on colour coding of the fluorescence signal helped assess differences in fluorescence intensity along the nerve (Fig. 6C insert). Back-table imaging of the excised specimen confirmed the fluorescence of the nerve (Fig. 6D).

Discussion
By using the P0-derived synthetic peptide Cy5-P0 101-125 , we were able to explore the homotypic P0 protein as molecular target that is widely expressed in myelin in the PNS. Specific binding was demonstrated in vitro, in 3D DRG nerve cultures, ex vivo, as well as in vivo. Truncation of the homotypical P0-protein into peptides yielded Cy5-P0 101-125 as lead compound. In vitro and in vivo studies indicate that this compound is able to provide nerve-specific staining with nanomolar affinity, which is in the same range as reported affinities for other fluorescently labelled targeted tracers [36,47,48]. Specificity of Cy5-P0 101-125 for P0 was shown both in vitro ( Fig. 2 and Additional file 1: Figure SI3) and in vivo (Fig. 4). An approximate 90% decrease in fluorescence intensity after pre-incubation with a P0-specific antibody was seen (Fig. 2CIII), while no staining was observed for the non-P0-specific control (Cy5-NP41) and the free dye (Cy5-Maleimide). As the Cy5-Maleimide dye variant was used for functionalization of both P0 101-125 and NP41, these results also exclude a targeting effect of the dye itself. This was corroborated by a markedly different staining pattern using free dye alone [30]. An additional beneficial factor for Cy5-P0 101-125 is that the production is scalable and can be done at reasonably low cost. Moreover, the peptide benefits from the superior pharmacokinetics that have been claimed for peptides over proteins such as antibodies [49,50].
In line with P0 expression, biodistribution studies performed with the radiolabelled analogue [ 111 In]In-DTPA-P0 101-125 helped rule out accumulation of P0 101-125 (MW = 3024 Da) in the CNS. Obviously, the chance of toxic side effects is also impacted by dosing and uptake in non-target organs. In nuclear medicine, disease-specific tracers are therefore applied using a micro-dosing regimen (< 100 µg/patient). Although the ability of using fluorescence at micro-dosing levels looks promising [51], this topic remains a subject of debate [52], and many studies still use high dosing regimens to realize in vivo functionalization of molecular targets [16,[53][54][55]. Local tracer deposition (an image-guided surgery concept that has proven valuable in e.g. lymphatic mapping and during occult lesion localization) limits dosing to 100 µg/ patient [12,51]. Recent experimental studies underscore that local deposition may also be valid when targeting peripheral nerves within a certain surgical anatomy [24,37]. Substantiated by previous reports [28] Fig. 4 AIII and B indicate that local tracer administration is feasible for nerve imaging applications.
While most image-guided surgery studies promote the use of near-infrared (NIR) Cy7 analogues [53,54,56], far-red Cy5-labels are also increasingly being applied in clinical trials [39,[57][58][59]. In fact, fluorescence-guided surgery trials have been reported for the full fluorescent light spectrum [60]. Uniquely, in a head-to-head comparison, Cy5 analogues even were shown to outperform Cy7-analogies in terms of signal intensity [42] and impact on tracer kinetics [36]. Moreover, Cy5 analogues, both a free dye and conjugated to a peptide, have shown to have a high fluorescence brightness in the presence of human serum albumin (Additional file 1: Table SI2; [36,41,61]). These factors combined with the fact that most groups are creating tumour-receptor-targeted tracers using NIR Cy7 analogues support the future implementation of multi-wavelength imaging applications, a concept that is gaining traction in the clinic [60].
Although intraneural injection provides a perfect proof of principle in both mice and pigs (Figs. 4AII, C and Additional file 1: Figure SI6), administration into a blood vessel near the target organ, such as the femoral  (Fig. 4A), still provides further optimization before it can be used in a human setting. Hence, the exact route for local tracer administration in large animals, minimization of the dose and the specificity of targeting remain the subject of ongoing studies. Another potential limitation of the approach presented is that myelinization of nerves in the PNS may vary, where sensory nerves are highly myelinated (and conductive), the level of myelination decreases substantially in autonomous nerves. Despite the fact that myelin is one of the most widely explored targets for nerve imaging, it is therefore not clear if myelin-specific tracers will help address all surgical nerve imaging demands.

Conclusion
By truncating the P0 protein, we have been able to successfully create a nerve-specific fluorescent tracer that is able to specifically stain P0/myelin expression in the PNS.