Realizing real-time optical molecular imaging in peripheral nerve tissue via Rhodamine B

Abstract

Background: Iatrogenic nerve injury is a consequential complication during surgery. Thus, real-time imaging of peripheral nerve (PN) possesses significant clinical implications. In recent years, the rapid advancements in optical molecular imaging (OMI) technology have provided essential technical foundations for the implementation of PN fluorescence imaging. This study aimed to realize real-time OMI of PNs via Rhodamine B.

Methods: Phosphate buffered saline (PBS), normal saline (NS), 5% glucose solution (GS), and fetal bovine serum (FBS) were selected for measuring the fluorescence spectra of Rhodamine B solutions prepared in each formulation. Rhodamine B solutions, with varying doses dissolved in 100 μL of each formulation, were prepared and applied to the exposed PNs of the mice for incubation later. To ascertain the optimal formulation and dose of Rhodamine B, an analysis was performed on the signal-to-background ratio (SBR) of the nerves. Based on the experimental results, we proceeded to incubate Rhodamine B solution on the PN tissue of mice and human subjects, as well as on neuronal cells, to verify the binding sites of Rhodamine B with nerve. Subsequently, histological studies were conducted to validate the binding site between Rhodamine B and the nerves. Finally, we injected the optimal combination of Rhodamine B solution into mice via the tail vein and collected the SBR of mouse nerve tissues at different time intervals to determine the optimal pre-injection time. Fluorescence images of various tissues were collected, and Hematoxylin and Eosin (H&E) staining results were observed to determine the metabolism of Rhodamine B in mice and its toxicity.

Results: The excitation peak of Rhodamine B in PBS, NS, 5% GS, and FBS formulations was 554 nm, and the emission peak was 576 nm. In PBS group, the maximum SBR was 15.37 ± 0.68 while the dose of Rhodamine B was 8 nmol. Through ex-vivo validation on fresh human nerve tissue and verification using mouse and human tissue sections, we observed fluorescent signals of Rhodamine Bin the regions of nerve tissue and the fluorescence signals were all concentrated on the neuronal cell membranes. After injection, the fluorescent signal in nerve tissue reached its peak at 24 hours (h), coinciding with the highest SBR (5.93 ± 0.92) in mouse nerve tissues at this time point. Additionally, the fluorescence signal could be maintained for at least 48 h. Within 24 h, lung dilation and fusion of alveoli occurred. Then these pathological manifestations gradually diminished, returning to normal at 2 weeks (w), with no significant acute or chronic adverse reactions observed in other tissues.

Conclusion: Rhodamine B enables fluorescence imaging of PNs and has the potential for clinical translation.

Keywords: OMI technology; Rhodamine B; SBR; peripheral nerve; real-time fluorescence imaging.

Figure 1

The self-developed Multi-modal Small Animal Live Imaging System. (A) Appearance of the imaging system. (B) Light path diagrams of the imaging system.

Figure 2

Fluorescence spectra of 1 nmol Rhodamine B dissolved in 100 μL different formulations. (A) Dissolved in PBS. (B) Dissolved in NS. (C) Dissolved in 5% GS. (D) Dissolved in FBS.

Figure 3

Representative image of different dose and formulation with Rhodamine B. We collected fluorescence images of nerve tissue, merge images, and white light images of mice in each group. All images acquired using the In Vivo Xtreme Imaging System (Bruker BioSpin Corp., Billerica, MA). (A) Dissolved in PBS. (B) Dissolved in NS. (C) Dissolved in 5% GS. (D) Dissolved in FBS.

Figure 4

Results of optimal formulation and dose of Rhodamine B. The analysis results were collected from 3 mice (12 nerves) in each group. SBR = intensity of nerve tissue to intensity of background tissue ratio. The fluorescence intensity of nerve tissue and background tissue in different groups can be seen in (A) dissolved in PBS, (B) dissolved in NS, (C) dissolved in 5% GS, and (D) dissolved in FBS. * = p < 0.05, *** = p < 0.001, and **** = p < 0.0001.

Figure 5

Analysis results of SBR in different formulation and dose groups. ** = p < 0.01, *** = p < 0.001, and **** = p < 0.0001.

Figure 6

Fluorescence images, pseudo-color images, and white light images of human ex-vivo nerve tissue.

Figure 7

Histological confirmation of nerve tissue. We confirmed the brachial plexus and sciatic nerve in mice, and the isolated tibial nerve and sciatic nerve in human from transverse and longitudinal section, respectively, using paraffin and frozen sections, respectively. Each set of sections were derived from three consecutive sections. (A) Mice nerve tissue, paraffin section. (B) Mice nerve tissue, frozen section. (C) Mice nerve tissue, paraffin section. (D) Human nerve tissue, frozen section.

Figure 8

Fluorescence microscopy image of ND7/23 neuronal cells.

Figure 9

Result of time dependent imaging. (A) Analysis results of SBR in different time of 8 nmol Rhodamine B dissolved into 100 μL PBS and injected through a mice tail vein. (B) Fluorescence images, optimized images, and real-time anatomy images. (C) Anatomical images of mouse nerve tissues result at 24 h post-operation based on fluorescence signal guidance.

Figure 10

Metabolic results of Rhodamine B in mice. (A) Fluorescence images, pseudo-color images, and white light images of different organs at different time. ① Heart. ② Liver. ③ Spleen. ④ Nerve. ⑤. Lung. ⑥ Kidney. ⑦ Brain. (B) Fluorescence intensity changes of different organs at different time.

Figure 11

Histological changes of different organs at different time after 100 μL Rhodamine B solution and PBS were injected into mice via the tail vein H&E.

Figure 12

Chemical structure of Rhodamine B. The molecular formula for Rhodamine B is C₂₈H₃₁ClN₂O₃ and the molecular weight is 479.01.