Nerve Suture Combined With ADSCs Injection Under Real-Time and Dynamic NIR-II Fluorescence Imaging in Peripheral Nerve Regeneration in vivo

Abstract

Peripheral nerve injury gives rise to devastating conditions including neural dysfunction, unbearable pain and even paralysis. The therapeutic effect of current treatment for peripheral nerve injury is unsatisfactory, resulting in slow nerve regeneration and incomplete recovery of neural function. In this study, nerve suture combined with ADSCs injection was adopted in rat model of sciatic nerve injury. Under real-time visualization of the injected cells with the guidance of NIR-II fluorescence imaging in vivo, a spatio-temporal map displaying cell migration from the proximal injection site (0 day post-injection) of the nerve to the sutured site (7 days post-injection), and then to the distal section (14 days post-injection) was demonstrated. Furthermore, the results of electromyography and mechanical pain threshold indicated nerve regeneration and functional recovery after the combined therapy. Therefore, in the current study, the observed ADSCs migration in vivo, electrophysiological examination results and pathological changes all provided robust evidence for the efficacy of the applied treatment. Our approach of nerve suture combined with ADSCs injection in treating peripheral nerve injury under real-time NIR-II imaging monitoring in vivo added novel insights into the treatment for peripheral nerve injury, thus further enhancing in-depth understanding of peripheral nerve regeneration and the mechanism behind.

Keywords: ADSCs; NIR-II; fluorescence imaging; nerve regeneration; nerve suture; peripheral nerve injury.

Figure 1

Schematic of the experimental process.

Figure 2

Fluorescence properties of PbS QDs+Tat and the labeling of ADSCs. (A) Schematic of synthesis of PbS QDs+Tat and labeling of ADSCs. (B) NIR-II imaging of PbS QDs labeled ADSCs in the state of suspension and (C) after centrifugation. (D) NIR-II imaging (λex: 808 nm, exposure time: 1 ms) of the same PbS QDs+Tat in a time course (0, 7, 14, 21, and 28 d post-preparation). (E) PL intensity of the same PbS QDs+Tat after storage. (F) PL intensity of PbS QDs and PbS QDs+Tat under different exposure time. (G) Normalized PL spectra of PbS QDs and PbS QDs +Tat.

Figure 3

Detection of sensitivity and stability of PbS QDs labeled ADSCs in vivo. (A,D,G,J) NIR-II imaging of PbS QDs labeled ADSCs (1 × 10⁵, 5 × 10⁴, 5 × 10³, and 5 × 10² cells) and PBS in vivo in a time course (0, 7, 14, and 21 d post-injection). (B,E,H,K) PL intensity measured from (A,D,G,J). (C,F,I,L) Fluorescence area measured from (A,D,G,J). (M) Longitudinal comparison of PL intensity measured from (A,D,G,J). (N) Longitudinal comparison of fluorescence area measured from (A,D,G,J).

Figure 4

In vivo distribution and migration of PbS QDs labeled ADSCs in a rat model of sciatic nerve injury. (A) Schematic illustration of the in vivo NIR-II imaging strategy. (B) Bright field photograph of the region of interest and NIR-II imaging of the experimental group (injected with PbS QDs labeled ADSCs) in a time course (0 d, 1 h, 1 d, 5 d, 7 d, 10 d, and 14 d post-injection). (C) Bright field photograph of the region of interest and NIR-II imaging of the control group (injected with pure PbS QDs) in a time course (0 d, 1 h, 1 d, 5 d, 7 d, 10 d, and 14 d post-injection). (D) Fluorescence area and (E) PL intensity of the proximal and distal sections of the sciatic nerve. (F) The ratio of PL intensity in distal/proximal nerve sections in the experimental group and the control group.

Figure 5

Functional examination through electromyography and mechanical pain threshold. Electromyography of (A) healthy rats, (B) rats with PbS QDs injection, and (C) rats with PbS QDs labeled ADSCs injection at 3 m post-injection. (D) Histogram of signal intensity measured from (A–C). (E) Pattern diagram of Electronic Von Frey in rat. (F) Measurements of the mechanical withdrawal threshold of rat taken after injury (***P < 0.001, vs. PbS QDs).

Figure 6

Biosafety of PbS QDs labeled ADSCs. (A1–D1) Bright field photographs and (A2–D2) NIR-II fluorescence images of major organs harvested from the rats in the control group and experimental group at 1 d, 1 m, and 2 m post-injection. (E) Representative HandE staining of major organs (collected and at 1 d, 1 m, and 2 m post-injection) of the rats injected with PbS QDs labeled ADSCs and the control group (scale bars represent 100 μm).