In Vivo Photoacoustic Imaging of Brain Injury and Rehabilitation by High-Efficient Near-Infrared Dye Labeled Mesenchymal Stem Cells with Enhanced Brain Barrier Permeability

Abstract

Stem cell migration and interaction with pathology are critical to understand the complexity and status of disease recovery progress. However, the dynamic visualization still remains a great challenge due to imaging technical limitation, cell labeling difficulty, or blood-brain barrier (BBB). Herein, fast photoacoustic tomography (PAT) with optical molecular probes is applied to noninvasively monitor traumatic brain injury (TBI) and its rehabilitation. The vascular distribution and TBI hemorrhage are clearly imaged, longitudinally monitored, and quantified. Bone mesenchymal stem cells (BMSCs) labeled with modified Prussian blue particles (PBPs), excellent near-infrared dyes and photoacoustic contrasts, are intravenously injected to the mice for improved observation and efficient therapy. BMSCs are demonstrated to be capable of overcoming BBB with enhanced delivery of PBPs to the brain parenchyma. Notably, the versatile BMSCs are observed by PAT to home to the damage region and repair the ruptured vasculature. Moreover, the wound treated by BMSCs exhibits much faster recovery speed than that without treatment. These findings can potentially provide a new noninvasive and high-resolution approach to image TBI, monitor recovery process, and especially trace BMSCs. This study will stimulate extensive researches on brain diseases and provide promising strategies of dye labeled BMSCs in regenerative medicine.

Keywords: Prussian blue; blood–brain barrier; bone mesenchymal stem cells; photoacoustic tomography; traumatic brain injury.

Scheme 1

In vivo photoacoustic monitoring of brain injury and rehabilitation by modified Prussian blue particles‐labeled mesenchymal stem cells.

Figure 1

A) TEM image of Prussian blue particles. B) Spectra profiles of PBPs, Hb, and HbO₂. C) Normalized PA signal amplitude with increased concentrations of the PBPs solution calculated from Figure S2 (Supporting Information). D) Viability of BMSCs treated with different concentrations of PBPs for 24 h. E) Labeling capacity of BMSCs incubated with PBPs for different periods of time. F) Stability of well‐labeled stem cells during 48 h cultivation. The photomicrographs of BMSCs G) before and H,I) after labeling with PBPs. PBPs encapsulated in the BMSCs was indicated by red arrows.

Figure 2

A) PA images of the mouse brain before, 3, 15, 30, 60, 120, 270, and 360 min after needle injury, respectively. The injury area is identified by the red arrow. B) The SBR of the damaged location at the predetermined time points. Error bars represent SEM (n = 6). C) The bleeding area change in the damaged region. Error bars represent SEM (n = 6). D) 3D PA image of the mouse brain after TBI. E) CT image of the mouse brain after cerebral injury. The wound hole that marked by the red dotted line circle was induced by the steel needle. F) The MRI image (axial plane) of the mouse brain after the brain injury.

Figure 3

A) PA images of the mouse brain at predetermined time intervals of injury after 3 min, 1, 3, 5, 7, 9, 11, 13, and 15 d, respectively. B) The SBR change of the damaged location at the predetermined time points. Error bars represent SEM (n = 6). C) The area change of blood clot in the damaged region at the predetermined time points. Error bars represent SEM (n = 6).

Figure 4

A) Mouse brain PA images of control group, B) PBP‐labeled BMSC‐treated, and C) BMSC‐treated groups at the predetermined time intervals of the recovery process. D) Normalized PA signal intensities of the damaged location at the predetermined time points. Error bars represent SEM (n = 6 per group). *P < 0.05 compared to PBS group. E) The blood clot area in the damaged region. Error bars represent SEM (n = 6 per group). *P < 0.05 compared to PBS group. F) PA images of the mouse brain (i) before and (ii) after a single injection of BMSCs (1 × 10⁶, 200 µL) labeled with PBPs (0.1 mg mL⁻¹). The image (iii) was obtained by subtracting the before injection image from the after injection image (iii = ii − i).

Figure 5

A) Photographs of the whole brain tissues before and 7 d after TBI. The dashed white box indicated the injured area. B) DAPI and immunohistochemical staining of brain sections for CD31 and CD44 in injured region after BMSC therapy of TBI. Blue, aubergine, and green fluorescence correspond to DAPI, CD44, and CD31, respectively. C) H&E staining of brain tissue slices during the recovery progress of TBI with or without intravenous administration of BMSCs.

Figure 6

A) Fluorescence imaging of the mice injected with Cy5.5‐labeled BMSCs. B) Quantitative fluorescence intensity of the damage region in TBI mice after intravenous injection of Cy5.5‐labeled BMSCs. Error bars represent SEM (n = 6). C) Quantitative fluorescence intensity of major organs including heart, liver, spleen, lung, kidney, and brain 72 h postinjection of Cy5.5‐labeled BMSCs. Error bars represent SEM (n = 6). *P < 0.05 compared to heart or spleen.