The administration time is a critical but long-neglected point in cell therapy based on macrophages because the incorrect time of macrophage administration could result in diverse outcomes regarding the same macrophage therapy. In this work, the second near-infrared (NIR-II) fluorescence imaging in vivo tracking of M2 macrophages during a pro-healing therapy in the mice model of rotator cuff injury revealed that the behavior of administrated macrophages was influenced by the timing of their administration. The delayed cell therapy (DCT) group had a longer retention time of injected M2 macrophages in the repairing tissue than that in the immediate cell therapy (ICT) group. Both Keller–Segel model and histological analysis further demonstrated that DCT altered the chemotaxis of M2 macrophages and improved the healing outcome of the repaired structure in comparison with ICT. Our results offer a possible explanation of previous conflicting results on reparative cell therapy and provoke reconsideration of the timing of these therapies.

The in vivo spatio-temporal patterns of neovascularization are still poorly understood because it is limited to multi-scale techniques from the cellular level to living animal level. Owing to deep tissue-penetration and zero autofluorescence background, the second near-infrared (NIR-II, 1,000-1,700 nm) fluorescence imaging recently shows promise in breaking through this dilemma by dynamically tracking the pathophysiological process of neovascularization in vivo. Here, NIR-II fluorescence imaging was recruited for monitoring blood vessels in order to visualize the vascular injury and quantitively assess neovascularization in mouse models of acute skeleton muscle contusion and hindlimb ischemia. The temporal analysis of real-time NIR-II fluorescence intensity demonstrated that the blood flow perfusion of ischemia area was able to rapidly restore to 96% of pre-ischemic state within one week. Moreover, the spatial analysis revealed that the lower and outer quadrants of ischemia area in the mouse model of hindlimb ischemia always had relatively high blood flow perfusion compared with other quadrants during three weeks post-ischemia, and even exceeded pre-ischemic quantity at 21 days post-ischemia. In conclusion, this in vivo imaging technique has significant potential utility for studying the spatio-temporal patterns of neovascularization in vivo.

The peripheral nervous system (PNS) is essential for performing and maintaining various motor and sensory functions. Abnormalities can lead to a series of peripheral neurological conditions, such as paraesthesia, pain, or spasms, which are debilitating and lowering the quality of life. The current guidelines for diagnosis rely predominantly on clinical symptoms resulting from PNS dysfunction, which occur already at an advanced stage. There are currently no effective methods that visually reflect the extent of peripheral neuropathy. In our study, we present a novel in vivo and in situ real-time imaging of peripheral nerves based on the second near-infrared window (NIR-II) fluorescence. In NIR-II system, PbS Qds with NIR-II fluorescence specifically bound to motor neuron-specific protein agrin, acting as image contrast. In mice model, peripheral nerves were visible as soon as after 2 h post injection. We provide evidence for the efficacy of this approach, which allows to directly demonstrate peripheral nerves, their structure, and potential damage sites and degree. Furthermore, our products were of good biocompatibility, while the neural fluorescence signal was solid, bright and stable for 4 h in vivo. Thus, overall, our results suggest that NIR-II is an effective new method for direct imaging of peripheral nerves in vivo, opening new horizons on early, improved and more precise, targeted diagnosis. A resulting more rapid installation of personalized therapy facilitates a better prognosis of clinical peripheral neuropathy.