Quantitative analysis by in vivo imaging of the dynamics of vascular and axonal networks in injured mouse spinal cord

Abstract

Understanding the endogenous repair capacity of spinal cord is pivotal to develop strategies to improve it. Here we design a paradigm of spinal cord lesion in the dorsal column using a 2-photon microscopy technique to dynamically and chronically monitor simultaneous changes of vascular and axonal networks in living mice up to 4 months postinjury. High-resolution images showed that early explorative sprouting of surviving injured axons resulted in extensive regrowth until and past the lesion site within 2 months. Blood vessel density was transiently up-regulated and most neurovascular interactions occurred within 2 weeks. Timelapse analysis showed that neovessels exerted a potent growth stimulating action, but no guidance effect on neighboring sprouts, possibly because of their geometry and plasticity. Nevertheless, if reconnection depends on axon sprout density, stimulation of angiogenesis would probably be beneficial to repair. More generally, this imaging approach is showing promise to aid in monitoring brain diseases and the efficacy of potential treatments.angiogenesis ͉ axon regeneration ͉ in vivo 2-photon imaging ͉ spinal cord injury ͉ time lapse S pinal cord injury (SCI) induces dramatic cellular and molecular changes in the neuronal environment resulting overall in impaired axonal regeneration (1-3). Posttraumatic disturbance of blood perfusion is, for example, responsible for secondary damage (3), and reports suggest that stimulation of posttraumatic angiogenesis with vascular endothelial growth factor (VEGF) improves functional recovery of injured rodents (4, 5). Optimizing treatments to target vascularization might then be a valuable therapeutic strategy in the context of SCI, but the finding that VEGF signals on axons in addition to blood vessels (6) challenges the importance of the vascular response in the recovery process.The conclusion of these studies is that the dynamics of the interactions occurring between axons and blood vessels and the influence of vascularization on axon regrowth have to be characterized thoroughly at the single axon level. Investigative tools are scarce, however, and poorly compatible with the determination of kinetics as most of the acquired knowledge comes from histological studies on spinal cord sections sampled from different subjects at different time points. A pioneering in vivo study combining wide-field imaging and the use of transgenic mice with fluorescent spinal cord axons reported observations of individual axons over 1 week after SCI (7). However, only a few axons were sampled per mouse and they were observed for a limited time. We sought to design a more dedicated imaging protocol on the basis of 2-photon microscopy (8, 9) to resolve populations of injured axons in 3 dimensions (3D) in the same mouse and to optimize the method to allow dynamic studies over a period of several months. Dual-color imaging allowed simultaneous visualization of posttraumatic vascular responses and for the first time neurovascular interactions were characterized in vivo at a cellul...

Astrocytic and Vascular Remodeling in the Injured Adult Rat Spinal Cord after Chondroitinase ABC Treatment

“…Further, we noticed that MAP1B-P-positive axons were often closely apposed to laminin-positive tubules (Supplementary data S4; see online supplementary material at ftp.liebertpub.com), suggesting a role of blood vessels in axonal growth, as previously shown by live imaging in adult rat. 51 Laminin antibodies also labeled hypertrophied blood vessels, mainly within the scar surrounding the injury site (Fig. 7A), as we had reported previously.…”

In-vivo single neuron axotomy triggers axon regeneration to restore synaptic density in specific cortical circuits

“…4b). In addition to regeneration from the cut ends we also looked for evidence of collateral sprouting, the appearance of de novo branch formation along the primary axon shaft, which is common in the spinal cord after corticospinal 29 and sensory neuron damage 30,31 . Collateral sprouting was not observed in our experiments, again indicating differences between axonal populations in different parts of the CNS in their response to injury.…”

Quantification of vascular perfusion in the spinal cord after injury.

“…This is an expected result since, at day 7, the injury epicenter is still experiencing a high level of angiogenesis; the body would not be working hard to rebuild the vascular bed if tissue were already well perfused at this time point (Loy, Crawford et al 2002;Dray, Rougon et al 2009). …”

“…Neuroregeneration and neurodegenerative diseases have been shown to correlate with microscopic angiogenic patterns so closely that the conceptual "neurovascular unit" is an increasingly popular target for research and intervention therapies (Dray, Rougon et al 2009). …”

“…Vascular damage is associated with numerous issues related to secondary injury and neuronal cell death (Awad, Ankeny et al 2010). Angiogenesis near the injury epicenter is closely correlated with repair and regrowth of neuronal tissue (Dray, Rougon et al 2009). In turn, population of the injury site with healthy cells is correlated with functional recovery (Urdzíková, Jendelová et al 2006).…”