The present study established a transient rat MCAO model receiving ADSC treatment and attempted to explore whether netrin-1 and DCC are involved in the neuroprotection of the stem cell-based therapy. The results showed that intra-arterially transplanted ADSCs effectively migrated toward the peri-infarct area and improved the neurological functions of MCAO rats. We also show that ADSC transplantation promoted the regeneration of neuronal fibers and blood vessels in the peri-infarct cortex, and that ADSC transplantation increased the level of netrin-1 and DCC protein in the peri-infarct cortex which were mainly expressed by neuronal perikaryal and fibers, respectively. These findings provide new clues about how ADSC transplantation promotes neurological recovery after stroke.

The available evidence from experimental animal models confirms that ADSC therapy can substantially improve neurological recovery after stroke [7, 10, 11, 34] indicating that this therapeutic scheme is a promising candidate for clinical application to stroke patients. Two important aspects need to be considered: the transplantation route of ADSCs and the optimal time point for ADSC therapy in acute ischemic stroke. As described in previous studies, a number of delivery routes for stem cells, including intra-arterial, intravenous, intraventricular, intracerebral, and intrastriatal, have been documented in experimental stroke models [4]. Du et al. [11] compared the therapeutic effects of the three most commonly used transplantation routes of ADSCs (intra-arterial, intravenous, and intraventricular) and found that, compared with intraventricular transplantation, intra-arterial or intravenous transplantation allowed higher dose injections with fewer invasions and appeared to be optimal in terms of therapeutic efficacy, safety, and feasibility [11]. Otero-Ortega et al. found that, after intravenous transplantation of ADSCs, migration and implantation of ADSCs were observed not in the brain, but in the liver, lung, and spleen, suggesting that most cells may be captured by peripheral organs with this delivery route [35].

Although ADSC therapy has a wider therapeutic window, which ranges from 30 min up to 14 days after stroke, early administration of ADSCs can achieve an efficient result for neuronal protection and repair [4]. Many studies have reported satisfactory results when the ADSCs were administered at 24 h after the induction of ischemic stroke [11, 34, 36]. Moreover, intra-arterial transplantation of ADSCs at 24 h after stroke showed the highest engraftment rate in the damaged brain of stroke rats [31]. Accordingly, the current study adopted the intra-arterial transplantation of ADSCs to rats at 24 h after MCAO. Consistent with previous studies, we demonstrated that ADSC transplantation significantly improved the neurological recovery of MCAO rats. We also provide evidence that ADSCs can effectively migrate toward the peri-infarct area via intra-arterial transplantation. Nevertheless, ADSCs were minimally detectable in the infarct core and other brain areas, which is probably due to the adverse environmental conditions of the infarct core where ADSCs are incapable of surviving, and the intact blood–brain barrier of other brain areas which makes it difficult for ADSCs to cross. We also noticed that, compared with the condition at day 7, ADSCs did not show a significant migration in the brain at day 14. This observation may be attributed to the following two factors: the rich chemokines in the peri-infarct area during the acute ischemic phase, which facilitates the recruitment of stem cells to the injury site [37], and the insufficient observation time in the current study. It has been reported that ADSCs are distributed on both hemispheres of the brain 8 weeks after intracerebral transplantation which indicates their robust in vivo migration [7]. More recently, the advent of molecular imaging techniques provides new and better means for noninvasive, repeated, and quantitative tracking of stem cells after transplantation, which is very useful in detecting, localizing, and examining the stem cells in vivo at both molecular and cellular levels [38, 39]. Therefore, molecular imaging techniques will help us better understand the behavior of stem cells and remedy the deficiency of this study.

Previous studies have demonstrated the beneficial effects of stem cell therapy on improving functional outcome after stroke through mechanisms implicated in brain plasticity, such as axonal sprouting, synaptic plasticity, remyelination, angiogenesis, and so on [4, 40]. In this study, we show that ADSC transplantation accelerated the regeneration of neuronal fibers and blood vessels in the peri-infarct cortex. It is generally believed that these plastic processes are stimulated by tropic factors (including brain-derived neurotrophic factor, vascular endothelial growth factor, hepatocyte growth factor, etc.) and other proteins (including synaptophysin, oligodendrocyte 2, microtubule associated protein 2, etc.) which are released or regulated by the ADSCs [4, 5, 9, 41], but the underlying molecular mechanisms remain only partially understood. In view of the facts that netrin-1 and DCC have also been found to be involved in these plastic processes [23], and that the overexpression of netrin-1 improves the functional recovery and reduces the infarct size in animal stroke models [42, 43], we measured the temporal and spatial expression patterns of netrin-1 and DCC in rat brains after ADSC transplantation. Western blot analysis revealed that the expression of netrin-1 and DCC increased in the peri-infarct cortex at days 7 and 14 after MCAO, which is consistent with results of previous studies [25]. We found that ADSC transplantation further enhanced the increased expression of netrin-1 and DCC at the same time point. Futhermore, immunofluorescence staining confirmed strong immunoreactivity for both netrin-1 and DCC in the peri-infarct cortex, but weak immunoreactivity in the contralateral cerebral cortex and no immunoreactivity in the infarct core, which bear great similarities to the locations of transplanted ADSCs in the rat brain. As the brain region adjacent to the infarct core, i.e., the peri-infarct area, is critical for the neurological rehabilitation due to its heightened neuroplasticity [44], the upregulation of netrin-1 and DCC in the peri-infarct cortex may reflect their potential role in the functional recovery induced by ADSC transplantation, discussed in greater detail below.

As we know, axonal injury and degeneration are prominent components resulting in reduced neuronal connectivity after acute ischemic stroke, which is largely responsible for the consequent impairment of neurological function [45]. Axonal regeneration and reorganization among surviving neurons take place simultaneously after stroke, trying to repair and re-establish lost connections, but this process is exceedingly difficult due to the limited neuronal intrinsic potential for axonal sprouting and regeneration in the central nervous system [45, 46]. Fortunately, netrin-1 and its receptor DCC have been well documented to not only enhance the capability of axons to sprout and regenerate, but also facilitate the generation of appropriate neural circuits by navigating these axons toward their ultimate targets, where synapses are formed [15, 21]. Moreover, netrin-1 has been found to affect dendritic outgrowth and targeting, and regulate synaptic function and plasticity in the adult mammalian brain via a DCC-dependent mechanism [47, 48]. In this study, the enhanced expression of netrin-1 and DCC was detected in the peri-ischemic cortex, and immunofluorescence staining showed that netrin-1 was expressed mainly in the neuronal perikarya while DCC was expressed extensively in neuronal fibers of this area. These findings suggest that netrin-1 may be secreted by neurons and then triggers axonal regeneration and reorganization to restore neuronal circuits after the cerebral ischemia by binding to its receptor DCC expressed in neuronal fibers. Furthermore, ADSC transplantation may promote this neuronal network remodeling by upregulating their expression.

As the cross-talk between nervous and vascular systems has been documented to occur at the molecular level during brain development and injury, both systems not only are similar in their anatomical form and structure but also follow the same paths [49]. In view of this, studies have investigated the role of netrin-1 in the vascular system and reported that netrin-1 promotes both developmental and therapeutic angiogenesis due to its ability to regulate the proliferation, differentiation, migration, and survival of endothelial cells [5052]. Fan et al. demonstrated that netrin-1 induced blood vessel formation in the brain of adult mice and ameliorated cerebrovascular development and remodeling [52]. Other studies have reported that netrin-1 accelerated revascularization and reperfusion in mice suffering from hindlimb ischemia [20, 53], and that netrin-1 augmented the angiogenesis of mesenchymal stem cells and improved the function of the ischemic hindlimb [51]. Also, netrin-1 is found to be superior to vascular endothelial growth factor in restoring nerve conduction velocity, possibly due to its strong biological effects on both the nervous and vascular system [20]. Thus, the beneficial effects of ADSC transplantation on promoting angiogenesis after stroke may involve netrin-1 in the repair and re-establishment of the vascular network in the peri-infarct cortex. In this study, we did not detect DCC immunoreactivity in blood vessels, suggesting that the proangiogenic effect of netrin-1 in the brain might act through other receptors; this awaits further study. Nevertheless, it is noteworthy that obvious DCC immunoreactivity was observed to wrap around the blood vessels, which can be attributed to the end-feet of the perivascular astrocytes. Perivascular astrocytes have been found to engage in promoting brain neovascularization, vessel differentiation, and stabilization [52, 54], suggesting that angiogenesis induced by netrin-1 may be mediated partly through the DCC-expressing perivascular astrocytes. Moreover, as perivascular astrocytes have a supportive role in the maintenance of the brain–blood barrier [55], and netrin-1 has been found to support blood–brain barrier integrity and protect the central nervous system against injury [56, 57], we speculate that netrin-1 and DCC may also play a positive role in the restoration of the brain–blood barrier after cerebral ischemia, which also needs further clarification.