Postconditioning represents a translationally exciting and relevant approach for cerebrovascular disease. The neuroprotection induced by early reperfusion interruption is probably associated with changes in cerebral blood flow. Subsequent events as free radical production, BBB integrity, inflammation and endothelial function have also been shown to be involved (Gao et al., 2008; Zhao, 2011; Zhao et al., 2012; Joo et al., 2013). The majority of experimental studies have focused on the acute mechanisms of ischemic postconditioning. The effect of ischemic postconditioning in brain for longer periods post-stroke remains to be fully elucidated. In a previous study we suggested that the therapeutic effects of postconditioning may involve the promotion of neurogenesis and angiogenic remodeling during the recovery phase after focal cerebral ischemia (Esposito et al., 2015). However, mechanisms for long-term outcomes and delayed stroke recovery remain unclear.

After a National Institutes of Health workshop in 2001, where the concept of neurovascular unit (NVU) was first proposed (Lo et al., 2003), CNS injury and disease are now well known to be the result from the dysfunction and death not only of neurons but of signaling among different cell types. Together these cell types, including astrocytes, pericytes, smooth muscle cells, endothelial cells, oligodendrocytes, microglia and neural and glial precursor cells, make up the neurovascular unit. Astrocytes comprise the major of non-neuronal cell population in the mammalian neurovascular unit and can produce many factors for protecting and restoring neurons (Tiedt et al., 2022).

Interestingly, astrocytes normally express BDNF, although at lesser levels than neurons (Saha et al., 2006; Giralt et al., 2010; Fulmer et al., 2014; Hong et al., 2016; de Pins et al., 2019). BDNF is known to increase with oxidative stress occurring during ischemia–reperfusion (I/R) injury (Qin et al., 2022) as part of an antioxidant defense and it has been previously showed that postconditioning stimulus can increase BDNF (Wu et al., 2015). Moreover, BDNF can stimulate neurogenesis (Schäbitz et al., 2007) and angiogenesis (Kermani and Hempstead, 2007).

Our current study tests the hypothesis that postconditioning affects astrocyte signaling via BDNF in order to promote neurovascular remodeling. Our results support this hypothesis by showing that after using fluorocitrate, an astrocyte inhibitor, the postconditioning neuroprotective effect together with the postconditioning dependent new angiogenesis and neurogenesis, were no longer observed.

Interactions among endothelial cells, glia, neurons and matrix (i.e., the neurovascular unit, NVU) underlie the acute pathophysiology of stroke (Lo and Rosenberg, 2009; Arai et al., 2011; Xing et al., 2012). It is likely that long-term effects of postconditioning are also mediated by cell–cell interactions in the NVU.

In the present study we investigated some of the underlying mechanisms consistent with improvements in poststroke neurogenesis and angiogenesis. Our hypothesis is that ischemic postconditioning can, in part, promote recovery by increasing the capability of the brain to “help itself” via astrocytic production of BDNF.

In a previous study we suggested that the beneficial effects of ischemic postconditioning can be maintained up to 2 weeks post ischemia, in part by promoting neurogenesis and angiogenesis (Esposito et al., 2015). For neural remodeling, astrocytes play an important role by releasing neurotrophic factors such as BDNF. For vascular remodeling, astrocytes may be an important source of pro-angiogenic molecules such as MMP9. Is it possible that ischemic postconditioning improves recovery by augmenting these endogenous processes? Our current data test the hypothesis that post-conditioning affects astrocyte signaling via BDNF in order to promote neurovascular remodeling. Our western blot data show changes in BDNF and MMP9 protein expression levels consistent with the neuroprotective, angiogenic and neurogenetic effect of postconditioning. We also measured VEGF, since it is one of the major factors involved in angiogenesis (Zhang et al., 2000), however, no difference in protein expression was observed after postconditioning. MMP2, together with MMP9, has been shown to be crucial for the “angiogenic switch” that occurs upon the initiation of neurogenesis (Akol et al., 2022), again no change in protein level was observed.

Collectively the results of the present paper suggest that during the recovery phase, postconditioning upregulates the ability of astrocytes to produce BDNF. BDNF, which is a member of the neurotrophin family, is involved in neuroprotection, neurogenesis, neuroplasticity and angiogenesis (Béjot et al., 2011; Grade et al., 2013) and during ischemic preconditioning, BDNF expression is stimulated (Truettner et al., 2002). Our data show that postconditioning also activates this BDNF pathway. Importantly, we also observed MMP9 increase, MMP9 and BDNF responses are related. BDNF can upregulate MMP9 production in rat primary cortical neurons (Kuzniewska et al., 2013), and MMP9 can cleave pro-BDNF converting it to mature BDNF (Mizoguchi et al., 2011). Thus, these coordinated mechanisms may allow postconditioning to amplify and integrate neurogenesis (Jiang et al., 2001; Parent et al., 2002; Ming and Song, 2005) and angiogenesis (Rosell et al., 2013).

While reactive astrocytes were traditionally thought as detrimental, it is now accepted that they can be beneficial under some conditions (Hayakawa et al., 2010; Qu et al., 2010; Barreto et al., 2011). Our data suggest that astrocytes may secrete BDNF and MMP9, which promote neuron survival, synaptic plasticity and angiogenic remodeling during stroke recovery. Indeed, not only the expression of BDNF seems specifically higher in astrocytes, by immunostaining analysis, but by using an astrocyte inhibitor we observed that the neuroprotective effect together with angiogenesis and neurogenesis were partially prevented.

Hence, the ability of postconditioning to amplify these endogenous mechanisms in the neurovascular unit should lead to novel ways to promote stroke recovery.

Nevertheless, there are several issues that should require further investigation. (1) What about other astrocyte factors? Our data point only to BDNF and MMP9, however, other factors such as FGF, HMGB1 and SDF-1 might be released by astrocytes and contribute to the neuroprotective mechanism induced by postconditioning. (2) How can we be sure benefits will disappear after even longer times? Indeed, overall infarcts tend to be smaller over time as brain remodels and shrinks. However previous studies have already showed that ischemic postconditioning neuroprotection lasts up to 1 month (Ren et al., 2008). Although in the present paper, postconditioning was found to be protective at 3 days and 2 weeks, studies exploring longer effect are needed. (3) What about other types of glia? We analyzed the interaction between astrocytes and neurons after postconditioning. Certainly, after stroke, dysfunctions are manifested at the level of cell–cell signaling between neuronal, glial and vascular elements (Arai and Lo, 2010; Ji et al., 2013). Probably a vascular or endothelial event due to reperfusion is also involved. We believe that all the NVU components may ultimately be involved in the benefits of postconditioning, although our data suggest that astrocytes may comprise a key rate-limiting step.

From a clinical perspective, ischemic postconditioning may be attractive as it can be translatable (Vinciguerra et al., 2018), especially for patients subjected to surgery and to endovascular therapy associated with blood vessel occlusion and revascularization. However, most studies so far are focused at 24 h. Additional investigation of postconditioning effects in a chronic phase after stroke may be desirable. Our work aims to define some of the molecular mechanisms responsible of postconditioning neuroprotection during the recovery phase after stroke.

The original contributions presented in the study are included in the article/Supplementary material, further inquiries can be directed to the corresponding authors.

The animal study was approved by Massachusetts General Hospital Institutional Animal Care and Use Committee in accordance with the National Institute of Health Guide for the Care and Use of Laboratory Animals. The study was conducted in accordance with the local legislation and institutional requirements.

EE: Conceptualization, Data curation, Writing – original draft. EL: Methodology, Writing – review & editing. OC: Conceptualization, Writing – review & editing. EHL: Conceptualization, Supervision, Writing – review & editing. KH: Supervision, Writing – review & editing. GP: Conceptualization, Writing – review & editing.

The author(s) declare financial support was received for the research, authorship, and/or publication of this article. This work was supported in part by grants from the National Institutes of Health, and the Rappaport Foundation.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision.

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