Time-dependent contribution of non neuronal cells to BDNF production after ischemic stroke in rats

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Abstract

Although brain-derived neurotrophic factor (BDNF) plays a central role in recovery after cerebral ischemia, little is known about cells involved in BDNF production after stroke. The present study testes the hypothesis that neurons are not the unique source of neosynthesized BDNF after stroke and that non neuronal-BDNF producing cells differ according to the delay after stroke induction. For this purpose, cellular localization of BDNF and BDNF content of each hemisphere were analysed in parallel before and after (4 h, 24 h and 8 d) ischemic stroke in rats. Stroke of different severities was induced by embolization of the brain with variable number of calibrated microspheres allowing us to explore the association between BDNF production and neuronal death severity. The main results are that (a) unilateral stroke increased BDNF production in both hemispheres with a more intense and long-lasting effect in the lesioned hemisphere, (b) BDNF levels either of the lesioned or unlesioned hemispheres were not inversely correlated to neuronal death severity whatever the delay after stroke onset, (c) in the unlesioned hemisphere, stroke resulted in increased BDNF staining in neurons and ependymal cells (at 4 h and 24 h), (d) in the lesioned hemisphere, beside neurons and ependymal cells, microglial cells (at 24 h), endothelial cells of cerebral arterioles (at 4 h and 24 h) and astrocytes (at 8 d) exhibited a robust BDNF staining as well. Taken together, overall data suggest that non neuronal cells are able to produce substantial amount of BDNF after ischemic stroke and that more attention should be given to these cells in the design of strategies aimed at improving stroke recovery through BDNF-related mechanisms.

Research highlights

▶ Unilateral ischemic stroke increases BDNF content in both hemispheres ▶ In both hemispheres, BDNF content was not inversely correlated to the intensity of neuronal death ▶ Non neuronal cells such as ependymal, endothelial cells, microglia and astrocytes are essential contributor in post-stroke BDNF production ▶ Neuronal cells implicated in BDNF synthesis differ according to the delay after stroke.

Introduction

The brain derived neurotrophic factor (BDNF), a member of neurotrophin family protein, exerts strong survival and differentiation function during the development of the nervous system (Cohen-Cory et al., 2010). However, BDNF is still present in mature brain where it is stored and released from neurons in a use-dependent fashion and has been implicated in long term potentiation, learning and memory formation (Greenberg et al., 2009, Nagappan and Lu, 2005). BDNF also represents a crucial signalling molecule in adaptative brain plasticity after stroke (Cowansage et al., 2010, Lipsky and Marini, 2007, Mattson, 2008). Interventions that improve recovery of function are most often associated with increased BDNF levels in perilesional areas (Chen et al., 2005b, Kim et al., 2005, Vaynman et al., 2004). Conversely, attenuating BDNF levels or its effects following cerebral ischemia reduces neuroplastic changes or recovery of function either spontaneous or induced by rehabilitation (Chen et al., 2005a, Madinier et al., 2009, Ploughman et al., 2009). From these data, pharmacological strategies aimed at increasing post-ischemic cerebral BDNF production appear to be a promising option in the treatment of stroke. A first step in the design of such approaches is the characterization of BDNF-producing-cells according to the delay after stroke onset.

Despite the central role of BDNF in recovery after stroke, the proper effect of stroke on cerebral BDNF production has been surprisingly poorly investigated. Available studies support increased BDNF production after stroke as suggested by increased BDNF levels in areas collected either at the lesion site or around the lesion for at least one week following stroke (Kokaia et al., 1998, Madinier et al., 2009, Sulejczak et al., 2007). Because increased levels of BDNF mRNA were observed in degenerating and surviving neurons in the acute post-stroke period (Comelli et al., 1992, Kokaia et al., 1995, Rickhag et al., 2007, Zhao et al., 2000), it has been generally accepted that neurons are the predominant source of neosynthesized BDNF in the ischemic brain. However, the contribution of non neuronal cells is suspected from in vitro studies that report the ability of microglial cells, cerebral endothelial cells and astrocytes to express and secrete BDNF when exposed to conditions mimicking ischemia (Bayas et al., 2002, Jean et al., 2008, Lai and Todd, 2008, Miklic et al., 2004, Saha et al., 2006). In vivo, data reported BDNF expression by non neuronal cells such as astrocytes (Sato et al., 2009, Uchida et al., 2010) or microglia (Batchelor et al., 1999, Madinier et al., 2009, Nagamoto-Combs et al., 2007) but, to the best of our knowledge, studies specifically designed to assess their exact contribution to BDNF production are lacking.

In this context, the objective of our study was to investigate the contribution of neuronal versus non neuronal cells in stroke-induced BDNF production with respect of the delay after stroke onset. For this purpose, identification of BDNF-reactive cerebral cells was coupled to the measurement of BDNF levels in the whole hemispheres before and after (4 h, 24 h and 8 d) ischemic stroke in rats. Stroke was performed by means of intracarotid injection of calibrated microspheres (Bralet et al., 1979), a model which allows the modulation of neuronal death severity by varying the amount of injected microspheres (Demougeot et al., 2001). Thus, using this specific model of ischemia, if neurons are the predominant cellular source of BDNF, BDNF content of the lesioned hemisphere, which include both infarcted and surrounding non infarcted areas, should be inversely correlated to the degree of embolization. A first set of experiments was designed to investigate the relationship between individual hemispheric BDNF levels and degree of embolization, and the impact of embolization on hemispheric BDNF production. In a second set of experiments, cellular BDNF localization was examined before and after embolization. Then, based on the simultaneous analysis of changes in cells expressing BDNF and hemispheric BDNF levels, the time-dependent contribution of non neuronal versus neuronal cells to stroke-induced BDNF production was deduced.

Section snippets

Materials

Acrylamide and bis-acrylamide, sodium dodecylsulfate (SDS) and tween-20 were obtained from Bio-Rad (Ivry sur Seine, France). The polyvinylidene difluoride (PVDF) membranes were purchased from GE Healthcare (Orsay, France). All other reagents were purchased from Sigma–Aldrich except where otherwise noted. The rabbit polyclonal antibody raised against BDNF (AB1779SP) and the mouse monoclonal antibody recognizing NeuN (MAB377) were purchased from Chemicon (Molsheim, France). The mouse monoclonal

Relationship between the individual BDNF levels and degree of embolization

Fig. 2 shows the relationship between BDNF levels and the degree of embolization. For both hemispheres, no relationship was observed between the two parameters in groups E 4 h (Fig. 2A) and E 24 h (Fig. 2B). In contrast, a positive correlation was observed between BDNF levels of the lesioned hemisphere and the degree of embolization after 8 d of ischemia (group E 8 d, tau = 0.556, P = 0.0187, Fig. 2C). Thus, the BDNF content of either the lesioned or the unlesioned hemisphere did not inversely

Discussion

Using an ischemic stroke model that offers the unique opportunity to assess the relationship between cerebral BDNF production and neuronal death severity, our study provides evidence that both neurons and non neuronal cells contribute to increase BDNF production after stroke and that the implicated-non-neuronal-cells differ according to the delay after stroke onset.

Increases in BDNF levels have been reported in pieces of tissue collected at the infarct or peri-infarct level (Kokaia et al., 1998

Acknowledgments

We thank Dr. Christine Arnould for her technical assistance at the microscopy center, INRA, Dijon. This work was financially supported by Burgundy and the Faculty of Medicine of Dijon.

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