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Long-term effects of pre and post-ischemic exercise following global cerebral ischemia on astrocyte and microglia functions in hippocampus from Wistar rats
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Research Report
Long-term effects of pre and post-ischemic exercise following global cerebral ischemia on astrocyte and microglia functions in hippocampus from Wistar rats Gisele Agustini Lovatela,d, Karine Bertoldib,d, Viviane Rostirol Elsnerbb,d, Francele Valente Piazzac,d, Carla Giovana Bassob,d, Felipe dos Santos Moyse´sb,d, Paulo Valdeci Wormb,d, Carlos Alexandre Nettob,d, Simone Marcuzzoc,d, Ionara Rodrigues Siqueirab,d,n a
Universidade Federal de Santa Catarina, Curso de Fisioterapia, Rua Pedro João Pereira, 150 Mato Alto, EP 88900-000 Araranguá, SC, Brazil b Universidade Federal do Rio Grande do Sul, Programa de Pós-Graduação em Ciências Biológicas: Fisiologia, Instituto de Ciências Básicas da Saúde, Instituto de Ciências Básicas da Saúde, Rua Sarmento Leite, 500, CEP 90050-170 Porto Alegre, RS, Brazil c Universidade Federal do Rio Grande do Sul, Programa de Pós-Graduação em Neurociências, Instituto de Ciências Básicas da Saúde, Instituto de Ciências Básicas da Saúde, Rua Sarmento Leite, 500, CEP 90050-170 Porto Alegre, RS, Brazil d Universidade Federal do Rio Grande do Sul, Departamento de Farmacologia, Instituto de Ciências Básicas da Saúde, Rua Sarmento Leite, 500, CEP 90050-170 Porto Alegre, RS, Brazil
art i cle i nfo
ab st rac t
Article history:
Persistent effects of pre- and postischemic exercise on glial cells activation after global
Accepted 25 August 2014
cerebral ischemia remains poorly understood. Here, we investigated the effect of both pre and postischemic treadmill exercise protocols (20 min/day during 2 weeks) on glial cells
Keywords:
immunostaining in the hippocampus of Wistar rats submitted to global ischemia. A
Treadmill exercise
synergistic effect between ischemia and postischemic exercise on the astrocytic area was
Pre-ischemic exercise
demonstrated. Postischemic exercise partially reversed the ischemia-induced increase on
Postischemic exercise
the area occupied by microglia, without any effect of pre-ischemic protocol. In conclusion,
Astrocyte
postischemic exercise distinctly modulates astrocyte and microglia immunostaining in the
Microglia
hippocampal dentate gyrus following global cerebral ischemia in Wistar rats.
Global ischemia
& 2014 Published by Elsevier B.V.
n Correspondence to: Laboratório de Neuropsicofarmacologia, Departamento de Farmacologia, Instituto de Ciências Básicas da Saúde, UFRGS. Rua Sarmento Leite, 500, CEP 90050-170 Porto Alegre, RS, Brazil. Fax:þ55 51 3308 3121. E-mail address: [email protected] (I.R. Siqueira).
http://dx.doi.org/10.1016/j.brainres.2014.08.068 0006-8993/& 2014 Published by Elsevier B.V.
Please cite this article as: Lovatel, G.A., et al., Long-term effects of pre and post-ischemic exercise following global cerebral ischemia on astrocyte and microglia functions in hippocampus from Wistar rats. Brain Research (2014), http://dx.doi.org/10.1016/ j.brainres.2014.08.068
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1.
Introduction
Epidemiological studies have shown neuroprotective effects of moderate physical activity (Yamada et al., 2012; Freiberger et al., 2012). In this context, several promising findings obtained in animal models of cerebral ischemia have been demonstrated. Pre-ischemic treadmill exercise is able to reduce infarct volume and inhibit inflammatory injury induced by the focal ischemia model (Guo et al., 2008; Ding et al., 2006; Jia et al., 2009). Indeed, pre-ischemic voluntary exercise in running wheel reduced neuronal damage induced by global cerebral ischemia (Stummer et al., 1994). It is interesting to note that postischemic treadmill exercise was able to diminish the global ischemia-induced apoptotic neuronal cell death (Sim et al., 2004; Lee et al., 2003). Besides, the traumatic brain injury-induced impairment of recognition memory, as well as the neuronal loss and activation of microglia in mice, was reversed by the postinjury exercise protocol (Chen et al., 2013). In addition, physical training improved perinatal hypoxic-ischemic encephalopathy-induced brain damage and memory impairments in Sprague-Dawley rats (Chen and Jiang, 2010). To our knowledge, there are no studies comparing the effects of pre- and postischemic exercise on cerebral ischemia outcomes. The mechanisms by which physical activity alters brain function are not yet defined, however it has been suggested that exercise might activate cellular and molecular pathways that contribute to neuroprotection. A growing body of data demonstrates that glial cells play a multifaceted and complex role in ischemia outcomes; astrocyte and microglia can have both harmful and supportive effects on cell proliferation and survival (Ekdahl et al., 2009). Astrogliosis is recognized as a cellular response following a wide variety of insults to the CNS, such as global ischemia (Valentim et al., 1999; Petito and Halaby, 1993), however, it is important to highlight that physical exercise itself can induce this phenomenon in different brain areas, including cortex, striatum and hippocampus (Li et al., 2005; Uda et al., 2006; Saur et al., 2014). Interestingly, Lee et al. (2009) demonstrated that high intensity postischemic exercise reduced the extent of reactive astrocytosis in the peri-infarct region induced by focal ischemia, without any effect of mild and moderate intensities. Global ischemia also triggers microglial outcomes, which was studied specifically on postischemic days 4–7 in the dentate
gyrus and CA1 subfield of the hippocampus in rats submitted to global ischemia (Marioka et al., 1991; Gehrmann et al., 1992); Q2 however, to our knowledge there are no studies evaluating persistent effects of global ischemia. Besides, divergent results about the influence of exercise per se on microglia function have been found. Voluntary exercise increased microglia proliferation in mice hippocampi with access to a free running wheel (Olah et al., 2009), while Kohman et al. (2012) found that 8 weeks wheel running exercise reduces the microglia proliferation in aged. Although there are no studies, to our knowledge, reporting the exercise effect on microglia function in global ischemia. Considering that reports describing persistent effects of exercise and/or ischemia as well as comparing pre- and postischemic exercise protocols were rarely performed. The aim of the present study was to compare the effect at long term of pre- and postischemic treadmill exercise protocols (20 min/day during 2 weeks) on glial cells immunostaining in the hippocampus of Wistar rats submitted to global ischemia.
2.
Results
Persistent effects of global cerebral ischemia and exercise on astrocyte and microglia functions in the hippocampal dentate gyrus were evaluated. We evaluated the long term effects of transient global cerebral ischemia in Wistar rats using the four-vessel occlusion, besides it was compared the impact of pre- and postischemic exercise protocols (Fig. 1). The GFAP immunostaining in the dentate gyrus is illustrated in Fig. 2. Two-way ANOVA revealed a significant effect of both factors, ischemia and exercise, on the quantification of the total percentage area (%) occupied by GFAPþ soma and astrocytic processes (F(1,29)¼ 297.73; po0.001; F(2,29)¼29.16; po0.001, respectively). The ischemic insult increased % of GFAP area in dentate gyrus subfield compared to sham groups. Two-way ANOVA revealed an effect of ischemia factor on GFAPþ soma and astrocytic processes in the stratum radiatum of hippocampus (F(1,29)¼39.72; po0.001; Fig. 3). Furthermore, this exercise protocol per se increased the area occupied by GFAPþ cells in the dentate gyrus (Fig. 2), while both exercise protocols did not affect this parameter in the stratum radiatum of the hippocampus (Fig. 3). Interestingly, there was a significant interaction between ischemia
Fig. 1 – Schematic representation of the experimental design. Please cite this article as: Lovatel, G.A., et al., Long-term effects of pre and post-ischemic exercise following global cerebral ischemia on astrocyte and microglia functions in hippocampus from Wistar rats. Brain Research (2014), http://dx.doi.org/10.1016/ j.brainres.2014.08.068
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Fig. 2 – Effects of cerebral ischemia and exercise on quantification of GFAPþ astrocyte in the dentate gyrus of the hippocampus. (A) Digital images of coronal sections of the dentate gyrus stained for GFAP immunofluorescence. (B) % of area occupied by astrocytes in the dentate gyrus in different groups. Columns represent mean 7S.D. (n ¼ 4–5). Two-way ANOVA followed by Duncan test; # significantly different from the sham groups; n significantly different from the sedentary ischemic and pre-ischemic exercise. Scale bar: 50 μm.
and exercise factors (F(2,29)¼7.57; p¼ 0.003). The Duncan post hoc test showed a significant increase in the % area occupied by astrocytes in the post-ischemic exercise group when compared to all groups. Two-way ANOVA showed the effect of both factors, ischemia and exercise, on the quantification of the total percentage area (%) occupied by Iba-1þ soma and microglial processes in the dentate gyrus (respectively, F(1,27)¼ 273.09; po0.001; F(2,27)¼8.27; p¼ 0.002, Fig. 4). Rats submitted to ischemia showed higher % area occupied by microglia when compared to sham groups. Moreover, there was a significant interaction between ischemia and exercise factors (F(2,27)¼ 23.03; po0.001). Duncan post hoc test revealed a significant decrease in the % area occupied by microglia in the post-
ischemic exercise groups when compared to other ischemic groups (Fig. 4).
3.
Discussion
The present study demonstrated that postischemic exercise may modulate glial functions, specifically can increase GFAP expression and decrease Iba-1 staining from rats submitted to global ischemia, without any effect of pre-ischemic protocol. An important finding that emerged from this study is that there was a synergistic effect between ischemia and exercise on the area occupied by astrocytes.
Please cite this article as: Lovatel, G.A., et al., Long-term effects of pre and post-ischemic exercise following global cerebral ischemia on astrocyte and microglia functions in hippocampus from Wistar rats. Brain Research (2014), http://dx.doi.org/10.1016/ j.brainres.2014.08.068
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Fig. 3 – Effects of cerebral ischemia and exercise on quantification of GFAPþ astrocyte in the stratum radiatum (SR) of the hippocampus. (A) Digital images of coronal sections of the SR stained for GFAP immunofluorescence. (B) % of area occupied by astrocytes in the SR in different groups. Columns represent mean7S.D. (n ¼4–5). Two-way ANOVA revealed an effect of ischemia factor; # significantly different from the sham groups. Scale bar: 50 μm.
Although some reports are in disagreement with our findings, since treadmill exercise suppressed formation of reactive astrocytes in other CNS insult models, such as Parkinson's disease (Dutra et al., 2012) and forebrain traumatic brain injuries (Seo et al., 2010), we infer that the robust increase in the % area occupied by astrocytes observed in the exercised ischemic rats may play a role in providing a favorable microenvironment, or even necessary for neuronal growth and restructuring after CNS insult. This possibility is supported by a previous study that rehabilitation training (complex environment housing) produces enhanced functional recovery, dendritic growth, synaptogenesis, and an increased number of synapses associate with astrocytic activation in the hippocampus after cerebral ischemia (Biernaskie and Corbett, 2001; Briones et al., 2006). Indeed, the involvement of astrocytes in angiogenesis has been recognized, contributing to induce a
favorable state to neurovascular unit (consisting of microvascular endothelium, astroglia, neurons and the extracellular matrix), what would protect the blood-brain-barrier function following ischemic damage. In this context, Huang et al. (2013) suggested that exercise-induced cognitive improvement of middle-aged rats might be associated with an increase on the capillaries density in the rat cortex. The exercise-induced astrogliosis here observed in the sham group is supported by previous studies which demonstrated an increase in astrocytic activation and proliferation of GFAP positive cells, in the subgranular zone of the hippocampus, in healthy exercised rats (Uda et al., 2006; Li et al., 2005; Saur et al., 2014). Besides, our findings are in accordance with Valentim et al. (1999) that observed an early and late increase in both immunocontent and GFAP phosphorylation rate in the hippocampus from ischemic rats,
Please cite this article as: Lovatel, G.A., et al., Long-term effects of pre and post-ischemic exercise following global cerebral ischemia on astrocyte and microglia functions in hippocampus from Wistar rats. Brain Research (2014), http://dx.doi.org/10.1016/ j.brainres.2014.08.068
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Fig. 4 – Effects of cerebral ischemia and exercise on quantification of Iba-1þ microglia in the dentate gyrus of the hippocampus. (A) Digital images of coronal sections of the dentate gyrus stained for Iba-1 immunofluorescence. (B) % of area occupied by microglia in the dentate gyrus in different groups. Columns represent mean7S.D. (n¼ 4–5). Two-way ANOVA followed by Duncan test; # significantly different from the sham groups; n significantly different from the sedentary ischemic and pre-ischemic exercise. Scale bar: 50 μm.
pointing to a long-term glial phenomenon. Moreover, Petito and Halaby (1993) demonstrated a persistent increase in GFAP immunocontent until five weeks in the CA1 area after ischemic insult. An early increase of Iba-1 expression was previously described in the dentate gyrus and CA1 of ischemic rats (Morioka et al., 1991; Gehrmann et al., 1992), and in our study an increase in the % area occupied by Iba-1 was observed 5 weeks after the ischemic event, pointing to a long-term microglial phenomenon. Interestingly, postischemic exercise reversed partially the microglia activation. To our knowledge, we provide here, for the first time, evidence of a long term effect of ischemic insult as well as exercise modulation on microglia activation after global cerebral ischemia. This effect
can be related to previous described neuroprotective properties, since some studies suggest a detrimental role of the microglial action by producing neurotoxic molecules and proinflammatory cytokines (Wang et al., 2007; Kaushal and Schlichter, 2008). Recently, it was described that the exercise decreased pro-inflammatory markers in the hippocampus from aged rats without any effect in young rats (Lovatel et al., 2013). Indeed, the exercise-induced reduction of the Iba-1 staining here observed could be related to decline on proinflammatory cytokines observed in aged rats (Lovatel et al., 2012), since these cytokines are frequently produced by activated microglia (Hetier et al., 1988; Giulian et al., 1986). Taken together, we can suppose that exercise is able to modulate the pro-inflammatory state against injury conditions.
Please cite this article as: Lovatel, G.A., et al., Long-term effects of pre and post-ischemic exercise following global cerebral ischemia on astrocyte and microglia functions in hippocampus from Wistar rats. Brain Research (2014), http://dx.doi.org/10.1016/ j.brainres.2014.08.068
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Summarizing, our results support the hypothesis that the exercise modulates glial cells functions; specifically postischemic exercise induced an increase of astrocytes imunostaining associated to a decrease in microglia function in the dentate gyrus from rats following global cerebral ischemia.
taken as VO2 max (Arida et al., 1999; Scopel et al., 2006). The sedentary group was handled exactly as the experimental animals and was left on the treadmill for 5 min without any stimulus to run. Neither electric shock nor physical prodding was used in this study and all the procedures took place between 14:00 and 17:00 h.
4.
Experimental procedure
4.4.
4.1.
Animals
In order to evaluate long term effects of ischemia and exercise, the animals were perfused at 38 days after ischemic insult (Fig. 1). Animals were anesthetized with ketamine (90 mg/kg i.p.) and xylazine (10 mg/kg i.p). Heparin (Cristalia, Brazil) was injected into the cardiac ventricle, and the animals were transcardially perfused (Milan, Brazil) with a saline solution, followed by a fixative solution 4% paraformaldehyde (Synth, Brazil). The brains were dissected, postfixed and cryoprotected by immersion in a sucrose solution (Synth, Brazil). The brains were frozen in isopentane (Merck, Germany) cooled in liquid nitrogen and kept in a freezer ( 70 1C) for further analyses. Coronal sections (40 μm) of the dorsal hippocampus were obtained using a cryostat (Leica, Germany) and serially collected on gelatin coated slides. This area corresponded to a distance of approximately 2.30– 4.52 mm posterior to the bregma (Paxinos and Watson, 1982). Every sixth section (240 μm apart) was processed for each immunostaining (Malberg et al., 2000; Veena et al., 2009; Piazza et al., 2014).
Three months-old male Wistar rats were used. Animals were provided by the Centro de Reprodução de Animais de Laboratório (CREAL) from the Universidade Federal do Rio Grande do Sul (UFRGS) and were maintained under standard conditions (12-h light/dark, 2272 1C) with food and water ad libitum at Unidade de Experimentação Animal do Hospital de Clínicas de Porto Alegre (UEA/HCPA). The NIH “Guide for the Care and Use of Laboratory Animals” (NIH publication no. 80-23, revised 1996) was followed in all experiments. The Local Ethics Committee (Comissão de Ética no Uso de Animais – CEUA/HCPA, no. 09-630) approved all handling and experimental conditions.
4.2.
Surgery
Transient global cerebral ischemia was induced in Wistar rats the four-vessel occlusion method described by Pulsinelli et al. (1982), with minor modifications (Netto et al., 1993). The vertebral arteries were permanently electrocoagulated under halothane anesthesia, and silastic ties were placed around each common carotid artery without the interruption of blood flow (Fig. 1). On the next day, the ties were tightened and clamped for 10 min. Sham animals were operated, but the ties were not tightened. The body temperature was maintained at 3770.5 1C during ischemia and reperfusion by means of a rectal probe and heating blanket.
Q3 using
4.3.
Training
Rats were randomly divided into sham sedentary, sham pre-surgery exercise, sham postsurgery exercised, sham ischemic, pre ischemic exercise, pos ischemic exercise. Preischemic exercised group was submitted to 20 min running sessions per day for 2 weeks before global cerebral ischemia, while the post ischemic exercised group was submitted to the running protocol after ischemia (Fig. 1). We demonstrated that a daily moderate intensity exercise (20 min once a day for two-weeks) reduces the damage in the hippocampal slices from Wistar rats submitted to in vitro ischemia (Scopel et al., 2006). The exercise training consisted of running sessions in an adapted motorized rodent treadmill (INBRAMED TK 01, Porto Alegre, Brazil), with individual Plexiglas lanes, at 60% of the animals maximal oxygen uptake (Brooks and White, 1978). Peak oxygen uptake (VO2) was measured indirectly in all animals before training. Each rat ran on a treadmill at a low initial speed with the speed being increased by 5 m/min every 3 min until the point of exhaustion (i.e., failure of the rat to continue running). The time to fatigue and workload were taken as indexes of exercise capacity, which was in turn
4.5.
Histological procedures
Iba-1 and GFAP immunofluorescence
Sections were fixated in ice cold acetone (10 min), followed by washing in ice cold phosphate buffer saline (PBS). Antigen retrieval was performed by heating sections in 0.01 M sodium citrate buffer (pH 6.0) in a thermostatic bath at 92 1C for 20 min. The tissues were washed in PBS and pre-incubated with 1% bovine serum albumin (BSA; Sigma, USA) in PBS, containing 0.1% Triton X-100 (PBS–Tx) for 30 min. Sections were incubated with the primary monoclonal rabbit antiIba-1 (ionized calcium binding adapter molecule 1) antibody to detect microglial cells (1:500; Wako Chemicals, USA) or the primary polyclonal rabbit anti-GFAP (Glial Fibrillary Acidic Protein) antibody to detect astrocytes (1:1000; Dako, UK) at 4 1C, for 48 h. Both Iba-1 and GFAP are upregulated during the activation of these cells (Piazza et al., 2014). Sections were washed in PBS–Tx and were incubated with a secondary antibody anti-rabbit Alexa Fluor 488 (1:500, Molecular Probes, Invitrogen, USA) for 1 h at room temperature in a dark chamber. Then, sections were washed in PBS and the slides were covered with an aqueous mounting medium (FluorSave, Calbiochem, Germany) and coverslips. The primary antibody was omitted in the negative controls.
4.6.
Quantification of Iba-1 and GFAP
A confocal microscope (Zeiss) was used to visualize the fluorescence immunostaining (excitation wavelength of 488 nm) of the sections. Images from the dentate gyrus and stratum radiatum of CA1 of the hippocampus (200 ) were acquired by z-axis analysis in a series of stacks (x and
Please cite this article as: Lovatel, G.A., et al., Long-term effects of pre and post-ischemic exercise following global cerebral ischemia on astrocyte and microglia functions in hippocampus from Wistar rats. Brain Research (2014), http://dx.doi.org/10.1016/ j.brainres.2014.08.068
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y ¼320.09 μm, z¼ 1.00 μm). The total area occupied by soma and processes of astrocytes and microglia was determined to reveal a cellular activation using Image J Software 1.42q. From each animal, 8–10 images were analyzed, 4–5 animals per group (Viola et al., 2009; Ferreira et al., 2011; Mestriner et al., 2011; Piazza et al., 2014). All images containing the region of interest were turned into binary (black and white) and a single threshold value was established for Iba-1 and GFAP staining. Then, each threshold was kept constant for all animal groups and was used to measure the total percentage area (%) occupied by soma and microglial and astrocytic processes, indicating the occurrence or not of cell hypertrophy in ischemia and/or exercise conditions, a characteristic of cellular activation (Ayoub and Salm, 2003; Estrada et al., 2012), according to the procedure previously described (Biscaro et al., 2012; Centenaro et al., 2011; Piazza et al., 2014).
4.7.
Statistical analysis
The data were analyzed using Two-way analysis of variance (ANOVA) with cerebral ischemia and exercise as independent variables. When there were statistically significant F values (po0.05), the Duncan post hoc test was used. All data are represented by the mean7S.D.
Conflict of interest The authors declare that the research was conducted in absence of any commercial or financial relationships that could be construed as potential conflicts of interest. The Local Ethics Committee approved all procedures with the animals
Acknowledgments This work received financial support from FIPE-Hospital de Clínicas de Porto Alegre (grant # 09-639). The project was also supported by the Brazilian funding agencies: Conselho NacioQ4 nal de Desenvolvimento Científico e Tecnológico – CNPq (Dr. I.R. Siqueira; G.A. Lovatel; V.R. Elsner; Piazza, F.V.); Coordenação de Aperfeiçoamento de Pessoal de Nível Superior – CAPES (K. Bertoldi; Basso, C.; F. Moysés). The authors thank to Electron Microscopy Center of the UFRGS for the microscopy analyzes. The authors also thank to Henrique B. Biehl for technical assistance.
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Please cite this article as: Lovatel, G.A., et al., Long-term effects of pre and post-ischemic exercise following global cerebral ischemia on astrocyte and microglia functions in hippocampus from Wistar rats. Brain Research (2014), http://dx.doi.org/10.1016/ j.brainres.2014.08.068
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Petito, C.K., Halaby, I.A., 1993. Ischemic neuronal necrosis to astrocyte expression of glial fibrillary acidic protein. Int. J. Dev. Neurosci. 11 (2), 239–247. Piazza, F.V., Segabinazi, E., Centenaro, L.A., Nascimento, P.S., Achaval, M., Marcuzzo, S., 2014. Enriched environment induces beneficial effects on memory deficits and microglial activation in the hippocampus of type 1 diabetic rats. Metab. Brain Disord. 29, 93–104. Pulsinelli, W.A., Brierley, J.B., Plum, F., 1982. Temporal profile of neuronal damage in a model of transient forebrain ischemia. Ann. Neurol. 11, 491–498. Saur, L., Baptista, P.P.A., Senna, P.N., Paim, M.F., Nascimento, P., Ilha, J., Bagatini, P.B., Achaval, M., Xavier, L.L., 2014. Physical exercise increases GFAP expression and induces morphological changes in hippocampal astrocytes. Brain Struct. Funct. Scopel, D., Fochesatto, C., Cimarosti, H., Rabbo, M., Bello´-Klein, A., Salbego, C., Netto, C.A., Siqueira, I.R., 2006. Exercise intensity influences cell injury in rat hippocampal slices exposed to oxygen and glucose deprivation. Brain Res. Bull. 71 (1-3), 155–159. Seo, T.B., Kim, B.K., Ko, I.G., Kim, D.H., Shin, M.S., Kim, C.J., Yoon, J.H., Kim, H., 2010. Effect of treadmill exercise on Purkinje cell loss and astrocytic reaction in the cerebellum after traumatic brain injury. Neurosci. Lett. 481 (3), 178–182. Sim, Y.J., Kim, S.S., Kim, J.Y., Shin, M.S., Kim, C.J., 2004. Treadmill exercise improves short-term memory by suppressing ischemia-induced apoptosis of neuronal cells in gerbils. Neurosci. Lett. 372 (3), 256–261. Stummer, W., Weber, K., Tranmer, B., Baethmann, A., Kempski, O., 1994. Reduced mortality and brain damage after locomotor activity in gerbil forebrain ischemia. Stroke 25, 1862–1869. Uda, M., Ishido, M., Kami, K., Masuhara, M., 2006. Effects of chronic treadmill running on neurogenesis in the dentate gyrus of the hippocampus of adult rat. Brain Res. 1104, 64–72. Valentim, L.M., Michalowski, C.B., Gottardo, S.P., Pedroso, L., Gestrich, L.G., Netto, C.A., Salbego, C.G., Rodnight, R., 1999. Effects of transient cerebral ischemia on glial fibrillary acidic protein phosphorylation and immunocontent in rat hippocampus. Neuroscience 91 (4), 1291–1297. Veena, J., Srikumar, B.N., Mahati, K., Bhagya, V., Raju, T.R., Rao, B.S.S., 2009. Enriched environment restores hippocampal cell proliferation and ameliorates cognitive deficits in chronically stressed rats. J. Neurosci. Res. 87, 831–843. Viola, G.G., Rodrigues, L., Ame´rico, J.C., Hansel, G., Vargas, R.S., Biasibetti, R., Swarowsky, A., Goncalves, C.A., Xavier, L.L., Achaval, M., Souza, D.O., Wessels, A.M., Scheltens, P., Barkhof, F., Heine, R.J., 2009. Morphological changes in hippocampal astrocytes induced by environmental enrichment in mice. Brain Res. 1274, 47–54. Wang, M.J., Lin, S.Z., Kuo, J.S., Huang, H.Y., Tzeng, S.F., Liao, C.H., Chen, D.C., Chen, W.F., 2007. Urocortin modulates inflammatory response and neurotoxicity induced by microglial activation. J. Immunol. 179 (9), 6204–6214. Yamada, M., Arai, H., Sonoda, T., Aoyama, T., 2012. Community based exercise program is cost-effective by preventing care and disability in Japanese frail older adults. J. Am. Med. Dir. Assoc. 13, 507–511.
Please cite this article as: Lovatel, G.A., et al., Long-term effects of pre and post-ischemic exercise following global cerebral ischemia on astrocyte and microglia functions in hippocampus from Wistar rats. Brain Research (2014), http://dx.doi.org/10.1016/ j.brainres.2014.08.068
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Research Report
Long-term effects of pre and post-ischemic exercise following global cerebral ischemia on astrocyte and microglia functions in hippocampus from Wistar rats Gisele Agustini Lovatela,d, Karine Bertoldib,d, Viviane Rostirol Elsnerbb,d, Francele Valente Piazzac,d, Carla Giovana Bassob,d, Felipe dos Santos Moyse´sb,d, Paulo Valdeci Wormb,d, Carlos Alexandre Nettob,d, Simone Marcuzzoc,d, Ionara Rodrigues Siqueirab,d,n a
Universidade Federal de Santa Catarina, Curso de Fisioterapia, Rua Pedro João Pereira, 150 Mato Alto, EP 88900-000 Araranguá, SC, Brazil b Universidade Federal do Rio Grande do Sul, Programa de Pós-Graduação em Ciências Biológicas: Fisiologia, Instituto de Ciências Básicas da Saúde, Instituto de Ciências Básicas da Saúde, Rua Sarmento Leite, 500, CEP 90050-170 Porto Alegre, RS, Brazil c Universidade Federal do Rio Grande do Sul, Programa de Pós-Graduação em Neurociências, Instituto de Ciências Básicas da Saúde, Instituto de Ciências Básicas da Saúde, Rua Sarmento Leite, 500, CEP 90050-170 Porto Alegre, RS, Brazil d Universidade Federal do Rio Grande do Sul, Departamento de Farmacologia, Instituto de Ciências Básicas da Saúde, Rua Sarmento Leite, 500, CEP 90050-170 Porto Alegre, RS, Brazil
art i cle i nfo
ab st rac t
Article history:
Persistent effects of pre- and postischemic exercise on glial cells activation after global
Accepted 25 August 2014
cerebral ischemia remains poorly understood. Here, we investigated the effect of both pre and postischemic treadmill exercise protocols (20 min/day during 2 weeks) on glial cells
Keywords:
immunostaining in the hippocampus of Wistar rats submitted to global ischemia. A
Treadmill exercise
synergistic effect between ischemia and postischemic exercise on the astrocytic area was
Pre-ischemic exercise
demonstrated. Postischemic exercise partially reversed the ischemia-induced increase on
Postischemic exercise
the area occupied by microglia, without any effect of pre-ischemic protocol. In conclusion,
Astrocyte
postischemic exercise distinctly modulates astrocyte and microglia immunostaining in the
Microglia
hippocampal dentate gyrus following global cerebral ischemia in Wistar rats.
Global ischemia
& 2014 Published by Elsevier B.V.
n Correspondence to: Laboratório de Neuropsicofarmacologia, Departamento de Farmacologia, Instituto de Ciências Básicas da Saúde, UFRGS. Rua Sarmento Leite, 500, CEP 90050-170 Porto Alegre, RS, Brazil. Fax:þ55 51 3308 3121. E-mail address: [email protected] (I.R. Siqueira).
http://dx.doi.org/10.1016/j.brainres.2014.08.068 0006-8993/& 2014 Published by Elsevier B.V.
Please cite this article as: Lovatel, G.A., et al., Long-term effects of pre and post-ischemic exercise following global cerebral ischemia on astrocyte and microglia functions in hippocampus from Wistar rats. Brain Research (2014), http://dx.doi.org/10.1016/ j.brainres.2014.08.068
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1.
Introduction
Epidemiological studies have shown neuroprotective effects of moderate physical activity (Yamada et al., 2012; Freiberger et al., 2012). In this context, several promising findings obtained in animal models of cerebral ischemia have been demonstrated. Pre-ischemic treadmill exercise is able to reduce infarct volume and inhibit inflammatory injury induced by the focal ischemia model (Guo et al., 2008; Ding et al., 2006; Jia et al., 2009). Indeed, pre-ischemic voluntary exercise in running wheel reduced neuronal damage induced by global cerebral ischemia (Stummer et al., 1994). It is interesting to note that postischemic treadmill exercise was able to diminish the global ischemia-induced apoptotic neuronal cell death (Sim et al., 2004; Lee et al., 2003). Besides, the traumatic brain injury-induced impairment of recognition memory, as well as the neuronal loss and activation of microglia in mice, was reversed by the postinjury exercise protocol (Chen et al., 2013). In addition, physical training improved perinatal hypoxic-ischemic encephalopathy-induced brain damage and memory impairments in Sprague-Dawley rats (Chen and Jiang, 2010). To our knowledge, there are no studies comparing the effects of pre- and postischemic exercise on cerebral ischemia outcomes. The mechanisms by which physical activity alters brain function are not yet defined, however it has been suggested that exercise might activate cellular and molecular pathways that contribute to neuroprotection. A growing body of data demonstrates that glial cells play a multifaceted and complex role in ischemia outcomes; astrocyte and microglia can have both harmful and supportive effects on cell proliferation and survival (Ekdahl et al., 2009). Astrogliosis is recognized as a cellular response following a wide variety of insults to the CNS, such as global ischemia (Valentim et al., 1999; Petito and Halaby, 1993), however, it is important to highlight that physical exercise itself can induce this phenomenon in different brain areas, including cortex, striatum and hippocampus (Li et al., 2005; Uda et al., 2006; Saur et al., 2014). Interestingly, Lee et al. (2009) demonstrated that high intensity postischemic exercise reduced the extent of reactive astrocytosis in the peri-infarct region induced by focal ischemia, without any effect of mild and moderate intensities. Global ischemia also triggers microglial outcomes, which was studied specifically on postischemic days 4–7 in the dentate
gyrus and CA1 subfield of the hippocampus in rats submitted to global ischemia (Marioka et al., 1991; Gehrmann et al., 1992); Q2 however, to our knowledge there are no studies evaluating persistent effects of global ischemia. Besides, divergent results about the influence of exercise per se on microglia function have been found. Voluntary exercise increased microglia proliferation in mice hippocampi with access to a free running wheel (Olah et al., 2009), while Kohman et al. (2012) found that 8 weeks wheel running exercise reduces the microglia proliferation in aged. Although there are no studies, to our knowledge, reporting the exercise effect on microglia function in global ischemia. Considering that reports describing persistent effects of exercise and/or ischemia as well as comparing pre- and postischemic exercise protocols were rarely performed. The aim of the present study was to compare the effect at long term of pre- and postischemic treadmill exercise protocols (20 min/day during 2 weeks) on glial cells immunostaining in the hippocampus of Wistar rats submitted to global ischemia.
2.
Results
Persistent effects of global cerebral ischemia and exercise on astrocyte and microglia functions in the hippocampal dentate gyrus were evaluated. We evaluated the long term effects of transient global cerebral ischemia in Wistar rats using the four-vessel occlusion, besides it was compared the impact of pre- and postischemic exercise protocols (Fig. 1). The GFAP immunostaining in the dentate gyrus is illustrated in Fig. 2. Two-way ANOVA revealed a significant effect of both factors, ischemia and exercise, on the quantification of the total percentage area (%) occupied by GFAPþ soma and astrocytic processes (F(1,29)¼ 297.73; po0.001; F(2,29)¼29.16; po0.001, respectively). The ischemic insult increased % of GFAP area in dentate gyrus subfield compared to sham groups. Two-way ANOVA revealed an effect of ischemia factor on GFAPþ soma and astrocytic processes in the stratum radiatum of hippocampus (F(1,29)¼39.72; po0.001; Fig. 3). Furthermore, this exercise protocol per se increased the area occupied by GFAPþ cells in the dentate gyrus (Fig. 2), while both exercise protocols did not affect this parameter in the stratum radiatum of the hippocampus (Fig. 3). Interestingly, there was a significant interaction between ischemia
Fig. 1 – Schematic representation of the experimental design. Please cite this article as: Lovatel, G.A., et al., Long-term effects of pre and post-ischemic exercise following global cerebral ischemia on astrocyte and microglia functions in hippocampus from Wistar rats. Brain Research (2014), http://dx.doi.org/10.1016/ j.brainres.2014.08.068
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Fig. 2 – Effects of cerebral ischemia and exercise on quantification of GFAPþ astrocyte in the dentate gyrus of the hippocampus. (A) Digital images of coronal sections of the dentate gyrus stained for GFAP immunofluorescence. (B) % of area occupied by astrocytes in the dentate gyrus in different groups. Columns represent mean 7S.D. (n ¼ 4–5). Two-way ANOVA followed by Duncan test; # significantly different from the sham groups; n significantly different from the sedentary ischemic and pre-ischemic exercise. Scale bar: 50 μm.
and exercise factors (F(2,29)¼7.57; p¼ 0.003). The Duncan post hoc test showed a significant increase in the % area occupied by astrocytes in the post-ischemic exercise group when compared to all groups. Two-way ANOVA showed the effect of both factors, ischemia and exercise, on the quantification of the total percentage area (%) occupied by Iba-1þ soma and microglial processes in the dentate gyrus (respectively, F(1,27)¼ 273.09; po0.001; F(2,27)¼8.27; p¼ 0.002, Fig. 4). Rats submitted to ischemia showed higher % area occupied by microglia when compared to sham groups. Moreover, there was a significant interaction between ischemia and exercise factors (F(2,27)¼ 23.03; po0.001). Duncan post hoc test revealed a significant decrease in the % area occupied by microglia in the post-
ischemic exercise groups when compared to other ischemic groups (Fig. 4).
3.
Discussion
The present study demonstrated that postischemic exercise may modulate glial functions, specifically can increase GFAP expression and decrease Iba-1 staining from rats submitted to global ischemia, without any effect of pre-ischemic protocol. An important finding that emerged from this study is that there was a synergistic effect between ischemia and exercise on the area occupied by astrocytes.
Please cite this article as: Lovatel, G.A., et al., Long-term effects of pre and post-ischemic exercise following global cerebral ischemia on astrocyte and microglia functions in hippocampus from Wistar rats. Brain Research (2014), http://dx.doi.org/10.1016/ j.brainres.2014.08.068
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Fig. 3 – Effects of cerebral ischemia and exercise on quantification of GFAPþ astrocyte in the stratum radiatum (SR) of the hippocampus. (A) Digital images of coronal sections of the SR stained for GFAP immunofluorescence. (B) % of area occupied by astrocytes in the SR in different groups. Columns represent mean7S.D. (n ¼4–5). Two-way ANOVA revealed an effect of ischemia factor; # significantly different from the sham groups. Scale bar: 50 μm.
Although some reports are in disagreement with our findings, since treadmill exercise suppressed formation of reactive astrocytes in other CNS insult models, such as Parkinson's disease (Dutra et al., 2012) and forebrain traumatic brain injuries (Seo et al., 2010), we infer that the robust increase in the % area occupied by astrocytes observed in the exercised ischemic rats may play a role in providing a favorable microenvironment, or even necessary for neuronal growth and restructuring after CNS insult. This possibility is supported by a previous study that rehabilitation training (complex environment housing) produces enhanced functional recovery, dendritic growth, synaptogenesis, and an increased number of synapses associate with astrocytic activation in the hippocampus after cerebral ischemia (Biernaskie and Corbett, 2001; Briones et al., 2006). Indeed, the involvement of astrocytes in angiogenesis has been recognized, contributing to induce a
favorable state to neurovascular unit (consisting of microvascular endothelium, astroglia, neurons and the extracellular matrix), what would protect the blood-brain-barrier function following ischemic damage. In this context, Huang et al. (2013) suggested that exercise-induced cognitive improvement of middle-aged rats might be associated with an increase on the capillaries density in the rat cortex. The exercise-induced astrogliosis here observed in the sham group is supported by previous studies which demonstrated an increase in astrocytic activation and proliferation of GFAP positive cells, in the subgranular zone of the hippocampus, in healthy exercised rats (Uda et al., 2006; Li et al., 2005; Saur et al., 2014). Besides, our findings are in accordance with Valentim et al. (1999) that observed an early and late increase in both immunocontent and GFAP phosphorylation rate in the hippocampus from ischemic rats,
Please cite this article as: Lovatel, G.A., et al., Long-term effects of pre and post-ischemic exercise following global cerebral ischemia on astrocyte and microglia functions in hippocampus from Wistar rats. Brain Research (2014), http://dx.doi.org/10.1016/ j.brainres.2014.08.068
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Fig. 4 – Effects of cerebral ischemia and exercise on quantification of Iba-1þ microglia in the dentate gyrus of the hippocampus. (A) Digital images of coronal sections of the dentate gyrus stained for Iba-1 immunofluorescence. (B) % of area occupied by microglia in the dentate gyrus in different groups. Columns represent mean7S.D. (n¼ 4–5). Two-way ANOVA followed by Duncan test; # significantly different from the sham groups; n significantly different from the sedentary ischemic and pre-ischemic exercise. Scale bar: 50 μm.
pointing to a long-term glial phenomenon. Moreover, Petito and Halaby (1993) demonstrated a persistent increase in GFAP immunocontent until five weeks in the CA1 area after ischemic insult. An early increase of Iba-1 expression was previously described in the dentate gyrus and CA1 of ischemic rats (Morioka et al., 1991; Gehrmann et al., 1992), and in our study an increase in the % area occupied by Iba-1 was observed 5 weeks after the ischemic event, pointing to a long-term microglial phenomenon. Interestingly, postischemic exercise reversed partially the microglia activation. To our knowledge, we provide here, for the first time, evidence of a long term effect of ischemic insult as well as exercise modulation on microglia activation after global cerebral ischemia. This effect
can be related to previous described neuroprotective properties, since some studies suggest a detrimental role of the microglial action by producing neurotoxic molecules and proinflammatory cytokines (Wang et al., 2007; Kaushal and Schlichter, 2008). Recently, it was described that the exercise decreased pro-inflammatory markers in the hippocampus from aged rats without any effect in young rats (Lovatel et al., 2013). Indeed, the exercise-induced reduction of the Iba-1 staining here observed could be related to decline on proinflammatory cytokines observed in aged rats (Lovatel et al., 2012), since these cytokines are frequently produced by activated microglia (Hetier et al., 1988; Giulian et al., 1986). Taken together, we can suppose that exercise is able to modulate the pro-inflammatory state against injury conditions.
Please cite this article as: Lovatel, G.A., et al., Long-term effects of pre and post-ischemic exercise following global cerebral ischemia on astrocyte and microglia functions in hippocampus from Wistar rats. Brain Research (2014), http://dx.doi.org/10.1016/ j.brainres.2014.08.068
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Summarizing, our results support the hypothesis that the exercise modulates glial cells functions; specifically postischemic exercise induced an increase of astrocytes imunostaining associated to a decrease in microglia function in the dentate gyrus from rats following global cerebral ischemia.
taken as VO2 max (Arida et al., 1999; Scopel et al., 2006). The sedentary group was handled exactly as the experimental animals and was left on the treadmill for 5 min without any stimulus to run. Neither electric shock nor physical prodding was used in this study and all the procedures took place between 14:00 and 17:00 h.
4.
Experimental procedure
4.4.
4.1.
Animals
In order to evaluate long term effects of ischemia and exercise, the animals were perfused at 38 days after ischemic insult (Fig. 1). Animals were anesthetized with ketamine (90 mg/kg i.p.) and xylazine (10 mg/kg i.p). Heparin (Cristalia, Brazil) was injected into the cardiac ventricle, and the animals were transcardially perfused (Milan, Brazil) with a saline solution, followed by a fixative solution 4% paraformaldehyde (Synth, Brazil). The brains were dissected, postfixed and cryoprotected by immersion in a sucrose solution (Synth, Brazil). The brains were frozen in isopentane (Merck, Germany) cooled in liquid nitrogen and kept in a freezer ( 70 1C) for further analyses. Coronal sections (40 μm) of the dorsal hippocampus were obtained using a cryostat (Leica, Germany) and serially collected on gelatin coated slides. This area corresponded to a distance of approximately 2.30– 4.52 mm posterior to the bregma (Paxinos and Watson, 1982). Every sixth section (240 μm apart) was processed for each immunostaining (Malberg et al., 2000; Veena et al., 2009; Piazza et al., 2014).
Three months-old male Wistar rats were used. Animals were provided by the Centro de Reprodução de Animais de Laboratório (CREAL) from the Universidade Federal do Rio Grande do Sul (UFRGS) and were maintained under standard conditions (12-h light/dark, 2272 1C) with food and water ad libitum at Unidade de Experimentação Animal do Hospital de Clínicas de Porto Alegre (UEA/HCPA). The NIH “Guide for the Care and Use of Laboratory Animals” (NIH publication no. 80-23, revised 1996) was followed in all experiments. The Local Ethics Committee (Comissão de Ética no Uso de Animais – CEUA/HCPA, no. 09-630) approved all handling and experimental conditions.
4.2.
Surgery
Transient global cerebral ischemia was induced in Wistar rats the four-vessel occlusion method described by Pulsinelli et al. (1982), with minor modifications (Netto et al., 1993). The vertebral arteries were permanently electrocoagulated under halothane anesthesia, and silastic ties were placed around each common carotid artery without the interruption of blood flow (Fig. 1). On the next day, the ties were tightened and clamped for 10 min. Sham animals were operated, but the ties were not tightened. The body temperature was maintained at 3770.5 1C during ischemia and reperfusion by means of a rectal probe and heating blanket.
Q3 using
4.3.
Training
Rats were randomly divided into sham sedentary, sham pre-surgery exercise, sham postsurgery exercised, sham ischemic, pre ischemic exercise, pos ischemic exercise. Preischemic exercised group was submitted to 20 min running sessions per day for 2 weeks before global cerebral ischemia, while the post ischemic exercised group was submitted to the running protocol after ischemia (Fig. 1). We demonstrated that a daily moderate intensity exercise (20 min once a day for two-weeks) reduces the damage in the hippocampal slices from Wistar rats submitted to in vitro ischemia (Scopel et al., 2006). The exercise training consisted of running sessions in an adapted motorized rodent treadmill (INBRAMED TK 01, Porto Alegre, Brazil), with individual Plexiglas lanes, at 60% of the animals maximal oxygen uptake (Brooks and White, 1978). Peak oxygen uptake (VO2) was measured indirectly in all animals before training. Each rat ran on a treadmill at a low initial speed with the speed being increased by 5 m/min every 3 min until the point of exhaustion (i.e., failure of the rat to continue running). The time to fatigue and workload were taken as indexes of exercise capacity, which was in turn
4.5.
Histological procedures
Iba-1 and GFAP immunofluorescence
Sections were fixated in ice cold acetone (10 min), followed by washing in ice cold phosphate buffer saline (PBS). Antigen retrieval was performed by heating sections in 0.01 M sodium citrate buffer (pH 6.0) in a thermostatic bath at 92 1C for 20 min. The tissues were washed in PBS and pre-incubated with 1% bovine serum albumin (BSA; Sigma, USA) in PBS, containing 0.1% Triton X-100 (PBS–Tx) for 30 min. Sections were incubated with the primary monoclonal rabbit antiIba-1 (ionized calcium binding adapter molecule 1) antibody to detect microglial cells (1:500; Wako Chemicals, USA) or the primary polyclonal rabbit anti-GFAP (Glial Fibrillary Acidic Protein) antibody to detect astrocytes (1:1000; Dako, UK) at 4 1C, for 48 h. Both Iba-1 and GFAP are upregulated during the activation of these cells (Piazza et al., 2014). Sections were washed in PBS–Tx and were incubated with a secondary antibody anti-rabbit Alexa Fluor 488 (1:500, Molecular Probes, Invitrogen, USA) for 1 h at room temperature in a dark chamber. Then, sections were washed in PBS and the slides were covered with an aqueous mounting medium (FluorSave, Calbiochem, Germany) and coverslips. The primary antibody was omitted in the negative controls.
4.6.
Quantification of Iba-1 and GFAP
A confocal microscope (Zeiss) was used to visualize the fluorescence immunostaining (excitation wavelength of 488 nm) of the sections. Images from the dentate gyrus and stratum radiatum of CA1 of the hippocampus (200 ) were acquired by z-axis analysis in a series of stacks (x and
Please cite this article as: Lovatel, G.A., et al., Long-term effects of pre and post-ischemic exercise following global cerebral ischemia on astrocyte and microglia functions in hippocampus from Wistar rats. Brain Research (2014), http://dx.doi.org/10.1016/ j.brainres.2014.08.068
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y ¼320.09 μm, z¼ 1.00 μm). The total area occupied by soma and processes of astrocytes and microglia was determined to reveal a cellular activation using Image J Software 1.42q. From each animal, 8–10 images were analyzed, 4–5 animals per group (Viola et al., 2009; Ferreira et al., 2011; Mestriner et al., 2011; Piazza et al., 2014). All images containing the region of interest were turned into binary (black and white) and a single threshold value was established for Iba-1 and GFAP staining. Then, each threshold was kept constant for all animal groups and was used to measure the total percentage area (%) occupied by soma and microglial and astrocytic processes, indicating the occurrence or not of cell hypertrophy in ischemia and/or exercise conditions, a characteristic of cellular activation (Ayoub and Salm, 2003; Estrada et al., 2012), according to the procedure previously described (Biscaro et al., 2012; Centenaro et al., 2011; Piazza et al., 2014).
4.7.
Statistical analysis
The data were analyzed using Two-way analysis of variance (ANOVA) with cerebral ischemia and exercise as independent variables. When there were statistically significant F values (po0.05), the Duncan post hoc test was used. All data are represented by the mean7S.D.
Conflict of interest The authors declare that the research was conducted in absence of any commercial or financial relationships that could be construed as potential conflicts of interest. The Local Ethics Committee approved all procedures with the animals
Acknowledgments This work received financial support from FIPE-Hospital de Clínicas de Porto Alegre (grant # 09-639). The project was also supported by the Brazilian funding agencies: Conselho NacioQ4 nal de Desenvolvimento Científico e Tecnológico – CNPq (Dr. I.R. Siqueira; G.A. Lovatel; V.R. Elsner; Piazza, F.V.); Coordenação de Aperfeiçoamento de Pessoal de Nível Superior – CAPES (K. Bertoldi; Basso, C.; F. Moysés). The authors thank to Electron Microscopy Center of the UFRGS for the microscopy analyzes. The authors also thank to Henrique B. Biehl for technical assistance.
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Please cite this article as: Lovatel, G.A., et al., Long-term effects of pre and post-ischemic exercise following global cerebral ischemia on astrocyte and microglia functions in hippocampus from Wistar rats. Brain Research (2014), http://dx.doi.org/10.1016/ j.brainres.2014.08.068
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Please cite this article as: Lovatel, G.A., et al., Long-term effects of pre and post-ischemic exercise following global cerebral ischemia on astrocyte and microglia functions in hippocampus from Wistar rats. Brain Research (2014), http://dx.doi.org/10.1016/ j.brainres.2014.08.068