Behavioural Brain Research 278 (2015) 257–261
Contents lists available at ScienceDirect
Behavioural Brain Research journal homepage: www.elsevier.com/locate/bbr
Short communication
Astrocyte morphology after ischemic and hemorrhagic experimental stroke has no influence on the different recovery patterns Régis Gemerasca Mestriner a,b,c,d,∗ , Lisiani Saur d , Pamela Brambilla Bagatini d , Pedro Porto Alegre Baptista d , Sabrina Pereira Vaz c,d , Kelly Ferreira c,d , Susane Alves Machado c,d , Léder Leal Xavier d , Carlos Alexandre Netto a,b a
Programa de Pós-Graduac¸ão em Ciências Biológicas: Fisiologia, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS, Brazil Departamento de Bioquímica, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS, Brazil c Faculdade de Enfermagem, Nutric¸ão e Fisioterapia, Pontifícia Universidade Católica do Rio Grande do Sul, Porto Alegre, RS, Brazil d Laboratório de Biologia Celular e Tecidual, Departamento de Ciências Morfofisiológicas, Pontifícia Universidade Católica do Rio Grande do Sul, Porto Alegre, RS, Brazil b
h i g h l i g h t s • Long-term astrocyte morphology has no influence on the different recovery patterns of stroke. • Ischemic and hemorrhagic stroke subtypes have similar long-term astrocyte morphology in perilesional sensorimotor cortex and dorsolateral striatum. • Long-term GFAP immunoreactivity profile is similar between ischemic and hemorrhagic stroke.
a r t i c l e
i n f o
Article history: Received 26 July 2014 Received in revised form 29 September 2014 Accepted 2 October 2014 Available online 12 October 2014 Keywords: Ischemic stroke Hemorrhagic stroke Glial fibrillary acidic protein Astrocytes Reactive astrogliosis
a b s t r a c t Stroke, broadly subdivided into ischemic and hemorrhagic subtypes, is a serious health-care problem worldwide. Previous studies have suggested ischemic and hemorrhagic stroke could present different functional recovery patterns. However, little attention has been given to this neurobiological finding. Coincidently, astrocyte morphology could be related to improved sensorimotor recovery after skilled reaching training and modulated by physical exercise and environmental enrichment. Therefore, it is possible that astrocyte morphology might be linked to differential recovery patterns between ischemic and hemorrhagic stroke. Thus, we decided to compare long-term GFAP-positive astrocyte morphology after ischemic (IS, n = 5), hemorrhagic (HS, n = 5) and sham (S, n = 5) stroke groups (induced by endothelin1, collagenase type IV-S and salina, respectively). Our results showed ischemic and hemorrhagic stroke subtypes induced similar long-term GFAP-positive astrocyte plasticity (P > 0.05) for all evaluated measures (regional and cellular optical density; astrocytic primary processes ramification and length; density of GFAP positive astrocytes) in perilesional sensorimotor cortex and striatum. These interesting negative results discourage similar studies focused on long-term plasticity of GFAP-positive astrocyte morphology and recovery comparison of stroke subtypes. © 2014 Elsevier B.V. All rights reserved.
Stroke, broadly subdivided into ischemic and hemorrhagic subtypes, is a serious health-care problem worldwide [1]. Some clinical studies have shown that by the time of hospital discharge hemorrhagic stroke presents a better functional improvement compared to ischemic stroke [2]. Moreover, ischemic stroke patients showed
∗ Corresponding author at: Faculdade de Enfermagem, Nutric¸ão e Fisioterapia, Pontifícia Universidade Católica do Rio Grande do Sul, Avenida Ipiranga, 6681, Prédio 12/8◦ andar, Porto Alegre CEP 90619-900, RS, Brazil. Tel.: +55 51 33203646; fax: +55 51 33203646. E-mail address: [email protected] (R.G. Mestriner). http://dx.doi.org/10.1016/j.bbr.2014.10.005 0166-4328/© 2014 Elsevier B.V. All rights reserved.
a longer functional recovery time window than those with the hemorrhagic etiology [3]. Yet, these recovery differences are not completely understood due to stroke heterogeneity [4]. Animal models aid researchers to control some factors and provide an unbiased analysis of stroke subtypes. We have recently published a study, using two controlled lesion “site and size” rat models, showing the spontaneous recovery pattern is better in hemorrhagic than in ischemic stroke [5]. Unfortunately, the neurobiological explanations for this finding remain poorly understood. Coincidentally, our research group has also shown astrocyte morphology could be related to improved sensorimotor recovery after a rehabilitation protocol [6] and modulated by physical
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exercise and environmental enrichment [7,8]. For example, skilled reaching training after collagenase-induced intracerebral hemorrhage increases the length of primary astrocytic GFAP-positive processes in perilesional tissue. This finding was correlated with functional forelimb recovery [6]. Moreover, enriched environment [8] and physical exercise [7] induce changes in the polarization of GFAP-positive astrocytes in healthy animals. Therefore, it is possible that astrocyte morphology might be linked to differential recovery patterns between ischemic and hemorrhagic stroke. Thus, we decided to compare long-term GFAP-positive astrocyte morphology after ischemic (IS) and hemorrhagic (HS) stroke using coronal sections left over from the study “Behavior outcome after ischemic and hemorrhagic stroke, with similar brain damage, in rats”, previously published in Behavioural Brain Research [5]. This was done in an effort to reduce the animals needed in research, according to the replacement, reduction and refinement principle (3R’s) [9]. The sections were taken randomly from 15 brains of Wistar rats divided into the following groups: Sham (n = 5), ischemic (IS) (n = 5) or hemorrhagic (HS) (n = 5). Brain ischemia or hemorrhage was induced by endotelin-1 (ET-1) and collagenase type IV-S microinjections, respectively (see Mestriner et al., 2013 for detailed description) [5]. The following morphological parameters were evaluated in GFAP positive astrocytes: (1) semi-quantitative analysis of GFAP imunohistochemistry intensity, measuring regional and cellular optical density; (2) astrocytic morphology and polarization (primary processes ramification and length), using Sholl concentric circles analysis; (3) density of GFAP positive astrocytes (number of GFAP positive astrocytes/mm2 ), using planar morphometry. All measurements were made in the perilesional sensorimotor cortex and striatum at 30 days post-surgery (Fig. 1). For immunohistochemistry, coronal brain sections (50 m) were obtained using a cryostat (Leica, Germany). They approximately ranged (relative to Bregma) from +1.80 mm (rostrally) to −0.70 mm (caudally). Briefly, brain sections were post-fixed in 4% PF for 15 min. After three washes in cold phosphate buffered saline (PBS, pH 7.4), GFAP immunohistochemistry was performed as previously described [7]. Sections were blocked for endogenous peroxidases (3% hydrogen peroxide in PBS) for 30 min, washed in PBS containing 0.4% Triton X-100 (PBS-Tx) and blocked with 2% bovine serum albumin (BSA) in PBS-Tx for 30 min. Sections were then incubated with anti-GFAP polyclonal antibody raised in rabbit (Z033401-2 – Dako), diluted 1:500 in PBS-Tx for 48 h at 4 ◦ C. After two washes in PBS-Tx, the sections were incubated in peroxidaseconjugated goat anti-rabbit IgG antibody (A0545 – Sigma–Aldrich), diluted 1:200 in PBS-Tx at room temperature for 2 h. Sections were then washed two times in PBS, and the GFAP immunostaining was performed by incubating the sections in a medium containing 0.06% 3,3 -diaminobenzidine (DAB, Sigma–Aldrich) dissolved in PBS for 10 min, and in the same solution containing 1 L of 3% H2 O2 per mL of DAB medium for an additional 10 min. After the DAB + H2 O2 revelation, the sections were rinsed in PBS, dehydrated in series of increasing ethanol concentrations (70, 90 and 100%), cleared with xylene and covered with Entellan (Merck) and coverslips. As a control to rule out unspecific binding, in a few sections the primary antibody was omitted and replaced by PBS-Tx. In order to minimize differences in the staining of astrocytes and in background levels, the brains in all experimental groups were fixed, cryoprotected and post-fixed in identical solutions for the same length of time, processed at the same time and incubated in the same immunostaining medium for the same period of time. The number of GFAP-immunoreactive astrocytes per mm2 in the surrounding damaged tissue was estimated using an Olympus BX 50 microscope coupled to a Motic Images Plus 2.0 camera and Image Pro Plus (Image Pro-Plus 6.1, Media Cybernetics, Silver Spring, EUA)
software. Our interested regions were sensorimotor cortex and dorsolateral striatum. For this analysis, four digitized images (20×) from surrounding injured tissue (two for each cortex and striatum) were obtained from each section (Fig. 1). Three sections from each animal were analyzed (a total of 12 images analyzed per animal – six from cortex and six from striatum). Two randomized squares measuring 5828 m2 and named areas of interest (AOIs) were overlaid on each image (Fig. 1). The astrocytes located inside this square or intersected by the upper and/or right edges of the square were counted. Astrocytes intersected by the lower and/or left edges of the square were not counted [7,8]. We established an area of analysis beginning at approximately 50 m laterally to lesion border for all morphological measurements to avoid errors related to complex overlapping of astrocytes bodies and process, blood vessels and artifacts in an immediately surrounding tissue to lesion (Fig. 1). The intensity of GFAP immunoreactivity was measured using semi-quantitative densitometric analysis [7,8] with the same software employed to estimate the astrocytic density. The same images and AOIs (5828 m2 ) used to estimate astrocytic density were used in the analysis of regional optical density (OD). The images were converted to an 8-bit gray scale (256 gray levels) and the AOIs were overlaid on each image. For the analysis of cellular OD, two astrocytes GFAP-positive located inside the first AOI (5828 m2 ) were randomly selected to cellular OD assessment. Thus, a new AOI measuring 10.37 m2 was placed over analyzed astrocytic soma in each image (processes were not measured). A number of 12 astrocytes per structure of interest were analyzed by animal. All lighting conditions and magnifications were kept constant during the process of capturing the images. Blood vessels and other artifacts were avoided and the background correction was performed according to the formula previously described [10]. The morphological analysis was done using the same images employed to measure cellular optical density. For the analysis of astrocytic ramification, an adaptation of Sholl’s concentric circles technique was used [6–8]. Briefly, seven virtual circles with 3.91 m intervals were drawn around each astrocyte. The degree of ramification of the astrocytes was measured by counting the number of times the astrocytic processes intersected with each virtual circle around the astrocytes. Primary process quantification was performed by counting the processes extending directly from the soma in both the lateral and central quadrants of astrocytes in the same sections. The longest primary process in each quadrant was measured by tracing the process with a manual measurement tool found in the Image Pro Plus software. All morphological data were obtained and analyzed by researchers blind to group identity (images capture and measurements). Data normality distribution was tested using the Kolmogorov–Smirnov test and showed a parametric profile. Data was analyzed using one-way ANOVA followed by Bonferroni post hoc test, when appropriate. Pearson correlation coefficient and analysis of co-variance (ANCOVA) were performed to assess the influence of astrocyte features on ladder walk performance. Behavioral data was obtained from our previous study [5]. All variables were expressed as mean ± standard error of the mean (SEM). Results were considered significant when P ≤ 0.05. SPSS 16.0 (Statistical Package for the Social Sciences, Inc., Chicago, USA) and G*Power 3.1 software were used for data analysis. In our study, GFAP-positive astrocytes surrounding damaged sensorimotor cortex and dorsolateral striatum (approximately from 50 m to 210 m laterally to border of tissue lost) were analyzed. In the qualitative analysis, it was possible to observe individual astrocyte’s soma and proximal processes for all groups. As expected, a highly complex network of GFAP-positive cells was observed in the tissue adjacent to the core of damage for both stroke
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Fig. 1. GFAP-positive astrocytic endpoints (regional and cellular optical density; primary processes number and length; and astrocytic density) and figure showing the morphological assessment (A–M). Fields 1–4 represent the evaluated surrounding tissue. Brain slice with damaged sensorimotor cortex and dorsolateral striatum (gray areas). (M) Amplified fields with areas of interest (AOIs) used to measure astrocytic density (mm2 ) and regional optical density. Dashed lines indicated the included edges. (L) Example of astrocyte with overlaid AOI (in white) used to evaluate optical cellular density and concentric circles (Sholl’s concentric circles method). (D) Dorsal; (V) ventral; (R) right; (L) left; (OD) optical density. Difference between sham and both stroke groups at *P ≤ 0.05; **P ≤ 0.01 and ***P ≤ 0.001. No differences were found between stroke groups. Data are presented as mean ± standard error of the mean (SEM).
groups. This intricate network was less complex slightly distal to lesion border. Then, individual astrocyte morphology was distinguishable (Fig. 2). In order to confirm our qualitative morphological findings, quantitative and semi-quantitative evaluations were performed, respectively, involving an estimation of lesion volume and length, astrocytic density, primary processes characteristics (number and length) and measurements of regional and cellular optical density. One-way ANOVA revealed main effects for “lesion volume” (F2,14 = 39.13/P < 0.001) and “lesion length” (F2,14 = 21.35/P < 0.001). The achieved power (1 − ˇ) was 0.99 for both factors. Bonferroni post hoc tests showed differences between sham and both stroke subtypes for lesion volume (P < 0.001) and lesion length (P < 0.001). No additional differences were found between ischemic and hemorrhagic stroke groups (P = 1.00 for lesion volume and lesion length), similarly to previously published [5]. Moreover, in order to ensure similar behavior as shown previously, we compared skilled walking in the current sample with Mestriner et al., 2013 full data [5]. ANOVA revealed main effects for “Group” (F5,45 = 20.98/P < 0.001). The achieved power (1 − ˇ) was 1.00. Bonferroni post hoc tests showed both Sham groups (current sample vs original sample) were different from all stroke groups (P ≤ 0.02). Additionally, no differences were found between both Sham groups (P = 1.00) as well as no differences were revealed
between all stroke groups (current vs original sample) (P > 0.07). Thus, present findings corroborate with our previously published study in terms of skilled walking performance [5]. Analysis of astrocytic density revealed significant main effects for sensorimotor cortex (F(2,14) = 9.18, P < 0.01, 1 − ˇ = 0.98), dorsolateral striatum (F(2,14) = 6.95, P ≤ 0.01, 1 − ˇ = 0.97) and combined (cortex and striatum) analysis (F(2,14) = 8.42, P ≤ 0.01, 1 − ˇ = 0.97). Major group differences were found between S and stroke rats (IS and HS) in the sensorimotor cortex (P ≤ 0.01), dorsolateral striatum (P ≤ 0.05 for IS and P ≤ 0.01 for HS) and combined structures (P ≤ 0.01). No differences were found between IS and HS groups in any analysis (P = 1.0). These findings showed that stroke induced an increased density of GFAP-positive astrocytes, as expected; both stroke subtypes have similar astrocytic density (Fig. 1). One-way ANOVA showed significant main effects in terms of the regional and cellular optical density for sensorimotor cortex (F(2,14) = 46.46, P ≤ 0.001, 1 − ˇ = 1.00/F(2,14) = 29.69, P ≤ 0.001, 1 − ˇ = 0.99), dorsolateral striatum (F(2,14) = 27.86, P ≤ 0.001, 1 − ˇ = 0.99/F(2,14) = 24.64, P ≤ 0.001, 1 − ˇ = 0.99) and combined (cortex and striatum) analysis (F(2,14) = 29.30, P ≤ 0.001, 1 − ˇ = 0.99/F(2,14) = 55.59, P ≤ 0.001, 1 − ˇ = 0.99), respectively. Bonferroni post hoc tests revealed significant differences between S and both stroke subtypes (IS and HS) (P ≤ 0.001) for all analysis. No differences between the stroke subtypes were found for
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Fig. 2. Digitized images after GFAP immunohistochemistry (20×). Sensorimotor cortex of (A) sham, (B) ischemic and (C) hemorrhagic stroke; dorsolateral striatum of (D) sham, (E) ischemic and (F) hemorrhagic stroke. Scale bar: 35 m (20×).
sensorimotor cortex (OD regional P = 0.51/OD cellular P = 0.15); dorsolateral striatum (OD regional P = 1.0/OD cellular P = 0.58) and these combined structures (OD regional P = 0.78/OD cellular P = 1.0) suggesting that both stroke subtypes were comparable in terms of GFAP imunorreactivity. There are GFAP-positive astrocytic ramification differences (F(2,14) = 8.59, P ≤ 0.01, 1 − ˇ = 0.95 for sensorimotor cortex/F(2,14) = 9.14, P ≤ 0.01, 1 − ˇ = 0.97 for dorsolateral striatum) as well as primary processes length main effects (F(2,14) = 10.06, P ≤ 0.01, 1 − ˇ = 0.97 for sensorimotor cortex/F(2,14) = 12.41, P ≤ 0.001, 1 − ˇ = 0.99 for dorsolateral striatum). As expected, we observed differences between sham vs stroke subtypes for analyzed structures and these morphological characteristics (P < 0.01). However, no differences were found between IS and HS for astrocytic ramification (P > 0.68) or processes length (P > 0.23) for both cortex and striatum. These findings revealed no longterm differences in GFAP-positive astrocytes ramification and/or primary processes length after ischemic or hemorrhagic stroke subtypes. Moreover, our results showed significant correlations between ladder walk performance and most features evaluated in astrocytes: cortical cellular density (r = 0.77, P = 0.001); striatal cellular density (r = 0.71, P = 0.03); regional optical density in the cortex (r = 0.68, P = 0.005); regional optical density in the striatum (r = 0.61, P = 0.01); cellular optical density in the striatum (r = 0.61, P = 0.01); number of cortical primary process (r = 0.53, P = 0.01); number of striatal primary process (r = 0.73, P = 0.002); cortical primary process (length) (r = 0.77, P = 0.001); and striatal primary process (length) (r = 0.54, P = 0.03). Non-significant correlation was observed only for cellular optical density in the cortex (r = 0.46, P = 0.08). However, ANCOVA analyses showed skilled walking errors and astrocyte morphology did not influence each other, as follow: cortical astrocyte density (F1,14 = 0.123, P = 0.76); striatal astrocyte density (F1,14 = 0.76, P = 0.81); regional
optical density in the cortex (F1,14 = 0.72, P = 0.81); regional optical density in the striatum (F1,14 = 0.005, P = 0.95); cellular optical density in the cortex (F1,14 = 0.52, P = 0.55); cellular optical density in the striatum (F1,14 = 0.28, P = 0.65); cortical primary process (number) (F1,14 = 0.16, P = 0.91); striatal primary process (number) (F1,14 = 0.35, P = 0.87); cortical primary process (length) (F1,14 = 0.19, P = 0.90); and striatal primary process (length) (F1,14 = 0.26, P = 0.66). Altogether, our findings revealed astrocytes morphology and skilled walking behavior did not influence each other. Stroke probably acted as a confounding factor in these significant correlations”. Rodent stroke models are largely used to provide in vivo mechanisms of neuroprotection and neural repair after stroke [5,6,11,12]. Several preclinical models should be properly designed and compared to understand plasticity mechanisms [11,12]. Accordingly, our research group has recently shown a comparative study design to evaluate ischemic and hemorrhagic stroke subtypes [5]. This design was thought to control experimental lesion volume, length and brain compromised areas; thus, the impact of stroke subtype on several pathophysiological mechanisms could be better understood in a comparative manner. Reactive astrocytes in neurotrauma, stroke, or neurodegeneration are thought to undergo cellular hypertrophy [13]. The hallmark of this reaction is an expressive upregulation of intermediate filaments, such as glial fibrillary acidic protein (GFAP) [14]. Reactive astrocytes might either reduce or exacerbate the damage to neurons depending on the time-point or post-injury stage as well as injury severity [15]. An overwhelming number of studies show any central nervous system injury increases astrocyte expression of intermediate filaments, such as GFAP [16]. However, to the best of our knowledge, this is the first study to evaluate GFAP-positive astrogliosis after ischemic and hemorrhagic stroke in a rational controlled and comparative design.
R.G. Mestriner et al. / Behavioural Brain Research 278 (2015) 257–261
On one hand, it is reasonable to consider brain repair is similar in all forms of stroke [11] despite the known differences in sensorimotor recovery between subtypes [5]. Astrocyte could contribute to damage by their role in spreading depression waves [17] or by sending apoptotic messengers or other deleterious molecules to otherwise healthy regions via gap junction channels [18]. They also inhibit regeneration by the glial scar [19,20]. These mechanisms may occur independently of injury etiology. On the other hand, morphological astrocytes profile at 30 days may not reflect earlier survival times, when differences may very well be strong. For example, reactive astrocytes could modulate the neurovascular unit differentially, at early and late stages after stroke [21]. Thus, its unknown if early astrocyte changes could be similar to all stroke subtypes. Moreover, early astrocyte changes may influence in longterm functional stroke recovery. This point is a limitation of our study design and could be matter of further investigation. As showed, the current findings could discourage similar studies focused on long-term astrocyte morphology and comparisons of spontaneous stroke subtypes recovery. Unfortunately, there were no previous studies comparing brain plasticity between controlled ischemic and hemorrhagic damage to expand this discussion. Our research group has previously shown GFAP-positive astrocytes modulated by skilled reaching training, which it was correlated with an improved sensorimotor behavior [6]. However, it is unknown if rehabilitative interventions, such as skilled reaching training, enriched rehabilitation, treadmill exercise, or pharmacological strategies could differentially modulate GFAP expression and behavior after each stroke subtype; it could be a subject for further investigation. Finally, these findings contribute toward the knowledge of the role of reactive astrogliosis and astrocyte morphology after different stroke. Conflict of interest The authors declare they have no conflict of interest. Acknowledgements The authors thank Ms. Raquel Matos for her technical assistance. This study was supported by the Brazilians Funding Agencies CNPq, CAPES and FAPERGS. Mestriner was a recipient of a PhD scholarship from CNPq. Léder Leal Xavier and Carlos Alexandre Netto are CNPq investigators.
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Contents lists available at ScienceDirect
Behavioural Brain Research journal homepage: www.elsevier.com/locate/bbr
Short communication
Astrocyte morphology after ischemic and hemorrhagic experimental stroke has no influence on the different recovery patterns Régis Gemerasca Mestriner a,b,c,d,∗ , Lisiani Saur d , Pamela Brambilla Bagatini d , Pedro Porto Alegre Baptista d , Sabrina Pereira Vaz c,d , Kelly Ferreira c,d , Susane Alves Machado c,d , Léder Leal Xavier d , Carlos Alexandre Netto a,b a
Programa de Pós-Graduac¸ão em Ciências Biológicas: Fisiologia, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS, Brazil Departamento de Bioquímica, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS, Brazil c Faculdade de Enfermagem, Nutric¸ão e Fisioterapia, Pontifícia Universidade Católica do Rio Grande do Sul, Porto Alegre, RS, Brazil d Laboratório de Biologia Celular e Tecidual, Departamento de Ciências Morfofisiológicas, Pontifícia Universidade Católica do Rio Grande do Sul, Porto Alegre, RS, Brazil b
h i g h l i g h t s • Long-term astrocyte morphology has no influence on the different recovery patterns of stroke. • Ischemic and hemorrhagic stroke subtypes have similar long-term astrocyte morphology in perilesional sensorimotor cortex and dorsolateral striatum. • Long-term GFAP immunoreactivity profile is similar between ischemic and hemorrhagic stroke.
a r t i c l e
i n f o
Article history: Received 26 July 2014 Received in revised form 29 September 2014 Accepted 2 October 2014 Available online 12 October 2014 Keywords: Ischemic stroke Hemorrhagic stroke Glial fibrillary acidic protein Astrocytes Reactive astrogliosis
a b s t r a c t Stroke, broadly subdivided into ischemic and hemorrhagic subtypes, is a serious health-care problem worldwide. Previous studies have suggested ischemic and hemorrhagic stroke could present different functional recovery patterns. However, little attention has been given to this neurobiological finding. Coincidently, astrocyte morphology could be related to improved sensorimotor recovery after skilled reaching training and modulated by physical exercise and environmental enrichment. Therefore, it is possible that astrocyte morphology might be linked to differential recovery patterns between ischemic and hemorrhagic stroke. Thus, we decided to compare long-term GFAP-positive astrocyte morphology after ischemic (IS, n = 5), hemorrhagic (HS, n = 5) and sham (S, n = 5) stroke groups (induced by endothelin1, collagenase type IV-S and salina, respectively). Our results showed ischemic and hemorrhagic stroke subtypes induced similar long-term GFAP-positive astrocyte plasticity (P > 0.05) for all evaluated measures (regional and cellular optical density; astrocytic primary processes ramification and length; density of GFAP positive astrocytes) in perilesional sensorimotor cortex and striatum. These interesting negative results discourage similar studies focused on long-term plasticity of GFAP-positive astrocyte morphology and recovery comparison of stroke subtypes. © 2014 Elsevier B.V. All rights reserved.
Stroke, broadly subdivided into ischemic and hemorrhagic subtypes, is a serious health-care problem worldwide [1]. Some clinical studies have shown that by the time of hospital discharge hemorrhagic stroke presents a better functional improvement compared to ischemic stroke [2]. Moreover, ischemic stroke patients showed
∗ Corresponding author at: Faculdade de Enfermagem, Nutric¸ão e Fisioterapia, Pontifícia Universidade Católica do Rio Grande do Sul, Avenida Ipiranga, 6681, Prédio 12/8◦ andar, Porto Alegre CEP 90619-900, RS, Brazil. Tel.: +55 51 33203646; fax: +55 51 33203646. E-mail address: [email protected] (R.G. Mestriner). http://dx.doi.org/10.1016/j.bbr.2014.10.005 0166-4328/© 2014 Elsevier B.V. All rights reserved.
a longer functional recovery time window than those with the hemorrhagic etiology [3]. Yet, these recovery differences are not completely understood due to stroke heterogeneity [4]. Animal models aid researchers to control some factors and provide an unbiased analysis of stroke subtypes. We have recently published a study, using two controlled lesion “site and size” rat models, showing the spontaneous recovery pattern is better in hemorrhagic than in ischemic stroke [5]. Unfortunately, the neurobiological explanations for this finding remain poorly understood. Coincidentally, our research group has also shown astrocyte morphology could be related to improved sensorimotor recovery after a rehabilitation protocol [6] and modulated by physical
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exercise and environmental enrichment [7,8]. For example, skilled reaching training after collagenase-induced intracerebral hemorrhage increases the length of primary astrocytic GFAP-positive processes in perilesional tissue. This finding was correlated with functional forelimb recovery [6]. Moreover, enriched environment [8] and physical exercise [7] induce changes in the polarization of GFAP-positive astrocytes in healthy animals. Therefore, it is possible that astrocyte morphology might be linked to differential recovery patterns between ischemic and hemorrhagic stroke. Thus, we decided to compare long-term GFAP-positive astrocyte morphology after ischemic (IS) and hemorrhagic (HS) stroke using coronal sections left over from the study “Behavior outcome after ischemic and hemorrhagic stroke, with similar brain damage, in rats”, previously published in Behavioural Brain Research [5]. This was done in an effort to reduce the animals needed in research, according to the replacement, reduction and refinement principle (3R’s) [9]. The sections were taken randomly from 15 brains of Wistar rats divided into the following groups: Sham (n = 5), ischemic (IS) (n = 5) or hemorrhagic (HS) (n = 5). Brain ischemia or hemorrhage was induced by endotelin-1 (ET-1) and collagenase type IV-S microinjections, respectively (see Mestriner et al., 2013 for detailed description) [5]. The following morphological parameters were evaluated in GFAP positive astrocytes: (1) semi-quantitative analysis of GFAP imunohistochemistry intensity, measuring regional and cellular optical density; (2) astrocytic morphology and polarization (primary processes ramification and length), using Sholl concentric circles analysis; (3) density of GFAP positive astrocytes (number of GFAP positive astrocytes/mm2 ), using planar morphometry. All measurements were made in the perilesional sensorimotor cortex and striatum at 30 days post-surgery (Fig. 1). For immunohistochemistry, coronal brain sections (50 m) were obtained using a cryostat (Leica, Germany). They approximately ranged (relative to Bregma) from +1.80 mm (rostrally) to −0.70 mm (caudally). Briefly, brain sections were post-fixed in 4% PF for 15 min. After three washes in cold phosphate buffered saline (PBS, pH 7.4), GFAP immunohistochemistry was performed as previously described [7]. Sections were blocked for endogenous peroxidases (3% hydrogen peroxide in PBS) for 30 min, washed in PBS containing 0.4% Triton X-100 (PBS-Tx) and blocked with 2% bovine serum albumin (BSA) in PBS-Tx for 30 min. Sections were then incubated with anti-GFAP polyclonal antibody raised in rabbit (Z033401-2 – Dako), diluted 1:500 in PBS-Tx for 48 h at 4 ◦ C. After two washes in PBS-Tx, the sections were incubated in peroxidaseconjugated goat anti-rabbit IgG antibody (A0545 – Sigma–Aldrich), diluted 1:200 in PBS-Tx at room temperature for 2 h. Sections were then washed two times in PBS, and the GFAP immunostaining was performed by incubating the sections in a medium containing 0.06% 3,3 -diaminobenzidine (DAB, Sigma–Aldrich) dissolved in PBS for 10 min, and in the same solution containing 1 L of 3% H2 O2 per mL of DAB medium for an additional 10 min. After the DAB + H2 O2 revelation, the sections were rinsed in PBS, dehydrated in series of increasing ethanol concentrations (70, 90 and 100%), cleared with xylene and covered with Entellan (Merck) and coverslips. As a control to rule out unspecific binding, in a few sections the primary antibody was omitted and replaced by PBS-Tx. In order to minimize differences in the staining of astrocytes and in background levels, the brains in all experimental groups were fixed, cryoprotected and post-fixed in identical solutions for the same length of time, processed at the same time and incubated in the same immunostaining medium for the same period of time. The number of GFAP-immunoreactive astrocytes per mm2 in the surrounding damaged tissue was estimated using an Olympus BX 50 microscope coupled to a Motic Images Plus 2.0 camera and Image Pro Plus (Image Pro-Plus 6.1, Media Cybernetics, Silver Spring, EUA)
software. Our interested regions were sensorimotor cortex and dorsolateral striatum. For this analysis, four digitized images (20×) from surrounding injured tissue (two for each cortex and striatum) were obtained from each section (Fig. 1). Three sections from each animal were analyzed (a total of 12 images analyzed per animal – six from cortex and six from striatum). Two randomized squares measuring 5828 m2 and named areas of interest (AOIs) were overlaid on each image (Fig. 1). The astrocytes located inside this square or intersected by the upper and/or right edges of the square were counted. Astrocytes intersected by the lower and/or left edges of the square were not counted [7,8]. We established an area of analysis beginning at approximately 50 m laterally to lesion border for all morphological measurements to avoid errors related to complex overlapping of astrocytes bodies and process, blood vessels and artifacts in an immediately surrounding tissue to lesion (Fig. 1). The intensity of GFAP immunoreactivity was measured using semi-quantitative densitometric analysis [7,8] with the same software employed to estimate the astrocytic density. The same images and AOIs (5828 m2 ) used to estimate astrocytic density were used in the analysis of regional optical density (OD). The images were converted to an 8-bit gray scale (256 gray levels) and the AOIs were overlaid on each image. For the analysis of cellular OD, two astrocytes GFAP-positive located inside the first AOI (5828 m2 ) were randomly selected to cellular OD assessment. Thus, a new AOI measuring 10.37 m2 was placed over analyzed astrocytic soma in each image (processes were not measured). A number of 12 astrocytes per structure of interest were analyzed by animal. All lighting conditions and magnifications were kept constant during the process of capturing the images. Blood vessels and other artifacts were avoided and the background correction was performed according to the formula previously described [10]. The morphological analysis was done using the same images employed to measure cellular optical density. For the analysis of astrocytic ramification, an adaptation of Sholl’s concentric circles technique was used [6–8]. Briefly, seven virtual circles with 3.91 m intervals were drawn around each astrocyte. The degree of ramification of the astrocytes was measured by counting the number of times the astrocytic processes intersected with each virtual circle around the astrocytes. Primary process quantification was performed by counting the processes extending directly from the soma in both the lateral and central quadrants of astrocytes in the same sections. The longest primary process in each quadrant was measured by tracing the process with a manual measurement tool found in the Image Pro Plus software. All morphological data were obtained and analyzed by researchers blind to group identity (images capture and measurements). Data normality distribution was tested using the Kolmogorov–Smirnov test and showed a parametric profile. Data was analyzed using one-way ANOVA followed by Bonferroni post hoc test, when appropriate. Pearson correlation coefficient and analysis of co-variance (ANCOVA) were performed to assess the influence of astrocyte features on ladder walk performance. Behavioral data was obtained from our previous study [5]. All variables were expressed as mean ± standard error of the mean (SEM). Results were considered significant when P ≤ 0.05. SPSS 16.0 (Statistical Package for the Social Sciences, Inc., Chicago, USA) and G*Power 3.1 software were used for data analysis. In our study, GFAP-positive astrocytes surrounding damaged sensorimotor cortex and dorsolateral striatum (approximately from 50 m to 210 m laterally to border of tissue lost) were analyzed. In the qualitative analysis, it was possible to observe individual astrocyte’s soma and proximal processes for all groups. As expected, a highly complex network of GFAP-positive cells was observed in the tissue adjacent to the core of damage for both stroke
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Fig. 1. GFAP-positive astrocytic endpoints (regional and cellular optical density; primary processes number and length; and astrocytic density) and figure showing the morphological assessment (A–M). Fields 1–4 represent the evaluated surrounding tissue. Brain slice with damaged sensorimotor cortex and dorsolateral striatum (gray areas). (M) Amplified fields with areas of interest (AOIs) used to measure astrocytic density (mm2 ) and regional optical density. Dashed lines indicated the included edges. (L) Example of astrocyte with overlaid AOI (in white) used to evaluate optical cellular density and concentric circles (Sholl’s concentric circles method). (D) Dorsal; (V) ventral; (R) right; (L) left; (OD) optical density. Difference between sham and both stroke groups at *P ≤ 0.05; **P ≤ 0.01 and ***P ≤ 0.001. No differences were found between stroke groups. Data are presented as mean ± standard error of the mean (SEM).
groups. This intricate network was less complex slightly distal to lesion border. Then, individual astrocyte morphology was distinguishable (Fig. 2). In order to confirm our qualitative morphological findings, quantitative and semi-quantitative evaluations were performed, respectively, involving an estimation of lesion volume and length, astrocytic density, primary processes characteristics (number and length) and measurements of regional and cellular optical density. One-way ANOVA revealed main effects for “lesion volume” (F2,14 = 39.13/P < 0.001) and “lesion length” (F2,14 = 21.35/P < 0.001). The achieved power (1 − ˇ) was 0.99 for both factors. Bonferroni post hoc tests showed differences between sham and both stroke subtypes for lesion volume (P < 0.001) and lesion length (P < 0.001). No additional differences were found between ischemic and hemorrhagic stroke groups (P = 1.00 for lesion volume and lesion length), similarly to previously published [5]. Moreover, in order to ensure similar behavior as shown previously, we compared skilled walking in the current sample with Mestriner et al., 2013 full data [5]. ANOVA revealed main effects for “Group” (F5,45 = 20.98/P < 0.001). The achieved power (1 − ˇ) was 1.00. Bonferroni post hoc tests showed both Sham groups (current sample vs original sample) were different from all stroke groups (P ≤ 0.02). Additionally, no differences were found between both Sham groups (P = 1.00) as well as no differences were revealed
between all stroke groups (current vs original sample) (P > 0.07). Thus, present findings corroborate with our previously published study in terms of skilled walking performance [5]. Analysis of astrocytic density revealed significant main effects for sensorimotor cortex (F(2,14) = 9.18, P < 0.01, 1 − ˇ = 0.98), dorsolateral striatum (F(2,14) = 6.95, P ≤ 0.01, 1 − ˇ = 0.97) and combined (cortex and striatum) analysis (F(2,14) = 8.42, P ≤ 0.01, 1 − ˇ = 0.97). Major group differences were found between S and stroke rats (IS and HS) in the sensorimotor cortex (P ≤ 0.01), dorsolateral striatum (P ≤ 0.05 for IS and P ≤ 0.01 for HS) and combined structures (P ≤ 0.01). No differences were found between IS and HS groups in any analysis (P = 1.0). These findings showed that stroke induced an increased density of GFAP-positive astrocytes, as expected; both stroke subtypes have similar astrocytic density (Fig. 1). One-way ANOVA showed significant main effects in terms of the regional and cellular optical density for sensorimotor cortex (F(2,14) = 46.46, P ≤ 0.001, 1 − ˇ = 1.00/F(2,14) = 29.69, P ≤ 0.001, 1 − ˇ = 0.99), dorsolateral striatum (F(2,14) = 27.86, P ≤ 0.001, 1 − ˇ = 0.99/F(2,14) = 24.64, P ≤ 0.001, 1 − ˇ = 0.99) and combined (cortex and striatum) analysis (F(2,14) = 29.30, P ≤ 0.001, 1 − ˇ = 0.99/F(2,14) = 55.59, P ≤ 0.001, 1 − ˇ = 0.99), respectively. Bonferroni post hoc tests revealed significant differences between S and both stroke subtypes (IS and HS) (P ≤ 0.001) for all analysis. No differences between the stroke subtypes were found for
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Fig. 2. Digitized images after GFAP immunohistochemistry (20×). Sensorimotor cortex of (A) sham, (B) ischemic and (C) hemorrhagic stroke; dorsolateral striatum of (D) sham, (E) ischemic and (F) hemorrhagic stroke. Scale bar: 35 m (20×).
sensorimotor cortex (OD regional P = 0.51/OD cellular P = 0.15); dorsolateral striatum (OD regional P = 1.0/OD cellular P = 0.58) and these combined structures (OD regional P = 0.78/OD cellular P = 1.0) suggesting that both stroke subtypes were comparable in terms of GFAP imunorreactivity. There are GFAP-positive astrocytic ramification differences (F(2,14) = 8.59, P ≤ 0.01, 1 − ˇ = 0.95 for sensorimotor cortex/F(2,14) = 9.14, P ≤ 0.01, 1 − ˇ = 0.97 for dorsolateral striatum) as well as primary processes length main effects (F(2,14) = 10.06, P ≤ 0.01, 1 − ˇ = 0.97 for sensorimotor cortex/F(2,14) = 12.41, P ≤ 0.001, 1 − ˇ = 0.99 for dorsolateral striatum). As expected, we observed differences between sham vs stroke subtypes for analyzed structures and these morphological characteristics (P < 0.01). However, no differences were found between IS and HS for astrocytic ramification (P > 0.68) or processes length (P > 0.23) for both cortex and striatum. These findings revealed no longterm differences in GFAP-positive astrocytes ramification and/or primary processes length after ischemic or hemorrhagic stroke subtypes. Moreover, our results showed significant correlations between ladder walk performance and most features evaluated in astrocytes: cortical cellular density (r = 0.77, P = 0.001); striatal cellular density (r = 0.71, P = 0.03); regional optical density in the cortex (r = 0.68, P = 0.005); regional optical density in the striatum (r = 0.61, P = 0.01); cellular optical density in the striatum (r = 0.61, P = 0.01); number of cortical primary process (r = 0.53, P = 0.01); number of striatal primary process (r = 0.73, P = 0.002); cortical primary process (length) (r = 0.77, P = 0.001); and striatal primary process (length) (r = 0.54, P = 0.03). Non-significant correlation was observed only for cellular optical density in the cortex (r = 0.46, P = 0.08). However, ANCOVA analyses showed skilled walking errors and astrocyte morphology did not influence each other, as follow: cortical astrocyte density (F1,14 = 0.123, P = 0.76); striatal astrocyte density (F1,14 = 0.76, P = 0.81); regional
optical density in the cortex (F1,14 = 0.72, P = 0.81); regional optical density in the striatum (F1,14 = 0.005, P = 0.95); cellular optical density in the cortex (F1,14 = 0.52, P = 0.55); cellular optical density in the striatum (F1,14 = 0.28, P = 0.65); cortical primary process (number) (F1,14 = 0.16, P = 0.91); striatal primary process (number) (F1,14 = 0.35, P = 0.87); cortical primary process (length) (F1,14 = 0.19, P = 0.90); and striatal primary process (length) (F1,14 = 0.26, P = 0.66). Altogether, our findings revealed astrocytes morphology and skilled walking behavior did not influence each other. Stroke probably acted as a confounding factor in these significant correlations”. Rodent stroke models are largely used to provide in vivo mechanisms of neuroprotection and neural repair after stroke [5,6,11,12]. Several preclinical models should be properly designed and compared to understand plasticity mechanisms [11,12]. Accordingly, our research group has recently shown a comparative study design to evaluate ischemic and hemorrhagic stroke subtypes [5]. This design was thought to control experimental lesion volume, length and brain compromised areas; thus, the impact of stroke subtype on several pathophysiological mechanisms could be better understood in a comparative manner. Reactive astrocytes in neurotrauma, stroke, or neurodegeneration are thought to undergo cellular hypertrophy [13]. The hallmark of this reaction is an expressive upregulation of intermediate filaments, such as glial fibrillary acidic protein (GFAP) [14]. Reactive astrocytes might either reduce or exacerbate the damage to neurons depending on the time-point or post-injury stage as well as injury severity [15]. An overwhelming number of studies show any central nervous system injury increases astrocyte expression of intermediate filaments, such as GFAP [16]. However, to the best of our knowledge, this is the first study to evaluate GFAP-positive astrogliosis after ischemic and hemorrhagic stroke in a rational controlled and comparative design.
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On one hand, it is reasonable to consider brain repair is similar in all forms of stroke [11] despite the known differences in sensorimotor recovery between subtypes [5]. Astrocyte could contribute to damage by their role in spreading depression waves [17] or by sending apoptotic messengers or other deleterious molecules to otherwise healthy regions via gap junction channels [18]. They also inhibit regeneration by the glial scar [19,20]. These mechanisms may occur independently of injury etiology. On the other hand, morphological astrocytes profile at 30 days may not reflect earlier survival times, when differences may very well be strong. For example, reactive astrocytes could modulate the neurovascular unit differentially, at early and late stages after stroke [21]. Thus, its unknown if early astrocyte changes could be similar to all stroke subtypes. Moreover, early astrocyte changes may influence in longterm functional stroke recovery. This point is a limitation of our study design and could be matter of further investigation. As showed, the current findings could discourage similar studies focused on long-term astrocyte morphology and comparisons of spontaneous stroke subtypes recovery. Unfortunately, there were no previous studies comparing brain plasticity between controlled ischemic and hemorrhagic damage to expand this discussion. Our research group has previously shown GFAP-positive astrocytes modulated by skilled reaching training, which it was correlated with an improved sensorimotor behavior [6]. However, it is unknown if rehabilitative interventions, such as skilled reaching training, enriched rehabilitation, treadmill exercise, or pharmacological strategies could differentially modulate GFAP expression and behavior after each stroke subtype; it could be a subject for further investigation. Finally, these findings contribute toward the knowledge of the role of reactive astrogliosis and astrocyte morphology after different stroke. Conflict of interest The authors declare they have no conflict of interest. Acknowledgements The authors thank Ms. Raquel Matos for her technical assistance. This study was supported by the Brazilians Funding Agencies CNPq, CAPES and FAPERGS. Mestriner was a recipient of a PhD scholarship from CNPq. Léder Leal Xavier and Carlos Alexandre Netto are CNPq investigators.
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