Recombinant Integrin β1 Signal Peptide Blocks Gliosis Induced by Aβ Oligomers

Glial cells participate actively in the early cognitive decline in Alzheimer’s disease (AD) pathology. In fact, recent studies have found molecular and functional abnormalities in astrocytes and microglia in both animal models and brains of patients suffering from this pathology. In this regard, reactive gliosis intimately associated with amyloid plaques has become a pathological hallmark of AD. A recent study from our laboratory reports that astrocyte reactivity is caused by a direct interaction between amyloid beta (Aβ) oligomers and integrin β1. Here, we have generated four recombinant peptides including the extracellular domain of integrin β1, and evaluated their capacity both to bind in vitro to Aβ oligomers and to prevent in vivo Aβ oligomer-induced gliosis and endoplasmic reticulum stress. We have identified the minimal region of integrin β1 that binds to Aβ oligomers. This region is called signal peptide and corresponds to the first 20 amino acids of the integrin β1 N-terminal domain. This recombinant integrin β1 signal peptide prevented Aβ oligomer-induced ROS generation in primary astrocyte cultures. Furthermore, we carried out intrahippocampal injection in adult mice of recombinant integrin β1 signal peptide combined with or without Aβ oligomers and we evaluated by immunohistochemistry both astrogliosis and microgliosis as well as endoplasmic reticulum stress. The results show that recombinant integrin β1 signal peptide precluded both astrogliosis and microgliosis and endoplasmic reticulum stress mediated by Aβ oligomers in vivo. We have developed a molecular tool that blocks the activation of the molecular cascade that mediates gliosis via Aβ oligomer/integrin β1 signaling.


Introduction
Alzheimer's disease (AD) is the most common form of dementia and the most prevalent neurodegenerative disease [1]. Given that the first description made by Alois Alzheimer about pre-senile dementia refers to the formation of senile amyloid plaques and neurofibrillary tangles (aggregates of hyperphosphorylated tau protein) these elements are key pathological hallmarks of AD [2][3][4][5][6][7]. The formation of neurofibrillary tangles follows wellestablished patterns, while senile plaques appear and distribute in a random manner. The predictable alteration in the pattern and severity of the pathology permits the distinction of initial, intermediate and advanced stages based on investigations carried out by Braak these data show that R s peptide diminish Aβ oligomer-induced gliosis by interfering with integrin β1 signaling.

Integrin β1 Signal Peptide Specifically Binds to Aβ Oligomers
First, we analyzed the amino acid sequence of integrin β1 and selected four regions from its extracellular domain. The first region was constituted by the first 20 amino acids (aa) and it was identified as R s , the second one, R w , included up to aa 139, the third region covered aa 1 to 378 including the VWA domain (R d ), and the last region included the whole extracellular domain (from aa 1 to 728, R t ) ( Figure 1A). Now, to determine what amino acid stretch could represent an effective binding domain for Aβ oligomers, four recombinant GST fusion proteins were generated (R s , R d , R w , and R t , fused to the GST protein), and their binding capacities to Aβ oligomers were determined by affinity chromatography as described in the Experimental procedures section. As shown in Figure 1B, the four fused proteins bound not only to the monomeric Aβ peptide (the most intense band) but also to the oligomeric forms, the strongest interaction being between the oligomeric forms with the GST-R s recombinant fusion protein ( Figure 1B, lane 7). On the other hand, in order to verify that GST protein (GST 0 ) was not involved in the interaction between Aβ and the fused proteins GST-R s , GST-R d , GST-R w , and GST-R t , we examined this possibility by affinity chromatography. As shown in Figure 1B (lane 2), GST 0 had no ability to bind either monomeric or oligomeric Aβ. Figure 1B (lane 1) represents the reconstitution of synthetic Aβ (as an internal control) in its different forms visualized by Western blotting. Together, these findings identified that the signal peptide (R s ) of the extracellular domain of integrin β1 was responsible for binding to Aβ oligomers in vitro.   After incubation, glutathione beads were washed and proteins separated by SDS-PAGE under nonreducing conditions and analyzed by Western blot using anti-Aβ1-42 antibody (6E10, from Covance). (C) ROS generation was measured by fluorimetry with 10 µM CM-H2DCFDA. Data are expressed as the relative fluorescence normalized to values of untreated or treated cells (100%). *** p < 0.001 compared to non-treated cells; # p < 0.05 compared to GST 0 ; unpaired one-way ANOVA.

R s Peptide Blocks Aβ Oligomer-Induced ROS Generation in Cultured Astrocytes
Next, we investigated whether GST-R s affected ROS generation mediated by Aβ oligomers in primary astrocyte cultures, as previously shown [38]. For that, we treated primary astrocyte cultures with 5 µM Aβ oligomers for 60 min alone or together with 5 µg/µL GST 0 (control) or 5 µg/µL GST-R s , and measured ROS levels by fluorimetry using 10 µM CM-H2DCFDA for 20 min. As expected, Aβ oligomers induced ROS generation ( Figure 1C, empty bar). Regarding GST 0 , this peptide did not interfere in Aβ oligomermediated ROS generation ( Figure 1C, gray bar). Nevertheless, GST-R s totally prevented ROS generation mediated by Aβ oligomers ( Figure 1C, solid bar). Taken together, these results show that integrin β1 signal peptide (R s ) binds in vitro to Aβ oligomers, and that it is able to prevent ROS generation induced by Aβ oligomers in primary astrocyte cultures.

Aβ Oligomers Trigger Gliosis in Mouse Hippocampus In Vivo
Aβ injection in mouse brain causes reactive astrogliosis in the dentate gyrus (DG) [38]. However, it is still unclear whether Aβ injection in mice brain also drives microgliosis. To investigate that possibility, we performed intrahippocampal injections of vehicle (control) or Aβ oligomers (Aβ) and examined astrocyte-and microglia-occupied areas by immunohistochemistry with astrocyte (GFAP and S100β) and microglia (Iba1) markers in dentate gyrus (DG). As expected, the intrahippocampal administration of Aβ strongly increased the presence of both the GFAP and S100β markers compared to control (Figure 2A). In addition, Aβ also boosted the presence of the Iba1 marker in DG compared to control (Figure 2A). Quantification of the immunohistochemical analysis showed significant increases in the GFAP, S100β and Iba1 markers in DG values due to Aβ treatment compared to control (Figure 2B, 1.00 ± 0.04 vs. 1.21 ± 0.04 for GFAP, 1.00 ± 0.04 vs. 1.46 ± 0.08 for S100β 1.00 ± 0.07 vs. 1.31 ± 0.08 for Iba1). These results confirm that Aβ induces astrogliosis and show that Aβ oligomers also lead to microgliosis in adult mouse DG. 2A). In addition, Aβ also boosted the presence of the Iba1 marker in DG compared to control (Figure 2A). Quantification of the immunohistochemical analysis showed significant increases in the GFAP, S100β and Iba1 markers in DG values due to Aβ treatment compared to control (Figure 2B, 1.00 ± 0.04 vs. 1.21 ± 0.04 for GFAP, 1.00 ± 0.04 vs. 1.46 ± 0.08 for S100β 1.00 ± 0.07 vs. 1.31 ± 0.08 for Iba1). These results confirm that Aβ induces astrogliosis and show that Aβ oligomers also lead to microgliosis in adult mouse DG.  Reactive astrocytes and microglia in the dentate gyrus (DG) of Aβ-injected mice. (A) Coronal sections of mouse brains were immunostained by DAB assay 7 days post -injection with Aβ or with vehicle (Ctrl). Photomicrographs show GFAP and S100β immunolabeling in astrocytes and Iba1 immunolabeling in microglia of the dentate gyrus. Scale bar: 100 µm and Scale bar in zoom is 50 µm. It is included in caption.Inset: 50 µm. (B) Box plot graphs show quantitative analysis of labelled areas for GFAP, S100β and Iba1 under Aβ and control conditions in the DG. Data are presented as the mean ± S.E.M. Fifteen slices from five animals were analyzed per condition. *** p < 0.001, ** p < 0.01, * p < 0.05 compared with Aβ-injected mice; unpaired Student's test.

R s Peptide Prevents Glia Reactivity in the DG of Aβ Oligomer-Injected Mice Brain
Before examining the functionality of the GST-R s fused protein in vivo, we evaluated whether GST 0 affected astrocyte and microglia reactivity in Aβ oligomer-injected brain. For that, we performed intrahippocampal injections of Aβ and Aβ with GST 0 (Aβ + GST 0 ) and quantified the changes in astrocyte and microglia morphology as described in the previous section. As shown in Figure 3A, the intrahippocampal administration of the combination of Aβ + GST 0 did not modify the area occupied by both the GFAP and S100β markers compared to Aβ alone. However, the area occupied by Iba1 staining appeared increased in the combination Aβ + GST 0 when it was compared to Aβ alone ( Figure 3A). Quantification of the immunohistochemical analysis showed that GST 0 in the presence of Aβ did not produce any significant change in GFAP and S100β staining ( Figure 3B; 0.94 ± 0.09 vs. 0.64 ± 0.03 for GFAP, 0.93 ± 0.08 vs. 0.67 ± 0.06 for S100β, whereas it caused microgliosis as compared to Aβ alone (1.00 ± 0.04 vs. 0.77 ± 0.03 for Iba1). These results suggest that the GST 0 protein did not reduce Aβ-dependent astrogliosis and/or microgliosis.
For that, we performed intrahippocampal injections of Aβ and Aβ with GST0 (Aβ + GST0) and quantified the changes in astrocyte and microglia morphology as described in the previous section. As shown in Figure 3A, the intrahippocampal administration of the combination of Aβ + GST0 did not modify the area occupied by both the GFAP and S100β markers compared to Aβ alone. However, the area occupied by Iba1 staining appeared increased in the combination Aβ + GST0 when it was compared to Aβ alone ( Figure 3A). Quantification of the immunohistochemical analysis showed that GST0 in the presence of Aβ did not produce any significant change in GFAP and S100β staining ( Figure 3B; 0.94 ± 0.09 vs. 0.64 ± 0.03 for GFAP, 0.93 ± 0.08 vs. 0.67 ± 0.06 for S100β, whereas it caused microgliosis as compared to Aβ alone (1.00 ± 0.04 vs. 0.77 ± 0.03 for Iba1). These results suggest that the GST0 protein did not reduce Aβ-dependent astrogliosis and/or microgliosis.  Based on that, we examined the ability of recombinant GST-R s peptide to prevent Aβ-mediated astrogliosis in brain. For that, we performed intrahippocampal injections of Aβ, and Aβ with GST-R s peptide (Aβ + GST-R s ) and the glial changes were analyzed and quantified. As shown in Figure 4A, the intrahippocampal administration of the combination of Aβ + GST-R s strongly reduced the presence of three-GFAP, S100β and Iba1-markers compared to Aβ.
Aβ + GST-Rs strongly reduced the presence of three-GFAP, S100β and Iba1-markers compared to Aβ.
Quantification of the immunohistochemical analysis showed a significant decrease in GFAP, S100β and Iba 1 ( Figure 4B) in the presence of Aβ + GST-Rs compared to Aβ (1.05 ± 0.10 vs. 1.30 ± 0.05 for GFAP, 1.03 ± 0.08 vs. 1.484 ± 0.167 for S100β 1.00 ± 0.05 vs. 1.22 ± 0.04 for Iba1). These results point out that integrin β1 signal peptide Rs blocks Aβ-induced not only astrogliosis but also in microgliosis in adult mouse DG. Photomicrographs show GFAP and S100β immunolabeling in astrocytes and Iba1 immunolabeling in microglia of the dentate gyrus. Scale bar: 100 µm and Scale bar in zoom is 50 µm. It is included in caption. 50 µm (B) Box plot graphs show quantitative analysis of labelled areas for GFAP, S100β and Iba1 under Aβ and Aβ + GST-Rs in the DG. Data are presented as the mean ± S.E.M. Fifteen slices from five animals were analyzed per condition. ** p < 0.01 compared with Aβ-injected mice; unpaired Student's test. Photomicrographs show GFAP and S100β immunolabeling in astrocytes and Iba1 immunolabeling in microglia of the dentate gyrus. Scale bar: 100 µm and Scale bar in zoom is 50 µm. It is included in caption. 50 µm (B) Box plot graphs show quantitative analysis of labelled areas for GFAP, S100β and Iba1 under Aβ and Aβ + GST-R s in the DG. Data are presented as the mean ± S.E.M. Fifteen slices from five animals were analyzed per condition. ** p < 0.01 compared with Aβ-injected mice; unpaired Student's test.

R s Peptide Reduces Endoplasmic Reticulum Stress in Astrocytes in DG of Aβ Oligomer-Injected Mice Brain
Acute injection of Aβ oligomers in mouse brain induces GRP78 chaperone protein overexpression particularly in astrocytes [39], being used as an endoplasmic reticulum stress marker. Therefore, we investigated whether recombinant R s fused protein to GST (GST-R s ) could also prevent endoplasmic reticulum stress in astrocytes after intrahippocampal Aβ injection. Accordingly, we carried out a double immunostaining assay for S100β and GRP78 of brain tissues previously injected with Aβ, Aβ + GST-R s and Aβ + GST 0 . Intrahippocampal administration of the combination of recombinant GST-R s peptide and Aβ oligomers strongly reduced GRP78 expression in S100β-positive astrocytes compared to Aβ oligomers alone ( Figure 5A,B). Furthermore, the combination of GST 0 and Aβ oligomers did not alter the effect induced by Aβ alone ( Figure 5B). Quantification of immunofluorescence staining showed a significant decrease in GRP78 in S100β values in DG from brains injected with GST-R s fusion protein compared to control (Aβ-injected mice) (26.95 ± 1.01 vs. 30.64 ± 1.24) ( Figure 5A). In contrast, GST 0 protein did not produce any effect in Aβ-induced endoplasmic reticulum stress in S100β ( Figure 5B) values compared to Aβ alone (21.42 ± 2.48 vs. 22.07 ± 1.36). These findings suggest that R s also prevents endoplasmic reticulum stress induced by Aβ oligomers.
overexpression particularly in astrocytes [39], being used as an endoplasmic reticulum stress marker. Therefore, we investigated whether recombinant Rs fused protein to GST (GST-Rs) could also prevent endoplasmic reticulum stress in astrocytes after intrahippocampal Aβ injection. Accordingly, we carried out a double immunostaining assay for S100β and GRP78 of brain tissues previously injected with Aβ, Aβ + GST-Rs and Aβ + GST0. Intrahippocampal administration of the combination of recombinant GST-Rs peptide and Aβ oligomers strongly reduced GRP78 expression in S100β-positive astrocytes compared to Aβ oligomers alone ( Figure 5A,B). Furthermore, the combination of GST0 and Aβ oligomers did not alter the effect induced by Aβ alone ( Figure 5B). Quantification of immunofluorescence staining showed a significant decrease in GRP78 in S100β values in DG from brains injected with GST-Rs fusion protein compared to control (Aβ-injected mice) (26.95 ± 1.01 vs. 30.64 ± 1.24) ( Figure 5A). In contrast, GST0 protein did not produce any effect in Aβ-induced endoplasmic reticulum stress in S100β ( Figure 5B) values compared to Aβ alone (21.42 ± 2.48 vs. 22.07 ± 1.36). These findings suggest that Rs also prevents endoplasmic reticulum stress induced by Aβ oligomers. Figure 5. GST-R s polypeptide reduces GRP78 expression in S100β-positive astrocytes of Aβ-injected mouse brains. Photomicrographs of double immunofluorescence staining for S100β (red) and GRP78 (green) on DG of animals injected with different: Aβ and Aβ + GST-R s (A) or Aβ and Aβ + GST 0 (B). Quantitative analysis of fluorescence intensity was performed for GRP78 levels in S100β-positive astrocytes in dentate gyrus after Aβ and Aβ + GST-Rs (A) or Aβ and Aβ + GST 0 (B). Scale bar in zoom area: 20 µm. Data are presented as the mean ± SEM. Fifteen slices from five animals were analyzed per condition. ns: non-significant; * p < 0.05 compared with Aβ-injected mouse; unpaired Student's test.

Discussion
Our study identifies the integrin β1 minimal region that binds to Aβ oligomers. This region spans from aa 1 to aa 20 and corresponds to integrin β1 signal peptide (R s peptide). From a functional point of view, this peptide is a very useful tool to block Aβ oligomer-induced ROS generation in primary astrocyte cultures and also in vivo when R s peptide in combination with Aβ oligomers is directly injected into the mice hippocampus. In this scenario, astroglial stress, astrogliosis and even microgliosis induced by Aβ oligomers are efficiently prevented.
Several investigations postulate that there are many potential receptors localized at neuronal synapses with both high affinity for Aβ peptide and the ability to intracellularly transduce the toxic instructions emanating from Aβ oligomers [40]. These include NMDA receptors that are directly activated by Aβ oligomers, altering its physiological function [41], although those that seem to be acquiring increasing relevance are integrins. In fact, the interaction between integrins and Aβ oligomers promotes neurotoxicity, inhibition of LTP and an increase in spine density [26,42]. In this regard, synthetic Aβ monomer binds through its amino acid sequence RHDS to the α2bβ3 integrin, being directly related to cerebral amyloid angiopathy, which contributes to dementia and AD [43].
Integrins control important cellular responses including proliferation, survival and cell migration [44]. All of them require the active participation of transducing molecules such as tyrosine kinases FAK, ILK and Src or small GTPases of the Rho family [44]. In addition, PKCs may also be involved in integrin-mediated signaling [45]. We have previously observed that Aβ oligomer-induced PKC phosphorylation is mediated by integrin β1 in astrocytes and in neurons [38]. Further, Aβ oligomers lead to NR2B subunit upregulation on neuronal membranes through the PKC signaling pathway [46]. Under these circumstances, integrin β1 transduces the message that Aβ oligomers brings, generating a cellular response which manifests itself in a higher permeability for calcium ions to alter cellular homeostasis [46]. Hence, depending on the stimulus or ligands, the same receptor along with its intracellular signaling molecules can switch on/off different pathways that lead to antagonistic cellular responses.
Currently, in addition to pharmacotherapy, gene therapeutic approaches for AD have entered phase I/II clinical trials [47]. The results of this preliminary study obtained with recombinant R s allow us to postulate a new pharmacological therapeutic alternative in AD. This recombinant peptide neutralizes Aβ oligomer activity from outside the cell ( Figure 6 panel B compared to panel A). In addition, Rs recombinant peptide is a useful tool that will aid understanding the molecular mechanisms of the deleterious actions initiated by Aβ oligomers both in vitro and in vivo.

Conclusions
We and others have described a key molecular relationship between integrin β1 and Aβ peptides required to modulate neuronal and glial biology [27,38,42,46]. The findings point out the molecular mechanism by which recombinant Rs peptide works in order to block Aβ oligomer intracellular signaling both in vitro and in vivo. The presence of this recombinant peptide in the extracellular medium interferes with binding between Aβ oligomers and its receptor, integrin β1, since the Rs peptide associates with Aβ oligomers, thus preventing it from binding to the endogenous integrin β1, and consequently avoiding the transmission of its toxic message. In fact, Rs peptide blocks ROS generation induced by Aβ oligomers and at the same time significantly reduces astroglial stress, astrogliosis and microgliosis. It is important to highlight that Rs peptide in turn protects the functional receptorial properties of integrin β1, allowing integrin β1 in the cell membrane to be accessible to physiological activators ( Figure 6). Future studies will allow us to investigate the efficacy of this peptide in preventing Aβ oligomers binding to other receptors.

Experimental Procedures
Animals. All experimental procedures (M20-2017-092) followed the European Directive 2010/63/EU and were approved by the ethics committee of the University of the

Conclusions
We and others have described a key molecular relationship between integrin β1 and Aβ peptides required to modulate neuronal and glial biology [27,38,42,46]. The findings point out the molecular mechanism by which recombinant R s peptide works in order to block Aβ oligomer intracellular signaling both in vitro and in vivo. The presence of this recombinant peptide in the extracellular medium interferes with binding between Aβ oligomers and its receptor, integrin β1, since the R s peptide associates with Aβ oligomers, thus preventing it from binding to the endogenous integrin β1, and consequently avoiding the transmission of its toxic message. In fact, R s peptide blocks ROS generation induced by Aβ oligomers and at the same time significantly reduces astroglial stress, astrogliosis and microgliosis. It is important to highlight that Rs peptide in turn protects the functional receptorial properties of integrin β1, allowing integrin β1 in the cell membrane to be accessible to physiological activators ( Figure 6). Future studies will allow us to investigate the efficacy of this peptide in preventing Aβ oligomers binding to other receptors.

Experimental Procedures
Animals. All experimental procedures (M20-2017-092) followed the European Directive 2010/63/EU and were approved by the ethics committee of the University of the Basque Country (UPV/EHU). Animals were housed in standard conditions under 12 h light/dark cycle and with ad libitum access to food water. All possible effort was made to minimize animal suffering and the number of animals used. Experiments were performed in C57BL6/J mice.
Preparation of Aβ 1-42 Oligomers. Aβ 1-42 oligomers were prepared as reported previously [48]. Briefly, Aβ1-42 was initially dissolved to 1 mM in hexafluoroisopropanol (Merck Life Science S.L.U., Madrid, Spain) and distributed aliquoted in sterile microcentrifuge tubes. Hexafluoroisopropanol was totally removed under vacuum in a speed vac system and the peptide film was stored at −80 • C. For the aggregation protocol, the peptide was first resuspended in anhydrous DMSO (Merck Life Science S.L.U., Madrid, Spain) to a concentration of 5 mM, to finally bring the peptide to a final concentration of 100 µM in Hams F-12 (Merck Life Science S.L.U., Madrid, Spain and to incubate it at 4 • C for 24 h. The preparation was then centrifuged at 14,000× g for 10 min, at 4 • C, to remove insoluble aggregates and the supernatants containing soluble Aβ 1-42 were transferred to clean tubes and stored at 4 • C. Plasmid Construct. The ITGβ1 fragments comprising amino acids 1-20 (R s ), 1-140 (R w ), 1-371 (R d ) and 1-728 (R t ) were generated by PCR amplification using pCMV6-XL5-ITGB1 (from Origene Technologies Inc. Rockville, MD, USA) as template (forward oligonucleotide, 5 -CGG AAT TCA TGA ATT TAC AAC C-3 and reverse oligonucleotides, 5 -CGG AAT TCA GCA AAC ACA CAG C-3 , 5 -CGG AAT TCG TCT TCA GCT CTC T-3 , 5 -CGG AAT TCA AGG GAA TTG TAT G-3 , 5 -CGG AAT TCG TCT GGA CCA GTG G-3 , each harboring EcoRI restriction sites (underlined). The EcoRI ITGβ1 extracellular fragments were subcloned into pGEX-4T3 (Merck Life Science S.L.U., Madrid, Spain) to generate the GST-R s , GST-R w , GST-R d and GST-R t fusion proteins. All GST-fused peptides were purified by affinity chromatography onto glutathione beads following standard procedures [49].
Binding Assay. In vitro binding assays with recombinant fusion proteins were performed as previously described [50]. Briefly, glutathione beads coated with recombinant fusion proteins (500 ng GST 0 , GST-R t , GST-R w , GST-R d or GST-R s ) were incubated with 100 pM Aβ oligomers in binding buffer (50 mM Tris-HCl pH7.5, 5 mM MgCl 2 , 20 mM KCl, 500 µg/mL BSA) for 1 h at RT. Immobilized GST beads were washed twice with binding buffer and five times with 50 mM Tris-HCl pH 7.5, 150 mM NaCl. Proteins were eluted adding sample buffer under non-reducing conditions and separated by SDS-PAGE followed by Western blot. Immunoreactive bands were visualized with anti-6E10 antibody and ECL.
Astrocyte Culture. Primary cultures of cerebral cortical astrocytes were prepared from P 0 -P 2 Sprague Dawley rats as previously described [51]. Cortical lobes were extracted and enzymatically digested with 400 µL of 2.5% trypsin and 40 µL of 0.5% deoxyribonuclease in Hank's Balanced Salt Solution (HBSS, Merck Life Science S.L.U., Madrid, Spain) for 15 min at 37 • C. The enzymatic reaction was stopped by adding IMDM medium supplemented with 10% FBS (Thermo Fisher Scientific, Madrid, Spain) and centrifuged at 300× g for 6 min. The cell pellet was resuspended in 1 mL of the same solution and mechanical dissociation was performed by using 21-and 23G-gauge cutting needles. The resulting cell suspension was centrifuged at 300× g for 6 min and plated onto 75 cm 2 flasks coated with 30 µg/mL Poly-D-Lysine. After 8 DIV, cells were plated onto PDL-coated plates and maintained for 2 days. The culture medium was replaced with IMDM with 1% FBS 24 h before Aβ treatment.
Image acquisition and analysis. Brightfield images were acquired with the Pannoramic MIDI II automated digital slide scanner (3DHistech Ltd., Budapest, Hungary). To analyze reactive gliosis, the area occupy by DAB divided by total area was measured.
Fluorescence immunostaining was observed with a Leica TCS SP8 microscope using a 63× oil-immersion objective to generate z-stack projections. For fluorescence intensity analysis, images were taken with the same settings for all experiment and the mean value along the stack profile was quantified with LAS AF Lite software, version 4.0, Leica Microsystems CMS GmbH, Shinjuku, Tokyo, Japan (Leica).
Statistical analysis. All data were expressed as the mean ± S.E.M. Statistical analyses were performed using absolute values. GraphPad Prism software (https://www.graphpad. com/scientific-software/prism/, accessed on 1 April 2022) was used applying one-way analysis of variance with post hoc Fisher's least significant difference (LSD) test for multiple comparisons and two-tailed, unpaired Student's t test for comparison of the two groups and control conditions.