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. Author manuscript; available in PMC: 2015 Jun 16.
Published in final edited form as: Stroke. 2011 Aug 11;42(11):3231–3237. doi: 10.1161/STROKEAHA.111.623280

Proliferating reactive astrocytes are regulated by Notch-1 in the peri-infarct area following stroke

Issei S Shimada 1,2, Alyssa Borders 1, Alexander Aronshtam 2, Jeffrey L Spees 2,*
PMCID: PMC4469355  NIHMSID: NIHMS316880  PMID: 21836083

Abstract

Background and Purpose

The formation of reactive astrocytes is common following CNS injuries such as stroke. However, the signaling pathway(s) that control reactive astrocyte formation or functions are poorly defined. Here we assess the affects of Notch 1 signaling in peri-infarct reactive astrocytes after stroke.

Methods

We examined reactive astrocyte formation in the peri-infarct area 3 days following distal Middle Cerebral Artery Occlusion (dMCAO), with or without Gamma-secretase inhibitor (GSI) treatment. To directly study the effects of inhibiting a GS cleavage target in reactive astrocytes, we generated GFAP-CreER™∷Notch 1 conditional knock out (GN) mice.

Results

GSI treatment after stroke decreased the number of proliferative GFAP-positive reactive astrocytes and RC2-positive reactive astrocytes directly adjacent to the infarct core. The decrease in reactive astrocytes correlated with an increased number of CD45-positive cells that invaded into the peri-infarct area. To study the influence of reactive astrocytes on immune cell invasion, ex vivo immune cell invasion studies were performed. We found that a gamma-secretase mediated pathway in astrocytes affected Jurkat cell invasion. Following Tamoxifen (TM) treatment, GN mice had a significantly decreased number of proliferating reactive astrocytes and RC2-positive reactive astrocytes. TM treatment also led to an increased number of CD45-positive cells that invaded the peri-infarct area.

Conclusions

Our results demonstrate that proliferating and RC2-positive reactive astrocytes are regulated by Notch 1 signal transduction and control immune cell invasion after stroke.

Keywords: RC2, MCAO, reactive astrocytes

INTRODUCTION

After brain injury, reactive astrocytes participate in the formation of a “glial scar”. Because the glial scar has been shown to inhibit axonal regeneration, reactive astrocytes have historically been considered as a barrier to neuronal repair1, 2. However, this negative view of reactive astrocytes is changing, as they have been shown to play several important roles in preserving neural tissue during the early phase of brain injuries2–4. Knockout mice lacking two intermediate filament proteins commonly expressed by astrocytes, GFAP and vimentin, exhibited attenuated reactive astrocyte formation, reduced glutamate transport, and increased infarct volumes 7 days following stroke5. Reactive astrocytes were also shown to limit cellular degeneration by maintaining and repairing the blood brain barrier (BBB) and decreasing immune cell infiltration after stab injury2, 6.

The emergence of reactive astrocytes in the peri-infarct area is one of the most obvious events in the brain after stroke. However, the signaling pathway(s) that control reactive astrocyte formation or their functions in the peri-infarct area are poorly defined. Gamma-secretase targeted proteins, such as Notch 1 and APP, are expressed by reactive astrocytes following brain injury7, 8. Here we determine the effects of Notch 1 signaling on a unique subpopulation of proliferative reactive astrocytes that localize adjacent to the infarct and that regulate the peri-infarct area after stroke.

METHODS

Mouse

All animal work was approved by the University of Vermont College of Medicine's Office of Animal Care in accordance with American Association for Accreditation of Laboratory Animal Care and National Institutes of Health guidelines. Focal cerebral ischemia was produced by permanently occluding the middle cerebral artery (MCA)9–11. Full methods are available online.

Please see http://stroke.ahajournals.org

RESULTS

RC2-positive reactive astrocytes express gamma-secretase cleavage products

To study reactive astrocytes in the brain after stroke, we performed distal middle cerebral artery occlusion (dMCAO) in mice. Reactive astrocytes expressed GFAP and/or RC2 in the cortical peri-infarct area after stroke (Figure 1A). We previously identified an RC2-positive subpopulation of reactive astrocytes that formed on the ipsilateral, but not the contralateral side of the brain following stroke11. At 3 days after stroke, the number of RC2–positive cells was significantly higher in the inner cortical layer adjacent to the infarct (200 μm radially from the infarct area) compared with the outer cortical layer (200 μm radially from the inner layer)(n = 3, p < 0.05; Figure 1B). Similarly, the number of GFAP/RC2-positive cells was significantly greater in the inner cortical layer compared with outer layer, demonstrating that RC2-positive reactive astrocytes appear directly adjacent to the infarct core after stroke (n = 3, p < 0.05; Figure 1B). Ki67/GFAP-positive proliferating reactive astrocytes were also observed adjacent to the infarct core after stroke (Figure 1C). The 3 day time point was chosen because the RC2 antigen and GFAP were highly expressed at this time relative to 1 day after stroke.

Figure 1. Heterogeneous reactive astrocytes exist in the peri-infarct area 3 days following stroke.

Figure 1

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(A, B) The innermost layer of GFAP-positive reactive astrocytes adjacent to the infarct expressed RC2. There were significantly more RC2-positive cells in the inner cortical layer compared with the outer cortical layer (n=3). Similarly, there were significantly more RC2/GFAP double-positive cells in the inner cortical layer (n=3). (C) Ki67/GFAP-positive proliferating reactive astrocytes located in the peri-infarct area 3 days following stroke. Nuclei were stained by DAPI. White boxes indicate the area of magnified view. Scale bars indicate 50 μm or 100 μm. * p < 0.05 compared with sham group.

Gamma-secretase activity is known to increase in the brain early after stroke7, 12. We examined the presence of gamma-secretase cleavage products APP Intracellular Domain (AICD) and Notch 1 Intracellular Domain (NICD1) in the peri-infarct area 3 days after stroke. By immunohistochemistry, GFAP-positive reactive astrocytes and RC2-positive reactive astrocytes both expressed AICD and NICD1 in the peri-infarct area (Figure 2A). We also observed expression and nuclear localization of AICD and NICD1 in NeuN-positive neurons (data not shown). These data suggested that gamma-secretase cleavage products may regulate reactive astrocytes and RC2-positive reactive astrocytes in the peri-infarct area after stroke.

Figure 2. Gamma-secretase inhibitor (GSI) treatment reduced AICD and GFAP expression levels in the peri-infarct area.

Figure 2

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(A) GFAP-positive cells and RC2-positive cells expressing AICD and NICD 1. Nuclei were stained by DAPI. Scale bars indicate 50 μm. (B) Time schedule of treatment. At the time of surgery, GSI or DMSO (vehicle) was injected. Mice were euthanized at 24 hours or 72 hours after treatment. (C) AICD expression levels increased in the peri-infarct area 1 day following stroke. In contrast, AICD expression levels decreased after GSI treatment compared with vehicle treatment. (n=6 mice for each group). (D) GFAP expression levels increased in the peri-infarct area 3 days following stroke. In contrast, GFAP expression levels decreased after GSI treatment compared with DMSO treatment (n=10–12 mice for each group). * p < 0.05 compared with DMSO treatment. ** p < 0.01 compared with sham group. *** p < 0.01 compared with DMSO treatment.

Formation of reactive astrocytes is disrupted by gamma-secretase inhibitor treatment

To determine whether gamma-secretase-mediated events controlled the formation of reactive astrocytes, we inhibited type I intracellular membrane protein cleavage with the gamma-secretase inhibitor, Dibenzazepine (GSI) (Figure 2B). In accordance with altered gamma-secretase activity early after stroke, we observed increased AICD expression levels in the peri-infarct area 1 day after stroke compared with AICD levels in sham-operated brains (n = 6, p <0.01, Figure 2C). Following GSI treatment, we observed decreased levels of AICD expression in the peri-infarct area compared with DMSO treatment (n = 6, p < 0.05; Figure 2C). Interestingly, overall NICD1 expression levels in the peri-infarct area did not increase following stroke compared to levels in sham animals. We did, however, observe a trend of decreased NICD1 expression levels in the peri-infarct area following GSI treatment compared with DMSO treatment (Sham, 106 ± 18.9 %; DMSO, 100 ± 15.4 %; GSI, 79 ± 7.4 %; mean ± s.e.m., n = 4–6 mice for each group). These data indicated that GSI treatment affected cleavage of type I intramembrane proteins in the peri-infarct area after stroke. As expected, GFAP protein expression levels were increased in the peri-infarct area 3 days after stroke compared with sham-operated brains. In contrast, GFAP protein expression levels were decreased 3 days after stroke following GSI treatment compared with DMSO treatment (n = 10–12, p < 0.01; Figure 2D).

We quantified the number of GFAP-positive reactive astrocytes after GSI treatment following stroke. The number of GFAP/Ki67-positive proliferating reactive astrocytes was significantly decreased by GSI treatment (n = 4, p < 0.05; Figure 3A). We quantified also the number of RC2-positive reactive astrocytes in the peri-infarct area after stroke and DMSO or GSI treatment. We observed a decreased number of RC2-positive reactive astrocytes in the cortical peri-infarct area after GSI treatment compared with vehicle treatment (n=3, p < 0.05; Figure 3B). These data indicated that gamma-secretase cleavage products play an important role in regulating the proliferating reactive astrocytes and the subpopulation of RC2-positive reactive astrocytes in the peri-infarct area following stroke. Notably, we did not observe a significant difference in the total numbers of GFAP-positive cells after stroke with GSI treatment compared with vehicle controls (DMSO, 5255.1 ± 437.9 cells/mm2; GSI, 5199.8 ± 231.8 cells/mm2; mean ± s.e.m., n = 4 mice for each group, p= 0.91).

Figure 3. The number of RC2-positive reactive astrocytes was significantly decreased following GSI treatment.

Figure 3

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(A) The number of Ki67- and GFAP-positive cells was decreased following GSI treatment compared with vehicle treatment 3 days after stroke (n=4 mice for each group). (B) The number of RC2-positive reactive astrocytes was decreased following GSI treatment 3 days after stroke (n=3 mice for each group). (C) CD45-positive cells in the peri-infarct area were surrounded by RC2- reactive astrocytes. The number of CD45-positive cells was decreased following GSI treatment. (n = 4–5 mice for each groups). Scale bars indicate 100 μm. * p < 0.05.

To examine whether DMSO or GSI treatment affected reactive astrocyte apoptosis/necrosis after stroke, we performed TUNEL assays for mice treated with PBS, DMSO or GSI. We observed many TUNEL-positive cells in the infarct areas of mice treated with PBS, DMSO and GSI (data not shown). However, we did not observe TUNEL- and GFAP-positive reactive astrocytes in the peri-infarct area 3 days following stroke (data not shown).

Reduced number of proliferating and RC2-positive reactive astrocytes and immune cell invasion into the peri-infarct area

One possible role of reactive astrocytes is to prevent immune cell invasion as reported in spinal cord injury and brain stab injury13, 14. In order to determine whether this was the case in our model, we quantified CD45-positive monocytes at the border of the infarct region following GSI treatment. Since monocytes and microglia express similar proteins, such as CD45 and CD11b, it is technically challenging to distinguish monocytes and microglia in the peri-infarct area. Therefore, we used CD45 as a marker and distinguished monocytes by their round morphology in the peri-infarct area. We observed a significant increase in the number of CD45-positive cells in the peri-infarct area following GSI treatment compared with the number in vehicle-treated controls (n = 4–5, p < 0.05; Figure 3D).

To study the effects of astrocytes on immune cell invasion, we isolated astrocytes from neonatal mouse brains. When cultured in serum-containing medium, the astrocytes were activated and expressed GFAP, Nestin and RC2, similar to protein expression patterns of RC2-positive reactive astrocytes after stroke (Figure 4A). Jurkat cells (T cell line) are commonly used to study cell invasion15. Jurkat cell invasion was determined with either 5% FBS or 5% FBS with astrocytes in the bottom wells of invasion chambers. In agreement with our observations in vivo, astrocytes cultured in 5% FBS significantly decreased Jurkat cell invasion compared with positive migration control (5% FBS medium alone)(n = 3, p < 0.05; Figure 4B). LDH assay revealed that astrocyte conditioned medium did not induce cell death in Jurkat cells (n = 4, p = 0.96; Figure 4B). To understand the effects of gamma-secretase mediated signaling in cultured astrocytes, invasion studies were performed using GSI-treated astrocytes and DMSO-treated astrocytes. GSI treatment significantly reduced the proliferation of astrocytes compared with DMSO treatment (n=4, Cyquant assay, Day 4, p < 0.01; Day 6, p < 0.05; BrDU incorporation assay, n = 4, p < 0.05; Figure 4C). LDH assay revealed that GSI treatment did not induce cell death in astrocytes (n = 3, p = 0.25; Figure 4C). Following GSI treatment, the number of invading Jurkat cells increased compared with DMSO treatment (n=4, p < 0.05; Figure 4C).

Figure 4. GSI treatment decreased the proliferation of astrocytes and increased Jurkat cell invasion ex vivo.

Figure 4

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(A) Cultured astrocytes expressed GFAP, Nestin and RC2. Western blotting of GFAP and Nestin in post-natal day 4 astrocytes over a time course in culture. (B) LDH assay showed that astrocyte conditioned medium did not induce Jurkat cell death (n=4). A diagram showing invasion assay. Astrocytes significantly decreased the invasion of Jurkat cells across invasion chambers over 24 hrs (n = 3 for each group). (C) Astrocyte proliferation was significantly decreased following GSI treatment (Cyquant assay; n=4 for each group). BrDU incorporation by proliferating astrocytes was significantly decreased following GSI treatment (n=4 for each group). GSI treatment did not induce cell death in astrocytes compared with DMSO treatment (LDH assay: n=3 for each group). GSI treatment significantly increased the invasion of Jurkat cells across invasion chambers over 24 hrs (n=3 for each group). (D) NICD 1-F2A-GFP or GFP was overexpressed in cultured astrocytes. The number of Ki67-positive proliferating GFP-positive astrocytes was quantified. NICD 1-F2A-GFP overexpression significantly increased cell proliferation compared with GFP overexpression (n=6). * p < 0.05, ** p < 0.01.

To determine whether NICD 1 levels alter astrocyte proliferation, we transfected cultured astrocytes with NICD 1-F2A-GFP or GFP control plasmid. Two days following transfection, we examined the number of proliferating astrocytes by immunocytochemistry for Ki67. We observed significantly more proliferative astrocytes following NICD 1-F2A–GFP transfection compared with GFP transfection alone (n=6, p < 0.05: Figure 4D).

Notch 1 inhibition in GFAP-CreER™∷Notch 1 cKO mice reduces reactive astrocyte formation

To determine whether Notch 1 regulates reactive astrocyte formation in vivo after stroke, we generated GFAP-CreER∷tdRFP mice (GR mice) and GFAP-CreER™∷Notch 1 cKO mice (GN cKO mice). In GR mice, tdRFP should be expressed exclusively in GFAP-positive reactive astrocytes following Tamoxifen (TM) treatment. In GN cKO mice, Notch 1 should be knocked out exclusively in GFAP-positive reactive astrocytes following TM treatment (Figure 5). First, to confirm the expression patterns of tdRFP in reactive astrocytes following stroke, we administered TM for 3 consecutive days and performed dMCAO surgery 7 days after the last TM administration. Three days following dMCAO surgery, reactive astrocytes were analyzed by immunohistochemistry. In GR mice, 70 % of GFAP-positive reactive astrocytes expressed tdRFP (Figure 6A). To confirm that NICD1 was no longer expressed in GFAP-positive cells following TM treatment of GN cKO mice, immunohistochemistry against NICD 1 was performed. As expected, following TM treatment, but not following corn oil treatment, NICD 1 was not expressed in GFAP-positive cells (Figure 6A). Quantifying GFAP positive cells, we observed a significantly reduced number of proliferative reactive astrocytes in GN cKO mice following TM treatment (n = 4, p < 0.01; Figure 6B). Notably, the number of RC2-positive reactive astrocytes was also significantly decreased (n = 4, p < 0.01; Figure 6C).

Figure 5. A diagram explaining inducible conditional knock out mouse model.

Figure 5

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Figure 6. Knockout of Notch1 in GFAP-CreER™∷Notch1 cKO mice (GN cKO mice) reduces reactive astrocyte proliferation.

Figure 6

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(A) tdRFP was expressed in GFAP-positive cells in GFAP-CreER™∷tdRFP mice. NICD 1 was not expressed in GFAP-positive reactive astrocytes after TM treatment compared with corn oil treatment in GN cKO mice. (B) The number of Ki67- and GFAP-positive cells decreased significantly following TM treatment compared with corn oil treatment 3 days after stroke (n=4 mice for each group). (C) CD45-positive immune cells in the peri-infarct area were surrounded by RC2-reactive astrocytes. The number of RC2-positive reactive astrocytes decreased significantly after TM treatment and CD45-positive cell invasion increased significantly following TM treatment compared with corn oil treatment 3 days after stroke (n = 4–5 mice for each group). * p < 0.01.

These data demonstrated that Notch 1 plays an important role in reactive astrocyte formation in the peri-infarct area following stroke. To determine whether the decreased number of proliferating reactive astrocytes would affect immune cell invasion, we quantified CD45-positive cell invasion into the peri-infarct area. In agreement with our pharmacological GSI treatment data and ex vivo invasion studies, we also observed a significantly increased number of CD45-positive cells in the peri-infarct area in TM-treated mice (n = 4–5, p < 0.05; Figure 6C). These data showed that proliferating reactive astrocytes, that include RC2-positive reactive astrocytes, are important for suppressing immune cell invasion after stroke.

DISCUSSION

Elucidation of the signaling mechanism(s) that regulate reactive astrocyte formation is important to understand and to treat CNS injury. Gamma-secretase mediated Notch signaling occurs through a conserved pathway that is important for stem cell proliferation and differentiation16. We found that reactive astrocytes expressed the gamma-secretase cleavage products, NICD 1 and AICD, in the peri-infarct area after stroke. To examine the effects of gamma-secretase activity and Notch 1 signaling on reactive astrocyte formation, we treated mice with GSI and generated GN cKO mice to exclusively knockout Notch 1 in GFAP-positive cells. The number of proliferating reactive astrocytes and RC2-positive reactive astrocytes were both significantly decreased following GSI treatment and also in GN cKO mice, demonstrating that Notch 1 plays a critical role in reactive astrocyte formation after stroke.

Reactive astrocytes were previously shown to prevent immune cell invasion and to reduce inflammation after brain stab injury and spinal cord injury2, 13. We found that the number of invading CD45-positive cells was increased in the peri-infarct area following GSI treatment and also in GN cKO mice. These data demonstrated that reactive astrocyte proliferation requires Notch 1 and that proliferating reactive astrocytes may have a specialized role in protecting the brain following stroke by decreasing immune cell invasion.

We did not observe an infarct size difference at 3 days after dMCAO in TM treated GN cKO mice compared with controls, suggesting that Notch 1 knock-out in reactive astrocytes may not alter neural protection early after stroke (ISS and JLS unpublished data). Arumugam et al reported that GSI treatment decreased infarct size in a reperfusion model of stroke (intraluminal) by reducing neuronal apoptosis and decreasing the immune response7. Notch 3 KO mice were reported to have larger stroke volumes than controls following transient MCAO17. Infusion of Delta-like 4, a Notch ligand, did not alter infarct volume after stroke18. Additional studies will be necessary to fully understand the roles of Notch signaling in stroke and where and when it is beneficial or detrimental to outcome.

We have shown that Notch 1 is a key factor required for reactive astrocyte proliferation in the peri-infarct area after stroke. Our data also indicate that the proliferative pool of reactive astrocytes surrounding the infarct core plays a role in suppressing immune cell invasion into peri-infarct tissues. Proliferating reactive astrocytes and the RC2-positive subpopulation of reactive astrocytes adjacent to the infarct core may provide important targets for treatment of stroke.

Supplementary Material

1

ACKNOWLEDGEMENTS

We thank Dr. Suzanne Baker for GFAP-CreER™ mice (St. Judes Children Hospital, Memphis, TN, USA) and Dr. Hans Joerg Fehling for ROSA-tdRFP mice (University Clinics Ulm, Germany). We thank Dr. Diane Jaworski, University of Vermont, for teaching us astrocyte isolation.

SOURCE OF FUNDING Issei S. Shimada is a recipient of the American Heart Association Postdoctoral Fellowship (10POST3730026). This work was supported by P20 RR016435 NIH/NCRR (Parsons R, COBRE PI, JLS, PI Project 3).

Footnotes

DISCLOSURES None

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