Ischemic stroke is a complex event in which the specific contribution of astrocytes to the affected brain region exposed to oxygen glucose deprivation (OGD) is difficult to study. This article introduces a methodology to obtain isolated astrocytes and study their reactivity and proliferation under OGD conditions.
Ischemic stroke is a complex brain injury caused by a thrombus or embolus obstructing blood flow to parts of the brain. This leads to deprivation of oxygen and glucose, which causes energy failure and neuronal death. After an ischemic stroke insult, astrocytes become reactive and proliferate around the injury site as it develops. Under this scenario, it is difficult to study the specific contribution of astrocytes to the brain region exposed to ischemia. Therefore, this article introduces a methodology to study primary astrocyte reactivity and proliferation under an in vitro model of an ischemia-like environment, called oxygen glucose deprivation (OGD). Astrocytes were isolated from 1-4 day-old neonatal rats and the number of non-specific astrocytic cells was assessed using astrocyte selective marker Glial Fibrillary Acidic Protein (GFAP) and nuclear staining. The period in which astrocytes are subjected to the OGD condition can be customized, as well as the percentage of oxygen they are exposed to. This flexibility allows scientists to characterize the duration of the ischemic-like condition in different groups of cells in vitro. This article discusses the timeframes of OGD that induce astrocyte reactivity, hypertrophic morphology, and proliferation as measured by immunofluorescence using Proliferating Cell Nuclear Antigen (PCNA). Besides proliferation, astrocytes undergo energy and oxidative stress, and respond to OGD by releasing soluble factors into the cell medium. This medium can be collected and used to analyze the effects of molecules released by astrocytes in primary neuronal cultures without cell-to-cell interaction. In summary, this primary cell culture model can be efficiently used to understand the role of isolated astrocytes upon injury.
Stroke is defined as "an acute neurological dysfunction of vascular origin with either sudden or rapid development of symptoms and signs, corresponding to the involvement of focal areas in the brain"1,2. There are two types of stroke: hemorrhagic and ischemic. When vascular dysfunction is caused either by an aneurysm or an arteriovenous malfunction, accompanied by weakening with posterior rupture of an artery, this is termed hemorrhagic stroke3 which, in most cases, leads to death. When a thrombus or an embolus obstructs blood flow, causing a temporary deprivation of oxygen and glucose to a brain region, it is called ischemic stroke4. Failure to nourish cells around the affected area or ischemic core leads to a homeostatic and metabolic imbalance, energetic dysfunction, neuronal death, and inflammation5, which can induce a life-long disability for patients6.
Ischemic stroke is a multifactorial injury involving several types of cells that react and exert their effects at different time points. Many interactions create a difficult environment to study the behavior of individual cells. So, how do we study the contribution of a specific cell type under such a complex environment? An accepted in vitro model of ischemia consists of exposing cells to oxygen and glucose deprivation (OGD), for a certain period, followed by the restoration of cells to a normoxic environment. This system simulates an ischemic stroke followed by blood reperfusion. In this method, cells or tissues are exposed to a glucose-free media in an environment purged of oxygen, using a specialized hypoxic chamber. The OGD incubation time can vary from a few minutes up to 24 h, depending on the hypothesis that wants to be tested. Studies have shown that depending on the times of OGD and normoxic environment, specific phenotypes of stroke (i.e., acute or subchronic) can be achieved. Primary isolated astrocytes, exposed to OGD with posterior restoration to normoxic conditions, is a well-studied cellular model to mimic stroke in vitro7. Using OGD is possible to reveal the independent molecular mechanisms of isolated cells under a stroke-like environment.
As our knowledge of astrocyte biology increases, it has become evident that they are crucial for maintaining synapses and sustaining neural repair, development, and plasticity8. Under normal conditions, astrocytes release and respond to cytokines, chemokines, growth factors and gliotransmitters, keeping metabolic balance and homeostasis within synapses5,9. In acute neuroinflammation, such as ischemic stroke, these cells can become reactive, show a long-term overexpression of Glial Fibrillary Acidic Protein (GFAP), and show hypertrophy in their morphology5,10,11,12. As the ischemic infarct develops, the homeostasis provided by astrocytes becomes affected, regarding normal glutamate uptake, energy metabolism, exchange of active molecules, and antioxidant activity13.
Reactivated astrocytes proliferate around the infarct tissue while leukocytes migrate towards the lesioned area14. Astrocytic proliferation can be measured using markers such as proliferating cell nuclear antigen (PCNA), Ki67, and bromodeoxyuridine (BrdU)15. This proliferative response is generated in a time-dependent manner and it helps forming the glial scar, an array of irreversibly reactive astrocytes along the parenchyma of the damaged site after an injury9. One of the initial functions of this scar is to limit the immune cell extravasation from this area. However, studies have shown that the scar becomes a physical impediment for axons to extend, as they release molecules inhibiting axonal growth, and create a physical barrier preventing axons from extending around the injured area16. Nevertheless, there is scientific evidence showing that after a spinal cord injury, completely preventing glial scar formation can impair the regeneration of axons17. Thus, the context in which the specific astrocytic response is measured, must be considered upon the framework of the injury studied.
The presented methodology can be applied to study the individualized function of astrocytes after oxygen glucose deprivation and it can be modified depending on the questions that the investigator wants to answer. For example, besides the morphological change and the markers expressed at different OGD times, the supernatants from astrocytes exposed to OGD can be further analyzed to identify soluble factors released by these cells, or used as a conditioned media to assess its effect in other brain cells. This approach enables studies on astrocyte reactivity that could lead to the elucidation of the factors that govern and modulate their response in an ischemic-stroke scenario.
Postnatal rats (Sprague Dawley) 1-4 days old are used to isolate cortices. The method of euthanasia is decapitation, as approved by NIH guidelines.
1. Preparation of Instruments and Materials for Surgery
2. Complete DMEM Preparation
3. Primary Astrocyte Culture
Note: After seeding, cell media must be changed every three days and cells can be grown up to confluency (11-13 days). On the third day, tap the flask several times to lift microglial cells and oligodendrocyte progenitor cells off the culture, remove all the 'old' media, wash twice using 10 mL of PBS, aspirate the PBS, then add fresh new media. See materials in Table of Materials.
4. Cultivating Astrocytes in 6-Well Plates
5. OGD Protocol for Primary Astrocytes
6. Protocol for Primary Astrocyte Immunofluorescence Preparation
Note: To assess astrocyte purity, different brain cell types such as neurons, microglia, and oligodendrocytes can be detected by immunofluorescence using different cell markers. Proliferation markers, PCNA, and propidium iodide (PI) can be used on primary astrocytes exposed to OGD followed by normoxic conditions. See materials in Table of Materials.
7. Cell Viability Assay
One of the main concerns of primary astrocytic culture is the presence of other cells such as neurons, oligodendrocytes, fibroblasts, and microglia. In Figure 1, isolated cells from rat cortices had media changes every 3 days and were either untreated or treated with added LME for 1 h. 24 h later, cells were immunostained for GFAP and counterstained with DAPI. Untreated cells showed an average of 39% non-GFAP positive cells, while LME-treated cells showed 8%. These results show how LME treatment and media change effectively decreased non-GFAP positive cells by 4.75 fold, producing an enriched-astrocyte culture. To determine if 1 h or 6 h OGD affects astrocyte viability after this methodology, Figure 2 shows an MTT assay of astrocyte cultures, 24 h after the insult. No significant difference in viability was found in any condition, showing that both times can be used to study astrocyte reactivity. Cell proliferation is one of the effects triggered by OGD in astrocytes. Proliferating cell nuclear antigen (PCNA) in Figure 3 increased from 10% in normoxic cells, to 30% after 1 h OGD, showing the expected in vivo astrocytic response15. Finally, astrocytes were exposed to 6 h of OGD followed by 24 h normoxic conditions. After this period of time, immunofluorescence against GFAP was performed. OGD-exposed cells were compared to astrocytes under normoxic conditions. Astrocytes without OGD show the traditional stellate morphology (Figure 4A) while cells that underwent OGD clearly present the characteristic hypertrophy of reactive astrocytes (Figure 4B). This hypertrophy can be visualized in the corresponding differential interference contrast (DIC) image of cortical rat astrocytes under normoxic and OGD conditions (Figure 5).
Figure 1. Immunofluorescence to validate primary cortical rat astrocytes culture purity with GFAP and DAPI. (A) The left panel represents GFAP positive cells (red), the middle panel shows nuclei (DAPI, blue), and the right panel shows merged images of non-LME-treated (upper panel) and LME-treated (lower panel) cells. White arrows show non-GFAP-positive stained cells. (B) Quantification of non-GFAP positive nuclei in LME and non-LME treated cells. An unpaired t-test was performed (p<0.0001) and the error bars represent the mean with SEM. Scale bars represent 50 microns and pictures were taken at 20x magnification. Please click here to view a larger version of this figure.
Figure 2. Viability assay of astrocytes with 24 h exposure to OGD. Astrocytes were cultured in 96-well plates and exposed to either normoxic, 1 h OGD, or 6 h OGD conditions. After treatment, cells were added to normoxic media for 24 h and viability was measured using MTT assay. Absorbance at 570 nm shows the cell viability of OGD-exposed cells in culture when compared to cells in normoxic conditions. Data was analyzed with one-way ANOVA (p=0.9669) and presented with error bars that represent the mean with SEM. Please click here to view a larger version of this figure.
Figure 3. Proliferation in rat astrocytes increases upon exposure to oxygen and glucose deprivation and can be detected using PCNA as a marker. (A) The left panel represents PCNA positive cells (green), the middle panel shows nuclei (DAPI, blue), and the right panel shows merged images of normoxic cells (upper panel) and 1 h OGD treated cells (lower panel). (B) Percent of PCNA positive cells in normoxic versus OGD treated cells. An unpaired-test was performed (p<0.0001) and the error bars represent the mean with SEM. Scale bars represent 50 microns and pictures were taken at 20x magnification. Please click here to view a larger version of this figure.
Figure 4. Expression of Glial Fibrillary Acidic Protein (GFAP) conjugated with Cy3 in primary rat cortical astrocyte culture. (A) Astrocytes in a normoxic environment show the characteristic stellate morphology. The intermediate filament protein, GFAP, fills the cell bodies and extends into the thin cytoplasmic processes. (B) After 6 h of OGD exposure astrocytes adopted a hypertrophic morphology. Scales represent 20 microns and pictures were taken at 40x magnification in a confocal microscope. Please click here to view a larger version of this figure.
Figure 5. Cortical rat astrocytes under normoxic and oxygen-glucose deprivation. Immunofluorescence of astrocytes under normoxic conditions (upper left panel) or 6 h OGD (lower left panel), stained using a Cy3-conjugated antibody against GFAP. The corresponding differential interference contrast (DIC) image is shown on the right panel. Scale bar represents 20 microns and pictures were taken at 20x magnification. Please click here to view a larger version of this figure.
Purpose | Antibody/Marker | Description |
Astrocyte Culture Validation | Iba-1 | Detects microglial cells |
NeuN | Identifies mature neurons | |
Olig1 | Detects mature oligodendrocytes | |
Olig2, NG2 | Detects oligodendrocyte precursor cells | |
GalC | Identifies immature or mature oligodendrocytes | |
Proliferation | PCNA | Binds p36 protein, expressed at high levels in proliferating cells |
Cell viability | Propidium iodide (PI) | Binds to the DNA, this marker indicates lack of cell viability |
MTT | Measures mitochondrial functionality | |
Reactivity | GFAP | Indicator of astrocyte reactivity |
Table 1. List of reagents used to stain various types of cells and cellular processes
This protocol describes the isolation of astrocytes from rat cortices. In this method, it is critical to decrease contamination with other cellular types such as microglia, oligodendrocytes, and fibroblasts. To reduce the number of microglia, several steps can be taken: changing the media, orbital shaking, and chemical treatments. Once culture purity is confirmed by immunofluorescence using selective cellular markers or for the most prominent cell contaminants, experiments can be performed. For instance, antibody against ionized calcium binding adaptor molecule 1 (Iba-1) can be used to detect microglia. Neuronal death starts early in the culture and these cells are eliminated together with oligodendrocyte precursor cells (OPCs) after flask tapping and media change at day 331. Oligodendrocytic precursor cells can be identified using antibodies such as olig2 or NG2. GalC can be used to identify immature or mature oligodendrocytes, but at this culture stage these are unlikely to be found. Alternatively, non-GFAP positive cells can be quantified and contaminants can be estimated. This last methodology does not discern between the specific cells, but will be a faster and more economic methodology to determine the percentage of non-astrocytic cells in the culture.
Once an astrocyte enriched culture is produced, OGD experiments can be conducted to study astrocyte reactivity. Purging a solution with nitrogen by bubbling to substitute oxygen is a commonly used protocol32. In our methodology, we use the bubbling method to purge oxygen with nitrogen gas (95% N2, 5% CO2) into the glucose-free media at a flow rate of 15 L/min, for 10 min, for a total volume of 0.025 L. To confirm that the oxygen is purged, we measured the oxygen concentration remaining in the media by using an oxygen meter for liquids and air. The oxygen concentration remaining in the media after purging for 10 min decreased from 7.9 mg/L to 0.3 mg/L. To determine how long the chamber must be purged to replace oxygen, purging with N2 was performed using no less than the chamber manufacturer's suggestion (20 L/min, 2-5 min). After 5 min of purging at 15 L/min, the oxygen concentration remaining in the chamber was 0%. Other methodologies using similar chambers have purged at an 8-20 L/min flow rate, and times of 5-10 min to substitute air oxygen with nitrogen33,34.
Like other cellular models, the OGD has limitations and results should be interpreted carefully. The OGD can be harmful to neurons, however, astrocytes can withstand these conditions for up to 24 h35. Another limitation is that astrocytes are isolated in this system, lacking signaling from other cells. One way to overcome this limitation is to use conditioned media from other cells under the same ischemic-like conditions. Alternatively, cytokines or other factors can be added to the media after OGD36.
An alternative method to study the effects of energy deficiency in cells, is to add ouabain to the media. This compound mainly inhibits the sodium/potassium pump, which depletes the intracellular production of ATP, similar to the effects of OGD37,38. However, this method has the disadvantage that all its mechanisms are not fully understood and it induces off-target effects, which introduces variables which constitute an unrealistic ischemic stroke scenario.
In summary, the OGD method is an isolated cell system which allows us to directly test the effects of different astrocytic drug targets in vitro that could further contribute to neuroprotection. It also provides a tool to study basic astrocyte biology. This model can create a strong basis of evidence to justify experimental conditions for the development of drugs using an in vivo model of stroke.
The authors have nothing to disclose.
The authors want to thank Paola López Pieraldi for the technical assistance. A.H.M. is grateful for the grants 8G12MD007600 and U54-NS083924 that supported this publication. We thank NIH-NIMHD-G12-MD007583 grant for the facility support. D.E.R.A. is grateful for the fellowship provided by NIHNIGMS-R25GM110513.We are grateful for the use of the Common Instrumentation Area and the aid of Dr. Priscila Sanabria for the use of the Optical Imaging Facility of the RCMI program by grant G12MD007583. In addition, we want to thank Jose Padilla for his outstanding role in filming and editing the visual protocol.
Instruments for Surgery – Step 1 | |||
Operating scissor 5.5” | Roboz Company | RS-6812 | Tools used to decapitate the rats. |
Curved forceps 7” | Roboz Company | RS-5271 | Holds the skin of the rat while the skull is removed. |
Micro-dissecting scissors 4” | Roboz Company | RS-5882 | Cuts both the skin and skull of the rat. |
Micro-dissecting forceps 4” angled, fine sharp | Roboz Company | RS-5095 | Holds the skin of the rat while the skull is removed. |
Micro-dissecting forceps 4” slightly curved 0.8 | Roboz Company | RS-5135 | Tool used to separate cortices. |
Micro-dissecting tweezers | Roboz Company | RS-4972 | Peels brain meninges. |
Dissection microscope | Olympus | SZX16 | Important for removing the meninge from the cortices. |
DMEM Preparation – step 2 | |||
Dulbecco’s Modified Eagle’s Medium (DMEM) | GibCo. Company | 11995-065 | Supports the growth of cells. |
Sodium bicarbonate | Sigma-Aldrich Company | S7277 | Supplement for the cell culture media. |
Fetal bovine serum (FBS) | GibCo. Company | 10437-010 | Serum-supplement for the cell culture. |
Penicillin-Streptomycin | GibCo. Company | 15140-148 | Inhibits the growth of bacterias in the cell culture. |
Filter System 1L with 0.22um pore | Corning | 431098 | |
Astrocyte culture – step 3 | |||
Serological pipets 5mL | VWR | 89130-896 | To pipette DMEM to containers with cells. |
Serological pipets 10mL | VWR | 89130-898 | To pipette DMEM to containers with cells. |
Serological pipets 25mL | VWR | 89130-900 | To pipette DMEM to containers with cells. |
Centrifuge conical tube 15mL | Santa Cruz Biotechnology | sc-200250 | |
Safe-lock tube 1.5mL | Eppendorf | 022363204 | |
Barrier Tips 200 uL | Santa Cruz Biotechnology | sc-201725 | |
Barrier Tips 1 mL | Santa Cruz Biotechnology | sc-201727 | |
Biohazard Orange Bag 14 x 19" | VWR | 14220-048 | |
60mm petri dishes | Falcon | 351007 | |
Sterile gauze pads | Honeywell Safety | 89133-086 | |
Stomacher 80 Biomaster | Sewar Lab System | 030010019 | Triturate the brain tissue. |
Stomacher 80 Blender Sterile Bags | Sewar Lab System | BA6040 | Sterile bag for the stomacher cell homogenizer. |
Beaker 400mL | Pyrex | 1000 | |
Sterile cell dissociation sieve, mesh #60 | Sigma-Aldrich Company | S1020 | To obtain a uniform single cell suspension. |
Sterile cell dissociation sieve, mesh #100 | Sigma-Aldrich Company | S3895 | To obtain a uniform single cell suspension. |
Invert phase microscope | Nikon | Eclypse Ti-S | Verify cells for contamination or abnormal cell growth. |
75cm2 sterile flasks | Falcon | 353136 | |
Multi-well plate | Falcon | 353046 | |
Micro cover glasses (coverslips), 18mm, round | VWR | 48380-046 | |
Bright-Line hemacytometer | Sigma-Aldrich Company | Z359629 | |
Pasteur pipettes | Fisher Scientific | 13-678-20D | |
Ethyl alcohol | Sigma-Aldrich Company | E7023 | |
L-leucine methyl ester hydrochloride 98% (LME) | Sigma-Aldrich Company | L1002 | Promotes the elimination of microglia cells in the primary cortical astrocyte cultutre. |
Cytosine β-D-arabinofuranoside (Ara-C) | Sigma-Aldrich Company | C1768 | |
Poly-D-Lysine Hydrobromide, mol wt 70,000-150,000 | Sigma-Aldrich Company | P0899 | |
Trypsin/EDTA | GibCo. Company | 15400-054 | |
Trypan Blue | Sigma-Aldrich Company | T8154 | |
Phosphate buffer saline (PBS) tablets | Calbiochem | 524650 | |
Sterile Water | Sigma-Aldrich Company | W3500 | |
OGD Medium Preparation – step 5 | |||
Centrifuge conical tube 50 mL | VWR | 89039-658 | |
Dulbecco’s modified Eagle’s medium-free glucose | Sigma-Aldrich Company | D5030 | Supports the growth of cells. |
Sodium bicarbonate | Sigma-Aldrich Company | S7277 | Supplement for the cell culture media. |
Penicillin-Streptomycin | GibCo. Company | 15140-148 | Inhibits the growth of bacterias in the cell culture. |
200mM L-glutamine | GibCo. Company | 25030-081 | Amino acid that supplements the growth of cells. |
Phospahet buffer saline (PBS) tablets | Calbiochem | 524650 | |
Filter System 50mL with 0.22um pore | Corning | 430320 | |
Centrifuge conical tube 50 mL | VWR | 89039-658 | |
Single Flow Meter | Billups-Rothenberg | SMF3001 | Measure gas flow in oxygen purge. |
Hypoxia Incubator Chamber | StemCell | 27310 | Generates a hypoxic environment for the cell culture. |
Traceable Dissolved Oxygen Meter | VWR | 21800-022 | |
95% N2/ 5% CO2 Gas Mixture | Linde | Purges the environment of oxygen. | |
primary astrocyte immunofluorescence – step 6 | |||
Phosphate buffer saline (PBS) tablets | Calbiochem | 524650 | |
Formaline Solution Neutral Buffer 10% | Sigma-Aldrich | HT501128 | Solution used to fix cells. |
Methanol | Fisher | A4544 | Solution used to fix cells. |
Non-ionic surfactant (Triton X-100) | Sigma-Aldrich | T8787 | |
Fetal bovine serum (FBS) | GibCo. Company | 10437-010 | Serum-supplement for the cell culture. |
Anti-NeuN | Cell Signaling | 24307 | Detects mature neurons, serves to validate the astrocytic culture. |
Anti-PCNA | Cell Signaling | 2586 | Detects proliferating cells. |
Propidium Iodide (PI) | Sigma-Aldrich Company | P4170 | Apoptosis staining. |
Anti-Olig1 | Abcam | AB68105 | Detects mature oligodendrocytes. |
Anti-Iba1+ | Wako | 016-20001 | Detects microglial cells. |
Anti-GFAP Conjugated with Cy3 | Sigma-Aldrich Company | C9205 | Detects reactive astrocytes in the treated cells. |
Alexa Fluor 488 | Molecular Probe Life Technology | A1101 | Anti-Mouse Secondary Antibody |
Alexa Fluor 555 | Molecular Probe Life Technology | A21428 | Anti-Rabbit Secondary Antibody |
4’,6’-diamidino-2-phenylindole (DAPI) | Sigma-Aldrich Company | D9542 | Nuclear staining |
Confocal microscope | Olympus |