**2. Materials and methods**

### **2.1 Ethical approval**

*Erythrocyte - A Peripheral Biomarker for Infection and Inflammation*

role in the regulation of blood pressure.

Scientific studies have established that prevalence of hypertension (HTN) needs to be reduced in order to control cardiovascular and cerebrovascular diseases [1]. At present, many laboratories around the world are exploring a plethora of biomarkers, as early indicators of HTN [2, 3], i.e., triglycerides, C-reactive protein, fibrinogen, serum albumin, uric acid, homocysteine and intracellular adhesion molecule-1(ICAM-1) [4]. All these biomarkers for hypertension are produced as a result of chronic disease-related comorbidities such as atherosclerosis, Type 2 diabetes and renal failure, therefore, they cannot be considered as 'true' predictive biomarkers for hypertension. Hence, this study is focusing on glial fibrillary acidic protein (GFAP), and a group of calcium-dependent proteases, such as calpain, calpastatin, cathepsin and mitogen activated protein kinase (MAPK), known to be strongly associated with HTN; hence could be potential early biomarkers for HTN. Similarly, an endogenous chemical, N-Methyl-D-Aspartic acid (NMDA), a type of glutamate is also documented as linked with HTN [5]. NMDA is a major excitatory neurotransmitter in the central nervous system, consisting of two types of receptors namely synaptic and extrasynaptic. Research indicates that the activation of the synaptic NMDA receptors is neuroprotective; whereas, the stimulation of extrasynaptic NMDA receptors (NMDARs) promotes cell death [6] and resultant HTN, which can be regulated by NMDA antagonists, such as 1-amino cyclo propane carboxylic acid (ACPC) [7]. NMDA type glutamate receptors are not only present in the neuronal cells but also in the non-neuronal cells such as astrocytes [8]. Recent studies have demonstrated a significant role of astrocytes in regulating blood flow due to the elevation in intracellular calcium (Ca2+) [9], which plays a significant

Astrocytes are present between blood vessels and neurons and are responsible for changes in the arterial blood pressure [10]. A decline in the cerebral blood supply activates the astrocytes to release a chemical signal to the nearby neurons that raises blood pressure, restoring blood flow and oxygen supply to the brain [10]. Thus, astrocytes perform a balancing role between brain perfusion and neuronal activities by mobilising their internal calcium [11], which in turn triggers the release of chemical transmitters such as glutamate [12]. Consequently, there is a calciumdependent bidirectional signalling pathway between astrocytes and neurons [13], which opens up the possibility of astrocytic involvement in the modulation of calcium-dependent molecules such as calpain, calpastatin, cathepsin and MAPK, which can potentially be considered as direct predictive biomarkers of HTN. Certain behavioural conditions, such as, stress also elevates blood pressure. Under stressful conditions, excessive release of corticotropin-releasing hormone (CRH) activates NMDA receptors, resulting in an influx of Ca2+ molecules, which enhance the activity of m-calpain [14]. Calpain is one of the major calcium-dependent proteolytic enzymes with various isoforms such as μ-calpain and m-calpain, which are activated by the synaptic and extra-synaptic NMDA receptors respectively [15, 16]. Studies have indicated that the activation of μ-calpain is important for cell-survival, whereas the stimulation of m-calpain initiates toxic effects and cell death [6], due to its interaction with NMDAR [17]. Activation of the type of NMDA

receptor defines the communication from the synapse to the nucleus [18].

Stimulation of synaptic NMDAR phosphorylates the intermediate filament proteins by extracellular signal regulated kinases 1 and 2 (ERK1/2) [19]. However, the activation of extrasynaptic NMDAR fails to phosphorylate and translocate intermediate filament proteins into the nucleus. The phosphorylated or non-phosphorylated state of intermediate filament proteins determine whether it promotes cell survival or induces cell death. These diverse functions that require the regulation

**1. Introduction**

**56**

HFAs were obtained from the Biobank of Macquarie University, after approval from the UTS Human Research Ethics Committee (ETH17–1883).

#### **2.2 Protocol for the cell culture of primary HFAs**

After getting a material transfer agreement (MTA # 17/979) between the two universities, the HFAs were tested for mycoplasma contamination and were found to be negative. The HFAs were then cultured in T75 flasks using Roswell Park Memorial Institute (RPMI 1640) media containing 10% heat-inactivated foetal calf serum (FCS) (**Table 1**), at 37°C, in a 5% CO2 humidified incubator. Freshly prepared media was used to feed the cells every five days.

The HFAs were seeded at 4x107 cells/T75-cm2 in tissue culture flasks containing RPMI (4.5 g/L glucose, L-glutamine, and 25 mM HEPES buffer), plus 10% FCS at 37°C, and incubated in a 5% CO2 humidified incubator.

For re-seeding, HFAs were detached from the flask by using 4 ml of trypsin, incubated for 3 minutes at 37°C, in a 5% CO2 humidified incubator. After incubation with trypsin, the cells were transferred to a 15 ml falcon tube and were centrifuged at 1600 g rpm, for 3 minutes. Trypsin was removed by washing three times with phosphate buffered saline (PBS). The cells were then resuspended in 2 ml of fresh media by gentle mixing and 1 ml of this cell solution was added to each T75 flask with 14 ml RPMI, labelled as A1 and A2 for reactive and normal astrocytes, respectively.

#### **2.3 Conversion of normal HFAs (A2) into reactive HFAs (A1)**

After achieving 95 to 100% confluency and three days before the proteomics experiment, the HFAs were incubated in the RPMI media with 1% FCS instead of 10% FCS, to avoid false positive proteomic results due to the proteins present in the 10% FCS. Twenty-four hours before the pellet formation for SP3 [22] protocol,


#### **Table 1.**

*Chemicals used for the tissue culture experiments.*

the cells were returned to RPMI media +10% FCS in both A1 and A2 flasks. Then, 100 μl of 1 mM adenosine triphosphate (ATP) was added to the A1 flask to convert the normal HFAs into reactive HFAs [23]. After 24 hours of incubation at 37°C, in a 5% CO2 humidified incubator, the cells were washed three times with PBS. The cells were subsequently detached from both flasks by adding 4 ml of trypsin and were incubated at 37°C in a humidified incubator, for 3 minutes. After incubation, the cells were transferred into two falcon tubes (15 ml tubes, labelled as A1 and A2) and were centrifuged at 1600 g rpm for 3 minutes. Trypsin was removed from the tubes by washing three times with 3 ml PBS. The cell samples from both tubes were transferred into 2 ml Eppendorf tubes and were microfuged for 1 minute, to remove PBS. Subsequently, the Eppendorf tubes containing cell pellets were snap frozen in liquid nitrogen and stored at -80°C for the proteomics experiment, using SP3 protocol.

#### **2.4 Immunocytochemistry of HFAs using anti-GFAP stain**

Six poly-L-lysine coated coverslips, three labelled as A1 and three as A2 were placed in a six well plate, containing 2 ml of fresh RPMI media with 10% FCS. The plates were incubated at 37°C, in a 5% CO2 humidified incubator to grow until ~70% confluency was achieved.

The cells in three of the six wells, were treated with 1 mM ATP to convert them into reactive (A1) astrocytes, whereas the other three coverslips with the HFAs (A2) were not treated with ATP. After 24 hours of incubation all the cells were washed three times with 0.1 M PBS, fixative (4% paraformaldehyde in PBS, 7.4 pH) was then added for 30 minutes, at room temperature. After fixation, both types of HFAs were gently washed three times with PBS and were ready for permeabilization and blocking of the unspecific binding of the proteins. To improve the permeabilization of the antibodies, the HFAs were incubated with 100 μl of 0.1% Triton X 100, for 30 minutes, at 37°C, in a 5% CO2 humidified incubator. The cells were gently washed three times with PBS, before being treated with the blocking solution (100 μl of 5% goat serum) and then the cells were incubated for 30 minutes.

**59**

**Table 2.**

*Reagents and equipment used for SP3 protocol.*

*Early Predictive Biomarkers for Hypertension Using Human Fetal Astrocytes*

The cells were incubated with a primary anti-GFAP antibody (1:100 dilution, ab7260 from ABCAM) at 4°C for 18 hours. Then, the HFAs were washed three times with PBS, and were incubated with the secondary antibody (1:100, ab150077 with AF488, ABCAM) in 1% BSA for 1 hour at the room temperature in the dark.

The HFAs were rinsed with PBS and were incubated with 1 μg/ml of nuclear stain [4, 6-diamidino-2-phenylindole (DAPI)] for 10 minutes followed by three rinses with PBS. Excess PBS was carefully wiped around the coverslips and the cells were mounted on the glass slides (75 mm x 25 mm and 1 mm thickness), which were clearly labelled with the type of cell and stain. Then a drop of mounting medium, Fluoroshield (ABCAM) was placed in the middle of the slide. The coverslip with the stained cells was carefully lifted using forceps and was placed in an inverted position on top of the slide, air bubbles were removed, and the cells were sealed with a clear nail polish to prevent drying and movement under the microscope. The cells were stored in the dark, at +4°C till image analysis was performed using the NIKON

**2.5 Protein extraction and digestion using single-pot, solid-phase-enhanced** 

The proteins of interest were extracted from A1 and A2 HFAs using the SP3 protocol, before performing mass spectrometric analysis. All the chemicals and equipment used for the preparation of lysis buffer and for protein extraction and

**Chemical name Company Cat. No.** Bovine serum albumin (BSA) Sigma 00A2153 Sodium dodecyl sulfate (SDS) Bio-Rad 1610302 Triton X-100 Sigma T8787 Nonidet-P40 (NP-40) Merck-Millipore 492016-100ML Tween 20 Sigma P1379 Deoxycholate Sigma 3970 Sodium chloride (NaCl) Sigma S7653 Glycerol Sigma G5516 HEPES sodium salt Sigma H3375 Iodoacetamide Bio-Rad 1632109 Dithiothreitol (DTT) Bio-Rad 1610611 Ammonium bicarbonate Sigma A6141 Trypsin + rLysC mix Promega V5073 1.5-mL Safe-Lock tubes Eppendorf 22363204 Tris-2-carboxyethyl phosphine Sigma 51805–45-9 Sera-Mag Speed Beads GE Healthcare 45152105050250 Magnetic rack, Magnetic Sphere Promega Z5342

digestion using SP3 protocol are shown in **Table 2** and **Figure 1**.

*DOI: http://dx.doi.org/10.5772/intechopen.98561*

*2.4.1 Nuclear staining and mounting*

A1 confocal microscope.

**sample-preparation (SP3) protocol**

The cells were incubated with a primary anti-GFAP antibody (1:100 dilution, ab7260 from ABCAM) at 4°C for 18 hours. Then, the HFAs were washed three times with PBS, and were incubated with the secondary antibody (1:100, ab150077 with AF488, ABCAM) in 1% BSA for 1 hour at the room temperature in the dark.
