Chapter 3: Materials and Methods Chapter Materials and Methods Department of Pharmacology, YLL School of Medicine 59 Chapter 3: Materials and Methods 3.1 Drug preparations 3.1.1 Purified Herba leonuri (pHL) The raw material of HL is originated from Sichuan province (China). Purified HL (pHL) was patented by our group (Zhu et al, 2006) and is commercially available in Singapore (known as ‘Kardigen’, Herbatis Pte. Ltd., Singapore). In this research, pHL powder was supplied by Herbatis Pte. Ltd. (Singapore). The extract contains mainly stachydrine (C7H13NO2), quercetin (C15H10O7), Kaempferol (C15H10O6), leonurine (C14H21N3O5) and apigenin (C15H10O5). The preparation procedures of pHL were previously described in the previous study (Sun et al, 2005). Briefly, the raw material of HL was extracted by boiling in water for 45 minutes, and the aqueous extract was concentrated under vacuum at 50°C (BUCHI Rotavapor R-144, waterbath B-480). After condensation overnight at 80°C, the liquid was made into freeze-dried powder. The extract was analyzed by LC (liquid chromatograph)-ESI (electrospray ionization)-MS (mass spectrometry) (API 365 LC-MS system, Applied Biosystems, USA) (Sun et al, 2005). The chemical structures of these compounds are shown in figure 2-10. 3.1.2 Leonurine Leonurine (molecular weight: 333) was synthesized and patented by our group (Zhu et al, 2008). Leonurine was synthesized from syringic acid by carbonylation, reaction with thionyl cloride (SOCl2), and the Gabriel reaction, as previously described (Zhu et al, 2005). Leonurine was confirmed to have 99% purity by high performance liquid chromatography (HPLC). Department of Pharmacology, YLL School of Medicine 60 Chapter 3: Materials and Methods 3.2 Animals Wistar rats (250-300g) were supplied from the Laboratory Animal Centre, National University of Singapore (NUS). In collaboration with Prof. Zhu YZ’s Laboratory, Fudan University to complete experiment protocol III, SD rats (250-300g) were supplied by Laboratory Animal Centre, Fudan University. Animals were housed under diurnal lighting conditions and fed standard rat chow and water ad libitum according to regulations of animal care by local committee. The experimental protocol was approved by the local ethical committee and confirmed to internationally accept ethical standards. 3.3 Middle cerebral artery occlusion (MCAO) Ischemic stroke was induced in the rats by MCAO. Before middle cerebral artery occlusion (MCAO) is carried out, surgical instruments will be autoclaved prior to the surgery.The rats were anaesthetized with ketamine + xylazine mixture (75mg/kg+10mg/kg (0.1ml/100g BW)) i.p.) and placed in the lateral position. 70% alcohol is applied to the region in between left orbit and external auditory canal to disinfect the region. A 2cm skin incision was made in the midpoint between and the left orbit and the external auditory canal. A small burr hole was made with a high speed microdrill through the outer surface of the skull and after removing the dura, the left middle cerebral artery (MCA) was exposed. Then, MCA was occluded by electrocauterization from the point where it crossed the inferior cerebral vein to a point proximal to the origin of the lenticulo-striate branches. The branches of the MCA between these points were occluded as well to ensure complete and permanent occlusion. The wound Department of Pharmacology, YLL School of Medicine 61 Chapter 3: Materials and Methods was closed with sutures and antibiotics (cephalexin, 15-20mg/kg) will be given to avoid possible post-operational infection. Rectal temperature was maintained at 37±0.5°C by means of a heating blanket throughout the surgery. Sham rats underwent similar procedures with the ommision of MCAO step (Lu et al, 2004). 3.4 Experimental protocols 3.4.1 Experimental protocol I 3.4.1.1 Objectives Generally, a pilot study to investigate the therapeutic effects of pHL on middle cerebral artery occluded rats was carried out. The aims of this part of study is to illustrate: 1. The effect of pHL on functional outcome of MCAO-induced rats. 2. The antioxidant and anti-apoptosis capacity of pHL on MCAO-induced rats. 3.4.1.2 Experimental design The experimental design is illustrated in figure 3-1. A total of 84 male Wistar rats were randomly divided into four groups: sham group with water treatment [Sham], sham group with pHL treatment (400mg/kg/day) [Sham + pHL], stroke group with water treatment as vehicle [Vehicle] and stroke group with pHL treatment (400mg/kg/day) [Stroke + pHL]. pHL was dissolved in water. The drugs were administrated orally once daily. After weeks of pre-surgery treatment, MCAO as described in section 3.3 was induced in stroke induced groups. Sham rats underwent similar procedures with the omission of MCAO. Department of Pharmacology, YLL School of Medicine 62 Chapter 3: Materials and Methods Animal treatments were continued for another week after surgery. During the period of post-surgery treatment, animal were assessed in terms of neurological deficit score. At day after surgery, animals were sacrificed and subsequent experiments were carried out to evaluate their therapeutic potentials. Sham Sham + pHL Vehicle Stroke + pHL weeks pre-surgery treatment MCAO/Sham-operated surgery Neurological Deficit Grading 4.3 System Results week post-surgery treatment TTC staining Total Antioxidant Assay DNA damage analysis Immunohistochemical staining TUNEL Assay Figure 3-1: A Flow chart represents the experimental outline in the pilot study of pHL. Department of Pharmacology, YLL School of Medicine 63 Chapter 3: Materials and Methods 3.4.2 Experimental protocol II 3.4.2.1 Objectives In experimental protocol II, the effects of pHL on modulation of mitochondrial function were elucidated: 1. Effect of pHL on mitochondrial ROS production in vitro. 2. Effects of pHL on mitochondrial function in vitro (ATP biosynthesis and oxygen consumption). 3. Effects of pHL on mitochondria isolated from MCAO-induced rats through oxygen consumption measurement and mitochondrial GSH pool. 3.4.2.2 In vitro mitochondrial studies 20 adult male Wistar rats were fasted overnight and anaesthetized with 0.2ml/100g ketamine/xylazine intraperitoneally (i.p) in order to collect the brain samples. Cortical tissues were separated for mitochondrial isolation. Before the experiments were to be carried out, quality of cortical mitochondrial preparations was assessed by JC-1 assay to ensure the quality of mitochondria is suitable for bioenergetics studies. Isolated mitochondria suspension was divided into groups as listed in Table 3-1 for subsequent experiments. ROS generation assay, ATP biosynthesis assay and mitochondrial respiration were done to evaluate the effect of pHL in isolated mitochondria (Figure 3-2). Department of Pharmacology, YLL School of Medicine 64 Chapter 3: Materials and Methods Table 3-1: Grouping for studies of effect of pHL on isolated mitochondria. Groups Control – No drug Control – H2O2 pHL-0.05mg/ml pHL-0.075mg/ml pHL-0.1mg/ml pHL-0.25mg/ml H2O2 + pHL-0.05mg/ml H2O2 + pHL-0.075mg/ml H2O2 + pHL-0.1mg/ml 10 H2O2 + pHL-0.25mg/ml 3.4.2.3 In vivo mitochondrial studies 28 male Wistar rats were randomly divided into four groups: sham group with water treatment [Sham], sham group with pHL treatment (400mg/kg/day) [Sham + pHL], stroke group with water treatment as vehicle [Vehicle] and stroke group with pHL treatment (400mg/kg/day) [Stroke + pHL]. The drugs were administrated orally once daily. After weeks of pre-surgery treatment, MCAO was induced in stroke induced groups. Sham rats underwent similar procedures with the omission of MCAO. Animals were sacrificed hours after MCAO/sham surgery. Left cortices and right cortices were collected for mitochondrial isolation. Effects of pHL on mitochondria isolated from each treatment group were evaluated in terms of mitochondrial respiration and glutathione assay hours after the surgery (Figure 3-2). Department of Pharmacology, YLL School of Medicine 65 Chapter 3: Materials and Methods In vitro experiments: In vivo experiments: Sham, Sham+pHL, Vehicle, Stroke+pHL Male Wistar Rat ~250g Fasting, overnight Mitochondrial Isolation JC-1 Assay weeks treatment Fast overnight before surgery With/ without ROS Inducer + Drug Treatment • • • ROS Generation Assay (Kinetic) ATP Biosynthesis Assay Mitochondrial Respiration Assay MCAO/Sham hours after surgery Mitochondrial Isolation • • Mitochondrial Respiration Assay Glutathione Assay Figure 3-2: Flow charts represent the general outlines of experimental protocol II. Department of Pharmacology, YLL School of Medicine 66 Chapter 3: Materials and Methods 3.4.3 Experimental protocol III 3.4.3.1 Objectives In experiment III, a key compound of pHL (Leonurine) was targeted to identify if it is one of the active ingredients of pHL for neuroprotection. The aims of this study are: 1. Identify the effect of Leonurine on MCAO-induced rats in terms of neurological recovery. 2. Identify if the effects of Leonurine act through antioxidant mechanisms 3. Identify the effects of Leonurine on mitochondrial function both in vitro and in vivo. 3.4.3.2 In vivo studies – animal treatment and MCAO 100 SD rats weighing 180-220g were randomly divided into five groups: sham-operation (Sham); MCAO group with water treatment (Vehicle); Stroke groups treated with 15mg/kg/day, 30mg/kg/day and 60 mg/kg/day of Leonurine (Leo15, Leo30, Leo60). Leonurine powder was dissolved in water. The drugs were administrated orally once daily. After week of pre-surgery treatment, stroke was induced in the rats by MCAO. Sham rats underwent similar procedure with the omission of MCAO. Rats were sacrificed 24 hours after surgery for sample collection (Figure 3-3). Experiments including TTC staining, neurological deficit grading system, SOD activity assay, GPx activity assay, and MDA assay was carried out and completed by Prof Zhu YZ’s Laboratory, Fudan University). Department of Pharmacology, YLL School of Medicine 67 Chapter 3: Materials and Methods Sham Vehicle Stroke + Leo (15, 30,60mg/kg/day) week pre-surgery treatment MCAO/Sham-operated surgery day after surgery TTC staining Neurological Score SOD Activity Assay MDA Assay GPx Activity Assay Figure 3-3: A Flow chart represents the experimental outline in the pilot study of Leonurine. 3.4.3.3 In vitro mitochondrial studies For second part of study, effects of Leonurine on mitochondrial ROS generation and its function were focused. Figure 3-4 summarizes the experimental design for this study. Similarly, 20 adult male Wistar rats were fasted overnight and anaesthetized with 0.2ml/100g ketamine/xylazine intraperitoneally (i.p) in order to collect the brain samples. Cortical tissues were separated for mitochondrial isolation. Isolated mitochondria suspension was divided into groups as listed in Table 3-2 for subsequent experiments. ROS generation assay, ATP biosynthesis assay and mitochondrial respiration were done to evaluate the effect of pHL in isolated mitochondria (Figure 3-4). Department of Pharmacology, YLL School of Medicine 68 Chapter 3: Materials and Methods of ABTSTM·+ can be monitored by reading the absorbance at 600nm. The antioxidants in the sample cause suppression of the absorbance at 600nm to a degree which is proportional to their concentration. 3.5.4 DNA oxidative damage analysis using GC/MS In order to evaluate the systemic oxidative damage after MCAO insult, blood was collected from heart (n=6) with needle washed with heparin (50U in saline). Collected blood was immediately subjected to centrifugation at 2000rpm for 10miutes to remove the plasma. Blood can be kept in -20°C until further usage. DNA was extracted from blood samples (n=6) using the phenol-chloroform method. Briefly, 1ml of solution A containing 10mM Tris-base, 10mM KCl and 10mM MgCl2.6H2O (pH7.6) was added into 1ml of blood sample followed by 33.33µl of Nonidet (Igepel, Sigma CA630). Vigorous shake was done to the samples to ensure a thorough mixing of the solutions. The samples were then further mixed for 15 minutes. After 15 minutes, the samples were centrifuged at 2000rpm for 10 minutes at 4ºC. Supernatants were removed after centrifugation. 160µl of solution B 10mM Tris-base, 10mM KCl, 10mM MgCl2.6H2O, 2mM EDTA and 0.5% SDS (pH7.6) was added to resolubilize the pellet. Next, 25µl of 2mg/ml RNase A was added to digest the contaminating RNA and the samples were allowed to incubate for 30 minutes at 37ºC, with gentle shaking. Subsequently, 80µl of phenol was added into each sample. This was followed by another vigorous vortex until the black beads were observed and sedimented, then the samples were centrifuged at 4000rpm for 10 minutes at 4ºC to collect supernatants. 40µl of phenol and 40µl of chloroform and isoamyl (24:1) Department of Pharmacology, YLL School of Medicine 73 Chapter 3: Materials and Methods were added into supernatant and allowed to vortex vigorously. Samples were centrifuged again at 4000rpm for 10 minutes at 4ºC. Supernatants were collected and added with 140µl 24:1 of chloroform and isoamyl. Next, vortex and centrifuge at 4000rpm for 10 minutes at 4ºC were done to the samples. Supernatants were collected and 3M sodium acetate and ethanol were added in the ratio of 1:0.1:2.5 (supernatant: sodium acetate: ethanol). Samples were then kept in at -20ºC for day. Next day, the samples were centrifuged at 3500rpm for 10 minutes to discard supernatants. Pellets were mixed and washed with 1ml of 70% ethanol followed by centrifugation at 10000rpm for 10 minutes. After the supernatants being removed, washing step was repeated. Resulting pellets were air dried and dissolved with 205µl of distilled water. Samples were then kept in -20ºC until analysis by GC/MS. The DNA samples were then analyzed by GC/MS (Agilent 6890 gas chromatograph and interfaced with an Agilent MSD 5973) as previously described (Whiteman, 2002). Briefly, aliquots of 100µg of DNA were freeze-dried under reduced pressure after addition of internal standards (0.5nmol, 6-azathymine and 2,6-diaminopurine). Samples and commercial standards were then subjected to hydrolysis by adding 0.5ml of 60% (v/v) formic acid. Subsequently, samples and standards were heated at 150°C for 45 minutes in evacuated glass hydrolysis tube. Samples were cooled, lyophilized and finally derivatized under a nitrogen atmosphere in poly(tetrafluoroethylene)-capped hypovials (Pierce) by adding 100µl of BSTFA (+1% TMCS/acetonitrile (4:1 v/v) mixture. Samples were again derivatized at 90°C for 45 minutes and analyzed by GC/MS. DNA oxidative adducts Department of Pharmacology, YLL School of Medicine 74 Chapter 3: Materials and Methods analyzed by GC/MS from each sample was summed up and the mean value of DNA adducts was obtained. 3.5.5 TUNEL (TdT-mediated dUTP Nick-End Labeling) assay At the end of treatment, the animals were perfused and fixed with 2% paraformaldehyde. The brain was then collected. Brains collected were post-fixed in 2% paraformaldehyde for two hours. They were then transferred into 15% sucrose in phosphate buffer overnight and subsequently 30% sucrose in phosphate buffer overnight to allow the sample dehydration. After dehydration step, brain tissues were sectioned at 20µm using a Leica® CM1510 Cryostat (Leica Microsystem, IL, USA). A DeadEndTM Fluorometric TUNEL System (G3250, Promega, USA) kit was chosen for detection of apoptosis-induced nuclear DNA fragmentation via fluorescence microscopy. This assay is based on the specific binding of TdT, which catalyzes the incorporation of fluorescein-dUTP at free 3’ ends of DNA. Specimens collected after cryostat from different rats (n=3) in each treatment group were used for TUNEL assay. Before starting the assay, all the slides were ensured to be dried properly. After proper drying of the slides, the slides were washed in 1xPBS for minutes. They were then fixed by immersing the slides in freshly prepared 4% methanol-free formaldehyde in PBS (pH7.4) in coplin jars for 15 minutes at room temperature. Next, the slides were washed in two changes of 1xPBS for five minutes each time. Subsequently, specimens were allowed to be permeabilized by incubating the slides in 20µg/ml Proteinase K solution for 10 Department of Pharmacology, YLL School of Medicine 75 Chapter 3: Materials and Methods minutes and washed by 1xPBS for minutes. The specimens were again fixed in 4% methanol-free formaldehyde solution in 1xPBS for minutes at room temperature and next, they were washed by immersing the slides in 1xPBS for minutes at room temperature. Subsequently, the specimens were incubated with 100µl of equilibration buffer, and equilibrate at room temperature for 10 minutes. While the specimens are equilibrating, nucleotide mix was thawed on ice in order to prepare rTdT incubation buffer. For each unit, the rTdT incubation buffer was made by 45µl of equilibration buffer, 5µl of nucleotide mix which is light sensitive and 1µl of rTdT enzyme. After the step of equilibration, the specimens were incubated with enough rTdT incubation buffer for 60 minutes in 37ºC humidified chamber. Care must be taken to ensure the specimens will not be dried out. Plastic coverslips were used to cover the cell to ensure equal distribution of the incubation buffer. Since the nucleotide mix is light sensitive, the slides were wrapped by aluminum foil to protect it from direct exposure to light. Also, coplin jars were wrapped by aluminum foil up upon this point to protect samples from light exposure. After one hour, the plastic coverslips were removed and the reactions were terminated by immersing the slides in 2x SSC for 15 minutes at room temperature. Then, the slides were washed in changes of 1xPBS for minutes each time at room temperature. This is to remove unincorporated flurescein-12-dUTP. Counterstaining was carried out by using DAPI which stains nucleus substances. Lastly, slides were viewed and photographed through a fluorescent Leica® microscope (Leica Microsystems, IL, USA) using standard fluorescein filter set to view the green fluorescence of fluorescein at 520 ± 20nm while blue DAPI was viewed at 460nm. Department of Pharmacology, YLL School of Medicine 76 Chapter 3: Materials and Methods 3.5.6 Immunohistochemical staining In addition, immunohistochemical staining (IHC) was also carried out to evaluate the locatization and expression level of apoptotic-related proteins, using frozen sections. A ready to use UltraVision Detection System Anti-Polyvalent, HRP/DAB kit (Lab Vision Corporation, CA, USA) was used for antibody staining. Brain samples from different rats (n=3) in each treatment group were used for antibody staining. Firstly, the slides were placed in coplin jars for washing in 0.2% 1x phosphate buffered saline-Triton X (0.2% PBS-Tx) for five minutes. Excess buffer was shaked off after washing. The slides were then incubated with hydrogen peroxide block for 10 minutes at room temperature. The blocking solution suppresses endogenous peroxidase and pseudoperoxidase activity in the tissue sections which could conceal the specific staining of the target antigens. After the process of blocking, slides were washed in two changes of 0.2% PBS-Tx for five minutes each time. Another minutes of incubation with Ultra V Block (provided reagent in the kit) was done to block the nonspecific background staining. Slides were rinsed in 0.2% PBS-Tx before primary antibody was added. Excess moisture around the sections was wiped off by paper towels. Tissues sections were then incubate with primary antibody for three hours. Polyclonal rabbit anti-Bax (N terminus) antibody (sc-493, Santa Cruz, CA, USA), polyclonal rabbit anti-Fas antibody (sc-715, Santa Cruz, CA, USA), polyclonal rabbit anti-Bcl-xL antibody (sc-492, Santa Cruz, CA, USA) and Bcl-2 (sc-7195, Santa Cruz, CA, USA) were used for the primary antibody incubation. All the antibody dilution was carried out by using 0.2% Department of Pharmacology, YLL School of Medicine 77 Chapter 3: Materials and Methods PBS-Tx. Dilution was done according to manufacturer’s instruction. Care was taken to ensure that the sections were not dried up during the incubation time by continual addition of specific antibodies when needed. After primary antibody incubation, the slides were then washed in changes of 1x phosphate buffered saline (1x PBS) for minutes each time. Following primary antibody incubation, tissue sections were incubated for one hour at room temperature with Biotinylated Goat Anti-Polyvalent. Also, care was taken to ensure the sections were not dried up during the incubation period. The slides were then washed in changes of 1x PBS for minutes each time. Subsequently, slides were incubated with streptavidin peroxidase which can conjugate with biotin present on the secondary antibody for 10 minutes. Slides were then rinsed in times of 1xPBS to remove excess streptavidin peroxidase. Colorimetric detection was performed with 3,3’- diaminobenzidine tetrahydrochloride (DAB) working solution to detect the specific antibody, secondary antibody and streptavidin-enzyme complex. The working solution is prepared by adding one drop of liquid DAB in 1ml of chromogen solution. The working solution was mixed well and the sections were covered by the solution for minute. The slides were then rinsed in 1x PBS for times. Counterstaininng was done by immersing the slides in a container containing distilled water for one minute. For counter-staining, slides were rinsed in hematoxylin times. Immediately, the slides were washed in running tap water to remove all traces of hematoxylin from the sections until water was clear. Following staining, dehydration of Department of Pharmacology, YLL School of Medicine 78 Chapter 3: Materials and Methods the tissue sections was carried out by immersing the slides in 70% ethanol for one minute, followed by 80% alcohol for one minute, then two changes of 100% ethanol for one minute each time, and finally in two changes of xylene for one minute each time. Hematoxylin stains nuclear substances, chromosomes, mitochondria and muscle striations blue to black and therefore display general structural features of the tissue. Lastly, the tissue sections were mounted by a drop of mounting medium Permount (Fisher Scientific International, PA, USA) by coverslip. The slides were left to dry overnight in the hood. Slides were viewed and photographed using a fluorescent Leica® microscope (Leica Microsystems, IL, USA). 3.5.7 Superoxide dismutase (SOD) activity assay Cortical tissues from eight rats of each group were collected 24 hours after MCAO and placed into an ice-cold RIPA lysis Buffer (Beyotime institute of biotechnology, Shanghai, China). The samples were then homogenized by Polytron homogenizer (Janke and Kunkel, Germany) followed by centrifugation at 3000rpm/minute for 15 min, supernatant was collected for SOD activity measurement. Superoxide Dismutase Detection Kit (A001, Nanjing Jiancheng Bioengineering Institute, Nanjing, China) was selected for SOD measurement. The assay was conducted according to the manufacturer’s instruction. Enzymatic activity of SOD was determined by competitive inhibition performed using xanthine–xanthine oxidase-generated superoxide to reduce the nitroblue tetrazolium (NBT) to blue diformazan at a constant rate. The rate of NBT reduction was monitored Department of Pharmacology, YLL School of Medicine 79 Chapter 3: Materials and Methods spectrophotometrically at 550 nm. One unit of SOD is defined as the amount of protein that inhibits the rate of NBT reduction by 50%. The results were given in unit per milligram of protein. 3.5.8 Glutathione peroxidase (GPx) activity assay Cortical tissues from eight rats of each group were collected 24 hours after MCAO and placed into an ice-cold RIPA lysis Buffer (Beyotime institute of biotechnology, Shanghai, China). The samples were then homogenized by Polytron homogenizer (Janke and Kunkel, Germany) followed by centrifugation at 3000rpm/minute for 15 min, supernatant was collected for GPx activity measurement. To determine the activity of GPx from each treatment group, Glutathione Peroxidase Detection Kit (A005, Nanjing Jiancheng Bioengineering Institute, Nanjing, China) was selected. The determination of GPx is based on the manufacturer’s instruction. Generally, GPx activity is measured by the oxidizing rate of GSH. In the presence of GPx, it can accelerate the reaction of the GSH and DTNB (5, 5’-dithiobis-2-nitrobenzoic acid) and produces a yellow colored 5-thio-2nitrobenzoic acid (TNB) which can be measured by spectrophotometer at 412nm. One unit of GPx is defined as the amount of protein that decreased the concentration of GSH by 1µM. The results were given in unit per milligram of protein. Department of Pharmacology, YLL School of Medicine 80 Chapter 3: Materials and Methods 3.5.9 Lipid peroxidation product measurement Cortical tissues from eight rats of each group were collected 24 hours after MCAO and placed into an ice-cold RIPA lysis Buffer (Beyotime institute of biotechnology, Shanghai, China). The samples were then homogenized by Polytron homogenizer (Janke and Kunkel, Germany) followed by centrifugation at 3000rpm/minute for 15 min, supernatant was collected for malondialdehyde (MDA) measurement. A MDA Detection Kit (A003, Nanjing Jiancheng Bioengineering Institute, Nanjing, China) was selected to determine the MDA level as a marker of lipid peroxidation. Assay was conducted according to manufacturer’s instruction. The kit is based on the MDA in the samples that forms 1:2 adduct with thiobarbituric acid (TBA). The MDA-TBA adduct formed from the reaction of MDA in samples with TBA can be measured spectrophotometrically at 532nm. The result was given as nanomole per milligram protein. 3.5.10 Preparation of intact rat brain mitochondria Brain mitochondria isolation was carried out as previously described (Sims, 1990). Briefly, adult Wistar Rats (n>60) weighed 280 – 300g were anaesthetized and brains were collected. The cortex was immediately excised and transferred into ice-cold isolation buffer containing 20mM Tris-HCl, 250mM sucrose, 40mM KCl, 2mM EGTA, and 1mg/ml bovine serum albumin, pH7.2. All the isolation procedures were carried out at 4°C. The cortex was homogenized using hand-held glass dounce tissue grinder with six tight upward and downward strokes, in isolation buffer. The crude homogenate was Department of Pharmacology, YLL School of Medicine 81 Chapter 3: Materials and Methods centrifuged at 2000 × g for minutes to remove cell debris and nuclei. The supernatant, containing the mitochondrial fraction was further centrifuged at 12000 × g for 11 minutes. Subsequently, the mitochondrial pellet was suspended in 3.5ml of 15% (v/v) Percoll. This suspension was overlain on two preformed percoll layers consisting of 3.5ml 40% and 3.5ml 23% (v/v) Percoll. The percoll gradient was established by centrifugation at 30700 × g for minutes. The distinct creamy layer in between 15% and 23% percoll was collected and resuspended in 500µl isolation buffer. The suspension was again subjected to centrifugation at 12000 × g for 11 minutes. Mitochondrial pellet was washed by resuspending the pellet with isolation buffer (without EGTA) and centrifugation at 12000 × g for 11 minutes. The resulting mitochondrial pellet was suspended in 200µl of isolation buffer to give the protein concentration of 25-35mg/ml as measured by nanodrop (Thermo Scientific). The mitochondrial suspension was kept on ice before the subsequent experiments. 3.5.11 Measurement of mitochondrial membrane potential – JC-1 assay In order to evaluate the isolated cortical mitochondria is suitable for bioenergetic studies, JC-1 assay was carried out (n=3). Mitochondrial probe JC-1 is a cationic carbocyanine dye which uptake into mitochondria is driven by transmembrane potential (Reser et al, 1995). JC-1 exists as green monomer/flurescence at low membrane potential. However, at higher membrane potential, it exists as red fluorescence of J aggregates which exhibit a braod excitation and emission spectrum. The mitochondrial membrane potential was Department of Pharmacology, YLL School of Medicine 82 Chapter 3: Materials and Methods measured on a PerkinElmer Life Sciences LS50B luminescence spectrometer (Buckinghamshire, UK) (Zhang et al, 2004). 2ml of respiratory buffer consisting of 20mM HEPES, 250mM sucrose, 10mM MgCl2 and 12.5mM KH2PO4, pH7.1 was introduced into a cuvette which was maintained at 37°C by an external waterbath. This was followed by the addition of 0.2µM JC-1 dye. The content in the cuvette was continuously stirred with a magnetic stirrer. After the system has reached equilibrium, 0.15mg of mitochondria and 125µM adenosine-di-phosphate (ADP) was added into the cuvette, followed by the addition of 5mM malate and 5mM glutamate to energize the mitochondria. Upon the establishment of optimal J-aggregate peak, 5µM of rotenone as complex I blocker was added reverse the previous reaction. Similar procedure was carried out for next that after the green monomer and red aggregate has reached the equilibrium in the buffer, 10mM succinate as complex II substrate was added to depolarize the mitochondria. This is again followed by complex II blocker using 250µM TTFA. 3.5.12 Measurement of ROS in isolated mitochondria ROS produced from mitochondria can be probed by dichloro fluorescein diacetate (DCFDA) (Wang and Joseph, 1999). DCFDA is a non-fluorescent ester of the dye fluoscein but in the presence of ROS, DCFDA is oxidized to the fluorescent product DCF. 0.1mg of mitochondria (n>6) was added into 200µl respiratory buffer consisting of 125mM KCl, 2mM KH2PO4, 1mM MgCl2·6H2O and 20mM HEPES, pH7.4, together with 1µM DCFDA. The reactions were started by measuring DCF fluorescence at Department of Pharmacology, YLL School of Medicine 83 Chapter 3: Materials and Methods excitation/emission wavelength of 485nm/526nm respectively for minutes as basal/background reading in a 96-well plate using a fluorescent microplate reader (Molecular Devices, Gemini XS, Sunnyvale, CA, USA), in the presence of 10mM succinate. Drug with multiple concentrations were added into the buffer after the basal reading (Table3-1, 2). Immediately after adding drugs, the reactions were measured again over 30 minutes to examine the effect of drugs added. Each actual reading was subtracted by the background fluorescence readings and the change of fluorescence intensity in each group was presented as percentage of increase as compared to the respective control group. To induce oxidative stress to isolated mitochondria, 1mM hydrogen peroxide H2O2 was added into the solution as for the H2O2-treated mitochondrial group before the basal reading was measured. 3.5.13 ATP biosynthesis in isolated mitochondria To test the effects of studied drugs on the mitochondrial function, ATP biosynthesis assay (n>6) was carried out based on luciferin-luciferase reaction as previously described (Vincent et al, 2004). The reaction takes place in two steps by luciferase, with the byproduct of light which can be monitored by spectrophotometer: luciferin + ATP → luciferyl adenylate + PPi luciferyl adenylate + O2 → oxyluciferin + AMP + light The buffer used consisting of 100mM KCl, 12.5mM KH2PO4 and 10mM Tris, pH7.6. 0.03mg of mitochondria was incubated with 1ml of buffer and 125µM ADP, complex II substrate 10mM succinate, complex I inhibitor 5µM rotenone and with the addition of Department of Pharmacology, YLL School of Medicine 84 Chapter 3: Materials and Methods drug in drug treated groups (Table 3-1, 2), under condition of continual shaking for minutes at 24°C. 1mM H2O2 was added into the solution as for the H2O2-treated mitochondrial group. The reactions were stopped by boiling for minutes and were immediately assayed or kept at -80°C for storage of ATP. Fresh or thawed samples were centrifuged at 14000rpm for 10 minutes at room temperature. The supernatant was collected and diluted 10 times so that the results could be extrapolated from a standard curve of 2-100pmol of ATP, a range that produced a linear response (Campbell, 1988). 25µl of diluted supernatants were added onto 96-well white plate. Samples have to be assayed together with standards for ATP determination. The FL-AAM (Adenosine 5’triphosphate (ATP) assay mix, Sigma) was diluted 250 times in FL-AAB solution (Adenosine 5’-triphosphate (ATP) assay mix dilution buffer, Sigma). 100µl of diluted FL-AAM was then added on to the standards and samples within minute. Chemiluminescence emitted by the luciferin-luciferase reaction was then measured using spectrophotometer (Varioskan Flash, Thermo Scientific). ATP biosynthesis among the treatment groups were expressed as percentage as compared to control. 3.5.14 Mitochondrial respiration measurement Mitochondrial respiration is another indicator of mitochondrial function. To evaluate the effects of studied drugs on mitochondrial respiration, mitochondrial oxygen consumption was measured (n>6) polarographically by monitoring the rate of oxygen consumption in an airtight chamber, equipped with a magnetic stirring device using a Clark type oxygen Department of Pharmacology, YLL School of Medicine 85 Chapter 3: Materials and Methods electrode (Hansatech, UK). Mitochondrial respiration was measured both in vitro and in vivo. For in vitro study, 1mg of mitochondria was added into a 2ml respiratory buffer consisting of 0.5mM EGTA, 3mM MgCl2·6H2O, 60mM K-lactobionate, 20mM Taurine, 10mM KH2PO4, 20mM HEPES, 110mM Sucrose and 1mg/ml bovine serum albumin, pH7.1. The study groups have been shown in Table 3-1. For in vivo study, mitochondrial isolation was done for both left and right cortex from each treatment group. Subsequently, 1mg of mitochondria was added into the respiration buffer for oxygen consumption measurement. After the addition of mitochondria, the suspension was stirred with magnetic stirrer to ensure the equal distribution of oxygen and contents within the solution. Once the system had reached the equilibrium, state respiration was initiated by the addition of 10mM succinate. Subsequently, 125µM of ADP was added into the chamber for state respiration, followed by state respiration by adding 0.5µg/ml of oligomycin. Oligomycin is a F0F1 ATP synthase inhibitor. During the oxygen consumption measurement, oligomycin will return the mitochondrial respiration to the basal rates. Respiratory control ratio (RCR) was then calculated as ratio of mean of state slope over state respiration slopes. Department of Pharmacology, YLL School of Medicine 86 Chapter 3: Materials and Methods 3.5.15 Glutathione level in isolated mitochondria Reduced glutathione (GSH) were determined by a glutathione assay kit (Cayman Chemical, 703002, Ann Arbor, MI) in order to examinine the effects of studied drug on mitochondrial antioxidant capacity. Glutathione level was measured both in vitro and in vivo. For in vitro studies, drugs (Table 3-1) were added into 0.5mg of mitochondrial (n=3) suspension for 30 minutes. 1mM H2O2 were added for H2O2 treated groups. In vivo study followed the details as shown in Table 3-2. In both cases, mitochondria suspension was incubated in the presence of 0.1% Triton X-100 on ice, for 10 minutes. It was then subjected to deproteination by addition of equal volume of freshly prepared 10% metaphosphoric acid, and allowed to stay in room temperature for minutes. This is followed by centrifugation of 5000×g for 10 minutes to remove the protein precipitate. The supernatant is collected and kept in -20°C. 50µl of 4M triethanolamine was added for each milliliter of thawed samples so to adjust the pH for the optimum condition for assay. GSH concentration was determined according to the manufacturer’s instruction. The assay utilizes the enzymatic recycling method, using glutathione reductase, for the quantitation of GSH. This is based on the principle that the sulfhydryl group of GSH reacts with DTNB (5, 5’-dithiobis-2-nitrobenzoic acid, Ellman’s reagent) and produces a yellow colored 5-thio-2-nitrobenzoic acid (TNB). The mixed disulfide, GSTNB (between GSH and TNB) that is concomitantly produced, is reduced by glutathione reductase to recycle the GSH and produce more TNB. The rate of TNB production is directly proportional to this recycling reaction which is in turn directly proportional to the Department of Pharmacology, YLL School of Medicine 87 Chapter 3: Materials and Methods concentration of GSH in the sample. Measurement of the absorbance of TNB at 405 or 414nm provides and accurate estimate of GSH in the sample. 3.6 Statistical analysis Data was represented as mean ± S.E.M of at least three independent preparations. Statistical analysis for neurological deficit grading system was measured by Chi Square. A difference with p[...]...Chapter 3: Materials and Methods Table 3- 2: Grouping for studies of effect of Leonurine on isolated mitochondria Groups 1 Control – No drug 2 Control – H2O2 3 Leonurine- 10µM 4 Leonurine- 100µM 5 Leonurine- 500µM 6 Leonurine- 1mM 7 H2O2 + Leonurine- 10µM 8 H2O2 + Leonurine- 100µM 9 H2O2 + Leonurine- 500µM 10 H2O2 + Leonurine- 1mM 3. 4 .3. 4 In vivo mitochondrial studies 28 male Wistar rats were randomly divided... of Medicine 73 Chapter 3: Materials and Methods were added into supernatant and allowed to vortex vigorously Samples were centrifuged again at 4000rpm for 10 minutes at 4ºC Supernatants were collected and added with 140µl 24:1 of chloroform and isoamyl Next, vortex and centrifuge at 4000rpm for 10 minutes at 4ºC were done to the samples Supernatants were collected and 3M sodium acetate and ethanol were... the mitochondrial fraction was further centrifuged at 12000 × g for 11 minutes Subsequently, the mitochondrial pellet was suspended in 3. 5ml of 15% (v/v) Percoll This suspension was overlain on two preformed percoll layers consisting of 3. 5ml 40% and 3. 5ml 23% (v/v) Percoll The percoll gradient was established by centrifugation at 30 700 × g for 5 minutes The distinct creamy layer in between 15% and 23% ... hours after MCAO/sham surgery Left cortices and right cortices were collected for mitochondrial isolation Effects of Leo on mitochondria isolated from each treatment group were evaluated in terms of mitochondrial respiration and glutathione assay 3 hours after the surgery (Figure 3- 4) Department of Pharmacology, YLL School of Medicine 69 Chapter 3: Materials and Methods In vitro experiments: In vivo... be measured spectrophotometrically at 532 nm The result was given as nanomole per milligram protein 3. 5.10 Preparation of intact rat brain mitochondria Brain mitochondria isolation was carried out as previously described (Sims, 1990) Briefly, adult Wistar Rats (n>60) weighed 280 – 30 0g were anaesthetized and brains were collected The cortex was immediately excised and transferred into ice-cold isolation... treatment Mitochondrial Isolation Fast overnight before surgery With/ without ROS Inducer + Drug Treatment • • • ROS Generation Assay (Kinetic) ATP Biosynthesis Assay Mitochondrial Respiration Assay MCAO/Sham 3 hours after surgery Mitochondrial Isolation • • Mitochondrial Respiration Assay Glutathione Assay Figure 3- 4: Flow charts represent the general outlines of mitochondrial studies for Leonurine. .. YLL School of Medicine 70 Chapter 3: Materials and Methods 3. 5 Experimental techniques 3. 5.1 Infarct volume measurement The infarct volume was assessed with TTC (2, 3, 5-triphenyltetrazolium chloride) staining After the brains had been collected, cerebellum and overlying membranes were removed It was then sectioned into eight pieces of 2mm thick coronal slices using a brain- sectioning block The coronal... chromatograph and interfaced with an Agilent MSD 59 73) as previously described (Whiteman, 2002) Briefly, aliquots of 100µg of DNA were freeze-dried under reduced pressure after addition of internal standards (0.5nmol, 6-azathymine and 2,6-diaminopurine) Samples and commercial standards were then subjected to hydrolysis by adding 0.5ml of 60% (v/v) formic acid Subsequently, samples and standards were... Chapter 3: Materials and Methods 3. 5.15 Glutathione level in isolated mitochondria Reduced glutathione (GSH) were determined by a glutathione assay kit (Cayman Chemical, 7 030 02, Ann Arbor, MI) in order to examinine the effects of studied drug on mitochondrial antioxidant capacity Glutathione level was measured both in vitro and in vivo For in vitro studies, drugs (Table 3- 1) were added into 0.5mg of mitochondrial. .. with Leonurine treatment (60mg/kg/day) [Sham + Leo], stroke group with water treatment as vehicle [Vehicle] and stroke group with Leonurine treatment (60mg/kg/day) [Stroke + Leo] The drugs were administrated orally once daily After 1 weeks of pre-surgery treatment, MCAO was induced in stroke induced groups Sham rats underwent similar procedures with the omission of MCAO Animals were sacrificed 3 hours . H 2 O 2 3 Leonurine- 10µM 4 Leonurine- 100µM 5 Leonurine- 500µM 6 Leonurine- 1mM 7 H 2 O 2 + Leonurine- 10µM 8 H 2 O 2 + Leonurine- 100µM 9 H 2 O 2 + Leonurine- 500µM 10 H 2 O 2 + Leonurine- 1mM. Figure 3- 3: A Flow chart represents the experimental outline in the pilot study of Leonurine. 3. 4 .3. 3 In vitro mitochondrial studies For second part of study, effects of Leonurine on mitochondrial. committee and confirmed to internationally accept ethical standards. 3. 3 Middle cerebral artery occlusion (MCAO) Ischemic stroke was induced in the rats by MCAO. Before middle cerebral artery