Purified herba leonuri and leonurine protect middle cerebral artery occluded rats from brain injury through antioxidative mechanism and mitochondrial protection 3

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

Chapter 3 Materials and Methods

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

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 electro-cauterization 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 2

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)

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 2 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

Animal treatments were continued for another 1 week after surgery During the period of post-surgery treatment, animal were assessed in terms of neurological deficit score At day 7 after surgery, animals were sacrificed and subsequent experiments were carried out

to evaluate their therapeutic potentials

4.3 Results

Figure 3-1: A Flow chart represents the experimental outline in the pilot study of pHL

2 weeks pre-surgery treatment MCAO/Sham-operated surgery

DNA damage analysis TUNEL Assay

chemical staining

Immunohisto-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)

Table 3-1: Grouping for studies of effect of pHL on isolated mitochondria

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 2 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 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 3 hours after the surgery (Figure 3-2)

Figure 3-2: Flow charts represent the general outlines of experimental protocol II

Sham, Sham+pHL, Vehicle, Stroke+pHL

Fast overnight before surgery

Mitochondrial Isolation

• Mitochondrial Respiration Assay

• Glutathione Assay

MCAO/Sham

2 weeks treatment

3 hours after surgery

Male Wistar Rat ~250g

Fasting, overnight

With/ without ROS Inducer

+ Drug Treatment

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 1 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)

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)

Stroke + Leo (15, 30,60mg/kg/day)

1 week pre-surgery treatment MCAO/Sham-operated surgery

1 day after surgery

TTC staining

Neurological Score

SOD Activity Assay

GPx Activity Assay

MDA Assay

Table 3-2: Grouping for studies of effect of Leonurine on isolated mitochondria

3.4.3.4 In vivo mitochondrial studies

28 male Wistar rats were randomly divided into four groups: sham group with water treatment [Sham], sham group 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 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)

Figure 3-4: Flow charts represent the general outlines of mitochondrial studies for

Leonurine

Sham, Sham+pHL, Vehicle, Stroke+pHL

Fast overnight before surgery

Mitochondrial Isolation

• Mitochondrial Respiration Assay

• Glutathione Assay

MCAO/Sham

1 week treatment

3 hours after surgery

Male Wistar Rat ~250g

Fasting, overnight

With/ without ROS Inducer

+ Drug Treatment

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 sections were stained with 0.1% TTC solution at 37ºC for 30 minutes and then preserved in 4% formalin solution The infarct size was analyzed with an image analyzer system (Scion image for windows) and converted by integration (including correction for edema and atrophy) to the true infarct size of ischemic damage using the equations (Lu et al, 2004):

S’= (S/ Ti) X Tc Percent infarct size= (ΣS’/ ΣTc) * 100 (S= stroke area, Tc= total area of contralateral hemisphere, Ti= total area of ipsilateral

hemisphere)

3.5.2 Evaluation of neurological deficit

To evaluate neuronal function impairment after stroke insult, neurological deficit grading system was carried out as described previously (Lu et al, 2005) A scale of zero to five was used to assess the behavioral and motor changes observed in the animal after the MCAO procedure This evaluation took place every day after the stroke surgery Rats were scored as zero when they were suspended from the tail, exhibited normal behavior

suspension and there was no other abnormality observed, they will be assigned a score of one The rats were then placed on the ground and allowed to move freely and were observed for circling behavior Rats that moved spontaneously in all directions but showed a monodirectional circling towards the paretic side when given a light jerk of the tail, they were assigned a score of two A score of three were assigned to rats that showed

a consistent spontaneous contralateral circling Rats that were very weak and walked only when stimulated were scored as 4 If the rats died at the day of assessment, they were assigned a score of five Rats that showed a higher clinical score exhibited all features of the lower grades Therefore, rats with higher scoring showed more severe neuronal function impairment

3.5.3 Total antioxidant assay

Plasma from each animal was collected for total antioxidant assay Blood was collected from heart with needle washed with heparin (50U in saline) Collected blood was immediately subjected to centrifugation at 2000rpm for 10miutes to collect the plasma Plasma can be kept in -20°C until further usage A Total Antioxidant Status Assay Kit

(615700, Calbiochem, Germany) kit was selected for the investigation of changes of

endogenous antioxidant system under the influence of pHL The assay was conducted according to manufacturer’s instruction The kit depends on the antioxidants in the sample inhibiting the oxidation of ABTSTM (2,2’-Azino-di-[3-ethylbenz-thiazoline sulphonate]) substrate to ABTSTM·+ product by a peroxidase, metmyoglobin The amount

of ABTS 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)