The role of agmatine and arginine

decarboxylase in ischemic tolerance after

transient cerebral ischemia in rat models

Jin Young Jung

Department of Medicine

The Graduate School, Yonsei University

The role of agmatine and arginine

decarboxylase in ischemic tolerance after

transient cerebral ischemia in rat models

Directed by Professor Seung Kon Huh

The Doctoral Dissertation submitted

to the Department of Medicine,

the Graduate School of Yonsei University

in partial fulfillment of the requirements for the degree of

Doctor of Philosophy

Jin Young Jung

May 2007

This certifies that the Doctoral Dissertation

of Jin Young Jung is approved.

__________________________________

Thesis Supervisor: Seung Kon Huh

__________________________________ Jong Eun Lee: Thesis Committee Member #1

__________________________________

Jin Woo Chang: Thesis Committee Member #2

__________________________________

Duck Sun Ahn: Thesis Committee Member #3

__________________________________

Ji Cheol Shin: Thesis Committee Member #4

The Graduate School

Yonsei University

May 2007

Acknowledgements

Some may consider this short section of the thesis trivial but for me it is

a chance to express my sincerest gratitude to those that I am truly thankful.

First of all, I would like to express my deepest gratitude to my thesis

supervisor and mentor Professor Seung Kon Huh. He has inspired me when

I was troubled and always gave me a warm heart. I would also like to thank

Professor Jong Eun Lee who shared her valuable time on the execution and

interpretation of this study, Professor Jin Woo Chang who always inspiring

me with passion and discerning insight. Professor Duck Sun Ahn whose

insightful comments were essential in completing this thesis, Professor Ji

Cheol Shin for the excellent suggestion for improvement in this thesis.

I wish to special thanks to Jae Hwan Kim for his many advises

concerning the experiment, Yong Woo Lee who gave me a great help for

completing this thesis.

I am deeply indebted to my parents, who always provided a solid

foundation for me to go my way. I feel a deep sense of gratitude for my

companion and wife, Ho Jung Kang and my lovely son, Jae Yoon Jung

who is the hope of my life.

May 2007

Jin Young Jung

i

TABLE OF CONTENTS

ABSTRACT---------------------------------------------------------------------- 1

I. INTRODUCTION-------------------------------------------------------------- 3

II. MATERIALS AND METHODS-------------------------------------------- 4

1. Animals and experimental protocols------------------------------------- 4

2. Induction of ischemic preconditioning and focal ischemia------------ 4

3. Morphometric measurement of brain edema and infarct volume----- 5

4. Agmatine analysis with HPLC-------------------------------------------- 6

4-1.Sample preparation ---------------------------------------------------- 6

4-2. Apparatus and chromatographic conditions------------------------ 6

5. Immunostaining for ADC, NOSs, phosphoERK1/2, and BMP-7-----6

6. Immunoblotting of ADC, Erk1/2 ----------------------------------------- 7

7. Statistical analysis----------------------------------------------------------- 7

III. RESULTS--------------------------------------------------------------------- 7

1. rCBF responses to experimental control group and ischemic preconditioning group in MCAO models--------------------------------

7

2. Brain edema and infarct volume after ischemic injury----------------- 8

3. The level of agmatine after ischemic injury----------------------------- 11

4. Assessment for level of ADC---------------------------------------------- 13

5. Assessment for level of nNOS and iNOS ------------------------------- 14

6. Assessment for level of ERK1/2, phosphoERK1/2, and BMP-7----- 17

IV. DISCUSSION----------------------------------------------------------------- 20

V. CONCLUSION---------------------------------------------------------------- 22

Ⅵ. REFERENCES---------------------------------------------------------------- 23

ABSTRACT (IN KOREAN) --------------------------------------------------- 28

ii

LIST OF FIGURES

Figure 1 Experimental protocol---------------------------------- 5

Figure 2 rCBF of experimental control group and ischemic preconditioning group in MCAO---------------------

8

Figure 3 Preconditioning reduces infarct size in a model of MCAO ---------------------------------------------------

9

Figure 4 Brain edema after ischemic injury with or without preconditioning------------------------------------------

11

Figure 5 Level of agmatine in rat brain tissue------------------ 12

Figure 6 Western blots of arginine decarboxylase------------- 13

Figure 7 Immunohistochemistry of arginine decarboxylase- 14

Figure 8 Immunohistochemistry of neuronal nitric oxide synthase in ischemic injured rat brain----------------

15

Figure 9 Immunohistochemistry of inducible nitric oxide synthase in ischemic injured rat brain----------------

16

Figure 10 Western blots of ERK1/2 in ischemic injured rat brain-------------------------------------------------------

17

Figure 11 Immunohistochemistry of phosphoERK1/2 in ischemic injured rat cerebral cortex------------------

17

Figure 12 Immunohistochemistry of phosphoERK1/2 in ischemic injured rat striatum--------------------------

18

Figure 13 Immunohistochemistry of BMP-7 at post-reperfusion 1hr------------------------------------------

19

LIST OF TABLES

Table 1. Infarct volume after ischemic injury------------------ 10

Table 2. Level of agmatine after ischemic injury-------------- 12

iii

LIST OF ABBREVIATIONS

ADC Arginine decarboxylase

BMP-7 Bone morphogenetic protein-7

EC Experimental control group

ERK1/2 Extracellular signal-regulated kinase1/2

HPLC high performance liquid chromatography

IP Ischemic preconditioning group

MCAO Middle cerebal artery occlusion

NO Nitric oxide

nNOS Neuronal nitric oxide synthase

iNOS Inducible nitric oxide synthase

rCBF Regional cerebral blood flow

1

Abstract

The role of agmatine and arginine decarboxylase in ischemic tolerance

after transient cerebral ischemia in rat models

Jin Young Jung

Department of Medicine

The Graduate School, Yonsei University

(Directed by Professor Seung Kon Huh)

Agmatine is an endogenous clonidine-displacing substance, an agonist for the α2-

adrenergic and imidazoline receptors, and an antagonist at N-methyl-D-aspartate

(NMDA) receptors. Agmatine was shown to protect neurons against glutamate toxicity

and this effect was mediated through NMDA receptor blockade, with agmatine

interacting at a site located within the NMDA channel pore. Furthermore, this

protection is associated with decreased nitric oxide synthase (NOS) activity and

expression, as well as NO generation.

Preconditioning describes a powerful sublethal treatment, which induces neurons to

become more resistant to a subsequent ischemic insult. Ischemic preconditioning is one

of the most important endogenous mechanisms for protecting cells against ischemic and

reperfusion injury.

In this study, the association of agmatine with ischemic preconditioning and ischemic

tolerance was investigated. The data obtained here have demonstrated that the

endogenous neuroprotective mechanisms are facilitated by ischemic preconditioning

through increasing ischemic tolerance by agmatine. The level of agmatine was increased

during the ischemic preconditioning and the increased level of agmatine also facilitates

the agmatine production during the ischemic injury. However, expression of arginine

decarboxylase (ADC) in preconditioning group was not demonstrable during the

2

ischemic injury and reperfusion injury.

Being structurally similar to L-arginine, agmatine has been considered as a nitric

oxide synthase (NOS) inhibitor, especially neuronal NOS. To investigate the

relationship between elevating levels of agmatine during ischemic preconditioning and

NOS expression, immunostaining against NOSs was performed. Results indicated that

the agmatine has a ischemic preconditioning decreased the expression of nNOS in the

cerebral cortex and striatum at 1 hr and 23 hr reperfusion following 1 hr ischemia.

The level of ERK which regulates various cellular processes such as cell growth and

differentiation was determined in ischemic brain with or without ischemic

preconditioning. The protein expression of ERK was increased in ischemic

preconditioning group than the experimental control group.

The expression of BMP-7 was also investigated in this study. The level of BMP-7

was induced in preconditioning group under MCA occlusion. Induced level of agmatine

may act by increasing the expression of BMP-7 and ERK which are involved in cell

survival.

These results indicated that neuroprotective mechanism of ischemic preconditioning

might be related with elevated level of agmatine and increasing BMP-7, ERK

expression.

______________________________________________________________________

Key words: agmatine, arginine decarboxylase, ischemic tolerance, preconditioning

3

The role of agmatine and arginine decarboxylase in ischemic tolerance

after transient cerebral ischemia in rat models

Jin Young Jung

Department of Medicine

The Graduate School, Yonsei University

(Directed by Professor Seung Kon Huh)

I. INTRODUCTION

Agmatine, formed by the decarboxylation of L-arginine by arginine decarboxylase (ADC), was

first discovered in 1910. It is hydrolyzed to putrescine and urea by agmatinase1. Recently,

agmatine, ADC, and agmatinase were found in mammalian brain2. Agmatine is an endogenous

clonidine-displacing substance, an agonist for the α2-adrenergic and imidazoline receptors, and

an antagonist at N-methyl-D-aspartate (NMDA) receptors2-4. Recent studies have shown that

agmatine may be neuroprotective in trauma and neonatal ischemia models1, 5-9. Agmatine was

shown to protect neurons against glutamate toxicity and this effect was mediated through

NMDA receptor blockade, with agmatine interacting at a site located within the NMDA channel

pore10. Despite this work, the mode and site(s) of action for agmatine in the brain have not been

fully defined.

Nitric oxide (NO) is known to trigger and a mediator cascades involved in inflammation and

apoptosis in ischemic injury and inducible Nitric oxide synthase (iNOS) is also involved in the

mechanisms by which ischemia-induced inflammation. Inducible NOS (iNOS) is expressed

predominantly in inflammatory cells infiltrating the ischemic brain and in cerebral blood

vessels11, 12. Delayed administration of iNOS inhibitors may be a useful therapeutic strategy to

target selectively the progression of ischemic brain injury.

Being structurally similar to L-arginine, agmatine is also a competitive nitric oxide synthase

(NOS) inhibitor13, 14. NOSs generate nitric oxide (NO) by sequential oxidation of the

4

guanidinium group in L-arginine, and agmatine is an L-arginine analogue with a guanidinium

group. This suggests that agmatine may protect the brain from ischemic injury by interfering

with NO signaling.

Ischemic tolerance is the phenomenon whereby ischemic preconditioning protects against a

subsequent lethal ischemia15. Endogenous mechanisms for protecting cells against ischemic

injury increases in the resistance of cells to ischemia arise after one or several transient episodes

of ischemia. Ischemic preconditioning has been shown to protect hippocampal CA1 pyramidal

cells from subsequent lethal ischemia16. Heat shock proteins, immediate early genes, anti-

oxidant enzyme, anti-apoptotic oncogene, interleukin-1h and adenosine might be involved in

ischemic tolerance. The protective mechanism of ischemic preconditioning are reported to

involve intracellular signal transduction pathway including endoplasmic reticulum and DNA

repairing function17.

The purpose of this study is to determine the effects of agmatine on ischemic tolerance after

transient focal ischemia model and assessment of level of agmatine and ADC during ischemic

injury with HPLC (High performance liquid chromatography) method. And the effect of

agmatine on ischemic preconditioning and tolerance was evaluated in this study.

II. MATERIALS & METHODS

1. Animals and experimental protocols

The protocol for these animal studies was approved by the Yonsei University Animal Care and

Use Committee in accordance with NIH guidelines. Adult male Sprague–Dawley rats (Sam Co.,

Osan, Korea) weighing 280 to 320 g were used for all experiments. Rats were allowed free

access to food and water before the experiment. Animals were anesthetized with ketamine (60

mg/kg, IP) before any surgery during which time body temperature was maintained at 36.5 ~

37.5 °C.

2. Induction of ischemic preconditioning and focal ischemia

Transient MCA occlusion was conducted as described earlier8. The MCA was occluded for 10

mins for ischemic preconditioning (IP) and 1 hr for ischemia. In IP, a 1 hr occlusion was

induced 3 days after a 10 mins occlusion and a 1hr occlusion was induced 3 days after sham

operation in experimental control (EC). In brief, a rat was intraperitoneally anesthetized with

ketamine, placed in a stereotaxic frame fitted. A craniectomy (3 mm in diameter, 6 mm lateral

and 2 mm caudal to bregma) was performed with extreme care over the MCA territory using a

5

trephine. The dura was left intact and a laser doppler flow meter probe was placed on the

surface of the ipsilateral cortex and fixed to the periosteum. The probe was connected to a laser

flow meter device (OMEGA FLOW, FLO-C1, Neuroscience, Tokyo, Japan) for continuous

monitoring of regional cerebral blood flow (rCBF). The right common carotid artery (CCA),

external carotid artery (ECA) and internal carotid artery (ICA) were exposed through a ventral

midline incision. A 4–0 monofilament nylon suture with a rounded tip (160 ㎛ in diameter)

was introduced into CCA lumen and gently advanced to ICA until rCBF was reduced to 15–

20% of the baseline (recorded by laser Doppler flow meter). After the desired period of

occlusion (10 mins or 1 hr), the suture was withdrawn to restore the blood flow (confirmed by

the return of rCBF to the baseline level). The wound was sutured and the rat was allowed to

recover from anesthesia before returning to the cage with free access to rat chow and water.

Figure 1. Experimental protocol. Diagram show the experimental protocol; EC (Experimental

control group), IP (Ischemic preconditioning group), MCAO(middle cerebral artery occlusion).

3. Morphometric measurement of brain edema and infarct volume

Animals were then decapitated at 0 hr, 0.5 hr, 1 hr, 2 hr, 4 hr, 7 hr, or 24 h after ischemia and

the brains rapidly removed and sectioned coronally at 2-mm intervals. 2nd, 4th, and 6th sections

of six serial slices were incubated for 15 mins in a 2 % solution of TTC at 37 °C and fixed by

immersion in 4 % paraformaldehyde solution. Using a computerized image analysis system

(Image J, NIH image, version 1.36), the area of infarction of each section was measured. The

volume of infarction in each animal was obtained from the product of slice thickness (2 mm)

and sum of infarction areas in all brain slices examined. Brain edema was determined from the

following formula:

Brain edema (%) = (the volume of ipsilateral hemisphere / the volume of contralateral

hemisphere) X 100 (%)

4. Agmatine analysis with HPLC

4-1.Sample preparation

Brain samples were prepared by a modification of the method of Reed and Belleroche ( Reed

6

LJ, 1990). The ipsilateral part of 3rd brain coronal section were quickly stored at -80 °C until

the time of processing and assay. For the HPLC method (Patchett ML, 1988), tissue samples

were weighed and homogenized using a sonicator for 10 sec in ice (setting 5; Sonifier Cell

Disruptor, Model W185; Plainview, L.I., NY, USA) in 0.5 ml of ice-cold 10% (w/v)

trichloroacetic acid per 150 mg tissue (wet weight). Sample homogenates were then left on ice

for 1 hr and then centrifuged at 20,000 g for 25 mins. The supernatant was washed 5 times using

an equal volume of diethyl-ether and the aqueous phase was saved. Any remaining ether was

evaporated at room temperature for 20 mins. A volume of 20 ul of sample plus 20 ul of the

OPA-ME derivatizing reagent was mixed for 2 mins at room temperature. Thereafter, 20 ul was

immediately injected into the HPLC system.

4-2. Apparatus and chromatographic conditions

The HPLC system consisted of a pump and multi-solvent delivery system (Shimadzu HPLC

CLASS-VP, Japan), a RF-10Axl fluorescence detector (excitation wavelength of 325 nm and

emission wavelength of 425 nm; Shimadzu, Japan) and a Hypersil GOLD 150 X 2.1, 5 ㎛

column (Thermo Electron). Potassium borate buffer (final 0.2 M, pH 9.4 at 20 °C) was prepared

by dissolving boric acid in water and adjusting the pH with a saturated solution of potassium

hydroxide in a final volume of 250 ml. The buffer was passed through a 0.22um filter (Gelman

Sciences, Ann Arbor, MI, USA) and stored at 4 °C. The OPA-ME derivatizing reagent was

prepared by dissolving 50 mg OPA in 1 ml of methanol, then adding 53 ㎕ of ME and 9 ml of

0.2 M potassium borate buffer (pH 9.4) and was stored at 4 °C for not more than three days

before use. The method of measuring agmatine utilized derivatization with OPA-ME. The

mobile phase consisted of a mixture of 46 % 10 mM potassium dihydrogen phosphate

containing 3 mM octylsulfate sodium salt in water (pH 5.93), 34 % acetonitrile and 20 %

methanol. The mobile phase was degassed before use.

5. Immunostaining for ADC, NOSs, phosphoERK1/2, and BMP-7

The 4th brain coronal section were quickly fixed with 4 % paraformaldehyde, and embedded

in paraffin. Brain sections were made by 6 ㎛. Sections were immunostained with antibodies

against ADC, nNOS (Upstate), iNOS (Calbiochem), phosphoERK1/2 (Cell signaling), or BMP-

7 (Santa Cruz), followed by an appropriate biotinylated secondary antibody. Stains were

visualized using the ABC kit (Vector, Burlingame, CA, USA) (Lee et al., 2002), then reacted

with diaminobenzidine (DAB, Sigma, St. Louis. MO, USA). Immunostaining controls were

prepared by tissue without primary antibodies. All incubation steps were performed in a

humidified chamber. The positive area was measured using a computerized image analysis

system (Image J, NIH image, version 1.36).

7

6. Immunoblotting of ADC and ERK1/2

Expressions of ADC and ERK1/2 proteins were estimated by immunoblotting in the ipsilateral

part of 5th brain coronal section. Immunoblotting was performed using anti-ADC, anti-ERK1/2

(Cell Signaling), and anti-actin (Santa Cruz) antibodies. Equal amounts of protein, 200 ㎍ per

condition, were separated on an 10 % polyacrylamide gel and electrotransferred onto

Immobilon-P membrane (Millipore, Bedford, MA, USA). Immunoreactive bands were

visualized with the ECL detection system using Kodak X-AR film.

7. Statistical analysis

Statistical tests to determine differences between groups were performed with student’s t test

using SPSS ver 13.0 (SPSS, Chicago, IL, USA). P value < 0.05 was considered significant. Data

are expressed as the mean ± standard deviation (SD).

III. RESULTS

1. rCBF responses to EC and IP in MCAO models

The relative rCBF pattern measured by laser Doppler flow meter over the ipsilateral parietal

cortex was presented in Figure 2. Baseline rCBF recorded before MCA occlusion under steady-

state conditions was defined as 100 % flow. After MCAO, CBF decreased to 20 % in both

goups, Ischemia was confirmed when the laser Doppler signal was reduced to 20 % of baseline.

Transient MCAO was performed in both EC and IP group with an hour of occlusion. During

reperfusion, rCBF returned to preischemic levels about 80 % of each reperfusion cycle. rCBF

levels were not significantly different between groups.

8

Figure 2. rCBF of EC and IP in MCAO. Relative rCBF measurements were made over the

ipsilateral brain cortex by laser Doppler flow meter. Baseline values before MCAO are defined

as 100 % flow. After the 10mins of preconditioning, rCBF was restored up to 80 % of

preischemic levels. Transient occlusion was performed in EC and IP group lasting 60 mins.

rCBF value was not significantly different in both groups; EC (Experimental control group), IP

(Ischemic preconditioning group), MCAO(middle cerebral artery occlusion).

2. Brain edema and infarct volume after ischemic injury

Infarct was significantly affected by preconditioning. Infarct volume was markedly reduced

in IP by approximately 47 % compared to EC (Figure 3-A, B and C). Preconditioning was

highly effective at protecting brain from ischemic injury. The infarct volume was summarized in

table 1. Preconditioning reduced the brain edema significantly 23 hr after reperfusion (R23)

following 1hr ischemia (Figure4).

9

A.

B.

10

C.

Figure. 3. Preconditioning reduced infarct size in a model of middle cerebral artery occlusion

(MCAO) in rat. (A) TTC staining of the ischemic injured brain of EC. (B) TTC staining of the

ischemic injured brain with IP. (C) Infarct volume after ischemic injury with and without

preconditioning. IP reduced the infarct volume significantly compared to EC in R23. EC

(Experimental control group), IP (Ishcemic preconditioning group), M1 (MCA occlusion 1 hr ),

R1 (Post reperfusion 1hr), R3 (3hr), R6 (6hr), R23 (23hr). (** P

11

Figure 4. Brain edema after ischemic injury with or without preconditioning. Preconditioning

group reduced the brain edema significantly in R23. EC (Experimental control group), IP

(Ischemic preconditioning group), M0 (MCA occlusion 0 hr), M0.5 (0.5hr), M1 (1hr), R1 (Post

reperfusion 1hr), R3 (3hr), R6 (6hr), R23 (23hr). (* P

12

Figure 5. Level of agmatine in rat brain tissue was measured at 0, 0.5, 1, 2, 4, 7, and 24 h after

ischemic injury. The highest peak was noted at 2 hr after injury. EC (Experimental control

group), IP (Ischemic preconditioning group).

Table 2. Level of agmatine after ischemic injury. EC (Experimental control group), IP

(Ischemic preconditioning group), M0 (MCA occlusion 0 hr), M0.5 (0.5hr), R1 (Post

reperfusion 1hr), R3 (3hr), R6 (6hr), R23 (23hr), (* P

13

4. Assessment for level of ADC

The expression of arginine decarboxylase (ADC) in IP group was not demonstrable during

the ischemic injury and reperfusion injury (Figure 6). In EC group, the level of ADC was

decreased during the ischemic reperfusion injury. In IP group, the expression of ADC slightly

decreased during the reperfusion period (R3-R23) however, the effect was minimized (Figure 6).

In immunostained brain sections with ADC antibodies, ADC-immunopositive area was

significantly increased in cerebral cortex protected by ischemic preconditioning 23 hr after

reperfusion (R23), but not in striatum (Figure 7).

Figure 6. Western blots of arginine decarboxylase (ADC) in ischemic rat brain. EC

(Experimental control group), IP (Ischemic preconditioning group), M0 (MCA Occlusion 0 hr),

M0.5 (0.5hr), M1 (1hr), R1 (Post reperfusion 1hr), R3 (3hr), R6 (6hr), R23 (23hr), (** P

14

Figure 7. Immunohistochemistry of arginine decarboxylase (ADC) in ischemic rat brain (A.

EC cortex B. IP cortex C. EC striatum D. IP striatum). Effect of preconditioning on the

expression of ADC in brain section. ADC-positive area (red or yellow) was increased in

ischemic preconditioning (IP) group (B) compared to experimental control (EC) group (A) at

23 hr after reperfusion. EC (Experimental control group), IP (Ischemic preconditioning group).

5. Assessment for level of nNOS and iNOS

It has been known that the neuroprotection of agmatine from ischemic injury was associated

with a reduction of nitric oxide (NO) and neuronal nitric oxide synthase (nNOS), but not

inducible NOS (iNOS). To investigate the effect of elevated level of agmatine by ischemic

15

preconditioning on NOSs expression, the expression of nNOS and iNOS was investigated. Our

data shows the number of nNOS-positive cells was significantly decreased in ischemic

preconditioning (IP) group in the cerebral cortex and striatum at 1hr and 23hr reperfusion

following 1 hr ischemia (Figure 8). However, the expression of iNOS was demonstrable at 1hr

and 23hr reperfusion in both groups (Figure 9).

Figure 8. Immunohistochemistry of nNOS in ischemic injured rat brain. (A. EC cortex B. IP

cortex C. EC striatum D. IP striatum). Micrographs of nNOS positive cells (brown) are

significantly decreased in IP group (B and D) compared to EC group (A and C) at 23 hr after

reperfusion. nNOS-positive area was decreased in ischemic preconditioning (IP) group (B)

compared to experimental control (EC) group (A) at 1hr and 23 hr after reperfusion. EC

(Experimental control group), IP (Ischemic preconditioning group).

16

Figure 9. Immunohistochemistry of iNOS in ischemic injured rat brain. (A. EC cortex B. IP

cortex C. EC striatum D. IP striatum). The expression of iNOS positive cells (brown) are

demonstrable and not significantly different in IP group (B and D) compared to EC group (A

and C) at 23 hr after reperfusion. EC (Experimental control group), IP (Ischemic

preconditioning group).

17

6. Assessment for level of ERK1/2, phosphoERK1/2, and BMP-7

Activation of the ERK1/2 pathway has been shown to be protective against brain ischemia.

The expression of ERK1/2 was increased during ischemic and reperfusion injury. The level of

ERK1/2 was higher in IP group than the EC group (Figure 10). phosphoERK1/2-positive cells

were increased in the cerebral cortex and striatum of ischemic injured rat (EC) at 1hr (R1) and

23hr (R23) after reperfusion. The positive cells were stained strongly at R1 more than at R23 in

EC. But the phosphoERK1/2-positive cells were decreased in the cerebral cortex and striatum of

preconditioned rat (IP) at 1hr and 23hr after reperfusion.

Figure 10. Western blots of ERK1/2 in ischemic injured rat brain. EC (Experimental control

group), IP (Ischemic preconditioning group), M0 (MCA occlusion 0 hr), M0.5 (0.5hr), M1 (1hr),

R1 (Post-reperfusion 1hr), R3 (3hr), R6 (6hr), R23 (23hr).

Figure 11. Immunohistochemistry of phosphoERK1/2 in ischemic injured rat cerebral cortex.

The expression of phosphoERK1/2 positive cells (brown) are significantly decreased in IP

group (B and D) compared to EC group (A and C) at 1hr (R1) and 23 hr (R23) after reperfusion.

EC (Experimental control group), IP (Ischemic preconditioning group).

18

Figure 12. Immunohistochemistry of phosphoERK1/2 in ischemic injured rat striatum. The

expression of phosphoERK1/2 positive cells (brown) are significantly decreased in IP group (B

and D) compared to EC group (A and C) at 1hr (R1) and 23 hr (R23) after reperfusion. EC

(Experimental control group), IP (Ischemic preconditioning group).

19

The expression of BMP-7 was also induced in IP group under MCA occlusion at post-

reperfusion 1hr in the protected cerebral cortex , however, there was not significant difference in

BMP-7 immunopositive area between IP and EC in cortex at post-reperfusion 23hr (Figure 13).

Figure 13. . Immunohistochemistry of BMP-7 at post-reperfusion 1hr. The expression of BMP-7

was increased in ipsilateral cortex of IP. (A. EC cortex B. IP cortex C. EC striatum D. IP

striatum).

20

ⅣⅣⅣⅣ. DISCUSSION

Ischemic preconditioning is one of the most important endogenous mechanisms for

neuroprotection and it has previously been shown to be protective effects against ischemic or

reperfusion injury18-21. Increases in the resistance of neuron to ischemia arise after one or several

transient episodes of ischemia/reperfusion. Previous reports suggest that heat shock proteins17, 23,

24, immediate early genes25, 26, antioxidant enzyme27, 28, antiapoptotic oncogene29, 30, interleukin-

1h31, 32, and adenosine33, 34 might be involved in the development of ischemic tolerance.

Recent reports indicated that agmatine has neuroprotective effects against ischemic injury in

neuronal cultures and experimental stroke in vivo8. Furthermore, this protection is associated

with decreased NOS activity and expression, as well as NO generation5. There are several

possible mechanisms of agmatine induced neuroprotection. First, agmatine has been shown to

reduce excitotoxicity in vitro by blocking NMDA receptor activation 1, 10. Second, agmatine, an

α-2 adrenoceptor agonist, and another α-2 adrenoceptor agonist, dexmedetomidine have been

shown to protect neurons from injury in vivo and in vitro 2, 22. Third, agmatine is a NOS

antagonist, and generation of NO has been implicated in ischemic brain injury23. Intracellulaly,

agmatine is reported to modulate the production of polyamines36 and is stored in synaptic

vesicles, accumulated by active uptake, released by depolarization, and inactivated by

agmatinase 37. It has been suggested that agmatine may modulate behavioral functions from

stress38. and reported that endogenous agmatine was increased in response to cold-restraint

stress 39.

In this study, the association of agmatine with ischemic preconditioning and ischemic

tolerance was investigated. The observed increases in the activities of agmatine following

preconditioning have not previously been reported. Chen et al.40 have reported that tolerance

was observed if the interval between the tolerizing paradigm and stroke was 2, 3, or 5 days, but

not 1 or 7 days. In this study, middle cerebral artery was occluded for 10 mins for ischemic

preconditioning (IP) and a 1 hr occlusion was induced 3 days after a 10 mins occlusion

according to Chen et al.40. The data obtained here demonstrate the endogenous; neuroprotective

mechanisms are facilitated by ischemic preconditioning thus result in increasing ischemic

tolerance. The level of agmatine was increased during the ischemic preconditioning and the

increased level of agmatine also facilitates the more amount of agmatine production during the

ischemic injury in this study. The effective concentration of agmatine in ischemic tolerance was

13.596 ± 3.069 ug/g protein (0.952 ± 0.215 ug/g tissue) in this study. The endogenous

concentration of agmatine in brain can be estimated at 0.331-1.105 ug/g tissue 4, 41. Ischemic

preconditioning yields levels of agmatine within the range in tolerance. However, expression of

arginine decarboxylase (ADC) in preconditioning group was not demonstrable during the

21

ischemic injury and reperfusion injury. The reason for this disparity between agmatine and

arginine decarboxylase expression is not clear. This might be result of negative inhibition

caused by first increase in agmatine during the ischemic preconditioning.

Agmatine possesses modest affinities for various receptors, including as an inhibitor of the

NMDA subclass of glutamate receptors 13 and of all isoforms of NOS 15, especially nNOS 11.

Nitric oxide (NO) is enzymatically formed from the terminal guanidinonitrogen of L-arginine

by nitric oxide synthase (NOS). NO and excitatory amino acids contribute to ischemic brain

injury. Inhibitors of nitric oxide synthase (NOS) and antagonists of N-methyl-D-aspartate

(NMDA) glutamate receptors are neuroprotective in ischemic brain injury5, 11, 12, 23. Nitric oxide

(NO) has been implicated in several models of cerebral preconditioning. Gidday et al 42 found

that hypoxic preconditioning of newborn rats induced protection against subsequent hypoxia 6

days later42. Puisieux et al.43 found that infarct size from middle cerebral artery occlusion

(MCAO) was reduced by preadministration of lipopolysaccharide (LPS) and that this effect was

blocked by the nonspecific NOS inhibitor NG-nitro-L-arginine methyl ester (L-NAME) 43.

However, the precise role of NO in IPC is also unclear. In this study, results indicated that the

ischemic preconditioning decreased the expression of nNOS in the cerebral cortex and striatum

at 1hr and 23hr reperfusion following 1 hr ischemia. The induction of agmatine by ischemic

preconditioning may suppress nNOS expression and reduce brain damage.

Several signaling proteins reportedly contribute to the induction of cerebral ischemic tolerance,

such as Akt and mitogen-activated protein kinases (MAPKs)44, 45 as well as neuronal nitric oxide

synthase (nNOS). However, the cellular signaling cascades are largely unknown.

The members of the mitogen-activated protein kinase (MAPK) which are characterized as

proline-directed serine-threonine-protein kinases, in particular, c-Jun NH2-terminal kinases

(JNK), p38 and extracellular signal-regulated kinases (ERK) play important roles in transducing

stress-related signals in eukaryotic cells24 and are thought to serve as important mediators of

signal transduction from cell surface to the nucleus. The alterations and involvement of

extracellular signal-regulated kinase (ERK) and c-Jun N-terminal protein kinase (JNK)

activation were reported in the hippocampal CA1 region in a rat model of global brain ischemic

tolerance25. In this study, the level of ERK1/2 was investigated by Western bloting. The protein

expression of ERK was increased in ischemic preconditioning group than the experimental

control group. The results suggest that ERK activation after preconditioning ischemia may

result in the prevention of JNK activation and thus be involved in the protective responses in

ischemic tolerance.

Bone morphogenetic protein-7 (BMP-7), a trophic factor in the TGF-β superfamily, was initially considered to be a trophic factor mainly for non-neuronal tissue48. Recent studies have

indicated that BMP-7 and receptors for BMP (BMPR) are expressed in neuronal tissue.

22

Especially BMP-7 is also expressed in perinatal neuronal tissues, including hippocampus, cortex,

and cerebellum26. Activated BMP receptors phosphorylate transcription factors Smad1, 5, or 8,

which in turn associate with a common mediator, Smad4. The resultant heteromeric Smad

complexes then translocate into the nucleus to regulate transcription50, 51. As increasing

information is obtained regarding the detailed molecular mechanism of Smad protein signaling,

a number of functional interactions between these proteins and MAPK signaling pathways have

been reported. Recent work has demonstrated positive functional interaction between the two

stress-activated protein kinase pathways and Smads. So, the expression of BMP-7 was

investigated in this study. The level of BMP-7 was induced in preconditioning group under

MCA occlusion, however, the expression was decreased 23 hr after reperfusion in both

experimental control and preconditioning group. Some researchers also reporeted bone

morphogenetic proteins (BMPs) are reducing ischemia-induced cerebral injury in rats26 and it

was reported that agmatine treatment increased the expression of BMP-7 around scar more than

experimental control in early period of spinal cord injury26. These survival effects by ischemic

preconditioning is accompanied by a marked induction of agmatine before severe ischemia.

ⅤⅤⅤⅤ. CONCLUSION

In this study, It has been demonstrated that the level of agmatine was increased during early

reperfusion period in the ischemic injured brain by ischemic preconditioning. This induced level

of agmatine may act in increasing the expression of BMP-7 and ERK1/2 which are involved in

cell survival, and in inhibiting the detrimental effects of nNOS during the ischemic insults. This

demonstrated that agmatine is a potentially promising treatment for cerebral ischemia.

23

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Abstract (in Korean)

일시적일시적일시적일시적 뇌뇌뇌뇌 허혈허혈허혈허혈 동물동물동물동물 모델에서모델에서모델에서모델에서 알기닌탈탄소알기닌탈탄소알기닌탈탄소알기닌탈탄소

효소효소효소효소 및및및및 아그마틴의아그마틴의아그마틴의아그마틴의 내성내성내성내성 강화강화강화강화 효과효과효과효과

연세대학교 대학원 의학과

정 진 영

아그마틴은 생체 내에서 자체적으로 생성되는 clonidine-displacing

substance 로, alpha 2-adrenergic 그리고 imidazoline 수용기에 결합하는

내재성 ligand 이며, NO 생성에 대한 endogenous regulator 로의 기능이

알려져 있다. 아그마틴은 L-arginine 과 구조적으로 유사하여 경쟁적

억제자(competitive inhibitor)로 작용할 수 있으며 일시적 국소 뇌허혈 손상

모델에서 일시적 뇌허혈 손상 후 재관류 4 시간 후에 투여하였을 때에도

허혈 손상에 대한 신경보호효과를 나타냄이 보고된 바 있다.

Ischemic preconditioning (IP) 이란 일종의 적응 반응으로, 조직을 약한

허혈(1 시간 미만)에 미리 노출시킨 경우, 그 후의 강한 지속적인

허혈(chronic ischemia) 손상을 받게 되었을 때, 손상이 줄어드는 현상을

말한다. 본 연구는 아그마틴이 preconditioning 에 관여하는 역할을

규명하고자 하였다. Ischemic preconditioning (IP)에 의해 뇌경색 부위와

부종이 줄어듦을 확인하였으며, ischemic preconditioning 후 agmatine 의

29

양이 실험대조군에 비해 유의하게 증가하였고, 그 양은 약 2 배 정도로

늘어났다. 또한 1 시간의 심각한 허혈손상 후 재관류 손상이 시작된 지

1 시간 후 역시 agmatine 은 허혈손상 시작 전 보다 약 2 배 이상 늘어났고

이와 같은 결과는 실험대조군과 ischemic preconditioning 군 에서 동일하게

관찰되었다. Agmatine 을 생성하는 알기닌탈탄소 효소(ADC) 의 발현을

조사한 결과 실험대조군과 ischemic preconditioning 군에서의 차이는

없었다. 다만 허혈손상 1 시간차에 실험대조군에서 더 많이 발현된 것으로

확인되었다. 재관류시의 ADC 의 발현은 실험대조군과 ischemic

preconditioning 군에서 차이가 거의 없었다. 면역조직화학 결과에서 보면

ADC 의 발현은 ischemic preconditioning 에 의해 보호된 부위에서는 그

발현이 실험대조군에 비해 증가함을 보여주고 반면, 손상된 부위에서는

ADC 의 발현이 별 차이가 나지 않음을 확인하였으며, 이는 ADC 에 의해

생성되는 agmatine 이 연관되어 있음을 간접적으로 보여주는 것이라

생각된다. 허혈후 재관류 손상시 cell death 에 중요한 역할을 하는 것으로

알려져 있는 nNOS 와 iNOS 발현을 조사한 결과, ischemic

preconditioning 으로 인해 nNOS 의 발현은 재관류 1 시간과 23 시간에

현격히 줄어들었으며, 반면 iNOS 는 실험대조군과 마찬가지로 발현됨을

확인하였다. 따라서 Ischemic preconditioning 에 의해 증가된 agmatine 이

nNOS 의 발현을 감소시킴으로써 신경보호작용을 나타내었을 것으로

생각되었다.

Ischemic preconditioning 시 세포생존에 밀접한 연관이 있는 것으로

알려진 ERK 의 발현을 확인한 결과, 허혈 및 재관류 손상 동안 ischemic

preconditioning 군에서 더 많이 발현되거나 비슷한 정도로 발현되는 것으로

관찰되었다. 특히 ischemic preconditioning 으로 허혈손상 시작 전에 이미

ERK 의 발현이 증가하였으며, 이러한 ERK 의 증가가 허혈 및 재관류 손상

시 세포 손상을 막아주는 역할을 하였을 것으로 판단된다. 또한 최근에

30

신경보호효과가 있는 것으로 보고되는 BMP-7 의 발현을 조직면역염색으로

확인한 결과 재관류 1 시간에 ischemic preconditioning 에 의해 보호된

부위에서 그 발현이 증가하였음을 확인하였다.

이상의 결과로부터 ischemic preconditioning 에 의해 agmatine 의 양이

증가함으로써 내재적 신경보호 효과가 증가 되었고, 이와 같은 agmatine 의

신경보호효과는 허혈손상에 대한 내성 증가와 연관성이 있을 것으로

생각되었다. 아그마틴의 농도는 ischemic preconditioning 시 증가 되었고,

이렇게 증가된 아그마틴은 허혈손상시 더 많은 양의 아그마틴을 생성하였다.

따라서 본 실험결과로부터 아그마틴은 ischemic preconditioning 과

연관되어 신경보호 효과를 갖고 있고, 이러한 결과는 향후 허혈성 뇌질환의

치료에 가능성을 갖고 있다고 할 수 있다.

핵심되는 말: 아그마틴, 알기닌 탈탄산효소, 뇌 허혈 손상적응, 내성

TABLE OF CONTENTSABSTRACTI. INTRODUCTIONII. MATERIALS AND METHODS1. Animals and experimental protocols2. Induction of ischemic preconditioning and focal ischemia3. Morphometric measurement of brain edema and infarct volume4. Agmatine analysis with HPLC4-1.Sample preparation4-2. Apparatus and chromatographic conditions

5. Immunostaining for ADC, NOSs, phosphoERK1/2, and BMP-76. Immunoblotting of ADC, Erk1/27. Statistical analysis

III. RESULTS1. rCBF responses to experimental control group and ischemic preconditioning group in MCAO models2. Brain edema and infarct volume after ischemic injury3. The level of agmatine after ischemic injury4. Assessment for level of ADC5. Assessment for level of nNOS and iNOS6. Assessment for level of ERK1/2, phosphoERK1/2, and BMP-7

IV. DISCUSSIONV. CONCLUSIONⅥ. REFERENCESABSTRACT (IN KOREAN)