The Role of Agmatine and Arginine Decarboxylase in Ischemic Tolerance After Transient Cerebral Ischemia in Rat Models

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.

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Thesis Supervisor: Seung Kon Huh

______Jong Eun Lee: Thesis Committee Member #1

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Jin Woo Chang: Thesis Committee Member #2

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Duck Sun Ahn: Thesis Committee Member #3

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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 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

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

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

iii 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

1 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

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 agmatinase 1. Recently, agmatine, ADC, and agmatinase were found in mammalian brain 2. 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 2-4. Recent studies have shown that agmatine may be neuroprotective in trauma and neonatal ischemia models 1, 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 pore 10 . 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 vessels 11, 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) inhibitor 13, 14 . NOSs generate nitric oxide (NO) by sequential oxidation of the

3 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 ischemia 15 . 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 ischemia 16 . Heat shock proteins, immediate early genes, antioxidant 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 function 17 . 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 earlier 8. 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

4 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

5 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 4 th 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).

6. Immunoblotting of ADC and ERK1/2 Expressions of ADC and ERK1/2 proteins were estimated by immunoblotting in the ipsilateral part of 5 th 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.

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).

8 A.

9 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<0.01)

3 Infarct volume (mm )

unit: EC PC

M0 0 0 Table 1. Infarct volume after M0.5 0 0 ischemic injury. EC (Experimental M1 19.830 11.922 control group), IP(Ischemic R1 49.252 7.120 preconditioning group). M0 (MCA R3 30.948 6.546 occlusion 0 hr), M0.5 (0.5hr), M1(1hr), R1 (Post reperfusion 1hr), R6 55.232 34.181 R3 (3hr), R6 (6hr), R23 (23hr). (** R23 142.271 66.756** P<0.01)

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<0.05)

3. The level of agmatine after ischemic injury Agmatine was detected in both tissue samples (preconditioning group and experimental control group). Electropherogram was obtained with HPLC method from rat brain samples. The peak corresponding to agmatine was well identified during the ischemic injury. By comparing the IP and EC traces showing in Figure 5, it can be seen the highest agmatine peak level at 2 hr after the injury. The level of agmatine was decreased dramatically after 2 hr and it shows plateau in preconditioning group. In experimental control group, the level of agmatine was increased gradually and it also showed plateau. The level of agmatine was summarized in Table 2.

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<0.05)

Agmatine (ug/g protein) EC IP M0 6.366 ± 1.250 13.596 ± 3.069* M0.5 12.946 ± 4.811 11.874 ± 1.356 M1 17.403 ± 7.821 12.617 ± 6.001 R1 14.072 ± 8.160 26.465 ± 13.130 R3 12.085 ± 5.614 9.409 ± 7.883 R6 17.210 ± 9.894 14.062 ± 6.608 R23 13.681 ± 3.568 8.827 ± 0.438

12 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<0.01)

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

14 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).

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).

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).

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).

18 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).

ⅣⅣⅣ. 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 injury 18-21 . Increases in the resistance of neuron to ischemia arise after one or several transient episodes of ischemia/reperfusion. Previous reports suggest that heat shock proteins 17, 23, 24 , immediate early genes 25, 26 , antioxidant enzyme 27, 28 , antiapoptotic oncogene 29, 30 , interleukin- 1h 31, 32 , and adenosine 33, 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 vivo 8. Furthermore, this protection is associated with decreased NOS activity and expression, as well as NO generation 5. 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 injury 23 . Intracellulaly, agmatine is reported to modulate the production of polyamines 36 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 stress 38. 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

20 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 injury 5, 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 later 42 . 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 cells 24 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 tolerance 25 . 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 tissue 48 . Recent studies have indicated that BMP-7 and receptors for BMP (BMPR) are expressed in neuronal tissue.

21 Especially BMP-7 is also expressed in perinatal neuronal tissues, including hippocampus, cortex, and cerebellum 26 . 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 transcription 50, 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 rats 26 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 injury 26 . These survival effects by ischemic preconditioning is accompanied by a marked induction of agmatine before severe ischemia.

ⅤⅤⅤ. 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.

22 ⅥⅥⅥ. REFERENCES

1. Hamada Y, Hayakawa T, Hattori H, Mikawa H. Inhibitor of nitric oxide synthesis reduces hypoxic-ischemic brain damage in the neonatal rat. Pediatric research 1994 Jan;35(1):10-4. 2. Higuchi Y, Hattori H, Kume T, Tsuji M, Akaike A, Furusho K. Increase in nitric oxide in the hypoxic-ischemic neonatal rat brain and suppression by 7-nitroindazole and aminoguanidine. European journal of pharmacology 1998 Jan 19;342(1):47-9. 3. Iadecola C. Bright and dark sides of nitric oxide in ischemic brain injury. Trends in neurosciences 1997 Mar;20(3):132-9. 4. Feng Y, Piletz JE, Leblanc MH. Agmatine suppresses nitric oxide production and attenuates hypoxic-ischemic brain injury in neonatal rats. Pediatric research 2002 Oct;52(4):606-11. 5. Yang XC, Reis DJ. Agmatine selectively blocks the N-methyl-D-aspartate subclass of glutamate receptor channels in rat hippocampal neurons. The Journal of pharmacology and experimental therapeutics 1999 Feb;288(2):544-9. 6. Li G, Regunathan S, Barrow CJ, Eshraghi J, Cooper R, Reis DJ. Agmatine: an endogenous clonidine-displacing substance in the brain. Science (New York, NY 1994 Feb 18;263(5149):966-9. 7. Piletz JE, Chikkala DN, Ernsberger P. Comparison of the properties of agmatine and endogenous clonidine-displacing substance at imidazoline and alpha-2 adrenergic receptors. The Journal of pharmacology and experimental therapeutics 1995 Feb;272(2):581-7. 8. Reynolds IJ. Arcaine uncovers dual interactions of polyamines with the N-methyl-D-aspartate receptor. The Journal of pharmacology and experimental therapeutics 1990 Dec;255(3):1001- 7. 9. Gilad GM, Gilad VH. Accelerated functional recovery and neuroprotection by agmatine after spinal cord ischemia in rats. Neuroscience letters 2000 Dec 22;296(2-3):97-100. 10. Gilad GM, Salame K, Rabey JM, Gilad VH. Agmatine treatment is neuroprotective in rodent brain injury models. Life sciences 1996;58(2):PL 41-6. 11. Kim JH, Yenari MA, Giffard RG, Cho SW, Park KA, Lee JE. Agmatine reduces infarct area in a mouse model of transient focal cerebral ischemia and protects cultured neurons from ischemia-like injury. Experimental neurology 2004 Sep;189(1):122-30. 12. Yu CG, Marcillo AE, Fairbanks CA, Wilcox GL, Yezierski RP. Agmatine improves locomotor function and reduces tissue damage following spinal cord injury. Neuroreport 2000 Sep 28;11(14):3203-7. 13. Olmos G, DeGregorio-Rocasolano N, Paz Regalado M, Gasull T, Assumpcio Boronat M, Trullas R, et al. Protection by imidazol(ine) drugs and agmatine of glutamate-induced neurotoxicity in cultured cerebellar granule cells through blockade of NMDA receptor.

23 British journal of pharmacology 1999 Jul;127(6):1317-26. 14. Auguet M, Viossat I, Marin JG, Chabrier PE. Selective inhibition of inducible nitric oxide synthase by agmatine. Japanese journal of pharmacology 1995 Nov;69(3):285-7. 15. Galea E, Regunathan S, Eliopoulos V, Feinstein DL, Reis DJ. Inhibition of mammalian nitric oxide synthases by agmatine, an endogenous polyamine formed by decarboxylation of arginine. The Biochemical journal 1996 May 15;316 ( Pt 1):247-9. 16. Weih M, Kallenberg K, Bergk A, Dirnagl U, Harms L, Wernecke KD, et al. Attenuated stroke severity after prodromal TIA: a role for ischemic tolerance in the brain? Stroke; a journal of cerebral circulation 1999 Sep;30(9):1851-4. 17. Kitagawa K, Matsumoto M, Tagaya M, Hata R, Ueda H, Niinobe M, et al. 'Ischemic tolerance' phenomenon found in the brain. Brain research 1990 Sep 24;528(1):21-4. 18. Stenzel-Poore MP, Stevens SL, Simon RP. Genomics of preconditioning. Stroke; a journal of cerebral circulation 2004 Nov;35(11 Suppl 1):2683-6. 19. Grabb MC, Choi DW. Ischemic tolerance in murine cortical cell culture: critical role for NMDA receptors. J Neurosci 1999 Mar 1;19(5):1657-62. 20. Gustavsson M, Anderson MF, Mallard C, Hagberg H. Hypoxic preconditioning confers long-term reduction of brain injury and improvement of neurological ability in immature rats. Pediatric research 2005 Feb;57(2):305-9. 21. Petrishchev NN, Vlasov TD, Sipovsky VG, Kurapeev DI, Galagudza MM. Does nitric oxide generation contribute to the mechanism of remote ischemic preconditioning? Pathophysiology 2001 Mar;7(4):271-4. 22. Vlasov TD, Korzhevskii DE, Polyakova EA. Ischemic preconditioning of the rat brain as a method of endothelial protection from ischemic/repercussion injury. Neuroscience and behavioral physiology 2005 Jul;35(6):567-72. 23. Kirino, T., Tsujita, Y., Tamura, A. Induced tolerance to ischemia in gerbil hippocampal neurons. Journal of Cerebral Blood Flow and Metabolism 1991;.11: 299– 307. 24. Nowak Jr., T.S. Synthesis of a stress protein following transient ischemia in the gerbil. Journal of Neurochemistry 1985; 45: 1635–1641. 25. Ikeda, J., Nakajima, T., Osborne, O.C., Mies, G., Nowak Jr., T.S. Coexpression of c-fos and hsp70 mRNAs in gerbil after ischemia: induction threshold, distribution and time course evaluated by in situ hybridization. Brain Research. Molecular Brain Research 1994; 26: 249– 258. 26. Sommer, C., Gass, P., Kiessling, M. Selective c-Jun expression in CA1 neurons of the gerbil hippocampus during and after acquisition of an ischemia-tolerant state. Brain Pathology 1995; 5: 135– 144. 27. Kato, H., Kogure, K., Araki, T., Liu, X.H., Kato, K., Itoyama, Y. Immunohistochemical

24 localization of superoxide dismutase in the hippocampus following ischemia in a gerbil model of ischemic tolerance. Journal of Cerebral Blood Flow and Metabolism 1995; 15: 60– 70. 28. Ohtsuki, T., Matsumoto, M., Kitagawa, K., Taguchi, A., Suzuki, K., Taniguchi, N., Kamada, T. Influence of oxidative stress on induced tolerance to ischemia in gerbil hippocampal neurons. Brain Research 1992; 599: 246– 252. 29. Katayama, Y., Muramatsu, H., Kamiya, T., McKee, A., Terashi, A. Ischemic tolerance phenomenon from an approach of energy metabolism and the mitochondrial enzyme activity of pyruvate dehydrogenase in gerbils. Brain Research 1997; 746: 126– 132. 30. Shimazaki, K., Ishida, A., Kawai, N. Increase in bcl-2 oncoprotein and the tolerance to ischemia-induced neuronal death in the gerbil hippocampus. Neuroscience Research 1994; 20: 95–99. 31. Barone, F.C., White, R.F., Spera, P.A., Ellison, J., Currie, R.W., Wang, X., Feuerstein, G.Z. Ischemic preconditioning and brain tolerance: temporal histological and functional outcomes, protein synthesis requirement, and interleukin-1 receptor antagonist and early gene expression. Stroke 1998; 29: 1937– 1951. 32. Ohtsuki, T., Ruetzler, C.A., Tasaki, K., Hallenbeck, J.M. Interleukin-1 mediates induction of tolerance to global ischemia in gerbil hippocampal CA1 neurons. Journal of Cerebral Blood Flow and Metabolism 1996; 16: 1137– 1142. 33. Kawahara, N., Ide, T., Saito, N., kawai, K., Kirino, T. Propentofylline potentiates induced ischemic tolerance in gerbil hippocampal neurons via adenosine receptor. Journal of Cerebral Blood Flow and Metabolism 1998; 18: 472– 475. 34. Hiraide, T., Katsura, K., Muramatsu, H., Asano, G., Katayama, Y. Adenosine receptor antagonists cancelled the ischemic tolerance phenomenon in gerbil. Brain Research 2001; 910: 94– 98. 35. Wei H, Jyvasjarvi E, Niissalo S, Hukkanen M, Waris E, Konttinen YT, et al. The influence of chemical sympathectomy on pain responsivity and alpha 2-adrenergic antinociception in neuropathic animals. Neuroscience 2002;114(3):655-68. 36. Demady DR, Jianmongkol S, Vuletich JL, Bender AT, Osawa Y. Agmatine enhances the NADPH oxidase activity of neuronal NO synthase and leads to oxidative inactivation of the enzyme. Mol Pharmacol. 2001 Jan;59(1):24-9. 37. Reis DJ, Regunathan S. Is agmatine a novel neurotransmitter in brain? Trends Pharmacol Sci. 2000 May;21(5):187-93. 38. Halaris A, Zhu H, Feng Y, Piletz JE. Plasma agmatine and platelet imidazoline receptors in depression. Ann N Y Acad Sci. 1999 Jun 21;881:445-51. 39. Feyza Aricioglu-Kartal and I. Tayfun Uzbay. Inhibitory effect of agmatine on naloxone-

25 precipitated abstinence syndrome in morphine dependent rats. Life Sci. 1997;61(18):1775-81. 40. Chen, J., Graham, S.H., Zhu, R.L., Simon, R.P., 1996. Stress proteins and tolerance to focal cerebral ischemia. Journal of Cerebral Blood Flow and Metabolism 16, 566– 577. 41. Abe K, Abe Y, Saito H. Agmatine suppresses nitric oxide production in microglia. Brain Res. 2000 Jul 28;872(1-2):141-8. 42. Gidday JM, Shah AR, Maceren RG, Wang Q, Pelligrino DA, Holtzman DM, et al. Nitric oxide mediates cerebral ischemic tolerance in a neonatal rat model of hypoxic preconditioning. J Cereb Blood Flow Metab 1999 Mar;19(3):331-40. 43. Puisieux F, Deplanque D, Pu Q, Souil E, Bastide M, Bordet R. Differential role of nitric oxide pathway and heat shock protein in preconditioning and lipopolysaccharide-induced brain ischemic tolerance. European journal of pharmacology 2000 Feb 11;389(1):71-8. 44. Centeno, J.M., Orti, M., Salom, J.B., Sick, T.J., Perez-Pinzon, M.A.Nitric oxide is involved in anoxic preconditioning neuroprotection in rat hippocampal slices. Brain Research 1999; 836 (1–2): 62–69. 45. Garcia, L., Burda, J., Hrehorovska, M., Burda, R., Martin, M.E., Salinas, M. Ischaemic preconditioning in the rat brain: effect on the activity of several initiation factors, Akt and extracellular signal-regulated protein kinase phosphorylation, and GRP78 and GADD34 expression. Journal of Neurochemistry 2004; 88 (1): 136–147. 46. Sugino T, Nozaki K, Takagi Y, Hattori I, Hashimoto N, Moriguchi T, et al. Activation of mitogen-activated protein kinases after transient forebrain ischemia in gerbil hippocampus. J Neurosci 2000 Jun 15;20(12):4506-14. 47. Gu Z, Jiang Q, Zhang G. Extracellular signal-regulated kinase and c-Jun N-terminal protein kinase in ischemic tolerance. Neuroreport 2001 Nov 16;12(16):3487-91. 48. Chang SC, Hoang B, Thomas JT, Vukicevic S, Luyten FP, Ryba NJ, et al. Cartilage-derived morphogenetic proteins. New members of the transforming growth factor-beta superfamily predominantly expressed in long bones during human embryonic development. The Journal of biological chemistry 1994 Nov 11;269(45):28227-34. 49. Mehler MF, Mabie PC, Zhang DM, Kessler JA. Bone morphogenetic proteins in the nervous system. Trends Neurosci. 1997;20:309 –317 50. Heldin CH, Miyazono K, ten Dijke P. TGF-beta signalling from cell membrane to nucleus through SMAD proteins. Nature 1997;390:465–71. 51. Derynck R, Zhang Y, Feng XH. Smads: transcriptional activators of TGF-beta responses. Cell 1998;95:737–40. 52. Chang CF, Morales M, Chou J, Chen HL, Hoffer B, Wang Y. Bone morphogenetic proteins are involved in fetal kidney tissue transplantation-induced neuroprotection in stroke rats. Neuropharmacology 2002 Sep;43(3):418-26.

26 53. Kim JH. The role of agmatine in CNS injury. Korea: Yonsei Univ.; 2006

27 Abstract (in Korean)

일시적 뇌 허혈 동물 모델에서 알기닌탈탄소

효소 및 아그마틴의 내성 강화 효과

<지도교수 허승곤>

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

28 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

29 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