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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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).
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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.
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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).
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A.
B.
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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
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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
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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
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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
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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
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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).
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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).
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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).
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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).
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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).
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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
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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.
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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.
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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
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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
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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)