II. MATERIALS AND METHODS
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).
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.
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).
A.
B.
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)
Table 1. Infarct volume after
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
4
. Assessment for level of ADCThe 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
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).
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).
Ⅳ
Ⅳ
ⅣⅣ. 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
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.
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.
Ⅴ
Ⅴ
ⅤⅤ. 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.
Ⅵ
Ⅵ
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