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Moderate cerebral venous congestion induces rapid cerebral protection via adenosine A1 receptor activation
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a v a i l a b l e a t w w w. s c i e n c e d i r e c t . c o m
w w w. e l s e v i e r. c o m / l o c a t e / b r a i n r e s
Research Report
Moderate cerebral venous congestion induces rapid cerebral protection via adenosine A1 receptor activation Keiichi Akaiwa a , Hidetoshi Akashi a,⁎, Hideki Harada b , Hideki Sakashita a , Shinichi Hiromatsu a , Tatsuhiko Kano b , Shigeaki Aoyagi a a
Department of Surgery, Kurume University School of Medicine, Kurume, Japan Department of Anesthesiology, Kurume University School of Medicine, Kurume, Japan
b
A R T I C LE I N FO
AB S T R A C T
Article history:
Stroke is a devastating complication in cardiovascular surgery, and neuronal damage is
Accepted 3 September 2006
worsened by intracranial pressure elevation caused by cerebral venous circulatory
Available online 24 October 2006
disturbances (CVCD). However, we have previously reported that CVCD before cerebral ischemia decreases the infarct area. In the present study, focal cerebral ischemia was
Keywords:
induced in spontaneously hypertensive rats by filament insertion through the carotid
Cerebral protection
artery. Rats were divided into the following four groups: sham-operated, mild or severe
Cerebral venous congestion
venous congestion (VC), and DPCPX. The DPCPX group received the adenosine A1 receptor
Adenosine A1 receptor
antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) prior to mild VC. Behavior, infarct
Cerebral venous circulatory
volume, edema and S-100 protein were evaluated among the four groups. The infarct
disturbance
volume rates in mild VC and severe VC groups were significantly less than that in sham-
Middle cerebral artery occusion
operated and DPCPX groups. However, the mortality of the severe VC group worsened in a
Cerebral infarct
time-dependent manner. We observed a significant decrease in edema in the mild VC group
Ischemia
compared to the DPCPX group. Behavioral scores also indicated that the mild VC group had
Neurological deficits
fewer neurological deficits than the other three groups, including the DPCPX group. We were
Rapid ischemic tolerance
able to induce rapid cerebral protection via adenosine A1 receptor activation by
Venous congestive preconditioning
administering an appropriate degree of VC prior to cerebral ischemia produced by middle
Transient MCAO
cerebral artery occlusion. Our work suggests possible mechanisms by which such effective
DPCPX
VC may lead to cerebral protection and adenosine A1 receptor activation. © 2006 Elsevier B.V. All rights reserved.
1.
Introduction
Stroke is a devastating complication in cardiovascular surgery and often makes the patient unable to gain from the benefits of the operation. Cardiovascular surgeons use several cerebral protection methods, such as retrograde cerebral perfusion (RCP) and selective cerebral perfusion (SCP) to prevent the development of such complications. Although remarkable
improvements have been made in cardiovascular surgery, cerebral complication still remains a major unsolved problem. Therefore, we have investigated the consequences of cerebral venous circulatory disturbances (CVCD) associated with cerebral ischemia, and have reported (Sakashita, 2004) that CVCD before cerebral ischemia decreased the infarct area compared with cerebral ischemia without CVCD, whereas CVCD after cerebral ischemia resulted in larger neuronal damage.
⁎ Corresponding author. Fax: +81 942 35 8967. E-mail address: [email protected] (H. Akashi). 0006-8993/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2006.09.019
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Recently, it has been reported that a sublethal ischemic insult might limit damage from a subsequent severe ischemic insult (Barone et al., 1998; Nakamura et al., 2002; Stagliano et al., 1999; Yano et al., 2001; Yoshida et al., 2004). This phenomenon was defined as ischemic tolerance. Depending on the latency between insults, two types of ischemic tolerance in the brain, rapid and delayed, have been documented in the literature (Barone et al., 1998; Kariko et al., 2004; Nishio et al., 2000; Yunoki et al., 2003). Although preconditioning with brief middle cerebral artery occlusion (MCAO) induced protection as rapid tolerance against neuronal damage, CVCD before cerebral ischemia may potentially produce ischemic tolerance in the same way as brief arterial cerebral ischemia. This possibility was discussed in one of our previous reports (Sakashita, 2004). In the present study, we conducted two experiments. The first determined the relation between the intensity of venous congestion (VC) and the protective effect against subsequent cerebral ischemia. The second experiment examined whether cerebral protection, if any, that is rapidly induced by pretreatment is modified by a highly selective adenosine A1 receptor antagonist, 8-cyclopentyl-1,3-dipropylxanthine (DPCPX). We also discuss possible mechanisms whereby such effective VC may lead to the development of a new cerebral protection paradigm.
Fig. 1 – Cerebral blood flow (CBF). CBF values for the MCA are expressed as a percentage of baseline values before MCAO. Successful occlusion and reperfusion of the MCA were examined using LDF. CBF changes of the MCA territory between during MCAO and after reperfusion are shown. There were no significant differences in the percentage change in CBF among the four groups.
2.2.
2.
Results
2.1.
Physiological variables and survival rates
Physiological parameters were monitored in all rats during the operation. Table 1 presents a summary of the physiological parameters immediately after reperfusion of the MCA. VC or DPCPX did not produce changes in body temperature, mean arterial pressure (BP), blood sugar (BS), hematocrit (Ht), pH, or blood gas data. Cerebral blood flow (CBF) changes are presented in Fig. 1. Baseline CBF before MCAO was defined as 100%, and changes in CBF are indicated as a percentage of the baseline CBF values. There were no significant differences in the rate of change of CBF among the four groups. The mortality rate was 0% among the sham-operated, mild VC, and DPCPX groups and 50% in the severe VC group; three of the six animals unexpectedly died before scheduled sacrifice (48 to 72 h post-ischemia).
Neurological deficits
The post-ischemic survival period was 72 h, and neurological deficits were evaluated immediately, 3 h, 24 h, and 72 h after reperfusion. Fig. 2 presents behavioral scores of post-ischemic neurological outcome for all rats. Behavioral scores were calculated from the totals of four-parameter scores (Fig. 3). Significant differences were observed among groups immediately, 24 h, and 72 h but not at 3 h after reperfusion of the MCA. Neurological deficits in the mild VC group were significantly fewer than those observed in the other two groups (shamoperated and DPCPX), during MCAO. Even in the severe VC group, the behavioral score was small in the early phase (during MCAO and 3 h after reperfusion). However, three of six rats in the severe VC group died between 48 h and 72 h after reperfusion, whereas the remaining three rats that survived also stopped moving 72 h after reperfusion. The severe VC group caused excess morbidity, resulting in an inability to assess the effect of this treatment on cerebral infarction.
Table 1 – Physiological variables immediately after reperfusion of the MCA Experimental groups Sham-op. Mild VC Severe VC DPCPX P value
Temperature (°C)
BP (mm Hg)
BS (mg/dl)
Ht
pH
PCO2 (mm Hg)
PO2 (mm Hg)
37.2 ± 0.2 37.0 ± 0.2 37.1 ± 0.3 37.1 ± 0.2 0.566
144 ± 5.2 146 ± 3.3 139 ± 1.2 143 ± 5.1 0.215
136 ± 16.0 136 ± 15.0 126 ± 19.3 137 ± 21.8 0.846
41.2 ± 2.6 40.3 ± 2.6 43.3 ± 1.2 40.5 ± 1.6 0.281
7.45 ± 0.03 7.46 ± 0.02 7.49 ± 0.01 7.45 ± 0.03 0.162
40.3 ± 5.1 40.2 ± 1.5 34.3 ± 2.3 37.6 ± 6.9 0.288
437.2 ± 81.3 463.3 ± 115.3 365.7 ± 151.6 444.1 ± 88.9 0.622
Physiological variables for all rats expressed as the mean ± SD. Differences among the four groups were compared by one-way ANOVA followed by a comparison using Fisher's LSD post hoc test. P values less than 0.05 were considered significant. There were no significant differences in any of the variables among the four groups. BP: mean arterial blood pressure, BS: blood sugar, temperature: rectal temperature.
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Fig. 2 – Behavioral scores in all rats. This figure is showing behavioral scores of in all rats at four points. Statistical analysis was conducted using the Kruskal–Wallis test followed by the post hoc Fisher's PLSD test. Significant differences among the four groups were observed at three points (during MCAO, and 24 and 72 h after reperfusion). *P < 0.05; **P < 0.01; ***P < 0.001.
These results suggest that mild VC group had the smallest neurological deficit and mild VC was the most effective at cerebral protection. However, these effects were reversed in the DPCPX group, which was also administered mild VC.
than those in the sham-operated group. These results suggested that mild VC was the most effective at cerebral protection because of the smaller infarct area and lower edema rates, and this effect was attenuated by DPCPX.
2.3.
2.4.
Infarct volume and cerebral edema
Fig. 4 shows the extent of ischemic area in these three groups using TTC staining. The colorless area was considered to correspond to the territory supplied by the occluded MCA. Infarct volume rates are presented as infarct area percentages of the right hemisphere, and these rates were compared among the all groups (Table 2, Fig. 5A). With regard to cerebral edema, the volume balance between the right hemisphere and the left hemisphere was calculated and edema rates are presented as right hemisphere volume percentages of the normal left hemisphere. These edema rates were compared among the four groups in the same manner as infarct area (Table 2, Fig. 5B). The infarct volume rates in the mild VC and severe VC groups were significantly less than those in the sham-operated and DPCPX groups (mild VC; 37.1 ± 2.6%, severe VC; 31.2 ± 5.7%, sham-operated; 44.8 ± 7.0%, DPCPX; 46.1 ± 5.5% P < 0.05). For edema rates, there was a significant difference between mild VC and DPCPX groups using the post hoc Fisher's PLSD test (Fig. 5B). Although the difference did not reach significance, edema rates in the mild VC group were less
CKBB and S-100 protein
Creatine kinase BB (CKBB) and S-100 protein were measured in order to evaluate cerebral damage immediately and 72 h after reperfusion of the MCA. There were no statistically significant differences in the values of CKBB and S-100 protein.
3.
Discussion
The results of the present study show that mild cerebral VC produces rapid cerebral protection against subsequent ischemic insults in rats, and that the beneficial effect of cerebral protection is attenuated by a selective adenosine A1 receptor antagonist, DPCPX. These results suggest that mild VCinduced rapid cerebral protection is mediated, at least in part, through adenosine A1 receptor-related mechanisms. Cerebral VC should logically cause an intracranial volume overload and an expansion of vascular beds, which might induce intracranial pressure elevation. The expanded and overloaded vascular beds cause vascular edema, which in turn
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Fig. 3 – Behavioral scores. Rats were evaluated neurologically using four parameters (motion of the forepaw, activity, motion, and posture). The behavioral score was defined as the total scores of four parameters that were classified into 12 grades (0 to 11 points).
brings a further increase in intracranial pressure, causing a drop in cerebral perfusion pressure and ultimately intensifying the degree of ischemia as well as potentiating cerebral edema itself. For this reason, although it is easy to understand how VC contributes to neuronal damage, it is more difficult to comprehend the mechanism by which VC has a protective role against subsequent ischemic insults. In the present experiments, VC loading immediately prior to lethal cerebral ischemia was protective against cerebral ischemia, although the optimal severity of VC loading before ischemia is an unresolved issue. It is conceivable that VC loading is responsible for expansion of cerebral vascular beds and increase of venous pressure, and the decrease in cerebral perfusion pressure might have the same conditioning as sublethal ischemia. In previous animal experiments (Sakashita, 2004), we examined the protective effects of VC preloading on the MCAO model in rats and found that it could shrink the ischemic infarct volume. However, if the same conditioning of VC was loaded after the induction of MCAO, the infarct volume was clearly expanded and led to death by the end of 24 h in the reperfusion period. Focusing on this, in the present study we examined whether any differences exist in the protective effects depending on the severity of preloaded VC. Based on the findings and results for neurological deficits, infarct volume, and cerebral edema, mild VC might be the most effective against cerebral protection. The mortality rate was 0% in the sham-operated, mild VC, and DPCPX groups. By
72 h after reperfusion, all of the rats in the severe VC group were either hardly moving or already dead, with a mortality rate of 50%. The morbidity in the severe VC group might be not merely ischemic injury, but excessive edematous damage caused by continuous severe VC, leading to poor survival beyond the 72 h of reperfusion. The method of VC in this experimental paradigm was permanent rather than transient, raising concerns about the effects of VC itself. We predicted that cerebral blood flow (CBF) should be affected and rats might show behavioral changes from VC itself. However, the rats did not exhibit any neurological defects and CBF revealed the same value with or without VC loading. In applying this protective effect of VC to clinical cases, e.g., to cardiac surgery using cardiopulmonary bypass (CPB), VC should be loaded transiently by increasing pressure of the superior vena cava. In these experiments we applied continual VC instead of transient VC due to the technical difficulty of producing this model. Moderate VC (mild VC in this study) could induce compensatory venous collaterals, resulting in a transient effect of VC, even though the ligation was permanent. Hence, mild VC in this study might mimic transient loading in the cerebral venous circulation. A cerebral protective effect was demonstrated in this experiment by mild VC loading prior to MCAO, however, the mechanism has not been elucidated. There have been many reports on the mechanism of cerebral protective effect of ischemic tolerance since the first one by Kitagawa et al. (1990). It has been reported that cross-tolerance against neuronal death is induced by many factors such as sublethal ischemia (Barone et al., 1998; Nakamura et al., 2002; Stagliano et al., 1999; Yano et al., 2001; Yoshida et al., 2004), hypoxia (Ballanyi, 2004; Centeno et al., 1999; Perez-Pinzon and Born, 1999), polyunsaturated fatty acids, lipopolysaccharides (Kariko et al., 2004; Rosenzweig et al., 2004), modified electroconvulsive shock (Mishima et al., 2005), electro-acupuncture (Wang et al., 2005), hyperthermia (Xu et al., 2002), and hypothermia (Nishio et al., 2000; Yunoki et al., 2003). Among these, ischemic and hypothermic preconditioning are well known to induce rapid tolerance and delayed tolerance, however, it has been reported that the mechanisms of rapid and delayed tolerances are different (Nishio et al., 2000). For example, delayed tolerance induced by hypothermia is inhibited by the protein synthesis inhibitor aminomycin (Nishio et al., 2000), whereas rapid tolerance was not (Yunoki et al., 2003). This finding suggests that de novo protein synthesis plays an important role in the induction of delayed tolerance, but is not involved in the induction of rapid tolerance. It has been reported for example that G-protein-coupled receptor (adenosine receptors, etc.) activity (Cohen et al., 2000; De Jonge and De Jong, 1999), an increase in protein kinase C activity (Perez-Pinzon and Born, 1999), an increase in mitogen-activated protein (MAP) kinase activity, Akt activity (Nakajima et al., 2004; Yano et al., 2001), nitric oxide (NO) production (Centeno et al., 1999; PerezPinzon and Born, 1999), and mitochondrial KATP channels (Ballanyi, 2004; Horiguchi et al., 2003; Perez-Pinzon and Born, 1999) are involved in the mechanism of rapid tolerance. It has been demonstrated that adenosine A1 receptor activity is strongly involved in the induction of rapid tolerance towards
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Fig. 4 – TTC staining, 72 h after reperfusion of the MCA. Infarct brain regions did not convert TTC and therefore remained unstained. The unstained area was measured in each slice by NIH image. The infarct volume rates in mild VC group were significantly lower than those in sham-operated and DPCPX groups. TTC: 2,3,5-triphenyltetrazolium chloride.
cerebral ischemia in rats (Hiraide et al., 2001; Nakamura et al., 2002; Wang et al., 2005). Adenosine A1 receptor activity is inhibited by treatment with the antagonist DPCPX, and in our experiments we confirmed whether or not the cerebral protective effect of mild VC was attenuated by inhibiting adenosine A1 receptor activity by preadministering DPCPX intraperitoneally before mild VC. The behavioral score was significantly worsened in the DPCPX group compared to the mild VC group during MCAO, although there were no significant differences at 3, 24, and 72 h after reperfusion. However, infarct volume and edema rates were significantly increased in the DPCPX group. These results suggest that the cerebral protective effect in mild VC group was at least partially inhibited by DPCPX preadministration, and strongly implicates the involvement of rapid cerebral protection involving adenosine A1 receptors. We did not see any pathological or behavioral effects of DPCPX administration alone in a pilot study (data not shown).
Even though we did not examine the direct effects of preadministered DPCPX on our MCAO model, this possibility has been already examined by other investigators (Yoshida et al., 2004), who showed DPCPX and DMSO had no apparent influence on infarct volume. Hence, we postulate that VC is mainly responsible for our results. Rapid cerebral protection was achieved by loading an appropriate degree of VC prior to MCAO in rats. Although the details of the exact mechanism of the cerebral protection remain unclear, we speculate that a steep drop in cerebral perfusion pressure due to the difference between decreased arterial and increased venous pressure might be a protective factor, which could mimic the conditioning as sublethal ischemia. While an appropriate intensity of loading to activate the adenosine A1 receptor is necessary, the severity of VC must not be so intense as to cause brain damage. These are future strategies that should be addressed in terms of clinical application.
Table 2 – Infarct volumes and edema volumes Volume (mm3)
Sham-op. Mild VC Severe VC DPCPX P value
Hemisphere
Infarct
353.3 ± 10.9 354.3 ± 17.9 368.3 ± 17.9 362.6 ± 30.6 0.666
158.5 ± 11.0 131.5 ± 9.3 115.3 ± 25.0 167.0 ± 24.6 0.010*
Infarct rate (%)
Edema volume (mm3)
44.8 ± 7.0 37.1 ± 2.6 31.2 ± 5.7 46.1 ± 5.5 0.003**
14.7 ± 12.4 6.7 ± 5.0 14.0 ± 1.0 23.2 ± 12.0 0.066
Right–Left
Edema rate (%) 104.4 ± 3.7 101.9 ± 1.4 104.0 ± 0.4 106.8 ± 3.4 0.050*
The data are expressed as the mean ± SD. The data were analyzed by one-way ANOVA followed by the post hoc Fisher's PLSD test. P < 0.05 was considered statistically significant. In the infarct and edema rates, significant differences were observed among the 4 groups. *P < 0.05; **P < 0.01.
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5.
Experimental procedures
5.1.
Animals
This study was approved by the Kurume University Ethics Committee for Animal Research, and the animals were cared for in accordance with Kurume University Experimental Animal Center guidelines. Twenty-four male spontaneously hypertensive rats (SHR) weighing 250 to 290 g were used in this study. The rats were allowed free access to food and water, and were housed in a room with a 12-h light–dark cycle. All animals were operated in the neuroanesthesia research laboratory at the Cognitive and Molecular Research Institute of Brain Diseases of Kurume University, followed by postsurgical management in the Experimental Animal Center.
5.2.
Fig. 5 – Infarct rates and edema rates. The infarct and edema rates (mean ± SD) are shown in panels A and B. The rates were analyzed by one-way ANOVA followed by the post hoc Fisher's PLSD test. A P < 0.05 was considered statistically significant. Panel A shows that the infarct rates in mild VC and severe VC groups were significantly less than those in sham-operated and DPCPX groups. Panel B shows that the edema rates in mild VC group were significantly less than those in DPCPX group. *P < 0.05; **P < 0.01.
In conclusion, mild VC by loading an appropriate degree of VC prior to cerebral ischemia produces rapid cerebral protection against subsequent ischemic insult in rats. Further, the beneficial effect of cerebral protection is attenuated by a selective adenosine A1 receptor antagonist, DPCPX. These results indicate that mild VC-induced rapid cerebral protection is mediated, at least in part, through adenosine A1 receptor-related mechanisms.
4.
Clinical inferences
Cerebral ischemia might unintentionally occur during surgeries of the aortic arch and carotid artery. A CVCD can occur during cardiovascular surgery as a result of inappropriate venous drainage during cardiopulmonary bypass (CPB) or prolonged retrograde cerebral perfusion (RCP). Conversely, surgeons could also intentionally control venous drainage during CPB by inducing VC through transient clamping of the SVC cannula, thereby increasing SVC pressure. With this point of view, cerebral VC might be implemented as a pre-ischemic therapy in case of impending cerebral ischemia.
Physiology
All rats were anesthetized with an induction mixture of 2% halothane in 70% nitrous oxide/30% oxygen and then maintained on 1% halothane in the same mixture using a face mask. The tail artery was exposed and cannulated for continuous monitoring of blood pressure and for arterial blood sampling throughout the experiment. Rectal temperatures were monitored and maintained between 36.8 °C and 37.2 °C using a heating pad (Animal Blanket Controller ATB1100, Nihon Kohden, Japan). Regional cerebral blood flow (CBF) was measured over the middle cerebral artery (MCA) territory by laser Doppler flowmetry (LDF) (ALF21, Advance Co, Inc, Japan). A rectangular thin probe (7.5 × 3.5 × 1.0 mm, Type-CS, Unique Medical, Japan) for the LDF was slid through a small scalp incision into the natural pocket between the temporal muscle and the lateral side on the skull over the temporal cortex as previously reported (Harada et al., 2005).
5.3. General preparation and middle cerebral artery occlusion (MCAO) With the rat in the supine position, a partial sternotomy and median incision of the neck were performed to allow MCAO, thereby inducing VC. MCAO was performed using a standard intraluminal procedure as described previously (Andrabi et al., 2004; Kabra et al., 2004; Nakamura et al., 2002; Yoshida et al., 2004). The right common carotid and external carotid arteries were exposed through a midline neck incision and the right common carotid artery was ligated at a site 5 mm proximal to the carotid bifurcation. The external carotid artery was ligated at two sites, which were proximal and distal to the first branch of the external carotid artery (the occipital artery). The internal carotid and pterygopalatine arteries were isolated and carefully separated from the adjacent vagus nerve, and the pterygopalatine artery was ligated close to its origin. The distal site of the internal carotid artery was temporally clamped using a microvascular clip. The common carotid artery was opened by a transverse incision at a site 2 mm proximal to the carotid bifurcation. A silicon-coated 30-mm 4-0 nylon monofilament was inserted into the internal carotid artery via the incision in the
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common carotid artery, and the abovementioned microvascular clip was de-clamped. The origins of the right MCA and the posterior communicating artery were occluded by the silicon-coated cylinder. MCAO was confirmed by LDF. The silk suture around the carotid artery was tightened around the intraluminal monofilament suture to prevent migration of the cylinder and bleeding. The neck incision was closed after fixation of the cylinder. The rats were allowed to recover from general anesthesia and their behavior was observed in order to evaluate them neurologically. After the MCA was occluded for 120 min, the cervical incision was reopened under brief general anesthesia, and the cylinder was removed to permit reperfusion of the MCA. All rats regained consciousness after closure of the incision.
5.4.
CVCD procedure
A partial sternotomy was necessary to expose the vertebral vein, the right superior vena cava, and the internal jugular vein for loading VC. After partial sternotomy and median incision of the neck, the vertebral veins, the anterior vena cava, and the internal jugular vein were carefully dissected and exposed bilaterally. The rats were divided into 3 groups according to the intensity of VC. None of the exposed veins in the sham-operated group were ligated. Both internal jugular veins and the right vertebral vein were ligated in the mild VC group, and in addition both external jugular veins were ligated in the severe VC group. Thereafter, rats in all groups underwent MCAO 40 min after VC. VC was maintained until sacrifice 3 days later. The mild VC treatment was used in DPCPX group, because the preliminary results suggested that mild VC group might show the most potent protection among the three groups (sham-operated, mild VC, and severe VC).
5.5.
53
Experimental groups
The rats were divided into four groups containing six animals each: sham-operated, mild VC, severe VC and DPCPX. Transient ischemia from MCAO lasted 120 min in all four groups, followed by 3 days of reperfusion till experiment termination. Rats in the sham-operated group underwent MCAO but did not have VC prior to MCAO. In the mild VC group, CVCD by ligations of the bilateral internal jugular veins and right vertebral vein were administered till 40 min before MCAO was performed. In the severe VC group, VC was produced by ligation of both internal and external jugular veins and right vertebral vein at the same time as in the mild VC group. In the DPCPX group, we used 8-cyclopentyl-1,3dipropylxanthine (DPCPX) as the selective adenosine A1 receptor antagonist in order to examine the role of adenosine A1 receptors in the action of VC prior to cerebral ischemia. DPCPX dissolved in dimethylsulfoxide (DMSO), was administered at a dose of 0.1 mg/kg by intraperitoneal injection 30 min before mild VC. All animals in this set of experiments survived for 72 h post-MCAO. The basic designs of this protocol are illustrated in Fig. 6.
5.6.
Evaluation of ischemic damage
5.6.1.
Neurological deficits
Neurological evaluation was performed 3, 24, and 72 h after ischemia by an investigator who was blinded to the experimental group design. The neurological findings were scored using modified original scales that have been previously described (Andrabi et al., 2004; Kabra et al., 2004; Nakamura et al., 2002). Four parameters were evaluated (forepaw motion, activity, motion, and posture). Briefly, motion scores were 0
Fig. 6 – Diagram of experimental protocols. Cerebral protective effects were evaluated in four groups (sham-operated, mild VC, severe VC, and DPCPX groups). VC was performed 40 min before MCAO and was maintained for 3 days. MCAO was performed for 120 min and then removed to permit reperfusion of the MCA in all rats. Rats of DPCPX group were intraperitoneally administered 0.1 mg/kg DPCPX dissolved in DMSO 30 min before mild VC. VC, venous congestion; MCA, middle cerebral artery; MCAO, middle cerebral artery occlusion; DPCPX, 8-cyclopentyl-1.3-dipropilxanthine; and DMSO, dimethylsulfoxide.
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(no observable neurological deficit), one (walk towards the paretic side only), two (decreased resistance to lateral push without circling), three (circling movements towards the paretic side) and four (no spontaneously walking). The other three parameters are shown in Fig. 3. Total scores of four parameters were 12 grades from 0 point to 11 points and defined as the behavioral score. These total scores were compared among each of the groups in this experiment.
5.6.2. Measurement of infarct volume, brain edema, and blood samples In this series, S-100 protein and creatine kinase BB (CKBB) were examined from blood samples obtained immediately following and 72 h after the end of MCAO. S-100 protein and CKBB were compared among groups in all experiments. Animals were anesthetized 72 h after reperfusion of the MCA by a lethal intraperitoneal injection of pentobarbital. Four milliliters of blood was immediately obtained for analysis of S-100 protein, blood sugar, and CKBB, following which brains were rapidly removed and sectioned coronally at 1-mm intervals. Tissue sections were subsequently immersed in 2% 2,3,5-triphenyltetrazolium chloride (TTC) dissolved in 0.9% saline, and incubated at 37 °C for 20 min. Infarct brain regions do not convert TTC and therefore remained unstained, whereas normal regions turned red (Horiguchi et al., 2003; Kabra et al., 2004; Wang et al., 2005; Xu et al., 2002). The unstained area was measured in each slice by NIH Image software (Macintosh version). The volume of the infarction was the sum of the unstained areas multiplied by the sum of the thickness of the slices. Edema volume was calculated from the difference between the right and left hemispheric volumes. Infarct volume and the extent of cerebral edema were compared among groups in all experiments.
5.7.
Statistical analysis
Physiological data, blood samples, infarct volumes and rates, and hemispheric volumes are expressed as the mean ± SD. The data were analyzed by one-factor analysis of variance (ANOVA) followed by the post hoc Fisher's Protected Least Significant Difference (PLSD) test. Behavioral scores, representing neurological deficit, were analyzed by nonparametric analyses using the Kruskal–Wallis test followed by the post hoc Fisher's PLSD test. These statistical analyses were performed with Statview for Macintosh version 5.0. A p value of < 0.05 was considered statistically significant.
Acknowledgments The authors thank Hideho Higashi, MD, and Eiichiro Tanaka, MD, for the helpful suggestions and reviewers for their helpful comments.
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(trans-2,3′,4,5′-tetrahydroxystilbene) is neuroprotective and inhibits the apoptotic cell death in transient cerebral ischemia. Brain Res. 1017, 98–107. Ballanyi, K., 2004. Protective role of neuronal KATP Channels in brain hypoxia. J. Exp. Biol. 207, 3201–3212. Barone, F.C., White, R.F., Spera, P.A., Ellison, J., Currie, R.W., Wang, X., Feuerstein, G.Z., 1998. Ischemic preconditioning and brain tolerance: temporal histological and functional outcomes, protein synthesis requirement, and interleukin-1 receptor antagonist and early gene expression. Stroke 29, 1937–1951. Centeno, J.M., Orti, M., Salom, J.B., Sick, T.J, Perez-Pinzon, M.A., 1999. Nitric oxide is involved in anoxic preconditioning neuroprotection in rat hippocampal slices. Brain Res. 836, 62–69. Cohen, M.V., Baines, C.P., Downey, J.M., 2000. Ischemic preconditioning: from adenosine receptor to KATP channel. Annu. Rev. Physiol. 62, 79–109. De Jonge, R., De Jong, J.W., 1999. Ischemic preconditioning and glucose metabolism during low-flow ischemia: role of the adenosine A1 receptor. Cardiovasc. Res. 43, 909–918. Harada, H., Wang, Y., Mishima, Y., Uehara, N., Makaya, T., Kano, T., 2005. A novel method of detecting rCBF with laser-Doppler flowmetry without cranial window through the skull for a MCAO rat model. Brain Res. Protoc. 14, 165–170. Hiraide, T., Katsura, K., Muramatsu, H., Asano, G., Katayama, Y., 2001. Adenosine receptor antagonists cancelled the ischemic tolerance phenomenon in gerbil. Brain Res. 910, 94–98. Horiguchi, T., Kis, B., Rajapakse, N., Shimizu, K., Busija, D.W., 2003. Opening of mitochondrial ATP-sensitive potassium channels is a trigger of 3-nitropropionic acid-induced tolerance to transient focal cerebral ischemia in rats. Stroke 34, 1015–1020. Kabra, D.G., Thiyagarajan, M., Kaul, C.L., Sharma, S.S., 2004. Neuroprotective effect of 4-amino-1, 8-napthalimide, a poly (ADP ribose) polymerase inhibitor in middle cerebral artery occlusion-induced focal cerebral ischemia in rat. Brain Res. Bull. 62, 425–433. Kariko, K., Weissman, D., Welsh, F.A., 2004. Inhibition of toll-like receptor and cytokine signaling—A unifying theme in ischemic tolerance. J. Cereb. Blood Flow Metab. 24, 1288–1304. Kitagawa, K., Matsumoto, M., Tagaya, M., Hata, R., Ueda, H., Niinobe, M., Handa, N., Fukunaga, R., Kimura, K., Mikoshiba, K., Kamada, T., 1990. ‘Ischemic tolerance’ phenomenon found in the brain. Brain Res. 528, 21–24. Mishima, Y., Harada, H., Sugiyama, K., Miyagawa, Y., Uehara, N., Kano, T., 2005. Induction of neuronal tolerance by electroconvulsive shock in rats subjected to forebrain ischemia. Kurume Med. J. 52 (4), 153–160. Nakajima, T., Iwabuchi, S., Miyazaki, H., Okumura, Y., Kuwabara, M., Nomura, Y., Kawahara, K., 2004. Preconditioning prevents ischemia-induced neuronal death through persistent Akt activation in the penumbra region of the rat brain. J. Vet. Med. Sci. 66 (5), 521–527. Nakamura, M., Nakakimura, K., Matsumoto, M., Sakabe, T., 2002. Rapid tolerance to focal cerebral ischemia in rats is attenuated by adenosine A1 receptor antagonist. J. Cereb. Blood Flow Metab. 22, 161–170. Nishio, S., Yunoki, M., Chen, Z.F., Anzivino, M.J., Lee, K.S., 2000. Ischemic tolerance in the rat neocortex following hypothermic preconditioning. J. Neurosurg. 93, 845–851. Perez-Pinzon, M.A., Born, J.G., 1999. Rapid preconditioning neuroprotection following anoxia in hippocampal slices: role of the K+ATP channel and protein kinase C. Neuroscience 89, 453–459. Rosenzweig, H.L., Lessov, N.S., Henshall, D.C., Minami, M., Simon, R.P., Stenzel-Poore, M.P., 2004. Endotoxin preconditioning prevents cellular inflammatory response during ischemic neuroprotection in mice. Stroke 35, 2576–2581.
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Sakashita, H., 2004. The consequences of cerebral venous circulatory disturbances associated with brain ischemia. Kurume Med. J. 51, 15–23. Stagliano, N.E., Perez-Pinzon, M.A., Moskowitz, M.A., Hung, P.L., 1999. Focal ischemic preconditioning induces rapid tolerance to middle cerebral artery occlusion in mice. J. Cereb. Blood Flow Metab. 19, 757–761. Yano, S., Morioka, M., Fukunaga, K., Kawano, T., Hara, T., Kai, Y., Hamada, J., Miyamoto, E., Ushio, Y., 2001. Activation of Akt/ protein kinase B contributes to induction of ischemic tolerance in the CA1 subfield of gerbil hippocampus. J. Cereb. Blood Flow Metab. 21, 351–360. Yoshida, M., Nakakimura, K., Cui, Y.J., Matsumoto, M., Sakabe, T., 2004. Adenosine A1 receptor antagonist and mitochondrial
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ATP-sensitive potassium channel blocker attenuate the tolerance to focal cerebral ischemia in rats. J. Cereb. Blood Flow Metab. 14, 771–779. Yunoki, M., Nishio, S., Ukita, N., Anzivino, M.J., Lee, K.S., 2003. Hypothermic preconditioning induces rapid tolerance to focal ischemic injury in the rat. Exp. Neurol. 181, 291–300. Wang, Q., Xiong, L., Chen, S., Liu, Y., Zhu, X., 2005. Rapid tolerance to focal cerebral ischemia in rats is induced by preconditioning with electroacupuncture: window of protection and the role of adenosine. Neurosci. Lett. 381, 158–162. Xu, H., Aibiki, M., Nagiya, J., 2002. Neuroprotective effects of hyperthermic preconditioning on infarcted volume after middle cerebral artery occlusion in rats: role of adenosine receptors. Crit. Care Med. 30, 1126–1130.
a v a i l a b l e a t w w w. s c i e n c e d i r e c t . c o m
w w w. e l s e v i e r. c o m / l o c a t e / b r a i n r e s
Research Report
Moderate cerebral venous congestion induces rapid cerebral protection via adenosine A1 receptor activation Keiichi Akaiwa a , Hidetoshi Akashi a,⁎, Hideki Harada b , Hideki Sakashita a , Shinichi Hiromatsu a , Tatsuhiko Kano b , Shigeaki Aoyagi a a
Department of Surgery, Kurume University School of Medicine, Kurume, Japan Department of Anesthesiology, Kurume University School of Medicine, Kurume, Japan
b
A R T I C LE I N FO
AB S T R A C T
Article history:
Stroke is a devastating complication in cardiovascular surgery, and neuronal damage is
Accepted 3 September 2006
worsened by intracranial pressure elevation caused by cerebral venous circulatory
Available online 24 October 2006
disturbances (CVCD). However, we have previously reported that CVCD before cerebral ischemia decreases the infarct area. In the present study, focal cerebral ischemia was
Keywords:
induced in spontaneously hypertensive rats by filament insertion through the carotid
Cerebral protection
artery. Rats were divided into the following four groups: sham-operated, mild or severe
Cerebral venous congestion
venous congestion (VC), and DPCPX. The DPCPX group received the adenosine A1 receptor
Adenosine A1 receptor
antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) prior to mild VC. Behavior, infarct
Cerebral venous circulatory
volume, edema and S-100 protein were evaluated among the four groups. The infarct
disturbance
volume rates in mild VC and severe VC groups were significantly less than that in sham-
Middle cerebral artery occusion
operated and DPCPX groups. However, the mortality of the severe VC group worsened in a
Cerebral infarct
time-dependent manner. We observed a significant decrease in edema in the mild VC group
Ischemia
compared to the DPCPX group. Behavioral scores also indicated that the mild VC group had
Neurological deficits
fewer neurological deficits than the other three groups, including the DPCPX group. We were
Rapid ischemic tolerance
able to induce rapid cerebral protection via adenosine A1 receptor activation by
Venous congestive preconditioning
administering an appropriate degree of VC prior to cerebral ischemia produced by middle
Transient MCAO
cerebral artery occlusion. Our work suggests possible mechanisms by which such effective
DPCPX
VC may lead to cerebral protection and adenosine A1 receptor activation. © 2006 Elsevier B.V. All rights reserved.
1.
Introduction
Stroke is a devastating complication in cardiovascular surgery and often makes the patient unable to gain from the benefits of the operation. Cardiovascular surgeons use several cerebral protection methods, such as retrograde cerebral perfusion (RCP) and selective cerebral perfusion (SCP) to prevent the development of such complications. Although remarkable
improvements have been made in cardiovascular surgery, cerebral complication still remains a major unsolved problem. Therefore, we have investigated the consequences of cerebral venous circulatory disturbances (CVCD) associated with cerebral ischemia, and have reported (Sakashita, 2004) that CVCD before cerebral ischemia decreased the infarct area compared with cerebral ischemia without CVCD, whereas CVCD after cerebral ischemia resulted in larger neuronal damage.
⁎ Corresponding author. Fax: +81 942 35 8967. E-mail address: [email protected] (H. Akashi). 0006-8993/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2006.09.019
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Recently, it has been reported that a sublethal ischemic insult might limit damage from a subsequent severe ischemic insult (Barone et al., 1998; Nakamura et al., 2002; Stagliano et al., 1999; Yano et al., 2001; Yoshida et al., 2004). This phenomenon was defined as ischemic tolerance. Depending on the latency between insults, two types of ischemic tolerance in the brain, rapid and delayed, have been documented in the literature (Barone et al., 1998; Kariko et al., 2004; Nishio et al., 2000; Yunoki et al., 2003). Although preconditioning with brief middle cerebral artery occlusion (MCAO) induced protection as rapid tolerance against neuronal damage, CVCD before cerebral ischemia may potentially produce ischemic tolerance in the same way as brief arterial cerebral ischemia. This possibility was discussed in one of our previous reports (Sakashita, 2004). In the present study, we conducted two experiments. The first determined the relation between the intensity of venous congestion (VC) and the protective effect against subsequent cerebral ischemia. The second experiment examined whether cerebral protection, if any, that is rapidly induced by pretreatment is modified by a highly selective adenosine A1 receptor antagonist, 8-cyclopentyl-1,3-dipropylxanthine (DPCPX). We also discuss possible mechanisms whereby such effective VC may lead to the development of a new cerebral protection paradigm.
Fig. 1 – Cerebral blood flow (CBF). CBF values for the MCA are expressed as a percentage of baseline values before MCAO. Successful occlusion and reperfusion of the MCA were examined using LDF. CBF changes of the MCA territory between during MCAO and after reperfusion are shown. There were no significant differences in the percentage change in CBF among the four groups.
2.2.
2.
Results
2.1.
Physiological variables and survival rates
Physiological parameters were monitored in all rats during the operation. Table 1 presents a summary of the physiological parameters immediately after reperfusion of the MCA. VC or DPCPX did not produce changes in body temperature, mean arterial pressure (BP), blood sugar (BS), hematocrit (Ht), pH, or blood gas data. Cerebral blood flow (CBF) changes are presented in Fig. 1. Baseline CBF before MCAO was defined as 100%, and changes in CBF are indicated as a percentage of the baseline CBF values. There were no significant differences in the rate of change of CBF among the four groups. The mortality rate was 0% among the sham-operated, mild VC, and DPCPX groups and 50% in the severe VC group; three of the six animals unexpectedly died before scheduled sacrifice (48 to 72 h post-ischemia).
Neurological deficits
The post-ischemic survival period was 72 h, and neurological deficits were evaluated immediately, 3 h, 24 h, and 72 h after reperfusion. Fig. 2 presents behavioral scores of post-ischemic neurological outcome for all rats. Behavioral scores were calculated from the totals of four-parameter scores (Fig. 3). Significant differences were observed among groups immediately, 24 h, and 72 h but not at 3 h after reperfusion of the MCA. Neurological deficits in the mild VC group were significantly fewer than those observed in the other two groups (shamoperated and DPCPX), during MCAO. Even in the severe VC group, the behavioral score was small in the early phase (during MCAO and 3 h after reperfusion). However, three of six rats in the severe VC group died between 48 h and 72 h after reperfusion, whereas the remaining three rats that survived also stopped moving 72 h after reperfusion. The severe VC group caused excess morbidity, resulting in an inability to assess the effect of this treatment on cerebral infarction.
Table 1 – Physiological variables immediately after reperfusion of the MCA Experimental groups Sham-op. Mild VC Severe VC DPCPX P value
Temperature (°C)
BP (mm Hg)
BS (mg/dl)
Ht
pH
PCO2 (mm Hg)
PO2 (mm Hg)
37.2 ± 0.2 37.0 ± 0.2 37.1 ± 0.3 37.1 ± 0.2 0.566
144 ± 5.2 146 ± 3.3 139 ± 1.2 143 ± 5.1 0.215
136 ± 16.0 136 ± 15.0 126 ± 19.3 137 ± 21.8 0.846
41.2 ± 2.6 40.3 ± 2.6 43.3 ± 1.2 40.5 ± 1.6 0.281
7.45 ± 0.03 7.46 ± 0.02 7.49 ± 0.01 7.45 ± 0.03 0.162
40.3 ± 5.1 40.2 ± 1.5 34.3 ± 2.3 37.6 ± 6.9 0.288
437.2 ± 81.3 463.3 ± 115.3 365.7 ± 151.6 444.1 ± 88.9 0.622
Physiological variables for all rats expressed as the mean ± SD. Differences among the four groups were compared by one-way ANOVA followed by a comparison using Fisher's LSD post hoc test. P values less than 0.05 were considered significant. There were no significant differences in any of the variables among the four groups. BP: mean arterial blood pressure, BS: blood sugar, temperature: rectal temperature.
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Fig. 2 – Behavioral scores in all rats. This figure is showing behavioral scores of in all rats at four points. Statistical analysis was conducted using the Kruskal–Wallis test followed by the post hoc Fisher's PLSD test. Significant differences among the four groups were observed at three points (during MCAO, and 24 and 72 h after reperfusion). *P < 0.05; **P < 0.01; ***P < 0.001.
These results suggest that mild VC group had the smallest neurological deficit and mild VC was the most effective at cerebral protection. However, these effects were reversed in the DPCPX group, which was also administered mild VC.
than those in the sham-operated group. These results suggested that mild VC was the most effective at cerebral protection because of the smaller infarct area and lower edema rates, and this effect was attenuated by DPCPX.
2.3.
2.4.
Infarct volume and cerebral edema
Fig. 4 shows the extent of ischemic area in these three groups using TTC staining. The colorless area was considered to correspond to the territory supplied by the occluded MCA. Infarct volume rates are presented as infarct area percentages of the right hemisphere, and these rates were compared among the all groups (Table 2, Fig. 5A). With regard to cerebral edema, the volume balance between the right hemisphere and the left hemisphere was calculated and edema rates are presented as right hemisphere volume percentages of the normal left hemisphere. These edema rates were compared among the four groups in the same manner as infarct area (Table 2, Fig. 5B). The infarct volume rates in the mild VC and severe VC groups were significantly less than those in the sham-operated and DPCPX groups (mild VC; 37.1 ± 2.6%, severe VC; 31.2 ± 5.7%, sham-operated; 44.8 ± 7.0%, DPCPX; 46.1 ± 5.5% P < 0.05). For edema rates, there was a significant difference between mild VC and DPCPX groups using the post hoc Fisher's PLSD test (Fig. 5B). Although the difference did not reach significance, edema rates in the mild VC group were less
CKBB and S-100 protein
Creatine kinase BB (CKBB) and S-100 protein were measured in order to evaluate cerebral damage immediately and 72 h after reperfusion of the MCA. There were no statistically significant differences in the values of CKBB and S-100 protein.
3.
Discussion
The results of the present study show that mild cerebral VC produces rapid cerebral protection against subsequent ischemic insults in rats, and that the beneficial effect of cerebral protection is attenuated by a selective adenosine A1 receptor antagonist, DPCPX. These results suggest that mild VCinduced rapid cerebral protection is mediated, at least in part, through adenosine A1 receptor-related mechanisms. Cerebral VC should logically cause an intracranial volume overload and an expansion of vascular beds, which might induce intracranial pressure elevation. The expanded and overloaded vascular beds cause vascular edema, which in turn
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Fig. 3 – Behavioral scores. Rats were evaluated neurologically using four parameters (motion of the forepaw, activity, motion, and posture). The behavioral score was defined as the total scores of four parameters that were classified into 12 grades (0 to 11 points).
brings a further increase in intracranial pressure, causing a drop in cerebral perfusion pressure and ultimately intensifying the degree of ischemia as well as potentiating cerebral edema itself. For this reason, although it is easy to understand how VC contributes to neuronal damage, it is more difficult to comprehend the mechanism by which VC has a protective role against subsequent ischemic insults. In the present experiments, VC loading immediately prior to lethal cerebral ischemia was protective against cerebral ischemia, although the optimal severity of VC loading before ischemia is an unresolved issue. It is conceivable that VC loading is responsible for expansion of cerebral vascular beds and increase of venous pressure, and the decrease in cerebral perfusion pressure might have the same conditioning as sublethal ischemia. In previous animal experiments (Sakashita, 2004), we examined the protective effects of VC preloading on the MCAO model in rats and found that it could shrink the ischemic infarct volume. However, if the same conditioning of VC was loaded after the induction of MCAO, the infarct volume was clearly expanded and led to death by the end of 24 h in the reperfusion period. Focusing on this, in the present study we examined whether any differences exist in the protective effects depending on the severity of preloaded VC. Based on the findings and results for neurological deficits, infarct volume, and cerebral edema, mild VC might be the most effective against cerebral protection. The mortality rate was 0% in the sham-operated, mild VC, and DPCPX groups. By
72 h after reperfusion, all of the rats in the severe VC group were either hardly moving or already dead, with a mortality rate of 50%. The morbidity in the severe VC group might be not merely ischemic injury, but excessive edematous damage caused by continuous severe VC, leading to poor survival beyond the 72 h of reperfusion. The method of VC in this experimental paradigm was permanent rather than transient, raising concerns about the effects of VC itself. We predicted that cerebral blood flow (CBF) should be affected and rats might show behavioral changes from VC itself. However, the rats did not exhibit any neurological defects and CBF revealed the same value with or without VC loading. In applying this protective effect of VC to clinical cases, e.g., to cardiac surgery using cardiopulmonary bypass (CPB), VC should be loaded transiently by increasing pressure of the superior vena cava. In these experiments we applied continual VC instead of transient VC due to the technical difficulty of producing this model. Moderate VC (mild VC in this study) could induce compensatory venous collaterals, resulting in a transient effect of VC, even though the ligation was permanent. Hence, mild VC in this study might mimic transient loading in the cerebral venous circulation. A cerebral protective effect was demonstrated in this experiment by mild VC loading prior to MCAO, however, the mechanism has not been elucidated. There have been many reports on the mechanism of cerebral protective effect of ischemic tolerance since the first one by Kitagawa et al. (1990). It has been reported that cross-tolerance against neuronal death is induced by many factors such as sublethal ischemia (Barone et al., 1998; Nakamura et al., 2002; Stagliano et al., 1999; Yano et al., 2001; Yoshida et al., 2004), hypoxia (Ballanyi, 2004; Centeno et al., 1999; Perez-Pinzon and Born, 1999), polyunsaturated fatty acids, lipopolysaccharides (Kariko et al., 2004; Rosenzweig et al., 2004), modified electroconvulsive shock (Mishima et al., 2005), electro-acupuncture (Wang et al., 2005), hyperthermia (Xu et al., 2002), and hypothermia (Nishio et al., 2000; Yunoki et al., 2003). Among these, ischemic and hypothermic preconditioning are well known to induce rapid tolerance and delayed tolerance, however, it has been reported that the mechanisms of rapid and delayed tolerances are different (Nishio et al., 2000). For example, delayed tolerance induced by hypothermia is inhibited by the protein synthesis inhibitor aminomycin (Nishio et al., 2000), whereas rapid tolerance was not (Yunoki et al., 2003). This finding suggests that de novo protein synthesis plays an important role in the induction of delayed tolerance, but is not involved in the induction of rapid tolerance. It has been reported for example that G-protein-coupled receptor (adenosine receptors, etc.) activity (Cohen et al., 2000; De Jonge and De Jong, 1999), an increase in protein kinase C activity (Perez-Pinzon and Born, 1999), an increase in mitogen-activated protein (MAP) kinase activity, Akt activity (Nakajima et al., 2004; Yano et al., 2001), nitric oxide (NO) production (Centeno et al., 1999; PerezPinzon and Born, 1999), and mitochondrial KATP channels (Ballanyi, 2004; Horiguchi et al., 2003; Perez-Pinzon and Born, 1999) are involved in the mechanism of rapid tolerance. It has been demonstrated that adenosine A1 receptor activity is strongly involved in the induction of rapid tolerance towards
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Fig. 4 – TTC staining, 72 h after reperfusion of the MCA. Infarct brain regions did not convert TTC and therefore remained unstained. The unstained area was measured in each slice by NIH image. The infarct volume rates in mild VC group were significantly lower than those in sham-operated and DPCPX groups. TTC: 2,3,5-triphenyltetrazolium chloride.
cerebral ischemia in rats (Hiraide et al., 2001; Nakamura et al., 2002; Wang et al., 2005). Adenosine A1 receptor activity is inhibited by treatment with the antagonist DPCPX, and in our experiments we confirmed whether or not the cerebral protective effect of mild VC was attenuated by inhibiting adenosine A1 receptor activity by preadministering DPCPX intraperitoneally before mild VC. The behavioral score was significantly worsened in the DPCPX group compared to the mild VC group during MCAO, although there were no significant differences at 3, 24, and 72 h after reperfusion. However, infarct volume and edema rates were significantly increased in the DPCPX group. These results suggest that the cerebral protective effect in mild VC group was at least partially inhibited by DPCPX preadministration, and strongly implicates the involvement of rapid cerebral protection involving adenosine A1 receptors. We did not see any pathological or behavioral effects of DPCPX administration alone in a pilot study (data not shown).
Even though we did not examine the direct effects of preadministered DPCPX on our MCAO model, this possibility has been already examined by other investigators (Yoshida et al., 2004), who showed DPCPX and DMSO had no apparent influence on infarct volume. Hence, we postulate that VC is mainly responsible for our results. Rapid cerebral protection was achieved by loading an appropriate degree of VC prior to MCAO in rats. Although the details of the exact mechanism of the cerebral protection remain unclear, we speculate that a steep drop in cerebral perfusion pressure due to the difference between decreased arterial and increased venous pressure might be a protective factor, which could mimic the conditioning as sublethal ischemia. While an appropriate intensity of loading to activate the adenosine A1 receptor is necessary, the severity of VC must not be so intense as to cause brain damage. These are future strategies that should be addressed in terms of clinical application.
Table 2 – Infarct volumes and edema volumes Volume (mm3)
Sham-op. Mild VC Severe VC DPCPX P value
Hemisphere
Infarct
353.3 ± 10.9 354.3 ± 17.9 368.3 ± 17.9 362.6 ± 30.6 0.666
158.5 ± 11.0 131.5 ± 9.3 115.3 ± 25.0 167.0 ± 24.6 0.010*
Infarct rate (%)
Edema volume (mm3)
44.8 ± 7.0 37.1 ± 2.6 31.2 ± 5.7 46.1 ± 5.5 0.003**
14.7 ± 12.4 6.7 ± 5.0 14.0 ± 1.0 23.2 ± 12.0 0.066
Right–Left
Edema rate (%) 104.4 ± 3.7 101.9 ± 1.4 104.0 ± 0.4 106.8 ± 3.4 0.050*
The data are expressed as the mean ± SD. The data were analyzed by one-way ANOVA followed by the post hoc Fisher's PLSD test. P < 0.05 was considered statistically significant. In the infarct and edema rates, significant differences were observed among the 4 groups. *P < 0.05; **P < 0.01.
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5.
Experimental procedures
5.1.
Animals
This study was approved by the Kurume University Ethics Committee for Animal Research, and the animals were cared for in accordance with Kurume University Experimental Animal Center guidelines. Twenty-four male spontaneously hypertensive rats (SHR) weighing 250 to 290 g were used in this study. The rats were allowed free access to food and water, and were housed in a room with a 12-h light–dark cycle. All animals were operated in the neuroanesthesia research laboratory at the Cognitive and Molecular Research Institute of Brain Diseases of Kurume University, followed by postsurgical management in the Experimental Animal Center.
5.2.
Fig. 5 – Infarct rates and edema rates. The infarct and edema rates (mean ± SD) are shown in panels A and B. The rates were analyzed by one-way ANOVA followed by the post hoc Fisher's PLSD test. A P < 0.05 was considered statistically significant. Panel A shows that the infarct rates in mild VC and severe VC groups were significantly less than those in sham-operated and DPCPX groups. Panel B shows that the edema rates in mild VC group were significantly less than those in DPCPX group. *P < 0.05; **P < 0.01.
In conclusion, mild VC by loading an appropriate degree of VC prior to cerebral ischemia produces rapid cerebral protection against subsequent ischemic insult in rats. Further, the beneficial effect of cerebral protection is attenuated by a selective adenosine A1 receptor antagonist, DPCPX. These results indicate that mild VC-induced rapid cerebral protection is mediated, at least in part, through adenosine A1 receptor-related mechanisms.
4.
Clinical inferences
Cerebral ischemia might unintentionally occur during surgeries of the aortic arch and carotid artery. A CVCD can occur during cardiovascular surgery as a result of inappropriate venous drainage during cardiopulmonary bypass (CPB) or prolonged retrograde cerebral perfusion (RCP). Conversely, surgeons could also intentionally control venous drainage during CPB by inducing VC through transient clamping of the SVC cannula, thereby increasing SVC pressure. With this point of view, cerebral VC might be implemented as a pre-ischemic therapy in case of impending cerebral ischemia.
Physiology
All rats were anesthetized with an induction mixture of 2% halothane in 70% nitrous oxide/30% oxygen and then maintained on 1% halothane in the same mixture using a face mask. The tail artery was exposed and cannulated for continuous monitoring of blood pressure and for arterial blood sampling throughout the experiment. Rectal temperatures were monitored and maintained between 36.8 °C and 37.2 °C using a heating pad (Animal Blanket Controller ATB1100, Nihon Kohden, Japan). Regional cerebral blood flow (CBF) was measured over the middle cerebral artery (MCA) territory by laser Doppler flowmetry (LDF) (ALF21, Advance Co, Inc, Japan). A rectangular thin probe (7.5 × 3.5 × 1.0 mm, Type-CS, Unique Medical, Japan) for the LDF was slid through a small scalp incision into the natural pocket between the temporal muscle and the lateral side on the skull over the temporal cortex as previously reported (Harada et al., 2005).
5.3. General preparation and middle cerebral artery occlusion (MCAO) With the rat in the supine position, a partial sternotomy and median incision of the neck were performed to allow MCAO, thereby inducing VC. MCAO was performed using a standard intraluminal procedure as described previously (Andrabi et al., 2004; Kabra et al., 2004; Nakamura et al., 2002; Yoshida et al., 2004). The right common carotid and external carotid arteries were exposed through a midline neck incision and the right common carotid artery was ligated at a site 5 mm proximal to the carotid bifurcation. The external carotid artery was ligated at two sites, which were proximal and distal to the first branch of the external carotid artery (the occipital artery). The internal carotid and pterygopalatine arteries were isolated and carefully separated from the adjacent vagus nerve, and the pterygopalatine artery was ligated close to its origin. The distal site of the internal carotid artery was temporally clamped using a microvascular clip. The common carotid artery was opened by a transverse incision at a site 2 mm proximal to the carotid bifurcation. A silicon-coated 30-mm 4-0 nylon monofilament was inserted into the internal carotid artery via the incision in the
BR A IN RE S E A RCH 1 1 22 ( 20 0 6 ) 4 7 –5 5
common carotid artery, and the abovementioned microvascular clip was de-clamped. The origins of the right MCA and the posterior communicating artery were occluded by the silicon-coated cylinder. MCAO was confirmed by LDF. The silk suture around the carotid artery was tightened around the intraluminal monofilament suture to prevent migration of the cylinder and bleeding. The neck incision was closed after fixation of the cylinder. The rats were allowed to recover from general anesthesia and their behavior was observed in order to evaluate them neurologically. After the MCA was occluded for 120 min, the cervical incision was reopened under brief general anesthesia, and the cylinder was removed to permit reperfusion of the MCA. All rats regained consciousness after closure of the incision.
5.4.
CVCD procedure
A partial sternotomy was necessary to expose the vertebral vein, the right superior vena cava, and the internal jugular vein for loading VC. After partial sternotomy and median incision of the neck, the vertebral veins, the anterior vena cava, and the internal jugular vein were carefully dissected and exposed bilaterally. The rats were divided into 3 groups according to the intensity of VC. None of the exposed veins in the sham-operated group were ligated. Both internal jugular veins and the right vertebral vein were ligated in the mild VC group, and in addition both external jugular veins were ligated in the severe VC group. Thereafter, rats in all groups underwent MCAO 40 min after VC. VC was maintained until sacrifice 3 days later. The mild VC treatment was used in DPCPX group, because the preliminary results suggested that mild VC group might show the most potent protection among the three groups (sham-operated, mild VC, and severe VC).
5.5.
53
Experimental groups
The rats were divided into four groups containing six animals each: sham-operated, mild VC, severe VC and DPCPX. Transient ischemia from MCAO lasted 120 min in all four groups, followed by 3 days of reperfusion till experiment termination. Rats in the sham-operated group underwent MCAO but did not have VC prior to MCAO. In the mild VC group, CVCD by ligations of the bilateral internal jugular veins and right vertebral vein were administered till 40 min before MCAO was performed. In the severe VC group, VC was produced by ligation of both internal and external jugular veins and right vertebral vein at the same time as in the mild VC group. In the DPCPX group, we used 8-cyclopentyl-1,3dipropylxanthine (DPCPX) as the selective adenosine A1 receptor antagonist in order to examine the role of adenosine A1 receptors in the action of VC prior to cerebral ischemia. DPCPX dissolved in dimethylsulfoxide (DMSO), was administered at a dose of 0.1 mg/kg by intraperitoneal injection 30 min before mild VC. All animals in this set of experiments survived for 72 h post-MCAO. The basic designs of this protocol are illustrated in Fig. 6.
5.6.
Evaluation of ischemic damage
5.6.1.
Neurological deficits
Neurological evaluation was performed 3, 24, and 72 h after ischemia by an investigator who was blinded to the experimental group design. The neurological findings were scored using modified original scales that have been previously described (Andrabi et al., 2004; Kabra et al., 2004; Nakamura et al., 2002). Four parameters were evaluated (forepaw motion, activity, motion, and posture). Briefly, motion scores were 0
Fig. 6 – Diagram of experimental protocols. Cerebral protective effects were evaluated in four groups (sham-operated, mild VC, severe VC, and DPCPX groups). VC was performed 40 min before MCAO and was maintained for 3 days. MCAO was performed for 120 min and then removed to permit reperfusion of the MCA in all rats. Rats of DPCPX group were intraperitoneally administered 0.1 mg/kg DPCPX dissolved in DMSO 30 min before mild VC. VC, venous congestion; MCA, middle cerebral artery; MCAO, middle cerebral artery occlusion; DPCPX, 8-cyclopentyl-1.3-dipropilxanthine; and DMSO, dimethylsulfoxide.
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(no observable neurological deficit), one (walk towards the paretic side only), two (decreased resistance to lateral push without circling), three (circling movements towards the paretic side) and four (no spontaneously walking). The other three parameters are shown in Fig. 3. Total scores of four parameters were 12 grades from 0 point to 11 points and defined as the behavioral score. These total scores were compared among each of the groups in this experiment.
5.6.2. Measurement of infarct volume, brain edema, and blood samples In this series, S-100 protein and creatine kinase BB (CKBB) were examined from blood samples obtained immediately following and 72 h after the end of MCAO. S-100 protein and CKBB were compared among groups in all experiments. Animals were anesthetized 72 h after reperfusion of the MCA by a lethal intraperitoneal injection of pentobarbital. Four milliliters of blood was immediately obtained for analysis of S-100 protein, blood sugar, and CKBB, following which brains were rapidly removed and sectioned coronally at 1-mm intervals. Tissue sections were subsequently immersed in 2% 2,3,5-triphenyltetrazolium chloride (TTC) dissolved in 0.9% saline, and incubated at 37 °C for 20 min. Infarct brain regions do not convert TTC and therefore remained unstained, whereas normal regions turned red (Horiguchi et al., 2003; Kabra et al., 2004; Wang et al., 2005; Xu et al., 2002). The unstained area was measured in each slice by NIH Image software (Macintosh version). The volume of the infarction was the sum of the unstained areas multiplied by the sum of the thickness of the slices. Edema volume was calculated from the difference between the right and left hemispheric volumes. Infarct volume and the extent of cerebral edema were compared among groups in all experiments.
5.7.
Statistical analysis
Physiological data, blood samples, infarct volumes and rates, and hemispheric volumes are expressed as the mean ± SD. The data were analyzed by one-factor analysis of variance (ANOVA) followed by the post hoc Fisher's Protected Least Significant Difference (PLSD) test. Behavioral scores, representing neurological deficit, were analyzed by nonparametric analyses using the Kruskal–Wallis test followed by the post hoc Fisher's PLSD test. These statistical analyses were performed with Statview for Macintosh version 5.0. A p value of < 0.05 was considered statistically significant.
Acknowledgments The authors thank Hideho Higashi, MD, and Eiichiro Tanaka, MD, for the helpful suggestions and reviewers for their helpful comments.
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