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Bradykinin preconditioning modulates aquaporin-4 expression after spinal cord ischemic injury in rats
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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
Bradykinin preconditioning modulates aquaporin-4 expression after spinal cord ischemic injury in rats Xu Wei-bing a,b , Gu Yan-ting c , Wang Yan-feng b , Lu Xu-hua d , Jia Lian-shun d , Lv Gang b,⁎ a
Department of Orthopaedics, Dalian Municipal Central Hospital, Da Lian 116033, Liaoning Province, P.R. China Department of Orthopaedics, The First Affiliated Hospital, China Medical University, Shen yang 110001, Liaoning Province, P.R. China c Department of Physiology, Life Science and Biology Pharmacopedia Institution, Shenyang Pharmaceutical University, Shen yang 110016, Liaoning Province, P.R. China d Department of Orthopaedics,Shanghai Changzheng Orthopaedic Hospital, Shanghai, 200003, China b
A R T I C LE I N FO
AB S T R A C T
Article history:
The study investigated whether bradykinin (BK) preconditioning could regulate the
Accepted 24 September 2008
expression of aquaporin-4 (AQP4) using an in vivo transient spinal cord ischemia model
Available online 14 October 2008
in rats. BK was infused continuously via the left femoral artery with infusion pump for 15 min (10 µg/kg/min) then we induced ischemia for 20 min and reperfusion for 24 and 72 h
Keywords:
respectively. The results demonstrated that the central part of the white matter exhibited
Bradykinin
loss of perivascular AQP4 and showed a partial recovery toward 72 h of reperfusion. The
Spinal cord ischemia
border zone of white matter was different from the central part of the white matter by
Aquaporin-4
showing no loss of perivascular AQP4 at 24 h of reperfusion but rather a slight increase. BK
Spinal cord edema
significantly reduced the expression level of AQP4 protein in the white matter, but it had none of this effect in the gray matter region at 72 h post-reperfusion. There was no difference in AQP4 protein levels between BK group and control group at the two abovementioned spinal cord regions at 24 h after reperfusion. In addition, the changes in AQP4 protein induced by BK preconditioning were obvious at 72 h after reperfusion, which were accompanied by a reduction of spinal cord edema. Our results demonstrated that the expression of AQP4 protein after spinal cord ischemia/reperfusion was region-specific, time-dependent and also indicated that the attenuation of AQP4 expression induced by BK could be one of the important molecular mechanisms in physiopathology of spinal cord ischemic edema. © 2008 Elsevier B.V. All rights reserved.
1.
Introduction
Spinal cord injury is one of the most devastating of all traumatic conditions that can be encountered by patients. A lot of studies have been performed on elucidating the mechanisms of spinal cord injury (Fujiki et al., 2005; Sharma
et al., 2005). The spinal cord edema is often long lasting and therapy resistant and thus poses a major challenge in the clinic. The molecular mechanisms that promote water flux across the spinal cord–blood interface in the generation phase and resolution phase of spinal cord edema should be further investigated.
⁎ Corresponding author. Fax: +86 2423256666. E-mail address: [email protected] (L. Gang). 0006-8993/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2008.09.087
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Ischemic preconditioning (IPC) is an endogenous protective mechanism invoked by a brief, sublethal ischemic insult, which can reduce cell injury caused by a subsequent lethal ischemic insult. The powerful endogenous neuroprotective mechanism has also been identified in the spinal cord (Huang et al., 2007). Studies have illustrated that many substances deteriorating spinal cord damage in ischemia can be protective in the process of spinal cord IPC (Orendacova et al., 2005). Bradykinin (BK) preconditioning induces protection against spinal cord ischemic injury, and this protection is likely due to the protection of the vasculature of the spinal cord and the promotion of neuronal survival (Wang et al., 2008). Our previous study also demonstrated that the spinal cord edema was significantly reduced by BK preconditioning after 72 h reperfusion. Thus, the molecular mechanisms of BK-induced edema clearance should be further verified, which could be helpful to the resolution of spinal cord ischemia-induced edema. Aquaporin-4 (AQP4) protein is very strongly expressed in glial cells of the spinal cord (Oshio et al., 2004; Rash et al., 1998). A recent study reported that changes in AQP4 expression in injured spinal cords parallel changes in spinal cord water content (Nesic et al., 2006), and Saadoun provided
direct evidence for involvement of AQP4 in spinal cord injuryrelated edema (Saadoun et al., 2008). However, the effect of BK on the expression of AQP4 in spinal cord ischemiainduced edema is poorly understood. Recently, a study demonstrates that the perivascular pool of AQP4 could become rate-limiting for water flux in pathophysiological conditions, such as in the reperfusion phase after a cerebral ischemic insult (Frydenlund et al., 2006; Lu and Sun, 2003; Meng et al., 2004), which supports the idea that the perivascular pool of AQP4 facilitates water flux across the brain–blood interface and offer an explanation for the reduction in brain edema formation and dissolution observed in AQP4-/-animals (Manley et al., 2000; Papadopoulos et al., 2004). Therefore, it is essential to know whether the perivascular pool of AQP4 is up- or down-regulated after an ischemic insult in the spinal cord, because such changes would determine the time course of edema formation. AQP4 is a response protein to brain injury and the disruption of the blood-brain barrier (BBB) is the most efficient inducer of AQP4 mRNA expression in astrocytes (Tomas-Camardiel et al., 2005). Previous studies have proved that BK preconditioning could help restore blood-spinal cord barrier (BSCB) integrity and reduce inflammatory cytokines (Wang et al., 2008). We
Fig. 1 – Immunogold analysis of AQP4 expression immediately (A, E), 24 h (B, F), 72 h (C, D, G, H) after the onset of reperfusion. Gray matter: A–D; white matter: E–H. At 24 h, there is a pronounced reduction in the number of gold particles (arrows) over perivascular membranes in ischemic gray matter (B) compared with the white matter (F). Compared with control group (G), BK group (H) had a significant decrease in AQP4-like immunoreactivity in the white matter at 72 h after reperfusion. In the ischemic gray matter, AQP4 labeling remains over the abluminal membrane of the perivascular endfeet (open arrows in C) at 72 h. End, endothelial cells; L, vessel lumen; P, pericyte. (Scale bar: 0.5 µm.)
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hypothesized that BK can reduce the degree of spinal cord edema induced by ischemia and improve the degree of BSCB injury, which might be related to its effect on the modulation of AQP4 expression. To test this hypothesis, we investigated the time course of AQP4 expression at the blood–spinal cord interface in a rat spinal cord ischemia injury model by immunogold studies. In addition, we studied whether preischemic administration of BK could have an effect on the expression of AQP4 at protein level in different regions of the spinal cord during reperfusion by immunohistochemistry and western blots methods.
2.
Results
2.1. Loss of perivascular AQP4 immunoreactivity in the ischemic gray matter In the central part of the ischemic gray matter, the abluminal membrane of the endfeet showed scattered gold particles (Fig. 1). The loss of perivascular AQP4 immunoreactivity in the gray matter lesion at 24 h of reperfusion was confirmed (Fig. 1C). Visual examination of earlier and later time points revealed no or more modest losses (Fig. 2). The normal spinal cord was used as an internal reference (calculated from the white matter and gray matter of normal spinal cord). The values for the ischemic white matter and gray matter are
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significantly different from the reference values for 24, 48 and 72 h after reperfusion (P < 0.05). Each of the four animals analyzed at 24 h of reperfusion showed a statistically significant loss of perivascular AQP4 in the ischemic gray matter, compared with 0 h group (Fig. 1). On average, the labeling decreased by 75%, with a minimum of 54% and a maximum of 92%.
2.2. Increase of perivascular AQP4 immunoreactivity in the border zone of white matter The central part of the white matter also exhibits loss of perivascular AQP4. This loss is of magnitude similar to that of the gray matter, but it shows a partial recovery toward 72 h of reperfusion. The abluminal membrane of the endfeet showed scattered gold particles (Fig. 1). None of the animals displayed any significant loss of AQP4 from perivascular membranes of the border zone of white matter, irrespective of reperfusion time. Indeed, the labeling in the border zone (the intensity of which was slightly depressed at the start of reperfusion) tended to increase toward 24 h and thence to decrease toward control level (Figs. 1 and 2).
2.3. BK preconditioning decreased the expression of perivascular AQP4 in white matter at 72 h after reperfusion AQP4 was strongly expressed in grey and white matter, around capillaries and in radial astrocytes (Fig. 3). There was no difference in AQP4 protein levels between BK group and control group at the two above-mentioned spinal cord regions at 24 h after reperfusion. After BK preconditioning, AQP4 labeling was significantly decreased in the white matter at 72 h after reperfusion (Fig. 1H). The BK group showed a significant attenuation in AQP4-like immunoreactivity compared with control group in the central part (Figs. 3A and D) and border zone of the white matter at 72 h after reperfusion (Figs. 3C and F). And the mean optical density value was 0.195 ± 0.007, 0.203 ± 0.004, lower than that in control group, which was 0.243 ± 0.008 (central part, P < 0.05), 0.254 ± 0.004 (border zone of white matter, P < 0.05) respectively (Figs. 3G, I).
2.4. BK-induced down-regulation of AQP4 proteins in white matter in rat spinal cord ischemia model
Fig. 2 – Time course of AQP4 expression after spinal cord ischemia. Values along the ordinate represent linear density of gold particles over perivascular membranes. Density values were obtained from the ischemic gray matter (yellow), the central part of ischemic white matter (red), and the border zone of white matter (blue). The horizontal black line indicates the reference level (calculated from the white matter and gray matter of normal spinal cord). The values for the ischemic white matter (red) and gray matter (yellow) are significantly different from the reference values for 24, 48 and 72 h after reperfusion. *P < 0.05 vs. reference values.
Compared with the control group, the expression level of AQP4 protein induced by BK preconditioning was not changed at 24 h but was markedly decreased in the white matter at 72 h after reperfusion (Fig. 4). The integrated density value (IDV) of the AQP4 protein was 46% lower than the control group in white matter. However, in the gray matter region, there was no significant change both 24 h and 72 h after reperfusion. The IDV of AQP4 with β-actin in control group and BK group at 24 h after reperfusion were 0.211 ± 0.037, 0.205 ± 0.028 (gray matter, P N 0.05), 0.225 ± 0.025, 0.217 ± 0.052 (white matter, P N 0.05) respectively. And the IDV of AQP4 with β-actin in control group and BK group at 72 h after reperfusion were 0.243 ± 0.025, 0.228 ± 0.023 (gray matter, P N 0.05), 0.874 ± 0.072, 0.465 ± 0.052 (white matter, P < 0.05) respectively.
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Fig. 3 – Effects of BK preconditioning on AQP4 expression in different spinal cord regions by immunohistochemical method at different time points after reperfusion. In control group (A–C): rats were induced ischemia for 20 min and reperfusion for 72 h. In BK group (D–F): rats were treated with BK (10 µg/kg/min for 15 min) prior to ischemic insults and 72-h reperfusion. (A, D): the central part of white matter; (B, E): gray matter; (C, F): the border zone of white matter. Compared with control group, BK group had a significant decrease in AQP4-like immunoreactivity in the white matter at 72 h after reperfusion. Imaged at × 20 and mean optical density were shown (n = 5, each) in the central part of white matter (G), gray matter (H), the border zone of white matter (I). *P < 0.05 vs. control group.
2.5. Effect of BK preconditioning on spinal cord water content
3.
Fig. 5 showed that the spinal cord water content in the ischemic hemisphere was significantly reduced in BK group compared with control group (78.27 ± 0.64% and 80.25 ± 0.69%, respectively, P < 0.05).
The study found that the perivascular AQP4 immunoreactivity in the ischemic gray matter displayed loss at 24 h of reperfusion. Visual examination of earlier and later time points revealed no or more moderate loss, suggesting that the
Discussion
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Fig. 4 – Western blot analysis reveals differing expression of AQP4 protein immunoreactivity in different regions and time points after reperfusion. (A, C) Lane1, 3: control group; lane 2, 4: BK group; lane 1, 2: gray matter; Lane3, 4: white matter. Representative Western blots illustrating differences in the 32-kDa band of AQP4. Changes of relative integrated density value (IDV) of AQP4 expression at 24 h (B) and 72 h (D) after reperfusion (n = 5, each). **P < 0.01 vs. control group.
perivascular AQP4 labeling reached minimum values at 24 h. The present work discloses that the expression level of the perivascular pool of AQP4 undergoes major changes after a transient ischemic insult. These results demonstrate directly the molecular mechanisms underlying the generation and dissolution of postischemic edema, because the perivascular pool of AQP4 allows bidirectional water flow and hence is likely to be rate-limiting for both water influx and efflux (Amiry-Moghaddam et al., 2005). Our data suggest a biphasic change in perivascular AQP4 expression in the central part of the white matter. The initial reduction in AQP4 expression will serve to delimit water influx, whereas the partial recovery of
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the AQP4 level (from 24 to 72 h) would be expected to help absorption of excess fluid. Meanwhile a tendency toward an elevated level of AQP4 expression in the white matter border zone was observed at a time when the AQP4 expression was decreased in the ischemic gray matter, which could explain the mechanism of an increased expression of AQP4 in the periinfarct zone after MCAO (Taniguchi et al., 2000). The present data clearly showed that the central part of the white matter by the ischemic insult was different from the gray matter region when it comes to changes in AQP4 expression in the reperfusion phase. Notably, unlike the gray matter region, the central part of white matter showed a partial recovery of perivascular AQP4 toward 72 h of reperfusion. The previous study demonstrated that ischemia disrupted the coupling between AQP4 and its anchoring complex (Puwarawuttipanit et al., 2006). Therefore, rapid changes in the size of the perivascular AQP4 pool can be supposed if the anchoring is severed, because of the high AQP4 concentration gradient between perivascular membranes and the abluminal endfoot membranes, which showed increased labeling after ischemia. Our findings are consistent with the idea that an ischemiainduced perturbation of AQP4 anchoring permits a lateral diffusion of AQP4, thereby draining the pool of AQP4 that normally resides in the perivascular membrane (Frydenlund et al., 2006). This explanation is in line with previous observations that truncated AQP4 disappears much more quickly from the membranes than wild type AQP4 (Neely et al., 2001). In contrast, there was no significant loss of AQP4 from perivascular membranes in the border zone of white matter for each group, irrespective of reperfusion time. Indeed, the labeling in the border zone (the intensity of which was slightly depressed at the start of reperfusion) began to increase toward 24 h and thence to decrease toward control level. The persistence of the perivascular AQP4 pool in the border zone of white matter suggests that partial ischemia is not sufficient to interfere significantly with anchoring of AQP4. Our findings are in agreement with a recent study which shows that osmotherapy with 7.5% hyperosmotic saline at 24 h after MCAO in rat leads to a significant decrease in the brain water
Fig. 5 – Effect of BK preconditioning on spinal cord edema after 20-min ischemia followed by 72-h reperfusion. In control group: rats were induced ischemia for 20 min and reperfusion for 72 h. In BK group: rats were treated with BK (10 µg/kg/min for 15 min) prior to ischemic insults and 72-h reperfusion. Data present means ± SD (n = 5, each), *P < 0.05 vs. control group.
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content in the cortical border zone but not in the central part of the lesion (Chen and Toung, 2007). AQP4 is strongly expressed in grey and white matter, around capillaries and in radial astrocytes. In the central part and border zone of white matter, the protein level of AQP4 expression was not significantly changed by BK preconditioning at 24 h after reperfusion but was markedly decreased at 72 h, suggesting that the regulation of AQP4 induced by BK preconditioning was time-dependent. However, in the gray matter region, there was no significant difference between control group and BK group both 24 h and 72 h after reperfusion. And water content of ischemia spinal cord was significantly reduced in the BK group compared with control group at 72 h after reperfusion. The up-regulation of the AQP4 expression in periinfarct zone edema region could be correlated with BBB disruption and inflammatory cytokines (Chen and Toung, 2007). Whereas the previous study demonstrated that BK preconditioning could help restore BBB integrity and reduce inflammatory cytokines (Wang et al., 2008), suggesting that the effect of BK on AQP4 expression might be related to this ability. In addition, BK can activate B2 receptor, release arachidonic acid and activate cyclo-oxygenase enzymes (Sobey, 2003). And it can also stimulate the release of excitatory amino acid neurotransmitters, induce the production of nitric oxide, and enhance the levels of Ca2+ in cell (Relton et al., 1997). Therefore, the effect induced by BK may be related to activation or strengthening of signal transmission pathway in spinal cord parenchymal cells and subsequent attenuation of AQP4 expression leading to protect neurons from edema. However, the role of BK receptors in different spinal cord regions should be further verified. Since “cytotoxic” brain edema is of decisive pathophysiological importance following spinal cord ischemia injury as it develops early and persists while BBB integrity is gradually restored (Lu and Sun, 2003). And AQP4 expression promotes brain swelling formation in cytotoxic brain edema models (Manley et al., 2000). Conceivably, the down-regulation of AQP4 expression by BK treatment could be beneficial to fluid clearance in ischemia-induced spinal cord edema. In conclusion, our data suggest that the size of the AQP4 pool controls the exchange of fluid between spinal cord and blood during edema formation and dissolution which is subject to large changes during the reperfusion phase. BK preconditioning contributed to spinal cord edema fluid clearance by reducing the expression of perivascular AQP4 in the white matter region of the spinal cord. The magnitude and direction of these changes are different between the gray matter and white matter of the ischemic lesion and should be taken into account in future strategies to develop new therapies. We believe that identifying interventions that could manipulate expression levels of AQP4 would be doubly useful.
4.
Experimental procedures
4.1.
Establishment of spinal cord ischemia model in rat
The adult male SD rats (300–350 g) were purchased from the Experimental Animals Center of China Medical University.
All animal experiments were conducted in accordance with the National Institute of Health Guide for the Care and Use of Laboratory Animals. Under pentobarbital anesthesia (45 mg/kg, intraperitoneally; Abbott Laboratories, North Chicago, IL), a 2F Fogarty catheter (Edwards Lifesciences) was inserted into the left femoral artery and the balloon was placed at the end of the aortic arch (Coston et al. 1983). To induce spinal cord ischemia, the balloon was inflated with 0.05 mL distilled water for 20 min. Complete occlusion of the descending aorta was evidenced by sustained loss of any detectable pulse measured by Doppler sonography in the right femoral artery. At the end of the ischemic period, the catheter was deflated and removed. This technique creates spinal cord ischemic injury in the lumbosacral segment, which causes paraplegia in a reproducible manner (Coston et al. 1983, Marsala and Yaksh, 1994). The rats were randomized into two groups: in control groups: we induced ischemia for 20 min and reperfusion for 0, 2, 6, 12, 24, 48, and 72 h respectively. In BK groups, BK was infused continuously via the left femoral artery with infusion pump for 15 min (10 µg/kg/min) then we induced ischemia for 20 min and reperfusion for 24 or 72 h. Five rats of 24 and 72 h groups after reperfusion and BK group were used in other experiments respectively.
4.2.
Electron microscopy
For quantitative immunogold studies, the tissue blocks did not exceed 1 mm in any dimension and were dissected from each of the following three regions: 1, central part of white matter; 2, gray matter; 3, border zone of white matter. Specimens were embedded in methacrylate resin (Lowicryl HM20; Polysciences, Warrington, PA) and polymerized by UV light below 0 °C. Ultra thin sections were incubated with antibodies to AQP4 (10 µg/ml, Santa Cruz Biotechnology) followed by goat anti-rabbit antibody coupled to 15-nm colloidal gold. The sections were examined in a Philips CM10 electron microscope (Netherlands) at 60 kV (Amiry-Moghaddam et al., 2005, Papadopoulos et al., 2004). Linear densities of gold particles over astrocyte membranes were determined by an extension of analysis (Soft Imaging Systems) as described (Amiry-Moghaddam et al., 2005).
4.3.
Immunohistochemistry
Rats of 24 and 72 h groups after reperfusion with or without BK preconditioning were fixed by transaortic perfusion with saline, followed by perfusion with 4% paraformaldehyde for the following immunochemistry study respectively. The lumbar spinal cords were removed, stored in fixative for 24 h, immersed in 30% sucrose solution in PBS for 24 h, then immediately frozen in liquid nitrogen and stored at − 70 °C for following studies. The sections were immunohistochemically stained with a rabbit polyclonal anti-AQP4 antibody (diluted 1:150, Santa Cruz Biotechnology) following standard procedures. The mean optical density values of AQP4 in the spinal cord were measured using a computer-assisted image analyzing system (Motic Images Advanced 3.2).
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4.4.
Western blot analysis
Western blot analysis was performed to investigate the expression of the AQP4 in different regions of spinal cords in the control group and BK group. Protein homogenates of brain samples were prepared by rapid homogenization in 10 volumes of lysis buffer (2 mM EDTA, 10 mM EGTA, 0.4%NaF, 20 mM Tris-Hcl, pH 7.5). Samples were centrifuged at 17,000 g for 1 h. The protein concentration of soluble materials was determined by the Coomassie G250 Binding method. The protein lysates (12 µg per lane for each sample) were fractioned on 12% SDS-polyacrylamide gels, followed by transferring to nitrocellulose membranes (Santa Cruz Biotechnology, Inc.). The membranes were blocked in blocking buffer (5% non-fat dairy milk dissolved in Tween-Tris-buffered saline, TTBS) overnight at 4 °C. The blots were then incubated with rabbit polyclonal antibody anti-AQP4 (dilution 1:400; Santa Cruz Biotechnology, Inc.) for 2 h. The AQP4 protein bands on these immunoblots were visualized using the enhanced chemiluminescene (ECL kit, Santa Cruz Biotechnology, Inc.). The AQP4 protein bands and β-actin bands were scanned using Chemi Imager 5500 V2.03 software, and IDVs were calculated by Fluor Chen 2.0 software and normalized with that of β-actin.
4.5.
Determination of spinal cord water content
Water content was determined according to the previous protocol (Li and Tator, 1999). The segments of lumbar spinal cord for 72 h groups after reperfusion and BK group were weighed on aluminum foil, dried at 100 °C for 24 h, and reweighed. The percent water content was calculated as the following calculation: water content (%) = [(wet weigh − dry weight) / wet weight] × 100%.
4.6.
Statistical analysis
Results were presented as the mean ± SD. One-way analyses of variance (ANOVA) were used to compare group differences in measurement of AQP4. Dunnett's post hoc tests were applied to compare specific group difference if the ANOVA revealed a significant difference. In other measurements, the data were assessed using paired Student's t test. P < 0.05 was considered statistically significant.
Acknowledgments This work was supported by Special fund for scientific research of postdoctoral subjects in China, no. 20060400292. Natural Science Foundation of Liaoning Province in China, no. 20052096. REFERENCES
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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
Bradykinin preconditioning modulates aquaporin-4 expression after spinal cord ischemic injury in rats Xu Wei-bing a,b , Gu Yan-ting c , Wang Yan-feng b , Lu Xu-hua d , Jia Lian-shun d , Lv Gang b,⁎ a
Department of Orthopaedics, Dalian Municipal Central Hospital, Da Lian 116033, Liaoning Province, P.R. China Department of Orthopaedics, The First Affiliated Hospital, China Medical University, Shen yang 110001, Liaoning Province, P.R. China c Department of Physiology, Life Science and Biology Pharmacopedia Institution, Shenyang Pharmaceutical University, Shen yang 110016, Liaoning Province, P.R. China d Department of Orthopaedics,Shanghai Changzheng Orthopaedic Hospital, Shanghai, 200003, China b
A R T I C LE I N FO
AB S T R A C T
Article history:
The study investigated whether bradykinin (BK) preconditioning could regulate the
Accepted 24 September 2008
expression of aquaporin-4 (AQP4) using an in vivo transient spinal cord ischemia model
Available online 14 October 2008
in rats. BK was infused continuously via the left femoral artery with infusion pump for 15 min (10 µg/kg/min) then we induced ischemia for 20 min and reperfusion for 24 and 72 h
Keywords:
respectively. The results demonstrated that the central part of the white matter exhibited
Bradykinin
loss of perivascular AQP4 and showed a partial recovery toward 72 h of reperfusion. The
Spinal cord ischemia
border zone of white matter was different from the central part of the white matter by
Aquaporin-4
showing no loss of perivascular AQP4 at 24 h of reperfusion but rather a slight increase. BK
Spinal cord edema
significantly reduced the expression level of AQP4 protein in the white matter, but it had none of this effect in the gray matter region at 72 h post-reperfusion. There was no difference in AQP4 protein levels between BK group and control group at the two abovementioned spinal cord regions at 24 h after reperfusion. In addition, the changes in AQP4 protein induced by BK preconditioning were obvious at 72 h after reperfusion, which were accompanied by a reduction of spinal cord edema. Our results demonstrated that the expression of AQP4 protein after spinal cord ischemia/reperfusion was region-specific, time-dependent and also indicated that the attenuation of AQP4 expression induced by BK could be one of the important molecular mechanisms in physiopathology of spinal cord ischemic edema. © 2008 Elsevier B.V. All rights reserved.
1.
Introduction
Spinal cord injury is one of the most devastating of all traumatic conditions that can be encountered by patients. A lot of studies have been performed on elucidating the mechanisms of spinal cord injury (Fujiki et al., 2005; Sharma
et al., 2005). The spinal cord edema is often long lasting and therapy resistant and thus poses a major challenge in the clinic. The molecular mechanisms that promote water flux across the spinal cord–blood interface in the generation phase and resolution phase of spinal cord edema should be further investigated.
⁎ Corresponding author. Fax: +86 2423256666. E-mail address: [email protected] (L. Gang). 0006-8993/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2008.09.087
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Ischemic preconditioning (IPC) is an endogenous protective mechanism invoked by a brief, sublethal ischemic insult, which can reduce cell injury caused by a subsequent lethal ischemic insult. The powerful endogenous neuroprotective mechanism has also been identified in the spinal cord (Huang et al., 2007). Studies have illustrated that many substances deteriorating spinal cord damage in ischemia can be protective in the process of spinal cord IPC (Orendacova et al., 2005). Bradykinin (BK) preconditioning induces protection against spinal cord ischemic injury, and this protection is likely due to the protection of the vasculature of the spinal cord and the promotion of neuronal survival (Wang et al., 2008). Our previous study also demonstrated that the spinal cord edema was significantly reduced by BK preconditioning after 72 h reperfusion. Thus, the molecular mechanisms of BK-induced edema clearance should be further verified, which could be helpful to the resolution of spinal cord ischemia-induced edema. Aquaporin-4 (AQP4) protein is very strongly expressed in glial cells of the spinal cord (Oshio et al., 2004; Rash et al., 1998). A recent study reported that changes in AQP4 expression in injured spinal cords parallel changes in spinal cord water content (Nesic et al., 2006), and Saadoun provided
direct evidence for involvement of AQP4 in spinal cord injuryrelated edema (Saadoun et al., 2008). However, the effect of BK on the expression of AQP4 in spinal cord ischemiainduced edema is poorly understood. Recently, a study demonstrates that the perivascular pool of AQP4 could become rate-limiting for water flux in pathophysiological conditions, such as in the reperfusion phase after a cerebral ischemic insult (Frydenlund et al., 2006; Lu and Sun, 2003; Meng et al., 2004), which supports the idea that the perivascular pool of AQP4 facilitates water flux across the brain–blood interface and offer an explanation for the reduction in brain edema formation and dissolution observed in AQP4-/-animals (Manley et al., 2000; Papadopoulos et al., 2004). Therefore, it is essential to know whether the perivascular pool of AQP4 is up- or down-regulated after an ischemic insult in the spinal cord, because such changes would determine the time course of edema formation. AQP4 is a response protein to brain injury and the disruption of the blood-brain barrier (BBB) is the most efficient inducer of AQP4 mRNA expression in astrocytes (Tomas-Camardiel et al., 2005). Previous studies have proved that BK preconditioning could help restore blood-spinal cord barrier (BSCB) integrity and reduce inflammatory cytokines (Wang et al., 2008). We
Fig. 1 – Immunogold analysis of AQP4 expression immediately (A, E), 24 h (B, F), 72 h (C, D, G, H) after the onset of reperfusion. Gray matter: A–D; white matter: E–H. At 24 h, there is a pronounced reduction in the number of gold particles (arrows) over perivascular membranes in ischemic gray matter (B) compared with the white matter (F). Compared with control group (G), BK group (H) had a significant decrease in AQP4-like immunoreactivity in the white matter at 72 h after reperfusion. In the ischemic gray matter, AQP4 labeling remains over the abluminal membrane of the perivascular endfeet (open arrows in C) at 72 h. End, endothelial cells; L, vessel lumen; P, pericyte. (Scale bar: 0.5 µm.)
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hypothesized that BK can reduce the degree of spinal cord edema induced by ischemia and improve the degree of BSCB injury, which might be related to its effect on the modulation of AQP4 expression. To test this hypothesis, we investigated the time course of AQP4 expression at the blood–spinal cord interface in a rat spinal cord ischemia injury model by immunogold studies. In addition, we studied whether preischemic administration of BK could have an effect on the expression of AQP4 at protein level in different regions of the spinal cord during reperfusion by immunohistochemistry and western blots methods.
2.
Results
2.1. Loss of perivascular AQP4 immunoreactivity in the ischemic gray matter In the central part of the ischemic gray matter, the abluminal membrane of the endfeet showed scattered gold particles (Fig. 1). The loss of perivascular AQP4 immunoreactivity in the gray matter lesion at 24 h of reperfusion was confirmed (Fig. 1C). Visual examination of earlier and later time points revealed no or more modest losses (Fig. 2). The normal spinal cord was used as an internal reference (calculated from the white matter and gray matter of normal spinal cord). The values for the ischemic white matter and gray matter are
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significantly different from the reference values for 24, 48 and 72 h after reperfusion (P < 0.05). Each of the four animals analyzed at 24 h of reperfusion showed a statistically significant loss of perivascular AQP4 in the ischemic gray matter, compared with 0 h group (Fig. 1). On average, the labeling decreased by 75%, with a minimum of 54% and a maximum of 92%.
2.2. Increase of perivascular AQP4 immunoreactivity in the border zone of white matter The central part of the white matter also exhibits loss of perivascular AQP4. This loss is of magnitude similar to that of the gray matter, but it shows a partial recovery toward 72 h of reperfusion. The abluminal membrane of the endfeet showed scattered gold particles (Fig. 1). None of the animals displayed any significant loss of AQP4 from perivascular membranes of the border zone of white matter, irrespective of reperfusion time. Indeed, the labeling in the border zone (the intensity of which was slightly depressed at the start of reperfusion) tended to increase toward 24 h and thence to decrease toward control level (Figs. 1 and 2).
2.3. BK preconditioning decreased the expression of perivascular AQP4 in white matter at 72 h after reperfusion AQP4 was strongly expressed in grey and white matter, around capillaries and in radial astrocytes (Fig. 3). There was no difference in AQP4 protein levels between BK group and control group at the two above-mentioned spinal cord regions at 24 h after reperfusion. After BK preconditioning, AQP4 labeling was significantly decreased in the white matter at 72 h after reperfusion (Fig. 1H). The BK group showed a significant attenuation in AQP4-like immunoreactivity compared with control group in the central part (Figs. 3A and D) and border zone of the white matter at 72 h after reperfusion (Figs. 3C and F). And the mean optical density value was 0.195 ± 0.007, 0.203 ± 0.004, lower than that in control group, which was 0.243 ± 0.008 (central part, P < 0.05), 0.254 ± 0.004 (border zone of white matter, P < 0.05) respectively (Figs. 3G, I).
2.4. BK-induced down-regulation of AQP4 proteins in white matter in rat spinal cord ischemia model
Fig. 2 – Time course of AQP4 expression after spinal cord ischemia. Values along the ordinate represent linear density of gold particles over perivascular membranes. Density values were obtained from the ischemic gray matter (yellow), the central part of ischemic white matter (red), and the border zone of white matter (blue). The horizontal black line indicates the reference level (calculated from the white matter and gray matter of normal spinal cord). The values for the ischemic white matter (red) and gray matter (yellow) are significantly different from the reference values for 24, 48 and 72 h after reperfusion. *P < 0.05 vs. reference values.
Compared with the control group, the expression level of AQP4 protein induced by BK preconditioning was not changed at 24 h but was markedly decreased in the white matter at 72 h after reperfusion (Fig. 4). The integrated density value (IDV) of the AQP4 protein was 46% lower than the control group in white matter. However, in the gray matter region, there was no significant change both 24 h and 72 h after reperfusion. The IDV of AQP4 with β-actin in control group and BK group at 24 h after reperfusion were 0.211 ± 0.037, 0.205 ± 0.028 (gray matter, P N 0.05), 0.225 ± 0.025, 0.217 ± 0.052 (white matter, P N 0.05) respectively. And the IDV of AQP4 with β-actin in control group and BK group at 72 h after reperfusion were 0.243 ± 0.025, 0.228 ± 0.023 (gray matter, P N 0.05), 0.874 ± 0.072, 0.465 ± 0.052 (white matter, P < 0.05) respectively.
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Fig. 3 – Effects of BK preconditioning on AQP4 expression in different spinal cord regions by immunohistochemical method at different time points after reperfusion. In control group (A–C): rats were induced ischemia for 20 min and reperfusion for 72 h. In BK group (D–F): rats were treated with BK (10 µg/kg/min for 15 min) prior to ischemic insults and 72-h reperfusion. (A, D): the central part of white matter; (B, E): gray matter; (C, F): the border zone of white matter. Compared with control group, BK group had a significant decrease in AQP4-like immunoreactivity in the white matter at 72 h after reperfusion. Imaged at × 20 and mean optical density were shown (n = 5, each) in the central part of white matter (G), gray matter (H), the border zone of white matter (I). *P < 0.05 vs. control group.
2.5. Effect of BK preconditioning on spinal cord water content
3.
Fig. 5 showed that the spinal cord water content in the ischemic hemisphere was significantly reduced in BK group compared with control group (78.27 ± 0.64% and 80.25 ± 0.69%, respectively, P < 0.05).
The study found that the perivascular AQP4 immunoreactivity in the ischemic gray matter displayed loss at 24 h of reperfusion. Visual examination of earlier and later time points revealed no or more moderate loss, suggesting that the
Discussion
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Fig. 4 – Western blot analysis reveals differing expression of AQP4 protein immunoreactivity in different regions and time points after reperfusion. (A, C) Lane1, 3: control group; lane 2, 4: BK group; lane 1, 2: gray matter; Lane3, 4: white matter. Representative Western blots illustrating differences in the 32-kDa band of AQP4. Changes of relative integrated density value (IDV) of AQP4 expression at 24 h (B) and 72 h (D) after reperfusion (n = 5, each). **P < 0.01 vs. control group.
perivascular AQP4 labeling reached minimum values at 24 h. The present work discloses that the expression level of the perivascular pool of AQP4 undergoes major changes after a transient ischemic insult. These results demonstrate directly the molecular mechanisms underlying the generation and dissolution of postischemic edema, because the perivascular pool of AQP4 allows bidirectional water flow and hence is likely to be rate-limiting for both water influx and efflux (Amiry-Moghaddam et al., 2005). Our data suggest a biphasic change in perivascular AQP4 expression in the central part of the white matter. The initial reduction in AQP4 expression will serve to delimit water influx, whereas the partial recovery of
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the AQP4 level (from 24 to 72 h) would be expected to help absorption of excess fluid. Meanwhile a tendency toward an elevated level of AQP4 expression in the white matter border zone was observed at a time when the AQP4 expression was decreased in the ischemic gray matter, which could explain the mechanism of an increased expression of AQP4 in the periinfarct zone after MCAO (Taniguchi et al., 2000). The present data clearly showed that the central part of the white matter by the ischemic insult was different from the gray matter region when it comes to changes in AQP4 expression in the reperfusion phase. Notably, unlike the gray matter region, the central part of white matter showed a partial recovery of perivascular AQP4 toward 72 h of reperfusion. The previous study demonstrated that ischemia disrupted the coupling between AQP4 and its anchoring complex (Puwarawuttipanit et al., 2006). Therefore, rapid changes in the size of the perivascular AQP4 pool can be supposed if the anchoring is severed, because of the high AQP4 concentration gradient between perivascular membranes and the abluminal endfoot membranes, which showed increased labeling after ischemia. Our findings are consistent with the idea that an ischemiainduced perturbation of AQP4 anchoring permits a lateral diffusion of AQP4, thereby draining the pool of AQP4 that normally resides in the perivascular membrane (Frydenlund et al., 2006). This explanation is in line with previous observations that truncated AQP4 disappears much more quickly from the membranes than wild type AQP4 (Neely et al., 2001). In contrast, there was no significant loss of AQP4 from perivascular membranes in the border zone of white matter for each group, irrespective of reperfusion time. Indeed, the labeling in the border zone (the intensity of which was slightly depressed at the start of reperfusion) began to increase toward 24 h and thence to decrease toward control level. The persistence of the perivascular AQP4 pool in the border zone of white matter suggests that partial ischemia is not sufficient to interfere significantly with anchoring of AQP4. Our findings are in agreement with a recent study which shows that osmotherapy with 7.5% hyperosmotic saline at 24 h after MCAO in rat leads to a significant decrease in the brain water
Fig. 5 – Effect of BK preconditioning on spinal cord edema after 20-min ischemia followed by 72-h reperfusion. In control group: rats were induced ischemia for 20 min and reperfusion for 72 h. In BK group: rats were treated with BK (10 µg/kg/min for 15 min) prior to ischemic insults and 72-h reperfusion. Data present means ± SD (n = 5, each), *P < 0.05 vs. control group.
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content in the cortical border zone but not in the central part of the lesion (Chen and Toung, 2007). AQP4 is strongly expressed in grey and white matter, around capillaries and in radial astrocytes. In the central part and border zone of white matter, the protein level of AQP4 expression was not significantly changed by BK preconditioning at 24 h after reperfusion but was markedly decreased at 72 h, suggesting that the regulation of AQP4 induced by BK preconditioning was time-dependent. However, in the gray matter region, there was no significant difference between control group and BK group both 24 h and 72 h after reperfusion. And water content of ischemia spinal cord was significantly reduced in the BK group compared with control group at 72 h after reperfusion. The up-regulation of the AQP4 expression in periinfarct zone edema region could be correlated with BBB disruption and inflammatory cytokines (Chen and Toung, 2007). Whereas the previous study demonstrated that BK preconditioning could help restore BBB integrity and reduce inflammatory cytokines (Wang et al., 2008), suggesting that the effect of BK on AQP4 expression might be related to this ability. In addition, BK can activate B2 receptor, release arachidonic acid and activate cyclo-oxygenase enzymes (Sobey, 2003). And it can also stimulate the release of excitatory amino acid neurotransmitters, induce the production of nitric oxide, and enhance the levels of Ca2+ in cell (Relton et al., 1997). Therefore, the effect induced by BK may be related to activation or strengthening of signal transmission pathway in spinal cord parenchymal cells and subsequent attenuation of AQP4 expression leading to protect neurons from edema. However, the role of BK receptors in different spinal cord regions should be further verified. Since “cytotoxic” brain edema is of decisive pathophysiological importance following spinal cord ischemia injury as it develops early and persists while BBB integrity is gradually restored (Lu and Sun, 2003). And AQP4 expression promotes brain swelling formation in cytotoxic brain edema models (Manley et al., 2000). Conceivably, the down-regulation of AQP4 expression by BK treatment could be beneficial to fluid clearance in ischemia-induced spinal cord edema. In conclusion, our data suggest that the size of the AQP4 pool controls the exchange of fluid between spinal cord and blood during edema formation and dissolution which is subject to large changes during the reperfusion phase. BK preconditioning contributed to spinal cord edema fluid clearance by reducing the expression of perivascular AQP4 in the white matter region of the spinal cord. The magnitude and direction of these changes are different between the gray matter and white matter of the ischemic lesion and should be taken into account in future strategies to develop new therapies. We believe that identifying interventions that could manipulate expression levels of AQP4 would be doubly useful.
4.
Experimental procedures
4.1.
Establishment of spinal cord ischemia model in rat
The adult male SD rats (300–350 g) were purchased from the Experimental Animals Center of China Medical University.
All animal experiments were conducted in accordance with the National Institute of Health Guide for the Care and Use of Laboratory Animals. Under pentobarbital anesthesia (45 mg/kg, intraperitoneally; Abbott Laboratories, North Chicago, IL), a 2F Fogarty catheter (Edwards Lifesciences) was inserted into the left femoral artery and the balloon was placed at the end of the aortic arch (Coston et al. 1983). To induce spinal cord ischemia, the balloon was inflated with 0.05 mL distilled water for 20 min. Complete occlusion of the descending aorta was evidenced by sustained loss of any detectable pulse measured by Doppler sonography in the right femoral artery. At the end of the ischemic period, the catheter was deflated and removed. This technique creates spinal cord ischemic injury in the lumbosacral segment, which causes paraplegia in a reproducible manner (Coston et al. 1983, Marsala and Yaksh, 1994). The rats were randomized into two groups: in control groups: we induced ischemia for 20 min and reperfusion for 0, 2, 6, 12, 24, 48, and 72 h respectively. In BK groups, BK was infused continuously via the left femoral artery with infusion pump for 15 min (10 µg/kg/min) then we induced ischemia for 20 min and reperfusion for 24 or 72 h. Five rats of 24 and 72 h groups after reperfusion and BK group were used in other experiments respectively.
4.2.
Electron microscopy
For quantitative immunogold studies, the tissue blocks did not exceed 1 mm in any dimension and were dissected from each of the following three regions: 1, central part of white matter; 2, gray matter; 3, border zone of white matter. Specimens were embedded in methacrylate resin (Lowicryl HM20; Polysciences, Warrington, PA) and polymerized by UV light below 0 °C. Ultra thin sections were incubated with antibodies to AQP4 (10 µg/ml, Santa Cruz Biotechnology) followed by goat anti-rabbit antibody coupled to 15-nm colloidal gold. The sections were examined in a Philips CM10 electron microscope (Netherlands) at 60 kV (Amiry-Moghaddam et al., 2005, Papadopoulos et al., 2004). Linear densities of gold particles over astrocyte membranes were determined by an extension of analysis (Soft Imaging Systems) as described (Amiry-Moghaddam et al., 2005).
4.3.
Immunohistochemistry
Rats of 24 and 72 h groups after reperfusion with or without BK preconditioning were fixed by transaortic perfusion with saline, followed by perfusion with 4% paraformaldehyde for the following immunochemistry study respectively. The lumbar spinal cords were removed, stored in fixative for 24 h, immersed in 30% sucrose solution in PBS for 24 h, then immediately frozen in liquid nitrogen and stored at − 70 °C for following studies. The sections were immunohistochemically stained with a rabbit polyclonal anti-AQP4 antibody (diluted 1:150, Santa Cruz Biotechnology) following standard procedures. The mean optical density values of AQP4 in the spinal cord were measured using a computer-assisted image analyzing system (Motic Images Advanced 3.2).
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4.4.
Western blot analysis
Western blot analysis was performed to investigate the expression of the AQP4 in different regions of spinal cords in the control group and BK group. Protein homogenates of brain samples were prepared by rapid homogenization in 10 volumes of lysis buffer (2 mM EDTA, 10 mM EGTA, 0.4%NaF, 20 mM Tris-Hcl, pH 7.5). Samples were centrifuged at 17,000 g for 1 h. The protein concentration of soluble materials was determined by the Coomassie G250 Binding method. The protein lysates (12 µg per lane for each sample) were fractioned on 12% SDS-polyacrylamide gels, followed by transferring to nitrocellulose membranes (Santa Cruz Biotechnology, Inc.). The membranes were blocked in blocking buffer (5% non-fat dairy milk dissolved in Tween-Tris-buffered saline, TTBS) overnight at 4 °C. The blots were then incubated with rabbit polyclonal antibody anti-AQP4 (dilution 1:400; Santa Cruz Biotechnology, Inc.) for 2 h. The AQP4 protein bands on these immunoblots were visualized using the enhanced chemiluminescene (ECL kit, Santa Cruz Biotechnology, Inc.). The AQP4 protein bands and β-actin bands were scanned using Chemi Imager 5500 V2.03 software, and IDVs were calculated by Fluor Chen 2.0 software and normalized with that of β-actin.
4.5.
Determination of spinal cord water content
Water content was determined according to the previous protocol (Li and Tator, 1999). The segments of lumbar spinal cord for 72 h groups after reperfusion and BK group were weighed on aluminum foil, dried at 100 °C for 24 h, and reweighed. The percent water content was calculated as the following calculation: water content (%) = [(wet weigh − dry weight) / wet weight] × 100%.
4.6.
Statistical analysis
Results were presented as the mean ± SD. One-way analyses of variance (ANOVA) were used to compare group differences in measurement of AQP4. Dunnett's post hoc tests were applied to compare specific group difference if the ANOVA revealed a significant difference. In other measurements, the data were assessed using paired Student's t test. P < 0.05 was considered statistically significant.
Acknowledgments This work was supported by Special fund for scientific research of postdoctoral subjects in China, no. 20060400292. Natural Science Foundation of Liaoning Province in China, no. 20052096. REFERENCES
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