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Temporal changes in expression of aquaporin3, -4, -5 and -8 in rat brains after permanent focal cerebral ischemia
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Research Report
Temporal changes in expression of aquaporin3, -4, -5 and -8 in rat brains after permanent focal cerebral ischemia Mei Yang a , Fei Gao b,1 , Hui Liu a,1 , Wei Hua Yu a,1 , Shan Quan Sun a,⁎ a
Institute of Neuroscience, Chongqing Medical University, Chongqing 400016, People's Republic of China Department of Urology, The First Affiliated Hospital of Chongqing Medical University, Chongqing 400016, People's Republic of China
b
A R T I C LE I N FO
AB S T R A C T
Article history:
Morbidity and mortality in stroke are attributed to cerebral edema. To date, six aquaporins
Accepted 8 July 2009
(AQPs) have been found in rat brains. Whereas studies have been focused on AQP1, -4 and -9,
Available online 16 July 2009
little is known about the expression of AQP3, -5 and -8. To clarify roles of AQP3, -5 and -8 in water movement, we examined the expression patterns of AQP3, -5 and -8 in ischemic
Keywords:
brains from the rats with permanent middle cerebral artery occlusion (pMCAO). We also
Aquaporin
investigated the expression of AQP4 after ischemia, which was used as a positive control.
Brain edema
We found that the expression of AQP4 increased continuously until 24 h after pMCAO in
Cerebral ischemia
both the ischemic core and the border region. The increased expression was correlated with
Astrocytes
brain swelling, whereas the expression of AQP3, -5 and -8 continued to increase until 24 h
Neurons
after pMCAO in the border region but decreased 6 h after pMCAO in the ischemic core. We also found AQPs were colocalized with GFAP-positive astrocytes and/or NeuN-positive neurons in rat brains. This is the first study describing the expression of AQP3, -5 and -8 in rat brains subjected to pMCAO. Our findings indicated that dynamic changes of AQP3, -4, -5 and -8 expression could contribute to the development of cerebral edema after brain ischemia. Besides, AQP3, -5 and -8 may be involved in the neuronal swelling. © 2009 Elsevier B.V. All rights reserved.
1.
Introduction
Brain edema is a leading cause of death after stroke, which disturbs homeostasis in the neuronal microenvironment and consequently impairs neuronal functions. In addition, brain edema increases intracranial pressure, which could impair vascular perfusion and further aggravate the edema. However, the mechanisms underlying brain edema are unclear. The discovery of aquaporins (AQPs) adds a new understanding of the brain water movement (Preston and Agre, 1991). To date, six aquaporins have been found in the brains, AQP1, -3, -4, -5, -8 and -9 (Yamamoto et al., 2001; Agre et al., 2002; King et al., 2004; Morishita et al., 2004; Verkman, 2005). It has been shown
that AQP1 is expressed in the choroid plexus and facilitates cerebrospinal fluid secretion (Speake et al., 2003). AQP4 and AQP9 have been implicated in the pathogenesis of brain edema by virtue of their variations under injury conditions (Badaut et al., 2001; Badaut et al., 2002; Ribeiro et al., 2006), however, the roles of AQP4 in the regulation of cerebral edema are controversial. Papadopoulos and Verkman have shown that reduced edema in AQP4 knock-out mice occurs after MCAO (Papadopoulos and Verkman, 2007), whereas a more recent work showing an upregulation of AQP4 after preconditioning correlating with attenuated edema (Hirt et al., 2009). Nevertheless, little is known about the functions of other AQPs such as AQP3, -5 and -8. Thus, we hypothesized that AQP3, -5
⁎ Corresponding author. Fax: +86 23 68485868. E-mail address: [email protected] (S.Q. Sun). 1 These authors contributed equally to this study. 0006-8993/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2009.07.018
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Fig. 1 – Changes of the brain water content of ischemic hemisphere at 1 h , 3 h , 6 h , 12 h and 24 h after pMCAO. * indicates p < 0.05 compared with sham-operated group. and -8 also played roles in the pathogenesis of brain edema in response to pMCAO. To clarify the expression patterns of AQP3, -4, -5 and -8 after pMCAO and to get a better understanding of the molecular mechanisms of brain edema formation, we examined the expression of AQP3, -4, -5 and -8 in the adult rat brains after pMCAO.
2.
Results
2.1. Brain water content in ischemic hemisphere was increased within 24 h in the rats subjected to pMCAO To examine whether pMCAO induces brain swelling, we quantified brain swelling by measurement of brain water content (BWC). We found that a rise in BWC on the ipsilateral side of the brains started at 1 hour (h) after pMCAO, and continued to increase with time and persisted until 24 h after pMCAO (Fig. 1). Moreover, an increase in BWC was time-dependent (Fig. 1). However, no significant difference of BWC was found on the contralateral side (Data not shown).
2.2.
Morphological examination of infarction after pMCAO
TTC staining and HE staining were used to evaluate the infarction after pMCAO. After focal cerebral ischemia, brain slices were divided into ischemic core and border regions in
the ipsilateral hemispheres according to the TTC staining (Fig. 2A) and HE staining (Fig. 2B). TTC, a colorless salt, was converted to red formazan product in the presence of a functioning mitochondrial electron transport chain. Thus, normal brain tissue was dyed to be bright red, while the infarct region appeared white. In addition, HE staining showed the morphological characteristic of neurons from the sham control, ischemic core and border region. In the brains of sham-operated rats, the morphology of the cells appeared intact. While 1 h after pMCAO, some cells became swollen, most cells in the ischemic core appeared shrunken 6 h after pMCAO, and 24 h after pMCAO, brain tissue became loose and most cells were broken.
2.3. Temporal changes of expression of AQPs mRNA in the brains after pMCAO To examine changes of expression of AQPs mRNA after pMCAO, we used Real-time RT-PCR to detect mRNA level. In general, we found that mRNA of AQP3, -4, -5 and -8 was expressed in sham-operated tissues and the ischemic brain tissues. Expression of mRNA of all AQPs in the ischemic core and the border region was significantly increased, compared with the sham-operated brain tissues (Fig. 3). Moreover, in the border region, the levels of AQP3, -4, -5 and -8 mRNA were all increased with the extension of ischemic time (Fig. 3—2A, B, CD). In the ischemic core region, AQP4 mRNA was increased from 1 h until 24 h after pMCAO (Fig. 3—1B). However, the levels of AQP3, -5 and -8 mRNA started to decrease 12 h and 24 h after the elevated expression reached their peaks at 6 h (Fig. 3—1A, CD). Besides, the expression of AQPs mRNA was not significantly different between the contralateral side (Data not shown) and the sham-operated brain tissues at different time points examined. In addition, we also found that the correlation between the level of AQP4 mRNA and BWC occurred both in the ischemic core (r = 0.701, n = 6, p < 0.05) and the border region (r = 0.677, n = 6, p < 0.05); the correlation between the level of AQP3, -5 and -8 mRNA and BWC occurred in the border region (AQP3: r = 0.624; AQP5 r = 0.702; AQP8 r = 0.663, n = 5, p < 0.05) and in the ischemic core during the earlier stage (AQP3: r = 0.631; AQP5 r = 0.746; AQP8 r = 0.638, n = 5, p < 0.05).
Fig. 2 – Cerebral infarction produced by pMCAO were detected by TTC staining and HE staining, respectively. (A) A representative image showing TTC staining of a brain section, which was divided into infarct core (I) in striatum and border zone (II) around cortex area in the ipsilateral hemisphere from the rat 6 h after pMCAO (B) A representative image showing HE staining of a brain section. Scale Bar: 150 μm.
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Fig. 3 – Temporal changes of expression of AQPs mRNA in ischemic core and border regions in ischemic and control hemisphere samples at 1, 3, 6, 12 and 24 h after pMCAO. Real-time PCR was used to detect mRNA level. The expression of AQP mRNA in ischemic core for figure 3 (1A–D) and the expression of AQP mRNA in border region for figure 3 (2A–D). A, B, C and D represent mRNA levels of AQP3, AQP4, AQP5 and AQP8, respectively. # indicates significantly different from the sham-operated group. * indicates significantly different from other ischemic time points.
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2.4. Temporal changes of expression of AQPs protein in the brains after pMCAO To examine the distribution of AQPs protein in brains from the rats subjected to pMCAO, we used immunohistochemical staining method to detect the location of AQPs in the control
and ischemic brains. AQP4 was expressed in the subpial processes of astrocytes, nucleus of olfactory, piriform cortex, ependyma, dentate gyrus in hippocampus, dorsal thalamus, subfornical organ, suprachiasmatic nucleus (SCN), supraoptic nucleus (SON) and white matter in normal brains (Fig. 4A). After ischemia, AQP4 labeling was not only markedly found in
Fig. 4 – (A) A representative image showing AQP4 immunoreactivity was expressed in subfornical organ from a normal brain. The sections were counterstained by BCIP/NBT. Scale Bar: 150 μm. (B) AQP4 immunoreactivity was expressed in subfornical organ of ischemic brain at 12 h after pMCAO. Scale Bar 300 μm. (C–E) Colabeling of AQP4 and GFAP in the choroid plexus of the lateral ventricle in contralateral hemisphere from ischemic brain at 12 h after pMCAO. C: AQP4 immunoreactivity (green) was present in choroid plexus. (D) GFAP immunoreactivity was present in choroid plexus. (E) AQP4 was co-localization with GFAP. F–H Colabeling of AQP4 and GFAP in the choroid plexus of the lateral ventricle in ipsilateral hemisphere from ischemic brain at 12 h after pMCAO. (F) AQP4 immunoreactivity (green) was present in choroid plexus. (G) GFAP immunoreactivity was present in choroid plexus. (H) AQP4 was co-localization with GFAP. Scale Bar: 75 μm in E, also applied to C and D; 75 μm in H, also applied to F and G.
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the ischemic hemisphere, but also on the contralateral side (Figs. 4C, D, E). Furthermore, the distribution of AQP4 expanded 12 h after pMCAO, AQP4 immunoreactivity was also found in choroid plexus (Figs. 4F, G, H). The increase in optical density of AQP4 immunoreactivity in the ischemic core and the border region began 1 h after pMCAO, continued to increase with occlusion time, and reached the highest level at 24 h (Figs. 4B, 8B). AQP3, -5 and -8 were found to be expressed in similar regions after pMCAO. AQP3, -5 and -8 immunoreactivity were found in the piriform cortex, hippocampus, dorsal thalamus, globus pallidus (GP), and choroid plexus (Figs. 6A, 7D–F). After ischemia, the levels of AQP3, -5 and -8 labeling were not only markedly induced in the ischemic hemisphere, but also on the contralateral side. In addition, the distribution of expression of AQP3, -5 and -8 expanded. Three hours after MCAO, the AQP3, -5 and -8 immunoreactivities could also been found in the subpial processes of astrocytes, dentate gyrus, ependyma, paraventricular nucleus, nucleus caudatus putamen (cp) and lateral hypothalamic nucleus. Moreover, the double-labeling immunofluorescence results showed that AQP3, -4, -5 and -8 labeling was colocalized with GFAP-positive profiles and with the neuro-
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nal marker NeuN (Figs. 5–7). The increase of AQP3, -5 and -8 labeling was observed both in neurons and astrocytes (Figs. 5–7). Optical density results showed that the expression of AQP3, -5 and -8 increased and continued with occlusion extension in the border region. However, AQP3, -5 and -8 increased rapidly in the earlier stage after ischemia onset and reached their peaks at 6 h after pMCAO, then decreased in the ischemic core region (Figs. 8A, C, D). To further quantitative analysis of the expression of AQPs protein, we used western blotting to detect levels of AQP3, -4, -5 and -8 proteins in both the sham-operated and ischemic brain specimens. The expression of AQPs protein was not significantly different on the contralateral side and the sham-operated brain tissues at different time point examined (p > 0.05, Data not shown). A correlation between the level of AQPs protein and mRNA was observed (AQP3: r = 0.671; AQP4: r = 0.826; AQP5 r = 0.752; AQP8 r = 0.858, n = 5, p < 0.05). In addition, a correlation between the level of AQP4 protein and BWC was observed in both the ischemic core region (r = 0.871, n = 5, p < 0.05) and the border region (r = 0.908, n = 5, p < 0.05). The correlation between the level of AQP3, -5 and -8 protein and BWC occurred in the border
Fig. 5 – (A–C) Colocalization of AQP3 with GFAP in caudatus putamen (CP) of ischemic hemisphere 6 h after pMCAO. (A) AQP3 immunoreactivity was present in CP. (B) GFAP-positive cells was found in CP. (C) AQP3 was seen to be co-localized with GFAP. (D–F) Colabeling of AQP3 with NeuN in the Gp of ischemic hemisphere at 6 h after MCAO. (D) AQP3 immunoreactivity was present in Gp. (E) NeuN immunoreactivity was present in Gp. (F) A merged image of AQP3 and NeuN. Scale Bar: 75 μm in C, also applied to A and B; 75 μm in F, also applied to D and E.
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Fig. 6 – (A) A representative image showing AQP5 immunoreactivity was expressed in the choroid plexus (arrow) of a normal brain. The section was counterstained by BCIP/NBT. Scale Bar: 150 μm. (B–D) Colocalization of AQP5 with GFAP in the CP of ischemic hemisphere 12 h after MCAO. (B) AQP5 immunoreactivity was present in CP. (C) GFAP immunoreactivity was present in CP. (D) Some AQP5-positive cells were co-localized with GFAP-positive cells (Arrow). Scale Bar: 75 μm in D also applied to B and C. (E–G) The GP of ischemic hemisphere 24 h after MCAO. (E) AQP5 immunoreactivity was present in GP. (F) NeuN immunoreactivity was present in GP. (G) AQP5 was co-localization with NeuN. Scale bar: 75 μm in G also applied to E and F.
region (AQP3: r = 0.591; AQP5 r = 0.647; AQP8 r = 0.529, n = 5, p < 0.05) and in the ischemic core during the earlier stage (AQP3: r = 0.630; AQP5 r = 0.588; AQP8 r = 0.746, n = 5, p < 0.05). Compared with the sham-operated brain tissues, the expression of AQPs protein in ischemic brain tissues was stronger (Fig. 4C–H). The levels of AQP4 protein in the ischemic core region and the border region were increased significantly with time after pMCAO (Fig. 9A and C). A similar phenomenon was observed in the expression of
AQP3, -5 and -8 protein in the border region (Fig. 9D, the bands showing the expression of AQP8 by western blotting were chosen as representative bands). However, the levels of AQP3, -5 and -8 increased rapidly and reached their peaks at 6 h , then decreased in ischemic core region (Fig. 9B). In summary, an increase in AQP4 expression coincided with the swelling of the ischemic hemisphere both in the ischemic core region and the border region, whereas expression of AQP3, -5 and -8 increased rapidly after ischemia and
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Fig. 7 – (A–C) Colocalization of AQP8 and GFAP in the nucleus caudatus putamen (cp) of the ischemic hemisphere 1 h after MCAO. (A) AQP8 immunoreactivity was present in CP. (B) GFAP immunoreactivity was present in CP. (C) Some AQP8-positive cells were co-localized with GFAP-positive cells. (D–F) Colocalization of AQP8 with NeuN in globus pallidus (GP) from a normal brain. Scale Bars 75 μm. (D) AQP8 immunoreactivity was present in normal GP. (E) NeuN immunoreactivity was present in normal GP. (F) AQP8 was co-localization with NeuN in normal GP. (G–I) Colocalization of AQP8 with NeuN in GP 24 h after pMCAO. (G) AQP8 immunoreactivity was present in GP. (H) NeuN immunoreactivity was present in GP. (I) AQP8 was co-localization with NeuN in GP. Scale bar in C, F and I 75 μm also applied to A and B, D and E, G and H.
reached their peaks at 6 h after pMCAO and then decreased in the ischemic core region.
3.
Discussion
Up to now, six aquaporin subtypes (AQP1, -3, -4, -5, -8 and -9) were found in the rodent brain. However, most of the data
came from studies on expression of AQP1, -4 and -9 (Frigeri et al., 1995a,b; Nielsen et al., 1995; Masseguin et al., 2000; Badran and Hermo, 2002; Aoki et al., 2003; Hasegawa et al., 2003; Kobayashi et al., 2006; Lee et al., 2008). Here, for the first time we have shown the expression of AQP-3, -5 and -8 in ischemic brains, which displayed both spatial and temporal variations after pMCAO. Up-regulation of AQP4 was clearly correlated with the period of brain swelling, and the time course of AQP3,
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Fig. 8 – Optical density of the expression of AQPs protein in normal brains and in the ischemic core from the ischemic brains at 1, 3, 6, 12, 24 h after pMCAO. A, B, C and D represent AQP3, AQP4, AQP5 and AQP8, respectively. IHC: immunohistochemistry. OD: optical density. IMF: immunofluorescence.
-5 and -8 expression was different from that of AQP4 in the ischemic core region.
3.1.
Expression of AQP4 after MCAO
Variations of AQP4 expression have been reported in injured spinal cords (Frydenlund et al., 2006; Nesic et al., 2006; Yamamoto et al., 2001). Here, we showed that pMCAO induced an increased expression of AQP4 in ischemic brains, and the expression was well correlated with the ischemic hemisphere swelling. Our finding suggests that AQP4 could be a major water channel participating in water movements after pMCAO. The increased AQP4 level could facilitate water diffusion through plasma membrane. Nevertheless, previous studies have shown that AQP4 expression decreased in the ischemic core after transient MCAO and under in vitro hypoxic conditions (Frydenlund et al., 2006; Yamamoto et al., 2001). The discrepancy could be related to the differences in the species (mouse vs. rat), the types of ischemia model (transient MCAO vs. permanent MCAO) and the conditions of ischemia (in vivo vs. in vitro).
3.2.
Expression of AQP3, -5 and -8 after MCAO
The profiles of expression of AQP3, -5 and -8 closely resembled each other in the ischemic core after pMCAO, however, their
expressions were quite different from AQP4, indicating that AQP3, -5 and -8 could cooperate with each other and mediate common functions. We have shown here that the distribution of AQP3, -5 and -8 expanded (Fig. 4) and the levels of AQP3, -5 and -8 expressions all increased continuously within the first 6 h after ischemia onset, and their expressions were found in both astrocytes and neurons. The maximum expression was observed in the ischemic core at 6 h after MCAO, followed by a decrease in the expression of AQP3, -5 and -8, but the expression was still higher than that in sham-operated group. It has been shown that edema begins to develop within the first hour of ischemia (Chen et al. 2005), and we showed here that an elevated expression of AQP3, -5 and -8 within the first 6 h of ischemia, suggesting that AQP3, -5 and -8 contribute to the early development of edema. The strongest rise in edema is between 1 h and 6 h , which correlates well with the strong increase in AQP3, -5 and -8 expression at first 6 h . While AQP4 expression is still strongest elevated between the hours 6 and 24; however, the edema formation is slowed during this time period. Therefore, our findings suggested that all AQPs investigated in the study could contribute to the development of edema.
3.3.
The possible functions of AQP3, -5 and -8
It has been demonstrated that differential cellular swelling occurs after brain injury (Garcia et al., 1994; Garcia et al.,
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Fig. 9 – Western blotting analysis of levels of AQP4 and AQP8 protein in ischemic core and border regions. (A–B) Western blotting analysis of levels of for AQP4 (A) and AQP8 (B) protein in the ischemic core region at 1, 3, 6, 12 and 24 h after pMCAO. (C–D) Western blotting analysis of levels of AQP4 (C) and AQP8 (D) protein at 1, 3, 6, 12 and 24 h after pMCAO in the ischemic border region. # indicates significantly different from the sham-operated group. * indicates significantly different from other ischemic time points.
1995; Dodson et al., 1997). Astrocytes swell first when brain cells are subjected to ischemia, and their end-feet are swollen within 5 min after an energy supply is interrupted. Astrocyte swelling persists for 24 h after MCAO, and then begins to undergo necrosis. While neurons seem to swell later but shrink earlier than astrocytes. This type accounts for 79% 6 h after ischemia in the ischemic hemisphere of rats. After 12 h to 24 h , most neurons (65%) are necrotic, whereas the proportion of swollen or shrunken neurons decreases to 21% (Garcia et al., 1995). Based on these findings showing the pathophysiological cause of cell swelling in the brains, it is possible that neurons are more susceptible than astrocytes under brain injury. Thus, 12 h after ischemia, neurons became necrotic. The levels of AQP3, -5 and -8 expression coincided with the swelling of the neurons in the ischemic core region, suggesting that the AQP3, -5 and -8 may provide key routes for water movement in neurons after pMCAO.
In summary, we have shown here the spatial and temporal profiles of AQP3, -4, -5 and -8 in ischemic brains after pMCAO. The levels of AQP4 correlated with brain swelling, suggesting that AQP4 could be a major route for astrocyte water movements after pMCAO. However, differential expression patterns of AQP3, -5, -8 and AQP4 were observed, suggesting that AQP3, -5 and -8 could be implicated in other functions in addition to brain edema formation. The upregulation of expression of AQP3, -5 and -8 after pMCAO in reactive neurons indicates that AQP3, -5 and -8 may play key roles in water homeostasis in neurons. Meanwhile, upregulation of AQP3, -5 and -8 was observed in astrocytes both in the ischemic core and the border region, suggesting these three AQPs could contribute to AQP4 in the edema formation after ischemia. Time-dependent difference in the progression of the AQPs suggests that AQPs play different roles after pMCAO. Inhibition of AQP3, -5 and -8 may rescue the neurons in the earliest phase of ischemia, whereas inhibition of AQP4 may decrease brain edema.
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4.
Experimental procedures
4.1.
Animals
Male Wistar rats, 10 weeks of age (body weight, 250 to 280 g), were obtained from the Animal Breeding Facility, Chongqing Medical University. All procedures complied with the recommendations of the National Institute of Health Guide for the Care and Use of Laboratory Animals (NIH Publications No. 80-23). Male adult Wistar rats were randomly divided into two groups, the ischemic group (n = 71) and the sham-operated group (n = 74). The animals were anesthetized using an intraperitoneal injection of chloral hydrate (350 mg/kg). 4.2.
Permanent middle cerebral artery occlusion
Permanent middle cerebral artery occlusion was induced in the male rats as previously described (Li et al., 2007). Briefly, this was, the surgical insertion of a nylon monofilament (40) through the external carotid artery into the internal carotid artery and advancing it. This resulted in occlusion of the ostium of the ipsilateral MCA. The length of the filament inserted into the vessel was adjusted according to body weight and varied from 18 to 20 mm. Further details of the surgical procedure were in accordance with methods published elsewhere, each rat had been fasted overnight and anesthetized with a mixture of halothane and nitrous oxide before inserting a nylon filament through the external carotid artery. MCA was occluded by inserting a filament into the internal carotid artery, which was advanced further until it closed the origin of the MCA. Rectal temperature was controlled and maintained at 36.5 °C with a temperature unit and a heating pad during the anesthesia period with heated fixed temperature operating table (Harvard Apparatus, USA). The animals were sacrificed at 1 h (n = 15), 3 h (n = 13), 6 h (n = 16), 12 h (n = 15), or 24 h (n = 12) after MCAO. The operation procedure of the sham-operated was the same as the ischemic group but without occluding unilateral MCA. For Real-time PCR and Western blot, tissues were sampled from the core of the ischemia including the striatal core and the overlying neocortex (the “central part of the ischemic cortex”). Samples were also collected from the neocortical border region (the neocortical border zone, defined as the most peripheral zone of the affected cortex and with distinct paler than the core) as previously described (Frydenlund et al., 2006). The contralateral side was also collected as an internal reference. 4.3.
Bain water content
Brain water content (BWC) was measured by using wet-dry weighing ratio method. The rats were anesthetized by an intraperitoneal injection of chloral hydrate (350 mg/kg). The brains were removed and divided into ischemic and contralateral hemispheres. The brain samples were weighed immediately after dissection (wet weight) and then dried in vacuum oven at 120 °C for 48 h . The dried brain was re-weighted. The percent of water content was calculated as ([wet weight − dry weight] / wet weight) × 100%.
4.4. Evaluation of infarct zone by TTC staining and H&E staining Rats were deeply anesthetized and sacrificed at 1 h , 3 h , 6 h , 12 h , or 24 h after MCAO. The brains were removed rapidly and sliced coronally at 2-mm intervals. All slices were incubated in 2% 2,3,5-triphenyltetrazolium chloride (TTC) (TaKaRa, China) at 37 °C for 20 min, then fixed in 4% paraformaldehyde solution for 24 h . The undamaged brain tissue appeared bright red, whereas the infarct zones appeared white or pale. For HE staining, the sections were prepared as the immunohistochemistry. Fixed tissues were dehydrated in graded alcohol and embedded in paraffin. Sections were stained with hematoxylin and eosin (HE) for general histology. 4.5.
Reverse transcription real-time PCR
Total RNA from tissue specimens was isolated using Tissue/cell RNA Mini kit at the different time points after pMCAO. The cDNAs were generated from 1 μg of total RNA by superscript II RNase H− reverse transcriptase with Oligo (dT) primer. Quantity of AQP mRNA levels was done by realtime PCR using Taqman probe. The primers were given in Table 1. All primer pairs were designed from rat AQP mRNA sequences retrieved from GenBank. The beta-actin primer sets were included as house-keeping control genes. Reactions were carried out in 20 μl volumes consisting of Premix Ex Taq (2×) 10 μl, PCR Forward Primer (10 μm) 0.4 μl, PCR Reverse Primer (10 μm) 0.4 μl, Taqman probe (20 μm) 0.8 μl, Rox reference Dye (50×) 0.4 μl, cDNA 2 μl and ddH2O 6 μl (TaKaRa, China). Each run consisted of serial dilution (10×) of standard preparation and rat cDNA samples to generate a standard curve. In each reaction, 2 μl cDNA was amplified. The amplification program was as follows: preincubation at 95 °C for 10 s (s), fast start polymerase action at 95 °C for 5 s, followed by 60 °C for 31 s. Taqman probe fluorescence was acquired at 60 °C in each amplification cycle. Changes in AQP mRNA expression were examined with ABI7000 Sequence Detection System (Perkin Elmer, USA). A standard curve was used to extrapolate the copy number of target cDNA in rat brain. 4.6. Tissue fixation, immunohistochemistry, and immunofluorescence Anesthetized rats were perfused transcardially with 4% paraformaldehyde in phosphate-buffered saline (PBS). Then brains were removed and immersed in a fixative solution overnight. Brains were further stored in PBS containing 30% sucrose for 48 h at 4 °C for cryoprotection. Brains were then sliced (10 μm thickness). Sections were blocked for 1 h at room temperature (RT) with normal goat serum, and then were incubated overnight at 4 °C with primary antibody dilution (1:400, rabbit anti-AQP, Alpha Diagnostic International, USA) in Tris buffered saline (TBS) plus 1% bovine serum albumin. After rinsing with TBS plus 0.1% Tween 20 (TTBS), tissues were incubated for 1 h at room temperature (RT) with secondary goat anti-rabbit IgG biotinylated antibody (SantaCruz, USA). After rinsing with TTBS, the tissues were
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Table 1 – Gene-specific Real-time PCR primers, TaqMan probes and their respective PCR fragment lengths.
β-actin AQP3 AQP4 AQP5 AQP8
Forward primer
Reverse primer
TaqMan-probe
Length (bp)
5′-CCCTGGCTCCTAGCACCAT-3′ 5′-TGCTGGGATTGTTTTTGGG-3′ 5′-CTGCAGTTATCAATGGGAACTGG-3′ 5′-GCCACATCAATCCAGCCATT-3′ 5′-GTTCATGCAGGCTCCAGAGAT-3′
5′-CACAGAGTACTTGCGCTCAGGA-3′ 5′-GCCGGAGACAACAAGCTCAT-3′ 5′-GCGCCTATGATTGGTCCAAC-3′ 5′-GGAGCAGCGAGATCTGGTTT-3′ 5′-CCATTGGTTTCTCTGTGTCATTGTG-3′
5′-FAAGATCAAGATCATTGCTCP-3′ 5′-FCTACTATGATGCAATCTGP-3′ 5′-FAAACCACTGGATATATTGP-3′ 5′-FTCTGGCCCTCTTAATAGP-3′ 5′-FCCACCACCTGCCAGGAP-3′
186 73 64 60 65
incubated in streptavidin–horseradish-peroxidase complexes for 1 h at RT. Tissues were further washed with TTBS, and color development was achieved by incubation for 15 min in BCIP/NBT (SantaCruz, USA). The reaction was stopped by washing tissue with tap water, followed by distilled water. Tissues were observed under an OLYMPUS labophot-2 microscope with white light illumination (Japan). For immunofluorescence, tissues were blocked with 10% goat serum and incubated with an antibody against AQPs (1:400, the source and the concentration of all AQP antibodies were the same. rabbit anti-AQP, Alpha Diagnostic International) and with an antibody against GFAP (astrocyte marker, 1:500, mouse anti-GFAP, Santa Cruz, USA) or NeuN (neuronal marker, 1:500, mouse anti-NeuN, Santa Cruz, USA). Immunofluorescence was revealed using FITC-labeled fragments of goat anti-rabbit IgG plus TRITClabeled fragments of goat anti-mouse IgG (Santa Cruz, USA). Tissue sections were mounted in 50% glycerol dissolved in PBS. Samples were observed under a confocal microscope equipped with argon and helio/neon lasers (Leica TCS SP2, Wetzlar, Germany). Controls were performed by omitting either one or both primary antibodies. All controls gave negative results with no detectable labeling. 4.7.
Quantification of immunoreactivity for AQPs
Immunoreactivity for AQPs was quantified in three different fields in the sham-operated brains and the ischemic and the contralateral hemispheres of ischemic brains. Optic density (OD) was measured using image analysis program (Leica TCS SP2, Wetzlar, Germany). A ratio between the OD in ischemic hemisphere and OD in contralateral hemisphere was calculated from a mean of three fields per section. These were cortex, GP and CP. We selected 5 sections from each brain and 5 brains in each group. 4.8.
Western blot analysis
Tissue specimens were homogenized at 4 °C in a homogenization buffer containing 50 mM ethylenediamine tetraacetic acid (EDTA), 2 μg/ml leupeptin, 2 μg/ml pepstatin A, 2 mM phenylmethysufonyl fluoride (PMSF), and 200 KIE/ml aprotinin. The homogenates were then centrifuged at 10,000 ×g for 20 min at 4 °C. The supernatant was collected and protein concentration was determined by a Bradford assay kit (Bio-Rad, Hercules, USA). Samples were separated by 12% SDS-PAGE gel and then transferred onto PVDF membranes. Blotted membranes were blocked with 5% skim milk and then incubated with primary antibodies against AQPs (1:500, rabbit anti-AQP, Alpha Diagnostic International, USA). Thereafter, they were incubated with
horse radish peroxidase (HRP)-conjugated second antibodies (Santa Cruz, USA). The optical densities of AQPs and betaactin bands were quantitatively analyzed with gel densitometry (Bio-Rad, Hercules, USA). The results were expressed as AQP/beta-actin. (Each point was repeated in triplicate.) The specific reaction was visualized by using a chemiluminescent substrate (Pierce, USA). We quantified western bands by gel densitometry (Bio-Rad, Hercules, USA). We divided the value of individual AQP protein band by the value for beta-actin for the same sample, a ratio of protein: beta-actin for each sample was obtained (Each point was repeated in triplicate). Bands were normalized with the beta-actin loading control, and each group was normalized to the ratio of the corresponding ischemia-matched sham control for analysis. 4.9.
Statistical analysis
All statistics were performed using the SPSS 11.0 software package (Chicago, IL, USA). Correlations were compared using analysis of bivariance. The level of AQPs protein expression and AQPs mRNA expression for specimens were expressed as means ± SD. Differences between individual groups were first compared using analysis of variance (one-way ANOVA); thereafter, data were analyzed with LSD multiple comparisons post hoc testing. All reported p values were two-sided, and a value of p < 0.05 was considered statistically significant. REFERENCES
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Dodson, R.F., Chu, L.W., Welch, K.M., Achar, V.S., 1977. Acute tissue response to cerebral ischemia in the gerbil: an ultrastructural study. J. Neurol. Sci. 33, 161–170. Frigeri, A., Gropper, M.A., Umenishi, F., Kawashima, M., Brown, D., Verkman, A.S., 1995a. Localization of MIWC and GLIP water channel homologs in neuromuscular, epithelial and glandular tissues. J. Cell Sci. 108, 2993–3002. Frigeri, A., Gropper, M.A., Turck, C.W., Verkman, A.S., 1995b. Immunolocalization of the mercurial-insensitive water channel and glycerol intrinsic protein in epithelial cell plasma membranes. Proc. Natl. Acad. Sci. U. S. A. 92, 4328–4331. Frydenlund, D.S., Bhardwaj, A., Otsuka, T., Mylonakou, M.N., Yasumura, T., Davidson, K.G., Zeynalov, E., Skare, O., Laake, P., Haug, F.M., Rash, J.E., Agre, P., Ottersen, O.P., Amiry-Moghaddam, M., 2006. Temporary loss of perivascular aquaporin-4 in neocortex after transient middle cerebral artery occlusion in mice. Proc. Natl. Acad. Sci. U. S. A. 103, 13526–13532. Garcia, J.H., Liu, K.F., Yoshida, Y., Chen, S., Lian, J., 1994. Brain microvessels: factors altering their patency after the occlusion of a middle cerebral artery (Wistar rat). Am. J. Pathol 145, 728–740. Garcia, J.H., Liu, K.F., Ho, K.L., 1995. Neuronal necrosis after middle cerebral artery occlusion in Wistar rats progress at different time intervals in the caudoputamen and the cortex. Stroke 26, 636–643. Hasegawa, T., Tanii, H., Suzuki, M., Tanaka, S., 2003. Regulation of water absorption in the frog skins by two vasotocin-dependent water-channel aquaporins, AQP-h2 and AQP-h3. Endocrinology 144, 4087–4096. Hirt, L., Ternon, B., Price, M., Mastour, N., Brunet, J.F., Badaut, J., 2009. Protective role of early aquaporin 4 induction against postischemic edema formation. J. Cereb. Blood Flow Metab. 29, 423–433. King, L.S., Kozono, D., Agre, P., 2004. From structure to disease: the evolving tale of aquaporin biology. Nat. Rev., Mol. Cell Biol. 5, 687–698. Kobayashi, H., Yokoo, H., Yanagita, T., Satoh, S., Kis, B., Deli, M., Niwa, M., Wada, A., 2006. Induction of aquaporin 1 by dexamethasone in lipid rafts in immortalized brain microvascular endothelial cells. Brain Res. 1123, 12–19. Lee, M., Lee, S.J., Choi, H.J., Jung, Y.W., Frøkiaer, J., Nielsen, S., Kwon, T.H., 2008. Regulation of AQP4 protein expression in rat
brain astrocytes: role of P2X7 receptor activation. Brain Res. 1195, 1–11. Li, Y., Xu, Z., Ford, G.D., Croslan, D.R., Cairobe, T., Li, Z., Ford, B.D., 2007. Neuroprotection by neuregulin-1 in a rat model of permanent focal cerebral ischemia. Brain Res. 1184, 277–283. Masseguin, C., Corcoran, M., Carcenac, C., Daunton, N.G., Güell, A., Verkman, A.S., Gabrion, J., 2000. Altered gravity downregulates aquaporin-1 protein expression in choroid plexus. J. Appl. Physiol. 88, 843–850. Morishita, Y., Sakube, Y., Sasaki, S., Wada, A., 2004. Molecular mechanisms and drug development in aquaporin water channel diseases: aquaporin superfamily (superaquaporins): expansion of aquaporins restricted to multicellular organisms. J. Pharmacol. Sci. 96, 276–279. Nesic, O., Lee, J., Ye, Z., Unabia, G.C., Rafati, D., Hulsebosch, C.E., Perez-polo, J.R., 2006. Acute and chronic changes in Aquaporin 4 expression after spinal cord injury. Neuroscience 143, 779–792. Nielsen, S., Pallone, T., Smith, B.L., Christensen, E.I., Agre, P., Maunsbach, A.B., 1995. Aquaporin-1 water channels in short and long loop descending thin limbs and in descending vasa recta in rat kidney. Am. J. Physiol. 268, F1023–F1037. Papadopoulos, M.C., Verkman, A.S., 2007. Aquaporin-4 and brain edema. Pediatr. Nephrol. 2007 (22), 778–784. Preston, G.M., Agre, P., 1991. Isolation of the cDNA for erythrocyte integral membrane protein of 28 kilodaltons: member of an ancient channel family. Proc. Natl. Acad. Sci. U. S. A. 88, 11110–11114. Ribeiro Mde, C., Hirt, L., Bogousslavsky, J., Regli, L., Badautl, J., 2006. Time course aquaporin expression after transient focal cerebral ischemia in mice. J. Neurosci. Res. 83, 1231–1240. Speake, T., Freeman, L.J., Brown, P.D., 2003. Expression of aquaporin 1 and aquaporin 4 water channels in rat choroid plexus. Biochim. Biophys. Acta 1609, 80–86. Verkman, A.S., 2005. Novel roles of aquaporins revealed by phenotype analysis of knockout mice. Rev. Physiol. Biochem. Pharmacol. 155, 31–55. Yamamoto, N., Kazuhiro, Y., Kiyofumi, A., Sobue, K., Tada, T., Fujita, Y., Katsuya, H., Fujita, M., Aihara, N., Mase, M., Yamada, K., Miura, Y., Kato, T., 2001. Alterations in the expression of the AQP family in cultured rat astrocytes during hypoxia and reoxygenation. Mole Brain Res. 90, 26–38.
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
Temporal changes in expression of aquaporin3, -4, -5 and -8 in rat brains after permanent focal cerebral ischemia Mei Yang a , Fei Gao b,1 , Hui Liu a,1 , Wei Hua Yu a,1 , Shan Quan Sun a,⁎ a
Institute of Neuroscience, Chongqing Medical University, Chongqing 400016, People's Republic of China Department of Urology, The First Affiliated Hospital of Chongqing Medical University, Chongqing 400016, People's Republic of China
b
A R T I C LE I N FO
AB S T R A C T
Article history:
Morbidity and mortality in stroke are attributed to cerebral edema. To date, six aquaporins
Accepted 8 July 2009
(AQPs) have been found in rat brains. Whereas studies have been focused on AQP1, -4 and -9,
Available online 16 July 2009
little is known about the expression of AQP3, -5 and -8. To clarify roles of AQP3, -5 and -8 in water movement, we examined the expression patterns of AQP3, -5 and -8 in ischemic
Keywords:
brains from the rats with permanent middle cerebral artery occlusion (pMCAO). We also
Aquaporin
investigated the expression of AQP4 after ischemia, which was used as a positive control.
Brain edema
We found that the expression of AQP4 increased continuously until 24 h after pMCAO in
Cerebral ischemia
both the ischemic core and the border region. The increased expression was correlated with
Astrocytes
brain swelling, whereas the expression of AQP3, -5 and -8 continued to increase until 24 h
Neurons
after pMCAO in the border region but decreased 6 h after pMCAO in the ischemic core. We also found AQPs were colocalized with GFAP-positive astrocytes and/or NeuN-positive neurons in rat brains. This is the first study describing the expression of AQP3, -5 and -8 in rat brains subjected to pMCAO. Our findings indicated that dynamic changes of AQP3, -4, -5 and -8 expression could contribute to the development of cerebral edema after brain ischemia. Besides, AQP3, -5 and -8 may be involved in the neuronal swelling. © 2009 Elsevier B.V. All rights reserved.
1.
Introduction
Brain edema is a leading cause of death after stroke, which disturbs homeostasis in the neuronal microenvironment and consequently impairs neuronal functions. In addition, brain edema increases intracranial pressure, which could impair vascular perfusion and further aggravate the edema. However, the mechanisms underlying brain edema are unclear. The discovery of aquaporins (AQPs) adds a new understanding of the brain water movement (Preston and Agre, 1991). To date, six aquaporins have been found in the brains, AQP1, -3, -4, -5, -8 and -9 (Yamamoto et al., 2001; Agre et al., 2002; King et al., 2004; Morishita et al., 2004; Verkman, 2005). It has been shown
that AQP1 is expressed in the choroid plexus and facilitates cerebrospinal fluid secretion (Speake et al., 2003). AQP4 and AQP9 have been implicated in the pathogenesis of brain edema by virtue of their variations under injury conditions (Badaut et al., 2001; Badaut et al., 2002; Ribeiro et al., 2006), however, the roles of AQP4 in the regulation of cerebral edema are controversial. Papadopoulos and Verkman have shown that reduced edema in AQP4 knock-out mice occurs after MCAO (Papadopoulos and Verkman, 2007), whereas a more recent work showing an upregulation of AQP4 after preconditioning correlating with attenuated edema (Hirt et al., 2009). Nevertheless, little is known about the functions of other AQPs such as AQP3, -5 and -8. Thus, we hypothesized that AQP3, -5
⁎ Corresponding author. Fax: +86 23 68485868. E-mail address: [email protected] (S.Q. Sun). 1 These authors contributed equally to this study. 0006-8993/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2009.07.018
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Fig. 1 – Changes of the brain water content of ischemic hemisphere at 1 h , 3 h , 6 h , 12 h and 24 h after pMCAO. * indicates p < 0.05 compared with sham-operated group. and -8 also played roles in the pathogenesis of brain edema in response to pMCAO. To clarify the expression patterns of AQP3, -4, -5 and -8 after pMCAO and to get a better understanding of the molecular mechanisms of brain edema formation, we examined the expression of AQP3, -4, -5 and -8 in the adult rat brains after pMCAO.
2.
Results
2.1. Brain water content in ischemic hemisphere was increased within 24 h in the rats subjected to pMCAO To examine whether pMCAO induces brain swelling, we quantified brain swelling by measurement of brain water content (BWC). We found that a rise in BWC on the ipsilateral side of the brains started at 1 hour (h) after pMCAO, and continued to increase with time and persisted until 24 h after pMCAO (Fig. 1). Moreover, an increase in BWC was time-dependent (Fig. 1). However, no significant difference of BWC was found on the contralateral side (Data not shown).
2.2.
Morphological examination of infarction after pMCAO
TTC staining and HE staining were used to evaluate the infarction after pMCAO. After focal cerebral ischemia, brain slices were divided into ischemic core and border regions in
the ipsilateral hemispheres according to the TTC staining (Fig. 2A) and HE staining (Fig. 2B). TTC, a colorless salt, was converted to red formazan product in the presence of a functioning mitochondrial electron transport chain. Thus, normal brain tissue was dyed to be bright red, while the infarct region appeared white. In addition, HE staining showed the morphological characteristic of neurons from the sham control, ischemic core and border region. In the brains of sham-operated rats, the morphology of the cells appeared intact. While 1 h after pMCAO, some cells became swollen, most cells in the ischemic core appeared shrunken 6 h after pMCAO, and 24 h after pMCAO, brain tissue became loose and most cells were broken.
2.3. Temporal changes of expression of AQPs mRNA in the brains after pMCAO To examine changes of expression of AQPs mRNA after pMCAO, we used Real-time RT-PCR to detect mRNA level. In general, we found that mRNA of AQP3, -4, -5 and -8 was expressed in sham-operated tissues and the ischemic brain tissues. Expression of mRNA of all AQPs in the ischemic core and the border region was significantly increased, compared with the sham-operated brain tissues (Fig. 3). Moreover, in the border region, the levels of AQP3, -4, -5 and -8 mRNA were all increased with the extension of ischemic time (Fig. 3—2A, B, CD). In the ischemic core region, AQP4 mRNA was increased from 1 h until 24 h after pMCAO (Fig. 3—1B). However, the levels of AQP3, -5 and -8 mRNA started to decrease 12 h and 24 h after the elevated expression reached their peaks at 6 h (Fig. 3—1A, CD). Besides, the expression of AQPs mRNA was not significantly different between the contralateral side (Data not shown) and the sham-operated brain tissues at different time points examined. In addition, we also found that the correlation between the level of AQP4 mRNA and BWC occurred both in the ischemic core (r = 0.701, n = 6, p < 0.05) and the border region (r = 0.677, n = 6, p < 0.05); the correlation between the level of AQP3, -5 and -8 mRNA and BWC occurred in the border region (AQP3: r = 0.624; AQP5 r = 0.702; AQP8 r = 0.663, n = 5, p < 0.05) and in the ischemic core during the earlier stage (AQP3: r = 0.631; AQP5 r = 0.746; AQP8 r = 0.638, n = 5, p < 0.05).
Fig. 2 – Cerebral infarction produced by pMCAO were detected by TTC staining and HE staining, respectively. (A) A representative image showing TTC staining of a brain section, which was divided into infarct core (I) in striatum and border zone (II) around cortex area in the ipsilateral hemisphere from the rat 6 h after pMCAO (B) A representative image showing HE staining of a brain section. Scale Bar: 150 μm.
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Fig. 3 – Temporal changes of expression of AQPs mRNA in ischemic core and border regions in ischemic and control hemisphere samples at 1, 3, 6, 12 and 24 h after pMCAO. Real-time PCR was used to detect mRNA level. The expression of AQP mRNA in ischemic core for figure 3 (1A–D) and the expression of AQP mRNA in border region for figure 3 (2A–D). A, B, C and D represent mRNA levels of AQP3, AQP4, AQP5 and AQP8, respectively. # indicates significantly different from the sham-operated group. * indicates significantly different from other ischemic time points.
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2.4. Temporal changes of expression of AQPs protein in the brains after pMCAO To examine the distribution of AQPs protein in brains from the rats subjected to pMCAO, we used immunohistochemical staining method to detect the location of AQPs in the control
and ischemic brains. AQP4 was expressed in the subpial processes of astrocytes, nucleus of olfactory, piriform cortex, ependyma, dentate gyrus in hippocampus, dorsal thalamus, subfornical organ, suprachiasmatic nucleus (SCN), supraoptic nucleus (SON) and white matter in normal brains (Fig. 4A). After ischemia, AQP4 labeling was not only markedly found in
Fig. 4 – (A) A representative image showing AQP4 immunoreactivity was expressed in subfornical organ from a normal brain. The sections were counterstained by BCIP/NBT. Scale Bar: 150 μm. (B) AQP4 immunoreactivity was expressed in subfornical organ of ischemic brain at 12 h after pMCAO. Scale Bar 300 μm. (C–E) Colabeling of AQP4 and GFAP in the choroid plexus of the lateral ventricle in contralateral hemisphere from ischemic brain at 12 h after pMCAO. C: AQP4 immunoreactivity (green) was present in choroid plexus. (D) GFAP immunoreactivity was present in choroid plexus. (E) AQP4 was co-localization with GFAP. F–H Colabeling of AQP4 and GFAP in the choroid plexus of the lateral ventricle in ipsilateral hemisphere from ischemic brain at 12 h after pMCAO. (F) AQP4 immunoreactivity (green) was present in choroid plexus. (G) GFAP immunoreactivity was present in choroid plexus. (H) AQP4 was co-localization with GFAP. Scale Bar: 75 μm in E, also applied to C and D; 75 μm in H, also applied to F and G.
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the ischemic hemisphere, but also on the contralateral side (Figs. 4C, D, E). Furthermore, the distribution of AQP4 expanded 12 h after pMCAO, AQP4 immunoreactivity was also found in choroid plexus (Figs. 4F, G, H). The increase in optical density of AQP4 immunoreactivity in the ischemic core and the border region began 1 h after pMCAO, continued to increase with occlusion time, and reached the highest level at 24 h (Figs. 4B, 8B). AQP3, -5 and -8 were found to be expressed in similar regions after pMCAO. AQP3, -5 and -8 immunoreactivity were found in the piriform cortex, hippocampus, dorsal thalamus, globus pallidus (GP), and choroid plexus (Figs. 6A, 7D–F). After ischemia, the levels of AQP3, -5 and -8 labeling were not only markedly induced in the ischemic hemisphere, but also on the contralateral side. In addition, the distribution of expression of AQP3, -5 and -8 expanded. Three hours after MCAO, the AQP3, -5 and -8 immunoreactivities could also been found in the subpial processes of astrocytes, dentate gyrus, ependyma, paraventricular nucleus, nucleus caudatus putamen (cp) and lateral hypothalamic nucleus. Moreover, the double-labeling immunofluorescence results showed that AQP3, -4, -5 and -8 labeling was colocalized with GFAP-positive profiles and with the neuro-
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nal marker NeuN (Figs. 5–7). The increase of AQP3, -5 and -8 labeling was observed both in neurons and astrocytes (Figs. 5–7). Optical density results showed that the expression of AQP3, -5 and -8 increased and continued with occlusion extension in the border region. However, AQP3, -5 and -8 increased rapidly in the earlier stage after ischemia onset and reached their peaks at 6 h after pMCAO, then decreased in the ischemic core region (Figs. 8A, C, D). To further quantitative analysis of the expression of AQPs protein, we used western blotting to detect levels of AQP3, -4, -5 and -8 proteins in both the sham-operated and ischemic brain specimens. The expression of AQPs protein was not significantly different on the contralateral side and the sham-operated brain tissues at different time point examined (p > 0.05, Data not shown). A correlation between the level of AQPs protein and mRNA was observed (AQP3: r = 0.671; AQP4: r = 0.826; AQP5 r = 0.752; AQP8 r = 0.858, n = 5, p < 0.05). In addition, a correlation between the level of AQP4 protein and BWC was observed in both the ischemic core region (r = 0.871, n = 5, p < 0.05) and the border region (r = 0.908, n = 5, p < 0.05). The correlation between the level of AQP3, -5 and -8 protein and BWC occurred in the border
Fig. 5 – (A–C) Colocalization of AQP3 with GFAP in caudatus putamen (CP) of ischemic hemisphere 6 h after pMCAO. (A) AQP3 immunoreactivity was present in CP. (B) GFAP-positive cells was found in CP. (C) AQP3 was seen to be co-localized with GFAP. (D–F) Colabeling of AQP3 with NeuN in the Gp of ischemic hemisphere at 6 h after MCAO. (D) AQP3 immunoreactivity was present in Gp. (E) NeuN immunoreactivity was present in Gp. (F) A merged image of AQP3 and NeuN. Scale Bar: 75 μm in C, also applied to A and B; 75 μm in F, also applied to D and E.
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Fig. 6 – (A) A representative image showing AQP5 immunoreactivity was expressed in the choroid plexus (arrow) of a normal brain. The section was counterstained by BCIP/NBT. Scale Bar: 150 μm. (B–D) Colocalization of AQP5 with GFAP in the CP of ischemic hemisphere 12 h after MCAO. (B) AQP5 immunoreactivity was present in CP. (C) GFAP immunoreactivity was present in CP. (D) Some AQP5-positive cells were co-localized with GFAP-positive cells (Arrow). Scale Bar: 75 μm in D also applied to B and C. (E–G) The GP of ischemic hemisphere 24 h after MCAO. (E) AQP5 immunoreactivity was present in GP. (F) NeuN immunoreactivity was present in GP. (G) AQP5 was co-localization with NeuN. Scale bar: 75 μm in G also applied to E and F.
region (AQP3: r = 0.591; AQP5 r = 0.647; AQP8 r = 0.529, n = 5, p < 0.05) and in the ischemic core during the earlier stage (AQP3: r = 0.630; AQP5 r = 0.588; AQP8 r = 0.746, n = 5, p < 0.05). Compared with the sham-operated brain tissues, the expression of AQPs protein in ischemic brain tissues was stronger (Fig. 4C–H). The levels of AQP4 protein in the ischemic core region and the border region were increased significantly with time after pMCAO (Fig. 9A and C). A similar phenomenon was observed in the expression of
AQP3, -5 and -8 protein in the border region (Fig. 9D, the bands showing the expression of AQP8 by western blotting were chosen as representative bands). However, the levels of AQP3, -5 and -8 increased rapidly and reached their peaks at 6 h , then decreased in ischemic core region (Fig. 9B). In summary, an increase in AQP4 expression coincided with the swelling of the ischemic hemisphere both in the ischemic core region and the border region, whereas expression of AQP3, -5 and -8 increased rapidly after ischemia and
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Fig. 7 – (A–C) Colocalization of AQP8 and GFAP in the nucleus caudatus putamen (cp) of the ischemic hemisphere 1 h after MCAO. (A) AQP8 immunoreactivity was present in CP. (B) GFAP immunoreactivity was present in CP. (C) Some AQP8-positive cells were co-localized with GFAP-positive cells. (D–F) Colocalization of AQP8 with NeuN in globus pallidus (GP) from a normal brain. Scale Bars 75 μm. (D) AQP8 immunoreactivity was present in normal GP. (E) NeuN immunoreactivity was present in normal GP. (F) AQP8 was co-localization with NeuN in normal GP. (G–I) Colocalization of AQP8 with NeuN in GP 24 h after pMCAO. (G) AQP8 immunoreactivity was present in GP. (H) NeuN immunoreactivity was present in GP. (I) AQP8 was co-localization with NeuN in GP. Scale bar in C, F and I 75 μm also applied to A and B, D and E, G and H.
reached their peaks at 6 h after pMCAO and then decreased in the ischemic core region.
3.
Discussion
Up to now, six aquaporin subtypes (AQP1, -3, -4, -5, -8 and -9) were found in the rodent brain. However, most of the data
came from studies on expression of AQP1, -4 and -9 (Frigeri et al., 1995a,b; Nielsen et al., 1995; Masseguin et al., 2000; Badran and Hermo, 2002; Aoki et al., 2003; Hasegawa et al., 2003; Kobayashi et al., 2006; Lee et al., 2008). Here, for the first time we have shown the expression of AQP-3, -5 and -8 in ischemic brains, which displayed both spatial and temporal variations after pMCAO. Up-regulation of AQP4 was clearly correlated with the period of brain swelling, and the time course of AQP3,
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Fig. 8 – Optical density of the expression of AQPs protein in normal brains and in the ischemic core from the ischemic brains at 1, 3, 6, 12, 24 h after pMCAO. A, B, C and D represent AQP3, AQP4, AQP5 and AQP8, respectively. IHC: immunohistochemistry. OD: optical density. IMF: immunofluorescence.
-5 and -8 expression was different from that of AQP4 in the ischemic core region.
3.1.
Expression of AQP4 after MCAO
Variations of AQP4 expression have been reported in injured spinal cords (Frydenlund et al., 2006; Nesic et al., 2006; Yamamoto et al., 2001). Here, we showed that pMCAO induced an increased expression of AQP4 in ischemic brains, and the expression was well correlated with the ischemic hemisphere swelling. Our finding suggests that AQP4 could be a major water channel participating in water movements after pMCAO. The increased AQP4 level could facilitate water diffusion through plasma membrane. Nevertheless, previous studies have shown that AQP4 expression decreased in the ischemic core after transient MCAO and under in vitro hypoxic conditions (Frydenlund et al., 2006; Yamamoto et al., 2001). The discrepancy could be related to the differences in the species (mouse vs. rat), the types of ischemia model (transient MCAO vs. permanent MCAO) and the conditions of ischemia (in vivo vs. in vitro).
3.2.
Expression of AQP3, -5 and -8 after MCAO
The profiles of expression of AQP3, -5 and -8 closely resembled each other in the ischemic core after pMCAO, however, their
expressions were quite different from AQP4, indicating that AQP3, -5 and -8 could cooperate with each other and mediate common functions. We have shown here that the distribution of AQP3, -5 and -8 expanded (Fig. 4) and the levels of AQP3, -5 and -8 expressions all increased continuously within the first 6 h after ischemia onset, and their expressions were found in both astrocytes and neurons. The maximum expression was observed in the ischemic core at 6 h after MCAO, followed by a decrease in the expression of AQP3, -5 and -8, but the expression was still higher than that in sham-operated group. It has been shown that edema begins to develop within the first hour of ischemia (Chen et al. 2005), and we showed here that an elevated expression of AQP3, -5 and -8 within the first 6 h of ischemia, suggesting that AQP3, -5 and -8 contribute to the early development of edema. The strongest rise in edema is between 1 h and 6 h , which correlates well with the strong increase in AQP3, -5 and -8 expression at first 6 h . While AQP4 expression is still strongest elevated between the hours 6 and 24; however, the edema formation is slowed during this time period. Therefore, our findings suggested that all AQPs investigated in the study could contribute to the development of edema.
3.3.
The possible functions of AQP3, -5 and -8
It has been demonstrated that differential cellular swelling occurs after brain injury (Garcia et al., 1994; Garcia et al.,
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Fig. 9 – Western blotting analysis of levels of AQP4 and AQP8 protein in ischemic core and border regions. (A–B) Western blotting analysis of levels of for AQP4 (A) and AQP8 (B) protein in the ischemic core region at 1, 3, 6, 12 and 24 h after pMCAO. (C–D) Western blotting analysis of levels of AQP4 (C) and AQP8 (D) protein at 1, 3, 6, 12 and 24 h after pMCAO in the ischemic border region. # indicates significantly different from the sham-operated group. * indicates significantly different from other ischemic time points.
1995; Dodson et al., 1997). Astrocytes swell first when brain cells are subjected to ischemia, and their end-feet are swollen within 5 min after an energy supply is interrupted. Astrocyte swelling persists for 24 h after MCAO, and then begins to undergo necrosis. While neurons seem to swell later but shrink earlier than astrocytes. This type accounts for 79% 6 h after ischemia in the ischemic hemisphere of rats. After 12 h to 24 h , most neurons (65%) are necrotic, whereas the proportion of swollen or shrunken neurons decreases to 21% (Garcia et al., 1995). Based on these findings showing the pathophysiological cause of cell swelling in the brains, it is possible that neurons are more susceptible than astrocytes under brain injury. Thus, 12 h after ischemia, neurons became necrotic. The levels of AQP3, -5 and -8 expression coincided with the swelling of the neurons in the ischemic core region, suggesting that the AQP3, -5 and -8 may provide key routes for water movement in neurons after pMCAO.
In summary, we have shown here the spatial and temporal profiles of AQP3, -4, -5 and -8 in ischemic brains after pMCAO. The levels of AQP4 correlated with brain swelling, suggesting that AQP4 could be a major route for astrocyte water movements after pMCAO. However, differential expression patterns of AQP3, -5, -8 and AQP4 were observed, suggesting that AQP3, -5 and -8 could be implicated in other functions in addition to brain edema formation. The upregulation of expression of AQP3, -5 and -8 after pMCAO in reactive neurons indicates that AQP3, -5 and -8 may play key roles in water homeostasis in neurons. Meanwhile, upregulation of AQP3, -5 and -8 was observed in astrocytes both in the ischemic core and the border region, suggesting these three AQPs could contribute to AQP4 in the edema formation after ischemia. Time-dependent difference in the progression of the AQPs suggests that AQPs play different roles after pMCAO. Inhibition of AQP3, -5 and -8 may rescue the neurons in the earliest phase of ischemia, whereas inhibition of AQP4 may decrease brain edema.
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4.
Experimental procedures
4.1.
Animals
Male Wistar rats, 10 weeks of age (body weight, 250 to 280 g), were obtained from the Animal Breeding Facility, Chongqing Medical University. All procedures complied with the recommendations of the National Institute of Health Guide for the Care and Use of Laboratory Animals (NIH Publications No. 80-23). Male adult Wistar rats were randomly divided into two groups, the ischemic group (n = 71) and the sham-operated group (n = 74). The animals were anesthetized using an intraperitoneal injection of chloral hydrate (350 mg/kg). 4.2.
Permanent middle cerebral artery occlusion
Permanent middle cerebral artery occlusion was induced in the male rats as previously described (Li et al., 2007). Briefly, this was, the surgical insertion of a nylon monofilament (40) through the external carotid artery into the internal carotid artery and advancing it. This resulted in occlusion of the ostium of the ipsilateral MCA. The length of the filament inserted into the vessel was adjusted according to body weight and varied from 18 to 20 mm. Further details of the surgical procedure were in accordance with methods published elsewhere, each rat had been fasted overnight and anesthetized with a mixture of halothane and nitrous oxide before inserting a nylon filament through the external carotid artery. MCA was occluded by inserting a filament into the internal carotid artery, which was advanced further until it closed the origin of the MCA. Rectal temperature was controlled and maintained at 36.5 °C with a temperature unit and a heating pad during the anesthesia period with heated fixed temperature operating table (Harvard Apparatus, USA). The animals were sacrificed at 1 h (n = 15), 3 h (n = 13), 6 h (n = 16), 12 h (n = 15), or 24 h (n = 12) after MCAO. The operation procedure of the sham-operated was the same as the ischemic group but without occluding unilateral MCA. For Real-time PCR and Western blot, tissues were sampled from the core of the ischemia including the striatal core and the overlying neocortex (the “central part of the ischemic cortex”). Samples were also collected from the neocortical border region (the neocortical border zone, defined as the most peripheral zone of the affected cortex and with distinct paler than the core) as previously described (Frydenlund et al., 2006). The contralateral side was also collected as an internal reference. 4.3.
Bain water content
Brain water content (BWC) was measured by using wet-dry weighing ratio method. The rats were anesthetized by an intraperitoneal injection of chloral hydrate (350 mg/kg). The brains were removed and divided into ischemic and contralateral hemispheres. The brain samples were weighed immediately after dissection (wet weight) and then dried in vacuum oven at 120 °C for 48 h . The dried brain was re-weighted. The percent of water content was calculated as ([wet weight − dry weight] / wet weight) × 100%.
4.4. Evaluation of infarct zone by TTC staining and H&E staining Rats were deeply anesthetized and sacrificed at 1 h , 3 h , 6 h , 12 h , or 24 h after MCAO. The brains were removed rapidly and sliced coronally at 2-mm intervals. All slices were incubated in 2% 2,3,5-triphenyltetrazolium chloride (TTC) (TaKaRa, China) at 37 °C for 20 min, then fixed in 4% paraformaldehyde solution for 24 h . The undamaged brain tissue appeared bright red, whereas the infarct zones appeared white or pale. For HE staining, the sections were prepared as the immunohistochemistry. Fixed tissues were dehydrated in graded alcohol and embedded in paraffin. Sections were stained with hematoxylin and eosin (HE) for general histology. 4.5.
Reverse transcription real-time PCR
Total RNA from tissue specimens was isolated using Tissue/cell RNA Mini kit at the different time points after pMCAO. The cDNAs were generated from 1 μg of total RNA by superscript II RNase H− reverse transcriptase with Oligo (dT) primer. Quantity of AQP mRNA levels was done by realtime PCR using Taqman probe. The primers were given in Table 1. All primer pairs were designed from rat AQP mRNA sequences retrieved from GenBank. The beta-actin primer sets were included as house-keeping control genes. Reactions were carried out in 20 μl volumes consisting of Premix Ex Taq (2×) 10 μl, PCR Forward Primer (10 μm) 0.4 μl, PCR Reverse Primer (10 μm) 0.4 μl, Taqman probe (20 μm) 0.8 μl, Rox reference Dye (50×) 0.4 μl, cDNA 2 μl and ddH2O 6 μl (TaKaRa, China). Each run consisted of serial dilution (10×) of standard preparation and rat cDNA samples to generate a standard curve. In each reaction, 2 μl cDNA was amplified. The amplification program was as follows: preincubation at 95 °C for 10 s (s), fast start polymerase action at 95 °C for 5 s, followed by 60 °C for 31 s. Taqman probe fluorescence was acquired at 60 °C in each amplification cycle. Changes in AQP mRNA expression were examined with ABI7000 Sequence Detection System (Perkin Elmer, USA). A standard curve was used to extrapolate the copy number of target cDNA in rat brain. 4.6. Tissue fixation, immunohistochemistry, and immunofluorescence Anesthetized rats were perfused transcardially with 4% paraformaldehyde in phosphate-buffered saline (PBS). Then brains were removed and immersed in a fixative solution overnight. Brains were further stored in PBS containing 30% sucrose for 48 h at 4 °C for cryoprotection. Brains were then sliced (10 μm thickness). Sections were blocked for 1 h at room temperature (RT) with normal goat serum, and then were incubated overnight at 4 °C with primary antibody dilution (1:400, rabbit anti-AQP, Alpha Diagnostic International, USA) in Tris buffered saline (TBS) plus 1% bovine serum albumin. After rinsing with TBS plus 0.1% Tween 20 (TTBS), tissues were incubated for 1 h at room temperature (RT) with secondary goat anti-rabbit IgG biotinylated antibody (SantaCruz, USA). After rinsing with TTBS, the tissues were
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Table 1 – Gene-specific Real-time PCR primers, TaqMan probes and their respective PCR fragment lengths.
β-actin AQP3 AQP4 AQP5 AQP8
Forward primer
Reverse primer
TaqMan-probe
Length (bp)
5′-CCCTGGCTCCTAGCACCAT-3′ 5′-TGCTGGGATTGTTTTTGGG-3′ 5′-CTGCAGTTATCAATGGGAACTGG-3′ 5′-GCCACATCAATCCAGCCATT-3′ 5′-GTTCATGCAGGCTCCAGAGAT-3′
5′-CACAGAGTACTTGCGCTCAGGA-3′ 5′-GCCGGAGACAACAAGCTCAT-3′ 5′-GCGCCTATGATTGGTCCAAC-3′ 5′-GGAGCAGCGAGATCTGGTTT-3′ 5′-CCATTGGTTTCTCTGTGTCATTGTG-3′
5′-FAAGATCAAGATCATTGCTCP-3′ 5′-FCTACTATGATGCAATCTGP-3′ 5′-FAAACCACTGGATATATTGP-3′ 5′-FTCTGGCCCTCTTAATAGP-3′ 5′-FCCACCACCTGCCAGGAP-3′
186 73 64 60 65
incubated in streptavidin–horseradish-peroxidase complexes for 1 h at RT. Tissues were further washed with TTBS, and color development was achieved by incubation for 15 min in BCIP/NBT (SantaCruz, USA). The reaction was stopped by washing tissue with tap water, followed by distilled water. Tissues were observed under an OLYMPUS labophot-2 microscope with white light illumination (Japan). For immunofluorescence, tissues were blocked with 10% goat serum and incubated with an antibody against AQPs (1:400, the source and the concentration of all AQP antibodies were the same. rabbit anti-AQP, Alpha Diagnostic International) and with an antibody against GFAP (astrocyte marker, 1:500, mouse anti-GFAP, Santa Cruz, USA) or NeuN (neuronal marker, 1:500, mouse anti-NeuN, Santa Cruz, USA). Immunofluorescence was revealed using FITC-labeled fragments of goat anti-rabbit IgG plus TRITClabeled fragments of goat anti-mouse IgG (Santa Cruz, USA). Tissue sections were mounted in 50% glycerol dissolved in PBS. Samples were observed under a confocal microscope equipped with argon and helio/neon lasers (Leica TCS SP2, Wetzlar, Germany). Controls were performed by omitting either one or both primary antibodies. All controls gave negative results with no detectable labeling. 4.7.
Quantification of immunoreactivity for AQPs
Immunoreactivity for AQPs was quantified in three different fields in the sham-operated brains and the ischemic and the contralateral hemispheres of ischemic brains. Optic density (OD) was measured using image analysis program (Leica TCS SP2, Wetzlar, Germany). A ratio between the OD in ischemic hemisphere and OD in contralateral hemisphere was calculated from a mean of three fields per section. These were cortex, GP and CP. We selected 5 sections from each brain and 5 brains in each group. 4.8.
Western blot analysis
Tissue specimens were homogenized at 4 °C in a homogenization buffer containing 50 mM ethylenediamine tetraacetic acid (EDTA), 2 μg/ml leupeptin, 2 μg/ml pepstatin A, 2 mM phenylmethysufonyl fluoride (PMSF), and 200 KIE/ml aprotinin. The homogenates were then centrifuged at 10,000 ×g for 20 min at 4 °C. The supernatant was collected and protein concentration was determined by a Bradford assay kit (Bio-Rad, Hercules, USA). Samples were separated by 12% SDS-PAGE gel and then transferred onto PVDF membranes. Blotted membranes were blocked with 5% skim milk and then incubated with primary antibodies against AQPs (1:500, rabbit anti-AQP, Alpha Diagnostic International, USA). Thereafter, they were incubated with
horse radish peroxidase (HRP)-conjugated second antibodies (Santa Cruz, USA). The optical densities of AQPs and betaactin bands were quantitatively analyzed with gel densitometry (Bio-Rad, Hercules, USA). The results were expressed as AQP/beta-actin. (Each point was repeated in triplicate.) The specific reaction was visualized by using a chemiluminescent substrate (Pierce, USA). We quantified western bands by gel densitometry (Bio-Rad, Hercules, USA). We divided the value of individual AQP protein band by the value for beta-actin for the same sample, a ratio of protein: beta-actin for each sample was obtained (Each point was repeated in triplicate). Bands were normalized with the beta-actin loading control, and each group was normalized to the ratio of the corresponding ischemia-matched sham control for analysis. 4.9.
Statistical analysis
All statistics were performed using the SPSS 11.0 software package (Chicago, IL, USA). Correlations were compared using analysis of bivariance. The level of AQPs protein expression and AQPs mRNA expression for specimens were expressed as means ± SD. Differences between individual groups were first compared using analysis of variance (one-way ANOVA); thereafter, data were analyzed with LSD multiple comparisons post hoc testing. All reported p values were two-sided, and a value of p < 0.05 was considered statistically significant. REFERENCES
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