Original Paper Received: April 16, 2014 Accepted after revision: July 21, 2014 Published online: October 18, 2014
Dev Neurosci 2014;36:542–551 DOI: 10.1159/000366058
Altered Expression of Aquaporin 1 and 5 in the Choroid Plexus following Preterm Intraventricular Hemorrhage Snjolaug Sveinsdottir a Magnus Gram a, b Magnus Cinthio c Kristbjörg Sveinsdottir a Matthias Mörgelin b David Ley a
Department of Pediatrics, b Division of Infection Medicine and c Department of Electrical Measurements, Lund University, Lund, Sweden
Key Words Intraventricular hemorrhage · Preterm birth · Hydrocephalus · Posthemorrhagic ventricular dilatation · Aquaporin · Choroid plexus
Abstract Intraventricular hemorrhage (IVH) with posthemorrhagic ventricular dilatation (PHVD) is a common cause of hydrocephalus in infants. Dysregulation of cerebrospinal fluid (CSF) production by the choroid plexus may contribute to the development of PHVD. The aquaporins (AQPs), transmural water transporting proteins, are believed to contribute to CSF production. The aim of the study was to characterize the expression and localization of AQP1, 4 and 5 in the choroid plexus following preterm IVH. Using a preterm rabbit pup model, the mRNA expression, protein level and localization of AQP1, 4 and 5 were investigated in the choroid plexus at 24 and 72 h following IVH with PHVD. Further, AQP1, 4 and 5 expression were characterized in primary human plexus epithelial cells exposed to CSF from preterm human infants with IVH and to hemoglobin metabolites. IVH with PHVD in the immature brain caused a downregulation of AQP1 mRNA, the key AQP in CSF production, but an upregulation of AQP1 protein level with apical epithelial cell localization. Notably, AQP5 was expressed in the choroid plexus with upregulated mRNA expression and protein levels during PHVD
© 2014 S. Karger AG, Basel 0378–5866/14/0366–0542$39.50/0 E-Mail [email protected] www.karger.com/dne
with apical epithelial cell localization. Analysis of human choroid plexus epithelial cells in vitro, following exposure to posthemorrhagic CSF and to hemin, displayed results concordant with those observed in vivo, i.e. downregulation of AQP1 mRNA and upregulation of AQP5 mRNA expression. AQP4 was neither detectable in vivo nor in vitro. The changes observed in AQP1 and AQP5 expression in the choroid plexus suggest an adaptive response following IVH with possible functional implications for the development of PHVD. © 2014 S. Karger AG, Basel
Introduction
Cerebral intraventricular hemorrhage (IVH) is observed in 15–20% of very preterm infants [1, 2] and is associated with a high mortality (20–50%). Up to 50% of the surviving infants develop posthemorrhagic ventricular dilatation (PHVD), which is the most common cause of hydrocephalus in children and is associated with poor neurodevelopmental outcome [3–5]. In order for the newborn brain to maintain normal function and maturation, a continuous equilibrium of ce-
Snjolaug Sveinsdottir and Magnus Gram contributed equally to this work.
Snjolaug Sveinsdottir Department of Pediatrics, Clinical Sciences Lund, Lund University SE–221 84 Lund (Sweden) E-Mail snjolaug.sveinsdottir @ med.lu.se
Downloaded by: University of Tokyo 157.82.153.40 - 5/19/2015 10:11:06 PM
a
rebrospinal fluid (CSF) volume and content is essential. However, following IVH and the development of PHVD there is a pathological accumulation and an altered composition of CSF in the ventricles. CSF is produced by the choroid plexus, which is a collection of villi composed of a single layer of highly specialized epithelial cells overlying a core of intercellular matrix and fenestrated capillaries. On the ventricular side of the epithelial cells there are a number of highly specialized transmembrane proteins that tightly regulate the composition of CSF regarding osmolarity, electrolyte and nutrient content [6]. Aquaporins (AQPs) are a family of channel proteins which facilitate the transport of water and solutes across membranes [7]. A number of AQPs have been described in the brain, e.g. AQP1, 3, 4, 5, 8 and 9 [8], and have been assigned to different cell types and physiological roles. AQP1 is described as the key AQP in the choroid plexus and has an important role as a water transporter in CSF production during both physiological and pathological conditions [8, 9]. However, there have been no studies on the effect of IVH with PHVD on AQP1 expression and localization in the choroid plexus. AQP5 has been described in rat brain astrocytes [10], but the role or presence of AQP5 in the choroid plexus has not been described. However, AQP5 is abundantly expressed in several other organs with secretory functions such as the lacrimal glands [11], salivary glands [12] and lungs [13]. The main function of AQP5 is to regulate water permeability [2], paracellular water transfer [14] and cell cytoskeleton organization and stabilization [15]. Furthermore, the expression of AQP5 in astrocytes is differentially regulated during metabolic and traumatic brain injury [10]. Consequently, characterizing the expression pattern of AQP5 in the choroid plexus following IVH with PHVD could be an important step towards understanding the development of hydrocephalus. AQP4 is one of the most abundant and well-studied AQPs in the brain and is mainly localized on the cytoplasmic membrane and end-feet of the astrocytes, where it plays a crucial role in regulating water flow into and out of the brain. AQP4 has been shown to be involved in brain swelling and edema following ischemia [16], and modulation of AQP4 expression is associated with improved functional outcome following cerebral hemorrhage [17]. Over time, there has been much speculation about AQP-targeted therapies in different forms of brain pathology. Although current research would suggest that this might be a feasible approach, it is of utmost importance to first understand the physiological role of AQPs in various areas and cell populations of the brain as well as changes in AQP expression after insult.
In this study we have characterized the expression and localization of AQP1, 4 and 5 in the choroid plexus following preterm IVH using a preterm rabbit pup model [18, 19]. The preterm rabbit pup exhibits a brain maturation corresponding to that of a human infant at 28–30 weeks of gestation [20], has a germinal matrix and develops spontaneous IVH [19]. Importantly, the hemorrhage is confined to the intraventricular space and results in a progressive ventricular enlargement very similar to that seen in preterm human infants [21]. In a recent study using this model and primary astrocyte cell cultures we showed that IVH is followed by a release of cell-free hemoglobin (Hb) in the intraventricular space and that accumulated Hb metabolites are causal inducers of inflammation [22]. In addition to the in vivo characterization of AQP1, 4 and 5, here we further investigated the effects of Hb metabolites on the expression of AQP1, 4 and 5 in primary human choroid plexus epithelial cells (HCPEpiC) and compared the response to that following exposure to posthemorrhagic CSF obtained from preterm human infants. We show, for the first time, that AQP5 is indeed expressed in the choroid plexus and that IVH with progressive PHVD is associated with an increased expression of AQP5 localized at the epithelial apical surface. Simultaneously, we observed a decrease in AQP1 mRNA expression following IVH. Corresponding changes in the expression of AQP1 and 5 were detected in HCPEpiC exposed to hemin and to posthemorrhagic CSF. We did not detect AQP4 in the choroid plexus or in the choroid plexus cell cultures, suggesting that AQP1 and 5 are key AQPs controlling the water flux across the choroid plexus barrier and in the production of CSF.
IVH Alters Aquaporin Expression and Distribution
Dev Neurosci 2014;36:542–551 DOI: 10.1159/000366058
Materials and Methods Animals The animal protocols were approved by the Swedish Animal Ethics Committee in Lund. The experiments were performed on a total of 39 rabbit pups from 10 litters delivered at gestational day 29 (term 32 days). A half-breed between New Zealand White and Lop was used. The pups were delivered by cesarean section after the does were anesthetized with propofol (5 mg/kg, i.v.) and with local infiltration of the abdominal wall using lidocaine with adrenaline (10 mg/ml + 5 μl/ml, 20–30 ml). After birth the pups were dried vigorously, weighed and placed in an infant incubator with a constant temperature of 36 ° C and 60% ambient humidity. At 2–3 h of age the pups were hand-fed with 1 ml of cat milk formula (KMR; PETAG Inc., USA) using a 3.5-Fr feeding tube and thereafter every 12 h, increasing each meal by 0.5 ml. At 2 h of age, the pups received an intraperitoneal injection of 50% glycerol (6.5 g/kg) to induce intracerebral hypotension due to hyperosmolality, thus causing rupture of the small vessels in the germinal matrix. Ultrasound imaging of the brain was performed at 6 h of age to
543
Downloaded by: University of Tokyo 157.82.153.40 - 5/19/2015 10:11:06 PM
3
2
a
PHVD 24 h
PHVD 72 h
Fig. 1. High-frequency ultrasound of normal brain, IVH and PHVD development. a Coronal images obtained by high-frequen-
cy ultrasound displaying a normal brain with no IVH and intraventricular IVH at 6 h and subsequent development of PHVD at 24 and 72 h of age in preterm rabbit pups. b Mean horizontal ventricular width at 6, 24, 48 and 72 h of age was determined by high-
detect and grade IVH and after that at 24, 48 and 72 h of age using the VisualSonics Vevo 2100 (VisualSonics Inc., Ont., Canada) with an MS-550D 40-MHz transducer (fig. 1a). Only animals with a large IVH at 6 h and with the development of PHVD at 24 and 72 h were used for data analysis; animals with no detectable IVH at all time points on cranial ultrasound were used as controls. All controls used in this study were glycerol-injected animals with no IVH as determined by cranial ultrasound. Measurements of ventricular size for the assessment of PHVD were obtained at the level of the midseptal nucleus in a coronal view at 6, 24, 48 and 72 h of age (fig. 1b). Each ventricle was measured horizontally from the midbrain plane to the lateral wall of the ventricle. The reproducibility and accuracy of ventricular measurements in this animal model using high-frequency ultrasound have been described previously [21].
*
4
0
6
24
b
48
72
Age (h)
frequency ultrasound in rabbit pup brains. Rabbit pups with IVH (black triangles) had higher mean measurements at all time points (p < 0.01) compared to pups with no IVH (control, black circles) at the same time points. Mean ventricular size was increased at 72 h compared to 24 h in rabbit pups with IVH. * p < 0.05. Vertical lines show means ± SD.
CSF Sampling from Preterm Infants CSF was sampled from preterm infants (gestational age at birth: 25–28 weeks) at 6–11 days after the detection of IVH by spinal tap or ventricular reservoir puncture according to clinical routine in the neonatal unit at Lund University Hospital. Immediately after sampling, the CSF was centrifuged (2,000 g, 20 ° C for 10 min) and stored at –80 ° C until further use, as described below. The sampled CSF was utilized for the study following written consent from the parents, and the study was approved by the ethical committee review board for studies in human subjects at Lund University, Sweden.
Preparation of Methemoglobin and Hemin Fetal Hb was purified as previously described [23] from freshly drawn human umbilical cord blood. Methemoglobin (metHb) was prepared by incubating the purified Hb solution at 37 ° C for 72 h. The metHb concentration was quantified as described previously [23]. Hemin (ferriprotoporphyrin IX chloride) was purchased from Porphyrin Products Inc. (Logan, Utah, USA), and a 10-mM stock solution was prepared using DMSO (Sigma). The metHb was purified from endotoxin contamination using the endotoxin-removing product EndoTrap (Hyglos GmbH, Germany) as described by the manufacturer. The absolute purity of metHb and hemin from contamination with endotoxin (0 EU/mg Hb/hemin) was determined using the QCL-1000TM Endpoint Chromogenic LAL Assay (Lonza, Switzerland) as described by the manufacturer.
Tissue Collection and Processing Rabbit pups were euthanized at 24 h (IVH + PHVD = 6, sham control = 6) and 72 h of age (IVH + PHVD = 9, sham control = 9), and the brains were removed from the skulls and sectioned at the level of the midseptal nucleus. The choroid plexus was carefully removed from the lateral ventricles, snap frozen and stored at –80 ° C until further mRNA and protein analysis as described below. For electron microscopy immunohistochemistry staining (EMIHC), the choroid plexus from 2 rabbit pups with IVH + PHVD and 2 sham controls at 72 h was fixed and prepared as described below. For IHC, perfusion fixation of brains was performed in 3 pups with IVH + PHVD and 2 sham controls under isoflurane anesthesia at 72 h of age by an infusion of saline solution followed by 4% paraformaldehyde in PBS. The perfused brains were then immersed in formaldehyde and prepared as described below.
544
Dev Neurosci 2014;36:542–551 DOI: 10.1159/000366058
Primary HCPEpiC HCPEpiC (ScienCell, Carlsbad, Calif., USA) were cultured in epithelial cell medium containing 2% fetal bovine serum, 1% epithelial cell growth supplement, 100 U/ml penicillin and 100 μg/ml
Sveinsdottir/Gram/Cinthio/Sveinsdottir/ Mörgelin/Ley
Downloaded by: University of Tokyo 157.82.153.40 - 5/19/2015 10:11:06 PM
IVH 6 h
Horizontal ventricular size (mm)
Control
IVH Normal
5
RNA Isolation and Real-Time PCR Total RNA was isolated from the choroid plexus and HCPEpiC cells using the acid guanidinium phenol chloroform method and RNeasy Mini Kit supplied by QIAGEN. The optical density ratio (optical density at 260 nm/280 nm) of RNA was always higher than 1.9. Reverse transcription was performed according to the manufacturer on 0.1–1 μg of total RNA using iScriptTM cDNA Synthesis Kit (Bio-Rad, Calif., USA) and RT2 First Strand Kit (QIAGEN). Real-time PCR was used to quantify the expression of AQP1, 4 and 5 mRNA. Data were normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The fold change values were calculated by normalizing against control samples from untreated animals or cells. RT2 PCR Profiler Array for analysis of HCPEpiC AQP1, 4 and 5 and GAPDH were purchased from QIAGEN. Primers for analysis of rabbit choroid plexus AQP1, 4 and 5 and GAPDH were designed accordingly, as follows: AQP1 forward primer 5′-GAT CGACTACACTGGCTGTGG-3′, reverse primer 5′-GGTTGT TGAAGTTGTGGGTGAG-3′; AQP4 forward primer 5′-CGTTTT AAAGAAGCCTTCAGCAA-3′, reverse primer 5′-CCTGTTG TCCTCCACCTCCAT-3′; AQP5 forward primer 5′-GGGCAA CCTGGCTGTCAA-3′, reverse primer 5′-AGCTGGAAGGTG AGGATCAACTC-3′, and GAPDH forward primer 5′-GAATC CACTGGCGTCTTCAC-3′, reverse primer 5′-CGTTGCTGAC AATCTTGAGAGA-3′. Expression was analyzed using the iTaq Universal SYBR Green Supermix (Bio-Rad) and RT2 SYBR Green Fluor qPCR Mastermix (QIAGEN). Amplification was performed at the respective adequate temperature for 40 cycles in an iCycler Thermal Cycler (Bio-Rad) and data were analyzed using iCycler iQ Optical System Software (Bio-Rad). Total Protein Analysis Total protein from the choroid plexus and HCPEpiC was determined by Pierce® BCA Protein Assay Kit (Thermo Scientific).
ml; Santa Cruz Biotechnology) and β-actin (monoclonal mouse anti-human β-actin; Abcam, Cambridge, UK). Western blot was performed using Alexa 647-conjugated secondary antibodies (Life Technologies). The bands were detected in a ChemiDoc XRS unit (Bio-Rad). The relative quantification of AQP1 and 5 bands were performed by densitometry using Image Lab software (Bio-Rad). Levels of AQP1were normalized against those of β-actin for respective samples. Immunohistochemistry IHC sections were deparaffinized by routine procedures and endogenous peroxidase activity was blocked with 3% H2O2 in methanol for 15 min. After washing with Triton-X-100 (0.25%) in PBS, the sections were blocked with normal goat serum (5%) for 1 h at room temperature. The slides were then incubated with AQP1 (1 μg/ml), AQP4 (1 ug/ml) and AQP5 (2 ug/ml) primary antibodies overnight at 4 ° C. Antibody detection was performed with a standard avidinbiotin complex detection system after which they were developed with 3,3-diaminobenzidine tetrahydrochloride as the chromogenic substrate (Vectastain avidin-biotin complex; Vector Laboratories, Burlingame, Calif., USA). The sections were mounted with Pertex (Histolab Products AB, Gothenburg, Sweden), examined and photographed (Olympus BHS photomicrographic system).
Transmission EM Immunolabeling of thin sections with gold-labeled anti-AQP1 and 5 were performed as described previously [24], with the modification that Aurion BSA was used as a blocking agent. Specimens were observed in a JEOL JEM 1230 electron microscope operated at an accelerating voltage of 80 kV. Images were recorded with a Gatan Multiscan 791 CCD camera. Statistics Comparisons between unrelated groups were performed with the unpaired Student t test or the Mann-Whitney U test as appropriate. Comparisons within groups were performed with the paired Student t test. Comparisons between multiple groups were performed by ANOVA with post hoc Bonferroni correction. p values
Dev Neurosci 2014;36:542–551 DOI: 10.1159/000366058
Altered Expression of Aquaporin 1 and 5 in the Choroid Plexus following Preterm Intraventricular Hemorrhage Snjolaug Sveinsdottir a Magnus Gram a, b Magnus Cinthio c Kristbjörg Sveinsdottir a Matthias Mörgelin b David Ley a
Department of Pediatrics, b Division of Infection Medicine and c Department of Electrical Measurements, Lund University, Lund, Sweden
Key Words Intraventricular hemorrhage · Preterm birth · Hydrocephalus · Posthemorrhagic ventricular dilatation · Aquaporin · Choroid plexus
Abstract Intraventricular hemorrhage (IVH) with posthemorrhagic ventricular dilatation (PHVD) is a common cause of hydrocephalus in infants. Dysregulation of cerebrospinal fluid (CSF) production by the choroid plexus may contribute to the development of PHVD. The aquaporins (AQPs), transmural water transporting proteins, are believed to contribute to CSF production. The aim of the study was to characterize the expression and localization of AQP1, 4 and 5 in the choroid plexus following preterm IVH. Using a preterm rabbit pup model, the mRNA expression, protein level and localization of AQP1, 4 and 5 were investigated in the choroid plexus at 24 and 72 h following IVH with PHVD. Further, AQP1, 4 and 5 expression were characterized in primary human plexus epithelial cells exposed to CSF from preterm human infants with IVH and to hemoglobin metabolites. IVH with PHVD in the immature brain caused a downregulation of AQP1 mRNA, the key AQP in CSF production, but an upregulation of AQP1 protein level with apical epithelial cell localization. Notably, AQP5 was expressed in the choroid plexus with upregulated mRNA expression and protein levels during PHVD
© 2014 S. Karger AG, Basel 0378–5866/14/0366–0542$39.50/0 E-Mail [email protected] www.karger.com/dne
with apical epithelial cell localization. Analysis of human choroid plexus epithelial cells in vitro, following exposure to posthemorrhagic CSF and to hemin, displayed results concordant with those observed in vivo, i.e. downregulation of AQP1 mRNA and upregulation of AQP5 mRNA expression. AQP4 was neither detectable in vivo nor in vitro. The changes observed in AQP1 and AQP5 expression in the choroid plexus suggest an adaptive response following IVH with possible functional implications for the development of PHVD. © 2014 S. Karger AG, Basel
Introduction
Cerebral intraventricular hemorrhage (IVH) is observed in 15–20% of very preterm infants [1, 2] and is associated with a high mortality (20–50%). Up to 50% of the surviving infants develop posthemorrhagic ventricular dilatation (PHVD), which is the most common cause of hydrocephalus in children and is associated with poor neurodevelopmental outcome [3–5]. In order for the newborn brain to maintain normal function and maturation, a continuous equilibrium of ce-
Snjolaug Sveinsdottir and Magnus Gram contributed equally to this work.
Snjolaug Sveinsdottir Department of Pediatrics, Clinical Sciences Lund, Lund University SE–221 84 Lund (Sweden) E-Mail snjolaug.sveinsdottir @ med.lu.se
Downloaded by: University of Tokyo 157.82.153.40 - 5/19/2015 10:11:06 PM
a
rebrospinal fluid (CSF) volume and content is essential. However, following IVH and the development of PHVD there is a pathological accumulation and an altered composition of CSF in the ventricles. CSF is produced by the choroid plexus, which is a collection of villi composed of a single layer of highly specialized epithelial cells overlying a core of intercellular matrix and fenestrated capillaries. On the ventricular side of the epithelial cells there are a number of highly specialized transmembrane proteins that tightly regulate the composition of CSF regarding osmolarity, electrolyte and nutrient content [6]. Aquaporins (AQPs) are a family of channel proteins which facilitate the transport of water and solutes across membranes [7]. A number of AQPs have been described in the brain, e.g. AQP1, 3, 4, 5, 8 and 9 [8], and have been assigned to different cell types and physiological roles. AQP1 is described as the key AQP in the choroid plexus and has an important role as a water transporter in CSF production during both physiological and pathological conditions [8, 9]. However, there have been no studies on the effect of IVH with PHVD on AQP1 expression and localization in the choroid plexus. AQP5 has been described in rat brain astrocytes [10], but the role or presence of AQP5 in the choroid plexus has not been described. However, AQP5 is abundantly expressed in several other organs with secretory functions such as the lacrimal glands [11], salivary glands [12] and lungs [13]. The main function of AQP5 is to regulate water permeability [2], paracellular water transfer [14] and cell cytoskeleton organization and stabilization [15]. Furthermore, the expression of AQP5 in astrocytes is differentially regulated during metabolic and traumatic brain injury [10]. Consequently, characterizing the expression pattern of AQP5 in the choroid plexus following IVH with PHVD could be an important step towards understanding the development of hydrocephalus. AQP4 is one of the most abundant and well-studied AQPs in the brain and is mainly localized on the cytoplasmic membrane and end-feet of the astrocytes, where it plays a crucial role in regulating water flow into and out of the brain. AQP4 has been shown to be involved in brain swelling and edema following ischemia [16], and modulation of AQP4 expression is associated with improved functional outcome following cerebral hemorrhage [17]. Over time, there has been much speculation about AQP-targeted therapies in different forms of brain pathology. Although current research would suggest that this might be a feasible approach, it is of utmost importance to first understand the physiological role of AQPs in various areas and cell populations of the brain as well as changes in AQP expression after insult.
In this study we have characterized the expression and localization of AQP1, 4 and 5 in the choroid plexus following preterm IVH using a preterm rabbit pup model [18, 19]. The preterm rabbit pup exhibits a brain maturation corresponding to that of a human infant at 28–30 weeks of gestation [20], has a germinal matrix and develops spontaneous IVH [19]. Importantly, the hemorrhage is confined to the intraventricular space and results in a progressive ventricular enlargement very similar to that seen in preterm human infants [21]. In a recent study using this model and primary astrocyte cell cultures we showed that IVH is followed by a release of cell-free hemoglobin (Hb) in the intraventricular space and that accumulated Hb metabolites are causal inducers of inflammation [22]. In addition to the in vivo characterization of AQP1, 4 and 5, here we further investigated the effects of Hb metabolites on the expression of AQP1, 4 and 5 in primary human choroid plexus epithelial cells (HCPEpiC) and compared the response to that following exposure to posthemorrhagic CSF obtained from preterm human infants. We show, for the first time, that AQP5 is indeed expressed in the choroid plexus and that IVH with progressive PHVD is associated with an increased expression of AQP5 localized at the epithelial apical surface. Simultaneously, we observed a decrease in AQP1 mRNA expression following IVH. Corresponding changes in the expression of AQP1 and 5 were detected in HCPEpiC exposed to hemin and to posthemorrhagic CSF. We did not detect AQP4 in the choroid plexus or in the choroid plexus cell cultures, suggesting that AQP1 and 5 are key AQPs controlling the water flux across the choroid plexus barrier and in the production of CSF.
IVH Alters Aquaporin Expression and Distribution
Dev Neurosci 2014;36:542–551 DOI: 10.1159/000366058
Materials and Methods Animals The animal protocols were approved by the Swedish Animal Ethics Committee in Lund. The experiments were performed on a total of 39 rabbit pups from 10 litters delivered at gestational day 29 (term 32 days). A half-breed between New Zealand White and Lop was used. The pups were delivered by cesarean section after the does were anesthetized with propofol (5 mg/kg, i.v.) and with local infiltration of the abdominal wall using lidocaine with adrenaline (10 mg/ml + 5 μl/ml, 20–30 ml). After birth the pups were dried vigorously, weighed and placed in an infant incubator with a constant temperature of 36 ° C and 60% ambient humidity. At 2–3 h of age the pups were hand-fed with 1 ml of cat milk formula (KMR; PETAG Inc., USA) using a 3.5-Fr feeding tube and thereafter every 12 h, increasing each meal by 0.5 ml. At 2 h of age, the pups received an intraperitoneal injection of 50% glycerol (6.5 g/kg) to induce intracerebral hypotension due to hyperosmolality, thus causing rupture of the small vessels in the germinal matrix. Ultrasound imaging of the brain was performed at 6 h of age to
543
Downloaded by: University of Tokyo 157.82.153.40 - 5/19/2015 10:11:06 PM
3
2
a
PHVD 24 h
PHVD 72 h
Fig. 1. High-frequency ultrasound of normal brain, IVH and PHVD development. a Coronal images obtained by high-frequen-
cy ultrasound displaying a normal brain with no IVH and intraventricular IVH at 6 h and subsequent development of PHVD at 24 and 72 h of age in preterm rabbit pups. b Mean horizontal ventricular width at 6, 24, 48 and 72 h of age was determined by high-
detect and grade IVH and after that at 24, 48 and 72 h of age using the VisualSonics Vevo 2100 (VisualSonics Inc., Ont., Canada) with an MS-550D 40-MHz transducer (fig. 1a). Only animals with a large IVH at 6 h and with the development of PHVD at 24 and 72 h were used for data analysis; animals with no detectable IVH at all time points on cranial ultrasound were used as controls. All controls used in this study were glycerol-injected animals with no IVH as determined by cranial ultrasound. Measurements of ventricular size for the assessment of PHVD were obtained at the level of the midseptal nucleus in a coronal view at 6, 24, 48 and 72 h of age (fig. 1b). Each ventricle was measured horizontally from the midbrain plane to the lateral wall of the ventricle. The reproducibility and accuracy of ventricular measurements in this animal model using high-frequency ultrasound have been described previously [21].
*
4
0
6
24
b
48
72
Age (h)
frequency ultrasound in rabbit pup brains. Rabbit pups with IVH (black triangles) had higher mean measurements at all time points (p < 0.01) compared to pups with no IVH (control, black circles) at the same time points. Mean ventricular size was increased at 72 h compared to 24 h in rabbit pups with IVH. * p < 0.05. Vertical lines show means ± SD.
CSF Sampling from Preterm Infants CSF was sampled from preterm infants (gestational age at birth: 25–28 weeks) at 6–11 days after the detection of IVH by spinal tap or ventricular reservoir puncture according to clinical routine in the neonatal unit at Lund University Hospital. Immediately after sampling, the CSF was centrifuged (2,000 g, 20 ° C for 10 min) and stored at –80 ° C until further use, as described below. The sampled CSF was utilized for the study following written consent from the parents, and the study was approved by the ethical committee review board for studies in human subjects at Lund University, Sweden.
Preparation of Methemoglobin and Hemin Fetal Hb was purified as previously described [23] from freshly drawn human umbilical cord blood. Methemoglobin (metHb) was prepared by incubating the purified Hb solution at 37 ° C for 72 h. The metHb concentration was quantified as described previously [23]. Hemin (ferriprotoporphyrin IX chloride) was purchased from Porphyrin Products Inc. (Logan, Utah, USA), and a 10-mM stock solution was prepared using DMSO (Sigma). The metHb was purified from endotoxin contamination using the endotoxin-removing product EndoTrap (Hyglos GmbH, Germany) as described by the manufacturer. The absolute purity of metHb and hemin from contamination with endotoxin (0 EU/mg Hb/hemin) was determined using the QCL-1000TM Endpoint Chromogenic LAL Assay (Lonza, Switzerland) as described by the manufacturer.
Tissue Collection and Processing Rabbit pups were euthanized at 24 h (IVH + PHVD = 6, sham control = 6) and 72 h of age (IVH + PHVD = 9, sham control = 9), and the brains were removed from the skulls and sectioned at the level of the midseptal nucleus. The choroid plexus was carefully removed from the lateral ventricles, snap frozen and stored at –80 ° C until further mRNA and protein analysis as described below. For electron microscopy immunohistochemistry staining (EMIHC), the choroid plexus from 2 rabbit pups with IVH + PHVD and 2 sham controls at 72 h was fixed and prepared as described below. For IHC, perfusion fixation of brains was performed in 3 pups with IVH + PHVD and 2 sham controls under isoflurane anesthesia at 72 h of age by an infusion of saline solution followed by 4% paraformaldehyde in PBS. The perfused brains were then immersed in formaldehyde and prepared as described below.
544
Dev Neurosci 2014;36:542–551 DOI: 10.1159/000366058
Primary HCPEpiC HCPEpiC (ScienCell, Carlsbad, Calif., USA) were cultured in epithelial cell medium containing 2% fetal bovine serum, 1% epithelial cell growth supplement, 100 U/ml penicillin and 100 μg/ml
Sveinsdottir/Gram/Cinthio/Sveinsdottir/ Mörgelin/Ley
Downloaded by: University of Tokyo 157.82.153.40 - 5/19/2015 10:11:06 PM
IVH 6 h
Horizontal ventricular size (mm)
Control
IVH Normal
5
RNA Isolation and Real-Time PCR Total RNA was isolated from the choroid plexus and HCPEpiC cells using the acid guanidinium phenol chloroform method and RNeasy Mini Kit supplied by QIAGEN. The optical density ratio (optical density at 260 nm/280 nm) of RNA was always higher than 1.9. Reverse transcription was performed according to the manufacturer on 0.1–1 μg of total RNA using iScriptTM cDNA Synthesis Kit (Bio-Rad, Calif., USA) and RT2 First Strand Kit (QIAGEN). Real-time PCR was used to quantify the expression of AQP1, 4 and 5 mRNA. Data were normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The fold change values were calculated by normalizing against control samples from untreated animals or cells. RT2 PCR Profiler Array for analysis of HCPEpiC AQP1, 4 and 5 and GAPDH were purchased from QIAGEN. Primers for analysis of rabbit choroid plexus AQP1, 4 and 5 and GAPDH were designed accordingly, as follows: AQP1 forward primer 5′-GAT CGACTACACTGGCTGTGG-3′, reverse primer 5′-GGTTGT TGAAGTTGTGGGTGAG-3′; AQP4 forward primer 5′-CGTTTT AAAGAAGCCTTCAGCAA-3′, reverse primer 5′-CCTGTTG TCCTCCACCTCCAT-3′; AQP5 forward primer 5′-GGGCAA CCTGGCTGTCAA-3′, reverse primer 5′-AGCTGGAAGGTG AGGATCAACTC-3′, and GAPDH forward primer 5′-GAATC CACTGGCGTCTTCAC-3′, reverse primer 5′-CGTTGCTGAC AATCTTGAGAGA-3′. Expression was analyzed using the iTaq Universal SYBR Green Supermix (Bio-Rad) and RT2 SYBR Green Fluor qPCR Mastermix (QIAGEN). Amplification was performed at the respective adequate temperature for 40 cycles in an iCycler Thermal Cycler (Bio-Rad) and data were analyzed using iCycler iQ Optical System Software (Bio-Rad). Total Protein Analysis Total protein from the choroid plexus and HCPEpiC was determined by Pierce® BCA Protein Assay Kit (Thermo Scientific).
ml; Santa Cruz Biotechnology) and β-actin (monoclonal mouse anti-human β-actin; Abcam, Cambridge, UK). Western blot was performed using Alexa 647-conjugated secondary antibodies (Life Technologies). The bands were detected in a ChemiDoc XRS unit (Bio-Rad). The relative quantification of AQP1 and 5 bands were performed by densitometry using Image Lab software (Bio-Rad). Levels of AQP1were normalized against those of β-actin for respective samples. Immunohistochemistry IHC sections were deparaffinized by routine procedures and endogenous peroxidase activity was blocked with 3% H2O2 in methanol for 15 min. After washing with Triton-X-100 (0.25%) in PBS, the sections were blocked with normal goat serum (5%) for 1 h at room temperature. The slides were then incubated with AQP1 (1 μg/ml), AQP4 (1 ug/ml) and AQP5 (2 ug/ml) primary antibodies overnight at 4 ° C. Antibody detection was performed with a standard avidinbiotin complex detection system after which they were developed with 3,3-diaminobenzidine tetrahydrochloride as the chromogenic substrate (Vectastain avidin-biotin complex; Vector Laboratories, Burlingame, Calif., USA). The sections were mounted with Pertex (Histolab Products AB, Gothenburg, Sweden), examined and photographed (Olympus BHS photomicrographic system).
Transmission EM Immunolabeling of thin sections with gold-labeled anti-AQP1 and 5 were performed as described previously [24], with the modification that Aurion BSA was used as a blocking agent. Specimens were observed in a JEOL JEM 1230 electron microscope operated at an accelerating voltage of 80 kV. Images were recorded with a Gatan Multiscan 791 CCD camera. Statistics Comparisons between unrelated groups were performed with the unpaired Student t test or the Mann-Whitney U test as appropriate. Comparisons within groups were performed with the paired Student t test. Comparisons between multiple groups were performed by ANOVA with post hoc Bonferroni correction. p values
