Neuroscience 275 (2014) 248–258
MATERNAL TREATMENT WITH GLUCOCORTICOIDS MODULATES GAP JUNCTION PROTEIN EXPRESSION IN THE OVINE FETAL BRAIN G. B. SADOWSKA AND B. S. STONESTREET *
Key words: connexins, brain, fetus, gap junctions, glucocorticoids, sheep.
Department of Pediatrics, The Alpert Medical School of Brown University, Women & Infants Hospital of Rhode Island, Providence, RI 02905, United States
INTRODUCTION Abstract—Gap junctions facilitate intercellular communication and are important in brain development. Connexins (Cx) comprise a transmembrane protein family that forms gap junctions. Cx-32 is expressed in oligodendrocytes and neurons, Cx-36 in neurons, and Cx-43 in astrocytes. Although single antenatal steroid courses are recommended for fetal lung maturation, multiple courses can be given to women at recurrent risk for premature delivery. We examined the effects of single and multiple glucocorticoid courses on Cx-32, Cx-36, and Cx-43 protein expressions in the fetal cerebral cortex, cerebellum, and spinal cord, and differences in connexin expression among brain regions under basal conditions. In the single-course groups, the ewes received dexamethasone (6 mg) or placebo as four intramuscular injections every 12 h over 48 h. In the multiple-course groups, the ewes received the same treatment, once a week for 5 weeks starting at 76–78 days of gestation. Connexins were measured by Western immunoblot on brain samples from 105 to 108-day gestation fetuses. A single dexamethasone course was associated with increases (P < 0.05) in cerebral cortical and spinal cord Cx-36 and Cx-43 and multiple courses with increases in cerebellar and spinal cord Cx-36, and cerebral cortical and cerebellar Cx-43. Cx-32 did not change. Cx-32 was higher in the cerebellum than cerebral cortex and spinal cord, Cx-36 higher in the spinal cord than cerebellum, and Cx-43 higher in the cerebellum and spinal cord than cerebral cortex during basal conditions. In conclusion, maternal glucocorticoid therapy increases specific connexins, responses to different maternal courses vary among connexins and brain regions, and connexin expression differs among brain regions under basal conditions. Maternal treatment with glucocorticoids differentially modulates connexins in the fetal brain. Ó 2014 IBRO. Published by Elsevier Ltd. All rights reserved.
Gap junctions are specialized membrane structures that mediate cell-to-cell communication via channels between adjacent cells. These junctions enable direct exchange of intercellular messengers that coordinate tissue homeostasis, proliferation, differentiation, metabolism, cell survival, and death (Bennett et al., 1991; Nagy and Rash, 2000; Bruzzone and Dermietzel, 2006; Eugenin et al., 2012). Connexins comprise a multigene family of transmembrane intercellular channel-forming membrane proteins (Herve et al., 2007). Intercellular communication via gap junctions is particularly important in organs with extensive tight junctions such as the brain (Michelle et al., 2012). Gap junctions couple neurons and glial cells in the central nervous system (CNS) in adults and during development (Rozental et al., 2000). Intercellular communication through gap junction channels is a prominent feature of the developing CNS (Bittman et al., 2002; Bruzzone and Dermietzel, 2006). Moreover, the expression of specific connexin proteins varies among different brain regions, developmental stages, and cell types (Orellana et al., 2009). Connexin-32, -36, and -43 are the major connexin proteins expressed in the developing brain (Bittman et al., 1997, 2002). Connexin-32 is found in Schwann cells and oligodendrocytes. It plays an important role in myelin forming cells and in interneuron synchronization (Dermietzel et al., 1989; Scherer et al., 1995; Li et al., 2004; Montoro and Yuste, 2004; Nagy et al., 2004). Gap junctions between neurons are formed by connexin-36 (Sutor and Hagerty, 2005). The principal gap junction protein in astrocytes is connexin-43 (Dermietzel et al., 1991), which has an organizational role during neural development (Katbamna et al., 2004; Bruzzone and Dermietzel, 2006). We have previously shown that connexin-32 and -43 proteins are expressed in the brain from early in fetal life and throughout ovine development. However, the developmental patterns of these connexins differ. Connexin-43 increases from 60% of gestation up to maturity in adults, whereas connexin-32 decreases during development (Sadowska et al., 2009). Furthermore, there is increasing evidence that unopposed connexin hemi-channels or connexons also could play
*Corresponding author. Address: Department of Pediatrics, The Alpert Medical School of Brown University, Women & Infants Hospital of Rhode Island, 101 Dudley Street, Providence, RI 02905-2499, United States. Tel: +1-(401)-274-1122x47429; fax: +1-(401)-4537571. E-mail address: [email protected] (B. S. Stonestreet). Abbreviations: ANOVA, analysis of variance; BCA, bicinchoninic acid protein assay; CNS, central nervous system; Cx, connexins; LSD, least-significant difference; TBST, Tris-buffered saline with 0.1% Tween-20. http://dx.doi.org/10.1016/j.neuroscience.2014.05.066 0306-4522/Ó 2014 IBRO. Published by Elsevier Ltd. All rights reserved. 248
G. B. Sadowska, B. S. Stonestreet / Neuroscience 275 (2014) 248–258
a role in perinatal brain injury by mediating release of paracrine molecules that propagate cell death signals (Davidson et al., 2012a,b, 2013a,b). Therefore, a greater understanding of connexins under various experimental conditions in the brain during fetal development is important. Antenatal glucocorticoids are routinely given to women at risk for premature labor to reduce the incidence of respiratory distress syndrome in preterm infants (Brownfoot et al., 2013). However, there is ongoing debate regarding the optimal dose, type of glucocorticoid, and regimen to accelerate pulmonary maturation versus potential harmful effects to other organs including the brain (Crowther and Harding, 2007; Stiles, 2007; Brownfoot et al., 2013). We have previously shown that both single and multiple courses of maternal glucocorticoids reduce blood–brain barrier permeability in the ovine fetus (Stonestreet et al., 2000; Sadowska et al., 2006). In addition, we have shown that this maternal treatment affects several proteins critical to CNS homeostasis in the fetal brain including tight junction proteins and Na+,K+-ATPase (Malaeb et al., 2009; Mehter et al., 2009; Sadowska et al., 2010). Tight junction proteins are well known to be vital constituents of the endothelial tight junction, which regulate blood–brain barrier function (Abbott et al., 2010). Connexins are increasingly recognized as important partners in the tight junctional complex (Meyer et al., 1992; Derangeon et al., 2009) and recent evidence suggests that connexins can also influence barrier function in the CNS (De Bock et al., 2014). Glucocorticoids have also been shown to affect connexins in several organ systems other than the brain (Kwiatkowski et al., 1994; Sasson and Amsterdam, 2002; Rafacho et al., 2007). Dexamethasone increased connexin-32 mRNA levels in rat hepatocyte cultures, connexin-36 mRNA and protein expression in pancreatic rat islets, and connexin-43 protein expression in human granulosa cells (Kwiatkowski et al., 1994; Sasson and Amsterdam, 2002; Rafacho et al., 2007). A recent report also indicates that dexamethasone treatment increases connexin-43 expression and the number of coupled astrocytes in primary glial cocultures of newborn rats (Hinkerohe et al., 2011). Taken together, there is sufficient evidence to consider that maternal treatment with glucocorticoids in doses, which are similar to those used to treat women in premature labor (Brownfoot et al., 2013), could also influence the expression of connexins within the fetal brain. The purpose of the current study was to examine the effects of maternal treatment with single and multiple courses of dexamethasone on connexin-32, -36, and -43 protein expressions in the cerebral cortex, cerebellum, and cervical spinal cord of the preterm fetal sheep brain. Full-term gestation in sheep is 145– 147 days. Therefore, the neurodevelopment of the sheep fetus in the current study at 107 days of gestation, is approximately similar to that of preterm human infants between 28 and 32 weeks of gestation (McIntosh et al., 1979; Back et al., 2006; Koome et al., 2013).
249
EXPERIMENTAL PROCEDURES The study was conducted after approval by the Institutional Animal Care and Use Committees of the Alpert Medical School of Brown University and Women and Infants Hospital of Rhode Island according to the National Institutes of Health Guidelines for the use of experimental animals.
Animal preparation and experimental design The samples of fetal brain in this study were obtained from animals enrolled in our previous studies (Hai et al., 2002; Kerzner et al., 2002; Stonestreet et al., 2004; Sadowska et al., 2006, 2010). The surgical and experimental procedures have been previously reported (Stonestreet et al., 1996; Sadowska et al., 2006). Surgery was performed under 1–2% halothane anesthesia on the 28 mixed breed pregnant ewes at 99–102 days of gestation as previously described in detail (Stonestreet et al., 1996). Briefly, polyvinyl catheters were placed into a brachial vein of the fetus for isotope administration and the thoracic aorta via the brachial artery for blood sample withdrawal for the studies designed to quantify blood– brain barrier permeability (Sadowska et al., 2006). Pregnant ewes were included in four groups as follows: (1) Single placebo course (n = 6) or (2) dexamethasone (n = 7) or (3) five placebo courses (n = 6) or (4) five dexamethasone courses (n = 9). The pregnant ewes assigned to the single-course groups were given four intramuscular injections of 6 mg of dexamethasone (Fujsawa USA, Deerfield, IL, concentration = 4 mg/ml) or placebo (0.9% NaCl, 1.5 ml) over 48 h every 12 h. The pregnant ewes assigned to the multiple-course groups were given the identical treatment once a week for five successive weeks beginning at 76–78 days of gestation as we previously described (Kerzner et al., 2002; Sadowska et al., 2006). After the pregnant sheep were given the fourth course of glucocorticoids, the fetuses and ewes were then catheterized at 99–101 days of gestation, so that the timing of surgery in the fetuses and ewes exposed to the multiple courses would be identical to those that were exposed to the single courses. The fifth and last course was begun on the fourth day after surgery at 104–107 days of gestation. The rationale for choosing this regimen (i.e. 6 mg of dexamethasone every 12 h for 48 h) was to use a schedule and doses of dexamethasone similar to those recommended to enhance fetal maturation in women at risk for preterm delivery (Consensus, 1995). In addition, pregnant women who are at continued risk for premature delivery can be exposed to several courses of glucocorticoids. The treatment of the pregnant ewes assigned to the multiple-course groups was begun at 76–78 days of gestation. Although this is slightly earlier than treatment with glucocorticoidswould begin in human pregnancies (22–23 weeks or 55–57% of gestation), the fetal sheep brain at 127 days of gestation (85% of gestation) is thought to be similar to the near term human brain (Barlow, 1969; Gunn et al., 1997). Hence, the fetal sheep brain is relatively more mature at
250
G. B. Sadowska, B. S. Stonestreet / Neuroscience 275 (2014) 248–258
full-term gestation e.g. 147–148 days of gestation compared with the human fetal brain at full term (39– 40 weeks). Consequently, the onset of administration of glucocorticoids at 52% of the ovine gestation does not differ substantially from that of the human fetus at 22–23 weeks of gestation (55–57%) of the human pregnancy. In our original studies (Sadowska et al., 2006), we selected this early time in gestation because we sought to measure blood–brain barrier permeability after these antenatal treatments in preterm fetal sheep (Sadowska et al., 2006). The ewes in single- and multiple-course groups stayed in the animal care facility for different durations, e.g. approximately 2 weeks in the single and 6 weeks in the multiple-course groups. As a result, the two groups of ewes could have received slightly different diets as previously described (Gray et al., 2006) and experienced different activity levels, i.e. potentially more activity in the single- than multiple-course groups, as the single-course groups remained on the farm for a longer duration than the multiple-course groups before the commencement of the studies. The fetal cerebral cortical, cerebellar, and cervical spinal cord samples were harvested and placed in liquid nitrogen 18 h after the final dose of dexamethasone or placebo was given to the ewes at 105–108 days (70%) of gestation (Sadowska et al., 2010). The cerebral cortical samples obtained from the parietal cortex most likely contained primarily gray matter and some white matter. The parietal cortex was examined because it represents a relatively large proportion of the fetal sheep cerebral cortex. Although the cerebral cortex contains mostly gray matter, based upon scanning and area fraction measurements (NIH Image J 1.45k, Softonic, International) of Luxol fast blue/H&E stained sections from our previous work (Petersson et al., 2002), we estimate that white matter represents approximately 27% of the parietal cortex in fetal sheep at 85% of gestation. We do not have slides from brains of fetal sheep at similar gestational ages to those in the current study. Therefore, we can only estimate that the frozen samples of the parietal cortex at 107 days (73%) of gestation could have contained approximately 20% white matter based upon the earlier gestational age when less white matter is present (Grever et al., 1996). The cerebellum contains both gray and white matter. In contrast, the cervical spinal cord contains mostly white matter. We selected these regions for analysis, as they are major regions from the rostral and hindbrain with different developmental trajectories (Barlow, 1969). The brain samples were kept at 80 °C until the analysis.
Protein extraction Cell membrane fractions from the regional brain samples were extracted as we previously described (Sadowska et al., 2010). Total protein concentrations of the homogenates were determined with a bicinchoninic acid protein assay (BCA, Pierce, Rockford, IL) and the extracted aliquots stored at 80 °C for later analysis.
Western immunoblot detection and quantification of proteins Fifty micrograms of total protein per well was fractionated using SDS–PAGE and transferred onto polyvinylidene diflouride membranes (Bio-Rad Laboratories, Hercules, CA) by the use of a semi-dry technique. After blocking the membranes with 10% non-fat milk for 1 h at room temperature, they were washed in Tris-buffered saline with 0.1% Tween-20 (TBST) 3 times for 10 min for each wash. Membranes were probed for connexin-32 (cat #13-8200, Zymed, San Francisco, CA), connexin-36 (cat #37-4600, Zymed), and connexin-43 (cat #13-8300, Zymed) with primary mouse monoclonal antibodies at a dilution of 1:5000 and for vinculin with primary mouse monoclonal antibodies (cat #MA1-83523, Thermo Scientificä Pierce, Waltham, MA) at a dilution of 1:10,000. After overnight incubation with primary antibodies at 4 °C, the immunoblots were washed in TBST three times and then incubated for 1 h at room temperature with goat anti-mouse (Zymed) horseradish peroxidase-conjugated secondary antibodies at a dilution of 1:10,000 for each of the proteins examined. Immunoblots were again washed in TBST 4 times. Secondary antibody binding was detected by the use of enhanced chemiluminescence (ECL plus, Western Blotting Detection reagents, Amersham Pharmacia Biotech, Inc., Piscataway, NJ) before exposing the film to autoradiography (Daigger, Vernon Hills, IL). The experimental samples were normalized to a protein standard obtained from a brain of fetal sheep at 70% of gestation that was not part of the current study. As an example, the study samples for connexin-43 from the cerebral cortex were normalized by the use of connexin-43 protein obtained from the cerebral cortex of a single fetal sheep that had not been exposed to any of the experimental interventions. These standard samples in the current report are designated as internal control samples. As we have previously reported (Ron et al., 2005; Kim et al., 2006; Malaeb et al., 2007; Sadowska et al., 2009, 2010), these samples serve as internal controls for loading quality, sample transfer in order to normalize the regional brain densitometric values. The use of the internal control standard is unique to our laboratory and allows for the comparison of large groups of animals over several different immunoblots. The experimental protein autoradiographic densitometrical values were expressed as a ratio to the internal control protein standards, thus facilitating normalized comparisons among the various groups and several different immunoblots. Vinculin was also used as a loading control to ensure that equal amounts of protein had been applied to each lane. However, the experimental protein bands were normalized to the internal control standards rather than to vinculin. All immunoblots contained samples from the four experimental groups and the three internal control standard samples that were placed as the first, middle, and last samples on each immunoblot. We calculated a coefficient of variation for the internal control standard samples on each immunoblot and accepted the
G. B. Sadowska, B. S. Stonestreet / Neuroscience 275 (2014) 248–258
experimental samples as valid only when the percent coefficient of variation for the internal control samples was
MATERNAL TREATMENT WITH GLUCOCORTICOIDS MODULATES GAP JUNCTION PROTEIN EXPRESSION IN THE OVINE FETAL BRAIN G. B. SADOWSKA AND B. S. STONESTREET *
Key words: connexins, brain, fetus, gap junctions, glucocorticoids, sheep.
Department of Pediatrics, The Alpert Medical School of Brown University, Women & Infants Hospital of Rhode Island, Providence, RI 02905, United States
INTRODUCTION Abstract—Gap junctions facilitate intercellular communication and are important in brain development. Connexins (Cx) comprise a transmembrane protein family that forms gap junctions. Cx-32 is expressed in oligodendrocytes and neurons, Cx-36 in neurons, and Cx-43 in astrocytes. Although single antenatal steroid courses are recommended for fetal lung maturation, multiple courses can be given to women at recurrent risk for premature delivery. We examined the effects of single and multiple glucocorticoid courses on Cx-32, Cx-36, and Cx-43 protein expressions in the fetal cerebral cortex, cerebellum, and spinal cord, and differences in connexin expression among brain regions under basal conditions. In the single-course groups, the ewes received dexamethasone (6 mg) or placebo as four intramuscular injections every 12 h over 48 h. In the multiple-course groups, the ewes received the same treatment, once a week for 5 weeks starting at 76–78 days of gestation. Connexins were measured by Western immunoblot on brain samples from 105 to 108-day gestation fetuses. A single dexamethasone course was associated with increases (P < 0.05) in cerebral cortical and spinal cord Cx-36 and Cx-43 and multiple courses with increases in cerebellar and spinal cord Cx-36, and cerebral cortical and cerebellar Cx-43. Cx-32 did not change. Cx-32 was higher in the cerebellum than cerebral cortex and spinal cord, Cx-36 higher in the spinal cord than cerebellum, and Cx-43 higher in the cerebellum and spinal cord than cerebral cortex during basal conditions. In conclusion, maternal glucocorticoid therapy increases specific connexins, responses to different maternal courses vary among connexins and brain regions, and connexin expression differs among brain regions under basal conditions. Maternal treatment with glucocorticoids differentially modulates connexins in the fetal brain. Ó 2014 IBRO. Published by Elsevier Ltd. All rights reserved.
Gap junctions are specialized membrane structures that mediate cell-to-cell communication via channels between adjacent cells. These junctions enable direct exchange of intercellular messengers that coordinate tissue homeostasis, proliferation, differentiation, metabolism, cell survival, and death (Bennett et al., 1991; Nagy and Rash, 2000; Bruzzone and Dermietzel, 2006; Eugenin et al., 2012). Connexins comprise a multigene family of transmembrane intercellular channel-forming membrane proteins (Herve et al., 2007). Intercellular communication via gap junctions is particularly important in organs with extensive tight junctions such as the brain (Michelle et al., 2012). Gap junctions couple neurons and glial cells in the central nervous system (CNS) in adults and during development (Rozental et al., 2000). Intercellular communication through gap junction channels is a prominent feature of the developing CNS (Bittman et al., 2002; Bruzzone and Dermietzel, 2006). Moreover, the expression of specific connexin proteins varies among different brain regions, developmental stages, and cell types (Orellana et al., 2009). Connexin-32, -36, and -43 are the major connexin proteins expressed in the developing brain (Bittman et al., 1997, 2002). Connexin-32 is found in Schwann cells and oligodendrocytes. It plays an important role in myelin forming cells and in interneuron synchronization (Dermietzel et al., 1989; Scherer et al., 1995; Li et al., 2004; Montoro and Yuste, 2004; Nagy et al., 2004). Gap junctions between neurons are formed by connexin-36 (Sutor and Hagerty, 2005). The principal gap junction protein in astrocytes is connexin-43 (Dermietzel et al., 1991), which has an organizational role during neural development (Katbamna et al., 2004; Bruzzone and Dermietzel, 2006). We have previously shown that connexin-32 and -43 proteins are expressed in the brain from early in fetal life and throughout ovine development. However, the developmental patterns of these connexins differ. Connexin-43 increases from 60% of gestation up to maturity in adults, whereas connexin-32 decreases during development (Sadowska et al., 2009). Furthermore, there is increasing evidence that unopposed connexin hemi-channels or connexons also could play
*Corresponding author. Address: Department of Pediatrics, The Alpert Medical School of Brown University, Women & Infants Hospital of Rhode Island, 101 Dudley Street, Providence, RI 02905-2499, United States. Tel: +1-(401)-274-1122x47429; fax: +1-(401)-4537571. E-mail address: [email protected] (B. S. Stonestreet). Abbreviations: ANOVA, analysis of variance; BCA, bicinchoninic acid protein assay; CNS, central nervous system; Cx, connexins; LSD, least-significant difference; TBST, Tris-buffered saline with 0.1% Tween-20. http://dx.doi.org/10.1016/j.neuroscience.2014.05.066 0306-4522/Ó 2014 IBRO. Published by Elsevier Ltd. All rights reserved. 248
G. B. Sadowska, B. S. Stonestreet / Neuroscience 275 (2014) 248–258
a role in perinatal brain injury by mediating release of paracrine molecules that propagate cell death signals (Davidson et al., 2012a,b, 2013a,b). Therefore, a greater understanding of connexins under various experimental conditions in the brain during fetal development is important. Antenatal glucocorticoids are routinely given to women at risk for premature labor to reduce the incidence of respiratory distress syndrome in preterm infants (Brownfoot et al., 2013). However, there is ongoing debate regarding the optimal dose, type of glucocorticoid, and regimen to accelerate pulmonary maturation versus potential harmful effects to other organs including the brain (Crowther and Harding, 2007; Stiles, 2007; Brownfoot et al., 2013). We have previously shown that both single and multiple courses of maternal glucocorticoids reduce blood–brain barrier permeability in the ovine fetus (Stonestreet et al., 2000; Sadowska et al., 2006). In addition, we have shown that this maternal treatment affects several proteins critical to CNS homeostasis in the fetal brain including tight junction proteins and Na+,K+-ATPase (Malaeb et al., 2009; Mehter et al., 2009; Sadowska et al., 2010). Tight junction proteins are well known to be vital constituents of the endothelial tight junction, which regulate blood–brain barrier function (Abbott et al., 2010). Connexins are increasingly recognized as important partners in the tight junctional complex (Meyer et al., 1992; Derangeon et al., 2009) and recent evidence suggests that connexins can also influence barrier function in the CNS (De Bock et al., 2014). Glucocorticoids have also been shown to affect connexins in several organ systems other than the brain (Kwiatkowski et al., 1994; Sasson and Amsterdam, 2002; Rafacho et al., 2007). Dexamethasone increased connexin-32 mRNA levels in rat hepatocyte cultures, connexin-36 mRNA and protein expression in pancreatic rat islets, and connexin-43 protein expression in human granulosa cells (Kwiatkowski et al., 1994; Sasson and Amsterdam, 2002; Rafacho et al., 2007). A recent report also indicates that dexamethasone treatment increases connexin-43 expression and the number of coupled astrocytes in primary glial cocultures of newborn rats (Hinkerohe et al., 2011). Taken together, there is sufficient evidence to consider that maternal treatment with glucocorticoids in doses, which are similar to those used to treat women in premature labor (Brownfoot et al., 2013), could also influence the expression of connexins within the fetal brain. The purpose of the current study was to examine the effects of maternal treatment with single and multiple courses of dexamethasone on connexin-32, -36, and -43 protein expressions in the cerebral cortex, cerebellum, and cervical spinal cord of the preterm fetal sheep brain. Full-term gestation in sheep is 145– 147 days. Therefore, the neurodevelopment of the sheep fetus in the current study at 107 days of gestation, is approximately similar to that of preterm human infants between 28 and 32 weeks of gestation (McIntosh et al., 1979; Back et al., 2006; Koome et al., 2013).
249
EXPERIMENTAL PROCEDURES The study was conducted after approval by the Institutional Animal Care and Use Committees of the Alpert Medical School of Brown University and Women and Infants Hospital of Rhode Island according to the National Institutes of Health Guidelines for the use of experimental animals.
Animal preparation and experimental design The samples of fetal brain in this study were obtained from animals enrolled in our previous studies (Hai et al., 2002; Kerzner et al., 2002; Stonestreet et al., 2004; Sadowska et al., 2006, 2010). The surgical and experimental procedures have been previously reported (Stonestreet et al., 1996; Sadowska et al., 2006). Surgery was performed under 1–2% halothane anesthesia on the 28 mixed breed pregnant ewes at 99–102 days of gestation as previously described in detail (Stonestreet et al., 1996). Briefly, polyvinyl catheters were placed into a brachial vein of the fetus for isotope administration and the thoracic aorta via the brachial artery for blood sample withdrawal for the studies designed to quantify blood– brain barrier permeability (Sadowska et al., 2006). Pregnant ewes were included in four groups as follows: (1) Single placebo course (n = 6) or (2) dexamethasone (n = 7) or (3) five placebo courses (n = 6) or (4) five dexamethasone courses (n = 9). The pregnant ewes assigned to the single-course groups were given four intramuscular injections of 6 mg of dexamethasone (Fujsawa USA, Deerfield, IL, concentration = 4 mg/ml) or placebo (0.9% NaCl, 1.5 ml) over 48 h every 12 h. The pregnant ewes assigned to the multiple-course groups were given the identical treatment once a week for five successive weeks beginning at 76–78 days of gestation as we previously described (Kerzner et al., 2002; Sadowska et al., 2006). After the pregnant sheep were given the fourth course of glucocorticoids, the fetuses and ewes were then catheterized at 99–101 days of gestation, so that the timing of surgery in the fetuses and ewes exposed to the multiple courses would be identical to those that were exposed to the single courses. The fifth and last course was begun on the fourth day after surgery at 104–107 days of gestation. The rationale for choosing this regimen (i.e. 6 mg of dexamethasone every 12 h for 48 h) was to use a schedule and doses of dexamethasone similar to those recommended to enhance fetal maturation in women at risk for preterm delivery (Consensus, 1995). In addition, pregnant women who are at continued risk for premature delivery can be exposed to several courses of glucocorticoids. The treatment of the pregnant ewes assigned to the multiple-course groups was begun at 76–78 days of gestation. Although this is slightly earlier than treatment with glucocorticoidswould begin in human pregnancies (22–23 weeks or 55–57% of gestation), the fetal sheep brain at 127 days of gestation (85% of gestation) is thought to be similar to the near term human brain (Barlow, 1969; Gunn et al., 1997). Hence, the fetal sheep brain is relatively more mature at
250
G. B. Sadowska, B. S. Stonestreet / Neuroscience 275 (2014) 248–258
full-term gestation e.g. 147–148 days of gestation compared with the human fetal brain at full term (39– 40 weeks). Consequently, the onset of administration of glucocorticoids at 52% of the ovine gestation does not differ substantially from that of the human fetus at 22–23 weeks of gestation (55–57%) of the human pregnancy. In our original studies (Sadowska et al., 2006), we selected this early time in gestation because we sought to measure blood–brain barrier permeability after these antenatal treatments in preterm fetal sheep (Sadowska et al., 2006). The ewes in single- and multiple-course groups stayed in the animal care facility for different durations, e.g. approximately 2 weeks in the single and 6 weeks in the multiple-course groups. As a result, the two groups of ewes could have received slightly different diets as previously described (Gray et al., 2006) and experienced different activity levels, i.e. potentially more activity in the single- than multiple-course groups, as the single-course groups remained on the farm for a longer duration than the multiple-course groups before the commencement of the studies. The fetal cerebral cortical, cerebellar, and cervical spinal cord samples were harvested and placed in liquid nitrogen 18 h after the final dose of dexamethasone or placebo was given to the ewes at 105–108 days (70%) of gestation (Sadowska et al., 2010). The cerebral cortical samples obtained from the parietal cortex most likely contained primarily gray matter and some white matter. The parietal cortex was examined because it represents a relatively large proportion of the fetal sheep cerebral cortex. Although the cerebral cortex contains mostly gray matter, based upon scanning and area fraction measurements (NIH Image J 1.45k, Softonic, International) of Luxol fast blue/H&E stained sections from our previous work (Petersson et al., 2002), we estimate that white matter represents approximately 27% of the parietal cortex in fetal sheep at 85% of gestation. We do not have slides from brains of fetal sheep at similar gestational ages to those in the current study. Therefore, we can only estimate that the frozen samples of the parietal cortex at 107 days (73%) of gestation could have contained approximately 20% white matter based upon the earlier gestational age when less white matter is present (Grever et al., 1996). The cerebellum contains both gray and white matter. In contrast, the cervical spinal cord contains mostly white matter. We selected these regions for analysis, as they are major regions from the rostral and hindbrain with different developmental trajectories (Barlow, 1969). The brain samples were kept at 80 °C until the analysis.
Protein extraction Cell membrane fractions from the regional brain samples were extracted as we previously described (Sadowska et al., 2010). Total protein concentrations of the homogenates were determined with a bicinchoninic acid protein assay (BCA, Pierce, Rockford, IL) and the extracted aliquots stored at 80 °C for later analysis.
Western immunoblot detection and quantification of proteins Fifty micrograms of total protein per well was fractionated using SDS–PAGE and transferred onto polyvinylidene diflouride membranes (Bio-Rad Laboratories, Hercules, CA) by the use of a semi-dry technique. After blocking the membranes with 10% non-fat milk for 1 h at room temperature, they were washed in Tris-buffered saline with 0.1% Tween-20 (TBST) 3 times for 10 min for each wash. Membranes were probed for connexin-32 (cat #13-8200, Zymed, San Francisco, CA), connexin-36 (cat #37-4600, Zymed), and connexin-43 (cat #13-8300, Zymed) with primary mouse monoclonal antibodies at a dilution of 1:5000 and for vinculin with primary mouse monoclonal antibodies (cat #MA1-83523, Thermo Scientificä Pierce, Waltham, MA) at a dilution of 1:10,000. After overnight incubation with primary antibodies at 4 °C, the immunoblots were washed in TBST three times and then incubated for 1 h at room temperature with goat anti-mouse (Zymed) horseradish peroxidase-conjugated secondary antibodies at a dilution of 1:10,000 for each of the proteins examined. Immunoblots were again washed in TBST 4 times. Secondary antibody binding was detected by the use of enhanced chemiluminescence (ECL plus, Western Blotting Detection reagents, Amersham Pharmacia Biotech, Inc., Piscataway, NJ) before exposing the film to autoradiography (Daigger, Vernon Hills, IL). The experimental samples were normalized to a protein standard obtained from a brain of fetal sheep at 70% of gestation that was not part of the current study. As an example, the study samples for connexin-43 from the cerebral cortex were normalized by the use of connexin-43 protein obtained from the cerebral cortex of a single fetal sheep that had not been exposed to any of the experimental interventions. These standard samples in the current report are designated as internal control samples. As we have previously reported (Ron et al., 2005; Kim et al., 2006; Malaeb et al., 2007; Sadowska et al., 2009, 2010), these samples serve as internal controls for loading quality, sample transfer in order to normalize the regional brain densitometric values. The use of the internal control standard is unique to our laboratory and allows for the comparison of large groups of animals over several different immunoblots. The experimental protein autoradiographic densitometrical values were expressed as a ratio to the internal control protein standards, thus facilitating normalized comparisons among the various groups and several different immunoblots. Vinculin was also used as a loading control to ensure that equal amounts of protein had been applied to each lane. However, the experimental protein bands were normalized to the internal control standards rather than to vinculin. All immunoblots contained samples from the four experimental groups and the three internal control standard samples that were placed as the first, middle, and last samples on each immunoblot. We calculated a coefficient of variation for the internal control standard samples on each immunoblot and accepted the
G. B. Sadowska, B. S. Stonestreet / Neuroscience 275 (2014) 248–258
experimental samples as valid only when the percent coefficient of variation for the internal control samples was
