Animals and Treatment
The experiments were performed on Sprague Dawley rats from the animal housing facility of the Jagiellonian University Medical College in Cracow. The animals were kept under a natural day-night cycle at 22 ± 2 °C with food and water available at libitum.
To determine estrous cycle phases, vaginal smears were taken from the females on a daily basis. On the proestrus day, the females were placed with males for 12 h, and the vaginal smears were subsequently examined for the presence of sperm.
BP-3 (purity-98%) obtained from Merck (Darmstadt, Germany) was formulated daily in Essex cream (Schering-Plough, Brussels, Belgium) at a final concentration of 10%, and administered from the first to the last day of pregnancy (approx. 22–23 days). Prior to treatment, the hair on the back of the neck to halfway towards the tail region was shaved and the animals were reshaved during treatment when the hair began to reappear. The examined group was treated with cream containing BP-3, at a dose of 100 mg/kg twice daily (at 8:00 and 17:00), whereas control female rats were treated with Essex cream without BP-3. We used a dose of 100 mg/kg BP-3, because after this dose, BP-3 blood levels in rats are comparable to those seen in humans using preparations containing this compound. It was shown that in humans after dermal application of a formulation containing 5% BP-3, the maximum concentration of these compound in plasma was 200–300 μg/l, and after 24 h about 80–200 μg/l (Tarazona et al., 2013). Similarly, Janjua et al. (2008) found that after a single application of BP-3, its concentration in the serum in men was about 250 ng/ml. We observed comparable BP-3 blood levels in the male rat, i.e. 216 ng/ml after the 100-mg/kg dose (Pomierny et al., 2019). After birth, the offspring were kept with the dam without any treatment. Twenty-one days after birth, male and female offspring were weaned and housed in groups of five per cage (with the males and females being separated) under standard conditions, without any treatment until the 43rd day. From 43 to 56 days of age, the female rats whose mothers received BP-3 were administered dermally with this compound, whereas cream without BP-3 was given to the control animals. The animals were observed daily for any abnormalities and their weights were recorded weekly. Two hours after the last morning BP-3 (or vehicle) application, the animals were subjected to the novel object recognition test or the novel location of the object recognition test. All tests were performed on 2-month-old females weighing 227.4 ± 15,5 g (control group) and 230.3 ± 12.8 g (BP-3 group). On the next day (24 h after the last BP-3 administration), the animals were euthanized via rapid decapitation. A small volume of the trunk blood was collected either in heparinized tubes for the hematological determination or the remaining volume in tubes containing EDTA, and these blood samples were centrifuged at 800×g, at 4 °C for 15 min and then plasma was stored at − 80 °C until used for biochemical assays. The brains and livers were rapidly removed, and the brain structures (hippocampus and frontal cortex) were dissected on ice-cold glass plates, frozen on dry ice, and stored at − 80 °C.
Novel Object and Novel Location Recognition Tests
To determine the impact of BP-3 on cognitive function (short-term and spatial memory), female rats from control and BP-3-treated groups were subjected to a novel object or novel location recognition test (Jabłoński et al. 2013). During habituation, the animals were allowed to explore an empty container and 24 h later they were exposed for 5 min to the same area containing two identical objects placed at an equal distance. Next, the animals were subdivided into two groups and 1 h after the pretest, rats from the first group were allowed to explore the same container for 5 min in the presence of the familiar object and a novel object, consistent in height and volume but different in shape and appearance. Animals from the second group were placed in the same container equipped with two identical familiar objects, but one of them was located in another place. The tests were recorded. After the tests were conducted, the films were manually checked to calculate the time that the animals spent exploring both objects. The results of the tests were presented as the preference index, i.e., the ratio of the time the animal spent exploring a novel object (for the novel object test) or exploring the object located in another place (for the novel location test) to the time spent exploring of both object.
LC/MS Analysis of BP-3
Tissues were homogenized in deionized water. Samples of homogenate or plasma mixed with internal standard were subjected to extraction by adding heptane and dichloromethane (1:1; v/v) and shaking for 10 min on an oscillating shaker. After centrifugation (10 min at 4000×g), the organic layer was obtained and evaporated under nitrogen stream at 37 °C.
Next, the chromatographic separation with mass spectrometric analysis was performed using Agilent 1100 liquid chromatograph (Agilent, Waldbronn, Germany) coupled to a mass spectrometer API 2000 (triple quadrupole) (Applied Biosystems MDS Sciex, Concord, Ontario, Canada) equipped with an electrospray ionization (ESI) interface. Chromatography equipment included a degasser, a binary pump, an autosampler and a column (Thermo Scientific BDS Hypersil C18; 100 × 3 mm I.D., 3 μm particle size) thermostated at 30 °C with its precolumn (100 × 3 mm I.D., 3 μm particle size) was used to separate samples—the volume of the sample injection was 40 μl with the flow rate of 0.4 ml/min. The mobile phase compositions were 0.025% glacial acetic acid in water (A) and methanol (B) with gradient: 0–1.5 min, isocratic gradient 40.0% (B); 1.5–2,5 min, linear gradient 40.0–95.0% (B); 2.5–6,5 min, isocratic gradient 95.0% (B); 6.5–8,0 min, linear gradient 95.0–40.0% (B); 8.0–10.0 min, and isocratic gradient 40.0% (B).
High-purity nitrogen (99.9%) from Peak NM20ZA as a curtain and collision gas and electrospray ionization in positive mode were used in the mass spectrometry analysis. The following parameters of ion source were applied: ion spray voltage (IS) 5000 V; nebulizer gas (gas 1) 20 psi; turbo gas (gas 2): 10 psi; temperature of the heated nebulizer (TEM) 250 °C; curtain gas (CUR) 20 psi, and the next ion path parameters for BP-3, BP-1, and BP-d10 were declustering potential (DP) 8 V; focusing potential (FP) 10 V; collision cell entrance potential (CEP) 13 V; and collision cell exit potential (CXP) 18 V. The following pairs of ions were monitored (values of m/z in brackets): BP-3 (229.0/151.1), BP-1 (215.0/107.0), and BP-d10 (193.0/110.0). For data analysis, Analyst software 1.6 (Perlan Technologies) was used. BP-3 and BP-1 concentrations were computed using calibration curves, constructed by linear regression analysis of the peak area versus concentrations.
Total Antioxidant Capacity
The total antioxidant capacity was determined using a modification of the ORAC method previously described by Sofic et al. (2002) and Prior and Cao (1999). Within this assay, the loss of fluorescein (as a probe) fluorescence is measured over time due to peroxyl-radical formation by the breakdown of 2,2′-azobis-2-methyl-propanimidamide dihydrochloride (AAPH). The tissue antioxidants inhibit this reaction. 6-Hydroxsy-2,5,7,8-tetramethylchroman-2-carboxylic acid (TROLOX) was used as a standard in the concentration range from 2.5 to 200 μM. The results were calculated according to Prior and Cao (1999), using the differences in areas under the fluorescein decay curves between the blank and a sample, and extrapolated to the TROLOX standard curve prepared in the same manner. Data are expressed as a percentage of the control group.
Lipid Peroxidation (MDA) Level
Fluorimetric assays for lipid peroxidation were performed using the Lipid Peroxidation (MDA) Colorimetric/Fluorometric Assays Kit (BioVision, USA). The assay is based on the reaction of the main product of lipid peroxidation, malondialdehyde (MDA) with thiobarbituric acid (TBA), at 95 °C for 60 min. The product of this reaction is MDA-TBA adduct, which can be quantified fluorometrically (Ex/Em = 532/553). The fluorescence was measured by a fluorescence plate reader (Fluoroskan Ascent FL, Thermo Labsystems). Lipid peroxidation in the samples was calculated from the standard curve and gathered as nanomoles of MDA per milligram of protein. The results were recalculated and expressed as a percentage of the control group.
Microdialysis of Frontal Cortex and Hippocampus
After 2.5% isoflurane anesthesia guide cannulas were stereotaxically implanted in the animals’ frontal cortex (anteroposterior (AP) −0.48 mm; mediolateral (ML) +2.0 mm; dorsoventral (DV) −1,2 mm—according to the atlas of Paxinos and Watson (2007), and the hippocampus (AP −4.36 mm; ML +1.8 mm; DV −2.5 mm)) using dental acrylic cement and cranial screws. Next, the cannulas were supported with obturators and 24 h later microdialysis was performed. Afterwards, the obturators were removed and, before collecting the first baseline sample, microdialysis probes (MAB 4, membrane with a molecular weight of 6 kDa cutoff, 2 mm length and 0.24 mm outer diameter, AgnTho’s AB, Sweden) were perfused with artificial cerebrospinal fluid (aCSF components [mM]: NaCl 147, KCl 4.0, MgCl2 1.0, CaCl2 2.2, pH 7.4) for 2 h at a constant flow rate of 2 μl per minute. Then, microdialysis probes were inserted into the guide cannulas of the frontal cortex and hippocampus before microdialysis samples were collected every 30 min for 3 h and immediately frozen and kept at a temperature of − 80 °C until further chromatographic analysis.
LC-MS Analysis of Glu
The chromatographic separation with mass spectrometric analysis was performed using the Agilent HPLC 1100 series system (Agilent, Waldbronn, Germany). Chromatographic separation parameters were based on Jastrzębska et al. (2015). Chromatography equipment included a degasser, a binary pump, an autosampler, and a thermostated column compartment. LiChrospher 60 RP-select B column (125 mm × 4.6 mm ID, 5 μm particle size) and a suitable guard column (4 mm × 4 mm, 5 μm particle size) (Merck, Germany) were used to separate aCSF samples. Following this, pairs of ions were monitored (values of m/z in brackets): Glu (148.0/84.1), Glu-d5 (153.22/89.1). For data analysis, Analyst software 1.4 (AB SCIEX, USA) was used. Glu concentrations in the noted brain structures were computed using calibration curves, constructed by linear regression analysis of the peak area versus concentrations. The presented data are expressed as the means of six samples collected every 30 min during 3 h of microdialysis for each animal.
RT-PCR Method
The female rats were decapitated 24 h after the last BP-3 administration; their brains were immediately removed, and the hippocampus and frontal cortex were isolated and immersed in RNAlater solution (Ambion, USA). According to the manufacturer’s protocol, the total RNA was extracted using TRI Reagent (Zymo Research, USA) and purified with Direct-zol RNA Miniprep Kit (Zymo Research, USA). Reverse transcription reactions and real-time PCR were conducted using a Transcriptor First Strand cDNA Synthesis kit and Fast Start Universal Probe Master (ROX) (Roche, Germany). According to the manufacturer’s protocol and using the PrimeQ real-time PCR system expression of the GLT-1, xc−, ERα, ERβ, GPR-30, AhR, C1qb, Cd40, Aif, and the reference gene GAPDH was analyzed. The fold change in expression was determined using the ΔΔc(t) method of relative quantification.
Western Blot Analysis
To obtain the membrane, nucleus, cytosol, and cytoskeleton fractions, the frontal cortex and hippocampus were subjected to subcellular fractionation using the Subcellular Protein Fractionation Kit (Roche, Germany). The protein concentrations were determined by the BCA Protein Assay Kit (Thermo, USA), and the individual cell fractions after dilution to the same protein concentration were mixed with a loading buffer (1:1) and heated for 5 min at 95 °C. Afterwards, samples containing 30–50 μg of the total protein in 15 μl were loaded on the stain-free gradient 4–15% SDS polyacrylamide gels (Bio-Rad, USA) and the electrophoresis was performed for 1 H at 150 V. The total amount of protein was evaluated in gels using the G:BOX Imaging System (Syngene, USA). Then, the proteins were transferred to PVDF membranes using a Trans-Blot Turbo Transfer System (Bio-Rad, USA). After several washes and blocking non-specific binding sites in 5% BSA, the membranes were incubated overnight with the following primary antibodies (in 1% BSA): anti-GLT-1 (EAAT2), anti-xc−, anti-ERα, anti-ERβ, anti-GPR30, anti-PR, anti-AhR, anti-Caspase 3 active, anti-Caspase 8, anti- Caspase 9, anti-Bax, anti-Bcl-2, anti-Iba1, anti-Caspase-1, anti-RIP, anti-glutathione (GPx) (Abcam (UK) or Santa Cruz Biotechnology (USA)). The membranes were then washed several times and incubated for 1 H at room temperature with proper secondary antibodies (in 1% BSA): goat anti-mouse-HRP, 1:7500, sc2005, Santa Cruz Biotechnology (USA); and goat anti-rabbit-HRP, 1:10000, ab6721, Abcam (UK) (Table 1). After washing, the signals were developed using the ECL method (Western Bright Quantum, Advansta, USA) and acquired by the G:BOX Imaging System (Syngene, USA). Making use of Gene Tools software (Syngene, USA), the protein expression was analyzed, taking into account the total amount of protein loaded into each well.
Table 1 List of antibodies with their concentrations and vendors used in this study Immunofluorescence Staining
For immunofluorescence staining, a separate group of animals was used and these animals were subjected to intracardiac perfusion for 24 h following the last administration of BP-3 or the vehicle. In order to do this, deep anesthesia was induced with ketamine (80 mg/kg) and xylazine (20 mg/kg). The rats were transcardially perfused with 250 ml of saline (0.9% NaCl, 32 °C) until all of the remaining blood was removed. Next, the animals were perfused with 500 ml of cold (4 °C) 4% PFA in 0.1 M phosphate-buffered saline (PBS). The brains were removed and immersed in the same 4% PFA solution overnight. At the start of the following day, the brains were immersed in sucrose solutions in 0.1 M PBS (concentration gradient 10%, 20%, and 30%) until they sank and stored (− 80 °C) until sectioning. The tissues were cut into coronal 20-μm sections using an automatic cryostat (Leica CM1860, Germany) and placed on microscopic slides. The slides were stored at − 20 °C until staining.
Immunostaining procedure: For active caspase-3 staining, trisodium citrate buffer solution (pH ≈ 9) was used as an antigen retriever buffer. The antigen retrieval was carried out in glass containers in a water bath for 30 min at 80 °C. Next, the containers were removed from the water bath and allowed to return to room temperature. All of the sections were washed twice in 0.2% Tween in PBS solution (PBST). Depending on the combination of secondary antibodies, 10% donkey normal serum (DNS) or 10% donkey and goat normal serum (1:1) (DNS + GNS) solution in PBST was used to block nonspecific antibody binding. The tissues were incubated in blocking medium for 1 h at room temperature. Next, the blocking medium was removed, and the tissues were immersed in appropriate primary antibody solutions (anti-Caspase 3 active, 1:300, ab2302, Abcam (UK)). The tissues were double-stained together with a neuronal marker (anti-MAP2, 1:1000, ab5392, Abcam (UK)), astroglial marker (anti-GFAP, 1:1000, ab4674, Abcam (UK)) or microglial marker (anti-Iba1, 1:200, ab5076, Abcam (UK)). Each mixture of two specific antibodies was dissolved in an appropriate 2% DNS or DNS + GNS serum in PBST. The slides were incubated with primary antibody solutions at 4 °C overnight. The following day, the slides were removed and incubated for 1 h on a shaker at room temperature. Next, the sections were washed twice in 2% DNS or DNS + GNS in PBST. The tissues were immersed in appropriate secondary antibodies (goat anti-chicken AF, 1:300, ab150173, Abcam (UK); donkey anti-goat AF, 1:500, ab150133, Abcam (UK); and donkey anti-rabbit TR, 1:300, ab6800, Abcam (UK)) solution in PBST and incubated for 1 h in the dark at room temperature. The antibodies used are shown in Table 1. Afterwards, the sections were washed three times in PBST, dried, and mounted in Fluoroshield mounting medium (Sigma, USA) and coverslipped. The staining was visualized using a Leica DMI8 (Germany) fluorescence inverted microscope. Images of motor-frontal cortex, as well as CA1, CA3, and DG fields of the hippocampus, were captured by a digital CCD camera (Leica DFC450, Germany) and expressed as the relative fluorescence unit (RFU).
Measurement of GPx Activity
GPx activity was estimated using method described by Lawrence and Burk (1976) based on the oxidation of NADPH to NADP+. Briefly, frozen samples of hippocampus were homogenized on ice and diluted in potassium sodium phosphate buffer (pH = 7). The working solution added to homogenates included NADPH, reduced glutathione (GSH), sodium azide, EDTA, and glutathione reductase (GR). Furthermore, hydrogen peroxide was used as a substrate, whose degradation in the presence of GSH was proceeded. GR catalyzed the NADPH-driven reduction of the generated oxidized glutathione (GSSG) followed by NADP+ formation. The decrease in samples’ absorbance proportional to the activity of GPx was measured at 340 nm for 30 min at 37 °C by multiwell plate reader POLARstar Omega (BMG Labtech, Germany). Results were expressed relative to the total amount of protein in samples using the BCA Protein Assay Kit (Thermo, USA).
Determination of Blood Hormone Levels
The concentration of 17β-estradiol (ab108667; Abcam, UK), progesterone (CSB-E07282r; Cusabio, China), testosterone (ab108666; Abcam, UK), prolactin (CSB-E06881r; Cusabio, China), fT3 (DKO037; DiaMetra, Italy), fT4 (DKO038; DiaMetra, Italy), and thyroid-stimulating hormone (TSH; CSB-E05115r; Cusabio, China) were determined using commercial ELISA kits. The assays were performed using undiluted plasma according to the manufacturers’ manuals. The absorbance was measured in a multiwell plate reader (TECAN Infinite M200 PRO), and hormone concentrations in the samples were calculated from a calibration curve.
Hematological Analyses
Immediately after collection, whole blood samples with heparin were used for hematological analyses. The leukocyte count (WBC), red blood cell count (RBC), platelet count (PLT), hemoglobin concentration (HGB), hematocrit (HCT), mean corpuscular volume (MCV), mean cell hemoglobin (MCH), and mean cell hemoglobin concentration (MCHC) were analyzed by means of a Cobas Micros (Roche, Palo Alto, CA, USA) analyzer. Hematological analyses were systematically checked as reported previously (Starek et al. 2008).
Statistical Analysis
All data were expressed as the means (±SEM—standard error of the mean) and analyzed in a GraphPad Prism (version 6.0, USA) program. Normal distribution of data sets was determined by a Shapiro-Wilk normality test, and t tests were used in further statistical analysis. The p value below 0.05 was considered statistically significant.