Materials
Sodium hydroxide was obtained from VWR chemicals (Leuven, Belgium), sodium carbonate from Panreac (Barcelona, Spain) and monosodium phosphate from J.T. Baker (Deventer, Holland). Perchloric acid (HClO4), potassium hydrogen carbonate and 4',6-diamidino-2-phenylindole (DAPI) were obtained from Fisher scientific (Loughborough, United Kingdom). Copper (II) sulphate, ethylenediaminetetraacetic acid, triton X-100, paraformaldehyde, immersion oil and FluorSave™ were obtained from Merck (Darmstadt, Germany). Isoflurane was obtained from Abbott Animal Health (North Chicago, IL, USA). Mouse monoclonal anti-ATP synthase β (ab14730), mouse monoclonal anti-endothelial nitric oxide synthase (eNOS) (ab76198), rabbit polyclonal anti-manganese superoxide dismutase (MnSOD) (ab13533), and rabbit polyclonal anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (ab9485) were obtained from Abcam (Cambridge, UK). Rabbit polyclonal anti-heat shock protein 27 (HSP27) (sc-9012), mouse monoclonal anti-B-cell lymphoma 2 (Bcl-2) antibody (sc-7382), rabbit polyclonal anti-Bax (sc-493), mouse monoclonal anti-apoptosis-inducing factor (AIF) antibody (sc-13116), and rabbit polyclonal anti-p53 (sc-6243) were obtained from Santa Cruz Biotechnology (Heidelberg, Germany). Rabbit polyclonal anti-glycogen synthase kinase 3 beta (GSK-3β), and protease inhibitor cocktail (P8340) were obtained from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany). Rabbit monoclonal anti-tumour necrosis alpha (TNF-α) (11948) was obtained from Cell Signaling Technology (Danvers, Massachusetts, USA). Anti-rabbit and anti-mouse horseradish peroxidase secondary antibodies (NA934V and NA931V, respectively) were obtained from GE Healthcare (UK). Clarity™ western enhanced chemiluminescence (ECL) substrate (Cat. #170–5061) and commercial kit DC™ Protein Assay (#5000112) were obtained from BIO-RAD Laboratories, Inc. (Hercules, CA, USA). The primary antibody polyclonal rabbit anti-glial fibrillary acidic protein (GFAP; Z0334) was obtained from Agilent Dako (CA, USA) and the primary polyclonal rabbit anti-pTau (pSER396; SAB4504557, phosphorylation site at serine 396) was obtained from Sigma-Aldrich (St. Louis, MO, USA). Histomount was obtained from National Diagnostics (Atlanta, GA, USA). All the other reagents used were obtained from Sigma-Aldrich (St. Louis, MO, USA) at the highest purity available.
Animals
Adult male CD-1 mice were 12 weeks old and weighed 38–48 g at the beginning of the experiment. They were purchased from Charles River Laboratories (L’Arbresle, France) and housed in IVC Sealsafe plus mouse Green Mouse 500 cages, in a temperature (22 ± 2ºC) and humidity-controlled environment, in a 12-hour light-dark cycle. Standard rodent 4RF21 certificate diet (Mucedola, Settimo Milanese, Italy) and water were offered ad libitum. Each cage held a maximum of 3 animals to allow socialization and maintain animal welfare. Mice were weighed every day in the week before the first injection of DOX, to allow them to be accustomed to the handler, minimize stress, and improve the animal welfare. Housing and experimental treatment of the animals were done following the guidelines defined by the European Council Directive (2010/63/EU) transposed into Portuguese law (Decreto-Lei no. 113/2013). The experiments were performed with the approval of the Portuguese National Authority for Animal Health (General Directory of Veterinary Medicine, process no. 0421/000/000/2016) and the local Committee Responsible for Animal welfare (ICBAS-UP, ORBEA).
The schedule of DOX administration was designed to mimic the human anticancer therapy that consists of multiple administrations of the drug in cycles followed by drug-free periods (O'Brien, Wigler et al. 2004). Therefore, mice received intraperitoneal injections (i.p.) twice a week, for three weeks. The animal received a total cumulative dose of 9 mg/kg (DOX 9) or 18 mg/kg (DOX 18) of DOX, which are equivalent to a human dose of 51.5 mg/m2 and 102.9 mg/m2, respectively (Reagan-Shaw, Nihal et al. 2008, Brandão, Reis-Mendes et al. 2021), thus well below the maximum recommended dose. DOX was dissolved in a sterile saline solution (NaCl 0.9%). The control group received injections of NaCl 0.9% in the same volume and schedule as the drug-treated mice. Throughout the experimental period, the mice well-being was monitored as described previously by the group (Brandão, Reis-Mendes et al. 2021, Dias-Carvalho, Ferreira et al. 2021). After the last administration, the animals were maintained drug-free for a week before euthanasia.
Samples Collection
One week after the last administration, the mice were placed in a closed chamber in an atmosphere enriched with 5% of the inhalant anaesthetic isoflurane until full sedation. When no reflex to stimuli was attained, exsanguination followed, with blood removal from the inferior vena cava. Afterwards, the mice were decapitated, and the brain was carefully removed and weighed. For some animals, the whole brain was homogenised in 5% (w/v) HClO4 and centrifuged at 16,060 g for 10 min (4⁰C). The supernatant was used for quantification of total glutathione (tGSH), reduced glutathione (GSH), oxidized glutathione (GSSG), and adenosine triphosphate (ATP), whereas the pellet was used for protein levels determination. For western blot analysis, brain hemispheres were homogenized in radio immune precipitation assay buffer (RIPA) and centrifuged at 16,060 g for 15 min (4⁰C), as previously described (Dias-Carvalho, Ferreira et al. 2022). Additionally, the brain hemispheres of the remaining animals were fixated in 4% paraformaldehyde for histological processing.
Quantification Of Tgsh, Gsh And Gssg
The tGSH and GSSG contents of the brain samples were quantified through the 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB)-GSSG recycling assay, as previously described (Dores-Sousa, Duarte et al. 2015). The levels of GSH were determined using the formula: GSH = tGSH – 2 x GSSG. The results of tGSH, GSH, and GSSG were normalized to the total protein content and expressed as a % of the control.
Quantification Of Brain Atp Levels
Whole brain levels of ATP were determined through the bioluminescent firefly luciferin-luciferase assay, as previously described (Costa, Silva et al. 2007). The ATP levels were normalized to the total protein content and expressed as a % of the control.
Protein Quantification
The protein contents of the collected brain samples were assayed with a commercial kit DC™ Protein Assay (Bio-Rad®, CA, USA), according to the manufacturer’s recommendations and using bovine serum albumin (BSA) as a standard.
Western Blot Analysis
Following Dias-Carvalho, A. et al (Dias-Carvalho, Ferreira et al. 2022), volumes of brain homogenate hemisphere samples equivalent to 50 µg or 70 µg of protein were reduced by Laemmli (Laemmli 1970) buffer [0.5 M Tris-HCl pH 6.8, 4% (w/v) SDS, 15% (v/v) glycerol, 0.04% (w/v) bromophenol blue and 20% (v/v) β-mercaptoethanol] in a ½ (v/v) ratio, and then incubated at 100°C for 5 minutes. A 12.5% or 15% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) was used to perform electrophoresis at 180 V in running buffer [25 mM Tris, 192 mM glycine and 0.1% (w/v) SDS] (Laemmli 1970). The blotting of the gels onto a nitrocellulose membrane (AmershamTM, Protan®, GE Healthcare) was performed for 2 hours at 200 mA with transfer buffer (25 mM Tris, 192 mM glycine and 20% methanol). As protein loading control, a Ponceau S staining was performed. To block nonspecific binding a solution of 5% (w/v) nonfat dry milk in TBS-T (100 mM Tris, pH 8.0, 1.5 M NaCl and 0.5% Tween 20) was used for 1 hour at room temperature with agitation. The incubation for the ligation with the corresponding primary antibody [anti-ATP synthase β (1:1000), anti-GSK-3β (1:1000), anti-SOD2/MnSOD (1:1000), and anti-HSP27 (1:500 and 1:1000)] was performed with constant agitation for 2 hours at room temperature or overnight at 4°C. Right after a washing step of 3 washes (10 minutes each) with TBS-T, the membranes were incubated with secondary antibody, anti-rabbit (1:5000) or anti-mouse (1:1000) horseradish peroxidase, for 2 hours at room temperature with agitation. A washing step followed and then, according to the manufacturer’s procedure, immunoreactive bands were detected with ECL reagents. Gel Doc XR system (Bio-Rad®, CA, USA) was used to record and scan the images, which were analyzed with Image Lab software (Bio-Rad®, CA, USA, version 6.0.1). The optical densities (OD) obtained were expressed in arbitrary units.
Mild stripping was performed when needed. This procedure included 2 incubations (10 minutes each) with a stripping solution (0.04 M glycine, 0.7 mM SDS, 10% Tween 20, pH 2.2), followed by two washing steps. The first included 2 x 10 minutes of wash with phosphate-buffered saline 1x (PBS) followed by a 5-minute wash with TBS-T. After the stripping procedure, the blocking step was performed once again, and the membrane was ready to a new probe.
Finally, data analysis and normalization were done by the control bands. The average of control bands of each membrane was obtained and then divided by the smallest average of membrane control bands, resulting in a ratio for each membrane by which the remaining samples were divided. All results were divided by 1000 to work with smaller absolute values.
Brain Tissue Processing For Histologic Analysis
Left-brain hemispheres were labelled for blind analysis and processed according to a previously published method (Dias-Carvalho, Ferreira et al. 2022). Succinctly, after a 24-hour fixation in 4% paraformaldehyde in 0.01 mM PBS (pH = 7.4) in low agitation at 4⁰C, the hemispheres were switched for 10% sucrose solution diluted in PBS and kept overnight at 4⁰C. The block of tissue obtained was then mounted on a Leica Vibratome VT1000S (Nuchloss, Germany) with the rostral surface up and consecutively sectioned in the coronal plane at 40 µm from the olfactory bulb to the caudal limit of the ventral hippocampus. The 40 µm sections were gathered in PBS and then kept at -20⁰C in the Olmos cryoprotectant solution [30% sucrose, 30% ethylene glycol and 1% polyvinylpyrrolidone-40 in PBS] until further processing.
Stereological Analysis Of The Volume Of The Left Hf
To identify the neuronal perikarya brain sections, they were stained with Giemsa solution following the protocol formerly published (Dias-Carvalho, Ferreira et al. 2022). The volume of the left HF was assessed through the Cavalieri’s Principle (Cruz-Orive 1999). The Giemsa-stained sections were analysed using a modified Olympus BH-2 microscope interfaced with a colour video camera and equipped with a Heidenhain ND 281 microcator (Traunreut, Germany), a computerized part, and an object rotator (Olympus, Albertslund, Denmark). A computer equipped with a frame grabber (Screen Machine II, FAST Multimedia, Germany) was connected to the monitor. Based on the Paxinos and Franklin’s mouse brain atlas (Paxinos and Franklin 2019), the outline of the HF and its subregions [(CA3, CA1, dentate gyrus (DG) and hilus) were marked at a total 100x magnification. The volumes of the subregions CA1 and CA2 were determined together due to the difficulty in accurately determining the boundary between these subregions. Assessments were carried out using C.A.S.T – Grid system software (Olympus Albertslund, Denmark) and a mean of 6 systematically sampled sections containing dorsal HF area were used per animal. In each section, the cross-sectional area of each subregion was estimated by point counting (Gundersen and Jensen 1987) using an appropriate grid of test points. The area of the counting frame in the DG and Hilus was 15979.95 µm2 and in CA1 and CA3 was 78913.35 µm2. The volume of each subregion was calculated from the total number of points that fell on each subregion and the thickness of the sections. The coefficient of error (CE) of the individual estimates was calculated according to Cruz-Orive (Cruz‐Orive 1999) and the mean value was 0.10.
Immunohistochemistry
The determination of the total number of GFAP-immunoreactive (GFAP-ir) astrocytes was performed following the previously described method (Dias-Carvalho, Ferreira et al. 2022). As a brief description, free-floating sections containing the HF region and sampled at regular intervals of 480 µm were washed with PBS (15 min x 4) and treated with 10% hydrogen peroxide (H2O2) in PBS for 10 min to inactivate endogenous peroxidase; and the sections were blocked with 5% normal goat serum (NGS) in PBS for 1 hour, at room temperature. Following this, the sections were incubated with the primary antibody polyclonal rabbit anti-GFAP at a 1:1000 dilution, for 72 hours at 4⁰C in PBS with 0.5% Triton X-100. The secondary antibody, biotinylated goat IgG anti-rabbit antibody, was used at a 1:400 dilution for 1 hour, followed by the incubation with avidin-biotin peroxidase complex at a 1:800 dilution for 1 hour. Afterwards, the sections were incubated for 1 min with 0.05% 3,3’-diaminobenzidine (DAB) to which 0.01% H2O2 was added and rinsed with PBS. The numerical density (Nv) of GFAP-ir astrocytes was estimated by applying the physical dissector method, in an average of 6 systematically sampled sections per animal containing the dorsal hippocampal formation. Six to 8 photographs per section were taken at a total 200x magnification in a Zeiss scope A1 Axio microscope coupled with a coloured camera and a computer with the software Carl Zeiss AxioVision Rel. 4.8 (New York, USA). The images were uploaded to the software ImageJ 1.52a and the area of interest was delimited and the number of GFAP-ir astrocytes present in one focal plane and not in the other was counted. The thickness of the sections was also measured, using a microcator applied to the microscope, and the volume was determined using the Cavalieri's Principle (Cruz-Orive 1999). The total number of GFAP-ir astrocytes was calculated by multiplying Nv by the volume of the hippocampal formation.
Immunofluorescence detection of p53, pTau, Bax, Bcl-2, AIF and TNF-α was performed in sections containing the prefrontal cortex (PFC) or the HF, sampled at regular intervals of 480 µm (one out of 12). In detail, the previously sliced sections were washed with PBS (15 min x 4) to remove the Olmos solution and then were incubated in 5% normal horse serum in PBS with 0.25% Triton X-100, for 1 hour at room temperature to block nonspecific binding sites. Sections were incubated with the primary antibodies diluted in PBS with 0.25% Triton X-100, for 72 hours at 4⁰C, in low agitation. The dilution ratio of primary antibodies was as follows: anti-p53 at a dilution of 1:250, anti-pTau at a dilution of 1:1500, anti-Bax at a dilution of 1:1000, anti-Bcl-2 at a dilution of 1:1000, anti-AIF at a dilution of 1:1000, and anti-TNF-α at a dilution of 1:500. The fluorescent secondary antibodies polyclonal anti-rabbit Alexa Fluor 488 (green; A32731) or polyclonal anti-rabbit Alexa Fluor 546 (red; A11035) were added to the samples at a dilution of 1:1000 in PBS with 0.25% Triton X-100 and incubated for 1 hour, at room temperature, protected from light. The slides were then washed in PBS (10 min x 3). The PBS-rinsed sections (10 min x 3) were coverslipped with FluorsafeTM mixed with DAPI at a 1:100 dilution. Photographs of p53, Bax and pTau immunestained sections were taken with identical exposure times, gain and offsets for image acquisition in a Carl Zeiss Axio Imager 2.0 microscope coupled with a coloured camera and a computer with the software Carl Zeiss AxioVision Rel. 4.8 (New York, USA) for image capture. Images were obtained by manually scanning the PFC and the HF at a 20x magnification and at the following excitation/emission wavelengths: 358/461 (for DAPI), 500/525 (for Alexa Fluor 488) and 555/565 (for Alexa Fluor 546). For the measurement of intensity per area, the software ImageJ 1.52a was used.
Statistical analysis
Results are presented as mean ± standard deviation (SD). Statistical analysis was conducted using GraphPad Prism version 8.0.2 (GraphPad Software, CA, USA). Outliers were identified by the ROUT test (Q = 1%) and removed before further statistical analyses. Statistical analysis between the three groups was done by a parametric analysis of variance (ANOVA), followed by the Tukey’s post hoc test, once a significant p was achieved. Statistical significance was accepted at p < 0.05. When p > 0.05 but < 0.1, a tendency is considered.