Reagents and antibodies
The Middlebrook 7H9 and 7H10 media and the OADC (oleic acid, albumin, dextrose and catalase) were obtained from Becton-Dickinson (Detroit MI, USA). The Rneasy® Mini Kit for RNA extraction, the Omniscript® Reverse Transcription Kit for obtaining complementary DNA and the QuantiTectTM SYBR® for RT-PCR were purchased from Qiagen (Germantown, MD, USA). The primers of the analysed genes and pierce BCA Protein Assay kit for protein quantification were got from InvitrogenTM Thermo Fisher Scientific (Waltham, MA, USA). The standards for dopamine, serotonin, epinephrine, and norepinephrine, as well as ascorbic acid, L-cysteine, bovine serum albumin (BSA), phenylmethylsulfonyl fluoride (PMSF) and α-tubulin antibody (T9026), were obtained from Sigma Chemical Co. (St. Louis, MO, USA). HPLC grade acetonitrile, perchloric acid, and EDTA were purchased from J.T. Baker (Mexico City, Mexico).
Primary antibodies against phospho-JNK (4671), JNK (9252), phospho-p38 (9215) and p38 (9212), were purchased from Cell Signaling Technology (Danvers, MA, USA). Primary antibodies against BDNF (ab72439) was obtained from Abcam Inc. (Cambridge, MA, USA). Secondary antibodies against rabbit (711-035-152) and mouse (715-035-150) were purchased from Enzo Life Sciences (Farmingdale, NY, USA). Primary antibodies against TNFα (SC-1351), IFNγ (SC-1379), IL1β (SC-1251), IL4 (SC-1260), iNOS (SC-651), TGFβ (SC-146) and indolamine 2-3 dioxygenase (IDO) (SC-13147) were purchased from Santa Cruz Biotechnology Inc. (Dallas, TX, USA).
Fluoro-jade B was purchased from Millipore (Bedford, MA, USA). All the other reagents were obtained from known commercial sources.
Animals
A total of 560 adult male BALB/c mice eight weeks old were obtained from the animal house facility of the National Institute of Medical Science and Nutrition Salvador Zubiran (INCMNSZ), Mexico. Mice were group-housed (n=5/cage) and randomly divided into two groups: control (CT, n=256) and infected (H37RV, n=304). All the animals were kept in an Accredited animal holding facility maintained at a controlled temperature (23 ± 1° C) and humidity (50 ± 20%) under a 12:12 h light: dark cycle (lights on at 07:00 h). Food and water were provided ad libitum. All the animal experiments were done according to the guidelines of the ARRIVE and Mexican Constitution law NOM 062–Z00-1999, and approval by the Ethical Committee for Experimentation in Animals of the INCMNSZ in Mexico protocol number: PAT-1865-16/19-1.
The experimental model of pulmonary TB
The murine model of progressive pulmonary TB was described previously [16]. Briefly, the reference Mtb strain H37Rv was cultured in 7H9 medium with OADC enrichment. Mid-log-phase cultures were used for all experiments. Mtb were counted and stored at -80°C until use. Bacterial aliquots were thawed and pulse-sonicated to remove clumps. After mice infection, the remnant of the bacterial inoculum was plated to confirm the number and viability of colony-forming units (CFU) administered to the animals. Male BALB/c mice, 8 weeks of age, were anaesthetized in a gas chamber using 0.1 mL per mouse of sevoflurane. A blunt stainless steel cannula with a small ball in its terminal end was inserted through the mouth and directed to the trachea, proper intratracheal placement of the cannula was verified by palpation of the small ball from the cannula rubbing the tracheal rings. Mice were infected through intratracheal instillation with 2.5 × 105 live bacilli.
Mice were maintained in a vertical position until spontaneous recovery. A total of 304 infected mice were maintained in groups of five in cages fitted with micro-isolators in a P-3 biosecurity level facility.
Experimental design
We analyzed the effects of pulmonary TB in the CNS inflammation and its relationship with behavioural changes. Following infection, mice were euthanized by exsanguination under anaesthesia at days 1, 3, 7, 14, 21, 28, 60 and 120 post-infection; lungs and brain were collected immediately to determine bacillary loads by colony-forming units counts (CFU). In selected areas of the brain were quantified cytokines gene and protein expression by quantitative reverse transcription-polymerase chain reaction (RT–PCR) and immunohistochemistry (IHC), respectively, the concentrations of the neurotransmitters by high-performance liquid chromatography (HPLC) and the histopathology alterations. A total of 256 non-infected mice (CT group) received only the vehicle (saline solution) by intratracheal route following the same procedure and were used as controls. Different behavioural tests were performed during pulmonary TB. These tests include the study of sickness behaviour (locomotor activity, food intake and weight loss), anxiety-like behaviour, neurological severity score, short and long-term memory and depression-like behaviour.
Determination of colony-forming units (CFU) in infected lungs and brain
Right lungs and brains from six mice at each time point of two independent experiments were used for bacterial colony counting. Lungs and brains were homogenised with a FastPrep homogeniser (MP Biomedicals) in sterile tubes containing 1 ml of isotonic saline solution. Four dilutions of each homogenate were spread onto duplicate plates containing Bacto Middlebrook 7H10 agar, enriched with OADC. Incubation time and CFU counting was at 21 days of plating [17].
Expression of cytokines by RT-PCR
Hippocampus, hypothalamus and cerebellums from six CT and infected animals at each time point were used to isolate mRNA using the Rneasy Mini Kit, according to recommendations of the manufacturer. Quality and quantity of RNA were evaluated through spectrophotometry (260/280) and on agarose gels. Reverse transcription of the mRNA was performed using 100μg RNA, oligo dT and the Omniscript kit. Real-time PCR was performed using the 7500 RT-PCR system (Applied Biosystems, USA) and Quantitec SYBR Green Mastermix kit (Qiagen). Standard curves of quantified and diluted PCR product, as well as negative controls, were included in each PCR run. Specific primers for genes encoding glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as housekeeping gene and for TNF-α, IFN-γ, Interleukin (IL) 12, IL-10, IL-4, TGFb, iNOS and IDO were designed using the program Primer Express (Applied Biosystems, USA). The gene expression of these cytokines was determined as previously described [17]. Cycling conditions used were: initial denaturation at 95 °C for 15 min, followed by 40 cycles at 95 °C for 20 s, 60 °C for 20 s, and 72 °C for 34 s. Quantities of the specific mRNA in the sample were measured according to the corresponding gene-specific standard.
Neurotransmitter quantification by HPLC
The neurotransmitters quantification was previously described [9]. Briefly, the hippocampus, cerebellum and the hypothalamus of six CT and infected animals were homogenised using 400 µl of a solution containing 5% ascorbic acid, 200mM sodium phosphate, 2.5 mM L-cysteine, and 2.5mM EDTA. Proteins were precipitated by the addition of 100 µl of 0.4 M perchloric acid, followed by incubation at 20 °C for 20 min. Supernatants containing norepinephrine (NE), epinephrine (EP), dopamine (DA), and serotonin (5-HT) were collected after centrifugation at 16128 g for 10min (4°C). NE, EP, DA, and 5-HT concentrations were determined by reversed-phase HPLC (RP-HPLC) in a system integrated by two 515 pumps (Waters™), degasser AF (Waters™), 717 autosampler (Waters™), and an X-LC™3120FP fluorescence detector (Jasco, Inc). Instruments were controlled by Millenium 32 software (Waters™). Chromatographic runs were performed using a Jupiter C18 column (300Å, five μ, 4.6×250mm, Phenomenex®) at 30°C. The column was equilibrated with the mobile phase A (MPA) containing 0.1% trifluoroacetic acid. Mobile phase B (MPB) containing 0.1% trifluoroacetic acid in acetonitrile was used to perform a linear gradient until reaching 20% MPB, from min 5 to min 15. Ten, 20% MPB was maintained until min 20; the flow rate was 0.8 ml/min. The fluorescence detector was set at gain 100, attenuation 32, response 20s, and 280nm and 315 nm for excitation and emission, respectively. The sample injection volume was 50μl. The concentration of each neurotransmitter was obtained by mg of protein in each sample. The concentration of total protein was obtained with the Pierce BCA protein assay kit.
Behaviour tests
In order to avoid potential habituation to the test, groups of mice were tested only once at the mentioned time points post-infection in control and infected mice. Animals were habituated to the test environment 24 h before that test was made. All behavioural trials were performed during the first 4 h of the dark phase of the light cycle.
Locomotor activity
The effect of Mtb lung infection on locomotor activity (LMA) was evaluated in mice individually positioned into a clean, novel cage similar to the household pen, but devoid of bedding or litter allowing the mouse to move between compartments freely. The box was divided into eight quadrants, animals were video recorded, and LMA was measured by counting the number of crossings for 10 min.
Food intake
In order to estimate the food intake, twice a week, the amount of food given to mice was weight, and the total ingesting of food by mice was calculated as is indicated in the next formulation where n is the number of mice per cage. Data are expressed as g/mouse/day.
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)
Weight loss
The weight loss of the animals infected with Mtb was estimated from day one post-infection until day 120. Each week the animals were weighted, and their loss weight recorded as a feature of sickness behaviour.
Depression-like behaviour
The tail suspension test [18] was carried out to evaluate depression-like- behaviour. Animals were suspended from the tail for 6 min in a tripod of 30 cm height, and their activity was recorded, focusing at the moment when the animal stopped moving. The time that the animal remains both mobile and immobile for 6 min was recorded.
Anxiety-like behaviour
The anxiety-like behaviour was evaluated studying the Stretched attend posture (forward elongation of the body) [19]. In this test was used a large circular platform raised at 35-40 cm from the floor, and a small circular awning fastened by the central pillar was placed at 4 cm. Each mouse was located in the covered area of the platform and filmed for 5 min. The number of stretched attend postures was counted as a measure of anxiety-like behaviour.
Neurological Severity Score (NSS)
Motor function and reflexes of the infected mice were evaluated using a neurological severity score [20]. It was valued regarding absent (0) or present (1), except for the hypomobility, motor impairment and balance that is rated as weak (1), moderate (2) or strong (3). The maximum rating of 31 (indicating neurological damage). Usual, from 3 to 6 ( For more details, see Additional File 1).
Evaluation of learning and memory.
Memory and learning after pulmonary infection with Mtb were assessed with the Object Recognition Test [21]. With this test, we evaluated short-term and long-term memory. In a first habituation phase, we placed the animal in the open field without any object for 10 min to become familiar with the environment. At 24 h, two identical objects (objects A) were placed in different positions, and the animal was left inside the box for 3 min. In the next phase, short-term memory was measured, for this we positioned an object A (familiar object) and placed a new object in the other position (object B), the interactions with both objects (the animal sniffs or touches the object with the front legs) were counted during 3 min. After 24 h, the long-term memory was measured, for which object B was changed to a novel object (C), and the same procedure was followed. The results are presented as the discrimination ratio, which is the difference in interactions expressed as a proportion of the total interactions with the two objects in both tasks.
Preparation of brain tissue for histological analysis
Four mice per selected time point were anaesthetised with sodium pentobarbital (100mg/kg, i.p.) and immediately perfused transcardially with isotonic saline solution, followed by cold 4% paraformaldehyde solution diluted with Sorensen’s phosphate buffer (0.133 M, pH 7.2). Brains were removed and postfixed in 4% paraformaldehyde for 24 hours and embedded in paraffin. Coronal sections 4μm thick mounted on glass slides, deparaffinised, and stained with hematoxylin and eosin. For quantification of tissue damage, five fields of regions CA1, CA2, CA3, and dentate gyrus (DG) of the hippocampus were analysed under a light microscope Q-win Leica 500 to estimate the number of neurons showing morphological damage (cell shrinking, condensed hyperchromatic nucleus, basophilic cytoplasmic material disappearance, or cytoplasmic vacuoles). The results are presented as a percentage of injured neurons in hippocampal regions.
Immunohistochemistry (IHC) analysis
The same brain tissue samples for the histological study were used for IHC. Coronal sections (4 μm thick) were mounted on poly-L-lysine-coated slides and then deparaffinized to detect activated microglia (IBA-1, 1:3000), gliosis (Glial fibrillary acidic protein GFAP, 1:3000), TNFa (1:250), IFNg (1:250), IL4 (1:250), and TGFb (1:200). After deparaffination, tissue sections were blocked for the unspecific activity of endogenous peroxidase with 3% hydrogen peroxide solution and methanol by 10 min. Subsequently, sections were incubated with each antibody and then incubated with goat anti-rabbit IgG-peroxidase diluted 1:500 for 30 min at room temperature. After extensive washings with PBS, the bound antibodies were detected with diaminobenzidine and counterstained with hematoxylin.
Localization of neuronal damage in the hippocampus by Fluoro-jade B (FJ-B) staining
FJ-B staining is a useful technique to determine damaged neurons that are still alive (degenerating neurons) and was done according to the published protocol [22]. Briefly, tissue sections 8 µm thick were deparaffinised and immersed in a solution containing 1% NaOH in 80% ethanol for 5 min, followed by 2 min in 70% ethanol and 2 min in distilled water. Afterwards, the sections were immersed in a 0.06% KMnO4 solution for 10 min under moderate moving and then cleaned in distilled water for 2 min. Tissue sections were stained with 0.0004% FJ-B solution for 20 min and washed three times with distilled water. The slides were dried in an oven at 50°C for 15 min and cleared with xylene before coverslipping. Two random fields were selected in each mouse from the different experimental groups. Results are expressed as the number of FJ-B positive cells per field.
Study of the activation of MAPK signalling by Western blot assays
In order to study if the mitogen activated protein kinases (MAPK) are involved in the cellular damage, the expression of JNK and p38 activated (phosphorylated) and non-activated was determined by western-blot, as well as the levels of the neurotrophin brain derived nerve growth factor (BDNF) using the same technique. The hippocampus, hypothalamus and cerebellum were dissected quickly and homogenized in 500 mL of lysis buffer pH 7.9 (containing 20 mM Tris HCl, 30 mM NaCl, 0.5 mM sucrose, 1 mg/mL leupeptin, 1 mg/mL aprotinin, 1 mg/mL pepstatin, 1 mg/mL PMSF and 1 mg/mL phosphatases inhibiting cocktail) and centrifuged at 20,800 g for 30 min at 4° C. The supernatants were used to determine the protein quantity using Lowry’s technique and in the western blots assay. Briefly, 50µg of the total lysate of protein were loaded and separated in 10% or 12% SDS polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Bedford, MA, USA). Membranes were blocked using 5% BSA for 2 h at room temperature with slight agitation. Blots were then incubated with anti-phospho-JNK (1:1,000) at room temperature for two hours, anti-JNK (1: 1,000), anti-phospho-p38 (1:1,000), anti-p38 (1:1,000), anti-BDNF (1:1,000), or anti-β-tubulin (1:8,000) at 4ºC for overnight. Membranes were washed three times (10 min) with TBS plus 0.1% Tween (TBS-T). A horseradish peroxidase-conjugated secondary polyclonal antibody anti-rabbit (1:10,000) and anti-mouse (1: 10,000) was then added for 2 h and after extensive washing. Bands were detected using the enhanced chemiluminescence of detection system (ECL, Amersham Pharmacia Biotech, USA). The membranes were washed with stripping solution (containing 0.2 M glycine, 0.1% SDS and 1% Tween 20, pH 2.2) for the detection of two or more proteins. The chemiluminescence imaging system Fusion Solo S (Eberhardzell, Biberach, Germany) was used. Areas values were obtained from the relationship between the optical densities (OD) of each band. Area values of each group were standardised to the area value of the control group (value= 1). Data are expressed as the OD ratio using ImageJ software.
Assessment of blood-brain barrier dysfunction using Evans Blue staining
We analysed the blood-brain barrier (BBB) dysfunction, according to a previously published protocol [23]. Briefly, mice were anaesthetised with sodium pentobarbital (100 mg/kg, i.p.) and injected with Evans blue (EB) (2%, 2 ml/kg) in the caudal vein 3 h before perfusion. Mice were perfused transcardially with isotonic saline solution and directly beheaded, stripping brain tissue on ice. Each brain was weighed and then homogenised with a FastPrep homogeniser (MP Biomedicals) in 0.25 ml of 100% TCA and 0.75 ml of PBS solution. Samples were cooled overnight at 4 °C and then centrifuged for 30 min at 1,000 x g at 4 °C. The EB in the supernatants of 100 μl of each sample was then measured at 620 nm using a 96-well plate reader. All measurements were within the range of detection established by a standard curve. The dye concentration was considered as the ratio of absorbance relative to the amount of tissue.
Statistical analysis
Data are expressed as the mean ± standard error of the mean (SEM) of two independent experiments. The statistical analysis was performed using Prism software version 7.0 (GraphPad, San Diego, CA, USA). For the survival curve, the Log-Rank and Kaplan-Meier test was applied. Cytokine expression by RT-PCR, behavioural assays, loss of body weight, food intake and neurotransmitter levels were analysed by Two Way ANOVA followed by Sidak multiple comparison tests (comparison of each group against the control). A value of p£0.05 was considered statistically significant.