Selected cannabis cultivars modulate glial activation: in vitro and in vivo studies | Journal of Cannabis Research

Plant cultivation: selected cannabis cultivars

Female plants from three cultivars of medical cannabis (CN2, CN4, CN6), 10 plants per cultivar, were produced from cuttings and grown simultaneously under uniform cultivation conditions in a controlled environment growing room as previously described (Shiponi & Bernstein 2021a, 2021b). In short, plants were cultivated in 4 L plastic pots in a perlite cultivation media (Agrekal, Habonim, Israel). During the vegetative growth phase, for 2 weeks, the plants were exposed to a 18/6 hrs light/dark photoperiod using Metal Halide bulbs (400 μmol m^-2 s^-1, Solis Tek Inc, Carson, California (Saloner & Bernstein 2020). For the remainder of the cultivation period, until flower maturation, the plants were grown under 12:12 light/dark photoperiod using High-Pressure Sodium bulbs (980 μmolm^-2 s^-1, Greenlab by Hydrogarden, Petah Tikva, Israel) (Saloner & Bernstein 2021). In the cultivation room, temperatures were kept at 26 and 24˚C day/night, respectively, and relative humidity at 44% and 60%, respectively. Irrigation was supplied via 1L h^-1 discharge-regulated drippers (Netafim, Tel-Aviv, Israel) and mineral nutrients were supplied by fertigation, dissolved in the irrigation solution (Shiponi & Bernstein 2021a, 2021b).

Drying

Inflorescences were harvested when about 30% of the trichome heads were of amber color, following the maturation stage acceptable for commercial harvesting. The inflorescences were immediately trimmed (wet-trimmed) to separate the protruding parts of the inflorescence leaves from the inflorescence. The trimmed inflorescences were then dried in an environmental-controlled chamber, in the dark, at 19˚C and 45% relative humidity until the plant material reached ~10% humidity, as is customary in the medical industry for material supplied to patients. Ten g of material sampled from 10 different primary inflorescences were ground thoroughly using a manual grinder, to create a homogenous mixture.

Extraction and quantitative analysis of cannabinoids and terpenes in the cannabis cultivars

One gram of ground plant material was extracted in a shaker for 60 min in 30 ml of absolute ethanol and concentrated under reduced pressure. Dried extracts were heated for 30 min at 110°C to decarboxylate the cannabinoids from their natural acidic state to their neutral form. Samples were redissolved in ethanol to a final concentration of 10 mg/ml for further chemical and biological analysis. Cannabinoid quantification was performed as indicated (Aizpurua-Olaizola et al. 2014). A Waters Alliance 2695 Separation Module with a Waters 996 Photodiode Array Detector together with a Micromass Quattro Micro Triple Quadrupole Mass Spectrometer using a Phenomenex Kinetex C18 column (2.6 μm, 150 mm×3 mm i.d.) with guard column and a binary A/B gradient (solvent A: water with 0.1% formic acid, and solvent B: MeOH with 0.1% formic acid). Initial conditions were 65% B for 10 mins, raised to 95% B over the next 20 min, held at 95% B for 15 min, decreased to 65% B over the next 5 min, and held at 65% B for 10 min for re-equilibration of the system. The flow rate was 0.2 mL/min and the column temperature was 30 °C. Mass spectra acquisition was carried out in the ESI positive ionization mode under the following conditions: capillary voltage – 3.5 kV, cone voltage – 45 V, extractor voltage – 3 V, RF lens – 0.2 V, source temperature – 120 °C, desolvation temperature – 350 °C, nitrogen flow rate of 700 L/h for desolvation and 50 L/h cone gas. Quantification of the selected cannabinoids was performed using single ion monitoring (SIR) mode. Standard solutions of the selected cannabinoids (THC, THCA, CBD, CBDA, cannabigerol (CBG), cannabigerolic acid (CBGA), and cannabinol (CBN) were obtained from Merck (Rehovot, Israel). For each compound, serial dilutions were performed, and calibrations curves generated for concentrations from 0.5-100 µg/ml using SIR of the molecular ion [M+H].

For terpene analysis, 1 µl of the ethanolic extract was injected in a Hewlett Packard G 1800B GCD system with a HP-5971 gas chromatograph using a SPB-5 (30 m × 0.25 mm × 0.25 um) column with an electron ionization detector, using GCD Plus ChemStation (Hewlett Packard, Santa Clara, CA, USA) and a helium flow rate of 1 ml/min. Initial conditions consisted of an inlet temperature of 250 °C; detector temperature of 280 °C; and an initial temperature of 50 °C. The initial temperature was held for 5 min before increasing at a rate of 5 °C/min to a final temperature of 280 °C. Terpenes were identified based on retention time and mass spectra and quantified based on calibration curves generated from standards.

Cell culture

BV2 murine microglial cells

Murine BV2 microglia were kindly provided by Professor Rosario Donato (Department of Experimental Medicine and Biochemical Sciences, University of Perugia, Italy) (Adami et al. 2004). Microglia were grown in RPMI-1640 medium with 10% fetal calf serum (FCS), streptomycin (100 µg/mL), penicillin (100 U/mL) and L-glutamine (4 mM) in 5% CO₂ humidified air incubator at 37 °C. The culture medium was replaced twice a week.

For each experiment, cells were grown overnight on 6-well and 24-well plates at a concentration of 1×10⁶ and 3×10⁵ cells per well, respectively. Serum-free medium (SFM) was added to the cells 4 h before initiation of the experiment. Subsequently, microglia were treated with SFM containing 0.1% bovine serum albumin (BSA), 4-(2-hydroxyethyl)piperazine-1-ethane-sulfonic acid (HEPES) buffer (10 mM at pH 7.4), and 1% FCS in the presence of Lipopolysaccharides (LPS) (7 ng/ml) with or without extracts from 3 cultivars (CN2, CN4, CN6) at different concentrations (1, 5, 10, 20 µg/ml) for 22 h. All culture media were purchased from Biological Industries (Kibbutz Beit-Haemek, Israel). LPS from Escherichia coli O55:B5, was purchased from Sigma Aldrich (Rehovot, Israel).

Primary neonatal rat glial cells

According to well-established protocols, primary glial cell cultures were prepared from whole brains of 1-day old Wistar rats (Adami et al. 2004; Levant et al. 2006). Cells were grown in 24-well plates coated with poly-L-lysine (0.25 mg/mL) at 37 °C in 5% CO₂. Dulbecco’s modified Eagle’s medium (4.5 mg glucose/mL), supplemented with 10% FCS, L-glutamine (2 mM), penicillin (100 U/mL), streptomycin (100 mg/mL) and insulin (0.2 U/mL) was used as the culture medium. As previously described (Filipovich-Rimon & Fleisher-Berkovich 2010), immunocytochemistry studies revealed that these cultures contain about 80% astrocytes and about 20% microglia. Before the experiment, cells were incubated in SFM for 4 h. LPS 500 ng/ml with or without extracts from 3 cultivars (CN2, CN4, CN6) (50 µg/ml) was added to SFM containing 0.1% BSA and HEPES (10 mM) for 22 h. At the end of each experiment, cells were collected with cold SFM (4 °C) and counted using the Z1 Coulter counter (Coulter Electronics, Miami, FL, USA).

Cell viability

Cells were seeded at a density of 1×10⁴ cells per well in 96-well plates and cultivated overnight in a complete RPMI-1640 medium (as described in 2.1.1). Increasing concentrations of CBG were given for 22 h following pre-incubation with SFM. Dimethylsulfoxide (DMSO) at a concentration of 0.026 % was used as a control. Actinomycin D, a transcription inhibitor at a concentration of 1 μM, was applied to inhibit cell proliferation. (2,3-Bis-(2-Methoxy-4-Nitro-5-Sulfophenyl)-2H-Tetrazolium-5-Carboxanilide) (XTT) reagent [2,3-Bis-(2- methoxy 4-nitro-5-sulfophenyl)-2H-tetrazolium5-carboxanilide] was mixed with the activation reagent, N-methyl dibenzopyrazine methyl sulfate, in a ratio of 50:1 according to the manufacture’s protocol (Biological Industries, Kibbutz Beit-Haemek, Israel). Subsequently, XTT solution mix was added to each well in a 1:2 ratio, and plates were incubated at 37 °C for an additional 1 h. Absorbance was measured at 450 nm against a reference wavelength at 650 nm using a microplate reader (Model 680, Bio-Rad, Hercules, CA, USA). Cell viability was calculated using the following formula: [A₄₅₀–A₆₅₀] of test cells × 100/[A₄₅₀-A₆₅₀] of control cells.

Determination of NO levels (Griess reaction)

As an indicator for NO release, nitrite levels in culture media were determined by an established assay using Griess reagent (Sigma Aldrich, Rehovot, Israel). A standard curve of sodium nitrite was used. 100 µL of culture medium and Griess reagent were mixed in a 96-well plate and incubated at room temperature for 15 min in the dark. Then, absorbance at 540 nm was measured using a microplate reader (Model 680, Bio-Rad, Hercules, CA, USA). Cells were harvested with cold SFM (4°C) and counted using the Z1 Coulter counter (Coulter Electronics, Miami, FL, USA).

Determination of TNFα levels (ELISA)

TNFα levels in the culture media were determined by enzyme-linked immunosorbent assay (ELISA) (BD Biosciences, San Diego, CA) according to the manufacturer’s protocol.

Western blot analysis

Proteins were separated on polyacrylamide-SDS (7.5%) gels and transferred to nitrocellulose membranes. After blocking with 4% BSA for 90 min at room temperature, membranes were incubated overnight at 4 °C with a specific rabbit anti-iNOS antibody (1:500, Cayman Chemicals, USA). Upon washing, the blots were incubated for 90 min at room temperature in the corresponding conjugated donkey anti-rabbit antibody (1:10,000, GE Healthcare, Buckinghamshire, UK). The position of the individual protein was detected using enhanced chemiluminescence (ECL) solution followed by exposure to the ChemiDocTM XRS+ (Bio-Rad Laboratories, Hercules, CA, USA) image system. Band intensity analysis was performed using a computerized image analysis system (ImageJ software, version 1.40C, NIH). Protein quantity was normalized to β-actin protein level measurements using mouse monoclonal anti-β-Actin−Peroxidase antibody (1:20,000, Sigma Aldrich, Rehovot, Israel).

Active MOG-induced EAE model

Female C57BL/6 mice, eight weeks old (~20 g) (Envigo, Jerusalem, Israel), were grown for one week and then immunized with myelin oligodendrocyte glycoprotein (MOG) [peptide 35–55] (AnaSpec, Fremont, CA, USA). Under anesthesia with isoflurane, each mouse was injected subcutaneously (SC) into two sites on the back, adjacent to each of the hind limbs (total volume 200 μL), with 200 μg MOG emulsified with a mixture of 200 μg/mL killed Mycobacterium tuberculosis H37RA (Difco, Detroit, MI, USA) in complete Freund’s adjuvant (BD Biosciences, USA). After that, each animal was injected i.p with reconstituted pertussis toxin (ENCO scientific services, Petach Tikvah, Israel) (400 ng/mL), which was repeated two days after the initial immunization.

Control EAE mice received a vehicle solution composed of Tween-20: ethanol: saline at a ratio of 1:1:8. Wet samples were dissolved in a vehicle solution. IP administration was used for seven consecutive days from day 7 to 14 post-immunization (p.i.). The mice were evaluated for a neurological score as follows: 0 normal; 0.5 mild ataxia of the hind limb; 1 decreased tail tone; 1.5 righting reflex within 3 s; 2 righting reflex between 4 and 7 s; 2.5 righting reflex between 7 and 10 s; 3 hind limbs paralysis or complete loss of righting reflex; 4 front and hind limbs paralysis; 4.5 moribund state; 5 death. At the end of the experiment, mice were anesthetized, and cardiac perfusion was performed. The spinal columns were fixed in 4% formaldehyde at 4 °C overnight and cryoprotected in 20% sucrose for 48 h at 4 °C. Then, spinal cords were dissected and mounted in OCT (Scigen Scientific Gardena, CA, USA), snap-frozen at -40 °C, and finally stored at – 80°C for further procedure. Free-floating sections (30 µm thick) were blocked for 1 h in blocking buffer [Antibody diluent (GBI Labs, Mukilteo, WA, USA) with 0.5% Triton], immunostained overnight at 4 °C with antibodies diluted in antibody diluent with 0.15% Triton. The following antibodies were used: monoclonal anti-glial fibrillary acidic protein (GFAP, clone GA5; 1:400) [MAB360, Millipore], rat anti-CD4 (clone RM4-5; 1:50) [BD Pharmingen] and rabbit anti-Iba1 (1:1,000) [FUJIFILM Wako Pure Chemical Corporation]. The next morning, sections were washed three times with 0.05 % Tween 20 in PBS, then incubated for 1 h at room temperature with a fluorescent-conjugated secondary anti-rat, anti-mouse, or anti-rabbit antibody (1:200, Alexa Fluor 647; Jackson). Subsequently, DAPI was applied for nuclear staining. Sections were mounted on slides with Immu-Mount (Thermo Scientific, MI, USA). Images were obtained using the Olympus FluoView FV1000 confocal microscope (Olympus, Hamburg, Germany) at a 1024×1024 pixel resolution.

Primary splenocytes

Splenocytes were isolated 15 days p.i by mashing the splenic tissue through a 2 μm filter and washing with PBS+EDTA 1mM. The collected fluid was once again passed through the strainer into a test tube and cells were centrifuged at 4 °C. The cell pellet was incubated with red blood cells (RBC) lysis buffer Ethylenediaminetetraacetic acid(EDTA) 10mM + NaHCO3 0.1M + NH4Cl 1.5M in DDW, 2 mL buffer/a spleen) for 10 min at room temperature. The RBC-lysed splenocytes were washed with PBS+EDTA 1mM and resuspended in the culture medium (DMEM) supplemented with 10% bovine calf serum, 1% penicillin/streptomycin, Sodium pyruvate (1%), HEPES buffer (10 mM at pH 7.4), 1% non-essential amino acids, and 0.02% 2-mercaptoethanol. Cells were seeded in a 96-well plate in U-shaped wells and incubated at 37 °C for 48 h, at which time the supernatants were removed for analysis by enzyme-linked immunosorbent assay (ELISA).

Imaging analysis

Glial fibrillary protein (GFAP), ionized calcium binding adapter molecule 1 (Iba1) and cluster of differentiation 4 (CD4) staining were quantified in lumbar sections from the spinal cord of each mouse using ImageJ software (version 1.40C, NIH) with the threshold function. An intensity threshold was set to mark only those areas showing significant staining. Identical laser‐scanning parameters were used for all samples. The indicated proteins’ averaged positive‐stained areas were calculated separately for each treated group and plotted as integrated density.

Statistical analysis

Experimental data are presented as means ±SEM. For significance assessment between groups, a one-way analysis of variance (ANOVA) and post hoc multiple comparison test (Tukey-Kramer Multiple Comparison Test) were performed using Prism version 5.00 for Windows, GraphPad Software, (San Diego, California, USA). Statistical significance was considered at p