Materials
Paclitaxel (Sigma Aldrich, MO, USA), synthetic Cannabidiol (PurisysTM, Athens, GA), Tetrahydrocannabivarin (Open Book Extracts, Roxboro, NC, USA), WAY100135 (Sigma Aldrich, MO, USA), Rimonabant (Sigma Aldrich, MO, USA), WAY100135 (Sigma Aldrich, MO, USA), AM630 (Sigma Aldrich, MO, USA), HAMS F12 media (Millipore Sigma, St. Louis, MO, USA), Neurobasal Media , N2 supplements, NGF, MITOSOX Stain, Neurite outgrowth assay kit, TPER, fetal bovine serum (Atlanta biological, MN, USA). Unless specified all the chemicals (GLP/GMP grade) were purchased from Sigma Aldrich, USA.
Invivo
Animals
C57BL/6J mice (4-5 weeks old) and male Sprague Dawley rats (7-8 weeks old) were provided by Envigo (Indianapolis, IN) for the current study on PIPN. FAMU has AAALAC-accredited animal facilities, and following NIH recommendations (Guide for the care and use of laboratory animals), the current protocol was evaluated and approved by Florida Agricultural and Mechanical University's Institutional Animal Use and Care Committee (protocol numbers: 020-06 & 021-04). The animals were sacrificed using the carbon dioxide (CO2) asphyxiation procedure.
Study design
To establish peripheral neuropathy, C57BL/6J female mice (4-5 weeks old) were given PTX (8 mg/kg, i.p.) every other day for four injections. In first set of experiments, animals were divided into three groups after they had developed neuropathy: a. untreated age-matched normal mice, b. PTX (8 mg/kg, i.p.) every other day for four injections, c. cannabidiol (CBD) group: animals were given 10 mg/kg CBD (i.p.) twice a week for six weeks following the previous PTX injection. d. Tetrahydrocannabivarin (THCV) group: animals were given 15 mg/kg of THCV (i.p.) twice a week for a total of six weeks after the last dose of PTX injection, e. Cannabidiol (CBD) + Tetrahydrocannabivarin (THCV) group: animals were given 10 mg/kg of CBD and 15 mg/kg of THCV (i.p.) and in the second set of experiments, the effects of CB1, CB2, and 5HT1A blockers on CBD and THCV-induced neurobehavioral changes in PTX-induced neuropathic mice were studied by dividing the animals into six groups, a &b: (PTX+CB1B+CBD or THCV): CB1 blocker (CB1B) 3 mg/kg/day, i.p., was given to mice for four weeks and three hours before administering CBD or THCV. c&d: (PTX+5HT1AB+CBD or THCV): 5HT1A blocker (5HT1AB), 10 mg/kg/day, i.p., was given to mice for four weeks and three hours before administering CBD or THCV e&f: (PTX+CB2B+CBD or THCV): CB2 blocker (CB2B) 1 mg/kg/day, i.p., was given to mice for four weeks and three hours before administering CBD or THCV. The PTX, CBD, THCV, AM630, Rimonabant and WAY10065 dosages were determined using existing literature reports [16-21]. After the animals were euthanized using CO2 asphyxiation and dorsal root ganglions (L1-L5) were extracted, biochemical and molecular parameters were examined.
Behavioral studies
Thermal hyperalgesia
Hargreaves Plantar Test: This test was carried out with some modifications [22,23]. Briefly, the animals were housed in 12 plexiglass enclosures kept at a constant temperature (30°C) above a horizontal glass surface. With a cut-off time of the 20s, the time it took a mouse to lift its right or left paw after being exposed to a radiant heat source of infrared irradiation (40 IR units) was measured. The results are reported in seconds as paw withdrawal latency, with a 10-minute time interval between each consecutive reading.
Hot and Cold plate method
Thermal hyperalgesia was measured using the hot and cold plate method, as previously reported [24]. Immediately after acclimatization, the mice were placed on a hot plate (55°C) and a cold plate (10°C) where the time latency for the animal to lick its right/left foot was measured with a cut-off time of 20s and an interval of 10 minutes at each reading, and the results were reported as paw withdrawal latency in seconds.
Mechanical Hyperalgesia
The Vonfrey and Randall Selitto tests were used to assess mechanical hypersensitivity in mice. The mice were poked with standard vonfrey fibres of varying weights (g), and the weight at which they lifted their paws was monitored using a digital electronic readout unit and reported as paw withdrawal threshold (g). The paw withdrawal pressure of mice was measured using Randall sellito pincture pressure on both paws. Each animal was tested five times, with a 10-15 minute delay between each reading [25].
RNA sequencing
RNA sequencing on DRG homogenates from control, PTX, CBD, and THCV-treated mice was performed by Novogene Corporation Inc (Sacramento, CA). Briefly, messenger RNA was isolated from total RNA using poly-T-oligo magnetic beads. This was followed by second strand cDNA synthesis using either dUTP or dTTP depending on the library type. Except in directed library preparation, user enzyme digestion was included after size selection. The library was quantified with Qubit, and the size distribution was detected with a bioanalyzer. Quantified libraries were pooled and sequenced on Illumina platforms. Clustering and sequencing were done following manufacturer's instructions. After generating clusters and paired-end reads, the library preparations were sequenced on Illumina. Initially, quality control of raw data in fastq format was handled using perl programs. Raw reads were cleaned by eliminating adaptor, poly-N, and low quality reads. The cleaned data Q20, Q30, and GC content were calculated and used for downstream analysis. For better mapping results, clean reads were mapped to the reference genomes using Hisat2 v2.0.5, which developed a database of splice junctions based on the gene model annotation file. Feature Counts v 1.5.0-p3 was used to count the reads mapped to each gene. In order to assess gene expression levels, FPKM (fragments per kilobase of exon per million mapped fragments) was calculated. DESeq2 R software was used to analyze differential gene expression (1.20.0). In order to reduce the false discovery rate, Benjamini and Hochberg's procedure was used. Genes with a P < 0.05 difference across groups were considered differentially expressed. The online Kyoto Encyclopedia of Genes and Genomes (KEGG) database (http://www.genome.jp/kegg/) was used to examine the statistical enrichment of differential expression genes in KEGG pathways. The effective genes and critical pathways in regulating neuronal function were predicted using Reactome, disease ontology, and DisGeNET databases.
Western blotting
For this study, separated DRGs were homogenized in TPER (1:100), centrifuged at 20,000 rpm for 20 minutes, and the supernatant was collected and evaluated for total protein content using a bicinchonic acid test kit. To denature the proteins, the sample was heated to 95°C for 10 minutes with 4X laemmli buffer containing 5% mercapto-ethanol. SDS PAGE gel electrophoresis resolved 40 g protein. Semi-dry transfer of resolved gel containing segregated proteins to PVDF/nitrocellulose membranes (Transbloto, Biorad, USA). Incubation with primary antibodies (P-38 MAPkinase, p-AMPK, PI3K, p-AKT, TFAM, HO-1, Catalase, Nrf2, Bax, caspase 3, caspase 1, caspase 9 and TGF-beta of rabbit or mouse origin) at 4°C overnight followed by three washes with PBST. We next probed the membranes for 2 hours at room temperature with HRP conjugated secondary anti-rabbit and anti-mouse antibodies. The ChemiDocTMXRS+ imaging system (BIO-RAD)was used to collect the luminescence signal which was quantified by using image J software (version 1.48, NIH, USA) [26].
In-vitro DRG Primary Cultures
Primary DRG neuronal cells were generated in vitro using adult rat dorsal root ganglions from the (L1-l5) lumbar region of the spinal cord with slight modifications as earlier reported [27]. Briefly, 9-10 week old rats were slaughtered and DRGs were separated aseptically into HAMS F12 medium with 10% FBS and 5% antibiotic/antimycotic solution. This was followed by centrifugation at 1200 rpm for 2 minutes before adding trypsin (0.25 percent) for 30 minutes and triturating with a glass pipette to dissociate into cells. This cell and tissue suspension was filtered through 70M nylon gauge. The monosuspended DRG cells were centrifuged for 3 minutes at 1200 rpm and resuspended in neurobasal media containing 10% FBS and 0.5 percent antibiotic and antimycotic solution. A 1:50 volume ratio of matrigel matrix to neurobasal media was used to grow the cells. The neurite extensions appeared two-three days after culture and were treated with various treatments at optimal concentrations.
Neurite outgrowth assay
Three days after DRG primary cultures in 12 well plates were treated with various concentrations of optimized drugs at specified intervals (48 hours), the cells were washed with fresh PBS and neurite outgrowth assay was performed using the manufacturer's kit-based protocol. Five fields were chosen at random and examined using a phase contrast microscope (Nikon ECLIPSE, Ti-U, Japan). The length of neurite outgrowths in 30 cells from each field was measured using Image J software (NIH, USA). The number of neurite outgrowths/axon-like extensions that are twice or more than the diameter of the cell body were counted [28].
Immunocytochemistry
DRG neurons were cultured on glass Coverslips in a 6-well plate at 5000 cells/well seeding density and fixed with 4% paraformaldehyde solution and permeabilized with 0.5 percent Triton-X 100 for 15 minutes at room temperature, as described elsewhere. These cells were blocked for 2 hours at room temperature with a 3 percent BSA solution in PBS. After blocking, the cells were incubated overnight at 4°C with primary antibodies (p-AMPK, Complex I, and TFAM) at 1:200 dilutions in 3 percent BSA solution. The following day, cells were washed with PBST and incubated with secondary anti-rabbit and anti-mouse antibodies conjugated with rhodamine and Alexa488 (Santa Cruz Biotechnology Inc., CA, USA) at room temperature for 2 hours. Finally, the coverslips were adhered to the glass slide with DAPI mounting medium (Sigma FluoroshieldTM). A confocal microscope was used to capture the images (Leica TCS SP8 Laser Scanning Spectral Confocal microscope, Germany) [29].
Assay for JC1
DRG primary cultures were stained with JC1 as previously described. Cultured DRG primary cells were incubated for 15 minutes with 5M JC-1 in PBS. The cells were washed with PBS to remove unbound red stain before being examined with a fluorescence microscope (Nikon ECLIPSE, Ti-U, Japan) with red and green filters. The mean red and green fluorescence intensity ratio was calculated using Image J software (NIH, USA), [30,31].
Mitosox Test
The Mitosox red assay was carried out in DRG primary cultures according to published protocols [32]. Briefly, the cultures were treated with various drug concentrations for 48 hours before being washed with PBS and incubated for 15 minutes at 37°C with 5M Mitosox reagent. The cells were then washed to remove any unbound Mitosox red reagent before being examined with a fluorescence microscope (Nikon ECLIPSE, Ti-U, Japan) with a green filter. Image J software was used to calculate the mean red fluorescence intensity (NIH, USA).
Statistical Analysis
Excel was used to calculate mean, SD, and SEM for each parameter. The results were analyzed with the newest version of graph pad prism program, with one way ANOVA to compare the groups (multiple comparison tests). When one way ANOVA demonstrated statistical significance, Bonferroni’s multiple comparisons test was used for post hoc analysis. Statistical significance was defined as P< 0.05 or less.