Cannabis sativa: In Vitro Production of Cannabinoids Jones Á, Veliky JA ( a) Growth of plant cell suspension cultures on glycerol as a sole source of. However, the production of medicines from hemp (Cannabis sativa L.) in Cell suspension cultures and hairy root cultures of hemp have been. (undifferentiated cell growth on solid media) and the production of THC and other important cannabinoids in cell suspension, hairy root and trichome cultures of.
of Production Cultures for Cannabinoids Suspension Cannabis the Cell
The staining of CB1 antibody appears specific for 2 reasons. Second, we could not detect the positive immunostaining or kDa protein band using the CB1 antibody preabsorbed with the antigen. We then examined adult naive rats sacrificed 2 hours after receiving a single dose of BrdU to label dividing cells.
Hoechst staining was conducted to reveal the total cultured cells. The arrow indicates the glial-like cells, located in the center of a neurosphere, with CB1 staining and without nestin staining. CB1 receptor; lane 3: PCR reaction without sample added. D Confocal microscopic assessments of costaining of BrdU and CB1 receptors in the SGZ located between the hilus and the granule cell layer granule of the dentate gyrus in an adult rat.
Error bars represent SEM. It measures cell proliferation by detecting dividing cells. This hypothesis was supported by further experiments in which U, a specific inhibitor of the ERK pathway, was employed.
The total cultured cells are labeled deep blue by Hoechst staining. Increased hippocampal cell proliferation following HU treatment in adult rats. BrdU labeling of dividing cells was used to test the acute effects of HU treatment on cell proliferation in adult hippocampus.
BrdU-labeled cells showed fusiform or irregular shape and were clustered or aggregated in the SGZ Figure 5 A throughout the whole hippocampus in all rats examined. We then examined the effects of chronic HU injection on cell proliferation in adult hippocampus. Cell proliferation was assessed by BrdU labeling of dividing cells.
Increased newborn hippocampal neurons following chronic HU treatment in adult rats. A recent study has demonstrated that newborn neurons in the dentate granule cell layer that had survived 4 weeks were stably integrated into the granule cell layer One month after the last HU, AM, or vehicle injection, the majority of BrdU-labeled cells migrated and dispersed into the granule cell layer and showed size and morphology indistinguishable from both their neighboring granule neurons and from different treatment Figure 6 A.
Fate and migration of BrdU-labeled cells in the dentate gyrus following chronic HU treatment. A Representative confocal microscopic images show costaining yellow of BrdU green and NeuN red in the dentate granule cell layer. The majority of BrdU-stained cells are doubly labeled with the neuronal marker NeuN and located within the granule cell layer. C There was no significant difference in the proportion of cells doubly labeled with BrdU and NeuN to the total cells singly labeled with BrdU.
No hippocampal neuronal death following chronic HU treatment in adult rats. Ample evidence has illustrated the increased hippocampal neurogenesis following ischemia, epileptic status, enriched environment, or exercise It is therefore possible that increased hippocampal neurogenesis following chronic HU treatment in adult rats may result from the toxic effects of chronic HU treatment on hippocampal neurons.
As depicted in Figure 7 A, HUtreated rats did not show detectable loss of NeuN-immunopositive neurons in the hippocampus, relative to naive control rats.
These results, however, do not exclude the possibility that some of NeuN-stain neurons following chronic HU treatment shown in Figure 7 A are dying. Accordingly, we used TUNEL stain and Fluoro-Jade B stain to examine the degenerating hippocampal neurons 31 in rats receiving chronic HU treatment, with the naive rats as negative control and kainic acid—treated rats as positive control We failed to detect any TUNEL- or Fluoro-Jade B—stained degenerating cells throughout the whole hippocampus in both naive rats and HUtreated rats, whereas kainic acid—injected rats showing epileptic status exhibited numerous dying cells in the CA3 pyramidal cell layer and even dentate granule cell layer Figure 7 , C and D.
Effects of chronic HU on neuronal survival. Anxiolytic and antidepressant effects of chronic HU Two recent studies employing novelty-suppressed feeding NSF tests and forced swimming test FSTs as measures of anxiety and depression have shown that chronic treatment with the antidepressant fluoxetine produced anxiolytic and antidepressant effects 18 , 19 , and the anxiolytic effects are likely achieved by promoting hippocampal neurogenesis Therefore, we employed the same behavioral tests to examine the effects of chronic HU treatment on measures of anxiety and depression.
Rats were subjected to behavioral testing 1 month later, based on the recent finding that hippocampal newborn neurons need 4 weeks to become functional Rats were killed for immunohistochemical staining after behavioral tests. The majority of BrdU-positive cells in vehicle-, AM, or HUtreated rats were located in the granule cell layer, suggesting that they became granule neurons. Post-hoc test showed results similar to those in Figure 5 D: Thus, these data together suggest that chronic HU treatment promoted hippocampal neurogenesis and exerted anxiolytic- and antidepressant-like effects.
A In the NSF test, rats receiving chronic HU but not AM showed significantly shortened latency to feed in a novel environment but not in their home cages, suggesting anxiolytic effects produced by HU C Among the rats receiving vehicle, AM, and HU, there was no significant difference in the number climbing in the first 5 minutes in the pretest sessions of the FST.
D Irradiation of the hippocampus prominently reduced cell proliferation in the SGZ. E Irradiation of the hippocampus blocked chronic HU—induced shortened latency of rats to feed in novel environment but not in their home cages in the NSF test. Association of hippocampal neurogenesis with anxiolytic- and antidepressant-like effects of chronic HU To determine the relationship between hippocampal neurogenesis and anxiolytic- and antidepressant-like effects produced by chronic HU, we examined the effects of a selective destruction of the hippocampal neural stem cells on the behavioral effects of chronic HU During the course of receiving chronic HU injections, 1 group of Long-Evans rats received two 5-Gy doses of x-rays confined to a limited brain region including the hippocampus, as previously described Four BrdU injections with hour intervals were given after the last HU injection.
Because two 5-Gy doses of x-rays were not found to alter the morphology and function of mature neurons in the hippocampus, hypothalamus, and amygdala 18 , our results together suggest that chronic HU treatment reduced anxiety and depression, likely via promoting hippocampal neurogenesis.
Natural selection has conserved cannabinoid receptors in various vertebrates and invertebrates that have been evolutionarily separate for million years 33 , indicating the importance of cannabinoid receptors to life. A recent study has shown CB1-immunoreactive newborn neurons in the adult rat hippocampus 1 week after BrdU injection This time interval allowed us to label mitotically active cells i. Similar results were also obtained in freely moving adult rats.
We also provided evidence indicating that the promoting effects of chronic HU treatment on adult hippocampal neurogenesis are not the outcome of hippocampal neuronal death, as we did not detect neuronal loss or dying hippocampal neurons following chronic HU injection. Our findings of cannabinoid-induced increase in hippocampal neurogenesis are in agreement with the recent observation that CB1 receptor—knockout mice display profound suppression of hippocampal neurogenesis In in vivo experiments, Rueda et al.
We observed, however, a significant increase in the hippocampal newborn neurons following twice-daily HU injection for 10 days. The differing regulatory effects of endocannabinoid shown in Rueda et al. In fact, some studies have shown that exo- and endocannabinoids have differential or opposing effects in many areas, including nociception 37 , the vascular system 38 , and epilepsy Following the observation that chronic HU treatment promoted neurogenesis in the dentate gyrus, we wondered whether chronic HU—induced newborn neurons are of functional significance.
Given the recent findings that chronic fluoxetine treatment produced antidepressant and anxiolytic effects 18 , 19 and the anxiolytic effects are likely achieved by promoting hippocampal neurogenesis 18 , we hypothesized that chronic HU—induced hippocampal neurogenesis may also correlate with anxiolytic and antidepressant effects. Our subsequent experiments supported this hypothesis. After 1 month of chronic HU treatment, rats deprived of food for 48 hours showed significantly reduced latency to eat food in a novel environment, suggesting that chronic HU treatment exerted anxiolytic effects.
These results are consistent with a recent study showing that once-daily injections of the cannabinoid receptor agonist CP55, for 11 days reduced anxiety in the elevated plus-maze test performed 30 days after the last CP55, injection Chronic HU—induced shortened latency to eat food in the novel environment is unlikely due to the well-known effects of HU on appetite 1 , because chronic HU treatment produced no significant effects on the latency to eat food or the amount of food consumed when rats were returned to the familiar environment of their home cages immediately after the test.
One week after undergoing NSF testing, the same rats receiving chronic HU treatment showed a significantly reduced duration of immobility in the FST, indicating that chronic HU also exerts antidepressant effects.
Because acute cannabinoid treatment profoundly affects motor function of humans and animals 1 , 10 , chronic HU—induced shortened immobility in the FST may be produced by its action in changing the motor activity of rats. This is unlikely, however, as we observed no significant difference in the number of rats climbing 41 , 42 among HU, AM, and vehicle-treated groups in the first 5 minutes of the pretest sessions of the FST.
The filtrate was concentrated to dryness by evaporation, and the residues were dissolved in methanol. The eluate was monitored by absorption at nm, and the peak intensities of these cannabinoids were determined with a Chromatocorder 21 TOSOH. Assay for Cell Death Activities of Cannabinoids —Young leaves where cuticles were removed from the lower surface were used to measure the cell death activity of cannabinoids.
After the leaf samples 1 - 1. For analyses of DNA in the cannabinoid-treated leaves, DNA was extracted from the above leaf samples 10 mg by the cetyltrimethylammonium bromide method and electrophoresed on a 1. The DNA was visualized with 0. The cell death activities of cannabinoids for suspension cells were measured as follows. Cannabis cells or tobacco cells each 0. For the evaluation of effects of cell death inhibitors, cells 0.
After h incubation, chlorophyll and cell viability in each sample was analyzed as described above. Concerning the effects of cyclosporin A on senescence naturally occurring in Cannabis leaves, mature leaves just before senescence were used. Cannabinoid Synthase Assay —Fresh Cannabis leaves mg at various stages were homogenized with 2 ml of m m sodium phosphate buffer pH 7. The supernatant was used as enzyme solution. The enzyme assay was carried out by a modification of the method described in our previous study The fixed cells were washed with PBS 1.
Suspension cells 30 g were homogenized in a mortar with isolation buffer pH 7. Further purification of mitochondria from the pellet was carried out using a modified method of Curtis and Wolpert The pellet was used as isolated mitochondria for further examinations.
Analysis of Mitochondrial Swelling —Mitochondria 0. TMRM staining of isolated mitochondria 0. Analyses of Mitochondrial Proteins —Isolated mitochondria 1. The nuclease assay was carried out by a modification of the method of Balk et al. The DNA in each sample was analyzed by agarose gel electrophoresis 1. The detection of cytochrome c in the same supernatant was carried out by immunoblotting as follows. The membranes were incubated with horseradish peroxidase-conjugated anti-mouse antibodies 1: For analyses of proteins nuclease and cytochrome c retained in mitochondria, the above pellets were used.
Isolation of Nuclei from Cannabis Cells — Cannabis nuclei were isolated from protoplasts of Cannabis cells. Cannabis cells 10 g were incubated in digestion solution ml consisting of 0. The protoplasts were resuspended in buffer pH 7. All further procedures for isolation of nuclei were performed as described by Balk et al.
Direct Visualization of Fluorescent Resin Causing Cell Death in Cannabis Leaves —In the present study, we discovered that fluorescence microscopy can directly visualize resin, which is assumed to induce cell death in Cannabis leaves.
As shown in Fig. Such fluorescence capsules on hemp leaves have not been reported, but we identified them as capitate-sessile glands, based on their morphological features sizes and shapes 30 , In intact leaves at the young L1 and L2 or mature L3 and L4 stage, the light bluish fluorescence was localized only within these glands Fig.
Furthermore, such leakage of fluorescence was observed in senescent tissues of more aged leaves L5 and L6 in Fig. Because nuclear DNA in the above damaged tissues and senescent leaves underwent extensive degradation supplemental Fig. S1 A , cell death was considered to occur in the tissues exposed to the fluorescent resin. Direct visualization of fluorescent glands on Cannabis leaves. A , fluorescent glands on Cannabis leaves. Leaves at various stages were collected from 7-week-old C.
B , distribution of fluorescent resin in Cannabis leaves having lesions. The same regions of the upper surface and lower surface of the damaged area were analyzed by light microscopy upper panel and by fluorescence microscopy lower panel. Young leaves right panels and mature leaves left panels were collected from plants at the stages L2 and L3, respectively.
We next investigated whether artificially induced leakage of the substance with light bluish fluorescence causes these cell death responses. Although resin composed of various secondary metabolites is known to be stored in glandular trichomes of plants 32 , methods to induce it to leak into host leaf tissues have not been established. We found that exfoliation of the cuticle from the lower surface of the mature leaves removed the glands but also quickly induced leakage of fluorescent resin into the leaf tissues Fig.
The disappearance of red fluorescence, which reflected chlorophyll degradation, was initiated within 24 h after inducing fluorescence leakage and thereafter expanded to a wide region Fig. When the viability of these leaf tissues was investigated using FDA 27 , green fluorescence characteristic of living cells extensively decreased with chlorophyll degradation Fig.
On the other hand, similar treatment of young leaves, which leaked little fluorescence, caused neither chlorophyll degradation Fig. These results suggest that secretion of the fluorescent resin from the glands into leaf tissues leads to cell death in Cannabis leaves. Cell death induced by artificial leakage of fluorescent resin. A , chlorophyll degradation by artificially induced leakage of fluorescent resin.
B and C , change of cell viability and chlorophyll by artificially induced leakage of fluorescent resin. Mature leaves B or young leaves C were incubated for 5 min, 24 h, or 48 h after removal of their cuticles.
Chlorophyll in each sample was investigated by fluorescent microscopy. The same sample was then stained with FDA, and its fluorescence was visualized by fluorescent microscopy. Identification and Characterization of Cell Death Regulators in Glandular Trichomes —Because the above results indicated the presence of cell death-mediating factors in the fluorescent resin, we attempted their identification by chemical and pharmacological analyses.
As a first step, the glands were isolated using our newly developed method Fig. In contrast, owing to its low fluorescence, it was impossible to confirm THCA leakage by fluorescence microscopy.
Identification of cell death regulators in the glands. A , glands isolated from Cannabis leaves. The glands were isolated by sonication of mature Cannabis leaves and then visualized by fluorescent microscopy.
B , HPLC of gland extracts. Methanol extracts of the isolated glands were analyzed by reverse-phase HPLC. C , TLC of extracts from senescent Cannabis leaves. Extracts prepared by n -hexane treatment of senescent leaves were loaded onto silica gel TLC. After TLC was developed using n -hexane-ethyl acetate 2: D , cell death activity of cannabinoids for Cannabis leaves. After young leaves from which the cuticles had been removed from the lower surface were incubated with various concentrations of cannabinoids for 24 h, chlorophyll in the samples was analyzed by fluorescent microscopy.
Thereafter, the same samples were stained with FDA and cell viability was analyzed by fluorescent microscopy. To accelerate the uptake of cannabinoids into leaf cells and to avoid influence arising from endogenous cannabinoids, we used the young leaves leaves corresponding to the lower panel shown in Fig.
Because Cannabis glands store, together with these cannabinoids, other minor secondary metabolites Fig. However, neither chlorophyll degradation nor a decrease of cell viability was found to be induced by this fraction data not shown. Judging from the contents of CBCA 0. Therefore, we concluded that secretion of both cannabinoids can induce cell death throughout Cannabis leaves.
Thus, both cannabinoids caused cell death in Cannabis leaves much more effectively than these known cell death regulators. Because little is known about the leaf stages in which their biosynthetic systems are activated, we measured the activities of these cannabinoid-synthesizing enzymes in the leaves at various stages. The young leaves showed much higher activities than other leaves, and both activities markedly decreased after leaf maturation supplemental Fig.
Features of Cell Death Catalyzed by Cannabinoids —To precisely characterize the mechanism of cell death mediated by cannabinoids, suspension-cultured cells derived from Cannabis leaves were also used. We first examined whether both cannabinoids induce cell death in Cannabis cells as well as Cannabis leaves. The cell death-inducing effects of these cannabinoids were evaluated by measuring the fluorescence intensity of the FDA-stained cells.
Cannabinoids have effects on cell receptors in the brain and body. They can change how those cells behave and communicate with each other. The most researched cannabinoid is deltatetrahydrocannabinol THC. THC is responsible for the way your brain and body respond to cannabis, including the high and intoxication.
THC has some therapeutic effects but it also has harmful effects. Harmful effects may be greater when the strength of THC is higher. The potency concentration or strength of THC in cannabis is often shown as a percentage of THC by weight or by volume of an oil. Cannabis that contains very low amounts of THC in its flowers and leaves less than 0.
Cannabidiol CBD is another cannabinoid. CBD is also being studied for its possible therapeutic uses. Terpenes are chemicals made and stored in the trichomes of the cannabis plant, with the cannabinoids.
Terpenes give cannabis its distinctive smell. The cannabis plant is used for its effects on the mind. It is also used for medical, social or religious purposes. Marijuana is a slang term for the dried flowers, leaves, stems and seeds of the cannabis plant. Results of the Canadian Cannabis Survey provide a snapshot of how much cannabis Canadians use, how often they use it, and in what form.
The application of plant in vitro cultures in cannabinoid production.
Further, production of THC as well as other important cannabinoids was achieved in cell suspension, hairy root and trichome cultures of. We have proposed a system of in vitro cell culture of C. sativa to produce cannabinoids, because potential drugs derived from cannabis would no longer be. Further, production of THC as well as other important cannabinoids was achieved in cell suspension, hairy root and trichome cultures of. Cannabis. The optimal.