Long-term effects on cardiorespiratory and behavioral responses in male and female rats prenatally exposed to cannabinoid.

Development of the respiratory system can be affected by the use of drugs during pregnancy, as the prenatal phase is highly sensitive to pharmacological interventions, resulting in long-term consequences. The deleterious effects of external cannabinoids during gestation may be related to negative interference in central nervous system formation, cardiorespiratory system function, and behavioral disorders. Nevertheless, the impact of external cannabinoids on cardiorespiratory network development, chemosensitivity, and its future consequences in adulthood is still unclear. We evaluated the effects of prenatal exposure to a synthetic cannabinoid (WIN 55,212-2, 0.5 mg·kg-1·day-1) on the cardiorespiratory control and panic-like behavior of male and female rats in adulthood. Exogenous cannabinoid exposure during pregnancy resulted in a sex-dependent difference in breathing control. Specifically, males showed increased chemosensitivity to CO2 and O2, whereas females exhibited decreased sensitivity. Altered cardiovascular control was evident, with prenatally treated males and females being more susceptible to hypertension and tachycardia under adverse environmental conditions. Moreover, WIN-treated males exhibited higher fragmentation of sleep episodes, whereas females displayed anxiolytic and panicolytic behavioral responses to CO2. However, no changes were observed in the mechanical component of the respiratory system, and there were no neuroanatomical alterations, such as changes in the expression of CB1 receptors in the brainstem or in the quantification of catecholaminergic and serotonergic neurons. These findings highlight that external interference in cannabinoid signaling during fetal development causes sex-specific, long-lasting effects for the cardiorespiratory system and behavioral responses in adulthood.NEW & NOTEWORTHY The surge in recreational cannabis use and cannabinoid-based medication prescription among pregnant women has been notable in recent years, fueled by the misconception that natural products are inherently safe. Significant gaps persist regarding the potential risks of maternal consumption of cannabinoids and the long-term effects on the cardiorespiratory system of their offspring, which may be determined by sex. Accordingly, this research aims to diminish this lack of information and raise a note of caution.

Repeated Administration of the Cannabinoid WIN Alters the Isoflurane-Sparing Effect of Morphine and Dexmedetomidine.

The impacts of morphine and dexmedetomidine on the MAC of isoflurane were studied in rats constantly medicated with the cannabinoid WIN 55,212-2. METHODS: Prior to the administration of morphine, the MAC was measured in both untreated rats (MAC (ISO)) and those treated with a cannabinoid (MAC (ISO + CANN)). The effects of morphine (MAC (ISO + MOR)) and dexmedetomidine (MAC (ISO + DEX)) on untreated rats and rats treated for 21 days with the cannabinoids (MAC (ISO + CANN + MOR)) and (MAC (ISO + CANN + DEX) were also studied. RESULTS: MAC (ISO) was 1.32 ± 0.06, and MAC (ISO + CANN) was 1.69 ± 0.09. MAC (ISO + MOR) was 0.97 ± 0.02 (26% less than MAC (ISO)). MAC (ISO + CANN + MOR) was 1.55 ± 0.08 (8% less than MAC (ISO + CANN)), MAC (ISO + DEX) was 0.68 ± 0.10 (48% less than MAC (ISO)), and MAC (ISO + CANN + DEX) was 0.67 ± 0.08 (60% less than MAC (ISO + CANN)). CONCLUSIONS: Medication with a cannabinoid for 21 days augmented the MAC of isoflurane. The sparing effect of morphine on isoflurane is lower in rats constantly medicated with a cannabinoid. The sparing effect of dexmedetomidine on the minimum alveolar concentration of isoflurane is greater in rats repeatedly medicated with a cannabinoid.

Sex- and age-specific respiratory alterations induced by prenatal exposure to the cannabinoid receptor agonist WIN 55,212-2 in rats.

BACKGROUND AND PURPOSE: Cannabis legalization has risen in many countries, and its use during pregnancy has increased. The endocannabinoid system is present in the CNS at early stages of embryonic development, and regulates functional brain maturation including areas responsible for respiratory control, data on the influence of external cannabinoids on the development of the respiratory system and possible consequences during postnatal life are limited. EXPERIMENTAL APPROACH: We evaluated the effects of prenatal exposure to synthetic cannabinoid (WIN 55,212-2 [WIN], 0.5 mg·kg-1 ·day-1 ) on the respiratory control system in neonatal (P0, P6-7 and P12-13) and juvenile (P27-28) male and female rats. KEY RESULTS: WIN administration to pregnant rats interfered sex-specifically with breathing regulation of offspring, promoting a greater sensitivity to CO2 at all ages in males (except P6-7) and in juvenile females. An altered hypoxic chemoreflex was observed in P0 (hyperventilation) and P6-7 (hypoventilation) males, which was absent in females. Along with breathing alterations, brainstem analysis showed an increase in the number of catecholaminergic neurons and cannabinoid receptor type 1 (CB1 ) and changes in tissue respiration in the early males. A reduction in pulmonary compliance was observed in juvenile male rats. Preexposure to WIN enhanced spontaneous apnoea and reduced the number of serotoninergic (5-HT) neurons in the raphe magnus nucleus of P0 females. CONCLUSIONS AND IMPLICATIONS: These data demonstrate that excess stimulation of the endocannabinoid system during gestation has prolonged and sex-specific consequences for the respiratory control system.

Minimum Alveolar Concentration of Isoflurane in Rats Chronically Treated with the Synthetic Cannabinoid WIN 55,212-2.

The minimum alveolar concentration MAC of isoflurane was measured in rats chronically treated with WIN 55,212-2. METHODS: The MAC of isoflurane was determined in 24 male rats from expiratory samples at time of tail clamping under the following conditions: without treatment MAC(ISO), in rats treated for 21 days with WIN 55,212-2 MAC(ISO + WIN55), and in rats 8 days after stopping treatment with WIN 55,212-2 (MACISO + WIN55 + 8D). RESULTS: The MAC(ISO) was 1.32 ± 0.06. In the MAC(ISO + WIN55) group, the MAC increased to 1.69 ± 0.09 (28%, p-value ≤ (0.0001). Eight days after stopping treatment with WIN55, the MAC did not decrease significantly, 1.67 ± 0.07 (26%, p-value ≤ 0.0001). CONCLUSIONS: The administration of WIN 55,212-2 for 21 days increases the MAC of isoflurane in rats. This effect does not disappear 8 days after discontinuation of treatment with the synthetic cannabinoid.

WIN 55,212-2 shows anti-inflammatory and survival properties in human iPSC-derived cardiomyocytes infected with SARS-CoV-2.

Coronavirus disease 2019 (COVID-19) is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which can infect several organs, especially impacting respiratory capacity. Among the extrapulmonary manifestations of COVID-19 is myocardial injury, which is associated with a high risk of mortality. Myocardial injury, caused directly or indirectly by SARS-CoV-2 infection, can be triggered by inflammatory processes that lead to damage to the heart tissue. Since one of the hallmarks of severe COVID-19 is the "cytokine storm", strategies to control inflammation caused by SARS-CoV-2 infection have been considered. Cannabinoids are known to have anti-inflammatory properties by negatively modulating the release of pro-inflammatory cytokines. Herein, we investigated the effects of the cannabinoid agonist WIN 55,212-2 (WIN) in human iPSC-derived cardiomyocytes (hiPSC-CMs) infected with SARS-CoV-2. WIN did not modify angiotensin-converting enzyme II protein levels, nor reduced viral infection and replication in hiPSC-CMs. On the other hand, WIN reduced the levels of interleukins six, eight, 18 and tumor necrosis factor-alpha (TNF-α) released by infected cells, and attenuated cytotoxic damage measured by the release of lactate dehydrogenase (LDH). Our findings suggest that cannabinoids should be further explored as a complementary therapeutic tool for reducing inflammation in COVID-19 patients.

Exposure to the cannabinoid agonist WIN 55, 212-2 in adolescent rats causes sleep alterations that persist until adulthood.

Cannabis and, to a lesser extent, synthetic cannabinoids are used during adolescence, a period in which multiple brain areas are still undergoing development. Among such areas is the hypothalamus, which is implicated in the control of sleep-wake cycle. In the present report, we show that exposing adolescent rats to the cannabinoid receptor agonist WIN 55, 212-2 (0.1, 0.3 or 1.0 mg/kg, i.p) for 14 days during adolescence (i.e., from post-natal day 30-44) resulted in significant sleep disturbances when the animals became adult (post-natal day 80). These included decreased wakefulness and enhanced rapid eye movement sleep. Furthermore, we found that labeling for NeuN, a marker of postmitotic neurons, was significantly increased the dorsomedial hypothalamic nucleus of rats treated with WIN 55, 212-2. The results suggest that excessive cannabinoid receptor activation during adolescence can persistently influence sleep patterns and neuronal activity later in life.

Characterization of Cancer-Induced Nociception in a Murine Model of Breast Carcinoma.

Severe and poorly treated pain often accompanies breast cancer. Thus, novel mechanisms involved in breast cancer-induced pain should be investigated. Then, it is necessary to characterize animal models that are reliable with the symptoms and progression of the disease as observed in humans. Explaining cancer-induced nociception in a murine model of breast carcinoma was the aim of this study. 4T1 (104) lineage cells were inoculated in the right fourth mammary fat pad of female BALB/c mice; after this, mechanical and cold allodynia, or mouse grimace scale (MGS) were observed for 30 days. To determine the presence of bone metastasis, we performed the metastatic clonogenic test and measure calcium serum levels. At 20 days after tumor induction, the antinociceptive effect of analgesics used to relieve pain in cancer patients (acetaminophen, naproxen, codeine or morphine) or a cannabinoid agonist (WIN 55,212-2) was tested. Mice inoculated with 4T1 cells developed mechanical and cold allodynia and increased MGS. Bone metastasis was confirmed using the clonogenic assay, and hypercalcemia was observed 20 days after cells inoculation. All analgesic drugs reduced the mechanical and cold allodynia, while the MGS was decreased only by the administration of naproxen, codeine, or morphine. Also, WIN 55,212-2 improved all nociceptive measures. This pain model could be a reliable form to observe the mechanisms of breast cancer-induced pain or to observe the efficacy of novel analgesic compounds.

Beta-adrenergic receptor activation increases GABA uptake in adolescent mice frontal cortex: Modulation by cannabinoid receptor agonist WIN55,212-2.

GABA transporters regulate synaptic GABA levels and dysfunctions in this system might result in psychiatric disorders. The endocannabinoid system (ECS) is the main circuit breaker in the nervous system and may alter noradrenaline (NA) communication, which in turn modulates the release of GABA. However, a close relationship between these systems has not been recognized. We asked whether NA and ECS might control extracellular GABA levels in slices of frontal cortex (FC) of adolescent Swiss mice with 40 days after birth (PN40). Here we show that NA and isoproterenol (ISO), a beta-adrenergic agonist, increased [3H]-GABA uptake in mice FC, while alpha1-adrenergic agonist phenylephrine had no effect. As GAT-1 is expressed and fully functional at the FC, addition of NO-711, a GAT-1 inhibitor, dose dependently blocked [3H]-GABA uptake. The increase of [3H]-GABA uptake induced by ISO was also blocked by NO-711. [3H]-GABA release induced by 80 mM KCl was reduced by NO-711, but not by removal of Ca2+. ISO also increased cyclic AMP (cAMP) levels and addition of WIN 55,212-2, a mixed CB1/CB2 receptor agonist, inhibited the effect of ISO in GABA uptake increase, GAT-1 expression and cAMP levels compared to control. Our data show that GABA transport increased by NA and ISO is negatively regulated by cannabinoid receptor agonist WIN55,212-2.

Experimental ischemia/reperfusion model impairs endocannabinoid signaling and Na+/K+ ATPase expression and activity in kidney proximal tubule cells.

LLC-PK1 cells, an immortalized epithelial cell line derived from pig renal proximal tubules, express all the major players of the endocannabinoid system (ECS) such as CB1, CB2 and TRPV1 receptors, as well as the main enzymes involved in the biosynthesis and degradation of the major endocannabinoids named 2-arachidonoylglycerol, 2-AG and anandamide, AEA. Here we investigated whether the damages caused by ischemic insults either in vitro using LLC-PK1 cells exposed to antimycin A (an inductor of ATP-depletion) or in vivo using Wistar rats in a classic renal ischemia and reperfusion (IR) protocol, lead to changes in AEA and 2-AG levels, as well as altered expression of genes from the main enzymes involved in the regulation of the ECS. Our data show that the mRNA levels of the CB1 receptor gene were downregulated, while the transcript levels of monoacylglycerol lipase (MAGL), the main 2-AG degradative enzyme, were upregulated in LLC-PK1 cells after IR model. Accordingly, IR was accompanied by a significant reduction in the levels of 2-AG and AEA, as well as of the two endocannabinoid related molecules, oleoylethanolamide (OEA) and palmitoylethanolamide (PEA) in LLC-PK1 cells. In kidney cortex homogenates, only AEA levels were significantly decreased. In addition, we found that in both the in vitro and in vivo model IR caused a reduction in the expression and activity of the Na+/K+ ATPase. These changes were reversed by the CB1/CB2 agonist WIN55,212, in a CB1-receptor dependent manner in the LLC-PK1 IR model. In conclusion, the ECS and Na+/K+ ATPase are down-regulated following IR in LLC-PK1 cells and rat kidney. We suggest that CB1 agonists might represent a potential strategy to reverse the consequences of IR injury in kidney tissues.