Elaine Aparecida Del Bel

Multiple mechanisms involved in the large-spectrum therapeutic potential of cannabidiol in psychiatric disorders

Cannabidiol (CBD) is a major phytocannabinoid present in the Cannabis sativa plant. It lacks the psychotomimetic and other psychotropic effects that the main plant compound Δ9-tetrahydrocannabinol (THC) being able, on the contrary, to antagonize these effects. This property, together with its safety profile, was an initial stimulus for the investigation of CBD pharmacological properties. It is now clear that CBD has therapeutic potential over a wide range of non-psychiatric and psychiatric disorders such as anxiety, depression and psychosis. Although the pharmacological effects of CBD in different biological systems have been extensively investigated by in vitro studies, the mechanisms responsible for its therapeutic potential are still not clear. Here, we review recent in vivo studies indicating that these mechanisms are not unitary but rather depend on the behavioural response being measured. Acute anxiolytic and antidepressant-like effects seem to rely mainly on facilitation of 5-HT1A-mediated neurotransmission in key brain areas related to defensive responses, including the dorsal periaqueductal grey, bed nucleus of the stria terminalis and medial prefrontal cortex. Other effects, such as anti-compulsive, increased extinction and impaired reconsolidation of aversive memories, and facilitation of adult hippocampal neurogenesis could depend on potentiation of anandamide-mediated neurotransmission. Finally, activation of TRPV1 channels may help us to explain the antipsychotic effect and the bell-shaped dose-response curves commonly observed with CBD. Considering its safety profile and wide range of therapeutic potential, however, further studies are needed to investigate the involvement of other possible mechanisms (e.g. inhibition of adenosine uptake, inverse agonism at CB2 receptor, CB1 receptor antagonism, GPR55 antagonism, PPARγ receptors agonism, intracellular (Ca2+) increase, etc.), on CBD behavioural effects.

Expression of neuronal nitric oxide synthase mRNA in stress-related brain areas after restraint in rats

The objective of the present study was to investigate the expression of neuronal nitric oxide synthase (nNOS) mRNA in stress-related areas after restraint. Male Wistar rats (n=4-6/group) submitted to 2 h of restraint during one (acute) or seven (chronic) days were sacrificed 24 h after the last restraint period. In situ hybridisation was performed with oligonucleotide probes radiolabeled with (35)S. Acute restraint induced a significant increase in nNOS mRNA in the paraventricular hypothalamic nucleus (PVN), medial parvocellular part, dorsolateral periaqueductal grey (DLPAG) and medial amygdaloid nucleus, but not in the hippocampal formation. This effect persisted after chronic restraint in the PVN and DLPAG. These results suggest that restraint stress induces changes in gene expression of nNOS in areas related to stress reactions.

Effects of acute and chronic fluoxetine treatments on restraint stress-induced Fos expression.

Chronic treatment with antidepressants has been shown to attenuate behavioral changes induced by uncontrollable stress. The mechanisms and brain sites of this effect, however, remain controversial. The objective of the present work was to investigate the effects of chronic and acute treatment with fluoxetine (FLX), a selective serotonin reuptake blocker, on Fos expression in animals submitted to restraint stress. Male Wistar rats (n = 3-9/group) received, during 1 or 21 days, intraperitoneal. Injections of vehicle (saline + 0.2% Tween-80, 1 ml/kg) or FLX (10 mg/kg). One hour after the last injection they were forced restrained for 2 h and sacrificed immediately after. Non-stressed animals were sacrificed 2 h after the last injection. The brains were removed and processed for immunohistochemistry. Fos-like immunoreactivity (FLI) was quantified by a computer system. In acutely treated animals FLX decreased stress-induced FLI in the medial amygdala (MeA), bed nucleus of the stria terminalis (BNST), ventrolateral part, and dorsolateral periaqueductal gray (PAG). After chronic treatment, however, the drug induced a significant increase in FLI in the BNST (ventrolateral and medial parts), lateral septal nucleus (LSN, dorsal part), dorsal raphe nucleus (DRN), and locus coeruleus in restrained group. In non-restrained animals chronic treatment with FLX increased FLI in the MeA, BNST (ventrolateral and dorsolateral parts), LSN (dorsal and intermediate parts), dorsolateral and dorsomedial PAG and in the DRN. The results suggest that chronic fluoxetine treatment induce plastic changes that result in a different regional pattern of Fos expression.

Behavioral and c-fos expression changes induced by nitric oxide donors microinjected into the dorsal periaqueductal gray.

The dorsolateral periaqueductal gray matter (DLPAG) is a key structure in behavioral and autonomic expression of defensive reactions. Inhibitors of nitric oxide (NO) synthase microinjected into the DLPAG are anxiolytic. The purpose of the present study was to evaluate the behavioral effects produced by administration of two NO donors, SIN-1 or DEA/NO, into the DLPAG. We also employed the detection of Fos-like immunoreactivity (FLI) to reveal brain areas activated by SIN-1 administration. Male Wistar rats (n = 7-11/group) received intra-DLPAG injection of SIN-1 (150 or 300 nmol), DEA/NO (150, 300, or 600 nmol), or saline (0.5 microl), and their behavior was observed in an open arena during 15 min. For the FLI assay, the animals (n = 3-5/group) were sacrificed 2.25 h after the drug. In a second experiment, rats (n = 5-7/group) received a first intra-DLPAG injection of saline or methylene blue (MB, 30 nmol), an NO antagonist, followed by saline or SIN-1 (300 nmol). SIN-1 (300 nmol) induced a flight response characterized by coordinated running and oriented jumps with escape attempts. Similar but short-lasting changes were seen after the administration of DEA/NO. FLI was dose-dependently induced by SIN-1 in several regions related to defensive reactions, including the periaqueductal gray, hypothalamic nuclei, medial amygdala, and cingulate cortex (analysis of variance, p < 0.05). A greater number of neurons showing FLI was found ipsilateral to the microinjection site. The drug effect was greater at this side in the bed nucleus of the stria terminalis, paraventricular and lateral hypothalamus, cingulate cortex, septohipoccampal nucleus, and horizontal limb of the diagonal band. The increase in SIN-1-induced FLI was attenuated by MB pretreatment in most regions. These results suggest that NO may participate in the modulation of defensive responses in the DLPAG.

Behavioral effects in the elevated plus maze of an NMDA antagonist injected into the dorsal hippocampus: influence of restraint stress

The objective of the study was to investigate the influence of restraint stress on the effects of 2-amino-7-phosphonoheptanoic acid (AP7), an NMDA receptor antagonist, injected into the hippocampus of rats submitted to the elevated plus maze (EPM). Male Wistar rats with cannulas aimed to the dorsal hippocampus were forced immobilized for 2 h. Twenty four hours later they received bilateral injections of saline or AP7 (10 nmol/0.5 microl), and were tested in the EPM. In another experiment the animals received the treatment immediately before or after the restraint period, and were tested in the EPM 24 h later. AP7 had no effect in any anxiety measure in non-stressed rats. In stressed animals the drug increased the percentage of open arm entries when injected before the test in the EPM. When administered immediately after the restraint period, AP7 increased the percentage of time spent in the open arms and tended to do the same with the percentage of entries in these same arms. The results suggest that interference with hippocampal NMDA receptors modify the anxiogenic effect of restraint stress in an EPM.

L-NOARG, an inhibitor of nitric oxide synthase, induces catalepsy in mice.

The objective of this study was to investigate possible motor effects of NG-nitro-l-arginine (l-NOARG), an inhibitor of nitric oxide synthase. Acute administration of l-NOARG (5–80 mg kg−1, i.p.) induced catalepsy in a dose-dependent manner in male albino-Swiss mice. The drug had an additive effect with haloperidol (0.125–0.5 mg kg−1, i.p.). The acute effect of l-NOARG (40 mg kg−1, i.p.) was attenuated by previous treatment with l-arginine (300 mg kg−1, i.p.). Sub-chronic treatment with l-NOARG (40 mg kg−1, i.p., twice a day for 4 days) was also able to attenuate the cataleptic effect of acute drug administration (40 mg kg−1, i.p.). These results show that acute systemic injections of l-NOARG induce significant motor effects.

A nitric oxide synthase inhibitor decreases 6-hydroxydopamine effects on tyrosine hydroxylase and neuronal nitric oxide synthase in the rat nigrostriatal pathway

There is evidence that nitric oxide plays a role in the neurotransmitter balance within the basal ganglia and in the pathology of Parkinsons disease. In the present work we investigated in striatal 6-hydroxydopamine (6-OHDA) lesioned rats the effects of a nitric oxide synthase (NOS) inhibitor, NG-nitro-l-arginine (L-NOARG), given systemically on both the dopaminergic (DA) neuronal loss and the neuronal NOS cell density. We analyzed the DA neuronal loss through tyrosine hydroxylase immunohistochemistry (TH). The nitrergic system was evaluated using an antibody against the neuronal NOS (nNOS) isoform. Treatment with the L-NOARG significantly reduced 6-OHDA-induced dopaminergic damage in the dorsal striatum, ventral substantia nigra and lateral globus pallidus, but had no effects in the dorsal substantia nigra and in the cingulate cortex. Furthermore, L-NOARG reduced 6-OHDA-induced striatal increase, and substantia nigra compacta decrease, in the density of neuronal nitric oxide synthase positive cells. These results suggest that nitric oxide synthase inhibition may decrease the toxic effects of 6-OHDA on dopaminergic terminals and on dopamine cell bodies in sub-regions of the SN and on neuronal nitric oxide synthase cell density in the rat brain.

A simple, inexpensive and easily reproducible model of spinal cord injury in mice: Morphological and functional assessment

Spinal cord injury (SCI) causes motor and sensory deficits that impair functional performance, and significantly impacts life expectancy and quality. Animal models provide a good opportunity to test therapeutic strategies in vivo. C57BL/6 mice were subjected to laminectomy at T9 and compression with a vascular clip (30g force, 1min). Two groups were analyzed: injured group (SCI, n=33) and laminectomy only (Sham, n=15). Locomotor behavior (Basso mouse scale-BMS and global mobility) was assessed weekly. Morphological analyses were performed by LM and EM. The Sham group did not show any morphofunctional alteration. All SCI animals showed flaccid paralysis 24h after injury, with subsequent improvement. The BMS score of the SCI group improved until the intermediate phase (2.037+/-1.198); the Sham animals maintained the highest BMS score (8.981+/-0.056), p<0.001 during the entire time. The locomotor speed was slower in the SCI animals (5.581+/-0.871) than in the Sham animals (15.80+/-1.166), p<0.001. Morphological analysis of the SCI group showed, in the acute phase, edema, hemorrhage, multiple cavities, fiber degeneration, cell death and demyelination. In the chronic phase we observed glial scarring, neuron death, and remyelination of spared axons by oligodendrocytes and Schwann cells. In conclusion, we established a simple, reliable, and inexpensive clip compression model in mice, with functional and morphological reproducibility and good validity. The availability of producing reliable injuries with appropriate outcome measures represents great potential for studies involving cellular mechanisms of primary injury and repair after traumatic SCI.

Decreased glial reactivity could be involved in the antipsychotic-like effect of cannabidiol.

NMDA receptor hypofunction could be involved, in addition to the positive, also to the negative symptoms and cognitive deficits found in schizophrenia patients. An increasing number of data has linked schizophrenia with neuroinflammatory conditions and glial cells, such as microglia and astrocytes, have been related to the pathogenesis of schizophrenia. Cannabidiol (CBD), a major non-psychotomimetic constituent of Cannabis sativa with anti-inflammatory and neuroprotective properties induces antipsychotic-like effects. The present study evaluated if repeated treatment with CBD (30 and 60 mg/kg) would attenuate the behavioral and glial changes observed in an animal model of schizophrenia based on the NMDA receptor hypofunction (chronic administration of MK-801, an NMDA receptor antagonist, for 28 days). The behavioral alterations were evaluated in the social interaction and novel object recognition (NOR) tests. These tests have been widely used to study changes related to negative symptoms and cognitive deficits of schizophrenia, respectively. We also evaluated changes in NeuN (a neuronal marker), Iba-1 (a microglia marker) and GFAP (an astrocyte marker) expression in the medial prefrontal cortex (mPFC), dorsal striatum, nucleus accumbens core and shell, and dorsal hippocampus by immunohistochemistry. CBD effects were compared to those induced by the atypical antipsychotic clozapine. Repeated MK-801 administration impaired performance in the social interaction and NOR tests. It also increased the number of GFAP-positive astrocytes in the mPFC and the percentage of Iba-1-positive microglia cells with a reactive phenotype in the mPFC and dorsal hippocampus without changing the number of Iba-1-positive cells. No change in the number of NeuN-positive cells was observed. Both the behavioral disruptions and the changes in expression of glial markers induced by MK-801 treatment were attenuated by repeated treatment with CBD or clozapine. These data reinforces the proposal that CBD may induce antipsychotic-like effects. Although the possible mechanism of action of these effects is still unknown, it may involve CBD anti-inflammatory and neuroprotective properties. Furthermore, our data support the view that inhibition of microglial activation may improve schizophrenia symptoms.

O trauma raquimedular

A medula espinhal dos mamiferos adultos nao permite a regeneracao de axonios. Por razoes ainda desconhecidas, as fibras neurais falham em cruzar o sitio da lesao, como se nao houvesse crescimento, desde a primeira tentativa. Quais mecanismos poderiam explicar a perda da capacidade de regeneracao? As cicatrizes formadas pelas celulas da glia seriam uma consequencia da falha na regeneracao ou a causa? Diversas linhas de evidencia sugerem que a regeneracao da medula espinhal seria impedida no sistema nervoso central pela acao de fatores locais no sitio da lesao, e que o sistema nervoso central nao-lesado e um meio permissivo para o crescimento axonal, na direcao de alvos especificos. Uma vez que os axonios sao induzidos adequadamente a cruzar a lesao com o auxilio de implantes, farmacos ou celulas indiferenciadas, as fibras em regeneracao podem encontrar a via especifica e estabelecer conexoes corretas. O que ainda nao se sabe e que combinacao de moleculas induz/inibe o potencial de regeneracao do tecido e que mecanismos permitem aos neuronios formarem conexoes especificas com os alvos com os quais sao programados a fazer.