Alterações nas respostas nociceptivas a estímulo mecânico e ao frio em dor neuropática induzida pela construção crônica do nervo ciático em ratos : envolvimento dos receptores TRPA1 e TRPM8

Orientador: Joice Maria da CunhaTrabalho de Conclusão de Curso (Bacharelado) - Universidade Federal do Paraná. Setor de Ciências Biológicas. Curso de Graduação em Biomedicin

Alterações nas respostas nociceptivas a estímulo mecânico e ao frio em dor neuropática induzida pela construção crônica do nervo ciático em ratos : envolvimento dos receptores TRPA1 e TRPM8

Orientador: Joice Maria da CunhaTrabalho de Conclusão de Curso (Bacharelado) - Universidade Federal do Paraná. Setor de Ciências Biológicas. Curso de Graduação em Biomedicin

Behavioral and neuroplastic effects of the combination of escitalopram and cannabinoids

Antidepressivos clássicos apresentam uma série de limitações na prática clínica, sendo uma das mais relevantes a latência de 4 a 8 semanas para indução de uma melhora significativa dos sintomas. Existem evidências que canabinóides, como o fitocanabinóide canabidiol (CBD) e o inibidor de degradação da anandamida URB597 (URB) geram efeitos comportamentais semelhantes aos antidepressivos. Canabinóides e antidepressivos podem, inclusive, apresentar mecanismos moleculares em comum. O presente estudo investigou os efeitos comportamentais e neuroplásticos de uma semana de tratamento com a combinação de uma dose do antidepressivo escitalopram (ESC) que gera efeitos comportamentais após 3 semanas de tratamento com doses sub-efetivas de canabinóides em camundongos estressados. Para isso, camundongos C57Bl6 machos foram submetidos ao protocolo de estresse crônico imprevisível (CUS) ou ao protocolo de estresse de derrota social (SDS) por 10 dias. Os animais foram divididos em 7 grupos experimentais (n=8-13; Controle (Não estressado); Veículo (Veí/Veí); ESC 10mg/kg (Esc/Veí); CBD 7,5 mg/kg (Veí/CBD); URB 0,1 mg/kg (Veí/CBD); ESC/CBD; ESC/URB) e foram tratados durante os 7 últimos dias do protocolo de estresse (via i.p.). O tratamento com a combinação de escitalopram e CBD gerou respostas preditivas de um efeito do tipo-antidepressivo no teste de suspensão pela cauda e de um efeito do tipo-ansiolítico no novelty suppressed feeding tanto no modelo de estresse homotípico (SDS) quanto no modelo de estresse heterotípico (CUS). Paralelamente às alterações comportamentais, foi observado que o grupo ESC/CBD apresentou uma maior expressão relativa de sinaptofisina no córtex pré-frontal, evidenciando um possível efeito pró-sinaptogênico da combinação. Esse mesmo tratamento promoveu aumento na expressão de sinaptotagmina e ?-actina no hipocampo. Os resultados encontrados no presente estudo evidenciam que a combinação com CBD pode otimizar a ação do antidepressivo escitalopram.Despite their widespread use, antidepressant drugs show some limitations in the clinical practice, including a delayed onset of action. Cannabinoid drugs, such as canabidiol (CBD) and the anandamide degradation inhibitor URB597 (URB), have shown behavioral effects that are similar to those induced by antidepressants in preclinical studies. Antidepressants and cannabinoids might even share common mechanisms. This study aimed to evaluate the behavioral and neuroplastic effects of one week of treatment of stressed animals with the combination of sub-effective doses of cannabinoids with 10mg/Kg of escitalopram (ESC)- dose that prevented- stressinduced behavioural changes only after 3 weeks of treatment. Male C57Bl6 mice were subjected to a 10-day protocol of either social defeat stress (SDS) or chronic unpredictable stress (CUS). Animals were divided in 7 experimental groups (n=8-13; Control (Non-stressed); Vehicle (Veh/Veh); ESC 10mg/kg (Esc/Veh); CBD 7,5mg/kg (Veh/CBD); URB 0,1mg/kg (Veh/CBD); ESC/CBD; ESC/URB) and the treatment was conducted during the 7 last days of the stress protocol (via i.p.). The treatment with the combination of ESC and CBD produced responses that are considered predictive of antidepressant-like effects in the tail suspension test and of anxiolytic-like effects in the novelty suppressed feeding in both the homotypic stress (SDS) and the heterotypic stress (CUS) models. In paralel with the bevavioral outcomes, molecular analysis revealed that the ESC/CBD group showed a higher relative expression of synaptophysin in the PFC, suggesting a pro-synaptogenic effect of the combination. Increased relative expression of synaptotagmin and ?-actin in the hippocampus following ESC/CBD treatment were also reported. The results presented in this study provide evidence that the combination with CBD can optimize the action of the antidepressant escitalopram

Behavioral and neuroplastic effects of the combination of escitalopram and cannabinoids

Antidepressivos clássicos apresentam uma série de limitações na prática clínica, sendo uma das mais relevantes a latência de 4 a 8 semanas para indução de uma melhora significativa dos sintomas. Existem evidências que canabinóides, como o fitocanabinóide canabidiol (CBD) e o inibidor de degradação da anandamida URB597 (URB) geram efeitos comportamentais semelhantes aos antidepressivos. Canabinóides e antidepressivos podem, inclusive, apresentar mecanismos moleculares em comum. O presente estudo investigou os efeitos comportamentais e neuroplásticos de uma semana de tratamento com a combinação de uma dose do antidepressivo escitalopram (ESC) que gera efeitos comportamentais após 3 semanas de tratamento com doses sub-efetivas de canabinóides em camundongos estressados. Para isso, camundongos C57Bl6 machos foram submetidos ao protocolo de estresse crônico imprevisível (CUS) ou ao protocolo de estresse de derrota social (SDS) por 10 dias. Os animais foram divididos em 7 grupos experimentais (n=8-13; Controle (Não estressado); Veículo (Veí/Veí); ESC 10mg/kg (Esc/Veí); CBD 7,5 mg/kg (Veí/CBD); URB 0,1 mg/kg (Veí/CBD); ESC/CBD; ESC/URB) e foram tratados durante os 7 últimos dias do protocolo de estresse (via i.p.). O tratamento com a combinação de escitalopram e CBD gerou respostas preditivas de um efeito do tipo-antidepressivo no teste de suspensão pela cauda e de um efeito do tipo-ansiolítico no novelty suppressed feeding tanto no modelo de estresse homotípico (SDS) quanto no modelo de estresse heterotípico (CUS). Paralelamente às alterações comportamentais, foi observado que o grupo ESC/CBD apresentou uma maior expressão relativa de sinaptofisina no córtex pré-frontal, evidenciando um possível efeito pró-sinaptogênico da combinação. Esse mesmo tratamento promoveu aumento na expressão de sinaptotagmina e ?-actina no hipocampo. Os resultados encontrados no presente estudo evidenciam que a combinação com CBD pode otimizar a ação do antidepressivo escitalopram.Despite their widespread use, antidepressant drugs show some limitations in the clinical practice, including a delayed onset of action. Cannabinoid drugs, such as canabidiol (CBD) and the anandamide degradation inhibitor URB597 (URB), have shown behavioral effects that are similar to those induced by antidepressants in preclinical studies. Antidepressants and cannabinoids might even share common mechanisms. This study aimed to evaluate the behavioral and neuroplastic effects of one week of treatment of stressed animals with the combination of sub-effective doses of cannabinoids with 10mg/Kg of escitalopram (ESC)- dose that prevented- stressinduced behavioural changes only after 3 weeks of treatment. Male C57Bl6 mice were subjected to a 10-day protocol of either social defeat stress (SDS) or chronic unpredictable stress (CUS). Animals were divided in 7 experimental groups (n=8-13; Control (Non-stressed); Vehicle (Veh/Veh); ESC 10mg/kg (Esc/Veh); CBD 7,5mg/kg (Veh/CBD); URB 0,1mg/kg (Veh/CBD); ESC/CBD; ESC/URB) and the treatment was conducted during the 7 last days of the stress protocol (via i.p.). The treatment with the combination of ESC and CBD produced responses that are considered predictive of antidepressant-like effects in the tail suspension test and of anxiolytic-like effects in the novelty suppressed feeding in both the homotypic stress (SDS) and the heterotypic stress (CUS) models. In paralel with the bevavioral outcomes, molecular analysis revealed that the ESC/CBD group showed a higher relative expression of synaptophysin in the PFC, suggesting a pro-synaptogenic effect of the combination. Increased relative expression of synaptotagmin and ?-actin in the hippocampus following ESC/CBD treatment were also reported. The results presented in this study provide evidence that the combination with CBD can optimize the action of the antidepressant escitalopram

Involvement of NAPE-PLD and neuroplastic mediated mechanisms in the ventral prefrontal cortex in the anti-stress effects of sub-effective doses of cannabidiol and escitalopram

Estresse crônico precipita a manifestação de sintomas de transtornos psiquiátricos como transtornos de humor e ansiedade. Inibidores seletivos de recaptação de serotonina (ISRS) são frequentemente utilizados para o tratamento desses transtornos psiquiátricos associados a estresse. Entretanto, uma das limitações de ISRSs é que requerem entre 4-8 semanas de tratamento para induzir uma melhora significativa nos sintomas. Previamente, relatamos que a combinação de escitalopram (ESC; 10mg/kg) com uma dose subefetiva de canabidiol (CBD; 7,5mg/kg) acelera o efeito do tipo-ansiolítico do ISRS em camundongos expostos a 10 dias de estresse. Aqui, nós hipotetizamos que o efeito antiestresse da combinação ESC+CBD é dependente da sinalização de mediadores lipídicos do endocanabinoidoma (ECBoma), um sistema que parece ser um alvo em comum entre CBD e ESC, no córtex pré-frontal (CPF) ventromedial (vm). Para testar isso, camundongos machos C57Bl6 (10-12 semanas) receberam a injeção intra- CPFvm de vetores virais que direcionavam a deleção mediada por um sistema CRISPR-Cas9 da enzima NAPE-PLD ou da enzima DAGLα, às quais estão envolvidas em vias de síntese de ligantes do ECBoma. Após 15 dias, os animais foram expostos a 10 dias de estresse crônico imprevisível (CUS) e tratados por 7 dias com veículo ou com ESC+CBD. Nós observamos que o efeito do tipo-ansiolítico de ESC+CBD no novelty-suppressed feeding (NSF) foi abolido em animais com deleção da enzima NAPE-PLD, mas não DAGLα, no CPFvm. Além disso, a combinação de ESC+CBD, apesar de não alterar significativamente a resposta comportamental de animais expostos a 10 dias de CUS no splash-test, induziu um efeito do tipo-ansiolítico em camundongos expostos a 21 dias de estresse e tratados com as combinações de Veículo ou ESC (10mg/kg) com Veículo ou CBD (7,5mg/kg) apenas durante os últimos 7 dias do protocolo de estresse. Ademais, estresse reduziu significativamente a expressão da enzima NAPE-PLD no CPF medial e o tratamento com ESC e CBD gerou um aumento na expressão da enzima e aumentou a porcentagem de interneurônios parvalbumina que expressavam NAPE-PLD no CPF medial. A exposição a CUS aumentou a densidade de células precursoras de oligodendrócitos (CPOs) e a sobrevivência de CPOs em atividade proliferativa no CPF medial. Os resultados indicam que 7 dias de tratamento com a combinação ESC+CBD induz um efeito do tipo-ansiolítico mesmo em animais expostos a 3 semanas de CUS. O efeito antiestresse do tratamento é dependente da expressão de NAPE-PLD no CPFvm e é caracterizado por alterações plásticas no CPF de camundongos estressados.Chronic stress precipitates the symptoms of psychiatric disorders, including mood and anxiety disorders. Selective serotonin reuptake inhibitors (SSRIs) are often used for treating these stress-associated psychiatric disorders. However, a main limitation of SSRIs is their late-onset of action (only after 4-8 weeks of treatment). Previously, we have shown that the combination of escitalopram (ESC; 10mg/kg) with a sub effective dose of cannabidiol (CBD; 7.5mg/kg) accelerates the anxiolytic-like effects of the SSRI in mice submitted to a 10-day stress protocol. Here, we hypothesize that the anti-stress effect of ESC+CBD is dependent on the signaling mediated by lipid ligands of the Endocannabinoidome (ECBome), a system that seems to be a common target for ESC and CBD, in the ventromedial prefrontal cortex (vmPFC). To test this, C57Bl6 male mice (10-12weeks) received an intra-vmPFC injection of viral vectors directing the CRISPR-Cas9-mediated deletion of the enzyme NAPE-PLD or the enzyme DAGLα, which are involved in the synthesis of ECBoma ligands. After 15 days, mice were exposed to 10 days of chronic unpredictable stress (CUS) and treated for 7 days with vehicle or with ESC+CBD. The anxiolytic-like effect of ESC+CBD in the novelty-suppressed feeding (NSF) test was abolished in mice with induced deletion of NAPE-PLD, but not DAGLα, in the vmPFC. Furthermore, the combination of ESC+CBD, despite not altering the behavior of mice exposed to a 10-day stress protocol in the splash-test, induced anxiolytic-like effect in mice exposed to 21 days of stress and treated with the combinations of vehicle or ESC (10mg/kg) with Vehicle or CBD (7.5mg/kg) only in the last 7 days of the stress protocol. Moreover, stress decreased the expression of NAPE-PLD in the medial PFC and the treatment with ESC+CBD increased the expression of the enzyme while also increased the percentage of parvalbumin (PV) interneurons that expressed NAPE-PLD in the medial PFC. In addition, CUS increased the density of oligodendrocyte precursor cells (OPCs) and the survival of proliferating OPCs in the medial PFC. Our results indicate that ESC+CBD induce an anxiolytic-like effect after 7 days of treatment even in mice exposed to 3 weeks of stress. The anti-stress effects of ESC+CBD rely on the expression of NAPEPLD in the vmPFC and are characterized by plastic alterations in the PFC of stressed mice

Enhanced proliferation of oligodendrocyte progenitor cells following retrovirus mediated Achaete-scute complex-like 1 overexpression in the postnatal cerebral cortex in vivo

The proneural transcription factor Achaete-scute complex-like 1 (Ascl1) is a major regulator of neural fate decisions, implicated both in neurogenesis and oligodendrogliogenesis. Focusing on its neurogenic activity, Ascl1 has been widely used to reprogram non-neuronal cells into induced neurons. In vitro, Ascl1 induces efficient reprogramming of proliferative astroglia from the early postnatal cerebral cortex into interneuron-like cells. Here, we examined whether Ascl1 can similarly induce neuronal reprogramming of glia undergoing proliferation in the postnatal mouse cerebral cortex in vivo. Toward this goal, we targeted cortical glia during the peak of proliferative expansion (i.e., postnatal day 5) by injecting a retrovirus encoding for Ascl1 into the mouse cerebral cortex. In contrast to the efficient reprogramming observed in vitro, in vivo Ascl1-transduced glial cells were converted into doublecortin-immunoreactive neurons only with very low efficiency. However, we noted a drastic shift in the relative number of retrovirus-transduced Sox10-positive oligodendrocyte progenitor cells (OPCs) as compared to glial fibrillary acidic protein (GFAP)-positive astrocytes. Genetic fate mapping demonstrated that this increase in OPCs was not due to Ascl1-mediated astrocyte-to-OPC fate conversion. Rather, EdU incorporation experiments revealed that Ascl1 caused a selective increase in proliferative activity of OPCs, but not astrocytes. Our data indicate that rather than inducing neuronal reprogramming of glia in the early postnatal cortex, Ascl1 is a selective enhancer of OPC proliferation

Enhanced proliferation of oligodendrocyte progenitor cells following retrovirus mediated Achaete-scute complex-like 1 overexpression in the postnatal cerebral cortex in vivo

Confocal raw microscopy images (czi) from every figue and analysis done in MS Excel. Zipped data folders include readme files explaining how to navigate them

Comparative study of cold hyperalgesia and mechanical allodynia in two animal models of neuropathic pain: different etiologies and distinct pathophysiological mechanisms.

Neuropathic pain (NP) affects more than 8% of the global population. The proposed action of the transient receptor potential ankyrin 1 (TRPA1) as a mechanosensor and the characterization of the transient receptor potential melastatin 8 (TRPM8) as a cold thermosensor raises the question of whether these receptors are implicated in NP. Our study aimed to evaluate the involvement of TRPA1 and TRPM8 in cold and mechanical signal transduction to obtain a comparative view in rat models of streptozotocin-induced diabetes (STZ) and chronic constriction injury of the sciatic nerve (CCI). The electronic von Frey test showed that STZ rats presented mechanical allodynia that was first evidenced on the 14th day after diabetes confirmation, and four days after CCI. This phenomenon was reduced by the intraplantar (ipl) administration of a TRPA1 receptor antagonist (HC-030031; 40 μL/300 μg/paw) in both NP models. Only CCI rats displayed cold hyperalgesia based on the cold plate test. The pharmacological blocking of TRPA1 through the injection of the antagonist attenuated cold hyperalgesia in this NP model. STZ animals showed a reduction in the number of flinches induced by the intraplantar injection of mustard oil (MO; TRPA1 agonist; 0.1%/50 μL/paw), or intraplantar injection of menthol (MT; TRPM8 agonist; 0.5% and 1%/50 μL/paw). The response induced by the ipl administration of MT (1%/50 µL/paw) was significantly different between the CCI and SHAM groups. Together, these data suggest a different pattern in nociceptive behavior associated with different models of NP, suggesting a variant involvement of TRPA1 and TRPM8 in both conditions

Video_2_Enhanced proliferation of oligodendrocyte progenitor cells following retrovirus mediated Achaete-scute complex-like 1 overexpression in the postnatal cerebral cortex in vivo.AVI

The proneural transcription factor Achaete-scute complex-like 1 (Ascl1) is a major regulator of neural fate decisions, implicated both in neurogenesis and oligodendrogliogenesis. Focusing on its neurogenic activity, Ascl1 has been widely used to reprogram non-neuronal cells into induced neurons. In vitro, Ascl1 induces efficient reprogramming of proliferative astroglia from the early postnatal cerebral cortex into interneuron-like cells. Here, we examined whether Ascl1 can similarly induce neuronal reprogramming of glia undergoing proliferation in the postnatal mouse cerebral cortex in vivo. Toward this goal, we targeted cortical glia during the peak of proliferative expansion (i.e., postnatal day 5) by injecting a retrovirus encoding for Ascl1 into the mouse cerebral cortex. In contrast to the efficient reprogramming observed in vitro, in vivo Ascl1-transduced glial cells were converted into doublecortin-immunoreactive neurons only with very low efficiency. However, we noted a drastic shift in the relative number of retrovirus-transduced Sox10-positive oligodendrocyte progenitor cells (OPCs) as compared to glial fibrillary acidic protein (GFAP)-positive astrocytes. Genetic fate mapping demonstrated that this increase in OPCs was not due to Ascl1-mediated astrocyte-to-OPC fate conversion. Rather, EdU incorporation experiments revealed that Ascl1 caused a selective increase in proliferative activity of OPCs, but not astrocytes. Our data indicate that rather than inducing neuronal reprogramming of glia in the early postnatal cortex, Ascl1 is a selective enhancer of OPC proliferation.</p

Image_1_Enhanced proliferation of oligodendrocyte progenitor cells following retrovirus mediated Achaete-scute complex-like 1 overexpression in the postnatal cerebral cortex in vivo.TIF

The proneural transcription factor Achaete-scute complex-like 1 (Ascl1) is a major regulator of neural fate decisions, implicated both in neurogenesis and oligodendrogliogenesis. Focusing on its neurogenic activity, Ascl1 has been widely used to reprogram non-neuronal cells into induced neurons. In vitro, Ascl1 induces efficient reprogramming of proliferative astroglia from the early postnatal cerebral cortex into interneuron-like cells. Here, we examined whether Ascl1 can similarly induce neuronal reprogramming of glia undergoing proliferation in the postnatal mouse cerebral cortex in vivo. Toward this goal, we targeted cortical glia during the peak of proliferative expansion (i.e., postnatal day 5) by injecting a retrovirus encoding for Ascl1 into the mouse cerebral cortex. In contrast to the efficient reprogramming observed in vitro, in vivo Ascl1-transduced glial cells were converted into doublecortin-immunoreactive neurons only with very low efficiency. However, we noted a drastic shift in the relative number of retrovirus-transduced Sox10-positive oligodendrocyte progenitor cells (OPCs) as compared to glial fibrillary acidic protein (GFAP)-positive astrocytes. Genetic fate mapping demonstrated that this increase in OPCs was not due to Ascl1-mediated astrocyte-to-OPC fate conversion. Rather, EdU incorporation experiments revealed that Ascl1 caused a selective increase in proliferative activity of OPCs, but not astrocytes. Our data indicate that rather than inducing neuronal reprogramming of glia in the early postnatal cortex, Ascl1 is a selective enhancer of OPC proliferation.</p