RESUMO
ETHNOPHARMACOLOGICAL RELEVANCE: Trichilia catigua A. Juss (Meliaceae) is used in Brazilian folk medicine to alleviate fatigue and emotional stress and improve memory. Previous studies from our laboratory reported that an ethyl-acetate fraction (EAF) of T. catigua that was given before cerebral ischemia in vivo prevented memory loss and reduced oxidative stress and neuroinflammation. Despite the value of these findings of a neuroprotective effect of T. catigua, treatment that was given immediately before or immediately after ischemia limits its clinical relevance. Thus, unknown is whether T. catigua possesses a specific time window of efficacy (TWE) when administered postischemia. AIM OF THE STUDY: Given continuity to previous studies, we investigated whether an EAF of T. catigua maintains its neuroprotective properties if treatment begins at different time windows of efficacy after ischemia. We also evaluated, for the first time, whether T. catigua possesses neuroplasticity/neurotrophic properties. MATERIAL AND METHODS: Rats were subjected to transient global brain ischemia (TGCI) and then given a single dose of the EAF (400 mg/kg) or vehicle (1 ml/kg) orally 1, 4, or 6 h postischemia. The levels of protein PCG, GSH, and GSSG, and activity of SOD and CAT were assayed as markers of oxidative stress on the day after ischemia. In another experiment, naive rats underwent spatial learning training in a radial maze task and then subjected to TGCI. Delayed treatment with the EAF began 4 or 6 h later and continued for 7 days. Retrograde memory performance was assessed 10, 17, and 24 days postischemia. Afterward, brains were examined for neurodegeneration and neuronal dendritic morphology in the hippocampus and cerebral cortex. Another group received the EAF at 4 h of reperfusion, and 4 days later their brains were examined for GFAP and Iba-1 immunoreactivity. Lastly, ischemic rats received the EAF 4 h after ischemia and neural plasticity-related proteins, BDNF, SYN, PSD 95, and NeuN were measured in the hippocampus 7 and 14 days after ischemia. RESULTS: A single EAF administration 1, 4, or 6 h postischemia alleviated oxidative stress that was caused by ischemia, expressed as a reduction of the amount of the PCG and GSSG, normalization of the GSH/GSSG ratio, and the restoration of SOD activity. Ischemia caused the persistent loss of memory (i.e., amnesia), an outcome that was consistently ameliorated by treatment with the EAF that was initiated 4 or 6 h postischemia. The 4 h delay in EAF treatment positively impacted dendritic morphology in neurons that survived ischemia. TGCI reduced BDNF, SYN, PSD-95, and NeuN protein levels in the hippocampus and cerebral cortex. The EAF normalized SYN and PSD-95 protein levels. Ischemia-induced neurodegeneration and glial cell activation were not prevented by EAF treatment. CONCLUSION: The present study corroborates prior data that demonstrated the neuroprotective potential of T. catigua and extends these data by showing that the delayed administration of EAF postischemia effectively prevented memory impairment and decreased oxidative stress, dendritic deterioration, and synaptic protein loss within a TWE that ranged from 1 to 6 h. This specific TWE in preclinical research may have clinical relevance by suggesting the possible utility of this plant for the development of neuroprotective strategies in the setting of ischemic brain diseases. Another innovative finding of the present study was the possible neurotrophic/neuroplastic properties of T. catigua.
Assuntos
Isquemia Encefálica , Meliaceae , Fármacos Neuroprotetores , Ratos , Animais , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Dissulfeto de Glutationa/metabolismo , Dissulfeto de Glutationa/farmacologia , Dissulfeto de Glutationa/uso terapêutico , Extratos Vegetais/farmacologia , Isquemia Encefálica/tratamento farmacológico , Estresse Oxidativo , Infarto Cerebral/tratamento farmacológico , Hipocampo , Transtornos da Memória/tratamento farmacológico , Acetatos/farmacologia , Superóxido Dismutase/metabolismo , Plasticidade Neuronal , Fármacos Neuroprotetores/farmacologiaRESUMO
Evidence for the clinical use of neuroprotective drugs for the treatment of cerebral ischemia (CI) is still greatly limited. Spatial/temporal disorientation and cognitive dysfunction are among the most prominent long-term sequelae of CI. Cannabidiol (CBD) is a non-psychotomimetic constituent of Cannabis sativa that exerts neuroprotective effects against experimental CI. The present study investigated possible neuroprotective mechanisms of action of CBD on spatial memory impairments that are caused by transient global cerebral ischemia (TGCI) in rats. Hippocampal synaptic plasticity is a fundamental mechanism of learning and memory. Thus, we also evaluated the impact of CBD on neuroplastic changes in the hippocampus after TGCI. Wistar rats were trained to learn an eight-arm aversive radial maze (AvRM) task and underwent either sham or TGCI surgery. The animals received vehicle or 10 mg/kg CBD (i.p.) 30 min before surgery, 3 h after surgery, and then once daily for 14 days. On days 7 and 14, we performed a retention memory test. Another group of rats that received the same pharmacological treatment was tested in the object location test (OLT). Brains were removed and processed to assess neuronal degeneration, synaptic protein levels, and dendritic remodeling in the hippocampus. Cannabidiol treatment attenuated ischemia-induced memory deficits. In rats that were subjected to TGCI, CBD attenuated hippocampal CA1 neurodegeneration and increased brain-derived neurotrophic factor levels. Additionally, CBD protected neurons against the deleterious effects of TGCI on dendritic spine number and the length of dendritic arborization. These results suggest that the neuroprotective effects of CBD against TGCI-induced memory impairments involve changes in synaptic plasticity in the hippocampus.
Assuntos
Canabidiol/uso terapêutico , Hipocampo/efeitos dos fármacos , Ataque Isquêmico Transitório/prevenção & controle , Plasticidade Neuronal/efeitos dos fármacos , Neuroproteção/efeitos dos fármacos , Sinapses/efeitos dos fármacos , Animais , Canabidiol/farmacologia , Modelos Animais de Doenças , Hipocampo/metabolismo , Hipocampo/patologia , Ataque Isquêmico Transitório/metabolismo , Ataque Isquêmico Transitório/patologia , Masculino , Plasticidade Neuronal/fisiologia , Neuroproteção/fisiologia , Técnicas de Cultura de Órgãos , Ratos , Ratos Wistar , Memória Espacial/efeitos dos fármacos , Memória Espacial/fisiologia , Sinapses/metabolismo , Sinapses/patologiaRESUMO
We previously found that fish oil (FO) facilitated memory recovery in the absence of pyramidal neuron rescue after transient, global cerebral ischemia (TGCI). Fish oil preserved the expression of microtubule-associated protein 2 (MAP-2), suggesting a relationship between dendritic plasticity and memory recovery that is mediated by FO after TGCI. The present study examined whether postischemic treatment with FO prevents ischemia-induced loss of dendritic processes in remaining pyramidal neurons. The effects of FO on neuroplasticity-related proteins were also examined after TGCI. Rats were subjected to TGCI (15 min, four-vessel occlusion model) and then received vehicle or FO (300 mg/kg docosahexaenoic acid) once daily for 7 days. The first dose was administered 4 h postischemia. Golgi-Cox staining was used to evaluate dentrict morphology in the pyramidal neurons of hippocampus (CA1 and CA3 subfields) and prefrontal cortex (PFC). Neuronal nuclei protein (NeuN), brain-derived neurotrophic factor (BDNF), growth-associated protein 43 (GAP-43), synaptophysin (SYP), and postsynaptic density protein 95 (PSD-95) levels were measured by Western blot in both structures. Fifteen minutes of TGCI reduced consistently the length of dendrites, number of dendritic branches and dendritic spine density (average of 25%, 43%, 32%, respectively) 7, 14, and 21 days postischemia, indicating that they did not recover spontaneously. This outcome of TGCI was reversed by FO treatment, an effect that was sustained even after treatment cessation. The NeuN and BDNF protein levels were reduced in both the hippocampus and PFC, which were recovered by FO treatment. GAP-43 protein levels decreased after ischemia in the PFC only, and this effect was also mitigated by FO. Neither SYP nor PSD-95 levels were altered by ischemia, but PDS-95 levels almost doubled after FO treatment in the ischemic group. These data support our hypothesis that synaptic plasticity at the level of dendrites may at least partially underlie the memory-protective effect of FO after TGCI and strengthen the possibility that FO has therapeutic potential for treating the sequelae of brain ischemia/reperfusion injury.
Assuntos
Dendritos/efeitos dos fármacos , Óleos de Peixe/farmacologia , Ataque Isquêmico Transitório/patologia , Plasticidade Neuronal/efeitos dos fármacos , Sinapses/efeitos dos fármacos , Animais , Dendritos/patologia , Ácidos Docosa-Hexaenoicos/farmacologia , Hipocampo/efeitos dos fármacos , Hipocampo/patologia , Masculino , Fármacos Neuroprotetores/farmacologia , Ratos , Ratos Wistar , Sinapses/patologiaRESUMO
Diabetes and aging are risk factors for cognitive impairments after chronic cerebral hypoperfusion (CCH). Cannabidiol (CBD) is a phytocannabinoid present in the Cannabis sativa plant. It has beneficial effects on both cerebral ischemic diseases and diabetes. We have recently reported that diabetes interacted synergistically with aging to increase neuroinflammation and memory deficits in rats subjected to CCH. The present study investigated whether CBD would alleviate cognitive decline and affect markers of inflammation and neuroplasticity in the hippocampus in middle-aged diabetic rats submitted to CCH. Diabetes was induced in middle-aged rats (14 months old) by intravenous streptozotocin (SZT) administration. Thirty days later, the diabetic animals were subjected to sham or CCH surgeries and treated with CBD (10 mg/kg, once a day) during 30 days. Diabetes exacerbated cognitive deficits induced by CCH in middle-aged rats. Repeated CBD treatment decreased body weight in both sham- and CCH-operated animals. Cannabidiol improved memory performance and reduced hippocampal levels of inflammation markers (inducible nitric oxide synthase, ionized calcium-binding adapter molecule 1, glial fibrillary acidic protein, and arginase 1). Cannabidiol attenuated the decrease in hippocampal levels of brain-derived neurotrophic factor induced by CCH in diabetic animals, but it did not affect the levels of neuroplasticity markers (growth-associated protein-43 and synaptophysin) in middle-aged diabetic rats. These results suggest that the neuroprotective effects of CBD in middle-aged diabetic rats subjected to CCH are related to a reduction in neuroinflammation. However, they seemed to occur independently of hippocampal neuroplasticity changes.
Assuntos
Encéfalo/irrigação sanguínea , Encéfalo/efeitos dos fármacos , Canabidiol/uso terapêutico , Circulação Cerebrovascular/efeitos dos fármacos , Diabetes Mellitus Experimental/tratamento farmacológico , Fatores Etários , Animais , Glicemia/efeitos dos fármacos , Glicemia/metabolismo , Encéfalo/metabolismo , Canabidiol/farmacologia , Circulação Cerebrovascular/fisiologia , Doença Crônica , Comorbidade , Diabetes Mellitus Experimental/sangue , Masculino , Ratos , Ratos Wistar , Resultado do TratamentoRESUMO
ETHNOPHARMACOLOGICAL RELEVANCE: Trichilia catigua A. Juss (Meliaceae) preparations have been used in folk medicine to alleviate fatigue, stress, and improve memory. Antinociceptive, antiinflammatory, and in vitro neuroprotective effects have been observed in animals. Cerebral ischemia/reperfusion (I/R) leads to severe neuropsychological deficits that are largely associated with oxidative stress, inflammation and neurodegeneration. We reported previously that an ethyl-acetate fraction (EAF) of T. catigua reduced brain ischemia-induced learning and memory impairments in the absence of histological protection. AIM OF THE STUDY: Continuing those studies, here we aimed to investigate the antioxidant and antiinflammatory properties of T. catigua in an in vivo model of I/R. MATERIAL AND METHODS: Rats were subjected to 15â¯min of brain ischemia (4-VO model) followed by up to 15 days of reperfusion. Vehicle was given by gavage 30â¯min before ischemia and at 1â¯h of reperfusion. In a first experiment, brain ischemia-induced changes in oxidative stress markers, i.e., reduced glutathione (GSH), oxidized glutathione (GSSG), superoxide dismutase (SOD), catalase (CAT), malondialdehyde (MDA), and protein carbonyl groups (PCGs) were measured on days 1, 3, and 5 post-ischemia. Similar time course analysis was done for neuroinflammation markers, i.e., microglia (OX42 immunorreactivity) and astrocytes (GFAP immunorreactivity), in the hippocampus. In a second experiment, the time points at which these markers of oxidative stress and neuroinflammation peaked were used to test the effects of T. catigua (400â¯mg/kg, p.o.). RESULTS: Oxidative stress markers peaked on day 1 post-ischemia. GSH decreased (-23.2%) while GSSG increased (+ 71.1%), which yielded a significant reduction in the GSH/GSSG ratio (-39.1%). The activity of CAT was largely reduced by ischemia (-54.6% to -65.1%), while the concentration of PCG almost doubled in the brain of ischemic rats (+99.10%) in comparison to sham. Treatment with the EAF of T. catigua normalized these changes in oxidative markers to the control levels (GSH: +27.5%; GSSG: -23.8%; GSH/GSSG: +44.6%; PCG: -80.3%). In the hippocampus, neuroinflammation markers peaked on day 5 post-ischemia, with microglial and astrocytic responses increasing to 54.8% and 37.1%, respectively. The elevation in glial cells response was completely prevented by EAF. CONCLUSION: These results demonstrate that T. catigua has both antioxidant and antiinflammatory activities after transient global cerebral ischemia in rats, which may contribute to the previously reported memory protective effect of T. catigua.
Assuntos
Anti-Inflamatórios/uso terapêutico , Antioxidantes/uso terapêutico , Isquemia Encefálica/tratamento farmacológico , Meliaceae , Fármacos Neuroprotetores/uso terapêutico , Extratos Vegetais/uso terapêutico , Traumatismo por Reperfusão/tratamento farmacológico , Acetatos/química , Animais , Anti-Inflamatórios/farmacologia , Antioxidantes/farmacologia , Isquemia Encefálica/metabolismo , Antígeno CD11b/metabolismo , Catalase/metabolismo , Proteína Glial Fibrilar Ácida/metabolismo , Glutationa/metabolismo , Hipocampo/efeitos dos fármacos , Hipocampo/metabolismo , Masculino , Fármacos Neuroprotetores/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Fitoterapia , Extratos Vegetais/farmacologia , Caules de Planta/química , Ratos Wistar , Traumatismo por Reperfusão/metabolismo , Solventes/química , Superóxido Dismutase/metabolismoRESUMO
We originally reported that an ethyl-acetate fraction (EAF) of Trichilia catigua prevented the impairment of water maze learning and hippocampal neurodegeneration after transient global cerebral (TGCI) in mice. We extended that previous study by evaluating whether T. catigua (i) prevents the loss of long-term retrograde memory assessed in the aversive radial maze (AvRM), (ii) confers hippocampal and cortical neuroprotection, and (iii) mitigates oxidative stress and neuroinflammation in rats that are subjected to the four vessel occlusion (4-VO) model of TGCI. In the first experiment, naive rats were trained in the AvRM and then subjected to TGCI. The EAF was administered orally 30min before and 1h after TGCI, and administration continued once per day for 7days post-ischemia. In the second experiment, the EAF was administered 30min before and 1h after TGCI, and protein carbonylation and myeloperoxidase (MPO) activity were assayed 24h and 5days later, respectively. Retrograde memory performance was assessed 8, 15, and 21days post-ischemia. Ischemia caused persistent retrograde amnesia, and this effect was prevented by T. catigua. This memory protection (or preservation) persisted even after the treatment was discontinued, despite the absence of histological neuroprotection. Protein carbonyl group content and MPO activity increased around 43% and 100%, respectively, after TGCI, which were abolished by the EAF of T. catigua. The administration of EAF did not coincide with the days of memory testing. The data indicate that antioxidant and/or antiinflammatory actions in the early phase of ischemia/reperfusion contribute to the long-term antiamnesic effect of T. catigua.
Assuntos
Amnésia Retrógrada/tratamento farmacológico , Amnésia Retrógrada/etiologia , Isquemia Encefálica/complicações , Inflamação/tratamento farmacológico , Inflamação/etiologia , Meliaceae/química , Estresse Oxidativo/efeitos dos fármacos , Extratos Vegetais/uso terapêutico , Animais , Biomarcadores/metabolismo , Córtex Cerebral/efeitos dos fármacos , Córtex Cerebral/metabolismo , Modelos Animais de Doenças , Hipocampo/metabolismo , Aprendizagem em Labirinto/efeitos dos fármacos , Extratos Vegetais/química , RatosRESUMO
A series of our previous studies demonstrated that fish oil (FO), equivalent to 300mg/kg docosahexahenoic acid (DHA), facilitates memory recovery after transient, global cerebral ischemia (TGCI) in the aversive radial maze (AvRM). The present study sought to address two main issues: (i) whether the memory-protective effect of FO that has been observed in the AvRM can be replicated in the passive avoidance test (PAT) and object location test (OLT) and (ii) whether FO at doses that are lower than those used previously can also prevent TGCI-induced memory loss. In Experiment 1, naive rats were trained in the PAT, subjected to TGCI (4-vessel occlusion model), and tested for retrograde memory performance 8 and 15days after ischemia. Fish oil (300mg/kg/day DHA) was given orally for 8days. The first dose was delivered 4h postischemia. In Experiment 2, the rats were subjected to TGCI, treated with the same FO regimen, and then trained and tested in the OLT. In Experiment 3, the rats were trained in the AvRM, subjected to TGCI, administered FO (100, 200, and 300mg/kg DHA), and tested for memory performance up to 3weeks after TGCI. At the end of the behavioral tests, the brains were examined for neurodegeneration and neuroblast proliferation. All of the behavioral tests (PAT, OLT, and AvRM) were sensitive to ischemia, but only the AvRM was able to detect the memory-protective effect of FO. Ischemia-induced neurodegeneration and neuroblast proliferation were unaffected by FO treatment. These results suggest that (i) the beneficial effect of FO on memory recovery after TGCI is task-dependent, (ii) doses of FO<300mg/kg DHA can protect memory function in the radial maze, and (iii) cognitive recovery occurs in the absence of neuronal rescue and/or hippocampal neurogenesis.
Assuntos
Óleos de Peixe/farmacologia , Hipocampo/efeitos dos fármacos , Ataque Isquêmico Transitório/tratamento farmacológico , Transtornos da Memória/tratamento farmacológico , Fármacos Neuroprotetores/farmacologia , Recuperação de Função Fisiológica/efeitos dos fármacos , Animais , Antioxidantes/farmacologia , Aprendizagem da Esquiva/efeitos dos fármacos , Aprendizagem da Esquiva/fisiologia , Complexo Nuclear Basolateral da Amígdala/efeitos dos fármacos , Complexo Nuclear Basolateral da Amígdala/patologia , Modelos Animais de Doenças , Hipocampo/patologia , Ataque Isquêmico Transitório/complicações , Ataque Isquêmico Transitório/patologia , Ataque Isquêmico Transitório/psicologia , Masculino , Transtornos da Memória/etiologia , Transtornos da Memória/patologia , Doenças Neurodegenerativas/tratamento farmacológico , Doenças Neurodegenerativas/etiologia , Doenças Neurodegenerativas/patologia , Doenças Neurodegenerativas/psicologia , Neurogênese/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Neurônios/patologia , Ratos Wistar , Recuperação de Função Fisiológica/fisiologia , Memória Espacial/efeitos dos fármacos , Memória Espacial/fisiologiaRESUMO
Cognitive and affective impairments are the most characterized consequences following cerebral ischemia. BAY 60-7550, a selective phosphodiesterase type 2 inhibitor (PDE2-I), presents memory-enhancing and anxiolytic-like properties. The behavioral effects of BAY 60-7550 have been associated with its ability to prevent hydrolysis of both cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) thereby interfering with neuronal plasticity. Here, we hypothesize that PDE2-I treatment could promote functional recovery after brain ischemia. Mice C57Bl/6 were submitted to bilateral common carotid artery occlusion (BCCAO), an experimental model of transient brain ischemia, for 20 min. During 21 days after reperfusion, the animals were tested in a battery of behavioral tests including the elevated zero maze (EZM), object location task (OLT) and forced swim test (FST). The effects of BAY 60-7550 were evaluated on neuronal nuclei (NeuN), caspase-9, cAMP response element-binding protein (CREB), phosphorylated CREB (pCREB) and brain-derived neurotrophic factor (BDNF) expression in the hippocampus. BCCAO increased anxiety levels, impaired hippocampus-dependent cognitive function and induced despair-like behavior in mice. Hippocampal neurodegeneration was evidenced by a decrease in NeuN and increase incaspase-9 protein levels in BCCAO mice. Ischemic mice also showed low BDNF protein levels in the hippocampus. Repeated treatment with BAY 60-7550 attenuated the behavioral impairments induced by BCCAO in mice. Concomitantly, BAY 60-7550 enhanced expression of pCREB and BDNF protein levels in the hippocampus of ischemic mice. The present findings suggest that chronic inhibition of PDE2 provides functional recovery in BCCAO mice possibly by augmenting hippocampal neuronal plasticity.
Assuntos
Isquemia Encefálica/tratamento farmacológico , Hipocampo/efeitos dos fármacos , Imidazóis/uso terapêutico , Plasticidade Neuronal , Inibidores de Fosfodiesterase/uso terapêutico , Triazinas/uso terapêutico , Animais , Fator Neurotrófico Derivado do Encéfalo/genética , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Caspase 9/genética , Caspase 9/metabolismo , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/genética , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Proteínas de Ligação a DNA , Exonucleases/antagonistas & inibidores , Hipocampo/irrigação sanguínea , Imidazóis/farmacologia , Masculino , Aprendizagem em Labirinto , Camundongos , Camundongos Endogâmicos C57BL , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Neurônios/fisiologia , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Inibidores de Fosfodiesterase/farmacologia , Triazinas/farmacologiaRESUMO
This study investigated the effects of cannabidiol (CBD), a non-psychotomimetic phytochemical present in Cannabis sativa, on the cognitive and emotional impairments induced by bilateral common carotid artery occlusion (BCCAO) in mice. Using a multi-tiered behavioral testing battery during 21days, we found that BCCAO mice exhibited long-lasting functional deficits reflected by increase in anxiety-like behavior (day 9), memory impairments (days 12-18) and despair-like behavior (day 21). Short-term CBD 10mg/kg treatment prevented the cognitive and emotional impairments, attenuated hippocampal neurodegeneration and white matter (WM) injury, and reduced glial response that were induced by BCCAO. In addition, ischemic mice treated with CBD exhibited an increase in the hippocampal brain derived neurotrophic factor (BDNF) protein levels. CBD also stimulated neurogenesis and promoted dendritic restructuring in the hippocampus of BCCAO animals. Collectively, the present results demonstrate that short-term CBD treatment results in global functional recovery in ischemic mice and impacts multiple and distinct targets involved in the pathophysiology of brain ischemic injury.
Assuntos
Isquemia Encefálica/complicações , Canabidiol/farmacologia , Canabidiol/uso terapêutico , Encefalite/tratamento farmacológico , Encefalite/etiologia , Plasticidade Neuronal/efeitos dos fármacos , Recuperação de Função Fisiológica/efeitos dos fármacos , Animais , Anti-Inflamatórios/farmacologia , Anti-Inflamatórios/uso terapêutico , Proteínas de Ligação ao Cálcio/metabolismo , Modelos Animais de Doenças , Proteínas do Domínio Duplacortina , Comportamento Exploratório/efeitos dos fármacos , Proteína Glial Fibrilar Ácida/metabolismo , Masculino , Aprendizagem em Labirinto/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Proteínas dos Microfilamentos/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Neuropeptídeos/metabolismo , Reconhecimento Psicológico/efeitos dos fármacos , Natação/psicologia , Fatores de TempoRESUMO
We reported that fish oil (FO) prevented the loss of spatial memory caused by transient, global cerebral ischemia (TGCI), provided the treatment covered the first days prior to and after ischemia. Continuing these studies, trained rats were subjected to TGCI, and FO was administered for 10days, with a time window of efficacy (TWE) of 4, 8 or 12h post-ischemia. Retrograde memory was assessed up to 43days after TGCI. In another experiment, ischemic rats received FO with a 4- or 12-h TWE, and dendritic density was assessed in the hippocampus and cerebral cortex. The brain lipid profile was evaluated in sham-operated and ischemic rats that were treated with FO or vehicle with a 4-h TWE. Ischemia-induced retrograde amnesia was prevented by FO administration that was initiated with either a 4- or 8-h TWE. Fish oil was ineffective after a 12-h TWE. Independent of the TWE, FO did not prevent ischemic neuronal death. In the hippocampus, but not cerebral cortex, TGCI-induced dendritic loss was prevented by FO with a 4-h TWE but not 12-h TWE. The level of docosahexaenoic acid almost doubled in the hippocampus in ischemic, FO-treated rats (4-h TWE). The data indicate that (i) the anti-amnesic effect of FO can be observed with a TWE of up to 8h, (ii) the stimulation of dendritic neuroplasticity may have contributed to this effect, and (iii) DHA in FO may be the main active constituent in FO that mediates the cognitive and neuroplasticity effects on TGCI.
Assuntos
Dendritos/efeitos dos fármacos , Óleos de Peixe/administração & dosagem , Hipocampo/efeitos dos fármacos , Ataque Isquêmico Transitório/tratamento farmacológico , Memória de Longo Prazo/efeitos dos fármacos , Fármacos Neuroprotetores/administração & dosagem , Amnésia Retrógrada/tratamento farmacológico , Amnésia Retrógrada/etiologia , Amnésia Retrógrada/metabolismo , Amnésia Retrógrada/patologia , Animais , Córtex Cerebral/efeitos dos fármacos , Córtex Cerebral/metabolismo , Córtex Cerebral/patologia , Dendritos/metabolismo , Dendritos/patologia , Modelos Animais de Doenças , Ácidos Docosa-Hexaenoicos/metabolismo , Hipocampo/metabolismo , Hipocampo/patologia , Ataque Isquêmico Transitório/metabolismo , Ataque Isquêmico Transitório/patologia , Ataque Isquêmico Transitório/psicologia , Masculino , Memória de Longo Prazo/fisiologia , Doenças Neurodegenerativas/tratamento farmacológico , Doenças Neurodegenerativas/metabolismo , Doenças Neurodegenerativas/patologia , Doenças Neurodegenerativas/psicologia , Plasticidade Neuronal/efeitos dos fármacos , Plasticidade Neuronal/fisiologia , Nootrópicos/administração & dosagem , Ratos Wistar , Fatores de TempoRESUMO
Chronic cerebral hypoperfusion (CCH) is a common condition associated with the development and/or worsening of age-related dementia.We previously reported persistent memory loss and neurodegeneration after CCH in middle-aged rats. Statin-mediated neuroprotection has been reported after acute cerebral ischemia. Unknown, however, is whether statins can alleviate the outcome of CCH. The present study investigated whether atorvastatin attenuates the cognitive and neurohistological outcome of CCH. Rats (1215 months old) were trained in a non-food-rewarded radial maze, and then subjected to CCH. Atorvastatin (10 mg/kg, p.o.) was administered for 42 days or 15 days, beginning 5 h after the first occlusion stage. Retrograde memory performance was assessed at 7, 14, 21, 28, and 35 days of CCH, and expressed by "latency," "number of reference memory errors" and "number of working memory errors." Neurodegeneration was then examined at the hippocampus and cerebral cortex. Compared to sham, CCH caused profound and persistent memory loss in the vehicle-treated groups, as indicated by increased latency (91.2% to 107.3%) and number of errors (123.5% to 2508.2%), effects from which the animals did not spontaneously recover across time. This CCH-induced retrograde amnesia was completely prevented by atorvastatin (latency: −4.3% to 3.3%; reference/working errors: −2.5% to 45.7%), regardless of the treatment duration. This effect was sustained during the entire behavioral testing period (5 weeks), even after discontinuing treatment. This robust and sustained memory-protective effect of atorvastatin occurred in the absence of neuronal rescue (39.58% to 56.45% cell loss). We suggest that atorvastatin may be promising for the treatment of cognitive sequelae associated with CCH.
Assuntos
Amnésia Retrógrada/tratamento farmacológico , Atorvastatina/farmacologia , Encéfalo/efeitos dos fármacos , Transtornos Cerebrovasculares/tratamento farmacológico , Memória/efeitos dos fármacos , Nootrópicos/farmacologia , Envelhecimento/efeitos dos fármacos , Envelhecimento/fisiologia , Amnésia Retrógrada/etiologia , Amnésia Retrógrada/patologia , Amnésia Retrógrada/fisiopatologia , Animais , Encéfalo/patologia , Encéfalo/fisiopatologia , Transtornos Cerebrovasculares/complicações , Transtornos Cerebrovasculares/patologia , Transtornos Cerebrovasculares/fisiopatologia , Doença Crônica , Modelos Animais de Doenças , Avaliação Pré-Clínica de Medicamentos , Aprendizagem em Labirinto/efeitos dos fármacos , Memória/fisiologia , Células Piramidais/efeitos dos fármacos , Células Piramidais/patologia , Células Piramidais/fisiologia , Ratos Wistar , Resultado do TratamentoRESUMO
ETHNOPHARMACOLOGICAL RELEVANCE: Trichilia catigua preparations have antinociceptive, antiinflammatory, and neuroprotective activity. Recently, a neuroprotective role for T. catigua was proposed using an in vitro model of ischemia-reperfusion in rat hippocampal slices. The aim of the present study was to evaluate the effects of an ethyl-acetate fraction (EAF) of T. catigua, which has potent antioxidant activity, in mice subjected to an in vivo model of cerebral ischemia. MATERIAL AND METHODS: Male Swiss mice were subject to the bilateral common carotid occlusion (BCCAO) model of cerebral ischemia. The animals were orally administered the T. catigua EAF (200, 400, or 800 mg/kg) 30 min before and once per day for 7 days after BCCAO. Histological and behavioral outcomes were assessed using Nissl staining and the Morris water maze test of cognition, respectively. RESULTS: Mice that were subjected to BCCAO exhibited cognitive impairments in the Morris water maze. The spatial cognitive deficits were counteracted by T. catigua EAF administration (200-800 mg/kg). The T. catigua EAF significantly increased the number of intact-appearing Nissl-stained cells in the hippocampus in BCCAO mice. CONCLUSIONS: These results show that the T. catigua EAF promoted functional recovery, decreased the delayed hippocampal cell loss, and mitigated the ongoing neurodegenerative processes induced by BCCAO in mice.
Assuntos
Transtornos Cognitivos/tratamento farmacológico , Meliaceae/química , Extratos Vegetais/farmacologia , Acetatos/química , Animais , Isquemia Encefálica/complicações , Isquemia Encefálica/tratamento farmacológico , Isquemia Encefálica/patologia , Artéria Carótida Primitiva/patologia , Estenose das Carótidas/complicações , Morte Celular/efeitos dos fármacos , Transtornos Cognitivos/etiologia , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Hipocampo/patologia , Masculino , Aprendizagem em Labirinto/efeitos dos fármacos , Camundongos , Extratos Vegetais/administração & dosagemRESUMO
We reported that fish oil (FO) abolishes retrograde amnesia consistently following transient global cerebral ischemia (TGCI) in young rats, provided it covered the first days prior to and after ischemia. Here, we further evaluated whether FO given post-ischemia in older rats (15-18 months old) is equally effective in facilitating memory recovery. We also tested whether the antiamnesic effect of FO observed after TGCI can be reproduced after chronic cerebral hypoperfusion (CCH). FO (300 mg/kg docosahexaenoic acid [DHA]) was delivered orally 4h after TGCI and continued once per day for 9 days. In the CCH group, FO treatment began soon after the first stage of 4-VO/ICA and continued daily for 43 days. Two weeks after surgery, the animals were tested for retrograde memory performance across 5 weeks. Both TGCI and CCH caused persistent memory impairment and hippocampal and cortical neurodegeneration. TGCI-induced retrograde amnesia was reversed by FO, an effect that was sustained for at least 5 weeks after discontinuing treatment. In contrast, the memory deficit caused by CCH remained unchanged after FO treatment. Both hippocampal and cortical damage was not alleviated by FO. We conclude that the FO-mediated antiamnesic effect following TGCI can be extended to older rats, even when the treatment begins 4h postischemia. Such efficacy was not reproduced after CCH. Therefore, the present results support the notion that FO may have therapeutic utility in treating learning/memory dysfunction after acute/transient cerebral ischemia and suggest that such benefits may not apply when a state of chronic cerebrovascular insufficiency is present.
Assuntos
Amnésia Retrógrada/tratamento farmacológico , Óleos de Peixe/uso terapêutico , Ataque Isquêmico Transitório/complicações , Fármacos Neuroprotetores/uso terapêutico , Amnésia Retrógrada/etiologia , Análise de Variância , Animais , Relação Dose-Resposta a Droga , Ataque Isquêmico Transitório/mortalidade , Degeneração Neural/tratamento farmacológico , Degeneração Neural/etiologia , Ratos , Ratos Wistar , Tempo de Reação/efeitos dos fármacosRESUMO
We previously reported that long-term treatment with fish oil (FO) facilitates memory recovery after transient, global cerebral ischemia (TGCI), despite the presence of severe hippocampal damage. The present study tested whether this antiamnesic effect resulted from an action of FO on behavioral performance itself, or whether it resulted from an anti-ischemic action. Different treatment regimens were used that were distinguished from each other by their initiation or duration with regard to the onset of TGCI and memory assessment. Naive rats were trained in an eight-arm radial maze, subjected to TGCI (4-VO model, 15 min), and tested for memory performance up to 6 weeks after TGCI. Fish oil (docosahexaenoic acid, 300 mg/kg/day) was given orally according to one of the following regimens: regimen 1 (from 3 days prior to ischemia until 4 weeks post-ischemia), regimen 2 (from 3 days prior to ischemia until 1 week post-ischemia), and regimen 3 (from week 2 to week 5 post-ischemia). When administered according to regimens 1 and 2, FO abolished amnesia completely. This effect persisted for at least 5 weeks after discontinuing the treatment. Such an effect did not occur, however, in the group treated according to regimen 3. Hippocampal and cortical damage was not alleviated by FO. The present results demonstrate that FO-mediated memory recovery (or preservation) following TGCI is a reproducible, robust, and long-lasting effect. Moreover, such an effect was found with a relatively short period of treatment, provided it covered the first days prior to and after ischemia. This suggests that FO prevented amnesia by changing some acute, ischemia/reperfusion-triggered process and not by stimulating memory performance on its own.
Assuntos
Ácidos Docosa-Hexaenoicos/uso terapêutico , Ataque Isquêmico Transitório/dietoterapia , Transtornos da Memória/dietoterapia , Animais , Córtex Cerebral/efeitos dos fármacos , Córtex Cerebral/patologia , Ácidos Docosa-Hexaenoicos/administração & dosagem , Esquema de Medicação , Hipocampo/efeitos dos fármacos , Hipocampo/patologia , Ataque Isquêmico Transitório/complicações , Ataque Isquêmico Transitório/patologia , Masculino , Aprendizagem em Labirinto/efeitos dos fármacos , Transtornos da Memória/complicações , Transtornos da Memória/patologia , Degeneração Neural/dietoterapia , Degeneração Neural/patologia , RatosRESUMO
ETHNOPHARMACOLOGICAL RELEVANCE: Trichilia catigua preparations have been popularly used in Brazil as a tonic for the treatment of fatigue, stress, impotence, and memory deficits. We recently demonstrated an antidepressant-like effect of acute administration of the Trichilia catigua ethyl-acetate fraction (EAF) in mice. The aim of the present study was to evaluate whether subchronic Trichilia catigua EAF administration maintains its antidepressant-like effects and whether these effects are related to hippocampal neurogenesis. MATERIAL AND METHODS: Trichilia catigua EAF (200 and 400mg/kg) was orally administered to mice for 14 day. The animals were tested in the forced swim test (FST) or tail suspension test (TST). After behavioral testing, the animals received bromodeoxyuridine (BrdU; 200mg/kg, i.p.) and were euthanized 24h, 7 day, or 15 day later. The brains were assayed for BrdU and doublecortin (DCX) immunohistochemistry to detect cell proliferation/survival and neurogenesis, respectively. RESULTS: Subchronic administration of 400mg/kg Trichilia catigua EAF promoted antidepressant-like effects in mice in both the FST and TST. The antidepressant-like effect was accompanied by an increase in cell proliferation in the dentate gyrus (DG) of the hippocampus 24h after the treatments were discontinued. This proliferative effect, however, did not influence cell survival or neurogenesis because no change in the number of BrdU- or DCX-positive cells was detected 7 or 15 day after the last EAF administration compared with controls. CONCLUSIONS: Trichilia catigua EAF produced antidepressant-like effects and induced hippocampal cell proliferation in mice. The results contribute information on the pharmacological and molecular mechanisms involved in the antidepressant-like effect of Trichilia catigua EAF.
Assuntos
Antidepressivos/uso terapêutico , Proliferação de Células/efeitos dos fármacos , Depressão/tratamento farmacológico , Hipocampo/efeitos dos fármacos , Meliaceae , Fitoterapia , Extratos Vegetais/uso terapêutico , Animais , Antidepressivos/administração & dosagem , Antidepressivos/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Depressão/etiologia , Proteína Duplacortina , Elevação dos Membros Posteriores , Hipocampo/citologia , Masculino , Camundongos , Camundongos Endogâmicos , Neurogênese/efeitos dos fármacos , Extratos Vegetais/administração & dosagem , Extratos Vegetais/farmacologia , NataçãoRESUMO
We investigated the effects of chronic administration of vitamin E on nitric oxide (NO)-producing neurons in the brains of streptozotocin (STZ)-induced diabetic rats using nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) histochemistry. We further evaluated the effects of diabetes and vitamin E treatment on experimental anxiety and memory processes using the elevated plus maze (EPM) Trial 1/2 protocol. Wistar rats were divided into four groups: normoglycemics (N), normoglycemics treated with vitamin E (NVE), diabetics (D), and diabetics treated with vitamin E (DVE). Diabetes mellitus was induced by a single intraperitoneal injection of STZ (35mg/kg). Vitamin E (100mg/kg) or vehicle was administered orally by gavage (1ml/kg) once each day for 7 weeks. After behavioral testing, the dentate gyrus of the hippocampus (DG), striatum, paraventricular nucleus of the hypothalamus (PVN), supraoptic nucleus (SON), and dorsolateral periaqueductal grey (DLPAG) were analyzed for NADPH-d histochemistry. STZ-induced diabetic rats exhibited decreased locomotor activity and cognitive impairment compared with normoglycemic controls. The number of NADPH-d-positive neurons was increased in the DG, striatum, and DLPAG of diabetic rats. An increase in soma area was detected in all structures analyzed (DG, striatum, PVN, SON, and DLPAG) of STZ-induced diabetic animals. The present study showed that chronic administration of vitamin E ameliorates memory in STZ-induced diabetic rats and revealed that NOS-producing neurons have an increased soma area which can be restored, at least partially, by vitamin E treatment. These results suggest the potential use of vitamin E as an adjuvant therapy for the prevention and treatment of diabetic conditions.
Assuntos
Encéfalo/metabolismo , Diabetes Mellitus Experimental/dietoterapia , Aprendizagem/fisiologia , Neurônios/metabolismo , Óxido Nítrico/metabolismo , Vitamina E/uso terapêutico , Animais , Ansiedade/tratamento farmacológico , Ansiedade/metabolismo , Encéfalo/patologia , Transtornos Cognitivos/dietoterapia , Transtornos Cognitivos/metabolismo , Transtornos Cognitivos/patologia , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/patologia , Masculino , Aprendizagem em Labirinto/fisiologia , Memória/fisiologia , Atividade Motora/fisiologia , NADP/metabolismo , Neurônios/patologia , Distribuição Aleatória , Ratos , Ratos Wistar , Vitamina E/metabolismoRESUMO
Cerebral ischemia leads to neurodegeneration and cognitive impairment. Fish oil (FO) constitutes a rich dietary source of omega-3 polyunsaturated fatty acids especially docosahexaenoic acid (DHA). The objective of the present study was to investigate whether long-term treatment with commercial, high concentration DHA-containing FO could be effective in alleviating both the cognitive and neurodegenerative deficits caused by transient, global cerebral ischemia (TGCI) in rats. Naive rats were trained for 10 days in an 8-arm radial maze task and then subjected to TGCI for 15 minutes (4-VO model) 3 days later (day 13). Retention of the previously acquired cognition (ie, memory) was assessed weekly on days 20, 27, 34, 41, 48, and 55 and measured by 3 behavioral parameters as follows: (i) latency to find the goal box, (ii) number of reference memory errors, and (iii) number of working memory errors. The extent of pyramidal cell death in the hippocampus was examined at the end of the behavioral analysis on day 43. Fish oil (300 mg/kg DHA, gavage) administration occurred once daily beginning 3 days before TGCI (the last day of training) and continued until day 41. Transient, global cerebral ischemia markedly disrupted memory performance measured by all 3 parameters (P < .0001 vs sham). This amnesic effect of ischemia persisted until the end of the behavioral analysis. Treatment with FO progressively reversed the TGCI-induced retention deficit until rats achieved control levels. This protective effect of FO on learning/memory function was clearly observed after both daily and cumulative data analysis (P < .001-0.01 vs vehicle). Such memory improvements remained statistically significant, even after cessation of FO treatment, indicating a sustained effect of FO. In contrast, FO failed to prevent ischemia-induced hippocampal damage in areas CA1, CA2, or CA4. Therefore, the present findings suggest that long-term FO treatment is able to facilitate functional recovery after ischemic brain damage, an effect that was distinct from hippocampal damage.
Assuntos
Amnésia/prevenção & controle , Óleos de Peixe/uso terapêutico , Hipocampo/patologia , Ataque Isquêmico Transitório/tratamento farmacológico , Animais , Suplementos Nutricionais , Ácidos Docosa-Hexaenoicos/farmacologia , Ácidos Docosa-Hexaenoicos/uso terapêutico , Óleos de Peixe/farmacologia , Hipocampo/efeitos dos fármacos , Ataque Isquêmico Transitório/patologia , Masculino , Aprendizagem em Labirinto , Células Piramidais/patologia , Ratos , Ratos WistarRESUMO
The effects of the Ginkgo biloba extract, EGb 761, on ischemia-induced learning/memory impairments and hippocampal damage were evaluated in a non-food motivated, aversive radial maze task (AvRM). Rats were subjected to 15 min transient, global cerebral ischemia (TGCI). In the first experiment, rats were rendered ischemic, and 23 days later were tested for acquisition performance (post-operative training). In a second experiment, rats were trained for 10 days and then subjected to ischemia (pre-operative training); the retention of cognition performance was assessed on days 31, 35 and 39 after ischemia. Acquisition and retention performances were expressed by (a) latency to find a goal box, (b) number of reference memory errors, and (c) number of working memory errors. EGb 761 (50 or 150 mg/kg) was given orally, starting before ischemia and continuing for up to 3 days after ischemia. TGCI markedly disrupted both acquisition and retention performance (p < 0.001-0.05). EGb 761 (150 mg/kg) completely reversed acquisition impairment as measured by the parameters 'latency' and 'number of reference errors' when performance was examined across sessions (p < 0.01-0.05). The total number of reference errors was also completely abolished by EGb 761 (150 mg/kg). However, EGb 761 did not statistically reduce the effects of TGCI on the parameter 'working memory errors' (across session and total). At 50 mg/kg, EGb 761 did not affect ischemia-induced acquisition impairment at all. The retention deficit caused by ischemia was not statistically reduced by EGb 761 (150 mg/kg), whatever examined across session or as total. EGb 761 reduced the extent of hippocampal CA1 cell loss (p < 0.01-0.001), an effect sustained at least up to 40 days after ischemia. These findings show that EGb 761 is effective in reducing, at least partially, both the cognitive impairments and hippocampal damage after TGCI in rats, and suggest that its effect on behavioral recovery may be dissociated from the neuroprotective effect on the hippocampus. The present results also validate the AvRM as an alternative, reliable behavioral test to assess the effects of drugs on behavioral recovery after ischemic brain damage.
Assuntos
Comportamento Animal/efeitos dos fármacos , Ataque Isquêmico Transitório/tratamento farmacológico , Fármacos Neuroprotetores/uso terapêutico , Extratos Vegetais/uso terapêutico , Recuperação de Função Fisiológica/efeitos dos fármacos , Análise de Variância , Animais , Contagem de Células/métodos , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Ginkgo biloba , Hipocampo/citologia , Ataque Isquêmico Transitório/patologia , Masculino , Aprendizagem em Labirinto/efeitos dos fármacos , Células Piramidais/efeitos dos fármacos , Ratos , Ratos Wistar , Tempo de Reação/efeitos dos fármacosRESUMO
There is much biochemical evidence, but very few studies in animal models of stroke in vivo, to suggest that Ginkgo biloba (EGb 761) may offer neuroprotection against regional, ischemic brain damage; additional investigations are needed to ensure future clinical trials. This study reports the effects of EGb 761 given acutely or chronically before ischemia. Rats were subjected to middle cerebral artery occlusion (MCAO) for 2 h and the brain infarct size was assessed 24 h later. Dipyrone (100 mg/kg, i.p.) was injected 30 min before ischemia, and 2.5 and 5.5 h after ischemia, to reduce ischemia-induced fever. EGb 761 (Tebonin) was given acutely (200 mg/kg, p.o., 60 min before ischemia) or chronically (100 mg/kg, p.o., once daily, for 14 days before ischemia). Acute or chronic treatment with EGb 761, either alone or in combination with dipyrone, did not reduce the infarct size compared with saline alone (p > 0.05). Dipyrone failed to prevent ischemia-induced fever during the intra-ischemic period (p > 0.05 vs saline; p < 0.001 vs sham). In the reperfusion phase, dipyrone reduced fever to normothermic levels in the group treated acutely with EGb 761 (p < 0.01 vs saline, p > 0.05 vs sham) but not after chronic EGb 761 (p < 0.01 vs sham), indicating possible pharmacokinetic interaction. In conclusion, within the context of unprevented, ischemia-induced fever, the present results demonstrate that EGb 761 has no significant effect on brain infarct size.
Assuntos
Isquemia Encefálica/tratamento farmacológico , Infarto da Artéria Cerebral Média/prevenção & controle , Fármacos Neuroprotetores/uso terapêutico , Extratos Vegetais/uso terapêutico , Animais , Anti-Inflamatórios não Esteroides/uso terapêutico , Isquemia Encefálica/complicações , Dipirona/uso terapêutico , Febre/tratamento farmacológico , Febre/etiologia , Ginkgo biloba , Masculino , Fitoterapia , Ratos , Ratos WistarRESUMO
A produção de fitoterápicos requer critérios técnicos de qualidade e avaliação de seus efeitos. Este estudo visa à avaliação de efeitos comportamentais de um extrato padronizado de Passiflora edulis. O extrato foi obtido de folhas por percolação. Em sua padronização determinou-se: a) teor de flavonóides totais (droga e extrato), b) resíduo seco, c) pH, d) densidade e e) teor alcoólico. Os efeitos farmacológicos da P. edulis foram avaliados sobre os seguintes parâmetros: 1) atividade exploratória, 2) tempo de sono induzido pelo pentobarbital e 3) catatonia. O extrato padronizado apresentou as seguintes propriedades: a) 3,83± 0,10g por cento (n=3) e 9,50± 0,27g por cento (n=3), respectivamente; b) 6,7± 0,30 por cento (n=6); c) 5,5 (n=3), d) 0,94± 0,01 g/mL (n=3); e) 50 °GL (n=3). O extrato de P. edullis exerceu efeitos dose-dependentes sobre os vários parâmetros analisados: 1) reduziu as atividades de comportamento exploratório (deambulação e autolimpeza: P<0,01) e, qualitativamente, diminuiu os comportamentos de levantamento ("rearing") e salto ("jumping"); 2) aumentou o tempo de hipnose induzida pelo pentobarbital (P<0,01); e 3) induziu catatonia (P<0,0001 extrato 80 mg/kg vs veículo). Através das investigações farmacológicas realizadas, o extrato de P. edulis demonstrou ser depressor do sistema nervoso central, sugerindo uma ação calmante tipo tranqüilizante maior.