RESUMO
Our previous studies showed that intranasal delivery of progesterone offers a good bioavailability and neuroprotective efficacy after experimental stroke. We have also demonstrated that progesterone receptors (PR) are essential for cerebroprotection by endogenous progesterone and by progesterone treatment. The identification of PR as a potential drug target for stroke therapy opens new therapeutic indications for selective synthetic progestins. Nestorone® (16-methylene-17α-acetoxy-19-nor-pregn-4-ene-3, 20-dione, also known as segesterone acetate) is a 19-norprogesterone derivative that more potently targets PR than progesterone. The objective of this study was to evaluate the cerebroprotective efficiency of intranasal administration of Nestorone after middle cerebral occlusion (MCAO) in mice. We show here that intranasal administration is a very efficient route to achieve a preferential delivery of Nestorone to the brain and confers a slow elimination and a sustained bioavailability. Furthermore, intranasal administration of Nestorone (at 0.08 mg/kg) improved the functional outcomes and decreased the ischemic lesion in male but not in female mice at 48 h post MCAO. Use of PRNesCre mice, selectively lacking expression of PR in neural cells, and their control PRloxP/loxP littermates showed that the cerebroprotective effects of Nestorone in male mice depended on neural PR as they were not observed in PRNesCre mice. Our findings show that intranasal delivery of Nestorone may be an efficient strategy to promote recovery after stroke in males and confirm the key role of PR in cerebroprotection. Furthermore, they point to sex differences in the response to Nestorone treatment and emphasize the necessity to include males and females in experimental studies.
Assuntos
AVC Isquêmico/tratamento farmacológico , Fármacos Neuroprotetores/administração & dosagem , Fármacos Neuroprotetores/uso terapêutico , Norprogesteronas/administração & dosagem , Norprogesteronas/uso terapêutico , Administração Intranasal , Animais , Comportamento Animal/efeitos dos fármacos , Encéfalo/metabolismo , Feminino , Infarto da Artéria Cerebral Média/prevenção & controle , Injeções Intraperitoneais , AVC Isquêmico/psicologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Fármacos Neuroprotetores/farmacocinética , Norprogesteronas/farmacocinética , Receptores de Progesterona/antagonistas & inibidores , Caracteres Sexuais , Resultado do TratamentoRESUMO
Intranasal administration is emerging as a very promising route to deliver therapeutics to the brain. We have recently shown that the intranasal delivery of progesterone at 8 mg/kg is neuroprotective after stroke in male mice. To explore the translational potential of intranasal progesterone treatment, we performed a dose-response study and analyzed outcomes at 48 h after middle cerebral artery occlusion (MCAO). The effects on functional outcomes at long-term were examined by using the optimal dose. In the first experiment, male C57BL/6JRj mice were treated with progesterone at 8, 16 or 24 mg/kg, or with placebo at 1, 6 and 24 h post-MCAO. Our results show that the dose of 8 mg/kg was optimal in counteracting the early histopathological impairments as well as in improving functional recovery. Steroid profiling in plasma showed that the dose of 8 mg/kg is the one that leads to sustained high levels of progesterone and its neuroactive metabolites. In the second experiment, the dose of 8 mg/kg was used and analyzes were performed at 2, 7 and 21 days post-MCAO. Progesterone increased survival, glycemia and body weight. Furthermore, progesterone decreased neurological deficits and improved performances of mice on the rotarod and pole as early as 2 days and up to 21 days post-MCAO. These findings show that intranasal administration of progesterone has a significant translational potential as a cerebroprotective treatment after stroke that can be effective to reduce mortality, to limit tissue and cell damage at the acute phase; and to confer a long-term functional recovery.
Assuntos
Isquemia Encefálica/tratamento farmacológico , Encéfalo/efeitos dos fármacos , Sistemas de Liberação de Medicamentos/métodos , AVC Isquêmico/tratamento farmacológico , Fármacos Neuroprotetores/administração & dosagem , Progesterona/administração & dosagem , Administração Intranasal , Animais , Encéfalo/metabolismo , Encéfalo/patologia , Isquemia Encefálica/sangue , Isquemia Encefálica/patologia , Relação Dose-Resposta a Droga , Géis , AVC Isquêmico/sangue , AVC Isquêmico/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fármacos Neuroprotetores/sangue , Progesterona/sangueRESUMO
Efforts are still needed regarding the research of therapeutics for ischemic stroke. While in experimental studies the protective effect of pharmacological agents is often highlighted by a reduction of the lesion size evaluated in the short term (days), in clinical studies a functional recovery of patients suffering from stroke is expected on the long-term (months and years). Long-term functional preclinical studies are highly recommended to evaluate potential neuroprotective agents for stroke, rather than an assessment of the infarction size at a short time point. The present study thus aimed to select among various behavioral tests those able to highlight long-term deficits (3 months) after cerebral ischemia in mice. Permanent focal cerebral ischemia was carried out in male Swiss mice by intraluminal occlusion of the left middle cerebral artery (MCA). Fourteen behavioral tests were assessed from 7 days to 90 days after ischemia (locomotor activity, neurological score, exit circle test, grip and string tests, chimney test, adhesive removal test, pole test, beam-walking tests, elevated plus maze, marble burying test, forced swimming test, novel object recognition test). The present study clearly identified a battery of behavioral tests able to highlight deficits up to 3 months in our mouse model of permanent MCA occlusion (locomotor activity, neurological score, adhesive removal test, pole test, beam-walking tests, elevated plus maze, marble burying test, forced swimming test and novel object recognition test). This battery of behavioral tests highlighting long-term deficits is useful to study future neuroprotective strategies for stroke treatment.
Assuntos
Isquemia Encefálica/complicações , Transtornos Mentais/diagnóstico , Transtornos Mentais/etiologia , Animais , Isquemia Encefálica/tratamento farmacológico , Modelos Animais de Doenças , Comportamento Exploratório/fisiologia , Locomoção/fisiologia , Masculino , Aprendizagem em Labirinto/fisiologia , Camundongos , Exame Neurológico , Oxigênio/uso terapêutico , Desempenho Psicomotor , Estatísticas não Paramétricas , NataçãoRESUMO
Progesterone has been shown to be cerebroprotective in different experimental models of brain injuries and neurodegenerative diseases. The preclinical data provided great hope for its use in humans. The failure of Phase 3 clinical trials to demonstrate the cerebroprotective efficiency of progesterone in traumatic brain injury (TBI) patients emphasizes that different aspects of the design of both experimental and clinical studies should be reviewed and refined. One important aspect to consider is to test different routes of delivery of therapeutic agents. Several studies have shown that the intranasal delivery of drugs could be used in different experimental models of central nervous system diseases. In this review, we will summarize the pharmacokinetic characteristics and practical advantages of intranasal delivery of progesterone. A special emphasis will be placed on describing and discussing our recent findings showing that intranasal delivery of progesterone after transient focal cerebral ischemia: 1) improved motor functions; 2) reduced infarct volume, neuronal loss, blood brain barrier disruption; and 3) reduced brain mitochondrial dysfunctions. Our data suggest that intranasal delivery of progesterone is a potential efficient, safe and non-stressful mode of administration that warrants evaluation for cerebroprotection in patients with brain injuries. This article is part of the Special Issue entitled "Novel Treatments for Traumatic Brain Injury".
Assuntos
Lesões Encefálicas/tratamento farmacológico , Fármacos Neuroprotetores/administração & dosagem , Progesterona/administração & dosagem , Administração Intranasal , Animais , Humanos , Fármacos Neuroprotetores/farmacocinética , Progesterona/farmacocinéticaRESUMO
Treatment with progesterone limits brain damage after stroke. However, the cellular bases of the cerebroprotective effects of progesterone are not well documented. The aims of this study were to determine neural cells and functions that are affected by progesterone treatment and the role of neural progesterone receptors (PR) after stroke. Adult male PRNesCre mice, selectively lacking PR in the central nervous system, and their control PRloxP/loxP littermates were subjected to transient ischemia by middle cerebral artery occlusion (MCAO) for 30â¯min. Mice received either progesterone (8â¯mg/kg) or vehicle at 1-, 6- and 24- hrs post-MCAO and outcomes were analyzed at 48â¯h post-MCAO. In PRloxP/loxP mice, progesterone exerted multiple effects on different neural cell types, improved motor functional outcomes and reduced total infarct volumes. In the peri-infarct, progesterone increased the density of neurons (NeuN+ cells), of cells of the oligodendroglial lineage (Olig2+ cells) and of oligodendrocyte progenitors (OP, NG2+ cells). Progesterone decreased the density of activated astrocytes (GFAP+ cells) and reactive microglia (Iba1+ cells) coexpressing the mannose receptor type 1 CD206 marker. Progesterone also reduced the expression of aquaporin 4 (AQP4), the water channel involved in both edema formation and resorption. The beneficial effects of progesterone were not observed in PRNesCre mice. Our findings show that progesterone treatment exerts beneficial effects on neurons, oligodendroglial cells and neuroinflammatory responses via PR. These findings demonstrate that progesterone is a pleiotropic cerebroprotective agent and that neural PR represent a therapeutic target for stroke cerebroprotection.
Assuntos
Hipóxia-Isquemia Encefálica/prevenção & controle , Microglia/citologia , Atividade Motora/efeitos dos fármacos , Fármacos Neuroprotetores/uso terapêutico , Oligodendroglia/citologia , Progesterona/uso terapêutico , Acidente Vascular Cerebral/tratamento farmacológico , Acidente Vascular Cerebral/patologia , Animais , Aquaporina 4/metabolismo , Hipóxia-Isquemia Encefálica/tratamento farmacológico , Infarto da Artéria Cerebral Média/patologia , Lectinas Tipo C/metabolismo , Masculino , Receptor de Manose , Lectinas de Ligação a Manose/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microglia/efeitos dos fármacos , Microglia/patologia , Oligodendroglia/efeitos dos fármacos , Oligodendroglia/patologia , Receptores de Superfície Celular/metabolismo , Receptores de Progesterona/metabolismoRESUMO
Both sex and steroid hormones are important to consider in human ischemic stroke and its experimental models. Stroke initiates a cascade of changes that lead to neural cell death, but also activates endogenous protective processes that counter the deleterious consequences of ischemia. Steroids may be part of these cerebroprotective processes. One option to provide cerebroprotection is to reinforce these intrinsic protective mechanisms. In the current review, we first summarize studies describing sex differences and the influence of steroid hormones in stroke. We then present and discuss our recent results concerning differential changes in endogenous steroid levels in the brains of male and female mice and the importance of progesterone receptors (PR) during the early phase after stroke. In the third part, we give an overview of experimental studies, including ours, that provide evidence for the pleiotropic beneficial effects of progesterone and its promising cerebroprotective potential in stroke. We also highlight the key role of PR signaling as well as potential additional mechanisms by which progesterone may provide cerebroprotection.
Assuntos
Progesterona/metabolismo , Acidente Vascular Cerebral/metabolismo , Animais , Feminino , Humanos , Masculino , Neuroproteção , Receptores de Progesterona/metabolismo , Caracteres Sexuais , Transdução de SinaisRESUMO
Treatment with progesterone protects the male and female brain against damage after middle cerebral artery occlusion (MCAO). However, in both sexes, the brain contains significant amounts of endogenous progesterone. It is not known whether endogenously produced progesterone enhances the resistance of the brain to ischemic insult. Here, we used steroid profiling by gas chromatography-tandem mass spectrometry (GC-MS/MS) for exploring adaptive and sex-specific changes in brain levels of progesterone and its metabolites after MCAO. We show that, in the male mouse brain, progesterone is mainly metabolized via 5α-reduction leading to 5α-dihydroprogesterone (5α-DHP), also a progesterone receptor (PR) agonist ligand in neural cells, then to 3α,5α-tetrahydroprogesterone (3α,5α-THP). In the female mouse brain, levels of 5α-DHP and 3α,5α-THP are lower and levels of 20α-DHP are higher than in males. After MCAO, levels of progesterone and 5α-DHP are upregulated rapidly to pregnancy-like levels in the male but not in the female brain. To assess whether endogenous progesterone and 5α-DHP contribute to the resistance of neural cells to ischemic damage, we inactivated PR selectively in the CNS. Deletion of PR in the brain reduced its resistance to MCAO, resulting in increased infarct volumes and neurological deficits in both sexes. Importantly, endogenous PR ligands continue to protect the brain of aging mice. These results uncover the unexpected importance of endogenous progesterone and its metabolites in cerebroprotection. They also reveal that the female reproductive hormone progesterone is an endogenous cerebroprotective neurosteroid in both sexes.SIGNIFICANCE STATEMENT The brain responds to injury with protective signaling and has a remarkable capacity to protect itself. We show here that, in response to ischemic stroke, levels of progesterone and its neuroactive metabolite 5α-dihydroprogesterone are upregulated rapidly in the male mouse brain but not in the female brain. An important role of endogenous progesterone in cerebroprotection was demonstrated by the conditional inactivation of its receptor in neural cells. These results show the importance of endogenous progesterone, its metabolites, and neural progesterone receptors in acute cerebroprotection after stroke. This new concept could be exploited therapeutically by taking into account the progesterone status of patients and by supplementing and reinforcing endogenous progesterone signaling for attaining its full cerebroprotective potential.
Assuntos
Neurônios , Progesterona/genética , Receptores de Progesterona/genética , Acidente Vascular Cerebral/genética , Acidente Vascular Cerebral/prevenção & controle , Envelhecimento , Animais , Química Encefálica/genética , Feminino , Deleção de Genes , Infarto da Artéria Cerebral Média/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fármacos Neuroprotetores , Pregnenolona/metabolismo , Progesterona/metabolismo , Receptores de Progesterona/metabolismo , Caracteres SexuaisRESUMO
This study investigated the effect of intranasal administration of progesterone on the early brain mitochondrial respiratory chain dysfunction and oxidative damage after transient middle cerebral occlusion in male and female mice. We showed that progesterone (8 mg/kg at 1 h post-middle cerebral occlusion) restored the mitochondrial reduced glutathione pool and the nicotinamide adenine dinucleotide-linked respiration in both sexes. Progesterone also reversed the decrease of the flavin adenine dinucleotide-linked respiration, which was only observed in females. Our findings point to a sex difference in stroke effects on the brain respiratory chain and suggest that the actions of progesterone on mitochondrial function may participate in its neuroprotective properties.
Assuntos
Encéfalo/efeitos dos fármacos , Infarto da Artéria Cerebral Média/tratamento farmacológico , Mitocôndrias/efeitos dos fármacos , Fármacos Neuroprotetores/uso terapêutico , Estresse Oxidativo/efeitos dos fármacos , Progesterona/uso terapêutico , Animais , Encéfalo/metabolismo , Encéfalo/patologia , Respiração Celular/efeitos dos fármacos , Feminino , Flavina-Adenina Dinucleotídeo/análogos & derivados , Flavina-Adenina Dinucleotídeo/metabolismo , Glutationa/metabolismo , Infarto da Artéria Cerebral Média/metabolismo , Infarto da Artéria Cerebral Média/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mitocôndrias/metabolismo , Mitocôndrias/patologia , NAD/metabolismo , Fármacos Neuroprotetores/administração & dosagem , Consumo de Oxigênio/efeitos dos fármacos , Progesterona/administração & dosagemRESUMO
Progesterone is a potential neuroprotective agent for cerebral stroke. One of the STAIR's recommendations is to test different routes of delivery of therapeutic agents. Here, we investigated the neuroprotective efficacy of intranasal delivery of progesterone in oleogel. Male mice were subjected to transient middle cerebral occlusion (MCAO) for 1 h. Mice received intranasal or intraperitoneal administrations of progesterone (8 mg/kg) at 1, 6, and 24 h post-MCAO. Plasma and brain levels of steroids were measured by gas chromatography-mass spectrometry 2 and 24 h after the last administration of progesterone. Behavioral and histopathological analyzes were performed at 48 h post-MCAO. For blood-brain barrier (BBB) permeability analysis, mice received one intranasal administration of progesterone or placebo at reperfusion and Evans Blue and sodium fluorescein extravasations were assessed at 4 h post-MCAO. Two hours after its nasal administration, progesterone reached elevated levels in brain and plasma and was bioconverted to its 5α-reduced metabolites and to 20α-dihydroprogesterone. However, brain levels of progesterone and its metabolites were about half those measured after intraperitoneal injections, whereas levels of 11-deoxycorticosterone and corticosterone were 5-times lower. In contrast, after 24 h, higher levels of progesterone were measured in brain and plasma after intranasal than after intraperitoneal delivery. Intranasal progesterone decreased the mortality rate, improved motor functions, reduced infarct, attenuated neuronal loss, and decreased the early BBB disruption. This study demonstrates a good bioavailability, a prolonged absorption and a good neuroprotective efficacy of intranasal delivery of progesterone, thus potentially offering an efficient, safe, non-stressful and very easy mode of administration in stroke patients.
Assuntos
Isquemia Encefálica/tratamento farmacológico , Encéfalo/efeitos dos fármacos , Fármacos Neuroprotetores/administração & dosagem , Progesterona/administração & dosagem , Acidente Vascular Cerebral/tratamento farmacológico , Administração Intranasal , Animais , Barreira Hematoencefálica/efeitos dos fármacos , Barreira Hematoencefálica/metabolismo , Encéfalo/metabolismo , Isquemia Encefálica/metabolismo , Isquemia Encefálica/mortalidade , Permeabilidade Capilar/efeitos dos fármacos , Permeabilidade Capilar/fisiologia , Corticosterona/metabolismo , Modelos Animais de Doenças , Infarto da Artéria Cerebral Média , Injeções Intraperitoneais , Masculino , Camundongos Endogâmicos C57BL , Distribuição Aleatória , Acidente Vascular Cerebral/metabolismo , Acidente Vascular Cerebral/mortalidadeRESUMO
OBJECTIVES: Traumatic brain injury causes deleterious brain edema, leading to high mortality and morbidity. Brain edema exacerbates neurologic deficits and may be attributable to the breakdown of endothelial cell junction protein, leukocyte infiltration, and matrix metalloproteinase activation. These all contribute to loss of blood-brain barrier integrity. The pleiotropic effects of statins, hydroxymethylglutaryl-coenzyme A reductase inhibitors, may inhibit posttraumatic brain edema. We therefore investigated the effect of acute simvastatin on neurologic deficits, cerebral edema, and its origins. DESIGN: Randomized laboratory animal study. SETTINGS: University-affiliated research laboratory. SUBJECTS: Male Sprague-Dawley rats. INTERVENTIONS: Rats were subjected to lateral fluid percussion traumatic brain injury. Our preliminary dose-effect study indicated that 37.5 mg/kg simvastatin, administered orally 1 hr and 6 hrs after traumatic brain injury, has the greatest anti-edematous effect. This dose was used to study its effects on brain edema and on its mechanisms. MEASUREMENTS AND MAIN RESULTS: We first assessed the effects of simvastatin 24 hrs after traumatic brain injury on brain edema, brain claudin-5 expression, and the vascular endothelial-cadherin (pTyr731)/total vascular endothelial-cadherin ratio, matrix metalloproteinase-9 activity, intercellular adhesion molecule-1 expression, and polymorphonuclear neutrophil infiltration. We also evaluated blood-brain barrier permeability by measuring Evans blue and fluorescein sodium salt extravasation into the cerebral parenchyma. We then investigated whether simvastatin reduces neurologic deficits, edema, and blood-brain barrier permeability earlier than 24 hrs; these effects were evaluated 6 hrs after traumatic brain injury. The anti-edematous effect of simvastatin 24 hrs after traumatic brain injury was associated with increased claudin-5 and decreased intercellular adhesion molecule-1, polymorphonuclear neutrophil infiltration, and blood-brain barrier permeability, with no effect on matrix metalloproteinase-9 activity or vascular endothelial-cadherin phosphorylation. Earlier, 6-hrs after traumatic brain injury, simvastatin reduced neurologic deficits, cerebral edema, and blood-brain barrier permeability. CONCLUSIONS: Simvastatin could be a new therapy for reducing posttraumatic edema by preventing damage to tight junctions and neutrophil infiltration into the parenchyma, thus preserving blood-brain barrier integrity.