RESUMO
Several studies have indicated that interrupted epigenetic reprogramming using Yamanaka transcription factors (OSKM) can rejuvenate cells from old laboratory animals and humans. However, the potential of OSKM-induced rejuvenation in brain tissue has been less explored. Here, we aimed to restore cognitive performance in 25.3-month-old female Sprague-Dawley rats using OSKM gene therapy for 39 days. Their progress was then compared with the cognitive performance of untreated 3.5-month-old rats as well as old control rats treated with a placebo adenovector. The Barnes maze test, used to assess cognitive performance, demonstrated enhanced cognitive abilities in old rats treated with OSKM compared to old control animals. In the treated old rats, there was a noticeable trend towards improved spatial memory relative to the old controls. Further, OSKM gene expression did not lead to any pathological alterations within the 39 days. Analysis of DNA methylation following OSKM treatment yielded three insights. First, epigenetic clocks for rats suggested a marginally significant epigenetic rejuvenation. Second, chromatin state analysis revealed that OSKM treatment rejuvenated the methylome of the hippocampus. Third, an epigenome-wide association analysis indicated that OSKM expression in the hippocampus of old rats partially reversed the age-related increase in methylation. In summary, the administration of Yamanaka genes via viral vectors rejuvenates the functional capabilities and the epigenetic landscape of the rat hippocampus.
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The analysis of how neural circuits function in individuals and change during evolution is simplified by the existence of neurons identified as homologous within and across species. Invertebrates, including leeches, have been used for these purposes in part because their nervous systems comprise a high proportion of identified neurons, but technical limitations make it challenging to assess the full extent to which assumptions of stereotypy hold true. Here, we introduce Minos plasmid-mediated transgenesis as a tool for introducing transgenes into the embryos of the leech Helobdella austinensis (Spiralia; Lophotrochozoa; Annelida; Clitellata; Hirudinida; Glossiphoniidae). We identified an enhancer driving pan-neuronal expression of markers, including histone2B:mCherry, which allowed us to enumerate neurons in segmental ganglia. Unexpectedly, we found that the segmental ganglia of adult transgenic H. austinensis contain fewer and more variable numbers of neurons than in previously examined leech species.
Assuntos
Sanguessugas , Animais , Sanguessugas/fisiologia , Sanguessugas/genética , Técnicas de Transferência de Genes , Neurônios/fisiologia , Gânglios dos Invertebrados/fisiologia , Animais Geneticamente Modificados/genética , TransgenesRESUMO
This study is based on the premise that the application of chemical synthesis strategies to structurally modify commercial drugs by complexation with biometals is a valid procedure to improve their biological effects. Our purpose is to synthesize a compound with greater efficacy than the original drug, able to enhance its antihypertensive and cardiac pharmacological activity. Herein, the structure of the coordination compound of Zn(II) and the antihypertensive drug olmesartan, [Zn(Olme)(H2O)2] (ZnOlme), is presented. After 8 weeks of treatment in SHR male rats, ZnOlme displayed a better blood pressure-lowering activity compared with olmesartan, with a noticeable effect even in the first weeks of treatment, while ZnCl2 showed similar results than the control. ZnOlme also reduced left ventricle (LV) weight and left ventricle/tibia length ratio (LV/TL), posterior wall thickness (PWT), and intraventricular septum in diastole (IVSd) suggesting its potential to prevent LV hypertrophy. Besides, ZnOlme reduced interstitial fibrosis (contents of collagen types I and III, responsible for giving rigidity and promoting vascular elasticity, respectively). The recovery of heart function was also evidenced by fractional shortening (diastolic left ventricular/systolic left ventricular) diameter determinations. Furthermore, ZnOlme increased the antioxidant capacity and prevented cardiac oxidative stress: it enhanced the reduction of reactive oxygen species generation, exerted a significant decrease in lipid peroxidation and enhanced glutathione contents in heart tissues compared to the control, Zn, and olmesartan treatments. Our results demonstrate that continuous oral administration of ZnOlme causes a better antihypertensive effect and grants enhancement of cardioprotection through antioxidant activity, in combination with hemodynamic improvement.
Assuntos
Anti-Hipertensivos , Hipertensão , Ratos , Animais , Masculino , Anti-Hipertensivos/farmacologia , Anti-Hipertensivos/uso terapêutico , Hipertensão/tratamento farmacológico , Ratos Endogâmicos SHR , Pressão Sanguínea , Hipertrofia Ventricular Esquerda/tratamento farmacológico , Hipertrofia Ventricular Esquerda/prevenção & controle , Zinco/farmacologia , Zinco/uso terapêuticoRESUMO
BACKGROUND: the antihypertensive drug α-methyldopa (MD) stands as one of the extensively used medications for managing hypertension during pregnancy. Zinc deprivation has been associated with many diseases. In this context, the synthesis of a Zn coordination complex [Zn(MD)(OH)(H2O)2]·H2O (ZnMD) provide a promising alternative pathway to improve the biological properties of MD. METHODS: ZnMD was synthesized and physicochemically characterized. Fluorescence spectral studies were conducted to examine the binding of both, the ligand and the metal with bovine serum albumin (BSA). MD, ZnMD, and ZnCl2 were administered to spontaneous hypertensive rats (SHR) rats during 8 weeks and blood pressure and echocardiographic parameters were determined. Ex vivo assays were conducted to evaluate levels of reactive oxygen species (ROS), thiobarbituric acid reactive substances (TBARS), and nitric oxide (NO). Cross-sectional area (CSA) and collagen levels of left ventricular cardiomyocytes were also assessed. Furthermore, the expression of NAD(P)H oxidase subunits (gp91phox and p47phox) and Superoxide Dismutase 1 (SOD1) was quantified through western blot analysis. RESULTS: The complex exhibited a moderate affinity for binding with BSA showing a spontaneous interaction (indicated by negative ΔG values) and moderate affinity (determined by affinity constant values). The binding process involved the formation of Van der Waals forces and hydrogen bonds. Upon treatment with MD and ZnMD, a reduction in the systolic blood pressure in SHR was observed, being ZnMD more effective than MD (122 ± 8.1 mmHg and 145 ± 5.6 mmHg, at 8th week of treatment, respectively). The ZnMD treatment prevented myocardial hypertrophy, improved the heart function and reduced the cardiac fibrosis, as evidenced by parameters such as left ventricular mass, fractional shortening, and histological studies. In contrast, MD did not show noticeable differences in these parameters. ZnMD regulates negatively the oxidative damage by reducing levels of ROS and lipid peroxidation, as well as the cardiac NAD(P)H oxidase, and increasing SOD1 expression, while MD did not show significant effect. Moreover, cardiac nitric oxide levels were greater in the ZnMD therapy compared to MD treatment. CONCLUSION: Both MD and ZnMD have the potential to be transported by albumin. Our findings provide important evidence suggesting that this complex could be a potential therapeutic drug for the treatment of hypertension and cardiac hypertrophy and dysfunction.
Assuntos
Anti-Hipertensivos , Hipertensão , Ratos , Animais , Anti-Hipertensivos/uso terapêutico , Metildopa/farmacologia , Metildopa/uso terapêutico , Espécies Reativas de Oxigênio/metabolismo , Superóxido Dismutase-1 , Óxido Nítrico/metabolismo , Hipertensão/tratamento farmacológico , Pressão Sanguínea , Ratos Endogâmicos SHR , Miócitos Cardíacos/metabolismo , Cardiomegalia , NADPH Oxidases , Zinco/farmacologia , Zinco/uso terapêuticoRESUMO
Hypertension is the most common cause of left ventricular hypertrophy, contributing to heart failure progression. Candesartan (Cand) is an angiotensin receptor antagonist widely used for hypertension treatment. Structural modifications were previously performed by our group using Zinc (ZnCand) as a strategy for improving its pharmacological properties. The measurements showed that ZnCand exerts a stronger interaction with the angiotensin II receptor, type 1 (AT1 receptor), reducing oxidative stress and intracellular calcium flux, a mechanism implied in cell contraction. These results were accompanied by the reduction of the contractile capacity of mesangial cells. In vivo experiments showed that the complex causes a significant decrease in systolic blood pressure after 8 weeks of treatment in spontaneously hypertensive rats (SHR). The reduction of heart hypertrophy was evidenced by echocardiography, the histologic cross-sectional area of cardiomyocytes, collagen content, the B-type natriuretic peptide (BNP) marker and connective tissue growth factor (CTGF) and the matrix metalloproteinase 2 (MMP-2) expression. Besides, the complex restored the redox status. In this study, we demonstrated that the complexation with Zn(II) improves the antihypertensive and cardiac effects of the parental drug.
Assuntos
Anti-Hipertensivos , Hipertensão , Hipertrofia Ventricular Esquerda , Zinco , Animais , Ratos , Anti-Hipertensivos/química , Anti-Hipertensivos/farmacologia , Compostos de Bifenilo/farmacologia , Pressão Sanguínea , Hipertensão/complicações , Hipertensão/tratamento farmacológico , Hipertrofia Ventricular Esquerda/tratamento farmacológico , Metaloproteinase 2 da Matriz , Miócitos Cardíacos , Ratos Endogâmicos SHR , Tetrazóis/farmacologia , Tetrazóis/uso terapêutico , Zinco/farmacologiaRESUMO
The main evolutionary milestone in the oviparity-viviparity transition is placentation. The placenta is an organ with great morphological diversity among eutherians. The expression of different glycosidic residues (Gr) in the near-term placenta constitutes its glycotype. In this study, the expression of different Gr was determined by lectin histochemistry in early, midterm, and near-term placentas of the plains viscacha (Lagostomus maximus), a caviomorph rodent with the highest poliovulatory rate and embryonic resorption rate among eutherians. Besides, a matrix with the expression of each Gr in the exchange trophoblast of viscacha and other eutherians was constructed to map and infer phylogenetic and evolutionary relationships. Between early, midterm, and near-term placentas, variations in the pattern expression of Gr were observed. The glycotype of the near-term placenta is composed of a high diversity of Gr. Reconstruction of the ancestral state for each Gr present in the near-term placenta showed a diverse scenario: some sugars were common to the species of Placentalia included in this study. In the analyzed species with synepitheliochorial and epitheliochorial placentas, no differential glycosylation patterns between them were observed. In species with invasive placentas, such as the endotheliochorial placentas of Carnivora, some common Gr were detected among them, while others were species-specific. In species with hemochorial placenta, the same Gr are shared. Particularly, in the viscacha greater differences with species of the Hominidae and even Muridae families were observed. Nevertheless, greater similarities with other caviomorph rodents were detected. Placental glycotype of each species constitutes an excellent tool to achieve phylogenetic and evolutionary inferences among eutherians.
Assuntos
Placenta , Roedores , Gravidez , Feminino , Animais , Placenta/anatomia & histologia , Filogenia , Placentação , TrofoblastosRESUMO
Vascular mineralization is a hallmark of enzootic calcinosis. Histopathological, ultrastructural, and immunohistochemical investigations were performed on the external carotid arteries of seven sheep naturally poisoned by Nierembergia veitchii. Histologically, moderate to marked hyperplasia of the tunica intima was observed without mineralization. The tunica media exhibited mild to severe mineralization and osteochondroid metaplasia. Sheep with enzootic calcinosis showed arterial overexpression of osteopontin and tissue-nonspecific alkaline phosphatase and immunolabeling for osteonectin and osteocalcin in both intima and media layers of the tested arteries. The main ultrastructural finding in the tunica media was a marked phenotypic change of vascular smooth muscle cells from a contractile phenotype (VSMC-C) into a synthetic phenotype (VSMC-S). In the tunica media, VSMC-S produced matrix and extracellular vesicles, forming mineralizable granules associated with arterial mineralization. VSMC-S were also present in the tunica intima, but matrix and extracellular vesicles and mineralization were not observed. The absence of matrix and extracellular vesicles in the intimal hyperplasia, even in the presence of noncollagenous bone proteins, tissue-nonspecific alkaline phosphatase, and vitamin D receptors, reinforces the hypothesis that the presence of matrix and extracellular vesicles are crucial for the development of vascular mineralization in enzootic calcinosis. It is proposed that the two different VSMC-S phenotypes in calcinosis are due to the expression of at least two genetically different types of these cells induced by the action of 1,25(OH)2D3.
Assuntos
Calcinose , Hiperplasia , Doenças dos Ovinos , Fosfatase Alcalina/metabolismo , Animais , Calcinose/veterinária , Células Cultivadas , Hiperplasia/patologia , Hiperplasia/veterinária , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/patologia , Miócitos de Músculo Liso/metabolismo , Miócitos de Músculo Liso/patologia , Ovinos , Doenças dos Ovinos/patologiaRESUMO
Cardiac cells depend on specific sarcolemmal ion transporters to assure the correct intracellular pH regulation. The sodium/bicarbonate cotransporter (NBC) is one of the major alkalinizing mechanisms. In the heart two different NBC isoforms have been described: the electroneutral NBCn1 (1Na+:1 HCO 3 - ) and the electrogenic NBCe1 (1Na+:2 HCO 3 - ). NBCe1 generates an anionic repolarizing current that modulates the action potential duration (APD). In addition to regulating the pH, the NBC is a source of sodium influx. It has been postulated that NBC could play a role in the development of hypertrophy. The aim of this research was to study the contribution of NBCe1 in heart electrophysiology and in the development of heart hypertrophy in an in vivo mouse model with overexpression of NBCe1. Heart NBCe1 overexpression was achieved by a recombinant cardiotropic adeno-associated virus (AAV9) and was evidenced by western-blot and qPCR. AAV9-mCherry was used as a transduction control. NBCe1 overexpression fails to increase heart growth. Patch clamp and electrocardiogram were performed. We observed a reduction on both, ventricular myocytes APD and electrocardiogram QT interval corrected by cardiac rate, emphasizing for the first time NBCe1 relevance for the electrical activity of the heart.
RESUMO
Five adult Saanen goats received a single oral dose of Heterophyllaea pustulata containing 42.25 µg/kg rubiadin (anthraquinone) and 3 adult goats were untreated controls. All goats were exposed to sunlight and sequential ear skin biopsies were collected before treatment and at 32 hours, 3 days, 8 days, and 15 days after treatment. Changes at 32 hours after dosing included epidermal spongiosis, single cell death and acantholysis, an increased BAX/BCL-2 protein ratio, and dermal edema. Lesions at day 3 included epidermal and adnexal necrosis, crust formation, and acanthosis. Acanthosis, hyperkeratosis, and dermal fibrosis and neovascularization were present at day 15. The pro-apoptotic (BAX)/anti-apoptotic (BCL-2) protein ratio increased at 32 hours, whereas epidermal and dermal PCNA immunolabeling increased between days 8 and 15 after treatment. The cutaneous lesions were consistent with sunlight-induced damage, and the occurrence in treated but not control goats indicates photosensitization.
Assuntos
Doenças das Cabras , Transtornos de Fotossensibilidade , Animais , Doenças das Cabras/induzido quimicamente , Cabras , Transtornos de Fotossensibilidade/induzido quimicamente , Transtornos de Fotossensibilidade/veterinária , PeleRESUMO
Embryonic death followed by resorption is a conserved process in mammals. Among the polyovular species, Lagostomus maximus (plains viscacha) constitutes a model of early and physiological embryonic death, since out of a total of 10-12 implants, 8-10 are resorbed during early/intermediate gestation, surviving are only the most caudal implantations of each uterine horn. This regular reproductive event is unique to this species, but many characteristics of the implantations during the early gestation of L. maximus, when embryonic death processes begin are unknown. The aim of the present work was to analyze the implantation sites of this species using morphological, morphometric, histochemical, lectinhistochemical, and immunohistochemical techniques to infer the possible causes of this event. Macroscopically, the length and width of the implantation sites significantly increased in a craniocaudal direction. Histochemically, the implantation sites did not differ in the expression of glycoconjugates and glycosidic residues. Furthermore, no variations were observed in cell renewal, hormone receptor expression, and decidualization. Both the glandular and vascular areas of the implantation sites significantly increased in the craniocaudal axis. Some necrotic cells and an inflammatory response with a predominance of lymphocytes and fibrin were observed in the cranial and middle but not in the caudal implantation sites. We conclude that signs of embryonic death and resorption are already observed in the early gestation of L. maximus. Our results reaffirm the hypothesis that postulates the key potential role of uterine glands and blood vessels in the gestation of the species, with emphasis on embryonic death. This pattern of embryonic death in L. maximus makes this species an unconventional mammalian model, which adds to the peculiarities of polyovulation (200-800 oocytes/estrus) and hemochorial placentation.
Assuntos
Eutérios , Roedores , Animais , Feminino , Hormônios , Placentação , Gravidez , ÚteroRESUMO
Previously, we have shown that an increased cGMP-activated protein Kinase (PKG) activity after phosphodiesterase 5 (PDE5) inhibition by Sildenafil (SIL), leads to myocardial Na+/H+ exchanger (NHE1) inhibition preserving its basal homeostatic function. Since NHE1 is hyperactive in the hypertrophied myocardium of spontaneous hypertensive rats (SHR), while its inhibition was shown to prevent and revert this pathology, the current study was aimed to evaluate the potential antihypertrophic effect of SIL on adult SHR myocardium. We initially tested the inhibitory capability of SIL on NHE1 in isolated cardiomyocytes of SHR by comparing H+ efflux during the recovery from an acid load. After confirmed that effect, eight-month-old SHR were chronically treated for one month with SIL through drinking water. Compared to their littermate controls, SIL-treated rats presented a decreased NHE1 activity, which correlated with a reduction in its phosphorylation level assigned to activation of a PKG-p38 MAP kinase-PP2A signaling pathway. Moreover, treated animals showed a decreased oxidative stress that appears to be a consequence of a decreased mitochondrial NHE1 phosphorylation. Treated SHR showed a significant reduction in the pro-hypertrophic phosphatase calcineurin, despite slight tendency to decrease hypertrophy was detected. When SIL treatment was prolonged to three months, a significant decrease in myocardial hypertrophy and interstitial fibrosis that correlated with a lower myocardial stiffness was observed. In conclusion, the current study provides evidence concerning the ability of SIL to revert established cardiac hypertrophy in SHR, a clinically relevant animal model that resembles human essential hypertension.
Assuntos
Cardiomegalia/prevenção & controle , Miócitos Cardíacos/efeitos dos fármacos , Músculos Papilares/efeitos dos fármacos , Inibidores da Fosfodiesterase 5/farmacologia , Citrato de Sildenafila/farmacologia , Trocador 1 de Sódio-Hidrogênio/metabolismo , Animais , Cardiomegalia/enzimologia , Cardiomegalia/etiologia , Cardiomegalia/fisiopatologia , Proteínas Quinases Dependentes de GMP Cíclico/metabolismo , Modelos Animais de Doenças , Regulação para Baixo , Fibrose , Hipertensão/complicações , Masculino , Miócitos Cardíacos/enzimologia , Miócitos Cardíacos/patologia , Músculos Papilares/enzimologia , Músculos Papilares/fisiopatologia , Fosforilação , Proteína Fosfatase 2/metabolismo , Ratos Endogâmicos SHR , Transdução de Sinais , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Some years ago, our group reported the presence of the female prostate in all the studied females of the plains viscacha (Lagostomus maximus). The goal of the present study was to characterize and compare the female prostate gland between adult pregnant and non-pregnant plains viscacha using histochemical, lectin-histochemical and immunohistochemical techniques, as well as optic and electron microscopy. Structurally, alveoli are lined by a simple epithelium formed by different cell types: basal cells, secretory cells in different stages of the secretory cycle and cells of clear cytoplasm. Secretory cells are the most abundant cell type, differing between them depending on the quantity and electron-density of their granules. The basal cells are less abundant and are responsible for the renewal of the alveolar epithelium. Likewise, other cells with secretory morphology were found in all the studied females; these have a clear cytoplasm, few granules and mitochondria. It could be considered that they are degranulated secretory cells or that they have partially released their granules. The stroma of the organ is formed by connective tissue and smooth muscle fibers, which are immunohistochemically evidenced against desmin. Histochemical and lectin-histochemical analysis revealed the presence of different glucidic residues in the different cell types. No structural, histochemical, lectin-histochemical, and ultrastructural differences were observed between pregnant and non-pregnant females of plain viscachas, except for the expression of some lectins. The paraurethral gland of Lagostomus maximus can be used as a model for studying the gland in other species since its structural and ultrastructural characteristics do not depend on the hormonal status of the female.
Assuntos
Próstata/ultraestrutura , Roedores/anatomia & histologia , Animais , Colágeno Tipo I/metabolismo , Colágeno Tipo III/metabolismo , Feminino , Lectinas/metabolismo , Masculino , Gravidez , Próstata/citologiaRESUMO
Physical training stimulates the development of physiologic cardiac hypertrophy (CH), being a key event in this process the inhibition of the Na+/H+ exchanger. However, the role of the sodium bicarbonate cotransporter (NBC) has not been explored yet under this circumstance. C57/Bl6 mice were allowed to voluntary exercise (wheel running) for five weeks. Cardiac mass was evaluated by echocardiography and histomorphometry detecting that training promoted the development of physiological CH (heart weight/tibia length ratio, mg/mm: 6.54 ± 0.20 vs 8.81 ± 0.24; interstitial collagen content, %: 3.14 ± 0.63 vs. 1.57 ± 0.27; and cross-sectional area of cardiomyocytes, µm2: 200.6 ± 8.92 vs. 281.9 ± 24.05; sedentary (Sed) and exercised (Ex) mice, respectively). The activity of the electrogenic isoform of the cardiac NBC (NBCe1) was estimated by recording intracellular pH under high potassium concentration and by measuring action potential duration (APD). NBCe1 activity was significantly increased in isolated cardiomyocytes of trained mice. Additionally, the APD was shorter and the alkalization due to high extracellular potassium-induced depolarization was greater in this group, indicating that the NBCe1 was hyperactive. These results are online with the observed myocardial up-regulation of the NBCe1 (Western Blot, %: 100 ± 13.86 vs. 202 ± 29.98; Sed vs. Ex, n = 6 each group). In addition, we detected a reduction in H2O2 production in the myocardium of trained mice. These results support that voluntary training induces the development of physiologic CH with up-regulation of the cardiac NBCe1 in mice. Furthermore, the improvement in the antioxidant capacity contributes to the beneficial cardiovascular consequences of physical training.
Assuntos
Miocárdio/metabolismo , Condicionamento Físico Animal , Simportadores de Sódio-Bicarbonato/metabolismo , Animais , Cardiomegalia Induzida por Exercícios/fisiologia , Peróxido de Hidrogênio/farmacologia , Masculino , Camundongos Endogâmicos C57BL , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Isoformas de Proteínas/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Regulação para CimaRESUMO
Development of alternative therapies for treating functional deficits after different neurological damages is a challenge in neuroscience. Insulin-like growth factor-1 (IGF-1) is a potent neurotrophic factor exerting neuroprotective actions in brain and spinal cord. It is used to prevent or treat injuries of the central nervous system using different administration routes in different animal models. In this study, we evaluated whether intracisternal (IC) route for IGF-1 gene therapy may abrogate or at least reduce the structural and behavioral damages induced by the intraparenchymal injection of kainic acid (KA) into the rat spinal cord. Experimental (Rad-IGF-1) and control (Rad-DsRed-KA) rats were evaluated using a battery of motor and sensory tests before the injection of the recombinant adenovector (day -3), before KA injection (day 0) and at every post-injection (pi) time point (days 1, 2, 3 and 7 pi). Histopathological changes and neuronal and glial counting were assessed. Pretreatment using IC delivery of RAd-IGF-1 improved animal's general condition and motor and sensory functions as compared to Rad-DsRed-KA-injected rats. Besides, IC Rad-IGF-1 therapy abrogated later spinal cord damage and reduced the glial response induced by KA as observed in Rad-DsRed-KA rats. We conclude that the IC route for delivering RAd-IGF-1 prevents KA-induced excitotoxicity in the spinal cord.
Assuntos
Ácido Caínico , Fármacos Neuroprotetores , Animais , Terapia Genética , Fator de Crescimento Insulin-Like I/genética , Ácido Caínico/toxicidade , Ratos , Ratos Sprague-Dawley , Medula EspinalRESUMO
The uterus is an organ with great plasticity due to the morphological and physiological changes it experiences during the estrous cycle and pregnancy. In mammals, pregnancy requires diverse sex hormones, growth factors and cytokines, among others, for promoting uterine remodeling to favor implantation, placentation, and embryo/fetus survival and growth. The hystricognathi rodent Lagostomus maximus (plains viscacha) has a high rate of embryonic resorption. The cranial and middle implants are reabsorbed 25-35 days after intercourse while the caudal embryos continue with their development until two precocial offspring are born. So far, no uterine studies of non-pregnant L. maximus females were performed to determine the possible existence of variations in the organ that could be related to the differential survival of the implants. We used ultrasonography, as well as morphological, morphometric, histochemical, lectinhistochemical, and immunohistochemical methods to study differences in the uterine glands (area), vasculature (area), and musculature (thickness) along the uterine horns in non-pregnant females. Along the uterus, all these structures were in more advanced developmental condition in the caudal region as compared to more anterior positions. These regional variations could be decisive in explaining the reason why only caudal implantations come to term. In contrast, no differences in the in the luminal and glandular epithelial cells, nor in the degree of cell proliferation and apoptosis, and hormonal receptor staining were found. These parameters could be related to implantation along the uterine horns, but not to the differential survival of the implants.
Assuntos
Embrião de Mamíferos/anatomia & histologia , Músculos/anatomia & histologia , Roedores/embriologia , Útero/anatomia & histologia , Útero/irrigação sanguínea , Animais , Apoptose , Proliferação de Células , Feminino , Hormônios/metabolismo , Lectinas/metabolismo , Receptores de Superfície Celular/metabolismo , Roedores/anatomia & histologia , Útero/diagnóstico por imagemRESUMO
Several countries have established self-help cryonics groups whose mission is to cryopreserve human bodies or brains after legal death and ship them to cryonics organizations. The objective of this study was to report the first case of human brain cryopreservation in Argentina and complementary experiments in rats. After legal death, the body of a 78-year-old Caucasian woman was transported to a funeral home where her head was submitted to intracarotid perfusion with 5 L cold physiologic saline followed by the same volume of cold saline containing 13% dimethyl sulfoxide and 13% glycerol. The brain was removed, temporarily frozen at -80°C, and shipped to a U.S. cryostasis facility. Three groups of rats were intracardially perfused with fixative but not frozen (Reference group), vitrification solution VM1 (Control group), or the cryoprotection solution used in the patient (Experimental group). Control and Experimental brains were stored at -80°C and subsequently assessed by immunohistochemistry for the adult neuron marker (NeuN), the immature neuron marker doublecortin (DCX), the dopaminergic neuron marker tyrosine hydroxylase, and the presynaptic marker synaptophysin (SYN). The number of NeuN-positive neurons remained unchanged in the experimental brain cortex, whereas the number of immature DCX neurons in the hippocampus fell markedly in the cryoprotected brains. The results were highly variable for hypothalamic dopaminergic neurons. Confocal microscopy for SYN revealed that cryopreservation did not affect the synaptic network in the hippocampus. To our knowledge, this is the first report correlating a human cryoprotection procedure with results in complementary experiments in laboratory animals.
Assuntos
Encéfalo , Criopreservação , Modelos Animais , Idoso , Animais , Encéfalo/anatomia & histologia , Cadáver , Criopreservação/métodos , Crioprotetores , Proteína Duplacortina , Feminino , Congelamento , Hipocampo , Humanos , Ratos , Fixação de Tecidos , VitrificaçãoRESUMO
Ventricular hypertrophy is a risk factors for arrhythmias, ischemia and sudden death. It involves cellular modifications leading to a pathological remodeling and is associated with heart failure. The activation of the G protein-coupled estrogen receptor (GPER) mediates beneficial actions in the cardiovascular system. Our goal was to prevent and regress the hypertrophy by the activation of GPER in neonatal cardiac myocytes (NRCM) and SHR male rats. Aldosterone increased the neonatal cardiomyocytes cell surface area after 48 h of incubation. The aldo-induced hypertrophy was blocked by the mineralocorticoid receptor (MR) inhibitor Eplererone or the reduction of MR expression by siRNA. The activation of GPER by the agonist G-1 totally prevented the increase surface area by Ald. The transfection of neonatal rat cardiac myocytes with a siRNA against GPER or the incubation with GPER blockers G-15 and G-36 inhibited the protection of G-1. The significant increase of cell surface area after 48 h of incubation with Ald was totally regressed in 24 h by the presence of G-1, indicating that the activation of GPER not only prevent the hypertrophy but also regress the hypertrophy when it is already established. In the in vivo model, G-1 or Vehicle was constantly infused via the minipump to SHR. The reduction of the hypertrophy by G-1 was evident by the cross-sectional area, BNP and ANP markers and by echocardiography. In this studied we demonstrated that the activation of GPER prevented and regressed the hypertrophy induced by Ald in NRCM and regressed hypertrophy in SHR rats.
Assuntos
Cardiomegalia/prevenção & controle , Receptores Acoplados a Proteínas G/metabolismo , Animais , Animais Recém-Nascidos , Western Blotting , Cardiomegalia/diagnóstico por imagem , Células Cultivadas , Ciclopentanos/farmacologia , Ecocardiografia , Eplerenona/farmacologia , Masculino , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Quinolinas/farmacologia , Ratos , Ratos Endogâmicos SHR , Ratos Wistar , Reação em Cadeia da Polimerase em Tempo Real , Receptores Acoplados a Proteínas G/antagonistas & inibidores , Receptores Acoplados a Proteínas G/fisiologiaRESUMO
Spinal cord injury (SCI) is a common pathological condition that leads to permanent or temporal loss of motor and autonomic functions. Kainic acid (KA), an agonist of kainate receptors, a type of ionotropic glutamate receptor, is widely used to induce experimental neurodegeneration models of CNS. Mesenchymal Stem Cells (MSC) therapy applied at the injured nervous tissue have emerged as a promising therapeutic treatment. Here we used a validated SCI experimental model in which an intraparenchymal injection of KA into the C5 segment of rat spinal cord induced an excitotoxic lesion. Three days later, experimental animals were treated with an intracerebroventricular injection of human umbilical cord (hUC) MSC whereas control group only received saline solution. Sensory and motor skills as well as neuronal and glial reaction of both groups were recorded. Differences in motor behavior, neuronal counting and glial responses were observed between hUC-MSC-treated and untreated rats. According to the obtained results, we suggest that hUC-MSC therapy delivered into the fourth ventricle using the intracerebroventricular via can exert a neuroprotective or neurorestorative effect on KA-injected animals.
Assuntos
Terapia Baseada em Transplante de Células e Tecidos , Transplante de Células-Tronco Mesenquimais , Traumatismos da Medula Espinal/terapia , Cordão Umbilical/transplante , Animais , Humanos , Infusões Intraventriculares , Ácido Caínico/farmacologia , Células-Tronco Mesenquimais/citologia , Neuroglia/metabolismo , Neuroglia/patologia , Neurônios/metabolismo , Neurônios/patologia , Ratos , Medula Espinal/patologia , Medula Espinal/transplante , Traumatismos da Medula Espinal/patologia , Cordão Umbilical/citologiaRESUMO
Pathological cardiac hypertrophy (PCH) can be triggered by epidermal growth factor receptor (EGFR) transactivation. Progression of PCH can be prevented by inhibition of hyperactive Na+/H+ exchanger isoform 1 (NHE1). We first aimed, to limit PCH of spontaneously hypertensive rats (SHR) by specific and localized silencing of cardiac EGFR, and second to study the connection of its activation pathway with cardiac NHE1 activity. Short hairpin RNA (shRNA) against EGFR was delivered with a lentivirus (l-shEGFR) in the cardiac left ventricle (LV) wall. Protein expression was analyzed by immunoblots, and NHE1 activity was indirectly measured in isolated papillary muscles by rate of pHi recovery from transient acidification. EGFR protein expression in the LV was reduced compared to the group injected with l-shSCR (Scrambled sequence) without changes in ErbB2 or ErbB4. Hypertrophic parameters together with cardiomyocytes cross sectional area were reduced in animals injected with l-shEGFR. Echocardiographic analysis exhibited a reduced fractional shortening in the l-shSCR group 30â¯days following treatment that was not observed in l-shEGFR group. l-shEGFR treated rats presented a reduced basal production of reactive oxygen species and decreased lipid peroxidation. NHE1 activity was significantly diminished in hearts with a partial EGFR silencing, without modification of its protein expression. We conclude that specifically silencing cardiac EGFR expression prevents progression of PCH through a pathway that involves a decrease in the NHE1 activity. Lentiviral vectors prove to be a valuable tool for long term expression of shRNA, bringing the possibility to extend its use in clinical area.
Assuntos
Cardiomegalia/metabolismo , Receptores ErbB/genética , Receptores ErbB/metabolismo , Inativação Gênica/fisiologia , Trocador 1 de Sódio-Hidrogênio/metabolismo , Animais , Cardiomegalia/patologia , Receptores ErbB/antagonistas & inibidores , Células HEK293 , Humanos , Masculino , Ratos , Ratos Endogâmicos SHR , Trocador 1 de Sódio-Hidrogênio/antagonistas & inibidoresRESUMO
Heart failure is the leading cause of death among diabetic people. Cellular and molecular entities leading to diabetic cardiomyopathy are, however, poorly understood. Coupling of cardiac carbonic anhydrase II (CAII) and Na+/H+ exchanger 1 (NHE1) to form a transport metabolon was analyzed in obese type 2 diabetic mice (ob-/-) and control heterozygous littermates (ob+/-). Echocardiography showed elevated systolic interventricular septum thickness and systolic posterior wall thickness in ob-/- mice at 9 and 16â¯weeks. ob-/- mice showed increased left ventricular (LV) weight/tibia length ratio and increased cardiomyocyte cross sectional area as compared to controls, indicating cardiac hypertrophy. Immunoblot analysis showed increased CAII expression in LV samples of ob-/-vs. ob+/- mice, and augmented Ser703 phosphorylation on NHE1 in ob-/- hearts. Reciprocal co-immunoprecipitation analysis showed strong association of CAII and NHE1 in LV samples of ob-/- mice. NHE1-dependent rate of intracellular pH (pHi) normalization after transient acid loading of isolated cardiomyocytes was higher in ob-/- mice vs. ob+/-. NHE transport activity was also augmented in cultured H9C2 rat cardiomyoblasts treated with high glucose/high palmitate, and it was normalized after CA inhibition. We conclude that the NHE1/CAII metabolon complex is exacerbated in diabetic cardiomyopathy of ob-/- mice, which may lead to perturbation of pHi and [Na+] and [Ca2+] handling in these diseased hearts.