RESUMO
Ischemia and reperfusion affect multiple elements of cardiomyocyte electrophysiology, especially within the mitochondria. We previously showed that in cardiac monolayers, upon reperfusion after coverslip-induced ischemia, mitochondrial inner membrane potential (ΔΨ) unstably oscillates between polarized and depolarized states, and ΔΨ instability corresponds with arrhythmias. Here, through confocal microscopy of compartment-specific molecular probes, we investigate the mechanisms underlying the postischemic ΔΨ oscillations, focusing on the role of Ca2+ and oxidative stress. During reperfusion, transient ΔΨ depolarizations occurred concurrently with periods of increased mitochondrial oxidative stress (5.07 ± 1.71 oscillations/15 min, N = 100). Supplementing the antioxidant system with GSH monoethyl ester suppressed ΔΨ oscillations (1.84 ± 1.07 oscillations/15 min, N = 119, t test p = 0.027) with 37% of mitochondrial clusters showing no ΔΨ oscillations (versus 4% in control, odds ratio = 14.08, Fisher's exact test p < 0.001). We found that limiting the production of reactive oxygen species using cyanide inhibited postischemic ΔΨ oscillations (N = 15, t test p < 10-5). Furthermore, ΔΨ oscillations were not associated with any discernable pattern in cell-wide oxidative stress or with the changes in cytosolic or mitochondrial Ca2+. Sustained ΔΨ depolarization followed cytosolic and mitochondrial Ca2+ increase and was associated with increased cell-wide oxidative stress. Collectively, these findings suggest that transient bouts of increased mitochondrial oxidative stress underlie postischemic ΔΨ oscillations, regardless of Ca2+ dynamics.
Assuntos
Mitocôndrias Cardíacas , Estresse Oxidativo , Humanos , Cálcio/metabolismo , Isquemia/metabolismo , Potencial da Membrana Mitocondrial , Mitocôndrias Cardíacas/metabolismo , Miócitos Cardíacos/metabolismo , Espécies Reativas de Oxigênio/metabolismo , ReperfusãoRESUMO
Stimulation of adult cardiomyocyte proliferation is a promising strategy for treating myocardial infarction (MI). Earlier studies have shown increased CCL2 levels in plasma and cardiac tissue both in MI patients and mouse models. In present study we investigated the role of CCL2 in cardiac regeneration and the underlying mechanisms. MI was induced in adult mice by permanent ligation of the left anterior descending artery, we showed that the serum and cardiac CCL2 levels were significantly increased in MI mice. Intramyocardial injection of recombinant CCL2 (rCCL2, 1 µg) immediately after the surgery significantly promoted cardiomyocyte proliferation, improved survival rate and cardiac function, and diminished scar sizes in post-MI mice. Alongside these beneficial effects, we observed an increased angiogenesis and decreased cardiomyocyte apoptosis in post-MI mice. Conversely, treatment with a selective CCL2 synthesis inhibitor Bindarit (30 µM) suppressed both CCL2 expression and cardiomyocyte proliferation in P1 neonatal rat ventricle myocytes (NRVMs). We demonstrated in NRVMs that the CCL2 stimulated cardiomyocyte proliferation through STAT3 signaling: treatment with rCCL2 (100 ng/mL) significantly increased the phosphorylation levels of STAT3, whereas a STAT3 phosphorylation inhibitor Stattic (30 µM) suppressed rCCL2-induced cardiomyocyte proliferation. In conclusion, this study suggests that CCL2 promotes cardiac regeneration via activation of STAT3 signaling, underscoring its potential as a therapeutic agent for managing MI and associated heart failure.
Assuntos
Insuficiência Cardíaca , Infarto do Miocárdio , Humanos , Camundongos , Animais , Ratos , Quimiocina CCL2/metabolismo , Infarto do Miocárdio/metabolismo , Miócitos Cardíacos , Insuficiência Cardíaca/metabolismo , Regeneração , Camundongos Endogâmicos C57BL , Apoptose , Fator de Transcrição STAT3/metabolismoRESUMO
Myocardial hypertrophy is a pathological thickening of the myocardium which ultimately results in heart failure. We previously reported that zonisamide, an antiepileptic drug, attenuated pressure overload-caused myocardial hypertrophy and diabetic cardiomyopathy in murine models. In addition, we have found that the inhibition of proteasome activates glycogen synthesis kinase 3 (GSK-3) thus alleviates myocardial hypertrophy, which is an important anti-hypertrophic strategy. In this study, we investigated whether zonisamide prevented pressure overload-caused myocardial hypertrophy through suppressing proteasome. Pressure overload-caused myocardial hypertrophy was induced in mice by trans-aortic constriction (TAC) surgery. Two days after the surgery, the mice were administered zonisamide (10, 20, 40 mg·kg-1·d-1, i.g.) for four weeks. We showed that zonisamide administration significantly mitigated impaired cardiac function. Furthermore, zonisamide administration significantly inhibited proteasome activity as well as the expression levels of proteasome subunit beta types (PSMB) of the 20 S proteasome (PSMB1, PSMB2 and PSMB5) and proteasome-regulated particles (RPT) of the 19 S proteasome (RPT1, RPT4) in heart tissues of TAC mice. In primary neonatal rat cardiomyocytes (NRCMs), zonisamide (0.3 µM) prevented myocardial hypertrophy triggered by angiotensin II (Ang II), and significantly inhibited proteasome activity, proteasome subunits and proteasome-regulated particles. In Ang II-treated NRCMs, we found that 18α-glycyrrhetinic acid (18α-GA, 2 mg/ml), a proteasome inducer, eliminated the protective effects of zonisamide against myocardial hypertrophy and proteasome. Moreover, zonisamide treatment activated GSK-3 through inhibiting the phosphorylated AKT (protein kinase B, PKB) and phosphorylated liver kinase B1/AMP-activated protein kinase (LKB1/AMPKα), the upstream of GSK-3. Zonisamide treatment also inhibited GSK-3's downstream signaling proteins, including extracellular signal-regulated kinase (ERK) and GATA binding protein 4 (GATA4), both being the hypertrophic factors. Collectively, this study highlights the potential of zonisamide as a new therapeutic agent for myocardial hypertrophy, as it shows potent anti-hypertrophic potential through the suppression of proteasome.
Assuntos
Anticonvulsivantes , Bloqueadores dos Canais de Cálcio , Cardiomegalia , Quinase 3 da Glicogênio Sintase , Complexo de Endopeptidases do Proteassoma , Zonisamida , Animais , Camundongos , Ratos , Proteínas Quinases Ativadas por AMP/metabolismo , Cardiomegalia/tratamento farmacológico , Quinase 3 da Glicogênio Sintase/farmacologia , Camundongos Endogâmicos C57BL , Miócitos Cardíacos , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Zonisamida/farmacologia , Zonisamida/uso terapêutico , Anticonvulsivantes/farmacologia , Anticonvulsivantes/uso terapêutico , Bloqueadores dos Canais de Cálcio/farmacologia , Bloqueadores dos Canais de Cálcio/uso terapêuticoRESUMO
PURPOSE: Cardiac tissue engineering is suggested as a promising approach to overcome problems associated with impaired myocardium. This is the first study to investigate the use of BC and gelatin for cardiomyocyte adhesion and growth. METHODS: Bacterial cellulose (BC) membranes were produced by Komagataeibacter xylinus and coated or mixed with gelatin to make gelatin-coated BC (BCG) or gelatin-mixed BC (mBCG) scaffolds, respectively. BC based-scaffolds were characterized via SEM, FTIR, XRD, and AFM. Neonatal rat-ventricular cardiomyocytes (nr-vCMCs) were cultured on the scaffolds to check the capability of the composites for cardiomyocyte attachment, growth and expansion. RESULTS: The average nanofibrils diameter in all scaffolds was suitable (~ 30-65 nm) for nr-vCMCs culture. Pore diameter (≥ 10 µm), surface roughness (~ 182 nm), elastic modulus (0.075 ± 0.015 MPa) in mBCG were in accordance with cardiomyocyte requirements, so that mBCG could better support attachment of nr-vCMCs with high concentration of gelatin, and appropriate surface roughness. Also, it could better support growth and expansion of nr-vCMCs due to submicron scale of nanofibrils and proper elasticity (~ 0.075 MPa). The viability of nr-vCMCs on BC and BCG scaffolds was very low even at day 2 of culture (~ ≤ 40%), but, mBCG could promote a metabolic active state of nr-vCMCs until day 7 (~ ≥ 50%). CONCLUSION: According to our results, mBCG scaffold was the most suitable composite for cardiomyocyte culture, regarding its physicochemical and cell characteristics. It is suggested that improvement in mBCG stability and cell attachment features may provide a convenient scaffold for cardiac tissue engineering.
RESUMO
Astrocytes are crucial in the regulation of neurotransmitter homeostasis, and while their involvement in the dopamine (DA) tripartite synapse is acknowledged, it necessitates a more comprehensive investigation. In the present study, experiments were conducted on primary astrocyte cultures from the striatum and cortex of neonatal rats. The pharmacological intricacies of DA uptake, including dependence on time, temperature, and concentration, were investigated using radiolabelled [3H]-DA. The mRNA expression of transporters DAT, NET, PMAT, and OCTs was evaluated by qPCR. Notably, astrocytes from both brain regions exhibited prominent mRNA expression of NET and PMAT, with comparatively lower expression of DAT and OCTs. The inhibition of DA uptake by the DAT inhibitor, GBR12909, and NET inhibitors, desipramine and nortriptyline, impeded DA uptake in striatal astrocytes more than in cortical astrocytes. The mRNA expression of NET and PMAT was significantly upregulated in cortical astrocytes in response to the DA receptor agonist apomorphine, while only the mRNA expression of NET exhibited changes in striatal astrocytes. Haloperidol, a DA receptor antagonist, and L-DOPA, a DA precursor, did not induce significant alterations in transporter mRNA expression. These findings underscore the intricate and region-specific mechanisms governing DA uptake in astrocytes, emphasizing the need for continued exploration to unravel the nuanced dynamics of astrocytic involvement in the DA tripartite synapse.
Assuntos
Astrócitos , Dopamina , Animais , Ratos , Animais Recém-Nascidos , Corpo Estriado , Proteínas de Membrana Transportadoras , RNA Mensageiro/genéticaRESUMO
Pain management in neonates continues to be a challenge. Diverse therapies are available that cause loss of pain sensitivity. However, because of side effects, the search for better options remains open. Dexmedetomidine is a promising drug; it has shown high efficacy with a good safety profile in sedation and analgesia in the immature nervous system. Though dexmedetomidine is already in use for pain control in neonates (including premature neonates) and infants as an adjunct to other anesthetics, the question remains whether it affects the neuronal activity patterning that is critical for development of the immature nervous system. In this study, using the neonatal rat as a model, the pharmacodynamic effects of dexmedetomidine on the nervous and cardiorespiratory systems were studied. Our results showed that dexmedetomidine has pronounced analgesic effects in the neonatal rat pups, and also weakly modified both the immature network patterns of cortical and hippocampal activity and the physiology of sleep cycles. Though the respiration and heart rates were slightly reduced after dexmedetomidine administration, it might be considered as the preferential independent short-term therapy for pain management in the immature and developing brain.
Assuntos
Animais Recém-Nascidos , Dexmedetomidina , Dexmedetomidina/farmacologia , Animais , Ratos , Analgésicos não Narcóticos/farmacologia , Analgesia/métodos , Manejo da Dor/métodos , Masculino , Ratos Sprague-Dawley , Dor/tratamento farmacológico , Frequência Cardíaca/efeitos dos fármacos , Feminino , Sistema Nervoso/efeitos dos fármacos , Sistema Nervoso/crescimento & desenvolvimentoRESUMO
OBJECTIVES: To observe the effects of melatonin on autophagy in cortical neurons of neonatal rats with hypoxic-ischemic brain damage (HIBD) and to explore its mechanisms via the PI3K/AKT signaling pathway, aiming to provide a basis for the clinical application of melatonin. METHODS: Seven-day-old Sprague-Dawley neonatal rats were randomly divided into a sham operation group, an HIBD group, and a melatonin group (n=9 each). The neonatal rat HIBD model was established using the classic Rice-Vannucci method. Neuronal morphology in the neonatal rat cerebral cortex was observed with hematoxylin-eosin staining and Nissl staining. Autophagy-related protein levels of microtubule-associated protein 1 light chain 3 (LC3) and Beclin-1 were detected by immunofluorescence staining and Western blot analysis. Phosphorylated phosphoinositide 3-kinase (p-PI3K) and phosphorylated protein kinase B (p-AKT) protein expression levels were measured by immunohistochemistry and Western blot. The correlation between autophagy and the PI3K pathway in the melatonin group and the HIBD group was analyzed using Pearson correlation analysis. RESULTS: Twenty-four hours post-modeling, neurons in the sham operation group displayed normal size and orderly arrangement. In contrast, neurons in the HIBD group showed swelling and disorderly arrangement, while those in the melatonin group had relatively normal morphology and more orderly arrangement. Nissl bodies were normal in the sham operation group but distorted in the HIBD group; however, they remained relatively intact in the melatonin group. The average fluorescence intensity of LC3 and Beclin-1 was higher in the HIBD group compared to the sham operation group, but was reduced in the melatonin group compared to the HIBD group (P<0.05). The number of p-PI3K+ and p-AKT+ cells decreased in the HIBD group compared to the sham operation group but increased in the melatonin group compared to the HIBD group (P<0.05). LC3 and Beclin-1 protein expression levels were higher, and p-PI3K and p-AKT levels were lower in the HIBD group compared to the sham operation group (P<0.05); however, in the melatonin group, LC3 and Beclin-1 levels decreased, and p-PI3K and p-AKT increased compared to the HIBD group (P<0.05). The correlation analysis results showed that the difference of the mean fluorescence intensity of LC3 and Beclin-1 protein in the injured cerebral cortex between the melatonin and HIBD groups was negatively correlated with the difference of the number of p-PI3K+ and p-AKT+ cells between the two groups (P<0.05). CONCLUSIONS: Melatonin can inhibit excessive autophagy in cortical neurons of neonatal rats with HIBD, thereby alleviating HIBD. This mechanism is associated with the PI3K/AKT pathway.
Assuntos
Animais Recém-Nascidos , Autofagia , Córtex Cerebral , Hipóxia-Isquemia Encefálica , Melatonina , Neurônios , Fosfatidilinositol 3-Quinases , Proteínas Proto-Oncogênicas c-akt , Ratos Sprague-Dawley , Transdução de Sinais , Animais , Melatonina/farmacologia , Hipóxia-Isquemia Encefálica/patologia , Hipóxia-Isquemia Encefálica/metabolismo , Ratos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Córtex Cerebral/patologia , Autofagia/efeitos dos fármacos , Fosfatidilinositol 3-Quinases/metabolismo , Neurônios/patologia , Neurônios/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Masculino , FemininoRESUMO
OBJECTIVES: To compare the repair effects of different doses of human umbilical cord mesenchymal stem cells (hUC-MSCs) on white matter injury (WMI) in neonatal rats. METHODS: Two-day-old Sprague-Dawley neonatal rats were randomly divided into five groups: sham operation group, WMI group, and hUC-MSCs groups (low dose, medium dose, and high dose), with 24 rats in each group. Twenty-four hours after successful establishment of the neonatal rat white matter injury model, the WMI group was injected with sterile PBS via the lateral ventricle, while the hUC-MSCs groups received injections of hUC-MSCs at different doses. At 14 and 21 days post-modeling, hematoxylin and eosin staining was used to observe pathological changes in the tissues around the lateral ventricles. Real-time quantitative polymerase chain reaction was used to detect the quantitative expression of myelin basic protein (MBP) and glial fibrillary acidic protein (GFAP) mRNA in the brain tissue. Immunohistochemistry was employed to observe the expression levels of GFAP and neuron-specific nuclear protein (NeuN) in the tissues around the lateral ventricles. TUNEL staining was used to observe cell apoptosis in the tissues around the lateral ventricles. At 21 days post-modeling, the Morris water maze test was used to observe the spatial learning and memory capabilities of the neonatal rats. RESULTS: At 14 and 21 days post-modeling, numerous cells with nuclear shrinkage and rupture, as well as disordered arrangement of nerve fibers, were observed in the tissues around the lateral ventricles of the WMI group and the low dose group. Compared with the WMI group, the medium and high dose groups showed alleviated pathological changes; the arrangement of nerve fibers in the medium dose group was relatively more orderly compared with the high dose group. Compared with the WMI group, there was no significant difference in the expression levels of MBP and GFAP mRNA in the low dose group (P>0.05), while the expression levels of MBP mRNA increased and GFAP mRNA decreased in the medium and high dose groups. The expression level of MBP mRNA in the medium dose group was higher than that in the high dose group, and the expression level of GFAP mRNA in the medium dose group was lower than that in the high dose group (P<0.05). Compared with the WMI group, there was no significant difference in the protein expression of GFAP and NeuN in the low dose group (P>0.05), while the expression of NeuN protein increased and GFAP protein decreased in the medium and high dose groups. The expression of NeuN protein in the medium dose group was higher than that in the high dose group, and the expression of GFAP protein in the medium dose group was lower than that in the high dose group (P<0.05). Compared with the WMI group, there was no significant difference in the number of apoptotic cells in the low dose group (P>0.05), while the number of apoptotic cells in the medium and high dose groups was less than that in the WMI group, and the number of apoptotic cells in the medium dose group was less than that in the high dose group (P<0.05). Compared with the WMI group, there was no significant difference in the escape latency time in the low dose group (P>0.05); starting from the third day of the latency period, the escape latency time in the medium dose group was less than that in the WMI group (P<0.05). The medium and high dose groups crossed the platform more times than the WMI group (P<0.05). CONCLUSIONS: Low dose hUC-MSCs may yield unsatisfactory repair effects on WMI in neonatal rats, while medium and high doses of hUC-MSCs have significant repair effects, with the medium dose demonstrating superior efficacy.
Assuntos
Animais Recém-Nascidos , Transplante de Células-Tronco Mesenquimais , Ratos Sprague-Dawley , Cordão Umbilical , Substância Branca , Animais , Ratos , Humanos , Cordão Umbilical/citologia , Substância Branca/patologia , Substância Branca/metabolismo , Proteína Glial Fibrilar Ácida/metabolismo , Proteína Glial Fibrilar Ácida/genética , Proteína Glial Fibrilar Ácida/análise , Células-Tronco Mesenquimais , Proteína Básica da Mielina/genética , Proteína Básica da Mielina/análise , Proteína Básica da Mielina/metabolismo , Masculino , Apoptose , Feminino , RNA Mensageiro/análise , RNA Mensageiro/metabolismoRESUMO
OBJECTIVES: To investigate the protective effects of 2-methoxyestradiol (2ME) against hypoxic pulmonary hypertension (HPH) in neonatal rats. METHODS: Ninety-six Wistar neonatal rats were randomly divided into a normoxia group, a hypoxia group, and a hypoxia + 2ME group, with each group further subdivided into 3-day, 7-day, 14-day, and 21-day subgroups, containing eight rats each. The hypoxia and hypoxia + 2ME groups received daily subcutaneous injections of saline and 2ME (240 µg/kg), respectively, while the normoxia group was raised in a normoxic environment with daily saline injections. Right ventricular systolic pressure (RVSP) was measured using the direct pressure method. Pulmonary vascular morphology was assessed using hematoxylin and eosin staining, with metrics including the percentage of medial thickness of small pulmonary arteries relative to the external diameter (MT%) and the cross-sectional area of the media of small pulmonary arteries relative to the total cross-sectional area (MA%). Immunohistochemistry was used to detect the expression levels of hypoxia-inducible factor-1α (HIF-1α) and proliferating cell nuclear antigen (PCNA) proteins, while real-time quantitative PCR was used to to assess HIF-1α and PCNA mRNA levels. RESULTS: Compared to the normoxia group, the hypoxia and hypoxia + 2ME groups showed increased RVSP and upregulated HIF-1α and PCNA protein and mRNA expression levels at 3, 7, 14, and 21 days after hypoxia (P<0.05). Furthermore, at 7, 14, and 21 days after hypoxia, the hypoxia group showed increased MT% and MA% (P<0.05). In comparison to the hypoxia group, the hypoxia + 2ME group exhibited reduced RVSP and downregulated HIF-1α and PCNA protein and mRNA expression levels, along with decreased MT% and MA% at 7, 14, and 21 days after hypoxia (P<0.05). CONCLUSIONS: 2ME may protect against HPH in neonatal rats by inhibiting the expression of HIF-1α and PCNA and reducing pulmonary vascular remodeling. Citation:Chinese Journal of Contemporary Pediatrics, 2024, 26(7): 757-764.
Assuntos
2-Metoxiestradiol , Animais Recém-Nascidos , Hipertensão Pulmonar , Subunidade alfa do Fator 1 Induzível por Hipóxia , Hipóxia , Antígeno Nuclear de Célula em Proliferação , Artéria Pulmonar , Ratos Wistar , Animais , 2-Metoxiestradiol/farmacologia , Ratos , Hipertensão Pulmonar/prevenção & controle , Hipertensão Pulmonar/tratamento farmacológico , Antígeno Nuclear de Célula em Proliferação/análise , Antígeno Nuclear de Célula em Proliferação/genética , Hipóxia/complicações , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Artéria Pulmonar/efeitos dos fármacos , Masculino , Feminino , Estradiol/farmacologia , Estradiol/análogos & derivados , RNA Mensageiro/análiseRESUMO
The developing entorhinal-hippocampal system is embedded within a large-scale bottom-up network, where spontaneous myoclonic movements, presumably via somatosensory feedback, trigger hippocampal early sharp waves (eSPWs). The hypothesis, that somatosensory feedback links myoclonic movements with eSPWs, implies that direct somatosensory stimulation should also be capable of evoking eSPWs. In this study, we examined hippocampal responses to electrical stimulation of the somatosensory periphery in urethane-anesthetized, immobilized neonatal rat pups using silicone probe recordings. We found that somatosensory stimulation in ~33% of the trials evoked local field potential (LFP) and multiple unit activity (MUA) responses identical to spontaneous eSPWs. The somatosensory-evoked eSPWs were delayed from the stimulus, on average, by 188 ms. Both spontaneous and somatosensory-evoked eSPWs (i) had similar amplitude of ~0.5 mV and half-duration of ~40 ms, (ii) had similar current-source density (CSD) profiles, with current sinks in CA1 strata radiatum, lacunosum-moleculare and DG molecular layer and (iii) were associated with MUA increase in CA1 and DG. Our results indicate that eSPWs can be triggered by direct somatosensory stimulations and support the hypothesis that sensory feedback from movements is involved in the association of eSPWs with myoclonic movements in neonatal rats.
Assuntos
Hipocampo , Uretana , Ratos , Animais , Animais Recém-Nascidos , Hipocampo/fisiologia , Estimulação ElétricaRESUMO
OBJECTIVES: To study the effect of gut microbiota on hematopoiesis in a neonatal rat model of necrotizing enterocolitis (NEC). METHODS: Neonatal Sprague-Dawley rats were randomly divided into a control group and a model group (NEC group), with 6 rats in each group. Formula milk combined with hypoxia and cold stimulation was used to establish a neonatal rat model of NEC. Hematoxylin and eosin staining was used to observe the pathological changes of intestinal tissue and hematopoiesis-related organs. Routine blood tests were conducted for each group. Immunohistochemistry was used to observe the changes in specific cells in hematopoiesis-related organs. Flow cytometry was used to measure the changes in specific cells in bone marrow. 16S rDNA sequencing was used to observe the composition and abundance of gut microbiota. RESULTS: Compared with the control group, the NEC group had intestinal congestion and necrosis, damage, atrophy, and shedding of intestinal villi, and a significant increase in NEC histological score. Compared with the control group, the NEC group had significantly lower numbers of peripheral blood leukocytes and lymphocytes (P<0.05), nucleated cells in the spleen, thymus, and bone marrow, and small cell aggregates with basophilic nuclei in the liver (P<0.05). The NEC group had significant reductions in CD71+ erythroid progenitor cells in the liver, CD45+ lymphocytes in the spleen and bone marrow, CD3+ T lymphocytes in thymus, and the proportion of CD45+CD3-CD43+SSChi neutrophils in bone marrow (P<0.05). There was a significant difference in the composition of gut microbiota between the NEC and control groups, and the NEC group had a significant reduction in the abundance of Ligilactobacillus and a significant increase in the abundance of Escherichia-Shigella (P<0.05), which replaced Ligilactobacillus and became the dominant flora. CONCLUSIONS: Multi-lineage hematopoietic disorder may be observed in a neonatal rat model of NEC, which may be associated with gut microbiota dysbiosis and abnormal multiplication of the pathogenic bacteria Escherichia-Shigella.
Assuntos
Enterocolite Necrosante , Microbioma Gastrointestinal , Doenças do Recém-Nascido , Ratos , Animais , Enterocolite Necrosante/etiologia , Ratos Sprague-Dawley , Animais Recém-NascidosRESUMO
OBJECTIVES: To study the effect of ligustrazine injection on mitophagy in neonatal rats with hypoxic-ischemic encephalopathy (HIE) and its molecular mechanism. METHODS: Neonatal Sprague-Dawley rats, aged 7 days, were randomly divided into a sham-operation group with 8 rats, a model group with 12 rats, and a ligustrazine group with 12 rats. The rats in the model group and the ligustrazine group were used to establish a neonatal rat model of HIE by ligation of the left common carotid artery followed by hypoxia treatment, and blood vessels were exposed without any other treatment for the rats in the sham-operation group. The rats in the ligustrazine group were intraperitoneally injected with ligustrazine (20 mg/kg) daily after hypoxia-ischemia, and those in the sham-operation group and the model group were intraperitoneally injected with an equal volume of normal saline daily. Samples were collected after 7 days of treatment. Hematoxylin and eosin staining and Nissl staining were used to observe the pathological changes of neurons in brain tissue; immunohistochemical staining was used to observe the positive expression of PINK1 and Parkin in the hippocampus and cortex; TUNEL staining was used to measure neuronal apoptosis; Western blotting was used to measure the expression levels of the mitophagy pathway proteins PINK1 and Parkin and the autophagy-related proteins Beclin-1, microtubule-associated protein 1 light chain 3 (LC3), and ubiquitin-binding protein (P62). RESULTS: Compared with the sham-operation group, the model group had a significant reduction in the number of neurons, an increase in intercellular space, loose arrangement, lipid vacuolization, and a reduction in Nissl bodies. The increased positive expression of PINK1 and Parkin, apoptosis rate of neurons, and protein expression levels of PINK1, Parkin, Beclin1 and LC3 (P<0.05) and the decreased protein expression level of P62 in the hippocampus were also observed in the model group (P<0.05). Compared with the model group, the ligustrazine group had a significant increase in the number of neurons with ordered arrangement and an increase in Nissl bodies, significant reductions in the positive expression of PINK1 and Parkin, the apoptosis rate of neurons, and the protein expression levels of PINK1, Parkin, Beclin1, and LC3 (P<0.05), and a significant increase in the protein expression level of P62 (P<0.05). CONCLUSIONS: Ligustrazine can alleviate hypoxic-ischemic brain damage and inhibit neuronal apoptosis in neonatal rats to a certain extent, possibly by inhibiting PINK1/Parkin-mediated autophagy.
Assuntos
Hipóxia-Isquemia Encefálica , Ratos , Animais , Hipóxia-Isquemia Encefálica/tratamento farmacológico , Hipóxia-Isquemia Encefálica/metabolismo , Animais Recém-Nascidos , Ratos Sprague-Dawley , Proteína Beclina-1 , Autofagia , Ubiquitina-Proteína Ligases/metabolismo , Proteínas Quinases/metabolismoRESUMO
OBJECTIVES: To study the effect of platelet-derived growth factor-BB (PDGF-BB) on pulmonary vascular remodeling in neonatal rats with hypoxic pulmonary hypertension (HPH). METHODS: A total of 128 neonatal rats were randomly divided into four groups: PDGF-BB+HPH, HPH, PDGF-BB+normal oxygen, and normal oxygen (n=32 each). The rats in the PDGF-BB+HPH and PDGF-BB+normal oxygen groups were given an injection of 13 µL 6×1010 PFU/mL adenovirus with PDGF-BB genevia the caudal vein. After 24 hours of adenovirus transfection, the rats in the HPH and PDGF-BB+HPH groups were used to establish a neonatal rat model of HPH. Right ventricular systolic pressure (RVSP) was measured on days 3, 7, 14, and 21 of hypoxia. Hematoxylin-eosin staining was used to observe pulmonary vascular morphological changes under an optical microscope, and vascular remodeling parameters (MA% and MT%) were also measured. Immunohistochemistry was used to measure the expression levels of PDGF-BB and proliferating cell nuclear antigen (PCNA) in lung tissue. RESULTS: The rats in the PDGF-BB+HPH and HPH groups had a significantly higher RVSP than those of the same age in the normal oxygen group at each time point (P<0.05). The rats in the PDGF-BB+HPH group showed vascular remodeling on day 3 of hypoxia, while those in the HPH showed vascular remodeling on day 7 of hypoxia. On day 3 of hypoxia, the PDGF-BB+HPH group had significantly higher MA% and MT% than the HPH, PDGF-BB+normal oxygen, and normal oxygen groups (P<0.05). On days 7, 14, and 21 of hypoxia, the PDGF-BB+HPH and HPH groups had significantly higher MA% and MT% than the PDGF-BB+normal oxygen and normal oxygen groups (P<0.05). The PDGF-BB+HPH and HPH groups had significantly higher expression levels of PDGF-BB and PCNA than the normal oxygen group at all time points (P<0.05). On days 3, 7, and 14 of hypoxia, the PDGF-BB+HPH group had significantly higher expression levels of PDGF-BB and PCNA than the HPH group (P<0.05), while the PDGF-BB+normal oxygen group had significantly higher expression levels of PDGF-BB and PCNA than the normal oxygen group (P<0.05). CONCLUSIONS: Exogenous administration of PDGF-BB in neonatal rats with HPH may upregulate the expression of PCNA, promote pulmonary vascular remodeling, and increase pulmonary artery pressure.
Assuntos
Hipertensão Pulmonar , Ratos , Animais , Becaplermina , Animais Recém-Nascidos , Antígeno Nuclear de Célula em Proliferação , Remodelação Vascular , Artéria Pulmonar/metabolismo , Hipóxia , Oxigênio , Proliferação de Células , Miócitos de Músculo Liso/metabolismoRESUMO
Schwann cells provide essential physical and chemical support for neurons and play critical roles in the peripheral nervous system. To acquire an enhanced understanding of the genetic characteristics of Schwann cells, we analyzed single-cell transcriptional profiling of Schwann cells in neonatal rat sciatic nerves, ordered the pseudotemporal states of Schwann cells, and determined the magnitude of RNA velocity vectors as well as cell cycle stages of Schwann cell subtypes. We discovered the cellular heterogeneity of Schwann cells in neonatal rat sciatic nerves, revealed the dynamic changes of Schwann cell subtypes, and pointed out the differentiation trajectory from Timp3- and Col5a3-expressing Schwann cell subtype 3 to other Schwann cell subtypes. The functional interpretation further indicated that subtype 3 Schwann cells display genetic signatures of DNA replication and the acquisition of mesenchymal traits. Our study presents a transcriptional summarization of the differentiation states of Schwann cell subtypes in neonatal rat sciatic nerves at single-cell resolution and may serve as a foundation for a deeper comprehension of the involvement of Schwann cells in the development and regeneration of peripheral nerves.
Assuntos
Células de Schwann , Análise de Célula Única , Animais , Animais Recém-Nascidos , Diferenciação Celular/genética , Regeneração Nervosa/fisiologia , Ratos , Células de Schwann/metabolismo , Nervo Isquiático/metabolismoRESUMO
Microglial cells are the main immune cells of the retina. The primary culture of the retinal microglia is critically important in investigating the cells' properties and behaviors in neurodegenerative and inflammatory retinal disease. Here, we described a modified protocol of a microglial cell culture from the neonatal rat retina. In our culture protocol, the retina was isolated from the neonatal rat eye from postnatal day 1 to day 3 and trypsinized into a single-cell suspension. The cells were seeded into a T75 flask, which was pre-coated with poly-D-lysine (PDL) and cultured with dulbecco's modified eagle medium-F12 (DMEM/F12) that contained 10% fetal bovine serum (FBS) with different concentrations. Small bright rounded cells were observed on the top of mixed glial cells on the seventh day, and attained the maximum cell number on the 14th day. Then, the isolation was performed by a shaking method and isolated cells were identified with microglia markers ionized calcium-binding adaptor molecule 1 (IBA1), transmembrane protein 119 (TMEM119), cluster of differentiation 11b (CD11b), as well as astrocyte marker glial fibrillary acidic protein (GFAP) by immunofluorescence staining. Additionally, the initial plating ratio of the mixed glial cell, culture period of isolation, procedures of the isolation, as well as the purification procedure, were optimized for our primary microglial cell culture. The morphological changes and phagocytic function were performed after lipopolysaccharide (LPS) stimulation. Moreover, the release of pro-inflammatory cytokines at different time points of LPS activation were measured. In the present study, we found that the concentration of one retina/T75 flask could harvest the largest number of microglial cells. Besides, we continuously cultured the mixed glial cells as long as one month and isolated the mixed glial cells as much as three times. In our study, we used an isolation-shaking rate of 200 rpm for 2h, which guaranteed the steady rate and resulted in high purification of the primary retinal-microglial cells, with no need of an additional purification procedure. In conclusion, we provided a high-producing protocol for the primary culture of purified rat retinal-microglial cells.
Assuntos
Lipopolissacarídeos , Microglia , Animais , Biomarcadores/metabolismo , Técnicas de Cultura de Células , Células Cultivadas , Microglia/metabolismo , Neuroglia/metabolismo , Ratos , Retina/metabolismoRESUMO
Cardiac hypertrophy, as one of the major predisposing factors for chronic heart failure, lacks effective interventions. Exploring the pathogenesis of cardiac hypertrophy will reveal potential therapeutic targets. S-nitrosylation is a kind of posttranslational modification that occurs at active cysteines of proteins to mediate various cellular processes. We here identified heat shock protein 90 (Hsp90) as a highly S-nitrosylated target in the hearts of rodents with hypertrophy, and the role of Hsp90 in cardiac hypertrophy remains undefined. The S-nitrosylation of Hsp90 (SNO-Hsp90) levels were elevated in angiotensin II (Ang II)- or phenylephrine (PE)-treated neonatal rat cardiomyocytes (NRCMs) in vitro as well as in cardiomyocytes isolated from mice subjected to transverse aortic constriction (TAC) in vivo. We demonstrated that the elevated SNO-Hsp90 levels were mediated by decreased S-nitrosoglutathione reductase (GSNOR) expression during cardiac hypertrophy, and delivery of GSNOR adeno-associated virus expression vectors (AAV9-GSNOR) decreased the SNO-Hsp90 levels to attenuate cardiac hypertrophy. Mass spectrometry analysis revealed that cysteine 589 (Cys589) might be the S-nitrosylation site of Hsp90. Delivery of the mutated AAV9-Hsp90-C589A inhibited SNO-Hsp90 levels and attenuated cardiac hypertrophy. We further revealed that SNO-Hsp90 led to increased interaction of glycogen synthase kinase 3ß (GSK3ß) and Hsp90, leading to elevated GSK3ß phosphorylation and decreased eIF2Bε phosphorylation, thereby aggravating cardiac hypertrophy. Application of GSK3ß inhibitor TWS119 abolished the protective effect of Hsp90-C589A mutation in Ang II-treated NRCMs. In conclusion, this study demonstrates a critical role of SNO-Hsp90 in cardiac hypertrophy, which may be of a therapeutic target for cardiac hypertrophy treatment.
Assuntos
Cardiomegalia , Insuficiência Cardíaca , Angiotensina II/metabolismo , Angiotensina II/farmacologia , Animais , Cardiomegalia/patologia , Glicogênio Sintase Quinase 3 beta/metabolismo , Proteínas de Choque Térmico HSP90/metabolismo , Insuficiência Cardíaca/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Miócitos Cardíacos/metabolismo , Ratos , Transdução de SinaisRESUMO
Celastrol is a quinone methide triterpenoid extracted from the root bark of Tripterygium wilfordii Hook F, and it exhibits extensive biological activities such as anti-cancer effects. However, narrow therapeutic window together with undesired side effects limit its clinical application. In this study, we explore celastrol's cardiotoxicity using the methods of histology and cell biology. The results show that celastrol administration dose-dependently induces cardiac dysfunction in mice as manifested by left ventricular dilation, myocardial interstitial fibrosis, and cardiomyocyte hypertrophy. Exposure to celastrol greatly decreases neonatal rat ventricular myocyte (NRVM) viability and promotes its apoptosis. More importantly, we demonstrate that celastrol exerts its pro-apoptotic effects through endoplasmic reticulum (ER) stress and unfolded protein response. Furthermore, siRNA targeting C/EBP homologous protein, a pivotal component of ER stress-mediated apoptosis, effectively prevents the pro-apoptotic effect of celastrol. Taken together, our results demonstrate the potential cardiotoxicity of celastrol and a direct involvement of ER stress in the celastrol-induced apoptosis of NRVMs. Thus, we recommend careful evaluation of celastrol's cardiovascular effects when using it in the clinic.
Assuntos
Cardiotoxicidade , Triterpenos , Animais , Apoptose , Cardiotoxicidade/etiologia , Cardiotoxicidade/metabolismo , Estresse do Retículo Endoplasmático , Camundongos , Triterpenos Pentacíclicos/farmacologia , RNA Interferente Pequeno , Ratos , Triterpenos/toxicidadeRESUMO
BACKGROUND: To explore the mechanism of endocannabinoid cannabinoid receptor 1 (CB1) receptor pathway that regulates synaptic plasticity in the dorsal horn of the spinal cord of rats with neuropathic pain at different ages. METHODS: Neonatal, juvenile, and adult male sprague dawley (SD) rats were divided into the spinal nerve preservation injury (SNI), SNI + Anandamide (AEA), SNI + D-AP5, SNI + CNQX, SNI + D-AP5 + AEA, SNI + CNQX + AEA, sham SNI, sham SNI + AEA, sham SNI + D-AP5, sham SNI + CNQX, sham SNI + D-AP5 + AEA, and sham SNI + CNQX + AEA groups, respectively. Paw withdrawal threshold (PWT) and long-term potentiation (LTP) of the spinal dorsal horn PS (field potential) were assessed to judge the spinal cord's functional state. Immunohistochemical staining and Western blot were conducted to detect CB1 protein levels in the spinal dorsal horn. RESULTS: The LTP response in the spinal cord was alleviated in the SNI + AEA group. After treatment with the N-methyl-D-aspartate (NMDA) receptor blocker D-AP5, the LTP of neonatal A nerve was relieved further. After treatment with the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor blocker CNQX, LTP change in the A nerve was not obvious. The LTP of the A and C nerves were relieved after D-AP5 or CNQX treatment in young and adult animals; however, the blocking effect of CNQX was obvious. The altered levels of PWT and CB1 support these results. CONCLUSIONS: The CB1 receptor activation produces analgesia in neonatal rats through NMDA receptor formation for PS inhibitory activity. In juvenile and adult rats, this phenomenon was effectuated through NMDA and AMPA receptors. This difference could be attributed to the varied number of NMDA and/or AMPA receptors activated during development and changes in the NMDA/AMPA receptor ratio.
Assuntos
N-Metilaspartato , Receptores de AMPA , 6-Ciano-7-nitroquinoxalina-2,3-diona/metabolismo , 6-Ciano-7-nitroquinoxalina-2,3-diona/farmacologia , Animais , Endocanabinoides/metabolismo , Endocanabinoides/farmacologia , Masculino , Ratos , Ratos Sprague-Dawley , Receptores de Canabinoides/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Medula Espinal , Corno Dorsal da Medula Espinal/metabolismo , SinapsesRESUMO
For isolation of neonatal rat cardiomyocytes (NRCM) the ventricular muscles of neonatal rats were treated with different digestive solutions: 0.06% trypsin (method I), 0.08% collagenase II (method II), 0.06% trypsin and 0.08% collagenase II for stepwise digestion (methods III and IV). After enzymatic dissociation of the tissue, the complete medium was added to stop this process. The cells suspensions obtained by methods I-III were collected and centrifuged. In contrast, the novel and improved method IV did not use centrifugation. Instead, various methods of adhesion were employed to separate non-myocardial cells. The isolation methods were compared by the quantity, survival rate, morphology, spontaneous pulsation rate, purity, and vitality of NRCM. These assessments showed that isolation method IV is a simple, efficient, and convenient way to obtain NRCM for culturing.
Assuntos
Ventrículos do Coração , Miócitos Cardíacos , Animais , Animais Recém-Nascidos , Células Cultivadas , Meios de Cultura , RatosRESUMO
OBJECTIVES: To investigate the effect of inhibiting miR-204 expression on the learning and memory abilities of neonatal rats with intrauterine growth restriction (IUGR) and related mechanism. METHODS: A rat model of IUGR was prepared by low-protein diet. The 3-day-old IUGR rats were divided into three groups: model, miRNA antagonist control and miR-204 antagonist, with 10 rats in each group. Ten normal neonatal rats served as the control group. Morris water maze test was used to measure the learning and memory abilities of the rats. Quantitative real-time PCR was used to measure the mRNA expression levels of miR-204 and brain-derived neurotrophic factor (BDNF) in the hippocampus. Nissl staining and TUNEL staining were used to observe the number of Nissl bodies and the apoptosis of cells in the hippocampus. Western blot was used to measure the expression levels of BDNF/TrkB signaling pathway-related proteins in the hippocampus. RESULTS: Compared with the control group, the model group had a significant increase in the escape latency and a significant reduction in the number of platform crossings (P<0.001). The model group also had significant increases in the apoptosis rate of cells and the expression level of miR-204 in hippocampal tissue (P<0.001), while the number of Nissl bodies, the mRNA expression level of BDNF, and the protein expression levels of BDNF, p-TrkB, and p-CREB in the model group were significantly reduced compared with the control group (P<0.001). After inhibition of the expression of miR-204, the number of Nissl bodies, the mRNA expression level of BDNF, and the protein expression levels of BDNF, p-TrkB, and p-CREB significantly increased, while the cell apoptosis rate and the expression level of miR-204 in the hippocampus significantly decreased. The escape latency was also reduced, while the number of platform crossings increased after inhibition of the expression of miR-204 (P<0.001). CONCLUSIONS: Inhibiting miR-204 can improve the learning and memory functions of neonatal rats with IUGR, possibly by targeted activation of the BDNF/TrkB signaling pathway.