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1.
Nat Commun ; 12(1): 5012, 2021 08 18.
Artigo em Inglês | MEDLINE | ID: mdl-34408142

RESUMO

Tendon self-renewal is a rare occurrence because of the poor vascularization of this tissue; therefore, reconstructive surgery using autologous tendon is often performed in severe injury cases. However, the post-surgery re-injury rate is relatively high, and the collection of autologous tendons leads to muscle weakness, resulting in prolonged rehabilitation. Here, we introduce an induced pluripotent stem cell (iPSC)-based technology to develop a therapeutic option for tendon injury. First, we derived tenocytes from human iPSCs by recapitulating the normal progression of step-wise narrowing fate decisions in vertebrate embryos. We used single-cell RNA sequencing to analyze the developmental trajectory of iPSC-derived tenocytes. We demonstrated that iPSC-tenocyte grafting contributed to motor function recovery after Achilles tendon injury in rats via engraftment and paracrine effects. The biomechanical strength of regenerated tendons was comparable to that of healthy tendons. We suggest that iPSC-tenocytes will provide a therapeutic option for tendon injury.


Assuntos
Tendão do Calcâneo/lesões , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/transplante , Traumatismos dos Tendões/terapia , Tenócitos/citologia , Tenócitos/transplante , Tendão do Calcâneo/citologia , Tendão do Calcâneo/fisiopatologia , Animais , Autorrenovação Celular , Terapia Baseada em Transplante de Células e Tecidos , Células Cultivadas , Humanos , Masculino , Ratos , Ratos Endogâmicos F344 , Recuperação de Função Fisiológica , Traumatismos dos Tendões/fisiopatologia
2.
J Neural Eng ; 18(4)2021 08 20.
Artigo em Inglês | MEDLINE | ID: mdl-34359056

RESUMO

Background. Robotic exoskeleton devices have become a promising modality for restoration of extremity function in individuals with limb loss or functional weakness. However, there exists no consistent or reliable way to record efferent motor action potentials from intact peripheral nerves to control device movement. Peripheral nerve motor action potentials are similar in amplitude to that of background noise, producing an unfavorable signal-to-noise ratio (SNR) that makes these signals difficult to detect and interpret. To address this issue, we have developed the muscle cuff regenerative peripheral nerve interface (MC-RPNI), a construct consisting of a free skeletal muscle graft wrapped circumferentially around an intact peripheral nerve. Over time, the muscle graft regenerates, and the intact nerve undergoes collateral axonal sprouting to reinnervate the muscle. The MC-RPNI amplifies efferent motor action potentials by several magnitudes, thereby increasing the SNR, allowing for higher fidelity signaling and detection of motor intention. The goal of this study was to characterize the signaling capabilities and viability of the MC-RPNI over time.Methods. Thirty-seven rats were randomly assigned to one of five experimental groups (Groups A-E). For MC-RPNI animals, their contralateral extensor digitorum longus (EDL) muscle was harvested and trimmed to either 8 mm (Group A) or 13 mm (Group B) in length, wrapped circumferentially around the intact ipsilateral common peroneal (CP) nerve, secured, and allowed to heal for 3 months. Additionally, one 8 mm (Group C) and one 13 mm (Group D) length group had an epineurial window created in the CP nerve immediately preceding MC-RPNI creation. Group E consisted of sham surgery animals. At 3 months, electrophysiologic analyses were conducted to determine the signaling capabilities of the MC-RPNI. Additionally, electromyography and isometric force analyses were performed on the CP-innervated EDL to determine the effects of the MC-RPNI on end organ function. Following evaluation, the CP nerve, MC-RPNI, and ipsilateral EDL muscle were harvested for histomorphometric analysis.Results. Study endpoint analysis was performed at 3 months post-surgery. All rats displayed visible muscle contractions in both the MC-RPNI and EDL following proximal CP nerve stimulation. Compound muscle action potentials were recorded from the MC-RPNI following proximal CP nerve stimulation and ranged from 3.67 ± 0.58 mV to 6.04 ± 1.01 mV, providing efferent motor action potential amplification of 10-20 times that of a normal physiologic nerve action potential. Maximum tetanic isometric force (Fo) testing of the distally-innervated EDL muscle in MC-RPNI groups producedFo(2341 ± 114 mN-2832 ± 102 mN) similar to controls (2497 ± 122 mN), thus demonstrating that creation of MC-RPNIs did not adversely impact the function of the distally-innervated EDL muscle. Overall, comparison between all MC-RPNI sub-groups did not reveal any statistically significant differences in signaling capabilities or negative effects on distal-innervated muscle function as compared to the control group.Conclusions. MC-RPNIs have the capability to provide efferent motor action potential amplification from intact nerves without adversely impacting distal muscle function. Neither the size of the muscle graft nor the presence of an epineurial window in the nerve had any significant impact on the ability of the MC-RPNI to amplify efferent motor action potentials from intact nerves. These results support the potential for the MC-RPNI to serve as a biologic nerve interface to control advanced exoskeleton devices.


Assuntos
Regeneração Nervosa , Nervos Periféricos , Animais , Eletromiografia , Contração Muscular , Músculo Esquelético , Ratos , Ratos Endogâmicos F344
3.
Int J Mol Sci ; 22(16)2021 Aug 09.
Artigo em Inglês | MEDLINE | ID: mdl-34445271

RESUMO

This study aimed to identify the proteomic changes produced by curcumin treatment following stimulation of the host immune system in a rat model of malignant mesothelioma. We analyzed the proteomes of secondary lymphoid organs from four normal rats, four untreated tumor-bearing rats, and four tumor-bearing rats receiving repeated intraperitoneal administrations of curcumin. Cross-comparing proteome analyses of histological sections of the spleen from the three groups first identified a list of eighty-three biomarkers of interest, thirteen of which corresponded to proteins already reported in the literature and involved in the anticancer therapeutic effects of curcumin. In a second step, comparing these data with proteomic analyses of histological sections of mesenteric lymph nodes revealed eight common biomarkers showing a similar pattern of changes in both lymphoid organs. Additional findings included a partial reduction of the increase in spleen-circulating biomarkers, a decrease in C-reactive protein and complement C3 in the spleen and lymph nodes, and an increase in lymph node purine nucleoside phosphorylase previously associated with liver immunodeficiency. Our results suggest some protein abundance changes could be related to the systemic, distant non-target antitumor effects produced by this phytochemical.


Assuntos
Biomarcadores Tumorais/metabolismo , Curcumina/farmacologia , Linfonodos/metabolismo , Mesotelioma , Proteínas de Neoplasias/metabolismo , Neoplasias Experimentais , Neoplasias Peritoneais , Proteoma/metabolismo , Animais , Masculino , Mesotelioma/tratamento farmacológico , Mesotelioma/metabolismo , Mesotelioma/patologia , Invasividade Neoplásica , Neoplasias Experimentais/tratamento farmacológico , Neoplasias Experimentais/metabolismo , Neoplasias Experimentais/patologia , Neoplasias Peritoneais/tratamento farmacológico , Neoplasias Peritoneais/metabolismo , Neoplasias Peritoneais/patologia , Ratos , Ratos Endogâmicos F344
4.
Toxicology ; 458: 152841, 2021 06 30.
Artigo em Inglês | MEDLINE | ID: mdl-34216699

RESUMO

The cardiotoxicity of various anticancer therapies, including radiotherapy, can lead to cardiovascular complications. These complications can range from damaging cardiac tissues within the irradiation field to increasing the long-term risks of developing heart failure, coronary artery disease, and myocardial infarction. We analyzed radiation-induced metabolites capable of mediating critical biological processes, such as inflammation, senescence, and apoptosis. Previously, by applying QTOF-MASS analysis to irradiated human fibroblasts, we identified that metabolite sets of lysophosphatidylcholine (LPC) were increased in these cells. In this study, radiation-induced LPC accumulation in human aortic endothelial cells (HAECs) increased reactive oxygen species (ROS) production and senescence-associated-beta-galactosidase staining, in addition to decreasing their tube-forming ability. Knockdown of lipoprotein-associated phospholipase A2 (Lp-PLA2) with small interfering RNA (siRNA) inhibited the increased LPC production induced by radiation, and reduced the radiation-induced cell damage produced by ROS and oxidized low-density lipoprotein (LDL). Lp-PLA2 depletion abolished the induction of proinflammatory factors, such as interleukin 1ß, tumor necrosis factor-alpha, matrix metalloproteinase 2, and matrix metalloproteinase 9, as well as adhesion molecules, such as intercellular adhesion molecule 1 (ICAM-1) and E-selection. Likewise, we showed that Lp-PLA2 expression was upregulated in the vasculature of irradiated rat, resulting in increased LPC production and LDL oxidation. Our data demonstrate that radiation-induced LPC production is a potential risk factor for cardiotoxicity that is mediated by Lp-PLA2 activity, suggesting that LPC and Lp-PLA2 offer potential diagnostic and therapeutic approaches to cardiovascular damage during radiotherapy.


Assuntos
1-Alquil-2-acetilglicerofosfocolina Esterase/metabolismo , 1-Alquil-2-acetilglicerofosfocolina Esterase/efeitos da radiação , Células Endoteliais/patologia , Células Endoteliais/efeitos da radiação , Lisofosfatidilcolinas/metabolismo , Fosfolipases A2/metabolismo , Fosfolipases A2/efeitos da radiação , Animais , Aorta/patologia , Aorta/efeitos da radiação , Citocinas/metabolismo , Feminino , Técnicas de Silenciamento de Genes , Humanos , Inflamação/metabolismo , Microtúbulos/efeitos dos fármacos , Microtúbulos/efeitos da radiação , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/efeitos da radiação , Radiação Ionizante , Ratos , Ratos Endogâmicos F344 , Espécies Reativas de Oxigênio/metabolismo
5.
Behav Processes ; 190: 104457, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34216685

RESUMO

Humans show distinct social behaviours when we recognise social similarity in opponents that are members of the same social group. However, little attention has been paid to the role of social similarity in non-human animals. In the Wistar subject rats, the presence of an unfamiliar Wistar rat mitigated stress responses, suggesting the importance of social similarity in this stress-buffering phenomenon. We subsequently found that the presence of unfamiliar Sprague-Dawley (SD) or Long-Evans (LE) rats, but not an unfamiliar Fischer 344 (F344) rat, similarly mitigated stress responses in the subject rats. It is therefore possible that the subject rats recognised social similarity to unfamiliar SD and LE rats. In this study, we demonstrated that the Wistar subject rats were capable of categorizing unfamiliar rats based on their strain, and that the Wistar subjects showed a preference for unfamiliar Wistar, SD, and LE rats over F344 rats. However, the subject rats did not show a preference among Wistar, SD, and LE rats. In addition, the results were not due to an aversion to F344 rats, and preference was not affected when anaesthetised rats were presented to the subject rats. The findings suggested that rats recognise social similarity to certain unfamiliar strains of rats.


Assuntos
Comportamento Social , Animais , Ratos , Ratos Endogâmicos F344 , Ratos Long-Evans , Ratos Sprague-Dawley , Ratos Wistar , Especificidade da Espécie
6.
J Vet Med Sci ; 83(9): 1435-1442, 2021 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-34305076

RESUMO

Properties of macrophages and lymphocytes appearing in renal fibrosis remains to be investigated. F344 rats were injected once a week with cisplatin (2 mg/kg body weight) for 8 weeks and examined at post-final injection weeks 1, 3, 6, 9, and 12. Rats developed progressive renal fibrosis at weeks 1 to 6 as fibrosis-progress phase, and subsequent amelioration at weeks 9 and 12. CD68+ M1-macrophages and major histocompatibility complex (MHC) class II+ macrophages remarkably increased persistently, whereas CD163+ M2-macrophages slightly increased. MHC class II+/CD68+ and MHC class II+/CD163+ macrophages were present, indicating that MHC class II+ macrophages might have both functions of M1- and M2-macrophages. In the fibrosis-progress phase, interleukin (IL)-6, tumor necrosis factor (TNF)-α, and interferon (IFN)-γ for M1-factors, and transforming growth factor (TGF)-ß1 and IL-10 for M2-factors tended to increase; tissue injury by M1 and fibrosis by M2 might have occurred simultaneously. Lots of CD4+ and CD8+ T cells appeared in close relation with MHC class II+ macrophages, and mainly CD4+ T cells formed aggregations. In the lymphocyte aggregates collected by laser microdissection, expression of IL-17A (for Th17 cells) and forkhead box P3 (FoxP3) (for Treg) significantly increased at weeks 1 and 6, respectively; presumably, Th17 cells might be involved in tissue injury, whereas Treg might be related to fibrosis amelioration. These results suggested that macrophages and T cells may contribute interrelatedly to renal fibrosis.


Assuntos
Cisplatino , Doenças dos Roedores , Animais , Linfócitos T CD8-Positivos , Cisplatino/toxicidade , Fibrose , Macrófagos/patologia , Ratos , Ratos Endogâmicos F344 , Doenças dos Roedores/patologia
7.
Int J Mol Sci ; 22(14)2021 07 17.
Artigo em Inglês | MEDLINE | ID: mdl-34299284

RESUMO

Forkhead box E1 (FOXE1) is a lineage-restricted transcription factor involved in thyroid cancer susceptibility. Cancer-associated polymorphisms map in regulatory regions, thus affecting the extent of gene expression. We have recently shown that genetic reduction of FOXE1 dosage modifies multiple thyroid cancer phenotypes. To identify relevant effectors playing roles in thyroid cancer development, here we analyse FOXE1-induced transcriptional alterations in thyroid cells that do not express endogenous FOXE1. Expression of FOXE1 elicits cell migration, while transcriptome analysis reveals that several immune cells-related categories are highly enriched in differentially expressed genes, including several upregulated chemokines involved in macrophage recruitment. Accordingly, FOXE1-expressing cells induce chemotaxis of co-cultured monocytes. We then asked if FOXE1 was able to regulate macrophage infiltration in thyroid cancers in vivo by using a mouse model of cancer, either wild type or with only one functional FOXE1 allele. Expression of the same set of chemokines directly correlates with FOXE1 dosage, and pro-tumourigenic M2 macrophage infiltration is decreased in tumours with reduced FOXE1. These data establish a novel link between FOXE1 and macrophages recruitment in the thyroid cancer microenvironment, highlighting an unsuspected function of this gene in the crosstalk between neoplastic and immune cells that shape tumour development and progression.


Assuntos
Quimiotaxia/fisiologia , Fatores de Transcrição Forkhead/genética , Macrófagos/patologia , Glândula Tireoide/patologia , Neoplasias da Glândula Tireoide/patologia , Animais , Células Cultivadas , Modelos Animais de Doenças , Fatores de Transcrição Forkhead/biossíntese , Fatores de Transcrição Forkhead/metabolismo , Humanos , Técnicas In Vitro , Macrófagos/citologia , Camundongos , Camundongos Knockout , Ratos , Ratos Endogâmicos F344 , Glândula Tireoide/metabolismo , Neoplasias da Glândula Tireoide/genética , Neoplasias da Glândula Tireoide/metabolismo
8.
Int J Mol Sci ; 22(12)2021 Jun 14.
Artigo em Inglês | MEDLINE | ID: mdl-34198710

RESUMO

Microglial activity in the aging neuroimmune system is a central player in aging-related dysfunction. Aging alters microglial function via shifts in protein signaling cascades. These shifts can propagate neurodegenerative pathology. Therapeutics require a multifaceted approach to understand and address the stochastic nature of this process. Polyphenols offer one such means of rectifying age-related decline. Our group used mass spectrometry (MS) analysis to explicate the complex nature of these aging microglial pathways. In our first experiment, we compared primary microglia isolated from young and aged rats and identified 197 significantly differentially expressed proteins between these groups. Then, we performed bioinformatic analysis to explore differences in canonical signaling cascades related to microglial homeostasis and function with age. In a second experiment, we investigated changes to these pathways in aged animals after 30-day dietary supplementation with NT-020, which is a blend of polyphenols. We identified 144 differentially expressed proteins between the NT-020 group and the control diet group via MS analysis. Bioinformatic analysis predicted an NT-020 driven reversal in the upregulation of age-related canonical pathways that control inflammation, cellular metabolism, and proteostasis. Our results highlight salient aspects of microglial aging at the level of protein interactions and demonstrate a potential role of polyphenols as therapeutics for age-associated dysfunction.


Assuntos
Envelhecimento/fisiologia , Suplementos Nutricionais , Microglia/metabolismo , Polifenóis/farmacologia , Transdução de Sinais , Animais , Dieta , Ontologia Genética , Masculino , Microglia/efeitos dos fármacos , Proteoma/metabolismo , Ratos Endogâmicos F344 , Transdução de Sinais/efeitos dos fármacos
9.
AAPS PharmSciTech ; 22(5): 187, 2021 Jun 21.
Artigo em Inglês | MEDLINE | ID: mdl-34155595

RESUMO

Several drugs have poor oral bioavailability due to low or incomplete absorption which is affected by various effects as pH, motility of GI, and enzyme activity. The gastroretentive drug delivery systems are able to deal with these problems by prolonging the gastric residence time, while increasing the therapeutic efficacy of drugs. Previously, we developed a novel technology to foam hot and molten dispersions on atmospheric pressure by a batch-type in-house apparatus. Our aim was to upgrade this technology by a new continuous lab-scale apparatus and confirm that our formulations are gastroretentive. At first, we designed and built the apparatus and continuous production was optimized using a Box-Behnken experimental design. Then, we formulated barium sulfate-loaded samples with the optimal production parameters, which was suitable for in vivo imaging analysis. In vitro study proved the low density, namely 507 mg/cm3, and the microCT record showed high porosity with 40 µm average size of bubbles in the molten suspension. The BaSO4-loaded samples showed hard structure at room temperature and during the wetting test, the complete wetting was detected after 120 min. During the in vivo study, the X-ray taken showed the retention of the formulation in the rat stomach after 2 h. We can conclude that with our device low-density floating formulations were prepared with prolonged gastric residence time. This study provides a promising platform for marketed active ingredients with low bioavailability.


Assuntos
Sulfato de Bário/síntese química , Sulfato de Bário/farmacocinética , Sistemas de Liberação de Medicamentos/métodos , Absorção Gastrointestinal/efeitos dos fármacos , Animais , Sulfato de Bário/administração & dosagem , Disponibilidade Biológica , Preparações de Ação Retardada/administração & dosagem , Preparações de Ação Retardada/síntese química , Preparações de Ação Retardada/farmacocinética , Formas de Dosagem , Mucosa Gástrica/efeitos dos fármacos , Mucosa Gástrica/metabolismo , Absorção Gastrointestinal/fisiologia , Masculino , Porosidade , Ratos , Ratos Endogâmicos F344
10.
Am J Physiol Gastrointest Liver Physiol ; 321(1): G87-G97, 2021 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-34075793

RESUMO

Aging can lead to rectoanal incontinence due to internal anal sphincter (IAS) dysfunction, which is characterized by a decrease in IAS tone and contractility and an increase in nonadrenergic noncholinergic (NANC) relaxation. We aimed to determine whether brain-derived neurotropic factor (BDNF) rescues this aging-associated IAS dysfunction (AAID). To do so, we studied the effects of BDNF on the basal and G protein-coupled receptors (GPCR)-stimulated IAS smooth muscle tone and on NANC relaxation in Fischer 344 rats representing different age groups [26-mo-old (aging) vs. 6-mo-old (young)], before and after tyrosine kinase receptor B (TrkB) antagonist K252a. We also used isolated smooth muscle cells (SMCs) to determine the effects of BDNF before and after different agonists. For some studies, we monitored NO release using smooth muscle perfusates. BDNF reversed AAID by rescuing the basal IAS tone and agonists [thromboxane A2 analog (U46619) and angiotensin II (ANG II)]-induced contractility, and NANC relaxation. These rescue effects of BDNF were selective as K252a attenuated the changes in the IAS without modifying the effects of K+depolarization. Because of the direct association between the basal and GPCR-stimulated IAS tone and RhoA/ROCK activation, we speculate that this pathway in the rescue effects of BDNF. Conversely, our data suggest that aging-associated increased NANC relaxation is reversed by decreased release of NO and decrease in the sensitivity of the released inhibitory neurotransmitter. In summary, BDNF rescue of AAID involves RhoA/ROCK and inhibitory neurotransmission. These data have direct implications for the role of BDNF in the pathophysiology and therapeutic targeting of aging-associated rectoanal motility disorders.NEW & NOTEWORTHY These studies demonstrate that brain-derived neurotropic factor (BDNF) rescues the aging-associated internal anal sphincter (IAS) dysfunction, characterized by a decrease in IAS tone, and increase in non-adrenergic noncholinergic relaxation. We determined the effects of BDNF on the basal and GPCR (TXA2 and ANG II)-stimulated IAS tone, and on NANC relaxation, before and after TrkB inhibitor K252a. BDNF may have an important role in the pathophysiology and therapeutic targeting of certain rectoanal motility disorders.


Assuntos
Envelhecimento/efeitos dos fármacos , Fator Neurotrófico Derivado do Encéfalo/farmacologia , Contração Muscular/efeitos dos fármacos , Músculo Liso/efeitos dos fármacos , Animais , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Masculino , Relaxamento Muscular/efeitos dos fármacos , Tono Muscular/fisiologia , Músculo Liso/metabolismo , Miócitos de Músculo Liso/metabolismo , Ratos , Ratos Endogâmicos F344 , Transdução de Sinais/fisiologia , Quinases Associadas a rho/metabolismo
11.
Int J Mol Sci ; 22(11)2021 May 23.
Artigo em Inglês | MEDLINE | ID: mdl-34071138

RESUMO

Schwann cells play an important role in peripheral nerve function, and their dysfunction has been implicated in the pathogenesis of diabetic neuropathy and other demyelinating diseases. The physiological functions of insulin in Schwann cells remain unclear and therefore define the aim of this study. By using immortalized adult Fischer rat Schwann cells (IFRS1), we investigated the mechanism of the stimulating effects of insulin on the cell proliferation and expression of myelin proteins (myelin protein zero (MPZ) and myelin basic protein (MBP). The application of insulin to IFRS1 cells increased the proliferative activity and induced phosphorylation of Akt and ERK, but not P38-MAPK. The proliferative potential of insulin-stimulated IFRS1 was significantly suppressed by the addition of LY294002, a PI3 kinase inhibitor. The insulin-stimulated increase in MPZ expression was significantly suppressed by the addition of PD98059, a MEK inhibitor. Furthermore, insulin-increased MBP expression was significantly suppressed by the addition of LY294002. These findings suggest that both PI3-K/Akt and ERK/MEK pathways are involved in insulin-induced cell growth and upregulation of MPZ and MBP in IFRS1 Schwann cells.


Assuntos
Insulina/farmacologia , Células de Schwann/efeitos dos fármacos , Animais , Divisão Celular/efeitos dos fármacos , Linhagem Celular Transformada , Cromonas/farmacologia , Neuropatias Diabéticas/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Flavonoides/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Quinases de Proteína Quinase Ativadas por Mitógeno/antagonistas & inibidores , Morfolinas/farmacologia , Proteínas da Mielina/biossíntese , Proteínas da Mielina/genética , Fosfatidilinositol 3-Quinases/efeitos dos fármacos , Fosforilação , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ratos , Ratos Endogâmicos F344 , Receptor de Insulina/biossíntese , Receptor de Insulina/genética , Transdução de Sinais/efeitos dos fármacos
12.
Int J Mol Sci ; 22(11)2021 May 21.
Artigo em Inglês | MEDLINE | ID: mdl-34064122

RESUMO

The σ2 receptor (transmembrane protein 97), which is involved in cholesterol homeostasis, is of high relevance for neoplastic processes. The upregulated expression of σ2 receptors in cancer cells and tissue in combination with the antiproliferative potency of σ2 receptor ligands motivates the research in the field of σ2 receptors for the diagnosis and therapy of different types of cancer. Starting from the well described 2-(4-(1H-indol-1-yl)butyl)-6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline class of compounds, we synthesized a novel series of fluorinated derivatives bearing the F-atom at the aromatic indole/azaindole subunit. RM273 (2-[4-(6-fluoro-1H-pyrrolo[2,3-b]pyridin-1-yl)butyl]-6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline) was selected for labelling with 18F and evaluation regarding detection of σ2 receptors in the brain by positron emission tomography. Initial metabolism and biodistribution studies of [18F]RM273 in healthy mice revealed promising penetration of the radioligand into the brain. Preliminary in vitro autoradiography on brain cryosections of an orthotopic rat glioblastoma model proved the potential of the radioligand to detect the upregulation of σ2 receptors in glioblastoma cells compared to healthy brain tissue. The results indicate that the herein developed σ2 receptor ligand [18F]RM273 has potential to assess by non-invasive molecular imaging the correlation between the availability of σ2 receptors and properties of brain tumors such as tumor proliferation or resistance towards particular therapies.


Assuntos
Encéfalo/metabolismo , Radioisótopos de Flúor/química , Radioisótopos de Flúor/metabolismo , Compostos Radiofarmacêuticos/química , Compostos Radiofarmacêuticos/metabolismo , Receptores sigma/metabolismo , Animais , Feminino , Humanos , Ligantes , Masculino , Camundongos , Neoplasias/metabolismo , Ratos , Ratos Endogâmicos F344 , Tetra-Hidroisoquinolinas/química , Tetra-Hidroisoquinolinas/metabolismo
13.
Am J Physiol Gastrointest Liver Physiol ; 320(6): G1081-G1092, 2021 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-33949202

RESUMO

Stress can trigger symptoms in patients with irritable bowel syndrome (IBS). Previously we demonstrated that chronic psychological stress induced microglial remodeling in the central nucleus of amygdala (CeA) and contributed to the development of visceral hypersensitivity via synaptic engulfment. However, the specific signaling mechanisms that microglia depend upon to recognize target neurons to facilitate visceral pain remain unknown. Here, we test the hypothesis that the microglia in the CeA contribute to chronic stress-induced visceral hypersensitivity via complement C1q/C3-CR3 signaling-mediated synaptic remodeling. In male and female Fischer-344 rats, micropellets of corticosterone (CORT) or cholesterol (control) were stereotaxically implanted bilaterally onto the CeA. After 7 days, microglial C1q, complement receptor 3 (CR3) expression, and microglia-mediated synaptic engulfment were assessed via RNAscope, quantitative PCR, and immunofluorescence. The microglial inhibitor minocycline, CR3 antagonist neutrophil inhibitory factor (NIF), or vehicle were daily infused into the CeA following CORT implantations. Visceral sensitivity was assessed via a visceromotor response (VMR) to graded pressures of isobaric colorectal distension (CRD). Our results suggest that chronic exposure to elevated CORT in the CeA induced visceral hypersensitivity and amygdala microglial morphological remodeling. CORT increased microglial C1q and CR3 expression and increased microglia-mediated synaptic engulfment. Both groups of animals with minocycline or NIF infusions reversed microglia-mediated synaptic remodeling and attenuated CORT-induced visceral hypersensitivity. Our findings demonstrate that C1q/C3-CR3 signaling is critical for microglia-mediated synaptic remodeling in the CeA and contributes to CORT-induced visceral hypersensitivity.NEW & NOTEWORTHY Patients with irritable bowel syndrome (IBS) show altered amygdala activity. We showed previously that stress induces visceral hypersensitivity partially through microglia-modulated synaptic plasticity in the central nucleus of the amygdala (CeA). Our current data suggest that the C1q/C3-CR3 cascade initiates microglia-mediated synaptic remodeling in the CeA. Blocking C3-CR3 interaction attenuates stress-induced visceral hypersensitivity. These findings uncover a role of microglia-synapse signaling in the brain-gut regulation and support a future therapeutic target to treat visceral pain.


Assuntos
Tonsila do Cerebelo/metabolismo , Complemento C1q/metabolismo , Complemento C3/metabolismo , Microglia/metabolismo , Plasticidade Neuronal/fisiologia , Dor Visceral/metabolismo , Animais , Colo/metabolismo , Corticosterona/sangue , Modelos Animais de Doenças , Feminino , Síndrome do Intestino Irritável/metabolismo , Masculino , Percepção da Dor/fisiologia , Ratos , Ratos Endogâmicos F344
14.
Am J Physiol Renal Physiol ; 320(6): F1174-F1190, 2021 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-33998295

RESUMO

Aberrant complement activation leads to tissue damage during kidney transplantation, and it is recognized as an important target for therapeutic intervention. However, it is not clear whether cold storage (CS) triggers the complement pathway in transplanted kidneys. The goal of the present study was to determine the impact of CS on complement activation in renal transplants. Male Lewis and Fischer rats were used, and donor rat kidneys were exposed to 4 h or 18 h of CS followed by transplantation (CS + transplant). To study CS-induced effects, a group with no CS was included in which the kidney was removed and transplanted back to the same rat [autotransplantation (ATx)]. Complement proteins (C3 and C5b-9) were evaluated with Western blot analysis (reducing and nonreducing conditions) and immunostaining. Western blot analysis of renal extracts or serum indicated that the levels of C3 and C5b-9 increased after CS + transplant compared with ATx. Quite strikingly, intracellular C3 was profoundly elevated within renal tubules after CS + transplant but was absent in sham or ATx groups, which showed only extratubular C3. Similarly, C5b-9 immunofluorescence staining of renal sections showed an increase in C5b-9 deposits in kidneys after CS + transplant. Real-time PCR (SYBR green) showed increased expression of CD11b and CD11c, components of complement receptors 3 and 4, respectively, as well as inflammatory markers such as TNF-α. In addition, recombinant TNF-α significantly increased C3 levels in renal cells. Collectively, these results demonstrate that CS mediates aberrant activation of the complement system in renal grafts following transplantation.NEW & NOTEWORTHY This study highlights cold storage-mediated aberrant activation of complement components in renal allografts following transplantation. Specifically, the results demonstrate, for the first time, that cold storage functions in exacerbation of C5b-9, a terminal cytolytic membrane attack complex, in renal grafts following transplantation. In addition, the results indicated that cold storage induces local C3 biogenesis in renal proximal cells/tubules and that TNF-α promotes C3 biogenesis and activation in renal proximal tubular cells.


Assuntos
Proteínas do Sistema Complemento/metabolismo , Rim/metabolismo , Animais , Linhagem Celular , Temperatura Baixa , Ativação do Complemento , Proteínas do Sistema Complemento/genética , Citocinas/administração & dosagem , Citocinas/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Transplante de Rim , Túbulos Renais Proximais/citologia , Masculino , Ratos , Ratos Endogâmicos F344 , Ratos Endogâmicos Lew
15.
Biomed Pharmacother ; 139: 111627, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-33965728

RESUMO

Lipids excess from an uterine environment can increase free radicals production of and thus induce oxidative status imbalance, a key factor for progression of non-alcoholic fatty liver disease (NAFLD) in offspring. Food antioxidant components in maternal diet may play an important role in preventing offspring metabolic disorders. The objective of the study was to evaluate the effects of açaí pulp supplementation on maternal high-fat diet, by assessing activity and expression of antioxidant enzymes and biomarkers of oxidative stress in the liver. Female Fisher rats were divided into four groups and fed a control diet (C), a high-fat diet (HF), a control diet supplemented with açaí (CA) and a high-fat diet supplemented with açaí (HFA) before mating, during gestation and lactation. The effects of açaí supplementation on oxidative stress biomarkers and antioxidant enzymes expression were evaluated in dams and male offspring after weaning. HFA diet increased body weight in dams, however reduced absolute and relative liver weight. There was a reduction in liver biomarkers of oxidative stress, malondialdehyde and carbonyl protein, as well as in catalase, glutathione peroxidase and superoxide dismutase activity. In offspring, HFA diet reduced liver weight and increased Gpx1, Gpx4 and Sod1 mRNA expression. These results suggest that açaí is able to restore redox status, preventing oxidative damage in dams by a direct mechanism and to promote beneficial effects on expression of antioxidant defences related genes in offspring.


Assuntos
Antioxidantes/metabolismo , Euterpe/química , Fígado/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Animais , Biomarcadores/metabolismo , Peso Corporal/efeitos dos fármacos , Dieta Hiperlipídica , Suplementos Nutricionais , Feminino , Expressão Gênica/efeitos dos fármacos , Glutationa Peroxidase/metabolismo , Lactação , Fígado/efeitos dos fármacos , Fígado/enzimologia , Camundongos , Hepatopatia Gordurosa não Alcoólica , Tamanho do Órgão/efeitos dos fármacos , Fosfolipídeo Hidroperóxido Glutationa Peroxidase/metabolismo , Gravidez , Ratos , Ratos Endogâmicos F344 , Superóxido Dismutase-1/metabolismo
16.
Toxicol Pathol ; 49(5): 1100-1108, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-33942680

RESUMO

The tolerability of single daily gavage doses of 0.5% or 2.0% (wt/vol) sodium lauryl sulfate (SLS) in 11- to 12-week-old male CD-1 mice was evaluated in a study of 3 months in duration. Live-phase, gross necropsy, and histopathologic parameters were evaluated. Mortality of 14% occurred in mice administered formulations containing SLS. Clinical observations in mice administered SLS included abnormal respiration (audible, irregular, and/or labored), swollen abdomen, rough haircoat, hunched appearance, and hypoactivity. Necropsy findings in mice administered SLS consisted of enlarged intestines containing abnormal contents with gas. There were no instances of mechanical gavage-related injury. Histologic evaluation of the respiratory tract revealed injury to the nasal passages and nasopharynx, including, but not limited to, inflammation, exudate, apoptosis/necrosis of epithelium, and atrophy of epithelium or olfactory nerves. Collectively, the data indicated that under the experimental conditions of our 3-month study in male CD-1 mice, once-daily gavage administration of vehicle formulations containing SLS at 0.5% or 2.0% resulted in nasal injury and 14% mortality supportive of gastroesophageal reflux. Sponsors utilizing formulations containing SLS in toxicity studies in CD-1 mice should exclude gastroesophageal reflux as a confounding factor in studies with morbidity or mortality associated with respiratory distress or evidence of aerophagia.


Assuntos
Testes de Carcinogenicidade , Administração Oral , Animais , Masculino , Camundongos , Camundongos Endogâmicos , Ratos , Ratos Endogâmicos F344 , Dodecilsulfato de Sódio/toxicidade
17.
Toxicol Sci ; 182(1): 10-28, 2021 07 16.
Artigo em Inglês | MEDLINE | ID: mdl-33944952

RESUMO

The tobacco-specific nitrosamine NNK [4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone] is found in tobacco products and tobacco smoke. NNK is a potent genotoxin and human lung carcinogen; however, there are limited inhalation data for the toxicokinetics (TK) and genotoxicity of NNK in vivo. In the present study, a single dose of 5 × 10-5, 5 × 10-3, 0.1, or 50 mg/kg body weight (BW) of NNK, 75% propylene glycol (vehicle control), or air (sham control) was administered to male Sprague-Dawley (SD) rats (9-10 weeks age) via nose-only inhalation (INH) exposure for 1 h. For comparison, the same doses of NNK were administered to male SD rats via intraperitoneal injection (IP) and oral gavage (PO). Plasma, urine, and tissue specimens were collected at designated time points and analyzed for levels of NNK and its major metabolite 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) and tissue levels of DNA adduct O6-methylguanine by LC/MS/MS. TK data analysis was performed using a non-linear regression program. For the genotoxicity subgroup, tissues were collected at 3 h post-dosing for comet assay analysis. Overall, the TK data indicated that NNK was rapidly absorbed and metabolized extensively to NNAL after NNK administration via the three routes. The IP route had the greatest systemic exposure to NNK. NNK metabolism to NNAL appeared to be more efficient via INH than IP or PO. NNK induced significant increases in DNA damage in multiple tissues via the three routes. The results of this study provide new information and understanding of the TK and genotoxicity of NNK.


Assuntos
Nitrosaminas , Espectrometria de Massas em Tandem , Animais , Carcinógenos , Cromatografia Líquida de Alta Pressão , Dano ao DNA , Exposição por Inalação , Injeções Intraperitoneais , Masculino , Nitrosaminas/toxicidade , Ratos , Ratos Endogâmicos F344 , Ratos Sprague-Dawley , Toxicocinética
18.
Neuropsychopharmacology ; 46(10): 1746-1756, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34007041

RESUMO

Repeated nicotine exposure leads to sensitization (SST) and enhances self-administration (SA) in rodents. However, the molecular basis of nicotine SST and SA and their biological relevance to the mounting genome-wide association study (GWAS) loci of human addictive behaviors are poorly understood. Considering a gateway drug role of nicotine, we modeled nicotine SST and SA in F1 progeny of inbred rats (F344/BN) and conducted integrative genomics analyses. We unexpectedly observed male-specific nicotine SST and a parental effect of SA only present in paternal F344 crosses. Transcriptional profiling in the ventral tegmental area (VTA) and nucleus accumbens (NAc) core and shell further revealed sex- and brain region-specific transcriptomic signatures of SST and SA. We found that genes associated with SST and SA were enriched for those related to synaptic processes, myelin sheath, and tobacco use disorder or chemdependency. Interestingly, SST-associated genes were often downregulated in male VTA but upregulated in female VTA, and strongly enriched for smoking GWAS risk variants, possibly explaining the male-specific SST. For SA, we found widespread region-specific allelic imbalance of expression (AIE), of which genes showing AIE bias toward paternal F344 alleles in NAc core were strongly enriched for SA-associated genes and for GWAS risk variants of smoking initiation, likely contributing to the parental effect of SA. Our study suggests a mechanistic link between transcriptional changes underlying the NIC SST and SA and human nicotine addiction, providing a resource for understanding the neurobiology basis of the GWAS findings on human smoking and other addictive phenotypes.


Assuntos
Comportamento Aditivo , Nicotina , Animais , Comportamento Aditivo/genética , Feminino , Estudo de Associação Genômica Ampla , Humanos , Masculino , Fenótipo , Ratos , Ratos Endogâmicos F344
19.
Stem Cell Res Ther ; 12(1): 312, 2021 05 29.
Artigo em Inglês | MEDLINE | ID: mdl-34051870

RESUMO

BACKGROUND: Small hepatocyte-like progenitor cells (SHPCs) appear to form transient clusters in rat livers treated with retrorsine (Ret) and 70% partial hepatectomy (PH). We previously reported that the expansion of SHPCs was amplified in Ret/PH-treated rat livers transplanted with Thy1+ cells derived from D-galactosamine-treated injured livers. Extracellular vesicles (EVs) produced by hepatic Thy1+ donor cells activated SHPCs via interleukin (IL)-17 receptor B signaling. As bone marrow-derived mesenchymal cells (BM-MCs) also express Thy1, we aimed to determine whether BM-MCs could also promote the growth of SHPCs. METHODS: BM-MCs were isolated from dipeptidyl-peptidase IV (DPPIV)-positive rats. BM-MCs or BM-MC-derived EVs were administered to DPPIV-negative Ret/PH rat livers, and the growth and the characteristics of SHPC clusters were evaluated 14 days post-treatment. miRNA microarrays and cytokine arrays examined soluble factors within EVs. Small hepatocytes (SHs) isolated from an adult rat liver were used to identify factors enhancing hepatocytic progenitor cells growth. RESULTS: The recipient's livers were enlarged at 2 weeks post-BM-MC transplantation. The number and the size of SHPCs increased remarkably in livers transplanted with BM-MCs. BM-MC-derived EVs also stimulated SHPC growth. Comprehensive analyses revealed that BM-MC-derived EVs contained miR-146a-5p, interleukin-6, and stem cell factor, which could enhance SHs' proliferation. Administration of EVs derived from the miR-146a-5p-transfected BM-MCs to Ret/PH rat livers remarkably enhanced the expansion of SHPCs. CONCLUSIONS: miR-146a-5p involved in EVs produced by BM-MCs may play a major role in accelerating liver regeneration by activating the intrinsic hepatocytic progenitor cells.


Assuntos
Vesículas Extracelulares , MicroRNAs , Animais , Medula Óssea , Células da Medula Óssea , Hepatócitos , MicroRNAs/genética , Ratos , Ratos Endogâmicos F344
20.
Skelet Muscle ; 11(1): 11, 2021 05 03.
Artigo em Inglês | MEDLINE | ID: mdl-33941271

RESUMO

BACKGROUND: Old age is associated with a significantly increased mortality in COVID-19 patients exposed to long-term controlled mechanical ventilation (CMV) and suggested to be due to the hyperinflammatory response associated with the viral infection. However, our understanding of age-related differences in the response to CMV in the absence of a viral infection remains insufficient. METHODS: Young (7-8 months) and old (28-32 months) F344 BN hybrid rats were exposed to the ICU condition for 5 days, i.e., complete immobilization, mechanical ventilation, and extensive monitoring. Transcriptomic (RNA-Seq) and proteomics (Proximity Extension Assay) analyses of the diaphragm and proteomics analysis of plasma were conducted to investigate the molecular differences between young and old rats exposed to the ICU condition. RESULTS: According to multi-omics analyses, significant differences were observed in the diaphragm between young and old rats in response to 5 days CMV and immobilization. In young rats, metabolic pathways were primarily downregulated in response to immobilization (post-synaptic blockade of neuromuscular transmission). In old rats, on the other hand, dramatic immune and inflammatory responses were observed, i.e., an upregulation of specific related pathways such as "IL-17 signaling pathway", along with a higher level of inflammatory factors and cytokine/chemokine in plasma. CONCLUSIONS: The dramatically increased mortality in old ICU patients with COVID-19-associated hyperinflammation and cytokine storm need not only reflect the viral infection but may also be associated with the ventilator induced diaphragm dysfunction (VIDD) and hyperinflammatory responses induced by long-term CMV per se. Although mechanical ventilation is a life-saving intervention in COVID-19 ICU patients, CMV should be cautiously used especially in old age and other means of respiratory support may be considered, such as negative pressure ventilation.


Assuntos
Diafragma/metabolismo , Mediadores da Inflamação/sangue , Proteoma , Respiração Artificial , Transcriptoma , Fatores Etários , Animais , Biomarcadores/sangue , Feminino , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Redes Reguladoras de Genes , Projetos Piloto , Mapas de Interação de Proteínas , Proteômica , Ratos Endogâmicos BN , Ratos Endogâmicos F344 , Transdução de Sinais
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