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Adipose-derived stem cells (ADSCs) have incredible potential as an avenue to better understand and treat neurological disorders. While they have been successfully differentiated into neural stem cells and neurons, most such protocols involve 2D environments, which are not representative of in vivo physiology. In this study, human ADSCs were cultured in 1.1 kPa polyethylene-glycol 3D hydrogels for 10 days with B27, CultureOne (C1), and N2 neural supplements to examine the neural differentiation potential of ADSCs using both chemical and mechanical cues. Following treatment, cell viability, proliferation, morphology, and proteome changes were assessed. Results showed that cell viability was maintained during treatments, and while cells continued to proliferate over time, proliferation slowed down. Morphological changes between 3D untreated cells and treated cells were not observed. However, they were observed among 2D treatments, which exhibited cellular elongation and co-alignment. Proteome analysis showed changes consistent with early neural differentiation for B27 and C1 but not N2. No significant changes were detected using immunocytochemistry, potentially indicating a greater differentiation period was required. In conclusion, treatment of 3D-cultured ADSCs in PEG-based hydrogels with B27 and C1 further enhances neural marker expression, however, this was not observed using supplementation with N2.
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Células-Tronco Neurais , Proteoma , Humanos , Tecido Adiposo , Células Cultivadas , Diferenciação Celular/fisiologia , Materiais Biocompatíveis , Hidrogéis/farmacologiaRESUMO
Neurological diseases are among the leading causes of disability and death worldwide and remain difficult to treat. Tissue engineering offers avenues to test potential treatments; however, the development of biologically accurate models of brain tissues remains challenging. Given their neurogenic potential and availability, adipose-derived stem cells (ADSCs) are of interest for creating neural models. While progress has been made in differentiating ADSCs into neural cells, their differentiation in 3D environments, which are more representative of the in vivo physiological conditions of the nervous system, is crucial. This can be achieved by modulating the 3D matrix composition and stiffness. Human ADSCs were cultured for 14 days in a 1.1 kPa polyethylene glycol-based 3D hydrogel matrix to assess effects on cell morphology, cell viability, proteome changes and spontaneous neural differentiation. Results showed that cells continued to proliferate over the 14-day period and presented a different morphology to 2D cultures, with the cells elongating and aligning with one another. The proteome analysis revealed 439 proteins changed in abundance by >1.5 fold. Cyclic nucleotide 3'-phosphodiesterase (CNPase) markers were identified using immunocytochemistry and confirmed with proteomics. Findings indicate that ADSCs spontaneously increase neural marker expression when grown in an environment with similar mechanical properties to the central nervous system.
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Hidrogéis , Proteoma , Humanos , Células Cultivadas , Hidrogéis/farmacologia , Tecido Adiposo , Diferenciação Celular , Materiais Biocompatíveis , Células-Tronco , Sistema NervosoRESUMO
Previous evidence shows that rapid changes occur in the brain following spinal cord injury (SCI). Here, we interrogated the expression of the neuropeptides pituitary adenylyl cyclase-activating peptide (PACAP), vasoactive intestinal peptides (VIP), and their binding receptors in the rat brain 24 h following SCI. Female Sprague-Dawley rats underwent thoracic laminectomy; half of the rats received a mild contusion injury at the level of the T10 vertebrate (SCI group); the other half underwent sham surgery (sham group). Twenty-four hours post-surgery, the hypothalamus, thalamus, amygdala, hippocampus (dorsal and ventral), prefrontal cortex, and periaqueductal gray were collected. PACAP, VIP, PAC1, VPAC1, and VPAC2 mRNA and protein levels were measured by real-time quantitative polymerase chain reaction and Western blot. In SCI rats, PACAP expression was increased in the hypothalamus (104-141% vs sham) and amygdala (138-350%), but downregulated in the thalamus (35-95%) and periaqueductal gray (58-68%). VIP expression was increased only in the thalamus (175-385%), with a reduction in the amygdala (51-68%), hippocampus (40-75%), and periaqueductal gray (74-76%). The expression of the PAC1 receptor was the least disturbed by SCI, with decrease expression in the ventral hippocampus (63-68%) only. The expression levels of VPAC1 and VPAC2 receptors were globally reduced, with more prominent reductions of VPAC1 vs VPAC2 in the amygdala (21-70%) and ventral hippocampus (72-75%). In addition, VPAC1 downregulation also extended to the dorsal hippocampus (69-70%). These findings demonstrate that as early as 24 h post-SCI, there are region-specific disruptions of PACAP, VIP, and related receptor transcript and protein levels in supraspinal regions controlling higher cognitive functions.
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Receptores do Hormônio Hipofisário , Traumatismos da Medula Espinal , Feminino , Ratos , Animais , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/genética , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/metabolismo , Receptores de Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/metabolismo , Ratos Sprague-Dawley , Receptores do Hormônio Hipofisário/genética , Receptores do Hormônio Hipofisário/metabolismo , Peptídeo Intestinal Vasoativo/genética , Peptídeo Intestinal Vasoativo/metabolismo , Receptores Tipo I de Polipeptídeo Intestinal Vasoativo/genética , Receptores Tipo I de Polipeptídeo Intestinal Vasoativo/metabolismo , Receptores Tipo II de Peptídeo Intestinal Vasoativo/genética , Receptores Tipo II de Peptídeo Intestinal Vasoativo/metabolismo , Receptores de Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/genética , Receptores de Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/metabolismo , Traumatismos da Medula Espinal/metabolismo , Encéfalo/metabolismoRESUMO
Background: Adipose-derived stem cells (ADSCs) are increasingly utilised in the field of neural regeneration due to their high accessibility and capacity for differentiation into neural like cells. Culturing ADSCs in the presence of various growth factors, small molecules and combinations thereof have shown promise in this regard; however, these protocols are generally complex, time-consuming and costly. The need for commercially available and chemically defined growth media/supplements is required to facilitate further developments in this area. Methods: In this study, we have examined the neural differentiation and proliferation potential of the commercially available supplements B27, CultureOne (C1) and N2 on human ADSCs (hADSCs). Through a combination of immunocytochemistry, cytokine analysis, and CNPase enzymatic assays, we provide novel insight into the neural differentiation effects of B27, C1 and N2 on hADSCs. Results: The study found that C1 and N2 supplements initiated neural differentiation of the cells, with C1 pushing differentiation towards an oligodendrocytic lineage and N2 initiating neuronal differentiation. This suggests that C1 and N2 supplements can be used to drive neural differentiation in hADSCs. However, B27 did not show significant differentiation in the time frame in which the experiments took place and therefore is unsuitable for this purpose. Conclusions: These findings highlight the utility of commercially available supplements in the neural differentiation of ADSCs and may assist in establishing simpler, more affordable differentiation protocols.
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Cardiovascular disease (CVD) is a leading cause of mortality worldwide, with cigarette smoking being a major preventable risk factor. Smoking cessation can be difficult due to the addictive nature of nicotine and the withdrawal symptoms following cessation. Electronic cigarettes (e-Cigs) have emerged as an alternative smoking cessation device, which has been increasingly used by non-smokers; however, the cardiovascular effects surrounding the use of e-Cigs remains unclear. This study aimed to investigate the effects of e-Cig aerosol condensate (EAC) (0 mg and 18 mg nicotine) in vitro on human coronary artery endothelial cells (HCAEC) and in vivo on the cardiovascular system using a mouse model of 'e-vaping'. In vitro results show a decrease in cell viability of HCAEC when exposed to EAC either directly or after exposure to conditioned lung cell media (p < 0.05 vs. control). Reactive oxygen species were increased in HCAEC when exposed to EAC directly or after exposure to conditioned lung cell media (p < 0.0001 vs. control). ICAM-1 protein expression levels were increased after exposure to conditioned lung cell media (18 mg vs. control, p < 0.01). Ex vivo results show an increase in the mRNA levels of anti-angiogenic marker, FKBPL (p < 0.05 vs. sham), and endothelial cell adhesion molecule involved in barrier function, ICAM-1 (p < 0.05 vs. sham) in murine hearts following exposure to electronic cigarette aerosol treatment containing a higher amount of nicotine. Immunohistochemistry also revealed an upregulation of FKBPL and ICAM-1 protein expression levels. This study showed that despite e-Cigs being widely used for tobacco smoking cessation, these can negatively impact endothelial cell health with a potential to lead to the development of cardiovascular disease.
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Doenças Cardiovasculares , Sistemas Eletrônicos de Liberação de Nicotina , Animais , Camundongos , Humanos , Nicotina/efeitos adversos , Molécula 1 de Adesão Intercelular , Células Endoteliais , Doenças Cardiovasculares/etiologia , Aerossóis , Proteínas de Ligação a TacrolimoRESUMO
Preeclampsia is a pregnancy-specific cardiovascular disorder, involving significant maternal endothelial dysfunction. Although inappropriate placentation due to aberrant angiogenesis, inflammation and shallow trophoblast invasion are the root causes of preeclampsia, pathogenic mechanisms are poorly understood, particularly in early pregnancy. Here, we first confirm the abnormal expression of important vascular and inflammatory proteins, FK506-binding protein-like (FKBPL) and galectin-3 (Gal-3), in human plasma and placental tissues from women with preeclampsia and normotensive controls. We then employ a three-dimensional microfluidic placental model incorporating human umbilical vein endothelial cells (HUVECs) and a first trimester trophoblast cell line (ACH-3P) to investigate FKBPL and Gal-3 signaling in inflammatory conditions. In human samples, both circulating (n = 17 controls; n = 30 preeclampsia) and placental (n ≥ 6) FKBPL and Gal-3 levels were increased in preeclampsia compared to controls (plasma: FKBPL, p < 0.0001; Gal-3, p < 0.01; placenta: FKBPL, p < 0.05; Gal-3, p < 0.01), indicative of vascular dysfunction in preeclampsia. In our placenta-on-a-chip model, we show that endothelial cells are critical for trophoblast-mediated migration and that trophoblasts effectively remodel endothelial vascular networks. Inflammatory cytokine tumour necrosis factor-α (10 ng/mL) modulates both FKBPL and Gal-3 signaling in conjunction with trophoblast migration and impairs vascular network formation (p < 0.005). Our placenta-on-a-chip recapitulates aspects of inappropriate placental development and vascular dysfunction in preeclampsia.
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Placenta , Pré-Eclâmpsia , Gravidez , Feminino , Humanos , Placenta/metabolismo , Galectina 3/genética , Galectina 3/metabolismo , Trofoblastos/metabolismo , Células Endoteliais da Veia Umbilical Humana/metabolismo , Proteínas de Ciclo Celular/metabolismo , Dispositivos Lab-On-A-Chip , Proteínas de Ligação a Tacrolimo/metabolismoRESUMO
Compression spinal cord injuries are a common cause of morbidity in people who experience a spinal cord injury (SCI). Either as a by-product of a traumatic injury or due to nontraumatic conditions such as cervical myelitis, compression injuries are growing in prevalence clinically and many attempts of animal replication have been described within the literature. These models, however, often focus on the traumatic side of injury or mimic short-term injuries that are not representative of the majority of compression SCI. Of this, nontraumatic spinal cord injuries are severely understudied and have an increased prevalence in elderly populations, adults, and children. Therefore, there is a need to critically evaluate the current animal models of compression SCI and their suitability as a method for clinically relevant data that can help reduce morbidity and mortality of SCI. In this review, we reviewed the established and emerging methods of animal models of compression SCI. These models are the clip, balloon, solid spacer, expanding polymer, remote, weight drop, calibrated forceps, screw, and strap methods. These methods showed that there is a large reliance on the use of laminectomy to induce injury. Furthermore, the age range of many studies does not reflect the elderly and young populations that commonly suffer from compression injuries. It is therefore important to have techniques and methods that are able to minimize secondary effects of the surgeries, and are representative of the clinical cases seen so that treatments and interventions can be developed that are specific.
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Compressão da Medula Espinal , Traumatismos da Medula Espinal , Animais , Modelos Animais de Doenças , Compressão da Medula Espinal/complicações , Traumatismos da Medula Espinal/complicações , PolímerosRESUMO
Traumatic brain injury (TBI) and obesity are two common conditions in modern society; both can impair neuronal integrity and neurological function. However, it is unclear whether the coexistence of both conditions will worsen outcomes. Therefore, in a rat model, we aimed to investigate whether the coexistence of TBI and a high-fat diet (HFD) has an additive effect, leading to more severe neurological impairments, and whether they are related to changes in brain protein markers of oxidative stress, inflammation, and synaptic plasticity. Sprague-Dawley rats (female, â¼250 g) were divided into HFD (43% fat) and diet (CD) (17% fat) groups for 6 weeks. Within each dietary group, half underwent a TBI by a weight-drop device, and the other half underwent sham surgery. Short-term memory and sensory function were measured at 24 h, 1 week, 3 weeks, and 6 weeks post-TBI. Brain tissues were harvested at 24 h and 6 weeks post-TBI, and markers of oxidative stress, apoptosis, inflammation, and synaptic plasticity were measured via immunostaining and Western blotting. In rats without TBI, HFD increased the pre-synaptic protein synaptophysin. In rats with TBI, HFD resulted in worsened sensory and memory function, an increase in activated macrophages, and a decrease in the endogenous antioxidant manganese superoxide dismutase (MnSOD). Our findings suggest that the additive effect of HFD and TBI worsens short term memory and sensation deficits, and may be driven by enhanced oxidative stress and inflammation.
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Lesões Encefálicas Traumáticas , Animais , Ratos , Feminino , Ratos Sprague-Dawley , Lesões Encefálicas Traumáticas/complicações , Lesões Encefálicas Traumáticas/metabolismo , Encéfalo/metabolismo , Estresse Oxidativo , Inflamação/metabolismo , Biomarcadores/metabolismoRESUMO
Evidence suggests that rapid changes to supporting glia may predispose individuals with spinal cord injury (SCI) to such comorbidities. Here, we interrogated the expression of astrocyte- and microglial-specific markers glial fibrillary acidic protein (GFAP) and ionized calcium binding adaptor molecule 1 (Iba1) in the rat brain in the first 24 hours following SCI. Female Sprague-Dawley rats underwent thoracic laminectomy; half of the rats received a mild contusion injury at the level of the T10 vertebral body (SCI group), the other half did not (Sham group). Twenty-four hours post-surgery the amygdala, periaqueductal grey, prefrontal cortex, hypothalamus, lateral thalamus, hippocampus (dorsal and ventral) in rats were collected. GFAP and Iba1 mRNA and protein levels were measured by real-time quantitative polymerase chain reaction and Western blot. In SCI rats, GFAP mRNA and protein expression increased in the amygdala and hypothalamus. In contrast, gene and protein expression decreased in the thalamus and dorsal hippocampus. Interestingly, Iba1 transcripts and proteins were significantly diminished only in the dorsal and ventral hippocampus, where gene expression diminished. These findings demonstrate that as early as 24 hours post-SCI there are region-specific disruptions of GFAP and Iba1 transcript and protein levels in higher brain regions. All procedures were approved by the University of Technology Sydney Institutional Animal Care and Ethics Committee (UTS ACEC13-0069).
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It has been reported that children may respond better than adults to a spinal cord injury (SCI) of similar severity. There are known biomechanical differences in the developing spinal cord that may contribute to this "infant lesion effect," but the underlying mechanisms are unknown. Using immunohistochemistry, we have previously demonstrated a different injury progression and immune cell response after a mild thoracic contusion SCI in infant rats, as compared to adult rats. Here, we investigated the acute inflammatory responses using flow cytometry and ELISA at 1 h, 24 h, and 1 week after SCI in neonatal (P7) and adult (9 weeks) rats, and locomotor recovery was examined for 6 weeks after injury. Adult rats exhibited a pronounced pro-inflammatory response characterized by neutrophils and M1-like macrophage infiltration and Th1 cytokine secretion. Neonatal rats exhibited a decreased pro-inflammatory response characterized by a higher proportion of M2-like macrophages and reduced Th1 cytokine responses, as compared to adults. These results suggest that the initial inflammatory response to SCI is predominantly anti-inflammatory in very young animals.
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Traumatismos da Medula Espinal , Animais , Animais Recém-Nascidos , Anti-Inflamatórios/uso terapêutico , Macrófagos , Ratos , Recuperação de Função Fisiológica , Medula Espinal , Traumatismos da Medula Espinal/tratamento farmacológicoRESUMO
Research has attributed tissue damage post-traumatic brain injury (TBI) to two-pronged effects, increased reactive oxygen species (ROS) and impairment of endogenous antioxidant defence systems, underpinned by manganese superoxide dismutase (MnSOD). Novel antioxidant nitroxides have been shown to mimic MnSOD to ameliorate oxidative stress related disorders. This study aimed to investigate the effects of two nitroxides, CTMIO and DCTEIO, on the neurological outcomes following moderate TBI in rats induced by a weight drop device. The rats were immediately treated with CTMIO and DCTEIO (40 mM in drinking water) post-injury for up to 2 weeks. The brains were histologically examined at 24 h and 6 weeks post injury. DCTEIO reduced the lesion size at both 24h and 6 weeks, with normalised performance in sensory, motor and cognitive tests at 24h post-injury. Astrogliosis was heightened by DCTEIO at 24h and still elevated at 6 weeks in this group. In TBI brains, cellular damage was evident as reflected by changes in markers of mitophagy and autophagy (increased fission marker dynamin-related protein (Drp)-1, and autophagy marker light chain 3 (LC3)A/B and reduced fusion marker optic atrophy (Opa)-1). These were normalised by DCTEIO treatment. CTMIO, on the other hand, seems to be toxic to the injured brains, by increasing injury size at 6 weeks. In conclusion, DCTEIO significantly improved tissue repair and preserved neurological function in rats with TBI possibly via a mitophagy mechanism. This study provides evidence for DCTEIO as a promising new option to alleviate lesion severity after moderate TBI, which is not actively treated.
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Antioxidantes/farmacologia , Lesões Encefálicas Traumáticas/tratamento farmacológico , Encéfalo/efeitos dos fármacos , Modelos Animais de Doenças , Transtornos Neurocognitivos/tratamento farmacológico , Óxidos de Nitrogênio/farmacologia , Animais , Antioxidantes/química , Encéfalo/metabolismo , Encéfalo/patologia , Lesões Encefálicas Traumáticas/metabolismo , Lesões Encefálicas Traumáticas/patologia , Feminino , Estrutura Molecular , Transtornos Neurocognitivos/metabolismo , Transtornos Neurocognitivos/patologia , Óxidos de Nitrogênio/química , Ratos , Ratos Sprague-DawleyRESUMO
Maternal smoking is currently a public health concern and has been associated with a number of complications in the offspring. E-cigarettes are gaining popularity as a "safer" alternative to tobacco cigarettes during pregnancy, however, there are a limited number of studies to suggest that it is actually "safe." Balb/C female mice were exposed to ambient air (n = 8; Sham), or tobacco cigarette smoke (n = 8; SE) before gestation, during gestation and lactation. A third group was exposed to cigarette smoke before gestation followed by e-cigarette aerosols during gestation and lactation (n = 8; Switch). Male offspring (12-week old, n = 10-14/group) underwent behavioral assessments to investigate short-term memory, anxiety, and activity using the novel object recognition and elevated plus maze tests. Brains were collected at postnatal day (P)1, P20, and Week 13 for global DNA methylation, epigenetic gene expression, and neuronal cell counts. The offspring from mothers switching to e-cigarettes exhibited no change in exploration/activity but showed a decrease in global DNA methylation, Aurora Kinase (Aurk) A and AurkB gene expression and a reduction in neuronal cell numbers in the cornu ammonis 1 region of the dorsal hippocampus compared with the SE group. Continuous tobacco cigarette smoke exposure during pregnancy resulted in marked neurological deficits in the offspring. Switching to e-cigarettes during pregnancy reduced these neurological deficits compared with cigarette smoke exposure. However, neurological changes were still observed, so we therefore conclude that e-cigarette use during pregnancy is not advised.
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Neural stem cells have become the focus of many studies as they have the potential to differentiate into all three neural lineages. This may be utilised to develop new and novel ways to treat neurological conditions such as spinal cord and brain injuries, especially if the stem cells can be modulated in vivo without additional invasive surgical procedures. This research is aimed at investigating the effects of the growth factors vascular endothelial growth factor, platelet-derived growth factor, brain-derived neurotrophic factor, and vascular endothelial growth factor/platelet-derived growth factor on hippocampal-derived neural stem cells. Cell growth and differentiation were assessed using immunohistochemistry and glutaminase enzyme assay. Cells were cultured for 14 days and treated with different growth factors at two different concentrations 20 ng/mL and 100 ng/mL. At 2 weeks, cells were fixed, and immunohistochemistry was conducted to determine cellular differentiation using antibodies against GFAP, nestin, OSP, and NF200. The cell medium supernatant was also collected during treatment to determine glutaminase levels secreted by the cells as an indicator of neural differentiation. VEGF/PDGF at 100 ng/mL had the greatest influence on cellular proliferation of HNSC, which also stained positively for nestin, OSP, and NF200. In comparison, HNSC in other treatments had poorer cell health and adhesion. HNSC in all treatment groups displayed some differentiation markers and morphology, but this is most significant in the 100 ng/ml VEGF/PDGF treatment. VEGF/PDGF combination produced the optimal effect on the HNSCs inducing the differentiation pathway exhibiting oligodendrocytic and neuronal markers. This is a promising finding that should be further investigated in the brain and spinal cord injury.
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There is a need for pharmaceutical agents that can reduce neuronal loss and improve functional deficits following traumatic brain injury (TBI). Previous research suggests that oxidative stress and mitochondrial dysfunction play a major role in neuronal damage after TBI. Therefore, this study aimed to investigate two drugs known to have antioxidant effects, L-carnitine and exendin-4, in rats with moderate contusive TBI. L-carnitine (1.5 mM in drinking water) or exendin-4 (15 µg/kg/day, ip) were given immediately after the injury for 2 weeks. Neurological function and brain histology were examined (24 h and 6 weeks post injury). The rats with TBI showed slight sensory, motor and memory functional deficits at 24 h, but recovered by 6 weeks. Both treatments improved sensory and motor functions at 24 h, while only exendin-4 improved memory. Both treatments reduced cortical contusion at 24 h and 6 weeks, however neither affected gliosis and inflammatory cell activation. Oxidative stress was alleviated and mitochondrial reactive oxygen species was reduced by both treatments, however only mitochondrial functional marker protein transporter translocase of outer membrane 20 was increased at 24 h post injury. In conclusion, L-carnitine and exendin-4 treatments immediately after TBI can improve neurological functional outcome and tissue integrity by reducing oxidative stress.
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Contusão Encefálica/tratamento farmacológico , Lesões Encefálicas Traumáticas/tratamento farmacológico , Carnitina/administração & dosagem , Exenatida/administração & dosagem , Estresse Oxidativo/efeitos dos fármacos , Animais , Antioxidantes/administração & dosagem , Encéfalo/efeitos dos fármacos , Encéfalo/fisiopatologia , Contusão Encefálica/metabolismo , Contusão Encefálica/fisiopatologia , Lesões Encefálicas Traumáticas/metabolismo , Lesões Encefálicas Traumáticas/fisiopatologia , Modelos Animais de Doenças , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/genética , Neurônios/efeitos dos fármacos , Neurônios/patologia , Ratos , Ratos Sprague-Dawley , Espécies Reativas de Oxigênio/metabolismoRESUMO
Electronic cigarette (e-cigarette) use is on the rise worldwide and is particularly attractive to young people and as a smoking substitute by pregnant woman. There is a perception in pregnant women and women of child-bearing age that the use of e-cigarettes (vaping) is safer than smoking tobacco cigarettes during pregnancy. However, there is little evidence to support this perception. Here, we examined the offspring from mouse dams that had been exposed during and after pregnancy to ambient air (sham) ( n = 8), e-cigarette aerosols with nicotine ( n = 8), or e-cigarette aerosols without nicotine ( n = 8). Offspring underwent cognitive testing at 12 weeks of age and epigenetic testing of brain tissues at 1 day, 20 days, and 13 weeks after birth. The findings showed deficits in short-term memory, reduced anxiety, and hyperactivity in offspring following maternal e-cigarette exposure using the novel object recognition and elevated plus maze tests. In addition, global DNA methylation was increased in the brains of offspring soon after birth. Using a quantitative-PCR array specific to chromatin modification enzymes on genomic DNA and histones,13 key genes were identified to be significantly altered in the offspring brains from the e-cigarette groups compared to the nonexposed groups. The changes to genes Aurka, Aurkb, Aurkc, Kdm5c, Kdm6b, Dnmt3a, Dnmt3b, and Atf2, all associated with modulating neurological activity, were validated using RT-qPCR. In conclusion, in a mouse model, maternal exposure to e-cigarette aerosols resulted in both cognitive and epigenetic changes in offspring. This suggests that the use of e-cigarettes during pregnancy may have hitherto undetected neurological consequences on newborns.
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Cognição , Sistemas Eletrônicos de Liberação de Nicotina , Epigênese Genética , Exposição Materna , Aerossóis/química , Animais , Comportamento Animal/efeitos dos fármacos , Encéfalo/metabolismo , Cognição/efeitos dos fármacos , DNA (Citosina-5-)-Metiltransferases/genética , DNA (Citosina-5-)-Metiltransferases/metabolismo , Metilação de DNA/efeitos dos fármacos , Modelos Animais de Doenças , Epigênese Genética/efeitos dos fármacos , Feminino , Camundongos , Camundongos Endogâmicos BALB C , Nicotina/toxicidade , Oxirredutases N-Desmetilantes/genética , Oxirredutases N-Desmetilantes/metabolismo , Gravidez , Efeitos Tardios da Exposição Pré-NatalRESUMO
Connexin43 (Cx43) hemichannels in spinal cord astrocytes are implicated in the maintenance of neuropathic pain following peripheral nerve injury. Peptide5 is a Cx43 mimetic peptide that blocks hemichannels. In this study, we investigated the effects of spinal delivery of Peptide5 on mechanical pain hypersensitivity in two mouse models of neuropathic pain, peripheral nerve injury and chemotherapy-induced peripheral neuropathy (CIPN). We demonstrated that 10days following a chronic constriction injury (CCI) of the sciatic nerve, Cx43 expression, co-localised predominantly with astrocytes, was increased in the ipsilateral L3-L5 lumbar spinal cord. An intrathecal injection of Peptide5 into nerve-injured mice, on day 10 when pain was well-established, caused significant improvement in mechanical pain hypersensitivity 8h after injection. Peptide5 treatment resulted in significantly reduced Cx43, and microglial and astrocyte activity in the dorsal horn of the spinal cord, as compared to control saline-treated CCI mice. Further in vitro investigations on primary astrocyte cultures showed that 1h pre-treatment with Peptide5 significantly reduced adenosine triphosphate (ATP) release in response to extracellular calcium depletion. Since ATP is a known activator of the NOD-like receptor protein 3 (NLRP3) inflammasome complex, a key mediator of neuroinflammation, we examined the effects of Peptide5 treatment on NLRP3 inflammasome expression. We found that NLRP3, its adaptor apoptosis-associated spec-like protein (ASC) and caspase-1 protein were increased in the ipsilateral spinal cord of CCI mice and reduced to naïve levels following Peptide5 treatment. In the models of oxaliplatin- and paclitaxel-induced peripheral neuropathy, treatment with Peptide5 had no effect on mechanical pain hypersensitivity. Interestingly, in these CIPN models, although spinal Cx43 expression was significantly increased at day 13 following chemotherapy, NLRP3 expression was not altered. These results suggest that the analgesic effect of Peptide5 is specifically achieved by reducing NLRP3 expression. Together, our findings demonstrate that blocking Cx43 hemichannels with Peptide5 after nerve injury attenuates mechanical pain hypersensitivity by specifically targeting the NLRP3 inflammasome in the spinal cord.
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Materiais Biomiméticos/administração & dosagem , Conexina 43/administração & dosagem , Hiperalgesia/tratamento farmacológico , Proteína 3 que Contém Domínio de Pirina da Família NLR/antagonistas & inibidores , Neuralgia/tratamento farmacológico , Fragmentos de Peptídeos/administração & dosagem , Animais , Hiperalgesia/metabolismo , Inflamassomos/antagonistas & inibidores , Inflamassomos/metabolismo , Injeções Espinhais , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Neuralgia/metabolismo , Resultado do TratamentoRESUMO
Traumatic spinal cord injury (SCI) is a detrimental condition that causes loss of sensory and motor function in an individual. Many complex secondary injury cascades occur after SCI and they offer great potential for therapeutic targeting. In this study, we investigated the response of endogenous neural progenitor cells, astrocytes, and microglia to a localized thoracic SCI throughout the neuroaxis. Twenty-five adult female Sprague-Dawley rats underwent mild-contusion thoracic SCI (n = 9), sham surgery (n = 8), or no surgery (n = 8). Spinal cord and brain tissues were fixed and cut at six regions of the neuroaxis. Immunohistochemistry showed increased reactivity of neural progenitor cell marker nestin in the central canal at all levels of the spinal cord. Increased reactivity of astrocyte-specific marker glial fibrillary acidic protein was found only at the lesion epicenter. The number of activated microglia was significantly increased at the lesion site, and activated microglia extended to the lumbar enlargement. Phagocytic microglia and macrophages were significantly increased only at the lesion site. There were no changes in nestin, glial fibrillary acidic protein, microglia and macrophage response in the third ventricle of rats subjected to mild-contusion thoracic SCI compared to the sham surgery or no surgery. These findings indicate that neural progenitor cells, astrocytes and microglia respond differently to a localized SCI, presumably due to differences in inflammatory signaling. These different cellular responses may have implications in the way that neural progenitor cells can be manipulated for neuroregeneration after SCI. This needs to be further investigated.
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Systemic administration of a Connexin43 mimetic peptide, Peptide5, has been shown to reduce secondary tissue damage and improve functional recovery after spinal cord injury (SCI). This study investigated safety measures and potential off-target effects of Peptide5 systemic administration. Rats were subjected to a mild contusion SCI using the New York University impactor. One cohort was injected intraperitoneally with a single dose of fluorescently labelled Peptide5 and euthanised at 2 or 4 h post-injury for peptide distribution analysis. A second cohort received intraperitoneal injections of Peptide5 or a scrambled peptide and was culled at 8 or 24 h post-injury for the analysis of connexin proteins and systemic cytokine profile. We found that Peptide5 did not cross the blood-spinal cord barrier in control animals, but reached the lesion area in the spinal cord-injured animals without entering non-injured tissue. There was no evidence that the systemic administration of Peptide5 modulates Connexin43 protein expression or hemichannel closure in the heart and lung tissue of SCI animals. The expression levels of other major connexin proteins including Connexin30 in astrocytes, Connexin36 in neurons and Connexin47 in oligodendrocytes were also unaltered by systemic delivery of Peptide5 in either the injured or non-injured spinal cords. In addition, systemic delivery of Peptide5 had no significant effect on the plasma levels of cytokines, chemokines or growth factors. These data indicate that the systemic delivery of Peptide5 is unlikely to cause any off-target or adverse effects and may thus be a safe treatment option for traumatic SCI.
Assuntos
Materiais Biomiméticos/farmacologia , Conexina 43/farmacologia , Traumatismos da Medula Espinal/tratamento farmacológico , Animais , Materiais Biomiméticos/administração & dosagem , Materiais Biomiméticos/efeitos adversos , Materiais Biomiméticos/farmacocinética , Conexina 43/administração & dosagem , Conexina 43/efeitos adversos , Conexina 43/farmacocinética , Modelos Animais de Doenças , Feminino , Ratos , Ratos Sprague-DawleyRESUMO
Blocking of Connexin43 hemichannels, the main gap junction protein located on astrocytes in the central nervous system, has been shown to reduce neural injury in a number of models. We demonstrated previously that local administration of a Connexin43 mimetic peptide, Peptide5, reduces secondary tissue damage after spinal cord injury (SCI). Here, we investigated whether acute systemic delivery of Peptide5 is also protective in a model of SCI. Rats were subjected to a mild spinal cord contusion using the Multicentre Animal Spinal Cord Injury Study impactor and were injected intraperitoneally with Peptide5 or a scrambled peptide immediately and at 2 h and 4 h post-injury. Rats were tested for locomotor recovery and pain hypersensitivity and euthanized at 8 h, 24 h, two weeks, or six weeks post-injury. Compared with control rats, Peptide5 treated rats showed significant improvement in hindlimb locomotor function between three and six weeks post-injury and reductions in at-level mechanical allodynia at weeks one and six post-injury. Immunohistochemistry showed that Peptide5 treatment led to a reduction in total Connexin43 and increased phosphorylated Connexin43 at 8 h compared with scrambled peptide. At two and six weeks, lesion size, the astrocytic and the activated macrophage, and/or microglial response were all decreased in the Peptide5 animals. In addition, neuronal cell numbers were higher in the Peptide5 animals compared with the scrambled peptide treated rats at two and six weeks. These results show for the first time that systemic administration of Peptide5 to block the pathological opening of Connexin43 hemichannels is a feasible treatment strategy in this setting, ameliorating the secondary SCI.