Calpains are ubiquitous proteases involved in cell proliferation, adhesion and motility. In the brain, calpains have been associated with neuronal damage in both acute and neurodegenerative disorders, but their physiological function in the nervous system remains elusive. During brain ischemia, there is a large increase in the levels of intracellular calcium, leading to the activation of calpains. Inhibition of these proteases has been shown to reduce neuronal death in a variety of stroke models. On the other hand, after stroke, neural stem cells (NSC) increase their proliferation and newly formed neuroblasts migrate towards the site of injury. However, the process of forming new neurons after injury is not efficient and finding ways to improve it may help with recovery after lesion. Understanding the role of calpains in the process of neurogenesis may therefore open a new window for the treatment of stroke. We investigated the involvement of calpains in NSC proliferation and neuroblast migration in two highly neurogenic regions in the mouse brain, the dentate gyrus (DG) and the subventricular zone (SVZ). We used mice that lack calpastatin, the endogenous calpain inhibitor, and calpains were also modulated directly, using calpeptin, a pharmacological calpain inhibitor. Calpastatin deletion impaired both NSC proliferation and neuroblast migration. Calpain inhibition increased NSC proliferation, migration speed and migration distance in cells from the SVZ. Overall, our work suggests that calpains are important for neurogenesis and encourages further research on their neurogenic role. Prospective therapies targeting calpain activity may improve the formation of new neurons following stroke, in addition to affording neuroprotection.
This unit describes two basic protocols for the detection of the proliferation of neural stem cells (NSC). The first one addresses cell proliferation in cultures, starting with primary cell cultures isolated from the mouse subventricular zone (SVZ), in which SVZ‐derived NSC are kept in culture as neurospheres. By using this culture system, we are able to study different stages of adult neurogenesis, such as proliferation, differentiation, migration, and survival. Thus, in the first basic protocol, we describe two different techniques to evaluate cell proliferation based on EdU incorporation: (a) immunocytochemistry and (b) flow cytometry. EdU, a new thymidine analog, which is detected by a reproducible and sensitive method based on click chemistry, does not require DNA denaturation, as is the case with BrdU. Thus, co‐labeling of EdU with other specific antibodies of extracellular or intracellular targets, as well as other DNA dyes, is possible. In the second basic protocol, we describe an in vivo assay to evaluate proliferation of NSC in the dentate gyrus of hippocampus of adult mice, by both BrdU and EdU detection. With this approach, it is also possible to study different stages of adult neurogenesis, by co‐labeling thymidine analogs with other specific markers, such as doublecortin (DCX) or neuronal nuclei protein (NeuN). Curr. Protoc. Stem Cell Biol. 24:2D.14.1‐2D.14.24. © 2013 by John Wiley & Sons, Inc.
In the adult mammalian brain, new neurons continue to be produced throughout life in two main regions in the brain, the subgranular zone (SGZ) in the hippocampus and the subventricular zone in the walls of the lateral ventricles. Neural stem cells (NSCs) proliferate in these niches, and migrate as neuroblasts, to further differentiate in locations where new neurons are needed, either in normal or pathological conditions. However, the endogenous attempt of brain repair is not very efficient. Calpains are proteases known to be involved in neuronal damage and in cell proliferation, migration and differentiation of several cell types, though their effects on neurogenesis are not well known. Previous work by our group has shown that the absence of calpastatin (CAST), the endogenous inhibitor of calpains, impairs early stages of neurogenesis. Since the hippocampus is highly associated with learning and memory, we aimed to evaluate whether calpain inhibition would help improve cognitive recovery after lesion and efficiency of post-injury neurogenesis in this region. For that purpose, we used the kainic acid (KA) model of seizure-induced hippocampal lesion and mice overexpressing CAST. Selected cognitive tests were performed on the 3rd and 8th week after KA-induced lesion, and cell proliferation, migration and differentiation in the dentate gyrus (DG) of the hippocampus of adult mice were analyzed using specific markers. Cognitive recovery was evaluated by testing the animals for recognition, spatial and associative learning and memory. Cognitive function was preserved by CAST overexpression following seizures, while modulation of post-injury neurogenesis was similar to wild type (WT) mice. Calpain inhibition could still be potentially able to prevent the impairment in the formation of new neurons, given that the levels of calpain activity could be reduced under a certain threshold and other harmful effects from the pathological environment could also be controlled.
Mesenchymal stromal cell (MSC) transplantation has been investigated as an advanced treatment of heart failure; however, further improvement of the therapeutic efficacy and mechanistic understanding are needed. Our previous study has reported that epicardial placement of fibrin sealant films incorporating rat amniotic membrane-derived (AM)-MSCs (MSC-dressings) could address limitations of traditional transplantation methods. To progress this finding toward clinical translation, this current study aimed to examine the efficacy of MSC-dressings using human AM-MSCs (hAM-MSCs) and the underpinning mechanism for myocardial repair. Echocardiography demonstrated that cardiac function and structure were improved in a rat ischemic cardiomyopathy model after hAM-MSC-dressing therapy. hAM-MSCs survived well in the rat heart, enhanced myocardial expression of reparative genes, and attenuated adverse remodeling. Copy number analysis by qPCR revealed that upregulated reparative genes originated from endogenous rat cells rather than hAM-MSCs. These results suggest hAM-MSC-dressing therapy stimulates a secondary release of paracrine factors from endogenous cells improving myocardial repair ("secondary paracrine effect"), and cardiac M2-like macrophages were identified as a potential cell source of repair. We demonstrated hAM-MSCs increased M2-like macrophages through not only enhancing M2 polarization but also augmenting their proliferation and migration capabilities via PGE 2 , CCL2, and TGF-b1, resulting in enhanced cardiac function after injury.
DMBA is a potent carcinogenic widely used in experimental oncology. It has a great mutagenic capacity after being metabolically activated in the liver and induces an accelerated tumor growth. In order to evaluate metabolic alterations associated with DMBA administration the metabolic profile of liver was analysed by high resolution magic angle spinning NMR spectroscopy (HRMAS), a powerful method to study how cellular physiology is affected by xenobiotics.Fifteen male wistar rats were divided into two experimental groups: control and DMBA administration Liver samples of male Wistar rats were collected and snap frozen in liquid nitrogen and later analyzed by high rotation magic angle NMR spectroscopy (HRMAS) in a in a 14.1 Tesla Varian spectrometer.HRMAS spectral analysis followed by principal component statistical analysis demonstrated that glycogen, glucose and insaturated lipids were significantly reduced in the DMBA group, while aspartate was increased.The results of this study suggest that DMBA reduces hepatic insaturated lipid contents and glycogen plus glucose. Increased glycolytic fluxes are characteristic of tumors and could explain the reductions in carbohydrate stores. Increased aspartate and reduced insaturated fatty acids could be the result of an alteration in cytosolic redox, which also characterizes tumorigenic activity.
Hyperthyroidism is caused by excess synthesis and secretion of thyroid hormone by the thyroid, which increases the metabolic rate. The experimental model of hyperthyroidism is conducted by an administration of an high dose of levothyroxine for two weeks. The aim of this study is to contribute to the histomorphometric characterization of thyroid follicles' areas on experimental hyperthyroidism.20 Wistar male rats were used, randomly distributed in four groups: control group, kept with no manipulation (Ctrl); group II, which received an administration of 250 mg/kg of levothyroxine by gavage 3 times a week (HT); group III received levothyroxine and acupuncture treatment (HT+AC); group IV, submitted only to the acupuncture treatment. After two weeks, all the animals were sacrificed and fragments from thyroid were collected and analyzed using ImageJ®. The points used for puncture were: ST09, ST36, HC4, RM23.When compared with the control group, we observed more frequently large follicles on group IV than in control group. The animals of group II and III have more often smaller follicles than those observed in control group.The results, although without statistical significance, suggest that acupuncture may have some effect on thyroid function.
Hyperthyroidism can be defined as the consequence of production and excessive action of the thyroid hormones. Acupuncture as been proven effective in the treatment of some pathologies, with minimum side effects.Twenty‐one male Wistar rats were divided into 3 groups: group I – control with no manipulation (Ctrl); group II – received levothyroxine (HT); group III ‐ received levothyroxine and acupuncture (HT+AC). Liver samples were snap frozen in liquid nitrogen and later analyzed by high rotation magic angle NMR spectroscopy (HRMAS) in a in a 14.1 Tesla Varian spectrometer. HRMAS spectral analysis followed by principal component statistical analysis demonstrated that lipids were significantly reduced in the HT group and increased in the HT+AC, the same happening with the aminoacids aspartate and glutamate. Glucose, as well as glycogen were significantly increased in both HT and HT+AC groups.The results suggest that HT reduces hepatic lipid contents, either by increased mobilization or reduced de novo lipogenesis, and that such reduction is reversed by AC treatment. The increase in glucose content is compatible with either an increase in glycogen breakdown or an increase in gluconeogenesis. Our results show an increase in glycogen content in both HT and HT+AC groups thus supporting the later. Accurate evaluation of lipogenesis and glucose turnover is currently underway using the 2H2O method.
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