Changes in number of neurons,astrocytes and microglia in brain after ischemic stroke assessed by immunohistochemistry and immunoblotting

It is known that the mechanisms of brain damage after a stroke are regulated by interaction within several cell types, primarily neurons, astrocytes, the endothelium, and microglia. Ischemic exposure disrupts the balance in the brain cellular content; thus, in the lesion, cells die by necrosis, while delayed induction of apoptosis occurs in the tissue surrounding the ischemic zone. Named cells die in the lesion and their ratio determines the clinical outcome of the disease. Thus, the detection of deaths within various cell types of the neurovascular unit is an important part of fundamental studies of the mechanisms of brain damage and preclinical studies of potential neuroprotective drugs. For this reason, we conducted a comparative study of the two most often used methods: immunohistochemical staining of brain sections, which allows to determine the number and localization of specific cells in the tissue among other types of cells, and immunoblotting, which detects specific proteins in the tissue homogenate. We found that, depending on the cell type, changes in their number and composition after a stroke can be localized in a limited part of the tissue or cover the entire hemisphere, which imposes restrictions on the use of any method of determining the number of cells in brain tissue. In general, the most preferable is the use of immunohistochemistry; however, with certain limitations, immunoblotting can be used to determine the proportion of astroglia and microglia.     

Engulfing astrocytes protect neurons from contact-induced apoptosis following injury

Clearing of dead cells is a fundamental process to limit tissue damage following brain injury. Engulfment has classically been believed to be performed by professional phagocytes, but recent data show that non-professional phagocytes are highly involved in the removal of cell corpses in various situations. The role of astrocytes in cell clearance following trauma has however not been studied in detail. We have found that astrocytes actively collect and engulf whole dead cells in an in vitro model of brain injury and thereby protect healthy neurons from bystander cell death. Time-lapse experiments showed that migrating neurons that come in contact with free-floating cell corpses induced apoptosis, while neurons that migrate through groups of dead cells, garnered by astrocytes, remain unaffected. Furthermore, apoptotic cells are present within astrocytes in the mouse brain following traumatic brain injury (TBI), indicating a possible role for astrocytes in engulfment of apoptotic cells in vivo. qRT-PCR analysis showed that members of both ced pathways and Megf8 are expressed in the cell culture, indicating their possible involvement in astrocytic engulfment. Moreover, addition of dead cells had a positive effect on the protein expression of MEGF10, an ortholog to CED1, known to initiate phagocytosis by binding to phosphatidylserine. Although cultured astrocytes have an immense capacity for engulfment, seemingly without adverse effects, the ingested material is stored rather than degraded. This finding might explain the multinuclear astrocytes that are found at the lesion site in patients with various brain disorders.     

Differential expression of microRNA patterns in planarian normal and regenerative tissues

MicroRNAs (miRNAs) are ~22-nt small non-coding RNAs that regulate the expression of specific target genes in many eukaryotes. miRNAs have been shown to play important roles in stem cell maintenance, cell fate determination, and differentiation. Planarians are capable of regenerating entire body plans from tiny fragments; this regenerative capacity is facilitated by a population of pluripotent stem cells known as neoblasts. Planarians have been a classic model system for the study of many aspects of stem cell biology. However, very limited knowledge on miRNA involved in this regulatory mechanism exists. This study profiles the expression of miRNAs in the normal and regenerative tissues of planarians using miRCURY LNA array technology. Thirteen miRNAs showed significant differences in expression between these two tissues. To further confirm our results, we examined the expression of two miRNAs by qRT-PCR. Results show that some known miRNAs may play key roles in the regulatory mechanisms of regeneration. Our findings can be utilized in future research on miRNA function.     

Mechanism of human immunodeficiency virus-induced complement expression in astrocytes and neurons

The cerebral complement system is hypothesized to contribute to neurodegeneration in the pathogenesis of AIDS-associated neurological disorders. Our former results have shown that the human immunodeficiency virus (HIV) strongly induces the synthesis of complement factor C3 in astrocytes. This upregulation explains in vivo data showing elevated complement levels in the cerebrospinal fluid of patients with AIDS-associated neurological symptoms. Since inhibition of complement synthesis and activation in the brain may represent a putative therapeutic goal to prevent virus-induced damage, we analyzed in detail the mechanisms of HIV-induced modulation of C3 expression. HIV-1 increased the C3 levels in astrocyte culture supernatants from 30 to up to 400 ng/ml; signal transduction studies revealed that adenylate cyclase activation with upregulation of cyclic AMP is the central signaling pathway to mediate that increase. Furthermore, activity of protein kinase C is necessary for HIV induction of C3, since inhibition of protein kinase C by prolonged exposure to the phorbol ester tetradecanoyl phorbol acetate partly abolished the HIV effect. The cytokines tumor necrosis factor alpha and gamma interferon were not involved in mediating the HIV-induced C3 upregulation, since neutralizing antibodies had no effect. Besides whole HIV virions, the purified viral proteins Nef and gp41 are biologically active in upregulating C3, whereas Tat, gp120, and gp160 were not able to modulate C3 synthesis. Further experiments revealed that neurons were also able to respond on incubation with HIV with increased C3 synthesis, although the precise pattern was slightly different from that in astrocytes. This strengthens the hypothesis that HIV-induced complement synthesis represents an important mechanism for the pathogenesis of AIDS in the brain.     

Endothelin-1 protects astrocytes from hypoxic/ischemic injury.

Under pathological conditions such as ischemia (I), subarachnoid hemorrhage, and Alzheimer's disease, astrocytes show a large increase in endothelin (ET) -like immunoreactivity. However, it is not clear whether ET is protective or destructive to these cells during brain injury. Using astrocytes from ET-1-deficient mice, we determined the effect of ET-1 on these cells under normal, hypoxic (H), and hypoxic/ischemic (H/I) conditions. Under normal culture conditions, astrocytes from wild-type and ET-1-deficient mice showed no difference in their morphology and cell proliferation rates. ET-3 and ETA receptor mRNAs were up-regulated whereas ETB receptor mRNA was down-regulated in ET-1-deficient astrocytes, suggesting that ET-1 and ET-3 may complement each other's functions and that the expressions of these endothelins and their receptors are regulated by a complex feedback mechanism. Under H and H/I conditions, ET-1 peptide and mRNA were up-regulated in wild-type astrocytes, and the astrocytes without ET-1 died faster than the wild-type astrocytes, as indicated by greater efflux of lactate dehydrogenase. The present study suggests that astrocytes without ET-1 are more vulnerable to H and H/I injuries and that the up-regulation of astrocytic ET-1 is essential for the survival of astrocytes.     

Gene expression patterns in in vivo normal adult astrocytes compared with cultured neonatal and normal adult astrocytes

This paper presents data on the basal gene expression patterns, determined by microarray analysis, for cultured neonatal and normal adult striatal astrocytes, and for comparison, for astrocytes isolated directly from adult rat striatum (in vivo adult astrocytes). Of the 1176 genes on the Clontech array, 1101 were expressed in one of the three types of astrocyte samples. Nineteen of the genes were expressed only in the astrocytes taken directly from adult rats (in vivo adult). The cultured neonatal astrocytes expressed many genes at a two-fold or greater level than their expression in cultured adult astrocytes, including genes in the adhesion, cytoskeleton, and extracellular matrix (ECM) family, signal transduction genes, and genes related to apoptosis, DNA-binding, and cell cycle regulation. Overall the results support the concept that cultured neonatal astrocytes are more "activated" than cultured adult cells, although the adult cells expressed higher levels of many metabolic enzyme and protease/protease inhibitor genes.     

Temporal patterns of cortical proliferation of glial cell populations after traumatic brain injury in mice

TBI (traumatic brain injury) triggers an inflammatory cascade, gliosis and cell proliferation following cell death in the pericontusional area and surrounding the site of injury. In order to better understand the proliferative response following CCI (controlled cortical impact) injury, we systematically analyzed the phenotype of dividing cells at several time points post-lesion. C57BL/6 mice were subjected to mild to moderate CCI over the left sensory motor cortex. At different time points following injury, mice were injected with BrdU (bromodeoxyuridine) four times at 3-h intervals and then killed. The greatest number of proliferating cells in the pericontusional region was detected at 3 dpi (days post-injury). At 1 dpi, NG2⁺ cells were the most proliferative population, and at 3 and 7 dpi the Iba-1⁺ microglial cells were proliferating more. A smaller, but significant number of GFAP⁺ (glial fibrillary acidic protein) astrocytes proliferated at all three time points. Interestingly, at 3 dpi we found a small number of proliferating neuroblasts [DCX⁺ (doublecortin)] in the injured cortex. To determine the cell fate of proliferative cells, mice were injected four times with BrdU at 3 dpi and killed at 28 dpi. Approximately 70% of proliferative cells observed at 28 dpi were GFAP⁺ astrocytes. In conclusion, our data suggest that the specific glial cell types respond differentially to injury, suggesting that each cell type responds to a specific pattern of growth factor stimulation at each time point after injury.     

间充质干细胞源性外泌体microRNA在心脏缺血性损伤修复中的研究进展

心脏缺血性损伤是危害人类健康的重要原因,过去的干细胞疗法具有重要的功能缺陷,如免疫排斥、致瘤性和输注毒性等问题。大量研究表明,间充质干细胞的主要治疗作用是由旁分泌因子所介导。最新研究发现,间充质干细胞来源的外泌体microRNA从移植的干细胞转移至缺血损伤的心脏细胞,调节细胞的增殖、凋亡、炎症和血管生成。本文对来源于间充质干细胞的外泌体及其内部microRNA在心脏缺血性损伤修复中的分子机制进行综述。     

Differential regulation of neuregulin 1 expression by progesterone in astrocytes and neurons

Glial-neuronal interactions are crucial processes in neuromodulation and synaptic plasticity. The neuregulin 1 family of growth and differentiation factors have been implicated as bidirectional signaling molecules that are involved in mediating some of these interactions. We have shown previously that neuregulin 1 expression is regulated by the gonadal hormones progesterone and 17beta-estradiol in the CNS, which might represent a novel, indirect mechanism of the neuromodulatory actions of these gonadal hormones. In the present study, we sought to determine the effects of progesterone and 17beta-estradiol on neuregulin 1 expression in rat cortical astrocytes and neurons in vitro. We observed that progesterone increased the expression of neuregulin 1 mRNA and protein in a dose-dependent manner in cultured astrocytes, which was blocked by the progesterone receptor antagonist RU-486. In contrast, 17beta-estradiol did not increase either neuregulin 1 mRNA or protein in astrocytes. We observed no effect of either progesterone or 17beta-estradiol on neuregulin 1 mRNA and protein in rat cortical neurons in vitro. Finally, we observed that treatment of cortical neurons with recombinant NRG1-beta1 caused PSD-95 to localize in puncta similar to that observed following treatment with astrocyte-conditioned medium. These results demonstrate that progesterone regulates neuregulin 1 expression, principally in astrocytes. This might represent a novel mechanism of progesterone-mediated modulation of neurotransmission through the regulation of astrocyte-derived neuregulin 1.     

Stretch-induced injury alters mitochondrial membrane potential and cellular ATP in cultured astrocytes and neurons

Energy deficit after traumatic brain injury (TBI) may alter ionic homeostasis, neurotransmission, biosynthesis, and cellular transport. Using an in vitro model for TBI, we tested the hypothesis that stretch-induced injury alters mitochondrial membrane potential (delta(psi)m) and ATP in astrocytes and neurons. Astrocytes, pure neuronal cultures, and mixed neuronal plus glial cultures grown on Silastic membranes were subjected to mild, moderate, and severe stretch. After injury, delta(psi)m was measured using rhodamine-123, and ATP was quantified with a luciferin-luciferase assay. In astrocytes, delta(psi)m dropped significantly, and ATP content declined 43-52% 15 min after mild or moderate stretch but recovered by 24 h. In pure neurons, delta(psi)m declined at 15 min only in the severely stretched group. At 48 h postinjury, delta(psi)m remained decreased in severely stretched neurons and dropped in moderately stretched neurons. Intracellular ATP content did not change in any group of injured pure neurons. We also found that astrocytes and neurons release ATP extracellularly following injury. In contrast to pure neurons, delta(psi)m in neurons of mixed neuronal plus glial cultures declined 15 min after mild, moderate, or severe stretch and recovered by 24-48 h. ATP content in mixed cultures declined 22-28% after mild to severe stretch with recovery by 24 h. Our findings demonstrate that injury causes mitochondrial dysfunction in astrocytes and suggest that astrocyte injury alters mitochondrial function in local neurons.     

Temporal and spatial patterns of endogenous danger signal expression after wound healing and in response to lymphedema

While acute tissue injury potently induces endogenous danger signal expression, the role of these molecules in chronic wound healing and lymphedema is undefined. The purpose of this study was to determine the spatial and temporal expression patterns of the endogenous danger signals high-mobility group box 1 (HMGB1) and heat shock protein (HSP)70 during wound healing and chronic lymphatic fluid stasis. In a surgical mouse tail model of tissue injury and lymphedema, HMGB1 and HSP70 expression occurred along a spatial gradient relative to the site of injury, with peak expression at the wound and greater than twofold reduced expression within 5 mm (P < 0.05). Expression primarily occurred in cells native to injured tissue. In particular, HMGB1 was highly expressed by lymphatic endothelial cells (>40% positivity; twofold increase in chronic inflammation, P < 0.001). We found similar findings using a peritoneal inflammation model. Interestingly, upregulation of HMGB1 (2.2-fold), HSP70 (1.4-fold), and nuclear factor (NF)-κβ activation persisted at least 6 wk postoperatively only in lymphedematous tissues. Similarly, we found upregulation of endogenous danger signals in soft tissue of the arm after axillary lymphadenectomy in a mouse model and in matched biopsy samples obtained from patients with secondary lymphedema comparing normal to lymphedematous arms (2.4-fold increased HMGB1, 1.9-fold increased HSP70; P < 0.01). Finally, HMGB1 blockade significantly reduced inflammatory lymphangiogenesis within inflamed draining lymph nodes (35% reduction, P < 0.01). In conclusion, HMGB1 and HSP70 are expressed along spatial gradients and upregulated in chronic lymphatic fluid stasis. Furthermore, acute expression of endogenous danger signals may play a role in inflammatory lymphangiogenesis.     

Cholesterol metabolism in neurons and astrocytes

Cells in the mammalian body must accurately maintain their content of cholesterol, which is an essential membrane component and precursor for vital signalling molecules. Outside the brain, cholesterol homeostasis is guaranteed by a lipoprotein shuttle between the liver, intestine and other organs via the blood circulation. Cells inside the brain are cut off from this circuit by the blood–brain barrier and must regulate their cholesterol content in a different manner. Here, we review how this is accomplished by neurons and astrocytes, two cell types of the central nervous system, whose cooperation is essential for normal brain development and function. The key observation is a remarkable cell-specific distribution of proteins that mediate different steps of cholesterol metabolism. This form of metabolic compartmentalization identifies astrocytes as net producers of cholesterol and neurons as consumers with unique means to prevent cholesterol overload. The idea that cholesterol turnover in neurons depends on close cooperation with astrocytes raises new questions that need to be addressed by new experimental approaches to monitor and manipulate cholesterol homeostasis in a cell-specific manner. We conclude that an understanding of cholesterol metabolism in the brain and its role in disease requires a close look at individual cell types.     

Regeneration of hippocampal pyramidal neurons after ischemic brain injury by recruitment of endogenous neural progenitors

The adult brain is extremely vulnerable to various insults. The recent discovery of neural progenitors in adult mammals, however, raises the possibility of repairing damaged tissue by recruiting their latent regenerative potential. Here we show that activation of endogenous progenitors leads to massive regeneration of hippocampal pyramidal neurons after ischemic brain injury. Endogenous progenitors proliferate in response to ischemia and subsequently migrate into the hippocampus to regenerate new neurons. Intraventricular infusion of growth factors markedly augments these responses, thereby increasing the number of newborn neurons. Our studies suggest that regenerated neurons are integrated into the existing brain circuitry and contribute to ameliorating neurological deficits. These results expand the possibility of novel neuronal cell regeneration therapies for stroke and other neurological diseases.     

Temporal and spatial profile of caspase 8 expression and proteolysis after experimental traumatic brain injury

Recent studies have demonstrated that the downstream caspases, such as caspase 3, act as executors of the apoptotic cascade after traumatic brain injury (TBI) in vivo. However, little is known about the involvement of caspases in the initiation phase of apoptosis, and the interaction between these initiator caspases (e.g. caspase 8) and executor caspases after experimental brain injuries in vitro and in vivo. This study investigated the temporal expression and cell subtype distribution of procaspase 8 and cleaved caspase 8 p20 from 1 h to 14 days after cortical impact-induced TBI in rats. Caspase 8 messenger RNA levels, estimated by semiquantitaive RT-PCR, were elevated from 1 h to 72 h in the traumatized cortex. Western blotting revealed increased immunoreactivity for procaspase 8 and the proteolytically active subunit of caspase 8, p20, in the ipsilateral cortex from 6 to 72 h after injury, with a peak at 24 h after TBI. Similar to our previous studies, immunoreactivity for the p18 fragment of activated caspase 3 also increased in the current study from 6 to 72 h after TBI, but peaked at a later timepoint (48 h) as compared with proteolyzed caspase 8 p20. Immunohistologic examinations revealed increased expression of caspase 8 in neurons, astrocytes and oligodendrocytes. Assessment of DNA damage using TUNEL identified caspase 8- and caspase 3-immunopositive cells with apoptotic-like morphology in the cortex ipsilateral to the injury site, and immunohistochemical investigations of caspase 8 and activated caspase 3 revealed expression of both proteases in cortical layers 2-5 after TBI. Quantitative analysis revealed that the number of caspase 8 positive cells exceeds the number of caspase 3 expressing cells up to 24 h after impact injury. In contrast, no evidence of caspase 8 and caspase 3 activation was seen in the ipsilateral hippocampus, contralateral cortex and hippocampus up to 14 days after the impact. Our results provide the first evidence of caspase 8 activation after experimental TBI and suggest that this may occur in neurons, astrocytes and oligodendrocytes. Our findings also suggest a contributory role of caspase 8 activation to caspase 3 mediated apoptotic cell death after experimental TBI in vivo.     

Leptin protects hippocampal CA1 neurons against ischemic injury

Leptin is an adipose hormone with well characterized roles in regulating food intake and energy balance. A novel neuroprotective role for leptin has recently been discovered; however, the underlying mechanisms are not clearly defined. The purpose of this study was to determine whether leptin protects against delayed neuronal cell death in hippocampal CA1 following transient global cerebral ischemia in rats and to study the signaling mechanism responsible for the neuroprotective effects of leptin. Leptin receptor antagonist, protein kinase inhibitors and western blots were used to assess the molecular signaling events that were altered by leptin after ischemia. The results revealed that intracerebral ventricle infusion of leptin markedly increased the numbers of survival CA1 neurons in a dose-dependent manner. Infusion of a specific leptin antagonist 10 min prior to transient global ischemia abolished the pro-survival effects of leptin, indicating the essential role of leptin receptors in mediating this neuroprotection. Both the Akt and extracellular signal-related kinase 1/2 (ERK1/2) signaling pathways appear to play a critical role in leptin neuroprotection, as leptin infusion increased the phosphorylation of Akt and ERK1/2 in CA1. Furthermore, pharmacological inhibition of either pathway compromised the neuroprotective effects of leptin. Taken together, the results suggest that leptin protects against delayed ischemic neuronal death in the hippocampal CA1 by maintaining the pro-survival states of Akt and ERK1/2 MAPK signaling pathways.