Therapeutic Prospects in Preeclampsia - A Mini-Review.

BACKGROUND: Preeclapmsia (PE) is characterized by early onset symptoms such as elevated blood pressure, proteinuria and edema in the pregnant woman, and may result in seizures in the affected female. Currently, there are no therapeutic drugs available to treat this condition, but there are interventions to regulate the symptoms based on the gestational period of the fetus, although the largely favored option is delivery of the fetus and placenta. OBJECTIVE: A search for biomolecules associated with PE was conducted so as to identify diagnostic markers and therapeutic leads. RESULTS: The literature search resulted in the identification of biomolecules such as Corin and Placental Protein 13 (PP13), among others that are associated with PE. Thereby, giving an insight into the various mechanistic pathways involved in the causation of PE. However, it is also evident that PE cannot be solely attributed to any single mechanism but is due to an interplay of different factors that have led to the development of this disease condition. CONCLUSION: The identified biomarkers would ultimately help in understanding this complex disease and perhaps lead to the discovery of potential effective molecular targets for clinical trials, thereby providing a valuable therapeutic option for affected pregnant women.

Role of dietary phenols in mitigating microglia-mediated neuroinflammation.

Chronic neuroinflammation is a pathological feature of a number of central nervous system (CNS) diseases and is mediated by sustained activation of microglial cells, the innate immune cells of the CNS. Studies have mainly focused on identifying the molecular and epigenetic mechanisms of microglial activation. This is crucial in designing therapeutic strategies for neuropathologies in which prolonged microglial activation is known to exacerbate disease condition. In recent years, increasing evidence show that naturally occurring compounds present in regular diet could function as "nutraceuticals," arresting microglial activation, and thus conferring neuroprotection. This review summarizes our understanding of the role of dietary phenolic nutraceuticals in mitigating microglia-mediated neuroinflammation. Studies show that these natural phenols inhibit key signaling pathways in activated microglia such as the NFκB, MAPK and JAK-STAT that trigger microglia-mediated inflammation in various neuropathological conditions such as injury, infection, stroke, autism and neurodegenerative diseases, i.e., Alzheimer's disease and Parkinson's disease. The anti-inflammatory and antioxidant effect exerted by these natural phenols have shown considerable success in improving disease condition in animal models of neuropathologies, and thus seem to be suitable candidates for developing therapeutic strategies.

Sirtuin 3 regulates Foxo3a-mediated antioxidant pathway in microglia.

Microglia are the prime cellular sources of reactive oxygen species (ROS) in the central nervous system (CNS). Chronic activation of microglia has been linked to aging-associated neurodegenerative diseases such as Alzheimer's disease (AD) and Parkinson's disease (PD) since they produce excessive amounts of ROS for a prolonged duration leading to oxidative stress. The present study was aimed at investigating the expression and role of Sirtuin 3 (Sirt3), a protein deacetylase which is implicated in regulating cellular ROS levels. It has been shown that Sirt3 reduces cellular ROS levels by deacetylating forkhead box O 3a (Foxo3a), a transcription factor which transactivates antioxidant genes, catalase (Cat) and manganese superoxide dismutase (mnSod). In the present study, Sirt3 immunoreactivity was localized in the ameboid microglial cells distributed in the corpus callosum (CC) of the early postnatal rat brain and diminished in the ramified microglial cells in the CC of the adult rat brain. A marked induction of Sirt3 expression was seen in lipopolysaccharide (LPS)-activated microglia in vivo and in vitro as well as in adult rat brains subjected to traumatic brain injury (TBI). Knockdown of Sirt3 in microglia led to an increase in the cellular and mitochondrial ROS and decrease in the expression of antioxidant, mnSod which is indicative of the function of Sirt3 in ROS regulation in microglia. Conversely, Sirt3 overexpression led to increase in the expression of antioxidants Cat and mnSod. Further, increase in the expression and nuclear translocation of Foxo3a was observed following Sirt3 overexpression, suggesting that Sirt3 regulates ROS by inducing the expression of antioxidants via activation of Foxo3a. The above results point to an antioxidant defense mechanism presented by Sirt3 through the activation of Foxo3a, in microglia.

Sphingosine kinase 2 and sphingosine-1-phosphate promotes mitochondrial function in dopaminergic neurons of mouse model of Parkinson's disease and in MPP+ -treated MN9D cells in vitro.

Dysregulation of sphingolipid metabolism has been shown to trigger the pathophysiology of many neurodegenerative disorders. The present study focuses on the role of one of the two sphingosine kinases, Sphk2 and its metabolite sphingosine-1-phosphate (S1P) signaling in Parkinson's disease (PD). Our study indicated a marked down regulation of Sphk2 expression in the substantia nigra region of the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced PD mouse model and in the cellular PD model. Localization studies indicated that Sphk2 was predominantly present in mitochondria, proposing for its potential role in mitochondrial functions. Since mitochondrial dysfunction has been described to be the major pathological event in PD, the present study focused on the role of Sphk2/S1P signaling in promoting mitochondrial functions in the MPTP-induced mouse model of PD and in 1-methyl-4 phenylpyridinium (MPP(+))-treated MN9D cells. Our study demonstrated that inhibition of Sphk2 decreased the expression of peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) and its downstream targets nuclear respiratory factor 1 (NRF-1) and mitochondrial transcription factor A (TFAM) which are the key genes regulating mitochondrial function. In addition, there was also a significant reduction in the total cellular adenosine triphosphate (ATP) and superoxide dismutase 2 (SOD 2) with an associated increase in levels of reactive oxygen species (ROS) in the absence of Sphk2. Interestingly, it was found that treating the cells with exogenous S1P along with MPP(+) exerted a neuroprotective effect by activation of p-CREB, PGC-1α and NRF-1 in the MN9D cells. Moreover, the level of ATP was unaffected in the MPP(+)-treated cells in the presence of S1P. It was also observed that levels of ROS were significantly decreased in the MPP(+)-treated cells in the presence of exogenous S1P. Our study also demonstrated that S1P exerted its protective effect through the S1P1 receptor. Taken together, these results show that Sphk2/S1P has an important role to play in the survival of the dopaminergic neurons, in the pathogenesis of PD.

Nanomedicine and its application in treatment of microglia-mediated neuroinflammation.

Nanomedicine, an emerging therapeutic tool in current medical frontiers, offers targeted drug delivery for many neurodegenerative disorders. Neuroinflammation, a hallmark of many neurodegenerative disorders, is mediated by microglia, the resident immunocompetent cells of the central nervous system (CNS). Microglial cells respond to various stimuli in the CNS resulting in their activation which may have a beneficial or a detrimental effect. In general, the activated microglia remove damaged neurons and infectious agents by phagocytosis, therefore being neuroprotective. However, their chronic activation exacerbates neuronal damage through excessive release of proinflammatory cytokines, chemokines and other inflammatory mediators which contribute to neuroinflammation and subsequent neurodegeneration in the CNS. Hence, controlling microglial inflammatory response and their proliferation has been considered as an important aspect in treating neurodegenerative disorders. Regulatory factors that control microglial activation and proliferation also play an important role in microglia-mediated neuroinflammation and neurotoxicity. Various anti-inflammatory drugs and herbal compounds have been identified in treating microglia-mediated neuroinflammation in the CNS. However, hurdles in crossing blood brain barrier (BBB), expression of metabolic enzymes, presence of efflux pumps and several other factors prevent the entry of these drugs into the CNS. Use of non-degradable delivery systems and microglial activation in response to the drug delivery system further complicate drug delivery to the CNS. Nanomedicine, a nanoparticle-mediated drug delivery system, exhibits immense potential to overcome these hurdles in drug delivery to the CNS enabling new alternatives with significant promises in revolutionising the field of neurodegenerative disease therapy. This review attempts to summarise various regulatory factors in microglia, existing therapeutic strategies in controlling microglial activation, and how nanotechnology can serve to improve the delivery of therapeutic drugs across the BBB for treating microglia- mediated neuroinflammation and neurodegeneration.

Downregulation of miR-124 in MPTP-treated mouse model of Parkinson's disease and MPP iodide-treated MN9D cells modulates the expression of the calpain/cdk5 pathway proteins.

Parkinson's disease (PD) is a debilitating neurodegenerative disorder causing severe motor disabilities resulting from the loss of dopaminergic neurons in the substantia nigra pars compacta (SNc) region of the midbrain. MicroRNAs (miRNAs) are small, non-coding RNAs which play a major role in several cellular processes in health and disease by regulating gene expression post-transcriptionally. Aberrant miRNA expression has been detected in post-mortem human PD brain samples, in vitro and in vivo PD models. However, none of the studies have focused on the role of the brain-abundant miR-124 in PD. In this study, we have evaluated the expression changes of miR-124 in the SN of the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced PD mouse model. MiRNA expression analysis by qPCR revealed a decrease in the expression of brain-enriched miR-124 in the SN of MPTP-treated mice as compared to controls. Further, in vitro study revealed a decrease in the expression of miR-124 in MN9D dopaminergic neurons treated with methyl phenyl pyridinium (MPP) iodide. The expression of calpains 1 and 2 which is modulated by miR-124 was increased in the SNc of MPTP-treated mice as observed at different time points after treatment and in the MN9D dopaminergic neurons treated with MPP iodide leading to increased expression of the p35 cleavage product, p25 and cyclin-dependent kinase 5 (cdk5). Calpain-p25-mediated increase in cdk5 expression leading to dopaminergic neuronal death has been demonstrated in human PD and MPTP-PD models. Increased expression of calpain 1/cdk5 pathway proteins was observed in anti-miR-124-transfected MN9D cells in our studies. Knockdown of miR-124 led to increased production of reactive oxygen species (ROS) and hydrogen peroxide (H2O2) both known to increase oxidative stress. Further, experiments with miR-124 target protector sequences specific to calpain 1 revealed an interaction of miR-124 with calpain 1. Overexpression of miR-124 after MPP iodide treatment on MN9D cells was found to attenuate the expression of the calpain 1/p25/cdk5 proteins while improving cell survival. These results suggest that miR-124 acts to modulate the expression of calpain/cdk5 pathway proteins in the dopaminergic neurons. A better understanding of the mechanisms controlling the expression of miR-124 will aid in targeting miR-124 for better treatment strategies for PD.

Dihydropyrimidinase-like 3 regulates the inflammatory response of activated microglia.

Microglia, the resident immune cells of the CNS, are known to respond to injuries, infection and inflammation in the CNS by producing proinflammatory cytokines and phagocytosing cell debris and pathogens. In this study, we investigated the expression pattern and role of dihydropyrimidinase-like 3 (Dpysl3), a member of collapsin response mediator protein family, on the inflammatory reaction of microglia. Microarray analysis comparing the global gene expression profile of ameboid and ramified microglia has shown that Dpysl3 is mainly expressed in ameboid microglia in the 5-day postnatal rat brain. Immunohistochemical analysis revealed that Dpysl3 was intensely expressed in ameboid microglial cells in the rat brain till postnatal 7th day and then gradually diminished in ramified microglia of 2 weeks postnatal rat brain. Further, in vitro analysis confirmed that Dpysl3 expression was induced in activated BV-2 microglia treated with lipopolysaccharide (LPS). It is well documented that microglial activation by LPS increased the expression of inducible nitric oxide synthase (iNOS) and proinflammatory cytokines through the activation of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) activity in BV-2 microglia. However, siRNA-mediated knockdown of Dpysl3 prevented the LPS-induced expression of iNOS and cytokines including interleukin-1 beta, and tumor necrosis factor-alpha as well as nuclear translocation of NF-κB in microglia. Remarkably, knockdown of Dpysl3 inhibited the migration of activated microglia coupled with deranged actin filament configuration (as revealed by F-actin cytoskeleton expression) in lamellipodia projecting from the cells. Knockdown of Dpysl3 also inhibited the phagocytic ability of activated microglia. These findings suggest that knockdown of Dpysl3 can inhibit activation, migration and phagocytic capability of microglia and consequently reduce neuroinflammation.

Nuclear factor-κB/p65 responds to changes in the Notch signaling pathway in murine BV-2 cells and in amoeboid microglia in postnatal rats treated with the γ-secretase complex blocker DAPT.

Microglial cells constitutively express Notch-1 and nuclear factor-kappaB/p65 (NF-kappaB/p65), and both pathways modulate production of inflammatory mediators. This study sought to determine whether a functional relationship exists between them and, if so, to investigate whether they synergistically regulate common microglial cell functions. By immunofluorescence labeling, real-time polymerase chain reaction (RT-PCR), flow cytometry, and Western blot, BV-2 cells exhibited Notch-1 and NF-kappaB/p65 expression, which was significantly up-regulated in cells challenged with lipopolysaccharide (LPS). This was coupled with an increase in expression of Hes-1, tumor necrosis factor-alpha (TNF-alpha), and interleukin-1beta (IL-1beta). In BV-2 cells pretreated with N-[N-(3,5-difluorophenacetyl)-1-alany1]-S-phenyglycine t-butyl ester (DAPT), a gamma-secretase inhibitor, followed by LPS stimulation, Notch-1 expression level was enhanced but that of all other markers was suppressed. Additionally, Hes-1 expression and NF-kappaB nuclear translocation decreased as shown by flow cytometry. Notch-1's modulation of NF-kappaB/p65 was also evidenced in amoeboid microglial cells (AMC) in vivo. In 5-day-old rats given intraperitoneal injections of LPS, Notch-1, NF-kappaB/p65, TNF-alpha, and IL-1beta immunofluorescence in AMC was markedly enhanced. However, in rats given an intraperitoneal injection of DAPT prior to LPS, Notch-1 labeling was augmented, but that of TNF-alpha and IL-1beta was reduced. The results suggest that blocking of Notch-1 activation with DAPT would reduce the level of its downstream end product Hes-1 along with suppression of NF-kappaB/p65 translocation, resulting in suppressed production of proinflammatory cytokines. It is concluded that Notch-1 signaling can trans-activate NF-kappaB/p65 by amplifying NF-kappaB/p65-dependent proinflammatory functions in activated microglia.

Sphingosine kinase 1 regulates the expression of proinflammatory cytokines and nitric oxide in activated microglia.

Microglial activation has been implicated as one of the causative factors for neuroinflammation in various neurodegenerative diseases. The sphingolipid metabolic pathway plays an important role in inflammation, cell proliferation, survival, chemotaxis, and immunity in peripheral macrophages. In this study, we demonstrate that sphingosine kinase1 (SphK1), a key enzyme of the sphingolipid metabolic pathway, and its receptors are expressed in the mouse BV2 microglial cells and SphK1 alters the expression and production of proinflammatory cytokines and nitric oxide in microglia treated with lipopolysaccharide (LPS). LPS treatment increased the SphK1 mRNA and protein expression in microglia as revealed by the RT-PCR, Western blot and immunofluorescence. Suppression of SphK1 by its inhibitor, N, N Dimethylsphingosine (DMS), or siRNA resulted in decreased mRNA expression of TNF-alpha, IL-1beta, and iNOS and release of TNF-alpha and nitric oxide (NO) in LPS-activated microglia. Moreover, addition of sphingosine 1 phosphate (S1P), a breakdown product of sphingolipid metabolism, increased the expression levels of TNF-alpha, IL-1beta and iNOS and production of TNF-alpha and NO in activated microglia. Hence to summarize, suppression of SphK1 in activated microglia inhibits the production of proinflammatory cytokines and NO and the addition of exogenous S1P to activated microglia enhances their inflammatory responses. Since the chronic proinflammatory cytokine production by microglia has been implicated in neuroinflammation, modulation of SphK1 and S1P in microglia could be looked upon as a future potential therapeutic method in the control of neuroinflammation in neurodegenerative diseases.

NG2, a member of chondroitin sulfate proteoglycans family mediates the inflammatory response of activated microglia.

Activation of microglial cells, the resident immune cells of the CNS causes neurotoxicity through the release of a wide array of inflammatory mediators including proinflammatory cytokines, chemokines and reactive oxygen species. In this study, we have investigated the expression of NG2 (also known as CSPG4), one of the members of transmembrane chondroitin sulfate proteoglycans family, in microglial cells and its role on inflammatory reaction of microglia by analyzing the expression of the proinflammation cytokines (interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha)), chemokines (stromal cell-derived factor-1alpha and monocyte chemotactic protein-1) and inducible nitric oxide synthase (iNOS). NG2 expression was not detectable in microglial cells expressing OX-42 in the brains of 1-day old postnatal rat pups and adult rats; it was, however, induced in activated microglial cells in pups and adult rats injected with lipopolysaccharide (LPS). In vitro analysis further confirmed that LPS induced the expression of NG2 in primary microglial cells and this was inhibited by dexamethasone. It has been well demonstrated that LPS induces the expression of iNOS and proinflammatory cytokines in microglia. However in this study, LPS did not induce the mRNA expression of iNOS and cytokines including IL-1beta, and TNF-alpha in microglial cells transfected with CSPG4 siRNA. On the contrary, mRNA expression of chemokines such as monocyte chemoattractant protein-1 (MCP-1) and stromal cell-derived factor-1alpha (SDF-1alpha) was significantly increased in LPS-activated microglial cells after CSPG4 siRNA transfection in comparison with the control. The above results indicate that NG2 mediates the induction of iNOS and inflammatory cytokine expression, but not the chemokine expression in activated microglia.

Aldose reductase is implicated in high glucose-induced oxidative stress in mouse embryonic neural stem cells.

Oxidative stress caused by hyperglycemia is one of the key factors responsible for maternal diabetes-induced congenital malformations, including neural tube defects in embryos. However, mechanisms by which maternal diabetes induces oxidative stress during neurulation are not clear. The present study was aimed to investigate whether high glucose induces oxidative stress in neural stem cells (NSCs), which compose the neural tube during development. We also investigated the mechanism by which high glucose disturbs the growth and survival of NSCs in vitro. NSCs were exposed to physiological d-glucose concentration (PG, 5 mmol/L), PG with l-glucose (25 mmol/L), or high d-glucose concentration (HG, 30 or 45 mmol/l). HG induced reactive oxygen species production and mRNA expression of aldose reductase (AR), which catalyzes the glucose reduction through polyol pathway, in NSCs. Expression of glucose transporter 1 (Glut1) mRNA and protein which regulates glucose uptake in NSCs was increased at early stage (24 h) and became down-regulated at late stage (72 h) of exposure to HG. Inhibition of AR by fidarestat, an AR inhibitor, decreased the oxidative stress, restored the cell viability and proliferation, and reduced apoptotic cell death in NSCs exposed to HG. Moreover, inhibition of AR attenuated the down-regulation of Glut1 expression in NSCs exposed to HG for 72 h. These results suggest that the activation of polyol pathway plays a role in the induction of oxidative stress which alters Glut1 expression and cell cycle in NSCs exposed to HG, thereby resulting in abnormal patterning of the neural tube in embryos of diabetic pregnancy.

Identification of differentially expressed genes in fetal rat forebrain exposed to a teratogen by cDNA microarray analysis.

In an attempt to understand the molecular basis underlying the neural tube defects induced by the teratogen, cyclophosphamide (CP), cDNA microarray analysis was carried out in neural tubes of embryos derived from normal and CP-treated rats. Genes found to have altered expression levels in CP-treated group were clustered into groups on the basis of their biological functions. The expression profile of different genes involved in transcription of molecules related to cell adhesion, inflammation, metabolism and neurotrophic factors pathways as well as in still undefined processes was differentially affected by the teratogen treatment. The most remarkable change was the up-regulation of genes related to an inflammatory process dominated by the fetal brain macrophages viz. amoeboid microglia. Amoeboid microglia/brain macrophage expansion, based on gene expression and histological analysis, was found to be vigorous at the subventricular region. The present results suggest that a vigorous inflammatory response involving amoeboid microglia/brain macrophages primarily is an important component in CP-induced prenatal development disorder.

High glucose alters the expression of genes involved in proliferation and cell-fate specification of embryonic neural stem cells.

AIMS/HYPOTHESIS: Maternal diabetes induces neural tube defects during embryogenesis. Since the neural tube is derived from neural stem cells (NSCs), it is hypothesised that in diabetic pregnancy neural tube defects result from altered expression of developmental control genes, leading to abnormal proliferation and cell-fate choice of NSCs. MATERIALS AND METHODS: Cell viability, proliferation index and apoptosis of NSCs and differentiated cells from mice exposed to physiological or high glucose concentration medium were examined by a tetrazolium salt assay, 5-bromo-2'-deoxyuridine incorporation, terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling and immunocytochemistry. Expression of developmental genes, including sonic hedgehog (Shh), bone morphogenetic protein 4 (Bmp4), neurogenin 1/2 (Neurog1/2), achaete-scute complex-like 1 (Ascl1), oligodendrocyte transcription factor 1 (Olig1), oligodendrocyte lineage transcription factor 2 (Olig2), hairy and enhancer of split 1/5 (Hes1/5) and delta-like 1 (Dll1), was analysed by real-time RT-PCR. Proliferation index and neuronal specification in the forebrain of embryos at embryonic day 11.5 were examined histologically. RESULTS: High glucose decreased the proliferation of NSCs and differentiated cells. The incidence of apoptosis was increased in NSCs treated with high glucose, but not in the differentiated cells. High glucose also accelerated neuronal and glial differentiation from NSCs. The decreased proliferation index and early differentiation of neurons were evident in the telencephalon of embryos derived from diabetic mice. Exposure to high glucose altered the mRNA expression levels of Shh, Bmp4, Neurog1/2, Ascl1, Hes1, Dll1 and Olig1 in NSCs and Shh, Dll1, Neurog1/2 and Hes5 in differentiated cells. CONCLUSIONS/INTERPRETATION: The changes in proliferation and differentiation of NSCs exposed to high glucose are associated with altered expression of genes that are involved in cell-cycle progression and cell-fate specification during neurulation. These changes may form the basis for the defective neural tube patterning observed in embryos of diabetic pregnancies.

Insulin-like growth factor I and II expression and modulation in amoeboid microglial cells by lipopolysaccharide and retinoic acid.

Insulin-like growth factors I and II are known to regulate the development of the CNS. We examined the developmental changes in insulin-like growth factor I and insulin-like growth factor II expression in the postnatal rat corpus callosum. Insulin-like growth factor I and insulin-like growth factor II mRNA expression increased at 3 days as compared with 1 day whereas the protein expression increased up to 7 days. Insulin-like growth factor I and insulin-like growth factor II immunoexpression was specifically localized in round cells confirmed by double immunofluorescence with OX-42 to be the amoeboid microglial cells. Insulin-like growth factor I expression was observed up to 7 days in amoeboid microglial cells while insulin-like growth factor II expression was detected in 1-3 day old rats. Exposure of primary rat microglial cell cultures to lipopolysaccharide increased insulin-like growth factor I and insulin-like growth factor II mRNA and protein expression significantly along with their immunoexpression in microglial cells. The lipopolysaccharide-induced increase in insulin-like growth factor I and insulin-like growth factor II mRNA and protein expression was significantly decreased with all-trans-retinoic acid. We conclude that insulin-like growth factor I and insulin-like growth factor II expression in amoeboid microglial cells in the developing brain is related to their activation. Once the activation is inhibited, either by transformation of the amoeboid microglial cells into ramified microglia regarded as resting cells or as shown by the effect of all-trans-retinoic acid administration, insulin-like growth factor I and insulin-like growth factor II mRNA and protein expression is downregulated.

Differential expression of cytokines in the rat heart in response to sustained volume overload.

OBJECTIVE: The present study aimed to investigate whether sustained volume overload is capable of inducing persistent upregulation of cardiac cytokines including tumor necrosis factor alpha (TNF)-alpha, interleukin (IL)-1beta, interleukin (IL)-6 and transforming growth factor (TGF)-beta(1). METHODS AND RESULTS: Volume overload-induced heart hypertrophy in rats was established by aortacaval fistula, and the cardiac cytokines were measured in the myocardium from 1 to 4 weeks after operation. In the post-fistula rats, cardiac IL-1beta and IL-6 gene and protein levels were upregulated throughout the time of measurement. Immunohistochemistry demonstrated that IL-1beta and IL-6 immunoreactive cells were widely distributed in the myocardium in the earlier time intervals, and mainly localized in the regions close to the endocardium in the later time intervals. The cardiac IL-1beta immunoreactive cells were mainly localized in the blood vessels whereas the IL-6 positive cells were composed of non-myocytes and cardiomyocytes. TGF-beta(1) positive staining was increased in the myocardium up to 3 weeks after aortacaval fistula and then decreased to basal levels thereafter. In contrast to the activation of cardiac IL-1beta and IL-6 in response to volume overload, TNF-alpha expression appeared unaltered in response to sustained volume overload in the transcription and protein levels. CONCLUSION: The results of the present study indicate that sustained volume overload is capable of inducing persistent upregulation of some cardiac cytokines. In addition, the differential expressions of TNF-alpha, IL-1beta and IL-6 suggest that the induction of IL-6 and IL-1beta is independent of TNF-alpha mediated pathways in this animal model.

Identification of RANKL in osteolytic lesions of the facial skeleton.

RANKL (receptor activator of nuclear factor kappaB ligand) promotes osteoclast differentiation, stimulates osteoclast activity, and prolongs osteoclast survival and adherence to bone. Abnormalities of the RANKL/RANK/osteoprotegerin system have been implicated in a range of diseases, including osteoporosis. To date, no work has been done in osteolytic lesions of the facial skeleton. In this study, specimens of ameloblastomas, dentigerous cysts, odontogenic keratocysts, and radicular cysts were subjected to immunohistochemical analysis for RANKL and tartrate-resistant acid phosphatase (TRAP). Immunofluorescence staining for TRAP was visualized under confocal microscopy. All specimens demonstrated distinct positive immunoreactivity to RANKL and TRAP. The TRAP-positive cells also stained with in situ hybridization for human calcitonin receptor, a definitive marker for osteoclasts. Mononuclear pre-osteoclasts were observed to migrate from blood to the connective tissue stroma and multinucleate toward the bone surface. It can be concluded that RANKL plays a role in bone resorption in osteolytic lesions of the facial skeleton.

Altered gene expression with abnormal patterning of the telencephalon in embryos of diabetic Albino Swiss mice.

AIMS/HYPOTHESIS: Several studies have shown that maternal diabetes increases the risk of congenital malformations in various organ systems including the neural tube. The present study analysed molecular and morphological changes in the forebrain of embryos from diabetic Albino Swiss mice. METHODS: Maternal diabetes-induced morphological changes in the forebrain were examined histologically. Cell proliferation index was assayed by BrdU labelling. In situ hybridisation and quantitative real-time PCR were used to analyse the expression of genes coding for sonic hedgehog ( Shh), Nkx2.1, brain factor-1 ( BF-1) and bone morphogenetic protein-4 ( Bmp4) that control forebrain patterning. RESULTS: There were no distinguishable abnormalities in the forebrain of embryos from diabetic pregnancies on embryonic day 0.5. At embryonic day 11.5, embryos of diabetic pregnancies displayed a fusion and thickening of the ventral telencephalic neuroepithelium and a partial absence of the dorsal telencephalon, indicating a severe patterning defect in the dorsoventral axis of the telencephalon. The cell proliferation index was also higher in the ventral telencephalon of these embryos. Molecular analyses indicated that expression of Shh, Nkx2.1 and BF-1 was increased and their expression domains expanded dorsally in the ventral telencephalon in embryos of diabetic mice at embryonic day 11.5. The expression of Bmp4 was reduced in the dorsal forebrain of these embryos. At embryonic day 8.5, only Shh expression was increased. CONCLUSIONS/INTERPRETATION: Altered expression of various genes involved in dorsoventral patterning of the forebrain is associated with forebrain malformations in embryos of diabetic mice.

Expression of macrophage colony-stimulating factor and its receptor in microglia activation is linked to teratogen-induced neuronal damage.

Prenatal exposure to teratogen agents is linked to the pathogenesis of neurodevelopment disorders, but the mechanisms leading to the neurodevelopmental disturbance are poorly understood. To elucidate this, an in vitro model of microglial activation induced by neuronal injury has been characterized. In this connection, exposure of primary microglial cells to the conditioned medium from the neuronal damage induced by teratogen, cyclophosphamide, is accompanied by a reactive microgliosis as assessed by reverse transcription-polymerase chain reaction, enzyme-linked immunosorbent assay, lectin histochemistry, double labeling immunohistochemistry and in situ hybridization. Our results showed that reactive microglia were capable of releasing various cytokines such as tumor necrosis factor-alpha, interleukin-1, interleukin-6, transforming growth factor-beta and nitric oxide. Also, we have shown that macrophage colony-stimulating factor (M-CSF) was in fact produced by the reactive microglia. Concomitant to this was the increased expression of M-CSF receptor in these cells following the teratogen-induced neuronal injury. The up-regulation of M-CSF receptor suggests that the cells are capable of responding to self-derived M-CSF in an autocrine fashion. Results with antibody neutralization further suggest that microglial proinflammatory response, as manifested by cytokine expression in culture, is mediated by M-CSF, which acts as a molecular signal that initiates a microglial reaction. We therefore suggest that microglial activation following cyclophosphamide treatment is not only a response to the neuronal damage, but is also a cause of the damage during pathogenesis of neurodevelopment disorders. To this end, the increased expression of M-CSF and its receptor on microglia would be directly linked to the active cell proliferation and proinflammatory response in the teratogen-induced injury.

Retinoic acid influences Phox2 expression of cardiac ganglionic cells in the developing rat heart.

We have studied the expression of the homeodomain transcriptional neuronal regulators Phox2a, Phox2b and the non-neuronal Schwann cell response using the marker S-100 in the differentiating phase of cardiac ganglionic cells in rat embryos following exogenous retinoic acid (RA) treatment of pregnant dams. In control embryos, the expression of Phox2b (E11) preceded that of Phox2a, which, along with the terminal neuronal differentiation marker PGP9.5, was expressed from E12 onwards. Phox2b expression remained unchanged in the differentiated phase of cardiac ganglionic cell development after RA treatment, whereas the population of cells expressing Phox2a, PGP9.5 and S-100 was diminished. These results suggest that RA disrupts the differentiation of cardiac neural crest cells into ganglionic cells destined to contribute to the parasympathetic innervation of the heart, by regulating the expression of Phox2a and Phox2b.

Retinoic acid influences the expression of the neuronal regulatory genes Mash-1 and c-ret in the developing rat heart.

We analyzed the expression of neuronal regulatory genes Mash-1 and c-ret by immunohistochemistry and reverse transcriptase-polymerase chain reaction in the developing heart of rat embryos following exogenous retinoic acid (RA) treatment of the pregnant dams. On E12, expression of Mash-1 and c-ret was confined to cells migrating via the common cardinal vein. On E16.5, Mash-1 and c-ret expression were restricted to cardiac ganglia around the great vessels and posterior atrial wall. While Mash-1 expression was down-regulated at birth, that of c-Ret was maintained. RA-treated hearts showed a down-regulation of both Mash-1 and c-Ret at the mRNA as well as at the protein level on E16.5. The present results show that differentiation of cardiac ganglionic cells is affected after RA treatment, by the down-regulation of Mash-1 and c-Ret.