The problem of cognitive impairment resulting from diabetes is gaining more acceptance and attention. Both type 1 and type 2 diabetes mellitus have been proved to be associated with reduced performance on numerous domains of cognitive function. Although the exact mechanisms of cognitive impairments in diabetes have not been completely understood, hyperglycemia and insulin resistance seem to play significant roles. And other possible risk factors such as hypoglycemia, insulin deficiency, vascular risk factors, hyperactive HPA axis, depression, and altered neurotransmitters will also be examined. In the meanwhile, this review analyzed the role of the active ingredient of Chinese herbal medicine in the treatment of diabetes cognitive impairments. Diabetes mellitus (DM), characterised by chronic hyperglycemia, is one of the most important and prevalent chronic diseases . The preDiabetes is associated with various risk factors that may influence cognitive functioning, including diabetes-specific factors , vascular risk factors that are linked to diabetes , and complications of (micro)vascular disease . Although the mechanisms underlying the development of cognitive dysfunction in diabetes have not been completely elucidated, hypotheses such as oxidative stress and insulin/insulin receptor-mediated signal transduction are gaining more attention. In this review, we will highlight the probable risk factors and the possibly involved mechanisms relating cognitive dysfunction in diabetes.It is well established that hyperglycemia, a shared characteristic in both type 1 diabetes mellitus (T1DM) and T2DM, plays an important role in cognitive decline in diabetes. There is a lot of cross-sectional and longitudinal studies that have shown that there is a close relationship between high blood glucose and cognitive decrements or dementia. A cross-sectional study related to young children with T1DM diagnosed in preschool age found similar performance between the diabetic and the healthy subjects in cognitive tests, but poor glycemic control with higher glycosylated hemoglobin (HbA1c) was related to lower general cognitive abilities, slower fine motor speed, and lower receptive language scores, indicating that young children with T1DM already demonstrate some negative cognitive effects in association with chronic hyperglycemia . StreptoSimilar evidence exists from cross-sectional studies in patients with type 2 diabetes. As a research found that T2DM was associated with diminished cognitive function in each cognitive domain except for memory functions and the cognitive decline was significantly associated with HbA1c and duration of DM . And othIn view of the effect of chronic hyperglycemia on brain function, structure and micrangium, it may be not too much to hypothesize that hyperglycemia is a determinant of cognitive decline in patients with diabetes . AlthougOxidative stress has been implicated as a contributor to both the onset and the progression of diabetes and its associated complications. Hyperglycemia can cause an increase in generation of reactive oxygen species (ROS), reactive nitrogen species (RNS), and oxidative stress markers, such as lipid peroxidation and protein oxidation, with an accompanying decrease in antioxidant levels, which have been suggested to be responsible for the induction of cognitive deficits often observed in diabetic rats , 22. AndHyperglycemia can also increase polyol pathway flux, enhancing the transformation of intracellular glucose into sorbitol via aldose reductase. Sorbitol cannot outflow the cell freely because of strong polarity, leading to the accumulation of sorbitol in cell . EvidencIn addition, hyperglycemia can contribute to the increased production of advanced glycated end products (AGEs) . The celα was shown to be significantly increased in brain, compared with the treated diabetic rat and control rats [Finally, hyperglycemia can lead to the activation of the diacylglycerol (DAG) protein kinase C (PKC) pathway . The PKCrol rats .However, the above mechanisms are not independent of each other. Oxidative stress may trigger a cascade of events leading to neuronal damage and ultimately affect the cognition in diabetes. For example, in STZ-induced diabetic rats, levels of prooxidant compounds were all increased in the hippocampus, including RAGEs, galectin-3 (a proatherogenic molecule), and aldose reductase (the key enzyme in polyol pathway); whereas activity of the glycolytic enzyme glyceraedehyde-3-phosphate dehydrogenase (an antioxidase) was decreased, indicating elevated superoxide levels. When antioxidant, dehydroepiandrosterone, was administered to diabetic rats, this oxidative imbalance was improved significantly, reinforcing that oxidative stress plays a role in diabetes-related neuronal damage .Otherwise, benefits of aggressive glucose management in type 2 diabetes are less clear. The Memory in Diabetes (MIND) substudy of the Action to Control Cardiovascular Risk in Diabetes (ACCORD) trial showed no benefit of intensive glucose-lowering therapy on cognitive function or total brain volume in a large population of people with type 2 diabetes during 40-month follow-up . SimilarHypoglycemia is a well-known complication of glucose-lowering therapy and severe hypoglycemic episodes may have detrimental effects on the brain. It is well established that an extended episode of profound hypoglycemia, such as one with a blood glucose level below 1.0 mmoL per liter, can induce massive cerebral energy failure with a corresponding development of neuronal necrosis . Less seIn contrast, The Diabetes Control and Complications Trial/Epidemiology of Diabetes Interventions and Complications (DCCT/EDIC) did not find evidence for an association between the frequency of severe hypoglycemic episodes and declined cognition in young adult patients (mean age 27 years) with type 1 diabetes . SimilarIt seems that patients who are exposed to chronic or recurrent hypoglycemia may become remarkably tolerant to the state, so that the long-term effects of repeated hypoglycemic episodes on cognition are controversial . In addiThe absolute deficiency of insulin and insulin resistance are the basic pathophysiologic changes of type 1 and type 2 diabetes, respectively. The role of insulin deficiency and insulin resistance in the development of cognitive impairment in diabetes is gaining an increasing concern.Inverse to traditional ideas, insulin crosses the blood-brain barrier and might even be produced locally in the brain . InsulinInsulin is an important neurotrophic factor, nevertheless, hyperinsulinism is not good news for diabetic patients. Furthermore, there is no doubt that insulin is neurotrophic in moderate concentration, but the whole circumstances become more complicated in conditions of hyperinsulinism in diabetes. There is increasing evidence linking insulin resistance to cognitive decline and dementia in diabetes , 52.β-peptide (Aβ) [β and tau protein, which are the main components of senile plaques and neurofibrillary tangles (NFTs), respectively, are the pathological hallmark of AD.For one thing, insulin resistance, resulting in compensatory hyperinsulinemia in type 2 diabetes, is associated with the abnormity of insulin/insulin receptor-mediated signal transduction. One of the key enzymes is glycogen synthase kinase 3 (GSK3), which is inactivated by phosphorylation in insulin/insulin receptor-mediated signal transduction, and generally opposes the actions of insulin. The impaired insulin/insulin receptor-mediated signaling pathway lead to activation of GSK3 by dephosphorylated, which is associated with the hyperphosphorylation of tau and increased production of amyloid-ide (Aβ) , 54. Aβ β clearance due to competition for common principal scavenger, insulin degrading enzyme (IDE) [β degradation. Since the affinity for the binding of insulin to IDE is much greater than the one for the Aβ peptide, hyperinsulinemia may lead to reduced Aβ peptide depurative mechanisms and accumulated concentration in the brain [For another, too much insulin in the brain may be associated with reduced Ame (IDE) . IDE is he brain .β peptide in brain indirectly via IDE or via insulin/insulin receptor signal pathway, leading to AD-like pathology and cognitive impairment. Actively improved insulin sensitivity may be favorable not only for blood glucose decreasing but also for cognitive improvement.To sum up, deficiency of insulin secretion, the characterization of type 1 diabetes, may lead to the declined nutrition of neurons in the brain and eventually cause neuronal atrophy, apoptosis, and necrosis and cognitive decline. However, too much complement of exogenous insulin increases the risk of hypoglycemia, which is dangerous to cognitive function. Insulin resistance in type 2 diabetes can result in high phosphorylation of tau protein and the accumulation of ADiabetes is often accompanied by vascular risk factors, such as hypertension, hypercholesterolemia, and obesity, which might also affect the brain and cognition. The Framingham study has shown that hypertension was a significant risk factor for poor cognitive performance in T1DM patients during the followed 28–30 years . AnotherHowever, not all the studies have the coincident results. In fact, there are studies that actually reported that high blood pressure and high cholesterol were associated with a decreased risk of dementia , 62. OneHowever, how these risk factors may exert their influence is not well understood. But it is well established that the vascular risk factors of diabetes are enhancing the risk of vascular diseases, decreasing cerebral blood flow, and activating inflammation, which may be responsible for the development and progression of cognitive disorders in diabetes.α (TNF-α) [Vascular risk factors are typically thought to produce deleterious effects on brain function via overt strokes. Patients with diabetes have a 2- to 6-fold increased risk in stroke . And dia (TNF-α) –70.In conclusion, the majority of cross-sectional and longitudinal studies have confirmed the association between vascular risk factors and cognitive decrements in diabetes. However, according to the complex circumstances in diabetes with vascular risk factors, it is not sure about how these factors contribute their role to the development of cognitive impairment.The hypothalamic-pituitary-adrenal (HPA) axis is a complex set of interactions between the hypothalamus, the pituitary gland, and the adrenal or suprarenal glands. While exposure to acute stress facilitates memory formation and consolidation, chronic stress or chronic exposure to stress levels of glucocorticoids impairs cognitive performance. Impairment in hypothalamic-pituitary-adrenal (HPA) axis function is proposed to participate in the experimental models of type 1 and type 2 diabetes, as well as diabetic subjects in poor glycemic control . AlthougFirstly, there is evidence that chronic overexposure to corticosterone, one kind of the glucocorticoids, can lead to impaired cognition . In diabIn addition, high corticosterone can impair synaptic plasticity and cause deficits in cognitive function , 76. TheFinally, there are several studies in human, which are with poorly controlled diabetes and cognition decline, show hyperactivation of the HPA axis and elevated circulating cortisol , 79. AndIt is therefore possible that HPA axis dysfunction and/or exposure to elevated glucocorticoid levels may contribute to the cognitive deficits in diabetes. Although the cause and effect have not been determined in many of these situations, these findings may place impairment in HPA axis function at the important area of diabetes-induced cognitive impairment.Researches show that patients with diabetes double the odds of comorbid depression compared to nondiabetic persons , 83, whiThe exact nature of the relation between diabetes and depression is unclear. There is study that suggests that depression in diabetes may disturb the balance of brain-derived neurotrophic factor (BDNF), causing cognitive decline or dementia . AnotherAlthough depressive symptoms may affect cognitive performance, the majority of studies assessed cognition in relation to diabetes controlled for the potential confounding effects of depression. It can be concluded that depression is not the sole risk factor in diabetes-associated cognitive decrements.Altered neurotransmitter function has been observed in diabetic rodent models and may also contribute to cognitive dysfunction. Correlations between lifetime glycemic control and increased frontal brain glucose and neurotransmitter concentrations measured with proton magnetic resonance spectroscopy in adults with type 1 diabetes do suggest an underlying pathophysiological change . People Cholinergic neurotransmission is a crucial process underlying memory and cognitive function. One of the most important mechanisms responsible for correct cholinergic function is performed by enzyme choline esterase (ChE). Several studies have found an increased ChE activity in brain and consequently can be associated with cognitive impairments in diabetic rats , 91. On Traditional Chinese Herbs are becoming the important sources for the development of antidiabetic drugs. A wealth of studies have described the effects of constituents from herbs or plants on diabetes-associated cognitive deficits in rodents by several aspect. There are more and more reports concerning the therapeutic function of the effective component of traditional Chinese herbs in diabetes cognitive impairments.Salvia miltiorrhiza Bunge, is widely used in China, Korea, Japan, and other Asian countries in the treatment of cerebrovascular diseases. C57BL/6 mice with streptozotocin injected intraperitoneally were administered with sodium Danshensu by gavage once a day for 12 weeks. The western blot analysis revealed that Danshensu partly blocked the expression of RAGE, p-p38, and COX-2 and NF-κB activation and inhibited the increase of TNF-α, IL-6, and prostaglandin E2 (PGE2) in the hippocampus tissues of the diabetic mice [Danshensu, a compound from tic mice . In the tic mice . This stAnemarrhena asphodeloides Bunge, is a well-known Chinese Materia Medica. The learning ability of the diabetic rats treated with total saponins from Rhizoma Anemarrhenae was significantly augmented in the Morris water maze test compared with the other diabetic rats. And the experimental rats showed both reduced Aβ(1–40) and Aβ(1–42) levels in brain. In the meanwhile, Rhizoma Anemarrhenae decreased the blood glucose level and increased the body weight of the diabetic rats [β(1–40), Aβ(1–42), and the blood glucose levels of STZ-induced diabetic rats, which maybe was one of the reasons for the prophylactic efficacy of Rhizoma Anemarrhenae to diabetes-associated cognitive decline.Rhizoma Anemarrhenae, the dried rhizome of tic rats . This reA growing body of literatures suggests that oxidative stress and inflammation are involved in the pathogenesis of late diabetes complications. Hyperglycemia reduces antioxidant levels and concomitantly increases the production of free radicals. Hyperglycemia is also associated with the enhanced inflammatory response , 98. EviPanax ginseng C. A. Mey., shows a remarkable antidiabetic effect. Chronic Re treatment decreased escape latency and increased percentage of time spent in the target quadrant of the diabetic animals. Furthermore, Re treatment reduced the levels of TNF-α and malondialdehyde  in brain areas, as well as enhanced GSH levels in serum of diabetic rats, compared to the untreated diabetic rats. Meanwhile, chronic Re treatment markedly decreases the fasting blood glucose level of diabetic rats [Ginsenoside Re (Re), a triterpenoid saponin compound derived from tic rats . In thisCoptis chinensis. Chronic treatment with berberine improved cognitive performance, lowered hyperglycemia, decreased lipid peroxidation, and increased the reduced glutathione of the diabetic animals [Withania somnifera and Aloe vera lowered the blood glucose level, reduced the oxidative damage, and improved memory impairment and motor dysfunction of diabetic rats [Berberine is an isoquinoline alkaloid, which is mainly found in  animals . Therefo animals . Chronic animals . Chronictic rats .Diabetes gradually impairs hippocampus-dependent cognitive functions and synaptic plasticity , 104. PaCholinergic neurotransmission is a crucial process underlying memory and cognitive function. Chronic treatment with lycopene could prevent the rise of AChE activity in the cerebral cortex of STZ-treated diabetic rats . Berberiβ in brain, anti-inflammation, antioxidation, restoring hippocampal synaptic plasticity, antiapoptotic, and protecting the cholinergic function. Most of the active ingredients of Traditional Chinese herbs could improve the blood glucose levels except Danshensu, which did not affect the levels of blood glucose of the diabetic animals. Since that chronic treatment with Danshensu had not regulate blood glucose in diabetic mice, partial restoration of cognitive functions observed in diabetic animals in that research may not be associated with the ability of attenuating hyperglycemia. At present the multifactorial pathogenesis of cognitive and memory impairments in diabetes has not yet completely been understood. Whether the other Traditional Chinese Herbs which have the inhibitory effect on the elevations of the blood glucose levels directly improved diabetes-induced cognitive impairments or indirectly through the correction of hyperglycemia have not been identified.The beneficial effects of the active ingredient of traditional Chinese herbs observed in all these studies above could be multivariate. The active ingredient of Traditional Chinese herbs could ameliorate the diabetes cognitive dysfunction by attenuating AGEs mediated neuroinflammation, reducing the accumulation of AIn conclusion, there have been significant gains in our understanding of the effect of diabetes on cognitive impairment. As substantial researches suggest that diabetes increases the risk of mild cognitive impairment and hastens the progression to dementia, cognitive impairment should be listed as one of the chronic complications of diabetes. Lots of risk factors such as hyperglycemia, hypoglycemia, insulin deficiency, insulin resistance, vascular risk factors, HPA axis dysfunction, depression, and altered neurotransmitters have been proved to be associated with cognitive decline in diabetes. It is unlikely that any single factor is solely responsible for the development of cognitive deficits in diabetic subjects. Rather, these factors may act in additive or synergistic ways to impair neuronal homeostasis, increase neuronal vulnerability, and eventually contribute to cognitive decline. The pathogenesis of cognitive dysfunction in diabetes is only partially understood. Chronic hyperglycemia-mediated oxidative stress and insulin resistance associated abnormity in insulin/insulin receptor signal pathway are the focus in the current researches.Although much research has been done on the influence of diabetes mellitus on cognitive function, still many questions remain. Firstly, it is not clear whether cognitive impairments seen in cognitive testing result in meaningful deficits in the daily life of the patients as studies show relatively light to mild decrements in cognitive functioning, which slowly progress over time. Given the subjective nature of assessing livelihood activities, it may be difficult to address this question. However, it can be inferred that the impact of cognitive impairment on diabetic patients will become more outstanding following the enhanced average life span, increased requirements for life quality, and raising prevalence of both diabetes and dementia around the world. Hence another key target for future studies is to find ways to prevent diabetes-associated cognitive dysfunction, particularly for dementia. Now that clinical and experimental researches have found some important risk factors in cognitive decline in diabetes; further researches need to verify if improving these risk factors, for example, improving insulin sensitivity, can decrease or prevent the occurrence of cognitive decline and dementia. Surprisingly, diabetes might not increase the rate of cognitive decline over time. When people aged 56–80 with diabetes were followed up for 4 years, their overall cognitive decline, measured by a comprehensive battery of cognitive tests, did not differ significantly from that of age-matched participants without diabetes ; it showMoreover, we should gain more insight into the etiology and mechanisms that drive accelerated cognitive decline in diabetes, which is important in treatment of dementia or cognitive decline in diabetes. Although oxidative stress is gaining acceptance as the key mechanism of peripheral neuropathy of diabetes, whether it is the same situation in central neuropathy complication of diabetes is unascertained. And as the association between AD and insulin resistance syndrome is gaining attention , insulin


We determined the characteristic features of synovial tissues of rheumatoid arthritis (RA) patients treated by TNF inhibitors in order to delineate their mechanism of action. Synovial tissues were obtained during the joint surgical operations from 12 RA patients who had been treated with TNF inhibitors in addition to disease modifying antirheumatic drugs (DMARDs) for at least 5 months (5–25 months) (RA-TNFinh), and from 12 RA patients who had been treated with DMARDs alone (RA-DMARD), and were evaluated under light microscopy. There were no significant differences in disease duration, serum CRP levels, DAS28, Steinbrocker’s stages on X-ray and treatment regimen except for TNF inhibitors between RA-TNFinh and RA-DMARD. The most prominent changes in the synovium from RA-TNFinh were discoid fibrosis in the subliming layers of the synovium with degeneration and detachment of synoviocytes and marked decrease in vasculatures. There was no significant difference in these synovial features between RA patients with infliximab and those with etanercept. Interestingly, appearance of osteoclasts was observed in RA-TNFinh (3 out of 12 patients) and in RA-DMARD (1 out of 12 patients). These results indicate that not only infliximab, but etanercept might have direct actions on synovial cells in the deep lining layers of the synovium, leading to the discoid fibrosis thereof. Moreover, the data confirm that the deep lining or sublining layers of the synovium are the most important portions that steer the disease process of RA synovitis. Rheumatoid arthritis (RA) is a chronic inflammatory disease characterized by hyperplasia of synovial tissues, leading to the destruction of joint structures . Thus, jTNF inhibitors, including anti-TNF-α antibodies and soluble TNF receptors, have brought epoch-making impacts on treatment for RA . SeveralSynovial tissues were obtained from 12 RA patients who had been treated with TNF inhibitors  for at least 5 months (5–25 months) during the joint surgical operations. As controls, synovial tissues were similarly obtained from 12 RA patients who had not received TNF inhibitors. Most of the operations were total joint replacement orthopedic surgery of hip, knee or elbow joints, as shown in Table Synovial tissues were fixed in formaldehyde and embedded in paraffin. The sections were stained by hematoxylin and eosin, Masson’s trichrome, immunohistochemistry with anti-CD68 monoclonal antibody (clone KP-1) and tartrate-resistant alkaline phosphatase (TRAP), followed by evaluation under the light microscopy.As shown in Table The most prominent change in the synovium from RA patients with TNF inhibitors (infliximab and etanercept) was discoid fibrosis in the sublining layers, which was almost absent in the synovium of control patients without TNF inhibitors Fig. . By contIn some portions, several characteristic changes appear to precede the formation of the discoid fibrosis in the synovium of patients with TNF inhibitors, including (1) pyknosis of surface synovial cells, (2) hydropic degeneration of surface synovial cells, 3) vacuolization in the deep lining layers and 4) sclerosis of small vasculature in the subsynovial space  and in those without TNF inhibitors (1 out of 12 patients). Notably, appearance of these giant cells is usually accompanied by cartilage debris clearance and bone formation Fig. d. The giTable The current studies have disclosed the characteristic features of synovial tissues of RA patients who had been treated with TNF inhibitors in comparison with those of RA patients with DMARDs alone. There were no significant differences in disease duration, serum CRP levels, DAS28 (DAS28-CRP with 3 variables), Steinbrocker’s stages on X-ray and Steinbrocker’s functional classes between both groups. It should be pointed out that all the patients in both groups had significant structure damages that required surgery. Therefore, it is most likely that both groups presented comparable RA disease activities. Moreover, there were no significant differences in treatment regimen between both groups, except for the use of TNF inhibitors. There is no difference between etanercept and infliximab, but similar changes could not be observed in patients given tocilizumab. Thus, discoid fibrosis in the sublining layers was observed in none of the 5 patients with tocilizumab (data not shown). Therefore, it is most likely that the histopathological features in the synovium of the RA patients with TNF inhibitors might be caused by TNF inhibitors themselves. Nonetheless, further studies with a larger number of patients with tocilizumab or abatacept would be important for the confirmation of the characteristic histopathological features in the synovium of the RA patients with TNF inhibitors. It would be also intriguing to compare the patients with TNF inhibitor alone and those with TNF inhibitors and MTX, since it is now well known that TNF inhibitor monotherapy is not enough to prevent joint destruction .The most prominent features of synovial tissues in RA patients treated with TNF inhibitors were discoid fibrosis in the sublining layers with various types of degeneration and detachment of synoviocytes in the deep lining layer, which could be observed in all the patients treated with TNF inhibitors. Discoid fibrosis appeared to be associated with vacuolation of degenerating synovial cells. There was no significant difference in the synovial histological features between RA patients with infliximab and those with etanercept. It is suggested that degenerating synoviocytes might be type B synoviocytes, which have been demonstrated to be located in the deep lining layers . NotablyThe other characteristic feature is the loss, narrowing or obstruction and fibrosis of vasculatures in the sublining layers, which could not be observed in the synovial tissues of RA patients treated with DMARDs alone. There, the preservation of vasculatures was clearly observed in the synovial tissues, but around them, synovial cell proliferation and inflammatory cell infiltration were markedly decreased or even disappeared and even covered with diffuse fibrosis. Therefore, the loss or obstruction of vasculatures due to TNF inhibitors could not be accounted for by decreased production of angiogenic cytokines, such as vascular endothelial growth factor (VEGF). In this regard, we previously demonstrated that angiogenesis took place prior to the proliferation of synovial lining cells or infiltration of inflammatory cells in the synovial tissues of very early stages of RA . The resWe have previously disclosed that abnormalities in bone marrow (BM) CD34+ cells are involved in the angiogenesis in the RA synovium. Thus, RA BM CD34+ cells have enhanced capacities to differentiate into endothelial cells in relation to synovial vascularization . The enhIn some of the RA synovial tissues, the formation of multinucleated giant cells was observed, especially in patients treated with TNF inhibitors. Some of the giant cells appeared in the synovial lining layers to be a result of degenerative process, but others with multiple nuclei were TRAP-positive, indicating that they are osteoclasts. The mechanism of the formation of osteoclasts especially in the synovium of patients with TNF inhibitors remains uncertain. It should be noted that infliximab, but neither anti-TNF receptor p55 antibody nor anti-TNF receptor p75 antibody, enhances osteoclast formation from human osteoclast progenitors in vitro . TherefoIn summary, the current studies have disclosed that TNF inhibitors result in the degeneration and disappearance of synovial cells in the deep lining layers, leading to the discoid fibrosis of characteristic morphology in the sublining layers. Moreover, the loss, narrowing or obstruction of vasculatures in the sublining layers is another characteristic feature of the synovium in RA patients treated with TNF inhibitors. These data confirm that the deep lining or sublining layers of the synovium are the most important portions that steer the disease process of RA. To elucidate the role of TNF-α in detail especially in these regions would be helpful for the complete understanding of the pathogenesis of RA.


BCL2 in osteoblasts reduces the number of osteocyte processes, probably due to the function of Bcl2 that modulates cytoskeletal reorganization, and induces the apoptosis of osteocytes, in which the transgene expression was reduced, presumably caused by an insufficient supply of oxygen, nutrients, and survival factors due to the reduced osteocyte processes. Our BCL2 transgenic mouse with accumulated dead osteocytes is a useful model to analyze the function of osteocytes, because a repair process, which replaces dead osteocytes with new osteocytes by bone resorption and formation, was not evident in the mice irrespective of the massive accumulation of dead osteocytesDisuse osteoporosis, which occurs commonly in prolonged bed rest and immobilization, is becoming a major problem in modern societies; however, the molecular mechanisms underlying unloading-driven bone loss have not been fully elucidated. Bone adjusts its shape and strength against mechanical stress. Osteocytes are the most abundant cells in bone and comprise the communication system through the processes and canaliculi throughout bone. The osteocyte network is considered to be an ideal mechanosensor and mechanotransduction system. We found that overexpression of BCL2 transgenic mice. Pyruvate dehydrogenase kinase isozymes  are negative regulators of pyruvate dehydrogenase complex (PDC), which converts pyruvate to acetyl-CoA in the mitochondria, linking glycolysis to the energetic and anabolic functions of the tricarboxylic acid (TCA) cycle. Pdk4 was upregulated in femurs and tibiae of wild-type mice but not of BCL2 transgenic mice after tail suspension. Bone in Pdk4-/- mice developed normally and was maintained. At unloading, however, bone mass was reduced due to enhanced osteoclastogenesis and Rankl expression in wild-type mice but not in Pdk4-/- mice. Osteoclast differentiation of Pdk4-/- bone marrow-derived monocyte/macrophage lineage cells (BMMs) in the presence of M-CSF and RANKL was suppressed, and osteoclastogenesis was impaired in the coculture of wild-type BMMs and Pdk4-/- osteoblasts, in which Rankl expression and promoter activity were reduced. Further, introduction of Pdk4 into Pdk4-/- BMMs and osteoblasts enhanced osteoclastogenesis and Rankl expression and activated Rankl promoter. These findings indicate that upregulation of Pdk4 expression in osteoblasts and bone marrow cells after unloading is, at least in part, responsible for the enhancement of osteoclastogenesis and bone resorption after unloading [We searched for the molecules responsible for disuse osteoporosis using


Metabolism of alcohol  is regulated by genetic and environmental factors as well as physiologic state. For a given alcohol intake, the rate of alcohol clearance, which ultimately determines tissue ethanol concentrations, may be the most significant risk factor for many of the detrimental effects of alcohol. Faster ethanol clearance would help minimize target tissue concentrations, and in pregnant women, mitigate fetal alcohol exposure. Much remains to be known about the effects of the altered endocrine milieu of pregnancy on alcohol metabolism and clearance in the mother. Research has shown that among pregnant rats allowed unrestricted access to alcohol and those fed alcohol containing liquid diets under experimental conditions via a feeding tube , urine ethanol concentrations (and thus blood and tissue ethanol concentrations) are lower in pregnant rats compared with non-pregnant females given the same dose of ethanol. Maternal nutritional status also is an important determinant of fetal alcohol toxicity. Research using the TEN system has demonstrated that alcohol-induced fetal growth retardation is potentiated by undernutrition in part via impaired alcohol metabolism and clearance. Although the harmful effects of alcohol  on the growing fetus have been recognized for nearly three decades, alcohol continues to be the most common malformation-causing chemical  ingested during pregnancy . One of Pregnancy presents a unique endocrine and metabolic circumstance not found in any other physiologic state with increased nutritional requirements. In addition, the physiologic and metabolic changes of pregnancy can result in altered metabolism of drugs and other chemicals that could result in altered drug efficacy . WhereasRodents serve as good experimental models of human metabolic and endocrine events of pregnancy. By far, most studies of chronic alcohol effects in rodents have been conducted using alcohol-containing liquid diets, such as that developed by Undernutrition is a particular problem in studies of alcohol consumption during pregnancy because of the increased nutrient requirements imposed by the growing fetal–placental unit. Feeding the Lieber–DeCarli diets to pregnant rodents resulted in an 18 to 50 percent reduction in gestational weight gain in pair-fed compared with ad libitum–fed mothers  . This ouTo circumvent these problems, investigators have turned to a method of feeding through a tube permanently inserted in the stomach  in which alcohol is infused as a part of a liquid diet a,b. ReseDiets can either be infused throughout the day (over 23 hours) or through the overnight period to mimic the usual eating patterns of the rat that occur during the dark cycle. The system also is amenable to precise caloric intake, leading to three distinct advantages over methods used in previous studies. First, it eliminates distorted findings resulting from pair-wise feeding regimens or lack of sufficient voluntary dietary intake. Second, it presents a robust, highly reproducible, and precise model to control dietary intake. Third, it makes it easy to estimate exposure to ethanol.Using the intragastric infusion model, Alcohol is predominantly broken down in the liver via the alcohol dehydrogenase (ADH) enzyme system. The ADH gene family produces  enzymes that metabolize a wide variety of substances , including ethanol, vitamin A, other simple alcohols , hydroxysteroids, and products of the degradation of fat compounds . At least five different classes (classes I through V) of ADH have been described in humans, and an additional two classes (classes VI and VII) have been found in other species such as the rat and chicken, respectively . These AAlcohol toxicity is strikingly evident in a variety of fetal tissues; the brain, skeleton, and liver are the most affected. No single mechanism has been sufficient to account for these varied effects, and it is likely that multiple factors are involved. Researchers have suggested a large number of possible mechanisms, including indirect effects of alcohol on maternal blood supply to the fetus ; direct effects of alcohol on cell division and cell death; the composition and fluidity of the cell membranes; and an imbalance in the cell’s oxidative status that can lead to cell damage, lipid peroxidation, effects on growth factors , and alcohol-mediated inhibition of cell adhesion. However, most studies have concluded that the toxic effects of alcohol are related to the circulating/peak concentrations of alcohol. Two lines of evidence support this.ADH2*2 gene variant  encodes the isozymes containing the, β2 subunit that oxidizes alcohol 40 times faster in vitro than the, β1 subunit (encoded by ADH2*1), people with the ADH2*2 allele have higher rates of alcohol clearance. A comparison of the ADH2*2 allele frequencies of mothers and FASD children revealed that mothers carrying the ADH2*2 allele had a lower incidence of FASD children than mothers without the allele. One of the mechanisms of this protection could be faster metabolism of alcohol attributed to the high-activity enzymes  in pregnant mice increased the embryotoxicity of alcohol, suggesting that alcohol, rather than its metabolites, is responsible for its toxicity . Further enzymes . TherefoResearch with animal models has investigated the effects of pregnancy on alcohol metabolism, the influence of nutrition on this effect, and the potential metabolic mechanisms involved. In one study, pregnant rats given an intravenous dose of 0.75 g/kg of ethanol showed an increased alcohol clearance rate compared with weight-matched nonpregnant rats . Similar3/4/d),3/4/d normally fed to non-pregnant rats. To address whether, this increased intake of calories affects alcohol metabolism pregnant rats received 220 Kcal/kg3/4/d, whereas nonpregnant rats received either 187 Kcal/kg3/4/d or 220 Kcal/kg3/4/d. While pregnant rats showed increased alcohol clearance, increased caloric intake in nonpregnant rats failed to alter the elimination of alcohol, indicating that changes in alcohol metabolism were due to pregnancy and not increased caloric intake  are lower in pregnant rats than nonpregnant females given the same dose of ethanol . Pregnan3/4/d) compared with none of the dams fed adequate nutrition and the same dose of alcohol. Undernourished pregnant dams also had greater fetal toxicity, as indicated by reduced pup numbers, full-litter resorptions, and reduced birth weight. These data suggested a remarkable alcohol–nutrition interaction (Nutrition plays an important role in assuring a normal pregnancy. Although it has been known for years that there is an important interaction between alcohol and nutrient intake during pregnancy on fetal development, the standard rodent models have not been adequate to study the role of maternal nutrition on the detrimental effects of alcohol on the growing fetus. Poor nutrition can have a number of deleterious effects on pregnancy. Alcohol interferes with nutritional supply to the fetal-placental unit  and nutreraction .Several possible mechanisms underlying alcohol–nutrition interactions have been explored. Two principal mechanisms are relevant in this context. First, it is clear that pregnant undernourished rats are unable to break down alcohol as efficiently as pregnant rats that receive adequate nutrition. This puts the fetus at greater risk for the toxic effects of alcohol, as evidenced by the higher amniotic fluid and blood alcohol concentrations. Second, liver ADH1, which is responsible for the majority of alcohol metabolism, also was significantly lower in the undernourished pregnant ethanol-fed dams . AlthougGene expression changes may underlie the combined contributions of alcohol and undernutrition and their interactions in causing fetal toxicity. Using large-scale microarray technology,


MicroRNAs are small non-coding RNAs that suppress gene expression through target mRNA degradation or translation repression. Recent studies suggest that miRNA plays an important role in multiple physiological and pathological processes in the nervous system. In this review article, we described what is currently known about the mechanisms in peripheral nerve regeneration on cellular and molecular levels. Recently, changes in microRNA expression profiles have been detected in different injury models, and emerging evidence strongly indicates that these changes promote neurons to survive by shifting their physiology from maintaining structure and supporting synaptic transmission towards a regenerative phenotype. We reviewed the putative mechanisms involved in miRNA mediated post-transcriptional regulation and pointed out several areas where future research is necessary to advance our understanding of how targeting miRNA machinery can be used as a therapeutic approach for treating nerve injuries. Nerve injuries induce severe disability and greatly compromise the quality of life. Injuries to peripheral nerves, occurring in approximately 2.8% of trauma patients, cause partial or total loss of motor, sensory and autonomic functions , the neuron must shift its physiology from synaptic transmission and maintenance of structure to the growth of the axon  and RNA interference (RNAi) have revolutionized our understanding of post-transcriptional regulation of gene expression and have provided powerful tools for targeted gene silencing.Caenorhabditis elegans by Andrew Fire and Craig Mello, which they termed “RNAi”  transactivating response RNA (TAR) binding protein (TRBP) and in D. melanogaster Dicer complexes with Loquacious  is degraded. Occasionally, both strands give rise to mature miRNA , a ribonucleoprotein complex associated with miRNA. Although, assembly of RISC is a very dynamic process and is not well understood, proteins of Argonaute (Ago) family are considered the most important components of RISC  is one of the conclusively identified subunits. Both miRNA and siRNA can co-immunoprecipitate with FMRP. The FMRP belongs to the fragile X gene family, which encodes three different proteins: FMRP, FXR1, and FXR2. The loss-of-function mutations in the FMRP gene results in fragile X syndrome (FXS), whereas the function of FXR1 and FXR2 remains unknown. However, it is suspected that all three fragile X protein functions similarly in regulating mRNA stability by binding with ribosomes, and FXR1 and FXR2 has the potential to associate with RISC as well  4E. When miRNA is bound to the mRNA 3′ UTR, Ago proteins interact with the 7-methylguanosine cap of the mRNA. The association of Ago, instead of eIF4E, with 7-methylguanosine cap prevents effective recruitment of ribosomes and therefore blocks the initiation of translation , particularly with respect to the regulation of β-amyloid precursor protein converting enzyme1 (BACE1)  is under the control of miRNAs as well. Conditional knockout of *, which target REST and co-REST, respectively  brains revealed dysregulated expression of several miRNAs in both mouse models and human patients , which in turn catalyzes the membrane association of Gα12/13. The membrane association of small G protein subunit Gα13 has been shown to be involved in the activation of RhoA signaling pathway (Siegel et al., Compared with the studies on dendritic miRNAs, there is less understanding in the role of miRNA in axonal functions. However, the studies investigating axonal miRNA pathway are growing. Pure axonal miRNAs have been obtained from superior cervical ganglia neurons cultured in compartmentalized Campenot cell culture chambers. In total, 130 mature miRNAs have been detected in distal axons, and a few, such as, miR-15b, miR-16, and miR-221, were highly enriched in axons (Natera-Naranjo et al., The involvement of the miRNA biosynthetic machinery in the regulation of intra-axonal local protein synthesis after injury was confirmed by the injury-regulated expression of biosynthetic enzymes, including components of RISC and P-bodies (Wu et al., Dicer impaired nerve regeneration according to functional behavioral tests, electrophysiological, and histological analyses in a mouse model of peripheral nerve crush. In addition, Dicer-deficient neurons failed to regenerate axons in DRG cultures after conditioning sciatic nerve lesion. Thus, this study demonstrated that the intact Dicer-dependent miRNA pathway is critical for the successful functional recovery in vivo and the regenerative axonogenesis in vitro (Wu et al., Further studies showed that inducible deletion of in vitro with robo2 and srGAP2 validated as its potential target genes (Zhang et al., Furthermore, a group of miRNAs that are specifically expressed in an injury-regulated pattern in the regenerating sciatic nerves and DRG were identified by miRNA microarrays and confirmed by qPCR (Strickland et al., in vivo and regenerative axon growth in vitro (Wu et al., In the past decade, as one of the major discoveries in the history of molecular and cell biology, the post-transcriptional regulation of gene expression has become a major focus of research. There has been rapid progress in deciphering the mechanisms underlying miRNA pathway. The understanding of miRNA pathway in neuroscience is growing through intensive investigation of a variety of neurological events. Temporally and spatially specific miRNA expression has been identified in neurodevelopment and in neurological diseases (Martino et al., The importance of local protein synthesis for nerve regeneration has been shown by an increase in local translation of proteins after peripheral nerve axotomy, and the observation that inhibiting this synthesis greatly reduces the reproduction of growth cones (Verma et al., The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Dendritic cells (DCs) are important cells of our innate immune system. Their role is critical in inducing adaptive immunity, tolerance, or allergic response in peripheral organs—lung and skin. The lung DCs are not developed prenatally before birth. The DCs develop after birth presumably during the first year of life; exposures to any foreign antigen or infectious organisms during this period can significantly affect DC developmental programming and generation of distinct DC phenotypes and functions. These changes can have both short-term and long-term health effects which may be very relevant in childhood asthma and predisposition for a persistent response in adulthood. An understanding of DC development at molecular and cellular levels can help in protecting neonates and infants against problematic environmental exposures and developmental immunotoxicity. This knowledge can eventually help in designing novel pharmacological modulators to skew the DC characteristics and immune responses to benefit the host across a lifetime. Asthma is a serious pulmonary disease that affects about 300 million people worldwide , and 8.2In allergic asthma, an immune reaction is caused by inhaled allergens with an overwhelming inflammatory response and obstruction in the airways. As a first step in sensitization, the antigen presenting cells  take up and process the inhalant allergens. The DCs are recognized as the key immune sentinel cell in the peripheral organs, including lung , and areWhile asthma phenotypes and endotypes are not fully characterized, resident lung DC types could be important , 18–22. Over the last several years, prominent research studies have demonstrated that the late fetal and early postnatal periods are phases of reduced immune competence , 32, 33.Particular events during early childhood can set the stage for specific developmental programming of DCs. The flexibility of a developing immune system and simultaneous exposure to allergens and other environmental stimuli can be important compounding factors for both the establishment and a long-term persistence of asthma. In addition to allergens, other risk factors for DC developmental programming towards particular DC phenotype and function during this “critical window period” may include environmental chemicals, drugs, certain dietary factors, infectious agents, and physical and psychological stressors. Not surprisingly, research studies suggest that the maturation and function of DCs are shifted by some of these predisposing risk factors. These include heavy metals, such as lead , and airChlamydia muridarum (an intracellular pathogen) [A number of recent studies have demonstrated that early exposures to athogen) –50, Baciathogen) , and infathogen)  alter thathogen) . In thisathogen) , 55. Respiratory syncytial virus infection is the most common cause of bronchiolitis and pneumonia in children under 1 year of age . Severe forms of the RSV lower respiratory tract infections (LRTI) are characterized by airway obstruction and prominent wheezing. Furthermore, RSV infection in infancy has been linked to the development of asthma in childhood. Thus, there has been great interest in determining whether the pathogenesis of RSV bronchiolitis in infancy induces a persistent Th2 bias, leading to the development of Th2-dependent asthma in later childhood.γ), and the Th2 cytokine, interleukin-4 (IL-4). Bendelja and colleagues studied the expression of IFN-γ and IL-4 in peripheral blood lymphocytes (PBL) of infants with various forms of RSV infection [γ, thus suggesting a Th2 bias. However, the expression of IL-4 was greater in subjects with mild upper respiratory tract infection (URTI) than in subjects with bronchiolitis or pneumonia. IFN-γ expression remained unaffected. Therefore, patterns of IFN-γ and IL-4 expression by PBL could not be associated with the severity of RSV infection. Some studies have focused on the expression of the prototype Th1 cytokine, interferon gamma  is critical to better manage immune maturation for a healthier life course.Histological details reveal that the MHC class II-positive cells start to appear in lung tissues of rat and human fetuses at 30–58% of term. Since the MHC class II is expressed ubiquitously by a variety of immune and nonimmune cells, the interpretation may not be DC specific. The appearance of MHC class II-positive DCs increases only after birth , 67–70. Importantly, the airway structures and epithelial cell system are also not fully developed at the time of birth or during the neonatal period. It is reasonable to believe that direct cell-cell interaction or chemical mediators and growth factors released by another cell type can have a significant impact on the establishment of normal lung DC infrastructure as the lung microenvironment and the normal lung physiology evolve , 71, 72.Rhesus monkeys. Similarities have been reported in asthmatic response among Rhesus monkeys and humans; the house dust mite antigen induces asthma conditions with clinical profiles; the biochemical and immunological markers are similar to those in human patients of asthma [Technical and ethical issues related to the availability of neonatal and infant lung tissues limit the enthusiasm to conduct studies that address the issues related to lung DC development. Studies are limited to first challenging the neonatal animal with infectious or allergenic stimuli and then investigating the DC types and functions later in life in the same animal. This approach may not adequately reflect the dynamic process of DC development or programming during early childhood in humans. Since DCs make <1% of total lung cells , it is nf asthma , 81, 82.Rhesus monkey, include the ease of timed pregnancies due to the estrogen-sensitive sex skin in cycling females, the availability (baboons breed year-round), and the relative ease of handling . MoreovPapio species) model [in vitro under DC-promoting conditions  model , 95–97. s) model . Since ws) model . We havenditions . A signiIn summary, our results indicate that the lung DCs are not developed at least until birth. We do not know when the DCs develop after birth during childhood, what triggers the lung DC development, and how the pathogenic stimuli affect the DC development leading to DC phenotypes with different functions. We speculate that pathogenic stimuli, such as allergens, may alter the lung DC developmental programming or SDPC→DC transition during early childhood in a way that an immunological memory is created for generation of asthma-promoting DC phenotypes or immune responses. Persistent reexposure to allergens may bolster the generation of these DC phenotypes so that an allergic response is maintained for a long time. in vivo, studies with baboon lung SDPCs and DCs can provide useful tools to unravel the molecular and immunologic basis of the lung DC development and develop novel pharmacological modulators. A focused effort in this direction can provide a unique opportunity to effectively manage the pediatric immune system for optimized maturation and reduced health risks. This would include opportunities to intervene at an early age and skew the DC programming towards normal DC phenotype and function using pharmacological modulators. Additionally, this information may be useful in better protecting neonates and infants from environmental insults that increase the later-life health risks. It is the magnitude and persistence of downstream immunoinflammatory effects of tissue DC function that position this topic as a central health issue for allergic and other chronic diseases.An understanding of the basic immunobiology of DC development and the early programming of asthma-promoting DC phenotypes and functions against allergenic stimuli can pave the way to identify the basis of childhood asthma. Although it can be challenging to study the molecular mechanisms for lung DC development


Drosophila, mouse, and zebrafish cohesinopathy models all show altered expression of developmental genes. However, there appears to be incomplete overlap among dysregulated genes downstream of mutations in different components of the cohesion apparatus. This is surprising because mutations in all cohesion proteins would be predicted to affect cohesin’s roles in cell division and gene expression in similar ways. Here we review the differences and similarities between genetic pathways downstream of components of the cohesion apparatus, and discuss how such differences might arise, and contribute to the spectrum of cohesinopathy disorders. We propose that mutations in different elements of the cohesion apparatus have distinct developmental outcomes that can be explained by sometimes subtly different molecular effects.The multi-subunit protein complex, cohesin, is responsible for sister chromatid cohesion during cell division. The interaction of cohesin with DNA is controlled by a number of additional regulatory proteins. Mutations in cohesin, or its regulators, cause a spectrum of human developmental syndromes known as the “cohesinopathies.” Cohesinopathy disorders include Cornelia de Lange Syndrome and Roberts Syndrome. The discovery of novel roles for chromatid cohesion proteins in regulating gene expression led to the idea that cohesinopathies are caused by dysregulation of multiple genes downstream of mutations in cohesion proteins. Consistent with this idea,  The cohesin complex and proteins that regulate its interaction with chromatin have multiple roles in cell division, DNA damage repair, gene transcription, and chromosome architecture. Proteins that make up the cohesin complex have been characterized in several model systems  subunits Smc1 and Smc3, which associate to form a tripartite ring incorporating an α-kleisin subunit, Mcd1/Scc1/Rad21. Smc1 and Smc3 are large rod-shaped proteins that dimerize at one end to form a “hinge” domain, and also interact at the other end via ATP-binding “heads,” which in turn interact with the α-kleisin subunit Figure . AlternaDrosophila and NIPBL in human) and Scc4/MAU2, recently dubbed “kollerin” , the Sororin protein is additionally required to establish and maintain cohesion  and Aurora B  through ATR and Chk1 pathway is important for cohesion and DSB repair  organization of chromatin  also termed Brachmann de Lange syndrome (BdLS), a broad spectrum disorder with multiple developmental and cognitive abnormalities  are dominantly inherited, and caused by mutations in the SMC1A (OMIM 300040) and SMC3 (OMIM 606062) also give rise to syndromes that fall within the CdLS spectrum, and account for about 5% of CdLS cases  mutations also cause a cohesinopathy syndrome  silencing -C) in Drosophila is exquisitely responsive to Rad21 and Nipped-B levels. Furthermore, when mRNA encoding these proteins is depleted in BG3 cells, the direction in which some E(spl)-C are regulated depends on the length of time of RNAi treatment, and the degree of Rad21 or Nipped-B knockdown. For example, E(spl)-C transcripts decrease after 3 days of Nipped-B RNAi, but increase by day 6  were dysregulated compared with controls. Dysregulated gene expression in the mutant cell lines was conserved, and correlated with disease severity and cohesin binding at misexpressed genes  fold changes in the expression of a great many genes. Liu et al.  analyzedNipbl locus show many features overlapping with CdLS, and microarray analyses indicated that reducing Nipbl dose resulted in small changes in expression of a great many genes. These mice also had severe developmental phenotypes, including craniofacial dysmorphology and heart defects, resembling CdLS. Of note was the altered expression of genes involved in fat metabolism, which could account for the lean habitus observed in mice and in CdLS patients , runx1 expression was retained in Rohon–Beard neurons, but was absent from a discrete population of cells in the hematopoietic mesoderm. Importantly, the hematopoietic mesoderm precursor cell population was still present in mutants, and expressed the dimerization partner for Runx1, cbfb, although not runx1 itself. Cohesin probably targets other runx genes in a cell type-specific manner, since rad21 mutants also lacked expression of runx3 in Rohon–Beard neurons and the lateral line primordia  in zebrafish embryos is too late to determine its involvement, since rad21 mutants arrest in development at 35 hpf. Like in Drosophila, cohesin is likely to regulate expression of genes in zebrafish brain; cohesin subunits are expressed in non-proliferating neurons of zebrafish brain implying a non-cell cycle role for cohesin in this tissue . Interestingly, we found that the eomes gene, a master regulator of endoderm formation, is consistently downregulated in rad21 and smc1a mutants, and nipbl morphants (FDR < 0.02), supporting a role for cohesin and Nipbl in endoderm formation. Eomes expression is regulated by pluripotency factors Nanog, Oct4, and Sox2  of zebrafish development in We expected our microarray analyses of zebrafish cohesinopathy mutants and morphants to result in similar lists of up- or downregulated genes, since embryos were analyzed at similar stages and cohesinopathy genes would be predicted to have similar roles in gene expression. Therefore we were surprised to find only modest overlap between regulated gene sets , the myc transcription factor (except in esco2 morphants), and downstream effectors of Notch signaling such as hey1, her4.2, and ascl1.Many genes that have altered regulation in response to depletion of cohesinopathy genes are different, raising the possibility that cohesin subunits and regulators have different functions in various pathways. However, genome-wide analyses of gene expression identified some commonly regulated pathways/genes such as those involved in endoderm development , block transcription of ribosomal RNA genes thereby directly influencing ribosome biogenesis, protein translation and the cell’s ability to grow , ascl1 is significantly downregulated . Such sensitivity could have high functional significance. Ascl1 is a potent neuronal lineage-specifying gene, being one of three genes sufficient to convert fibroblasts into iPN cells  were consistently affected in our zebrafish cohesinopathy models (Rhodes et al., Drosophila (Schaaf et al., Drosophila (Dorsett, We also found that certain Notch signaling targets of the Dorsett, , strong Pcdhα in a mouse neuroblastoma cell line (Monahan et al., In addition to neuronal cell fate, it appears that cohesin together with CTCF could contribute to maintaining neuronal identity. Several studies show that cohesin and CTCF regulate expression of protocadherin genes (Kawauchi et al., Drosophila mushroom body (Pauli et al., C. elegans (Seitan et al., Pds5b (Zhang et al., esco2 morphant zebrafish, we observed defects that were consistent with abnormal neural crest cell migration (Monnich et al., rad21, we observed that while the trigeminal ganglia of the brain are specified, the axons clump together rather than extending forward (Figure Evidence suggests that the widespread disruption of neuronal gene expression found in cohesinopathy mutants results in abnormal behavior and function of neurons. As discussed previously, localized disruption of cohesin subunits causes failure of axon pruning in the d Figure . It is vGrowth, metabolism, and development of the central nervous system appear to be processes that are universally sensitive to cohesinopathy mutations. Much of this pathology is likely to be caused by cohesin’s role in the regulation of gene expression. However, cohesin has another important role in the repair of DNA damage, and its loss is likely to trigger DNA damage checkpoints (Jessberger, When damaged DNA is detected, cells respond by coordinating cell cycle arrest, DNA repair, and programmed cell death (Ciccia and Elledge, ESCO2 is mutated. The syndrome, known as Warsaw Breakage Syndrome, is considered to reside at an interface between DNA damage repair and sister chromatid cohesion (van der Lelij et al., Interestingly, mutations in other genes responsible for the DNA damage response underlie human syndromes with phenotypes that overlap the cohesinopathies (Ciccia and Elledge, It is possible that the CoAT ESCO2 has a particularly crucial role in DNA damage repair, since mutations in ESCO2 appear to resemble mutations in DNA damage repair pathways more than the other cohesinopathies do. Indeed, ESCO2-depleted cells are hypersensitive to DNA damaging agents such as Mitomycin C (van der Lelij et al., Rad21 null mutation are hypersensitive to IR, and exhibit problems with integrity and maintenance of the gastrointestinal tract and hematopoietic system post-irradiation (Xu et al., RAD21 mutations also have impaired DNA damage repair (Deardorff et al., RAD21 sensitizes breast cancer cells to chemical agents that damage DNA (Atienza et al., Rad21 gene is crucial for DNA damage repair. In addition, depletion of SMC1 sensitizes HeLa cells to DNA damage (Bauerschmidt et al., Other cohesinopathy mutations also have potential to compromise DNA damage repair. Mice heterozygous for a It is not clear to what extent DNA damage repair defects contribute the pathology of cohesinopathies (Dorsett and Strom, most affected by a particular cohesinopathy mutation in a given population of cells (Figure We propose a model to explain the diverse phenotypes observed downstream of cohesinopathy genes, in which different phenotypes emerge according to the “phase” of the cohesin cycle that is s Figure . In thisall cohesin-related processes, including transcription regulation during interphase, chromatid cohesion during S phase, and DNA damage repair. Therefore mutations in genes encoding these proteins have potential to disrupt all the processes shown in the model (Figure It is important to note that cohesin subunits and the cohesin loading kollerin complex participate in l Figure . Howeverrad21 contain heavy maternal loading of Rad21 protein and are able to develop for about 20 h before cell cycle deficiencies halt growth. However, well before cell cycle defects have any impact, rad21 null embryos fail to activate runx1 expression in the hematopoietic mesoderm (Horsfield et al., runx1 expression that is below the level necessary to sustain cell division. The primary impact of suboptimal levels of Rad21 is that of altered gene expression in a subpopulation of cells, and the secondary impact of cell cycle arrest is not observed until Rad21 levels are further depleted. Radiation sensitivity observed in Rad21 heterozygous mice (Xu et al., For example, zebrafish embryos zygotic null for In summary, the phenotypic outcome of cohesinopathy mutations may differ between cell populations and in any given cell population, depend upon the degree of sensitivity of gene expression to cohesin levels, the requirement for cell proliferation, and the presence of environmental stressors such as DNA damaging agents. There is likely to be significant overlap in these contributing factors to cohesin-related developmental disorders.Although considerable progress has been made over the last 10 years in the understanding of cohesin function in the cell cycle, transcription, and human developmental disease, important questions remain. How is the transcriptional role of cohesin coordinated with its role in genome organization, cell division, and DNA repair? Why do some cohesinopathy mutations lead to developmental gene dysregulation, while others lead to chromosome segregation defects and cell death? Human syndromes and animal models have potential to lend important insight into the integration of cohesin functions in cell division and development. Continued research will be vital for understanding the pathology of cohesinopathy syndromes, and the development of future potential for clinical management or therapy.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Axotomy of central neurons leads to functional and structural alterations which largely revert when neural progenitor cells (NPCs) are implanted in the lesion site. The new microenvironment created by NPCs in the host tissue might modulate in the damaged neurons the expression of a high variety of molecules with relevant roles in the repair mechanisms, including neurotrophic factors. In the present work, we aimed to analyze changes in neurotrophic factor expression in axotomized neurons induced by NPC implants. For this purpose, we performed immunofluorescence followed by confocal microscopy analysis for the detection of vascular endothelial growth factor (VEGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3) and nerve growth factor (NGF) on brainstem sections from rats with axotomy of abducens internuclear neurons that received NPC implants (implanted group) or vehicle injections (axotomized group) in the lesion site. Control abducens internuclear neurons were strongly immunoreactive to VEGF and BDNF but showed a weak staining for NT-3 and NGF. Comparisons between groups revealed that lesioned neurons from animals that received NPC implants showed a significant increase in VEGF content with respect to animals receiving vehicle injections. However, the immunoreactivity for BDNF, which was increased in the axotomized group as compared to control, was not modified in the implanted group. The modifications induced by NPC implants on VEGF and BDNF content were specific for the population of axotomized abducens internuclear neurons since the neighboring abducens motoneurons were not affected. Similar levels of NT-3 and NGF immunolabeling were obtained in injured neurons from axotomized and implanted animals. Among all the analyzed neurotrophic factors, only VEGF was expressed by the implanted cells in the lesion site. Our results point to a role of NPC implants in the modulation of neurotrophic factor expression by lesioned central neurons, which might contribute to the restorative effects of these implants. Axotomy of adult CNS neurons leads to severe morphofunctional alterations which include synaptic stripping and reduced firing rate By using the oculomotor system as the experimental model, we have previously described that the transection of the medial longitudinal fascicle (MLF) induces profound alterations in the axotomized internuclear neurons of the abducens nucleus, which are left disconnected from their target As a further step, we have explored in our central axotomy model the effects of neural progenitor cell (NPC) implants based on the reported efficacy of this cell population in promoting functional improvement of damaged CNS neurons NPCs used in our study were obtained from the subventricular zone (SVZ) of postnatal animals. In their neurogenic niche, SVZ cells have been described to express the angiogenic factor vascular endothelial growth factor (VEGF) and the neurotrophic factors brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF) and neurotrophin-3 (NT-3) In the present work, we aimed to investigate possible modifications of neurotrophic factor expression in central injured neurons following NPC implants which could be involved in the physiological recovery induced by these implants. For this purpose, we transected the MLF in adult rats and implanted NPCs at the lesion site. Changes induced by the lesion and/or the implant in the expression of VEGF, BDNF, NT-3 and NGF were analyzed in the axotomized population of abducens internuclear neurons. In addition, we also evaluated the expression of these neurotrophic molecules by the implanted NPCs. The findings of this study indicate that axotomy induces changes in the expression of neurotrophic factors by injured central neurons that can be differentially modulated by NPC implants. We propose that the neurotrophic regulation promoted by NPCs might contribute to the restorative effects of NPC implants.Experiments were performed in adult and 7 day postnatal (P7) Wistar rats in accordance with the guidelines of the European Union (86/609/EU) and Spanish law  for the use and care of laboratory animals. Surgical procedures used in this study were approved by the ethics committee of Universidad de Sevilla.2, at 37°C. After 1–2 days, cell aggregates known as neurospheres were formed  and were expanded in the form of neurospheres essentially as reported e formed . Cells we formed .6 IFU/ml) with a multiplicity of infection of 3. Neurosphere-derived cells were incubated for 8 hours with virus-added DM in T25 flasks containing 250,000 cells. Virus-containing medium was then removed and replaced with fresh DM. After 48–72 hours of virus incubation, cells from neurospheres expressed GFP as confirmed by direct visualization in a Zeiss LSM 7 DUO confocal microscope  under the control of the cytomegalovirus (CMV) promoter , adult rats were placed on a stereotaxic frame and a window trephined in the occipital bone. The right MLF was transected by means of a 750-µm width microblade aimed with a 45° anterior angle at the following stereotaxic coordinates: 3.5 mm caudal to lambda and 0 mm lateral The unilateral transection of the right MLF disrupts the axons of the internuclear neurons of the left abducens nucleus. As the left MLF was not transected, right side abducens internuclear neurons remained unlesioned and were used as control neurons throughout this study.Eight weeks after lesion followed by cell implant or vehicle injection, animals were perfused transcardially under deep anesthesia  with 100 ml of physiological saline followed by 250 ml of 4% paraformaldehyde in 0.1 M sodium phosphate buffer, pH 7.4. The brainstem was removed and cryoprotected by immersion in a solution of 30% sucrose in sodium phosphate-buffered saline (PBS) until it sank. Then, for cryostat sectioning, the brainstem was cut in two pieces: the more rostral one, containing the site of lesion in the MLF, was sectioned sagitally  and the After washing in PBS, coronal sections containing the abducens nucleus were incubated with 1% sodium borohydride for 10 min for antigen retrieval. They were rinsed again in PBS and incubated for 1 hour in a blocking solution consisting of 5% normal donkey serum in PBS with 0.01% Triton X-100 (PBS-T). Following the blocking treatment, sections were incubated overnight at room temperature with one of the following primary antibodies: rabbit anti-VEGF , rabbit anti-BDNF  or rabbit anti-NT-3 , prepared in blocking solution. After several rinses in PBS, sections were incubated with a biotinylated secondary antibody in PBS-T , washed again and finally transferred for 45 minutes to a solution containing streptavidin-FITC . Then, in order to identify abducens internuclear neurons, subsequent immunohistochemistry for the detection of calretinin was performed since this protein is selectively expressed within the abducens nucleus by this population of neurons The immunohistochemical protocol for NGF varied slightly since a different antigen retrieval method was needed, which consisted of incubating the sections in 0.01 M sodium citrate buffer, pH 6, at 74°C for 40 min. The remaining protocol was the same as described above except that incubation with the primary antibody  lasted for 72 hours at 4°C. Finally, calretinin immunohistochemistry and mounting on slides were performed as described above.It is important to point out that, in order to avoid differences in immunostaining intensity due to methodological procedures, series of sections from implanted and axotomized groups were always processed simultaneously for each neurotrophic factor.To analyze neurotrophic factor expression by implanted cells, parasagittal sections containing the lesion and implant site were processed for single immunohistochemistry for VEGF, BDNF, NT-3 or NGF, following the same described procedure but a streptavidin-DyLight649 was used  instead of a FITC-coupled streptavidin, since implanted cells were already labeled in green by their GFP expression.For positive controls, the immunohistochemistry protocol was performed in brain sections that contained areas previously reported as immunopositive for each of the neurotrophic factors used in this study.Negative controls carried out by omission of the primary antibodies resulted in absence of staining in all cases. Additional negative controls were performed by preadsorption of the antibodies with their blocking peptides. Specifically, each antibody and its corresponding immunizing peptide were mixed for 2 hours at a 1∶20 proportion before being used for the immunohistochemical protocol. Sections incubated with this preadsorbed solution showed no specific staining.For the VEGF primary antibody, a blocking peptide was not available by the provider. Then, as negative control, the primary antibody incubation was replaced by incubation with normal rabbit IgG (Jackson ImmunoResearch) at the same concentration than the primary antibody. Again, no specific labeling was found.Confocal microscopy images of abducens neurons were captured in order to analyze differences between groups in the intensity of the VEGF, BDNF, NT-3 and NGF immunostainings.For each neurotrophic factor, all abducens neurons identified in one out of three coronal sections from each animal were captured with a confocal laser scanning microscope (Zeiss LSM 7 DUO) at 63X magnification and 8-bit resolution. Different focal planes containing the cell nucleus and separated 2 µm in the z axis were scanned by exciting the FITC and TRITC fluorophores with 488-nm argon and DPSS 561-nm lasers, respectively, with a pinhole aperture of 1 Airy Unit. Before scanning, gray scales were adjusted to maximize their dynamic range. Acquisition settings were kept constant during the image capture of neurons from the left (lesioned) and right (unlesioned) abducens nucleus of the same section.Captured images were analyzed with ImageJ (NIH). In order to determine the intensity of the immunostaining, we calculated the mean gray value of the cell immunolabeling  by manually outlining the cell cytoplasm in the corresponding scanned images. Optical density measurements have been demonstrated to correlate significantly with the amount of protein present in the brain tissue Comparisons between groups were carried out in Sigma Plot 11 (Systat Software) by using one-way ANOVA followed by post-hoc multiple comparisons (Dunńs method) at a significant level of p<0.05.Positive and negative controls were carried out to test the specificity of the antibodies used in this study for the immunoidentification of the neurotrophic factors BDNF, NT-3, NGF and VEGF.Initially, the immunohistochemistry protocol and the antibody titration were established in sections containing brain regions in which the expression of each factor has been previously described in the literature VEGF expression was conspicuous in Purkinje cells of the cerebellum  and in mAs previously described, calretinin constitutes a good marker of the internuclear neurons located in the abducens nucleus All calretinin-immunopositive neurons examined in the abducens nucleus also showed a strong immunoreactivity against BDNF, as observed in the three experimental groups . HoweverIn order to test whether the effects of central axotomy and NPC implants on VEGF and BDNF expression were restricted to the injured neuronal population within the abducens nucleus  we also examined VEGF and BDNF immunoreactivity in the motoneurons of the same nucleus, whose axons were left intact. Abducens motoneurons were identified by their ChAT immunostaining . Both VEAbducens internuclear neurons showed a weak immunostaining against NT-3 . The anaNGF immunoreactivity was mainly confined to the neuropil of the abducens nucleus, showing faintly-stained cell bodies . OpticalWe also aimed to explore whether implanted cells in the lesion site might constitute a retrograde source of neurotrophic factors for the axotomized abducens internuclear neurons, due to the close proximity between the proximal stump of sectioned axons and the implanted NPCs. For that, we performed VEGF, BDNF, NT-3 and NGF immunohistochemistry in brainstem parasagittal sections containing the lesion and NPC implantation site . ImplantIn this study, we have demonstrated that VEGF and BDNF expression in axotomized neurons can be modulated differentially by NPCs implanted in the site of injury. In addition, NPCs derived from the SVZ are able to express VEGF after implantation in the damaged brain. Based on the recently-demonstrated and relevant functions of VEGF in neuroprotection and synaptic modulation, we propose that this neurotrophic factor could play an important role in the benefitial effects of NPC implants on axotomized abducens internuclear neurons.Transection of the MLF leaves abducens internuclear neurons disconnected from their natural target in the oculomotor nucleus. The axotomized neurons exhibit a profound removal of inhibitory and excitatory synapses and a reduction in eye position and velocity sensitivities In the control situation, we observed that all analyzed abducens internuclear neurons displayed an intense immunoreactivity for VEGF and BDNF, whereas NT-3 and NGF revealed a faint staining. However, this was not the case for other brainstem neurons which showed an intense immunoreactivity for NT-3 and NGF (data not illustrated). Therefore, the intense labeling found in abducens internuclear neurons specifically after VEGF and BDNF immunoreactivity suggests that these two neurotrophic factors likely act as important molecules for the functional maintenance of this neuronal population, probably through autocrine or paracrine pathways, although the retrograde and/or anterograde routes can not be discarded.After axotomy, abducens internuclear neurons did not modify their immunoreactivity to VEGF. However, VEGF content was significantly increased in these neurons when NPCs were implanted after axotomy. Two possible explanations may account for this finding: i) VEGF might have been retrogradely transported by the sectioned axons and accumulated in the soma, and ii) damaged neurons might have increased their VEGF expression in implanted animals. Since some of the implanted cells were VEGF-immunoreactive and abducens internuclear neurons express the receptor for VEGF, Flk-1 (unpublished data), the first possibility is feasible. With regard to the second possibility, it is well established that the new microenvironment created by the cross-talk between implanted and host tissue cells can induce changes in the neuronal expression of certain molecules Abducens internuclear neurons of the axotomized group exhibited a significant increase in BDNF and NT-3 content eight weeks after MLF transection. We have previously demonstrated that abducens internuclear neurons express the trk receptors for BDNF and NT-3  A noticeably finding of the present study was that the motoneurons of the abducens nucleus, which lie intermingled with the internuclear neurons, did not show any change in the intensity of their immunostaining for either VEGF or BDNF. Since the same methodological protocol was used for both cell types, these data clearly validate the modifications found in the population of abducens internuclear neurons after NPC implants and, moreover, demonstrate that changes in neurotrophic factors were exclusive for those neurons that were axotomized by the lesion procedure.As stated before, implanted cells might constitute a new source of trophic factors for axotomized cells. In fact, NPCs from the SVZ express VEGF in the neurogenic niche In summary, this study demonstrates that NPCs implanted in the injured brain express VEGF eight weeks after implantation in the host lesioned tissue and are able to modulate VEGF and BDNF expression in lesioned neurons. We propose that the increased VEGF expression promoted by NPCs in injured neurons might be involved in the restorative effects of NPC implants.Figure S1Characterization of neurosphere cultures. A-B. Phase-contrast microscopy images of floating neurospheres (A) and dissociated neurosphere cells grown as a monolayer on a poly-lysine substrate (B). C, E, G. Fluorescence microscopy images of neurospheres immunostained with antibodies against nestin , βIII-tubulin , GFAP  and NG-2  and counterstained with DAPI (blue). D, F, H. Fluorescence microscopy images of adhered cells immunostained with antibodies against nestin , βIII-tubulin , GFAP  and NG-2  and counterstained with DAPI (blue). Scale bars: 50 µm in A, B and D; 25 µm in C and F; 15 µm in E, G and H.(TIF)Click here for additional data file.Supporting Information S1Characterization of neurosphere cultures.(DOCX)Click here for additional data file.


Journal of the American Heart Association (JAHA), Lugenbiel et al1 describe a novel gene-based approach for potentially treating heart failure by targeting discrete cell populations within the sino-atrial node.Heart failure is a major public health epidemic that claims hundreds of thousands of lives annually in the United States alone. Despite important advances in the management and prevention of many cardiovascular disorders, the incidence and prevalence of heart failure are increasing with the aging of the population. Existing treatments for patients with heart failure are far from being optimal and are often associated with proarrhythmic activity and risk of sudden cardiac death. Therefore, the search for novel, effective, and safe therapeutic options for these patients represents a critical area of unmet need for this country and the rest of the world. In an elegant proof of concept study published in this issue of the 2 In fact, a strong link between average heart rate and cardiovascular morbidity in patients with heart failure has been documented. In clinical trials the ability of a given heart failure treatment to reduce overall mortality is, in some cases, predicted by its heart rate lowering efficacy. Moreover, a causative relation between elevated heart rate and heart failure is epitomized by the fact that sustained tachycardia, in the absence of other risk factors or insults, is a well-established mechanism of ventricular remodeling that culminates in the onset of heart failure.3 In the experimental laboratory, numerous groups have exploited the relation between elevated heart rate and left ventricular dysfunction by developing highly reproducible small and large animal models of heart failure using chronic tachycardia pacing of the right atrium or ventricle. These experimental models have been instrumental in advancing our fundamental understanding of the mechanisms that underlie mechanical, structural, electrical, metabolic, ionic, and molecular dysfunction in the failing heart. Of note, cessation of rapid pacing following the onset of heart failure is typically associated with reversal of pathological electrical and structural remodeling and an improved cardiac function,4 further highlighting the role of heart rate control in the management of heart failure.Several lines of evidence derived from experimental and clinical studies indicate that a controlled heart rate is an important target for heart failure therapy.6 Indeed, chronic β-adrenergic receptor stimulation, which increases the energetic cost of contractility at a time of reduced oxygen supply, can exacerbate myocardial damage and accelerate mortality in heart failure patients. In contrast, β-adrenergic receptor blockers, which are mainstays of heart failure therapy, are distinguished by their heart rate lowering efficacy and their ability to hinder and/or reverse key features of pathological remodeling in the failing heart. Because β-adrenergic receptor blockers are confounded by negative inotropic properties that can further reduce the contractile function of an already compromised failing heart, their efficacy in the acute and subacute phases is often limited. To that end, considerable interest in recent years has been directed toward the development of specific heart rate lowering agents that selectively target pacemaker channels. Ideally, such agents would mimic the beneficial effects of β-blockers in terms of heart rate reduction while avoiding their confounding side effects on contractility.Hallmark features of the failing heart include a prominent reduction in contractile efficiency and the loss of contractile reserve. These deficits, caused by altered calcium signaling, excitation-contraction coupling, and decreased myofilament sensitivity to calcium can lead to hemodynamic insufficiency, compromise, and death. To enhance cellular contractility, β-adrenergic receptor agonists, such as isoproterenol and dobutamine, have been developed. The positive inotropic property of these agents is mediated, in part, by the enhancement in activator calcium through L-type calcium channels which are phosphorylated by cAMP-dependent protein kinase A. Unfortunately the chronic use of these agents has been severely hampered by adverse effects, including heart rate acceleration and promotion of ventricular arrhythmias.fI), was developed to suppress heart rate by targeting the hyperpolarization-activated cyclic nucleotide (HCN) gene family. In experimental and clinical studies, ivabradine improved myocardial energetics and left ventricular function. Specifically, in the SHIFT trial (Systolic Heart failure treatment with fI Inhibitor ivabradine), patients with symptomatic heart failure who were treated with ivabradine had a significant benefit (by >25%) in terms of cardiovascular mortality and hospitalization when compared with the placebo treated group.7 These highly encouraging findings have cemented the concept of heart rate modulation through pacemaker channel inhibition as an important strategy for the management of heart failure.9 As with any pharmacological therapy for a chronic disease, challenges related to dosing, bioavailability, selectivity, and adverse interactions with other drugs are expected. These complicating issues warrant further optimization, as alternative nonpharmacological approaches for modulating heart rate are explored.Ivabradine, a selective inhibitor of the pacemaker current , specifically in the sino-atrial node. In effect, these authors use highly sophisticated molecular and gene therapy tools to create a “biological beta blocker” that can be locally delivered.In this issue of 10 and “bio-beta blockers” for combating tachycardia.11 This is followed by a discussion of the unique advantages and potential pitfalls of the exciting approach undertaken by Lugenbiel et al1 for suppressing heart rate.In what follows, we provide a brief overview of recent efforts to develop cell- and gene-based therapies for heart rate control, including the creation of “bio-pacemakers” for combating bradycardia14 Our growing understanding of the molecular basis of cardiac pacemaking activity, including the revelation of a complex interplay between voltage and calcium clocks within the cardiac myocyte, has been instrumental in the identification of key targets that can be exploited for the creation of viable biological pacemakers.Major milestones have been recently achieved in our collective effort to develop novel, gene-based approaches for the treatment of cardiac rhythm disorders. Indeed, several groups have made great strides toward the creation of biological pacemakers that can one day (perhaps in the near future) either fully replace or at the very least assist existing electronic devices.14 used viral gene transfer of an engineered dominant negative construct of Kir2.1 (Kir2.1AAA) to focally inhibit the inward rectifier potassium current; thereby converting normally quiescent cells into ones that exhibited spontaneous depolarization and rhythmic activity. When injected into the left ventricle, these constructs could “capture” the myocardium resulting in premature ventricular beats that likely emanated from the site of injection.14 In other seminal studies, Rosen et al systematically developed HCN based approaches for the creation of robust biological pacemakers, both in vitro and in vivo.17 These biological pacemakers provided physiologically acceptable heart rates and exhibited positive chrontropic responses to emotional arousal and stress. Indeed, the efficacy and translatability of these HCN-based approaches were elegantly documented in preclinical large animal models by the Rosen group.17In proof of concept studies, Miake et alCa-LI),18 which mediates action potential propagation in specialized conducting tissues, such as the sino-atrial and atrio-ventricular nodes.As mentioned earlier, heart rate reduction is an important target for heart failure therapy. As such, novel molecular tools that can either lower resting heart rate or dampen the excessive acceleration of heart rate upon β-adrenergic stimulation are needed. To that end, Donahue and colleagues pioneered efforts to create gene therapy tools for rhythm disorders by targeting the L-type calcium current  suppression of heart rate. This surely cannot be achieved by a short-term adenoviral mediated gene transfer strategy coupled with a highly invasive electroporation technique. The development of clinically translatable gene delivery methods and cardiotropic vectors for long-term modulation of gene expression in the sino-atrial node and/or the myocardium would be required. Indeed, the use of various adeno-associated vector serotypes or adeno-associated vector chimera may prove essential in achieving this ambitious aim. As with any good study, the intriguing findings by Lugenbiel et al


Mouse models provide powerful tools for studying the mechanisms underlying the dysfunction of the autonomic reflex control of cardiovascular function and those involved in cardiovascular diseases. The established murine model of two-kidney, one-clip (2K1C) angiotensin II-dependent hypertension represents a useful tool for studying the neural control of cardiovascular function. In this paper, we discuss the main contributions from our laboratory and others regarding cardiac-autonomic imbalance and baroreflex dysfunction. We show recent data from the angiotensin-dependent hypertensive mouse demonstrating DNA damage and oxidative stress using the comet assay and flow cytometry, respectively. Finally, we highlight the relationships between angiotensin and peripheral and central nervous system areas of cardiovascular control and oxidative stress in the 2K1C hypertensive mouse. The sympathetic nervous system has an excitatory action on the heart and blood vessels, whereas the parasympathetic cardiovagal innervation has an inhibitory action on the heart . Cardiac As recently reviewed , conditiIn this paper, we will highlight the characteristics of the murine model of RAS-dependent hypertension, provide new insights into the role played by oxidative oxygen species (ROS) in the integrative brain areas, and discuss which findings are expected to be revealed next.  For decades, the rat has been used to study the relationship between RAS and the autonomic nervous system. However, genetic discoveries and advances in molecular biotechnologies have provided the opportunity to develop many mouse models for human diseases. Although a major disadvantage of this animal is the small size, advances in surgical techniques have overcome this limitation, allowing for studies of cerebral , 9, card In our laboratory, we used the procedure established by Wiesel et al.  to develTwo weeks after renal artery clipping, 2K1C mice already exhibit arterial hypertension  with simAn advantage to using the C57BL/6 mouse for the induction of RAS-dependent hypertension is that it is a prototype of strains with a single renin gene , that isIn some brain areas, including the rostral ventrolateral medulla (RVLM), hypothalamic paraventricular nucleus (PVN), and subfornical organ (SFO), a local RAS has been identified to act as a critical mediator of chronic hypertension in the 2K1C mouse model , 20, 21.An imbalance of the autonomic nervous system, as often occurs in conjunction with several cardiovascular diseases, affects BP and HR variability , 23, whiβ1-blocker (atenolol), a muscarinic, cholinergic receptor blocker (atropine methyl nitrate) or a double blockade of those receptors [The parasympathetic cardiovagal and sympathetic tones in the mouse have traditionally been assessed through pharmacological methods involving a eceptors , 29. Theeceptors , with a eceptors .As shown in Among the neural systems that control cardiovascular function, the baroreflex is a neural mechanism that acts moment-to-moment to maintain BP with minimal fluctuations . With eaIn our laboratory, the sensitivity of the baroreflex has been traditionally assessed through pharmacological approaches in conscious animals. An acute, phenylephrine- (Phe-) induced increase in BP leads to an increase in the number of action potentials generated at each discharge and, consequently, to a reflexive increase in parasympathetic and a decrease in sympathetic nerve activities. The opposite is observed during an acute, sodium nitroprusside- (SNP-) induced decrease in BP. Peak values of mean arterial pressure (MAP) and HR in response to Phe and SNP injections are fitted to a sigmoidal logistic equation, which is used to determine the gain (first derivative of the curve) and the maximum reflex tachycardia (upper plateau) and reflex bradycardia (lower plateau) , 35. Con1 receptors at the aortic arch (and probably at the carotid sinus) act by decreasing the sensitivity of aortic afferents during physiological changes in BP, thus contributing to the impairment of the baroreflex function in cardiovascular diseases [ A disruption in the balance between parasympathetic and sympathetic tones, as discussed above, can lead to an impairment in baroreflex sensitivity, as has been demonstrated by our laboratory in different models of hypertension , 36–38. diseases , 39, 40.diseases . Moreovediseases ; howeverThere is mounting evidence that increased oxidative stress contributes to increased cardiac and vascular sympathetic tone and decreased baroreflex sensitivity in cardiovascular diseases, including hypertension, as recently reviewed elsewhere , 21. BecThe intracellular oxidation of DHE to the fluorescent dye ethidium has been previously used as an indicator of superoxide generation . DHE is The most important, biologically active oxidant in the cardiovascular system, superoxide is a highly reactive and short-lived radical responsible for ROS generation. In addition, it can interact with nearby molecules such as DNA, and thus play a key role in inducing DNA oxidative damage , 53. TheAs illustrated in Our finding of increased DNA damage and ROS production in the 2K1C mouse is in agreement with other studies that found an accumulation of superoxide at the ganglionic level  and in dAlthough therapies have been aimed at nullifying the undesirable effects of angiotensin II, some investigators have focused on demonstrating the importance of the counterbalanced effects of angiotensin 1-7 , which iAs recently reviewed , there iSome gene therapies have also been tested in studies of 2K1C hypertensive mice, but up until now, no favorable results have been observed. For example, Gava et al.  used genIn the past few years, the mouse model of 2K1C hypertension has greatly contributed to the understanding of the relationships between RAS and neural control of cardiovascular function. In addition to the actions of systemic angiotensin II, it has been demonstrated thata local RAS in RVLM, PVN and SFO brain areas act as a critical mediator of chronic hypertension in this experimental model. The 2K1C hypertensive mouse exhibits a cardiac autonomic imbalance characterized by an increased sympathetic tone and a decreased vagal tone, beyond impaired baroreflex sensitivity. In addition to the demonstrations that ROS play a crucial role in RAS signaling at the ganglionic and central nervous system levels, there are growing evidences that DNA damage and increased oxidative stress contribute to the increased cardiac and vascular sympathetic tone and decreased baroreflex sensitivity in the renovascular hypertension. It is well known that angiotensin II increases superoxide production through the activation of NADPH oxidase. Gene therapies by delivering an adenovirus encoding eNOS or enzymes that prevent the elevation of superoxide anions have shown to improve the cardiac autonomic control of HR and baroreflex sensitivity and to prevent renovascular hypertension in the murine model. Although therapies have been aimed at nullifying the undesirable effects of angiotensin II, a putative target for the development of new cardiovascular drugs is the angiotensin 1-7 which induces the release of NO and diminishes NADPH oxidase activation, counteracting the effects of angiotensin II. Therefore, future studies should address potential strategies to decrease oxidative stress and to prevent or restore the cardiac autonomic balance and the baroreflex function in the mouse model of renovascular hypertension.