Inner Mongolia is among the most important regions in terms of contribution to the socio-economic development of China. Furthermore, its grassland is a major ecological barrier for Northern China. The present study evaluates the changes in ecosystem services availability and human wellbeing based on a survey on864 herdsmen of the grassland and 20 governmental officials. The survey provided the following results: (1) The supporting and provisioning services of the grassland have recently declined, thus affecting the herdsmen’s wellbeing. The intensity of grazing, coal exploitation and tourism development heavily limits the availability of ecosystem services, among which provisioning ones are perceived as the most important. Below a certain threshold, grazing and mining are likely to promote the improvement of herdsmen’s wellbeing, while trespassing that point, the enhancement of the herdsmen’s living standards is curbed. (2) The herdsmen’s cultural exchange, health conditions and social relations are better now than before; however, the threats on herdsmen’s health and safety have increased. (3) A better income is among the most urgent herdsmen’s needs. Livestock revenues did not increase at the same rate as the damage to the grassland and human risk exposure did. (4) The governmental ecological compensation policy did not entirely solve the problem of grassland desertification and environmental degradation, although it is also true that the degree of implementation and effectiveness of government policies is related to the still insufficient herdsmen’s understanding and acceptance of these policies. Over the last thirty years, the complex relationship between ecosystem services, economic development and human well-being has been widely acknowledged, even if it has not been completely defined–5. The dSince the MEA results where published, several researchers pointed out the existence of trade-offs between ecosystem services, so that increases in one category of ecosystem services may lead to a decline in other categories due to socio-ecological dynamics–10, whicAs a big prairie country, China has several typologies of natural grassland over about 400 million hectares, accounting for 40% of land area. After the implementation of the “returning cultivated land to forests” policy, a “returning grazing land to grassland” policy was launched in 2003. In fact, due to natural causes and human factors such as overgrazing, 90% of the available natural grasslands showed varying degrees of degradation, leading to intensified desertification, drying up of rivers and lakes, dust storms and other natural disasters occurring more frequently. Grassland degradation is a widely observed problem and estimations for Inner Mongolia’s grasslands reported 30–50% to be degraded . In thisThe grassland ecosystem provides a variety of service functions, among which climate regulation, water and soil conservation, conservation of biodiversity, nutrient cycling, leisure and entertainment–27. An aActually,in the past centuries, animal grazing was the main activity for herdsmen to gain an income.To some extent, more animal grazing means more income, but of course more grazing activity means more ecological pressure on the grassland, thus reducing thegrassland ability to provide direct and indirect ecosystem services.After thousands of years of nomadic animal husbandry activities, northern China temperate grassland shows widespread degradation, followed in the past half century by increased grain production in the 1970s, overgrazing since the 1980s, global warming and prairie drought since the 1990s, and intensified coal mining in the 21st century. State Statistics Bureau point out that livestock overloading rate in the grassland of the Inner Mongoliagot to 116.7% in 2012.If grazing activity were stopped, the grassland would grow much better in so increasing itsresilience, wild animals would come back and finally the hierarchy of the ecosystem food chain would be restored and would allow therecovery of ecosystem service functionsThe change in ecosystem services and human wellbeing has an effect on the landscape. Landscape sustainability science enhances the understanding of and improves the dynamic relationship between the ecosystem and human well-being in a changing landscape. Spatially explicit methods and experimental approaches are essential to coupling the dynamics of ecosystem services and human well-being.ConsiderThis research was carried out in Xilinguole League, which lies in the middle part of the Inner Mongolia Autonomous region, in northern China, ranging from East longitude115° 25' to 119° 58'and North latitude from 41° 35' to 46° 46' 29]. It . It 29].As one of the four major grasslands around the world, Xilinguole is rich with natural resources and species diversity. The national Grassland Nature Reserve of China was brought into the international monitoring system by UNESCO. The region also is the most important livestock product base in China. The number of head of livestock reached 12,881,900 in the year 2013, an increase of 9.3% over the previous year; the total livestock increase was 1,096,600 units, with a growth rate of 5.3% compared to total livestockin 2010. The herdsman’s production style has changed in the last decades. Now most of the herdsman use allocated grasslands. The herdsmen who keep the traditional nomadic translocation are less than 5%. In winter most of the herdsman feed their animals in permanent barns with dry grass that they harvest in summer.The total population of Xilinguole League was 1,002,600 persons in 2010 . The ecoThe region includes 13 natural reserves, out of which two National Nature Reserves and seven regional level protected areas. The two national protected areas cover a surface of 680,000 hectares, while the regional nature reserves cover an area of 970,000 ha. Environmental quality is monitored by 13 environmental monitoring stations. Also due to these protected areas, the number of tourists in the grassland increased to 11,106,100 in 2013,13% more than in 2012. A number of environmental protection policies were implemented, such as:combination of protection and construction, with special attention on protection aspects; returning cultivated land to forests and returning grazing land to grassland; pollution control at the source.The Xilinguole area is also rich with mineral resources, with more than 80 types of minerals extracted. Coal resources are extremely abundant. According to coal exploration data, the Xilinguole League has a total of 188.3 billion tons of coal reserves and contains the second largest reserve in Inner Mongolia .Coal proHowever, increasing large-scale human activities in the grassland are seriously affecting the local environment, in so also affecting the well-being of local populations. Understanding the potential impacts of present and future growth on both human wellbeing and natural systems is a challenging and timely topic to the local governments and herdsmen.We did not seek ethics approval in this case because the questionnaire did not involve any personal privacy or any ethical questions, or any animal experiment, we only need the views of herdsmen about changes in ecosystem services and human well-being, but we approached our research institution's ethics committee prior to the beginning of this study for approval and got approvement. We informed consent from the caretakers (students’ teachers) on behalf of the minors/children enrolled in my study. The consent was verbal since the aim of questionnaire was told by their teachers, and teachers sent the questionnaire to those students who were interested in attending and answering the questionnaire.A questionnaire (see Appendix for full text) was designed to investigate how responses from herdsmen and government officials about recent changes in the ecosystem services and human well-being were related to demographic and lifestyle factors . In September of 2011, four towns  in Xilinguole League were selected as study areas. Questionnaires were distributed and explained in one college and one middle school in Xilinguole city, asking the students to bring them home and help parents to fill them out. In so doing, problems with correct understanding and writing in Chinese were overcome, thanks to the students support and explanations. Of course, no instructions about the content and the values at stake were provided, not to alter the results of the survey. After one month, questionnaires were collected and processed. The involvement of students also increased the probability that the answers reflected the point of view of the entire family. According to Landeghem’s research, human wThe questionnaires were designed to ascertain the views of herdsmen about changes in ecosystem services and human wellbeing and how these changes have affected their behavior. Our survey includes data on the changing grassland ecosystem services, the respondent’s income, material needs for basic living, health condition, cultural exchange, education, freedom and choices rights. Our analysis integrates and compares data from the questionnaire and from Xilinguole League Statistical Yearbook .The evaluation of grassland ecosystem services could help people understand the value of these services and enhance the awareness of ecosystem protection.The main goal is making people realize that the direct and indirect ecosystem services of the grassland supply valuable base in maintaining human well-being.6ha, the area without degradationis 10.27×106ha, while areas presenting mild, moderate and severe degradation are respectively 3.68×106ha, 1.93×106ha and 2.08×106ha[7t/yr based on the consideration of the degree of the degradation of this ecosystem[7t/yr,responses were as follows:39.2% of surveyed herdsmen indicated provisioning services, 27.4% thought regulating services were the most important function, 25.5% of herdsmen suggested cultural services, while only 7.9% of respondents thought supporting service played the most important role. These dWhen asked “What is the relationship between pasture growth and herdsman life?”, 64.8% of the surveyed herdsmen said that their livelihoods would be better if the pasture grew well. Clearly,herdsmen still rely on grazing in their hope for improved living standards .RespondeIncome perspectives play an important role as a mechanism to contribute to poverty alleviation. IncreasRegarding the net annual income of the herdsmen, 47.3% reported that their household annual income was between 10 thousand RMB(about 1588$)and 30 thousand RMB(about 4762$). One quarter (23.6%)responded that their family annual income was over 30 thousand RMB(about 4762$).Finally, 29.1% of herdsmen reported that their household annual income was still less than 10 thousand RMB(about 1588$) . ResultsAt present, only 20.1% of herdsmen reported that they are better-off; 67.1%of the herdsmen thought they had enough food and clothing. However, 12.3%reported that they live in poverty. Thus, more than 85.0% of the herdsmen perceive that they have gotten out of poverty .2 emissions.It also reduces herdsmen’s electricity expenditures and the environmental damage brought about by coal mining in grassland and thus improves the herdsmen’s well-being.A number of basic material and energy needs are indispensable components of herdsmen daily life. Electricity is one of these. With the development and establishment of coal-fired and wind power plants, 98.6% of the herdsmen reported that they could obtain electricity. In this aspect, herdsmen’swell-being has dramatically improved. At present, 21.7% of herdsmen use the electricity from wind power. Wind enAmong the interviewed herdsmen, 49.0% complained that their grassland area is too small to meet their needs for grazing, 35.7% of the herdsmen’s pasture can barely meet their needs, and 15.3% of herdsmen were satisfied with their grassland area. This reflects that herdsmen still aim to obtain more grassland area to increase their livestock breeding proportion and add more income. The per capita area of one quarter (25.9%) of investigated herdsmen’s was less than 66.7 ha, while14.1% reported more than 133.4 ha, and most of them (60.0%) reported a per capita area between 66.7 ha and 133.4 ha.At present, the main vehicles herdsmen use are cars, trucks, motorcars, bikes and carriages.Among these, motorcar use accounted for 49.8%, and the proportion of cars is relatively high at 20.6% .The propCurrently, 90.6% of surveyed herdsmen settle in houses made of brick and cement, replacing the yurt.The change in dwellings has been a gigantic transformation of the herdsmen’s life-style, which aThe domain of health includes expenditures for personal health, life expectancy and mortality, and physical and mental health conditions . In addiThe life expectancy of Xilinguole League is 71.3 years.This expectancy is inferior than the 77.9 years of the average Chinese life.In this The index of safety in this area is frequently evaluated considering resource safety, property and life safety. Resource safety mainly means pasture and water quality and availability for livestock. Property and life safety involves the amount of livestock, the number of snowstorms that occur and violent crime and property crime rates.Overall, the trend in outbreaks of grassland pests has gotten worse, largely due to drought in recent years, but not excluding the possibility of an effect of grassland degradation caused by overgrazing. Grassland pests and diseases are a direct threat to pasture forage production and indirectly affect the development of the livestock, reduce the income of the herdsmen and pull down their well-being. Controlling grassland pests requires engineering and biological measures, such as building grassland food chains. When asked about the occurrence of locusts, pests and diseases in the grassland, 47.2% of the herdsmen thought that they were more serious than before, 36.7% reported that the situation had changed little, while 16.1% of the herdsman thought this phenomenon was not serious.Xilinguole League lies in a semiarid region; therefore,fresh water is notably important for local development. Xilinguole League provides 310 million cubic meters of water per year for industrial uses, in addition to supplying drinkable, ecological, and agricultural water with a guaranteed rate of 97%. According to Wang et al, one tonWhen asked whether there was enough water for daily life, 42.3% of the responses were positive, and 44.3% of surveyed herdsmen thought that the available water just meet their needs.The remaining 13.4% of responses reported that they lacked of fresh water in their daily life, and some areas have had water shortages. HoweverSnowstorms are one of the most important disasters in the grassland.If the livestock die in a snowstorm, it will have tremendous effect on herdsmen’s income. The results of the survey showed that over 55.8% herdsman deemed that blizzards influenced their livestock safety, as it destroyed herdsmen’s houses and barns and caused the loss of livestock, reducing herdsmen’s income. Over half (52.4%) of the herdsmen thought that snowstorms were a serious threat to their life safety and decreased their well-being. During the survey, 62.5% herdsman said that they built stronger barns and prepared enough forage for livestock to eat in the winter, while 22.7% herdsman had to ask for help from the government when snowstorms came. Herdsmen are highly dependent on forage, and snowstorm threatens human safety and their income, decreasing the level of human well-being. The incThe culture and education of herdsmen includes the infrastructures and availability of schools, the education that their children receive and the time herdsmen are able to invest to participate in cultural activities.When asked at what age children start going to school, 90.2% of responses reported that their children go to school at the legal age, although some go to school beyond 8yearsold. At present, most people graduate from middle school and high school.Fewer people drop out of primary school than in the past, which shows that herdsmen consider children’s education important and also indicates that the compulsory education system is more effectively carried out in this area . HoweverAlthough some problems still need to be addressed, the school directors try their best to build a good atmosphere for students, which improves the education conditions of the herdsmen and provides a critical foundation for the improvement of herdsmen’s well-being. Educational progress promotes other wellbeing domains, such as individual economic returns, achievements and accomplishments, enhanced worker productivity, lower crime rates and greater civic participation –43.Most of herdsmen now live in towns, replacing the previous solo living far away from each other. This can establish social norms that promote cohesion, repair and strengthen family cohesion, and provide safe, equitable labor environments that are beneficial to the development of healthy relationships.AccordinWhen asked “where would you like to live?”, 52.5% of herdsmen responded that they like the grassland and prefer to live in rural areas than in the city.They believe that the air is fresher and there is less pollution.On the other hand, 16.8% of the herdsman want to move to Xilinhot,the capital of Xilinguole League, and 30.7% would move to either a neighbouring town or country or elsewhere, because they think that transportation is much more convenient, it is easy for their children to go to school, and they can benefit from more information and opportunities for personal development. HoweverRegarding democracy rights , 73.9%of the herdsmen reported that the cadres of the towns and villages were selected by their democratic votes, while 9.1%and17.0% of the herdsmen thought that the cadres were appointed respectively by family or by higher authorities. These data indicate that herdsmen perceive to have exercised their democratic rights when selecting local political leaders. The perception that their own values are respected and lead to appoint the desired administration should be considered an improvement of personal herdsmen well-being.Ecosystem services in the Xilinguole region have undergone important changes under the influence of human activities. These changes are so important that people have recently become more aware of them. Human activities, including excess grazing, over-mining and land reclamation, contribute to environmental pollution and degradation of ecosystem services–49. ChanAs the magnitude of the impact of human activities on ecological systems becomes day-by-day more apparent, there is growing awareness of the link between the ecosystem integrity and human health, economic development, social justice and national security .PracticeOnly 30.7% of herdsmen reported that their grassland was seriously overgrazed, 43.2% of herdsmen thought that the amount of their livestock was suited for their grassland area, and 26.1% of herdsmen reported that their grassland area was enough for their livestock to survive . They thThe increased coal exploitation did not directly change the local herdsmen’s fuel use. Most of them still use cow dung and sheep manure as fuel. The mined coal, which is a nonrenewable resource, is mainly traded and used elsewhere. Only 9.8% of herdsmen reported that they benefited from grassland mining, but 50.1% herdsman reported that the coal mining could enhance their income. Considering that coal exploitation also affects the environment and local quietude, losses seem to outweigh gains. With the presence of coal enterprises, followed by road construction, the establishment of the transit stations and the laying of pipeline, the original and beautiful natural prairie landscape has been replaced by mining landscape, which changes the prairie pattern, damages the culture and quietude of the grasslands and reduces their tourism value. Coal exA total of 81.8% of government officials  deemed tWhen similar questions were asked to the local herdsmen, only 14.0% deemed that their income increased, 39.3% deemed that their income had not significantly changed, and 46.7% deemed that their income decreased . In contTourism as an ecosystem service requires the identification of biodiversity, ecosystem and landscape features or assets that drive tourism development as well as of related socio-economic changes–56,52. TPolicy decisions related to land use play a significant role in shaping the current state of ecosystems and human well-being in all contexts, thereby implying the need for more informed decision making by governments. The effSeveral economic and environmental assessments of the grassland ecosystem value to the economy and well-being of local population pointed out and quantified its direct and indirect contribution to the economic development of the region, so that any integrity decrease of the grassland ecosystem is likely to heavily affect the quality of life and the sustainability of the entire local economy and social structure. The survey conducted to test the life standard and environmental awareness of local herdsmen population shows that their life is already affected to some extent by the environmental degradation, but does not show their full awareness of the problems, the responsibilities and the possible solutions. The herdsmen still rely on traditional income patterns  which makes most of them hardly able to look out of the box and identify the new options offered by environmental protection and eco-compensation policies for grassland reclamation. Moreover, while coal mining and climate change are recognized among the major factors affecting decreased grassland availability and productivity, the same importance is not assigned to overgrazing due to intensive livestock farming. Changes in the supporting services largely relate to the intensity of grazing. At intensities less than the threshold value, grazing in grassland increases the biomass production and promotes biodiversity.Instead, overgrazing beyond the threshold value restrains grassland primary productivity, destroys the physical behavior of soil and aggravates water loss and soil erosion,62. ThusThe grassland regulating services have also decreased under the influence of human factors and natural factors in recent years. The decWith extensive tourism in grassland areas, the cultural services of grassland have been significantly improved, and herdsmen wellbeing in social relations and health has also obviously been enhanced. Landscape beauty in a well managed grassland attracts tourists from different countries, enhances the cultural exchange between herdsmen and the outside world, promotes the development of other services industries and provides more opportunities for people to choose their occupations. At the same time, it also improves herdsmen’s income and quality of life.Because of its special geographical location and important ecological functions, the scientific research value of the grassland has also increased. The culture of the grassland attracts a large number of researchers, which makes more people understand the grassland, enhances people’s awareness of the need to conserve its integrity, promotes the implementation of grassland sustainable development strategies and improves human wellbeing.Herdsmen are aware of the occurred losses due to environmental degradation. Some herdsmen have also transferred their attention from increasing income to environmental protection and are even willing to invest part of their income to protect the environment and so improve their quality of life. The study of the relationship between ecosystem services and human well-being is very important but also very complex. Clearly, human needs are the driving force behind changes in the ecosystem’s integrity. It is not a given that, once human demand increases beyond the ecosystem carrying capacity, humans stop plundering nature and damaging the ecosystem. A healthy natural environment not only meets the basic human needs but also helps to provide a sense of community by enhancing feelings of pride and a strong sense of kinship among its citizens, who share the common goal of making their community a better place to live .According to the responses to the survey, most herdsmen complained that their income had not significantly changed since 2005. This seems to contradict the data from the statistical office of the Xilinguole League, which report that the average net income of herdsmen was 3,581 RMB in 2005 and 7,471 RMB in 2010, an increase of47.9%. However, rising prices, inflation and the extreme disparity between the rich and the poor may have contributed to that perception, making people feel that their income is steady at the previous level or even decreased. Herdsmen still put basic needs first in terms of well-being and indicate that income dominates their concerns. The herdsmen perceive their income as unchanged or even less than it was before the application of the banning grazing policy. Since 2003, China government started the project of “returning grazing land to grassland”to strictly control and reduce the negative effect of overgrazing. Usually grazing ban mainly was carried out in the high grazing intensity grasslands, but actually it seems very difficult to be implemented since the government owns the grassland that make herdsman care more their animals growth rather than grassland protection. At present, the eco-compensation of grassland is 95.4 RMB(about 15$) per ha in Xilinguole League. The compensation is a way to partially increase income to previous levels, and herdsmen hope that the government will further improve the compensating standard. Based on our survey, government officials believe that the ecological compensation of banning grazing could cover the herdsmen’s losses and that the amount of the compensation is an important factor affecting regional sustainable development. What amount of increase could satisfy herdsmen, how much the government can pay and what type of alternative compensation might be available are topics worth further study and discussion.According to the herdsmen questionnaire survey, the main aspects of ecosystem services and human wellbeing changes in grassland are the following: (1) In the recent years, the supporting and provisioning services of the grassland have declined while the culture services of grassland has received more attention than before. Although the referred value of the regulating services is the largest according to literature, in herdsmen’s views the provisioning services were the most important factor of all the ecosystem services influencing human wellbeing. (2) The herdsmen’s cultural exchange, health, education and social relations are better than before, but the threat from different factors to herdsmen’s health and safety increased. (3) The government’s ecological compensation policy did not fully address the losses due to grassland desertification and environmental degradation.The implementation of government policies is still affected by insufficient understanding of their goals by the herdsmen.(4) The most urgent need of the herdsmen is raising their incomes. The herdsmen’s revenue did not increase, but the herdsmen’s exposure to increased survival risk due to grassland degradation and overexploitation did. (5) The influence of grazing, mining and tourism development on the ecosystem services of the grassland depend on the intensity of these activities. Within a certain range, grazing and mining are capable to promote the improvement of the herdsmen’s wellbeing, but once that line is crossed, the upgrading of the herdsman’s living standards is curbed.S1 Appendix(PDF)Click here for additional data file.


Ischemic stroke is the third leading cause of death and disability. Up until now, all clinical trials testing potential stroke neuroprotectants failed. For this reason attention of researchers has been focusing on the identification of brain endogenous neuroprotective mechanisms activated after cerebral ischemia. In this context, ischemic preconditioning and ischemic post-conditioning represent two neuroprotecive strategies to investigate in order to identify new molecular target to reduce the ischemic damage.Most of the current focus on developing neuroprotective therapies is aimed at preventing neuronal death. However, these approaches have not been successful despite many years of clinical trials mainly because the numerous side effects observed in humans and absent in animals used at preclinical level. Recently, the research in this field aims to overcome this problem by developing strategies which induce, mimic, or boost endogenous protective responses and thus do not interfere with physiological neurotransmission. Preconditioning is a protective strategy in which a subliminal stimulus is applied before a subsequent harmful stimulus, thus inducing a state of tolerance in which the injury inflicted by the challenge is mitigated. Tolerance may be observed in ischemia, seizure, and infection. Since it requires protein synthesis, it confers delayed and temporary neuroprotection, taking hours to develop, with a pick at 1–3 days. A new promising approach for neuroprotection derives from post-conditioning, in which neuroprotection is achieved by a modified reperfusion subsequent to a prolonged ischemic episode. Many pathways have been proposed as plausible mechanisms to explain the neuroprotection offered by preconditioning and post-conditioning. Although the mechanisms through which these two endogenous protective strategies exert their effects are not yet fully understood, recent evidence highlights that the maintenance of ionic homeostasis plays a key role in propagating these neuroprotective phenomena. The present article will review the role of protein transporters and ionic channels involved in the control of ionic homeostasis in the neuroprotective effect of ischemic preconditioning and post-conditioning in adult brain, with particular regards to the Na The blood–brain barrier was preserved thanks to the reduced activity of matrix metalloproteinases and cell adhesion molecules Gidday, .+-ATPase, acid-sensing ion channels, Na+/H+ exchange, Na+/K+/2Cl− cotransport and Na+/Ca2+ exchanger) and transporters for metabolites.Improved capacity to preserve cellular ionic and pH homeostasis: preconditioning regulates expression and activity of ion transport systems , the plasma membrane Ca2+-ATPase (PMCA), the Na+/H+ exchange (NHE), the Na+/K+/2Cl− cotransport (NKCC) and the acid-sensing cation channels (ASIC).Ischemic brain conditioning activates intracellular biological responses to a potential lethal insult; therefore it is predictable that organs reinforce their tolerance when exposed to a sublethal insult prior or after a harmful episode of ischemia by increasing energy metabolism or delaying anoxic depolarization after the onset of the subsequent ischemic insult  is a protein that aids in the active transport of sodium, potassium, and chloride into and out of cells; it is widely distributed throughout the body and it has important functions in organs that secrete fluids. NKCC2 is found specifically in the kidney, where it serves to extract sodium, potassium, and chloride from the urine so that they can be reabsorbed into the blood  mice the lack of NHE1 attenuated the damage induced by conditions mimicking cerebral ischemia in vitro, and this effect was confirmed also in vivo  is protective in adult myocardium in a model of ischemia followed by reperfusion; however, the effect is unclear in immature myocardium  during the first 2 min of reperfusion  during brain post-conditioning few information are available. In a recent work it has been demonstrated that in hippocampal neurons exposed to hypoxia conditioning mimiking an in vitro post-conditioning, NKCC1 is strongly activated through binding with STE20/SPS1-related proline/alanine-rich kinase (SPAK)  superfamily, which are Na+- selective cation channels sensitive to amiloride  are neuronal voltage-insensitive cationic channels activated by extracellular protons. ASIC proteins are a subfamily of the ENaC/Deg superfamily of ion channels. ASICs are potential drug targets for treating a wide variety of conditions linked to both the CNS and PNS. Acid-sensing cation channels (ASIC) have been extensively studied both in physiological and in pathological conditions for their wide distribution in the nervous system and since they represent key targets in many different aspects of acidosis, a condition that occurs during ischemic stroke, as previously analyzed  diseases. Indeed, ischemic brain pH falls to 6.0 due to the accumulation of lactic acid and to the protons produced by ATP hydrolysis , including Akt  is an antiporter membrane protein that removes calcium from cells. It consists of nine transmembrane segments that can mediate Ca2+ and Na+ fluxes across the plasma membrane. Depending on the intracellular concentrations of Ca2+, [Ca2+]i, and Na+, [Na+]i, NCX can operate either in the forward mode, coupling the uphill extrusion of Ca2+ to the influx of Na+ ions, or in the reverse mode, mediating the extrusion of Na+ and the influx of the Ca2+ ions  increase this exchanger mediates the entrance of sodium ions and the extrusion of calcium ions in tight dependence of their electrochemical gradient. The way in which the antiporter works (forward or reverse) depends on several factors such as membrane potential, Na+ and Ca2+ gradient. Therefore, NCX represents an important actor in the regulation of pathophysiological and physiological responses to increases of [Ca2+]i and [Na+]i  is a transport protein in the plasma membrane of cells that functions removing calcium (Ca2+) from the cell; so it is also an important regulator of the calcium concentration in the extracellular space. This protein is the only plasmamembrane high affinity Ca2+ transporting system. It removes Ca2+ from the cytosol in all eukaryotic cells, and for most of them it is the only Ca2+ exporting system together with Na+/Ca2+ exchanger which represent the principal Ca2+ exchanger in heart cells and neurons (Jensen et al., 2+ affinity of the pump, however renders it the only system capable of fine-tuning its exchanges with the external ambient (Brini et al., The plasma membrane Ca22+ -ATPase may export intracellular Ca2+ even with relatively lower transport capacity during prolonged membrane hyperpolarization and during conditions of increased intracellular Na+ concentrations, as occurred under ischemic conditions. Ohta et al. (2+ (Kato et al., Conversely to other plasma membrane buffering systems, such as NCX and ATPases, plasma membrane Caa et al.  suggesteMoreover, in gerbils, ischemic preconditioning by pre-exposure to 2-min ischemia is extremely effective in protecting against ischemic cell death since there is a modification of the electrophysiological properties of CA1 neurons (Kawai et al., The identification of the players involved in the control of ionic homeostasis during pre and post-conditioning should provide more direct opportunities for translational neuroprotection trials. Indeed, the present review, summarizing the specific effect of the plasmamembrane proteins involved in the maintenance of cellular ionic concentration, would provide information of which transporters needed to be activated or inhibited in order to protect the brain by the ischemic injury.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.


Puccinia triticina races 165 and 260 were used to constitute compatible and incompatible combinations to investigate the relationship between NO and H2O2 and between NO and calcium (Ca2+) signaling in the cell defense process by pharmacological means. The specific fluorescent probe DAF-FM DA was coupled with confocal laser scanning microscopy and used to label intracellular nitric oxide (NO) and monitoring the real-time NO dynamics during the processes of wheat defense response triggered by P. triticina infection. The results showed that at 4 h after inoculation, weak green fluorescence was observed in the stomatal guard cells at the P. triticina infection site in the incompatible combination, which indicates a small amount of NO production. Twelve hours after inoculation, the fluorescence of NO in- cell adjacent to the stomata gradually intensified, and the NO fluorescent area also expanded continuously; the green fluorescence primarily occurred in the cells undergoing a hypersensitive response (HR) at 24–72 h after inoculation. For the compatible combination, however, a small amount of green fluorescence was observed in stomata where the pathogenic contact occurred at 4 h after inoculation, and fluorescence was not observed thereafter. Injections of the NO scavenger c-PTIO prior to inoculation postponed the onset of NO production to 48 h after inoculation and suppressed HR advancement. The injection of imidazole, a NADPH oxidase inhibitor, or EGTA, an extracellular calcium chelator, in the leaves prior to inoculation, delayed the onset of NO production in the incompatible combination and suppressed HR advancement. Combined with our previous results, it could be concluded that, Ca2+ and hydrogen peroxide (H2O2) are involved in upstream of NO production to induce the HR cell death during P. triticina infection, and Ca2+, NO and H2O2 are jointly involved in the signal transduction process of HR in the interaction system.In this research, the wheat cultivar 'Lovrin 10' and  As a common intracellular messenger, Ca2+ is widely involved in intracellular biochemical reactions and signal transduction processes. In the interactions between plants and pathogens, an increase in cytosolic Ca2+ ([Ca2+]cyt) concentrations is often the early-stage cellular response to pathogens infection, and this increase is also required to induce the HR form of cell death at the pathogen infection site cyt is associated with cyclic nucleotide-gated channels (CNGCs) [2+]cyt was closely related to NO production cyt in response to the elicitor by the protoplasts of resistant wheat varieties was likely dependent on the influx of extracellular Ca2+, and a similar result was also observed in ultrastructure of wheat leaf infected by P. triticina cyt and H2O2 production cyt, and the signal is transduced and amplified through calcium-binding proteins such as calmodulin, calcium-dependent protein kinase and calcineurin cyt and NO signals in the signal transduction pathway of the host response to pathogen infections is still inconclusive.NO also has important regulatory functions in the interactions between plants and pathogens. Particularly, the HR form of cell death at the pathogen infection site and gene expression triggered by NO acting as a signaling substance has become a focus in researches related . Among pis of NO , with Cantration . Ma et antration  found th2+ in mesophyll cells and the changes of [Ca2+]cyt in the elicitor-protoplast system, our previous study has shown that the calcium messenger system mediates the interactions between wheat plants and P. triticina and that Ca2+ influx is essential for the HR defensive response to P. triticina infection [2+ has any influence on the formation of NO in these interactions, EGTA was used in this study. The results showed that NO green fluorescent was not observed at the infection site following the injection of EGTA at 4–48 h after inoculation, NO fluorescence was mostly concentrated in HR cells at 72 h after inoculation  and abscisic acid (ABA) treatments, NO synthesis relied on the H2O2 generated in the guard cells [2O2 generation and to induce the expression of relevant defense-related downstream genes, and in broad bean guard cells, NO can regulate the accumulation of H2O2 [2O2 was likely at upstream of NO and regulated the formation of NO by regulating NR activity, which ultimately leads to stomatal closure in Arabidopsis thaliana.When plant responses to biotic and abiotic stresses, NO and H enzymes –56. NO ard cells , 58. How of H2O2 . Liu et  of H2O2  reported2O2 can mediate HR in host cells in the incompatible combination between wheat plants and P. triticina. Pharmacological analysis indicated that in the interaction system, the influx of Ca2+ triggered H2O2 generation, which initiated hypersensitive cell death [2O2 and NO. In the incompatible combination, subsequent to the pre-injection of imidazole, NO production was inhibited (P. triticina infection, H2O2 acts at upstream of NO. It requires further study whether NO provides feedback regulation on the concentration of intracellular H2O2.Our previous results have demonstrated that Hll death . Imidazonhibited ; in addinhibited , and thenhibited . Therefo


The discovery of the STIM and ORAI proteins has allowed researchers to begin clarifying the mechanisms and physiological impact of store-operated Ca2+ entry, the major pathway for generating calcium signals in innate immune cells. Here, we review evidence from cell lines and mouse models linking STIM and ORAI proteins to the control of specific innate immune functions of neutrophils, macrophages, and dendritic cells.Phagocytic cells, such as neutrophils, macrophages, and dendritic cells, migrate to sites of infection or damage and are integral to innate immunity through two main mechanisms. The first is to directly neutralize foreign agents and damaged or infected cells by secreting toxic substances or ingesting them through phagocytosis. The second is to alert the adaptive immune system through the secretion of cytokines and the presentation of the ingested materials as antigens, inducing T cell maturation into helper, cytotoxic, or regulatory phenotypes. While calcium signaling has been implicated in numerous phagocyte functions, including differentiation, maturation, migration, secretion, and phagocytosis, the molecular components that mediate these Ca Intracellular Ca2+ signaling is based on the principle that cytosolic Ca2+ concentrations are maintained at levels ∼100,000-fold lower than the extracellular environment by energy-driven pumps that either extrude Ca2+ ions from the cell, or sequester them in membrane-bound intracellular compartments, termed Ca2+ stores. This large concentration gradient then allows an extremely rapid influx of Ca2+ upon regulated opening of Ca2+ channels, either at the plasma membrane (PM) or on the membranes of Ca2+ stores. The major intracellular store is the endoplasmic reticulum (ER), where pumps of the sarcoendoplasmic-reticulum ATPase family maintain luminal Ca2+ levels high under steady-state conditions, although the role of lysosomes as Ca2+ storage and signaling platforms has been increasingly recognized more recently . As the name implies, SOCE has been functionally defined as the opening of PM Ca2+ channels that occurs subsequent to and as a consequence of the release of Ca2+ from intracellular stores, and was historically also termed capacitive Ca2+ entry (2+-release activated current (CRAC). Although for decades the CRAC current could be identified through its unique electrophysiological properties, the molecular machinery underlying SOCE was not described until 2005–2006, with the characterization of STIM and ORAI proteins . InsP3 then diffuses from the site of activation to the surface of the ER, where it binds InsP3 receptors that allow Ca2+ to flow out of the ER and into the cytosol  activation domains can trap and directly gate PM Ca2+ channels. In addition to luminal ER Ca2+ content, the activity of STIM proteins are regulated by phosphorylation, ROS-dependent cysteine modification, as well as binding to regulatory proteins, including junctate (ASPH), partner of stromal interaction molecule 1 (POST/TMEM20/SLC35G1) and store-operated calcium entry associated factor (SARAF/TMEM66) , and additionally by their translocation to phosphatidylinositol , while the function of STIM2 is less understood. STIM2 has a higher sensitivity for Ca2+ and thus becomes activated before STIM1, but is less potent as a ligand for ORAI channels, and its highly conserved alternatively spliced isoform STIM2β is a potent inhibitor of SOCE  and pathogen- or danger-associated molecular pattern (PAMP and DAMP) receptors and migrate across the vascular endothelium toward infection sites to ingest and kill invading pathogens. Neutrophil emigration across inflamed endothelium is a multistep process initiated by the engagement of selectin and chemokine receptors that trigger a transition from cell rolling to integrin-dependent arrest and migration  by 70%, an effect that correlated with a decreased phosphorylation of Akt, Src, and Rac2  or the anaphylatoxin C5a in vitro, and normal neutrophil infiltration into lungs in a model of immune complex-induced pneumonitis. Whether this phenotype correlated with alterations in neutrophil Ca2+ signals is unclear, however, as SOCE was not measured in these Stim1−/− neutrophils. In a subsequent study, we used the myeloid-specific LysM-Cre promotor to delete the Stim1 gene in neutrophils from C57BL/6 mice . Stim1 ablation decreased phagocytosis in neutrophils by 50% and caused a corresponding 50% decrease in the frequency of the local Ca2+ elevations occurring near phagosomes (2+ readmission protocol was preserved in STIM1-deficient neutrophils (data not shown). Yet Stim1 deletion decreased the extent and length of cortical ER cisternae juxtaposed to phagosomes and to the PM by 50%. We, therefore, concluded that STIM1 recruits ER cisternae near phagosomes to generate Ca2+ microdomains that promote the phagocytic process. The prophagocytic Ca2+ signals were generated by the release of Ca2+ from the recruited ER stores and by the opening of phagosomal Ca2+ channels by STIM1, implying a dual role for STIM1 as an adaptor protein that delivers “calciosomes” to specific cellular locations and as ligand for intracellular channels. The study suggests that local Ca2+ signals might be more important than global Ca2+ signals in the control of neutrophil function.In a pioneering study, Braun et al.  used letagosomes . To our Stim1−/− mice to study the impact of Stim1 ablation on neutrophil function. The delayed phase of the Ca2+ elevations evoked by chemoattractant, Fcγ-receptor cross-linking, store depletion, and integrin-mediated adhesion were reduced in Stim1−/− neutrophils. The extent of inhibition was not quantified, but the representative traces suggest a near complete abrogation of Ca2+ entry triggered by receptor ligation in Stim1−/− neutrophils. Surface levels of adhesion molecules LFA-1  and Mac-1 (CD11b/CD18), adhesion to fibrinogen, PLC-γ phosphorylation, and InsP3 production were normal in Stim1−/− neutrophils, ruling out upstream defects in integrin signaling. Despite the SOCE defect, Stim1−/− neutrophils efficiently migrated toward chemoattractant in vitro and were recruited in the peritoneal cavity and in foot pads following injection of thioglycollate and MIP-2. Phagocytosis of opsonized S. aureus was reduced by 50–70%, and integrin and chemoattractant-mediated lactoferrin release, by 30%. A more profound defect was observed for superoxide production measured with the luminol-based chemiluminescence and cytochrome c reduction assays, the inhibition ranging from 25 to 50% for FcγR-mediated to 50–100% for integrin and chemoattractant-mediated responses. The authors concluded that STIM1 is dispensable for neutrophil adhesion and migration, and that the major cellular defects of Stim1 ablation in neutrophils are a defective respiratory burst, reduced phagocytosis, and a mild degranulation defect. The blunted superoxide production of Stim1−/− neutrophils was linked to reduced phosphorylation of the p40phox and p47phox NADPH oxidase subunits by PKC-α and -β. In vivo, mice engrafted with Stim1−/− bone marrow cells were more susceptible to S. aureus pneumonia and L. monocytogenesis septicemia and had reduced liver damage following ischemia-reperfusion injury, with levels of liver enzymes and of the inflammatory cytokine IL-6 comparable to those observed in p47phox−/− mice lacking a functional oxidase. This rather spectacular phenotype cannot be unambiguously attributed to defective neutrophil function, however, because SOCE deficiency in macrophages and DCs might contribute to the increased susceptibility of Stim1−/− chimera to bacterial infections, while impaired platelet function might account for the reduced tissue injury upon hepatic ischemia-reperfusion.More recently, Zhang et al.  used irrStim1fl/fl; LysMCre/Cre). The STIM1-deficient neutrophils isolated from the blood and bone marrow of these mice had ∼50% reduced Ca2+ entry following store depletion with thapsigargin and reduced chemotaxis to a range of chemoattractant substances across fibronectin-coated membranes. In vivo, a decreased neutrophil infiltration in the skin was observed following application of imiquimod to induce psoriasis-like lesions, despite normal epidermal production of chemoattractant by plasmacytoid DCs and keratinocytes. The reduced chemotaxis and emigration of Stim1−/− neutrophils observed in this study using conditional genetic ablation is thus at odds with the two previous studies using chimeric mice  studied the recruitment of neutrophils by the anaphylatoxin C5a in mice transplanted with bone marrow cells from Orai1+/− mice, Ca2+ elevations associated with the capture of neutrophils on ICAM-1- and E-selectin-coated surfaces, neutrophil arrest, polarization, and directional migration under shear flow were reduced (2+ elevations initiated by activation of high-affinity LFA-1 (CD11a/CD18) were more deficient than the response to chemotactic peptides, suggesting that ORAI1 is specifically required for integrin-mediated outside-in signaling. Accordingly, ORAI1 depletion reduced the size of LFA-1 clusters and F-actin polymerization, causing a defect in adhesion strengthening under shear stress. In vivo, the defect in adhesion strengthening translated into reduced neutrophil recruitment in skin wounds . Th. Th2+ reto ORAI1 . Again, ficiency . Thus, aMacrophages, like neutrophils, kill and clear invading pathogens through phagocytosis, via the acidification and release of enzymes within their phagosomes . Phagoso2+ elevations to the control of macrophage functions is disparate and somewhat contradictory. Early studies revealed that receptors for platelet-activating factor and for the chemotactic peptide fMLP induce SOCE, implicating SOCE in the control of macrophage function , while C5a bioactivity and cytokine secretion in the alveolar fluid were blunted in a model of hypersensitivity pneumonitis. Consequently, chimeric Stim1−/− mice were protected against AIHA as well as autoimmune thrombocytopenia and anaphylaxis induced by the injection of anti-platelet antibodies, an IgG-dependent response mediated by macrophages and mast cells. This study thus establishes STIM1 as an essential mediator of autoimmune inflammation that controls FcγR-mediated signaling in macrophages.Based on these premises, the diverging outcome of altered STIM1-ORAI coupling on macrophage functions in animal models is not too surprising . The firrophages . In addiStim1−/− mice together with global and chimeric Stim2−/− mice to study the contributions of STIM1 and STIM2 in macrophage functions. SOCE and store content were decreased to a similar extent in peritoneal macrophages isolated from chimeric Stim1−/− and global Stim2−/− mice, but the Ca2+ elevations evoked by cross-linking of FcγRIII and FcγRIV were only marginally decreased in Stim2−/− cells. This milder Ca2+ signaling defect correlated with a milder inhibition of phagocytosis that could be rescued by the addition of C5a or of liver supernatant from autoimmune mice to Stim2−/− macrophages. Unexpectedly and contrary to the mild Ca2+ phenotype, CCL2 and C5a-induced chemotaxis, as well as Toll-like receptor-4-mediated cytokine release were decreased in Stim2−/− and normal in Stim1−/− macrophages. Similar diverging cellular defects were observed in the RAW 264.7 macrophage cell line, where STIM1 silencing inhibited phagocytosis, while STIM2 but not STIM1 silencing reduced chemotaxis  under the control of the inducible Mx1-Cre or myeloid-specific Vav-Cre promotors to test the functional impact of SOCE in myeloid cells. Consistent with earlier studies in chimeric mice, SOCE evoked by store depletion or by FcγRII/III cross-linking was nearly absent in BMDMs from these mice. However, ablation of the two STIM proteins did not impact the intracellular Ca2+ store content or the differentiation and maturation of BMDMs. Despite the complete lack of SOCE, FcγR-independent phagocytic uptake of nonopsonized latex beads and bacterial phagocytosis of S. aureus were normal. Surprisingly, and in complete disagreement with the studies in chimeric mice, FcγR-dependent phagocytosis of opsonized particles and of red blood cells was also completely normal in BMDM and peritoneal macrophages from all of the STIM-deficient mice tested measured at different times and at different phagocytic indexes. In contrast, chelation of cytosolic Ca2+ with BAPTA-AM significantly reduced FcγR-dependent phagocytic uptake to a greater extent than nonopsonized targets, demonstrating that intracellular Ca2+ elevations positively regulate phagocytosis by macrophages. Phagosomal maturation was equally unaffected as lysosomal marker recruitment to phagosomes was comparable to wild-type mice. The authors then tested other macrophage functions and observed normal production of a battery of cytokines  in BMDM exposed to synthetic or natural ligands of PRRs known to mediate Ca2+ signals , indicating that STIM proteins and SOCE are dispensable for cytokine production by macrophages. Finally, IL-1β secretion, cleavage of procaspase 1 into active caspase 1, and intracellular levels of pro-IL-1β were normal in BMDMs from Stim1fl/flMx1-Cre mice and Stim1fl/fl; Stim2fl/flVav-Cre mice stimulated with LPS and ATP, and with monosodium urate crystals or bacterial flagellar toxin to induce the activation of the NLRP3 and NLRC4 inflammasome, respectively. Overall, this comprehensive study clearly demonstrates that STIM1 and STIM2 proteins are not required for phagocytosis, cytokine secretion, or inflammasome activation in macrophages.These conclusions were challenged by a recent study by Vaeth et al. , who useStim genes and radiation chimeric mice reconstituted with bone marrow cells from Stim1−/− mice result from differences in the genetic background of the mice used, or whether added radiation-induced stress can account for these differences remains to be determined, but it is conceivable that the additional defects in ER Ca2+ content may contribute in part to the discrepancies. Future studies confirming or disproving the differential effects of STIM2 ablation, or the effects of STIM protein on migration, will also be informative in discerning how Ca2+-dependent functions are regulated in macrophages.Whether the differences in findings obtained with inbred mice with conditional myeloid ablation of the two DCs are phagocytic immune cells whose fundamental role is to engage and instruct the adaptive immune system. It is generally believed that, unlike neutrophils and to a certain extent macrophages, the function of DCs is not to kill ingested particles. Instead they are specialized to integrate and use the ingested material as well as the signals received during the ingestion encounter to then present antigens to naive T cells, and, depending on the context, help initiate T-helper, cytotoxic, or immunosuppressive responses , 37, 80.2+ signaling to multiple DC functions, including cell-surface marker upregulation accompanying differentiation and maturation, phagocytosis, cytokine secretion, migration, and antigen presentation , and made the provocative suggestion that “pure” (what is now defined as STIM-ORAI mediated) SOCE may be the only major Ca2+ entry pathway in DCs  was either unchanged or even slightly improved, as judged by T-cell proliferation measured by carboxyfluorescein succinimydil ester dilution or by the higher numbers of IFN-γ-positive T cells. In contrast, phagocytosis, inflammasome activation, and antigen presentation were inhibited by intracellular Ca2+ chelation. Thus, while human DCs may rely largely on STIM1 and ORAI1-dependent Ca2+ signaling for some PRR-dependent responses, alternative Ca2+ signaling pathways may allow mouse DCs to function independently of STIM proteins. Currently, several questions still remain. For instance, as both STIM proteins were ablated in the Vaeth et al. .Author contributions: N.D. and P.N. drafted manuscript; N.D. and P.N. edited and revised manuscript; N.D. and P.N. approved final version of manuscript.


I would like to comment on the technique for intercostal nerve preservation. It is difficult to assess and quantify immediate postoperative pain clinically, which is why I enthusiastically read the article in which Marchetti-Filho et al. describThoracic surgeons should certainly be able to deal with acute and chronic post-thoracotomy pain. In the past, neuropathic pain occurred in approximately 50% of patients, being generally mild or moderate. However, it is estimated to persist in 5% of cases, leading to disability. In such cases, three-dimensional reconstruction of CT scans of the chest has been used in order to assist in the decision of whether to perform intercostal neurolysis.Surgical techniques to prevent intercostal nerve injury should be validated and used in order to reduce the incidence of acute and chronic post-thoracotomy pain.After an extensive thoracotomy, the intercostal space should be opened slowly and gradually with the Finochietto retractor in order to avoid rib fractures. However, the amount of rib spreading can suddenly overcome the resistance of one or more ribs, causing one or more fractures. A rib fracture can be accompanied by neurovascular bundle injury at the upper and lower ribs. This can result in chronic neuralgia and paresthesia in the postoperative period. Rib spreading with the Finochietto retractor is traumatic because the neurovascular bundle at the upper rib is crushed and remains so for a long period of time. In order to avoid intercostal nerve compression by the Finochietto retractor, it has been proposed that an intercostal muscle flap be harvested before placement of the retractor.In the past, circumcostal suture placement for rib approximation was widely used for thoracotomy closure. However, it often caused neuropathic pain as a result of the suture material compressing the neurovascular bundle at the lower rib. The current intercostal nerve preservation strategy requires that care be taken when closing a thoracotomy. There are currently three thoracotomy closure techniques: transosteal or transcostal suture closuretransperiosteal suture closurepericostal suture closure, whereby sutures are placed in the virtual space between the periosteum and the neurovascular bundleThree interrupted sutures are generally placed in order to close an extensive thoracotomy. When a rib fracture occurs, one or more sutures are needed in order to stabilize and align the fracture fragments. Synthetic absorbable 1-0 polyglactin 910 suture should be used whenever possible. For increased suture strength, a double-loop, U-shaped suture  is generally recommended. Below, I describe the thoracotomy closure technique whereby sutures are placed in the virtual space between the periosteum and the neurovascular bundle at the lower rib. The surgical needle with absorbable 1-0 suture is passed into the thoracic cavity through a point above the upper edge of the rib cranial to the thoracotomy. The needle inside the thoracic cavity is retrieved with the needle holder, and the suture is pulled until it reaches half of its length. The needle goes into the thoracic cavity again and out of the chest wall through a point located very close to the entry point. A U-shaped suture  is thus obtained, the strength of which is greater. The needle is removed, and the suture ends are trimmed with straight Kelly forceps. The suture end inside the thoracic cavity is pulled out and looped around the Kelly forceps so that the double suture is halved. The procedure described above is performed for each of the three or four sutures required for thoracotomy closure, thus concluding the first step of the closure technique. At the lower edge of the rib caudal to the thoracotomy, blunt dissection is performed with curved Halstead forceps so that the tip of the instrument enters the thoracic cavity . The disMinor bleeding from the blunt dissection should be managed so that hemostasis is achieved at this point. With the use of dissecting forceps, the suture end is led to the tips of the curved Halstead forceps, being clamped and pulled out of the thoracic cavity. This is done for all sutures passed through. Subsequently, each double suture is firmly tightened and tied with a surgeon's knot. Care must be taken to prevent the tied ribs from touching one another or from overlapping. After the remaining suture is cut and removed, the muscle layers are closed with sutures and thoracotomy closure is completed.


Therefore, we validated whether ADRP retinas experience a cytosolic Ca2+ overload, and whether sustained UPR in the wild-type retina could promote retinal degeneration through Ca2+-mediated calpain activation. We performed an ex vivo experiment to measure intracellular Ca2+ in ADRP retinas as well as to detect the expression levels of proteins that act as Ca2+ sensors. In separate experiments with the subretinal injection of tunicamycin (UPR inducer) and a mixture of calcium ionophore (A231278) and thapsigargin (SERCA2b inhibitor) we assessed the consequences of a sustained UPR activation and increased intracellular Ca2+ in the wild-type retina, respectively, by performing scotopic ERG, histological, and western blot analyses. Results of the study revealed that induced UPR in the retina activates calpain-mediated signaling, and increased intracellular Ca2+ is capable of promoting retinal degeneration. A significant decline in ERG amplitudes at 6 weeks post treatment was associated with photoreceptor cell loss that occurred through calpain-activated CDK5-pJNK-Csp3/7 pathway. Similar calpain activation was found in ADRP rat retinas. A twofold increase in intracellular Ca2+ and up- and downregulations of ER membrane-associated Ca2+-regulated IP3R channels and SERCA2b transporters were detected. Therefore, sustained UPR activation in the ADRP rat retinas could promote retinal degeneration through increased intracellular Ca2+ and calpain-mediated apoptosis.The molecular mechanism of autosomal dominant retinitis pigmentosa (ADRP) in rats is closely associated with a persistently activated unfolded protein response (UPR). If unchecked, the UPR might trigger apoptosis, leading to photoreceptor death. One of the UPR-activated cellular signaling culminating in apoptotic photoreceptor cell death is linked to an increase in intracellular Ca The ADRP progression in these animals is characterized by an activated unfolded protein response (UPR) and the mitochondrial dysfunction.2+, a second messenger, participating in a wide variety of physiological functions, including signal transduction, muscle contraction, protein, and hormone secretion. The normal ER functions to regulate and control intracellular Ca2+, thus governing protein synthesis, gene expression, secretion, metabolism, and apoptosis.6 Therefore, the ER disturbance results in a release of Ca2+ into the cytosol, after which free Ca2+ can be either transported to the mitochondria or directly activate cytotoxic cellular pathways.7 The findings support the fact that Ca2+ transport from the ER to mitochondria has a significant role in regulating cellular bioenergetics, the production of reactive oxygen species, the induction of autophagy, and apoptosis.10It is known that the ER serves as the primary store in cells for Ca2+ concentrations, perhaps associated with their varying functions, including the transduction of photon energy into an electrical signal and transcriptional, translational, metabolic, and synaptic properties.11 For example, the CNGCs channels regulated by the phototransduction cascade and the VGCCs channels regulated by light-induced cell membrane hyperpolarization are located in the outer segments (OS) and in the cell body and synaptic terminal, respectively. This differential localization results in high Ca2+ influx in the OS and low Ca2+ influx in the cell body and synaptic terminal in dark conditions. In addition, SOCE channels in the plasma membrane, SERCA transporters, IP3 receptors, and ryanodine receptors in the ER, and the VDAC channels in the mitochondria located in the inner segment (IS) have also been shown to contribute to the regulation of intracellular Ca2+ in photoreceptors.13It has been found that in photoreceptors, different cellular compartments demonstrate a marked variation in Ca2+.7 Thus, Ca2+-induced cell death in addition to apoptotic cell death has been proposed to occur in the outer nuclear layer (ONL) of the retina of transgenic animals mimicking human retinal degeneration, resulting in the massive activation of PKG, calpain, HDAC, and PARP activation.14Despite the fact that historically cell death associated with calcium ion perturbations has been primarily recognized as necrosis, recent evidence suggest that more complex cell death scenarios could be associated with changes in the concentrations of Ca2+-induced photoreceptor cell death has a crucial role in ADRP pathogenesis.18 Despite the fact that progress has been made in understanding the significance of Ca2+ overload, direct evidence of Ca2+ overload in ADRP photoreceptors has not been provided. Importantly, the role of an activated UPR in promoting Ca2+-mediated cytotoxicity in degenerating photoreceptors and the link between UPR-modulated IP3, ryanodine, and SERCA channel activities and retinal degeneration has not yet been established.There are several studies supporting the hypothesis that Ca2+ overload in ADRP retinas and a cytosolic Ca2+ overload triggers a loss of photoreceptor function, eventually leading to photoreceptor cell death in rat models of ADRP.Therefore, we hypothesized that a persistently activated UPR contributes into an intracellular Ca2 However, it has been demonstrated that activation of the calpain system requires intracellular Ca2+ concentrations of at least tens of μM,19 whereas the intracellular Ca2+ concentrations in dark-adapted photoreceptors range only from 300–500 nM.20 This made us question whether the perturbance of ER homeostasis could promote enough Ca2+ release to result in the uptake of Ca2+ by mitochondria. Other studies have demonstrated that inhibition of Ca2+ can be beneficial by delaying ERG amplitude attenuation.22 However, direct evidence of Ca2+ overload in photoreceptor cells has not yet been provided.Previous studies from our laboratory have shown that the S334ter and P23H Rho rats have retinal degeneration associated with persistently activated UPR and disrupted mitochondrial membrane potential (MMP), resulting in calpain activation and the release of cytochrome C and AIF from mitochondria.2+ load in ADRP retinas with ongoing UPR, we validated the method of Ca2+ measurement and provided proof of principle that the activated UPR causes the release of the Ca2+ into the cytosol. To that end, we performed subretinal injections of P30 SD rats with tunicamycin (Tn), which has been previously demonstrated to activate the UPR in the retina.4 Tn was injected into the right eye for detection of levels of intracellular Ca2+ in photoreceptors with an experimentally induced UPR response. Vehicle injection into the left eye served as a control. 2+ overload in the cytosol of photoreceptors.Before detection of the Ca2+ in S334ter and P23H Rho photoreceptors. Our data showed that photoreceptors had an overload of intracellular Ca2+ at P30 that was in agreement with the rate of retinal degeneration found in these animals http://www.ucsfeye.net/mlavailRDratmodels.shtml. Therefore, after registering a free cytosolic Ca2+ increase, we decided to dissect the sources of such overload by testing the expression of Ca2+ signaling proteins specific for different compartments of photoreceptor cells.Next, we measured cytosolic Ca2+ increase in S334ter and P23H RHO photoreceptors.14 Because VGCC expression is known to reflect the modulation in Ca2+ concentration,23 we tested the level of VGCC protein expression by western blot analysis and found no difference between control and experimental groups. These data suggested that the Ca2+ increase registered in transgenic photoreceptors is not generated by VGCC malfunctioning. Keeping that in mind, we then tested whether the source of the Ca2+ elevations was from intracellular compartments by measuring the expression levels of the cytosolic Ca2+-signaling proteins calpastatin and calcineurin, the ER resident calreticulin (CRN), and the mitochondrial VDAC protein.The diffuse accumulation of the cGMP and S334ter and P23H Rho rats has been previously shown in the ONL of the retina, suggesting a Ca2+-dependent cysteine protease inhibitor of calpains, was upregulated in both transgenic retinas. Both mRNA and protein levels were increased at P21 , a Ca2+- and calmodulin-dependent serine–threonine phosphatase was also upregulated in both transgenic retinas at P30 , a Cad at P21 . Interess at P30 . Increas2+, metabolites, and apoptosis24 and found that Vdac mRNA was significantly higher in both transgenic retinas at P21 and P30  elevated in both transgenic retinas at P21 and (over fourfold) in S334ter retinas at P40 (The ER resident Cas at P40 . Interes2+ (2+-sensing receptor proteins: phosphorylated (p) IP3R receptor, SERCA2b, and Bax inhibitor-1 (BI-1)  . We lear retinas . pIP3R p retinas . These c retinas . In genefor BI-1 . Our dat2+ from the cytosol of the cell to the lumen of the ER. Interestingly Serca2b mRNA was elevated in both transgenic retinas at P21 , targeting the SERCA2b activity to create a new experimental rat model of retinal degeneration. A23187, a divalent cation ionophore, is widely used in laboratories to increase intracellular Ca2+ in intact cells. The dose of A23187 (0.05 μg/eye) was chosen based on a previously published in vitro study.28 Tg is a non-competitive inhibitor of the SERCACa2+ ATPase. Therefore, injecting the SD rat retinas with these drugs would be expected to result in increased cytosolic Ca2+. Retinas were analyzed 2, 4, and 6 weeks after injection by ERG and protein analyses to validate the effect of increased cytosolic Ca2+ on photoreceptor function and cellular signaling. Other retinal cell types in this model would also be expected to have increased cytosolic Ca2+. However, by registering the photoreceptor-originated scotopic a-wave ERG amplitudes vs b-wave amplitude originating mostly from bipolar cells, we measured functional changes occurring directly or indirectly in these cells, and tested the direct effects of increased Ca2+ specifically in rod photoreceptor cells. Experimental data from Tg- and A23187-injected retinas are presented in 2+ results in retinal functional loss. B-wave amplitude also declined in this model by 39% and 47% at 2 and 6 weeks, correspondingly.Knowing that the compromised ER integrity persistently promotes Ca with Tn  showed tP<0.05), suggesting that injections resulted in Ca2+-induced retinal degeneration in the experimental rat model.To verify whether the decline in photoreceptor function was associated with photoreceptor cell loss, we further performed a histological analysis in which we stained retinal cryosections with H&E . We foun2+ in the rat retina. We analyzed downstream calpain-activated targets and tested the CDK5 expression, which is known to be governed by calpain's processing of a proteolytic cleavage of the CDK5's activator, p35. The CDK5 level was significantly higher in A23187+Tg–injected retinas than vehicle-injected retinas, pointing out the potential phosphorylation of the MEKK1 and activation of the JNK apoptotic pathway.29 To validate this hypothesis, we tested the pJNK level and found it to be 77% higher in drug-injected retinas. Altogether, these data indicate that the apoptotic pathway was elevated in treated retinas. Therefore, we then verified caspase-3/7 activity and found almost a ninefold elevation of the caspase-3/7 activity in retinal extract treated with drugs.To characterize the experimental rat model of retinal degeneration, we harvested injected (A23187+Tg) retinas 36 h post injection and analyzed the cellular signaling involved in photoreceptor cell loss. . We foun2+ overload in the nerve fiber layer and the inner nuclear layer in P23H Rho rat retinas (line 1) has been reported recently.30 However, indirect indications of increased intracellular Ca2+ in degenerating photoreceptors have been provided only by the therapeutic modulation of Ca2+ blockers or inhibitors in their retinas.22 Our study is the first molecular genetic evidence, demonstrating that in the retina of rat models of ADRP, Ca2+ release from the ER contributes to the mechanism of retinal degeneration. We first demonstrated an increase in intracellular Ca2+ during the ADRP progression in photoreceptors and linked this increase to a persistently activated UPR-induced ER Ca2+ depletion. Thus, we found that the modified expression of ER membrane Ca2+ channels and resident buffering proteins is associated with increased cytoplasmic Ca2+ and an elevation of mitochondrial VDAC. These events are in agreement with a previously reported cytochrome C and AIF1 release from the mitochondria in these rats.31 In addition, we demonstrated that the ER perturbance in the retina is associated with Ca2+-induced calpain activation capable of triggering retinal degeneration in an experimental rat model.The presence of a Ca30 Interestingly, although the rate of retinal degeneration in these two ADRP models differs, the level of the cytosolic Ca2+ increase in the P30 photoreceptors is similar. This suggests that the source of elevated intracellular Ca2+ in photoreceptors could be a result of malfunctioning ER and resulted extensive Ca2+ outflow from ER lumen. It is worth mentioning here that if for the pIP3R and BI-1 both the mRNA and the protein level were upregulated, the CRN protein was not altered as compared with elevated Crn mRNA. Perhaps, discrepancy in a gene and protein expression in both groups can be easily explained by the recently reported correlation between the CRN overproduction and the Ca2+ overload within the ER. Thus, Michalak et al.32 has found that 50% of all Ca2+ stored in the ER binds to CRN, and therefore, CRN protein overproduction indicates an elevation in the ER luminal Ca2+. Moreover, Waser et al.25 have demonstrated that overexpression of Crn mRNA is a sign of ER Ca2+ depletion. Therefore, the upregulation of Crn mRNA expression and unaltered protein production indicate ER Ca2+ reduction in both ADRP retinas.Both ADRP rat models demonstrate the activation of the UPR at P21 and P30.31 we observed the upregulation of calpastatin and calcineurin levels. In general, CN is known to regulate the activity of AKT through dephosphorylation.23 Therefore, we were not surprised to observe a marked increase in the CN protein in P30 P23H Rho retinas that was in agreement with previously detected decrease of pAKT and upregulation of mTOR.1 All these data highlight a cellular defense mechanism activated in photoreceptors from P21 to P40 that is trying to cope with a launched mechanism of photoreceptor degeneration. However, the strength of such signaling seems to be insufficient to block Ca2+-induced calpain activation and, consequently, intracellular Ca2+ affects the mitochondrial homeostasis in these animals. As proof of this hypothesis, the observed VDAC elevation indicates a disrupted molecule flux across the outer mitochondrial membrane and suggests cross-talk between these two organelles.Concomitantly with activated UPR and upregulated calpain activity have been previously shown,2+-induced signaling. We have previously tested Tn in vivo and have demonstrated that the activation of the UPR induces retinal degeneration in mouse retinas.4 In this study, we demonstrated sustained UPR-induced cytotoxicity in a primary photoreceptor culture and in vivo and analyzed a signaling responsible for cell loss. Both in vitro and in vivo, Tn induces a calpain activation that is associated with disrupted ER Ca2+-sensing receptor expression. This implies that UPR-induced photoreceptor cell death occurs via Ca2+ outflow from ER lumen and Ca2+-mediated calpain signaling. However, to obtain direct evidence that the intracellular Ca2+ increase induced by persistently activated UPR in photoreceptors as seen in 2+-induced retinal degeneration.Next, we were interested in testing whether sustained UPR activation is capable of triggering photoreceptor cell death via Ca2+-induced photoreceptor cell death. The rod-derived a-wave amplitude of the scotopic ERG was significantly downregulated. In this model, the functional loss of photoreceptors is in agreement with a number of dying cells. Therefore, the findings from these experiments permit us to conclude that we created an experimental model of Ca2+-induced retinal degeneration. The experimental model mimics the inherited retinal degeneration occurring in rats, not only by the reduction of the scotopic ERG amplitudes and photoreceptor cell death, but also by the mechanism responsible for apoptotic photoreceptor cell death found in ADRP rat retinas. Perhaps, the Ca2+-calpain-CDK5-JNK-caspase-3/7 signaling activated by the injection of drugs is not the only mechanism by which the execution of photoreceptor cell death occurs. However, the fact of activated calpain suggests that this signaling also triggers apoptotic cell death. It also implies that a future experiment with photoreceptor-specific knockout or the overexpression of calpain is necessary to understand the role of calpain activation in retinal degeneration. Despite existing limitations, such as the cell specificity of responses to drug injection in the retina, the importance of experimental model for the field of retinal cell biology can be appreciated by applying genetically modified animals with the modified expression of Ca2+-sensing genes involved in neurodegeneration.Subretinal injection of the combined A23187 and Tg drugs triggers Ca2+-activated cellular signaling in ADRP progression and indicated that manipulation with ER channels SERCA2b, IP3R, calpain, calpastatin and CDK5 expression during a UPR-induced cytosolic Ca2+ increase might be beneficial for patients with retinal degeneration.Therefore, our study revealed the involvement of CaThe animal protocol was carried out with approval from the Institutional Animal Care and Use Committee at the University of Alabama at Birmingham and in accordance with the guidelines of the Association for Research in Vision and Ophthalmology statement for the use of Animals in Ophthalmic and Vision Research. All efforts were made to minimize the number and the suffering of the animals used.Homozygous S334ter Rho (line 4) and P23H Rho (line 3) transgenic rats were maintained in the UAB housing facility and were bred with wild-type (WT) Sprague-Dawley (SD) rats to generate heterozygous S334ter-4 Rho and P23H-3 Rho rats. Therefore, the SD rats were used as WT controls in our experiments. The animals were killed on postnatal days (P) P13, P21, P30, P40, and P60 for RNA, protein analyses, calcium measurement, and photoreceptor isolation. All rats were maintained in specific pathogen-free conditions with a 12-h light–dark daily cycle.2+ indicator dye Fluo4 AM  and Calceine red–orange AM to detect viable cells (Life Technologies C34851- 5 μM) for 90 min in Ames media at 36 °C  and Pluronic acid (Life Technologies P6866-10 μM). Retinas were flat mounted to orient the photoreceptor cell side upwards in the perfusion chamber (perfused 2–4 ml/min with oxygenated Ames media at 36 ºC). Flat-mounted retinas were imaged using infrared light on a Zeiss Axioskop microscope equipped with a 40 × long working distance water-immersion objective. Fluo4 fluorescence was measured using a 488-nm excitation light from an Excite light source to detect fluorescence from free calcium. Viable cells were identified by Calceine fluorescence in response to a 577-nm excitation light, fluorescence images were acquired with an Axiocam hRM camera and Axiovision 4.6 software. Fluorescence levels of individual photoreceptors from all rat strains were measured and quantified from the fluorescence images of flat mount retinas using Image J. Only individual viable photoreceptors with both Calcein AM and Fluo4 AM fluorescence were counted.Retinas were harvested from S334ter, P23H, and SD rats under a dissecting microscope in dim red light. The retinas were incubated with CaN=5). cDNA was prepared using a cDNA Reverse transcription kit  from the RNA extracts of SD, S334ter-4 Rho, and P23H-3 Rho retinas. Each cDNA (20 ng) was subjected to qRT-PCR using Applied Biosystems TaqMan assays  on a One Step Plus instrument (Applied Biosystems) to compare the number of cycles (Ct) needed to reach the midpoint of the linear phase. All observations were normalized to the GAPDH-housekeeping gene. The replicated RQs (Relative Quantity) values for each biological sample were averaged. Biological samples from each strain were used for the qPCR data analysis.Retinas from SD, S334ter-4 Rho, and P23H-3 Rho rats were isolated at P13, P21, P30, P40, and P60. Total RNA was isolated from the individual retinas from each strain using Trizol . Antibodies against phosphorylated (p) IP3R (#S1756), Calpastatin (#4146S), M-Calpain (#2556S), and P-SAPK/JNK (46668P) are from Cell Signaling Technology. Serca2b (#ab2861), Calcineurin (ab3673), antibodies were purchased from Abcam . BI-1 was obtained from Novus Biologicals , antibodies against Bip (#sc-1050) and CDK5 (SC173) from Santa Cruz ; and B-actin from Sigma-Aldrich . All antibodies we used at a dilution of 1 : 1000.Retinal protein extracts were obtained from dissected retinas by sonication in a buffer containing 25 mM of sucrose, 100 mM of Tris-HCl, pH=7.8, and a mixture of protease inhibitors . The total protein concentration in the right and left retinas from individual rat pups was measured using a Biorad protein assay, and 40 Two different treatment groups were generated by intravitreal drug injections. In the 1st group, SD rats at P21 were injected intravitreally with UPR inducer Tn (2 μg Sigma T7765) and contralateral eyes were injected with vehicle. Retinas were extracted at post injection days 1, 4, and 8 from injected eyes and compared for the protein expression of the markers of calcium-induced apoptosis with western blot. In the next treatment, different groups of SD rats at P21 were injected intravitreally with calcium ionophore A23187 (500 ng Sigma #C7522) and thapsigargin (100 ng Sigma #T9033) together. Injected animals were analyzed for retinal functional testing (ERG) 2, 4, and 6 weeks following the injection as well as for the activation of calcium-induced apoptosis 36 h after injection.2), −10dB (0.25cd*s/m2), 0dB (2.5cd*s/m2), 5dB (7.91cd*s/m2), 10dB (25cd*s/m2), and 15dB (79.1cd*s/m2). Five scans were performed and averaged for each light intensity. The a-wave amplitudes were measured from the baseline to the peak in the cornea-negative direction, and the b-wave amplitudes were determined from the cornea-negative peak to the major cornea-positive peak. The signal was amplified, digitized, and stored using the LKC UTAS-3000 Diagnostic System .Rats were dark-adapted overnight, then anesthetized with ketamine (100 mg/kg) and xylazine (10 mg/kg). The pupils were dilated in dim red light with 2.5% phenylephrine hydrochloride ophthalmic solution . Scotopic ERGs were recorded using a wire contacting the corneal surface with 2.5% hypromellose ophthalmic demulcent solution (Akorn). The ERG was performed at the following light intensities: −20dB (0.025cd*s/m3 V).The detection of calpain activity was performed using the Calpain Activity Assay kit from BioVision according to the manufacturer's recommendations. The activation of calpains in intravitreal drug-injected  and vehicle-injected SD retinal tissues was compared. The detection of the cleavage substrate Ac-LLY-AFC was performed in a fluorometer that was equipped with a 400-nm excitation filter and 505-emission filter .Caspase-3/7 activity was measured using Caspase-3/7-Glo assay system kit from Promega  as per the manufacturer's instructions. The retinal protein extract of intravitreal drug-  and vehicle-treated SD animals were compared for caspase-3/7 activity. The luminescent signal generated from caspase cleavage by substrate was measured in a luminometer , and kept at 4 °C for 8 h. Then, the eyes were hemisected and the eyecups were transferred to fresh PBS to remove formaldehyde and then immersed in a 30% sucrose solution for cryoprotection. Eyecups were then embedded in a cryostat compound  and frozen at −80 °C. Twelve-micron sections were obtained using a cryostat. To count the nuclei of photoreceptors, we stained cryostat-sectioned retinas with H&E using an H&E stain Kit (Cat#3490). Other slides were used for immunohistochemistry. Digital images of the right and left retinas of individual rats were taken, and the outer segment length was analyzed in the central superior and inferior retinas, located equidistant from the ONH. Images were analyzed by an investigator blinded to the experimental conditions. All sections were examined on a microscope equipped with a digital camera .Rats were killed using a COTwelve-micron sections were obtained and fixed on polylysine-treated glass slides. Slides were warmed for 30 min at 37 °C and washed in 0.1 M PBS for 10 min three times. Slides were kept in blocking buffer with 10% normal goat serum and 0.3% Triton solution for 1 h at room temperature and washed with PBS three times. The sections were incubated with primary antibody for VDAC  at 4 °C overnight. The slides were then washed three times with PBS and incubated with secondary antibody for 1 h at room temperature. After washing, the sides were cover slipped using a mounting medium containing DAPI and allowed to dry for 1 h. Images were using a wide-field fluorescence microscope .μg/cm2 anti WGA directed against wheat-germ agglutinin (WGA) lectin . Anti-WGA was diluted in 25 mM of bicarbonate buffer (pH 8), with 0.9% NaCl and 2 mg/ml of BSA. After three washes with warm bicarbonate buffer, the dishes were consequently coated with 5 μg/cm2 of WGA lectin (vector laboratories) diluted in bicarbonate buffer and incubated for 2 h at 37 °C. After incubation, the petri dishes were washed with warm PBS and stored in 0.2% BSA in PBS until used.Petri dishes were incubated for 2 h at 37 °C with 2.5 l-Glutamine was added to the tissue. For the dissociation of the retinal cells, the treated retinas were manually pipetted (gently 5–6 times) using 1000 μl and 200 μl tips. The retinal suspension containing dissociated cells was collected.The retinas from 30-day-old SD rats were harvested under dim red light, and the retinas were suspended in cold Hybernate A media (Life Technologies). Each retina was transferred to a tube with 1 ml of HybA and warmed for 8 min at 37 °C in a water bath. The warmed HybA media was aspirated, and the retina was incubated with Papain  in HybA for 20 min at 37 °C in a water bath with gentle shaking every 10 min. Papain solution was aspirated and 1 ml of 2% FBS solution in HybA was added to the retina and incubated for 5 min at room temperature to stop the enzymatic reaction. FBS solution was aspirated and Neurobasal (NBA) media supplemented with 1 : 50 B27 and 0.5 mM of 5 cm2 in NBA media supplemented with B27 and Glutamine.The retinal suspension was placed on lectin-coated petri dishes and incubated for 30 min at 37 °C with gentle swirling every 10 min. After incubation, non-adherent cells were removed by washing them with serum-free NBA media. The attached cells were dissociated with pipetting and seeded with 4 × 10Semi-quantitative RT-PCR was performed with cDNAs prepared from photoreceptors isolated from the WT rats. PCR products were detected using 1% agarose gel and UV light based imager (Life Technologies). Primer sequences (Sigma-Aldrich) for different retinal cell specific markers are provided in 5 cells/well were seeded into 96-well micro-culture plates at 37 °C with 5% CO2 and allowed to attach for 24 h. Cells were treated with designated doses of Tn for 48 h and incubated with MTT at a final concentration of 0.5 mg/ml for 4 h before the completion of the exposure time at 37 °C. The formation of MTT to formazon crystals by viable cells was assessed using 200 μl/well of DMSO at room temperature for 15 min. Optical density was measured at 490 nm using a micro-plate reader model 680 . The reduction in viability of cells in each well was expressed as the percentage of control cells.Cell viability was assessed by a 3--2-5-diphenyl tetrazolium bromide (MTT) assay. Then, 1 × 102+ detection in ADRP photoreceptors. For comparisons of protein levels and Ca2+ detection in Tn-injected and controlled retinas we used the Student's t-test. To calculate the statistical significance of differences in the a- and b-wave ERG amplitudes, a two-way ANOVA was applied. For all experiments, a P-value lower than 0.05 was considered to be significant .Two-way ANOVA comparisons were used to calculate the statistical significance of differences in fold-change of mRNA expression and levels of pIP3R protein in rats. A one-way ANOVA test was used to calculate the statistical significance of differences in levels of normalized proteins in P21 ADRP retinas and Ca


The present study explored whether Baicalein stimulates eryptosis. To this end, forward scatter was taken for measurement of cell volume, annexin-V-binding for phosphatidylserine-exposure, Fluo3 fluorescence for [Ca2+]i and fluorescent antibodies for ceramide abundance. As a result, a 48 h exposure of human erythrocytes to Baicalein was followed by significant decrease of forward scatter (≥10 µM), significant increase of the percentage of annexin-V-binding cells (≥25 µM), significant increase of [Ca2+]i (50 µM) and significant increase of ceramide abundance (50 µM). The effect of Baicalein (50 µM) on annexin-V-binding was significantly blunted but not abrogated by removal of extracellular Ca2+. In conclusion, at the concentrations employed, Baicalein stimulates suicidal erythrocyte death or eryptosis, an effect at least in part due to the combined effects of Ca2+ entry and ceramide formation.The polyphenolic flavonoid Baicalein has been shown to trigger suicidal death or apoptosis of tumor cells and is thus considered for the prevention and treatment of malignancy. Similar to apoptosis of nucleated cells, erythrocytes may enter eryptosis, the suicidal erythrocyte death characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. Stimulators of eryptosis include increase of cytosolic Ca Scutellaria baicalensis i), which is followed by activation of Ca2+-sensitive K+ channels with subsequent K+ exit, hyperpolarization, Cl− exit and thus cellular loss of KCl and water with subsequent cell shrinkage i, as well as ceramide abundance determined utilizing flow cytometry.The present study thus tested whether Baicalein stimulates eryptosis. To this end, human erythrocytes were incubated in Ringer with or without presence of Baicalein and cell volume, phosphatidylserine abundance at the cell surface, i). Thus, a further series of experiments was performed to elucidate the effect of Baicalein on [Ca2+]i. Erythrocytes were loaded with Fluo3-AM and the Fluo3 fluorescence determined by flow cytometry following incubation for 48 h in Ringer solution without or with Baicalein (5–50 µM). As illustrated in Cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the cell surface are both known to be stimulated by increase of cytosolic Ca2+ ionophore ionomycin was followed by a strong increase of annexin-V-binding  required for those effects were in the range of the peak concentrations reported in Baicalein treated rats . However2+ activity ([Ca2+]i), an effect presumably due to stimulation of cation channels in the cell membrane. Earlier studies revealed that the erythrocyte cation channels involve TRPC6 [Baicalein increased cytosolic Cave TRPC6 .2+ entry with subsequent increase of [Ca2+]i, activation of Ca2+ sensitive K+ channels, K+ exit, cell membrane hyperpolarisation, Cl− exit and thus cellular loss of KCl accompanied by osmotically driven water [+ entry through the unselective cation channel.The cell shrinkage following Baicalein treatment was presumably the result of Caen water . The celen water . The swe2+]i. Accordingly, the effect of Baicalein on phosphatidylserine translocation is in part dependent on entry of extracellular Ca2+.The stimulation of cell membrane scrambling by Baicalein is similarly in part due to increase of [Ca2+, Baicalein treatment is still followed by a significant increase of phosphatidylserine exposure. The residual effect is in part due to stimulation of ceramide formation. Ceramide is a well-known stimulator of eryptosis [However, even in the absence of extracellular Caryptosis .Similar to what has been shown for several other stimulators of eryptosis , the effConsequences of excessive eryptosis include anemia, since phosphatidylserine exposing eryptotic erythrocytes are phagocytosed and thus rapidly cleared from circulating blood . Anemia At least in theory, phosphatidylserine exposing erythrocytes may further interfere with microcirculation ,75,76,772+-permeable erythrocyte cation channels [Elimination of phosphatidylserine exposing erythrocytes may protect against untoward effects of hemolysis . The remchannels ,82. Subschannels . Accordichannels ,83,84,85channels , and by channels , chlorprchannels  or NO sychannels . In theoin vitro at a hematocrit of 0.4% in Ringer solution containing (in mM) 125 NaCl, 5 KCl, 1 MgSO4, 32 N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid (HEPES), 5 glucose, 1 CaCl2; pH 7.4 at 37 °C for 48 h. Where indicated, erythrocytes were exposed to Baicalein  at the indicated concentrations. In Ca2+-free Ringer solution, 1 mM CaCl2 was substituted by 1 mM glycol-bis(2-aminoethylether)-N,N,N',N'-tetraacetic acid (EGTA).Leukocyte-depleted erythrocytes were kindly provided by the blood bank of the University of Tübingen. The study is approved by the ethics committee of the University of Tübingen (184/2003V). Erythrocytes were incubated 2 and then stained with Annexin-V-FITC  in this solution at 37 °C for 20 min under protection from light. In the following, the forward scatter (FSC) of the cells was determined, and annexin-V fluorescence intensity was measured with an excitation wavelength of 488 nm and an emission wavelength of 530 nm on a FACS Calibur .After incubation under the respective experimental condition, 50 µL cell suspension was washed in Ringer solution containing 5 mM CaCl2 and 5 µM Fluo-3/AM. The cells were incubated at 37 °C for 30 min and washed twice in Ringer solution containing 5 mM CaCl2. The Fluo-3/AM-loaded erythrocytes were resuspended in 200 µL Ringer. Then, Ca2+-dependent fluorescence intensity was measured with an excitation wavelength of 488 nm and an emission wavelength of 530 nm on a FACS Calibur .After incubation, erythrocytes were washed in Ringer solution and then loaded with Fluo-3/AM  in Ringer solution containing 5 mM CaClFor the determination of ceramide abundance, a monoclonal antibody-based assay was used. After incubation, cells were stained for 1 h at 37 °C with 1 µg/mL anti ceramide antibody  in PBS containing 0.1% bovine serum albumin (BSA) at a dilution of 1:5. The samples were washed twice with PBS-BSA. Subsequently, the cells were stained for 30 min with polyclonal fluorescein isothiocyanate (FITC) conjugated goat anti-mouse IgG and IgM specific antibody  diluted 1:50 in PBS-BSA. Unbound secondary antibody was removed by repeated washing with PBS-BSA. The samples were then analyzed by flow cytometric analysis with an excitation wavelength of 488 nm and an emission wavelength of 530 nm.t-test as appropriate. n denotes the number of different erythrocyte specimens studied. Since different erythrocyte specimens used in distinct experiments are differently susceptible to triggers of eryptosis, the same erythrocyte specimens have been used for control and experimental conditions.Data are expressed as arithmetic means ± SEM. As indicated in the figure legends, statistical analysis was made using ANOVA with Tukey’s test as post-test and 2+ entry and ceramide formation thus leading to subsequent erythrocyte shrinkage and erythrocyte cell membrane scrambling. Accordingly, Baicalein stimulates eryptosis, the suicidal erythrocyte death. The concentrations required for those effects are 10–50 µM.In conclusion, the polyphenolic flavonoid Baicalein stimulates Ca