Pathological high intraocular pressure induces glial cell reactive proliferation contributing to neuroinflammation of the blood-retinal barrier via the NOX2/ET-1 axis-controlled ERK1/2 pathway.

BACKGROUND: NADPH oxidase (NOX), a primary source of endothelial reactive oxygen species (ROS), is considered a key event in disrupting the integrity of the blood-retinal barrier. Abnormalities in neurovascular-coupled immune signaling herald the loss of ganglion cells in glaucoma. Persistent microglia-driven inflammation and cellular innate immune system dysregulation often lead to deteriorating retinal degeneration. However, the crosstalk between NOX and the retinal immune environment remains unresolved. Here, we investigate the interaction between oxidative stress and neuroinflammation in glaucoma by genetic defects of NOX2 or its regulation via gp91ds-tat. METHODS: Ex vivo cultures of retinal explants from wildtype C57BL/6J and Nox2 -/- mice were subjected to normal and high hydrostatic pressure (Pressure 60 mmHg) for 24 h. In vivo, high intraocular pressure (H-IOP) was induced in C57BL/6J mice for two weeks. Both Pressure 60 mmHg retinas and H-IOP mice were treated with either gp91ds-tat (a NOX2-specific inhibitor). Proteomic analysis was performed on control, H-IOP, and treatment with gp91ds-tat retinas to identify differentially expressed proteins (DEPs). The study also evaluated various glaucoma phenotypes, including IOP, retinal ganglion cell (RGC) functionality, and optic nerve (ON) degeneration. The superoxide (O2-) levels assay, blood-retinal barrier degradation, gliosis, neuroinflammation, enzyme-linked immunosorbent assay (ELISA), western blotting, and quantitative PCR were performed in this study. RESULTS: We found that NOX2-specific deletion or activity inhibition effectively attenuated retinal oxidative stress, immune dysregulation, the internal blood-retinal barrier (iBRB) injury, neurovascular unit (NVU) dysfunction, RGC loss, and ON axonal degeneration following H-IOP. Mechanistically, we unveiled for the first time that NOX2-dependent ROS-driven pro-inflammatory signaling, where NOX2/ROS induces endothelium-derived endothelin-1 (ET-1) overexpression, which activates the ERK1/2 signaling pathway and mediates the shift of microglia activation to a pro-inflammatory M1 phenotype, thereby triggering a neuroinflammatory outburst. CONCLUSIONS: Collectively, we demonstrate for the first time that NOX2 deletion or gp91ds-tat inhibition attenuates iBRB injury and NVU dysfunction to rescue glaucomatous RGC loss and ON axon degeneration, which is associated with inhibition of the ET-1/ERK1/2-transduced shift of microglial cell activation toward a pro-inflammatory M1 phenotype, highlighting NOX2 as a potential target for novel neuroprotective therapies in glaucoma management.

High-fat diet causes endothelial dysfunction in the mouse ophthalmic artery.

Obesity is a significant health concern that leads to impaired vascular function and subsequent abnormalities in various organs. The impact of obesity on ocular blood vessels, however, remains largely unclear. In this study, we examined the hypothesis that obesity induced by high-fat diet produces vascular endothelial dysfunction in the ophthalmic artery. Mice were subjected to a high-fat diet for 20 weeks, while age-matched controls were maintained on a standard diet. Reactivity of isolated ophthalmic artery segments was assessed in vitro. Reactive oxygen species (ROS) were quantified in cryosections by dihydroethidium (DHE) staining. Redox gene expression was determined in ophthalmic artery explants by real-time PCR. Furthermore, the expression of nicotinamide adenine dinucleotide phosphate oxidase 2 (NOX2), the receptor for advanced glycation end products (RAGE), and of the lectin-like oxidized low-density-lipoprotein receptor-1 (LOX-1) was determined in cryosections using immunofluorescence microscopy. Ophthalmic artery segments from mice on a high-fat diet exhibited impaired vasodilation responses to the endothelium-dependent vasodilator acetylcholine, while endothelium-independent responses to nitroprusside remained preserved. DHE staining intensity in the vascular wall was notably stronger in mice on a high-fat diet. Messenger RNA expression for NOX2 was elevated in the ophthalmic artery of mice subjected to high fat diet. Likewise, immunostainings revealed increased expression of NOX2 and of RAGE, but not of LOX-1. These findings suggest that a high-fat diet triggers endothelial dysfunction by inducing oxidative stress in the ophthalmic artery via involvement of RAGE and NOX2.

Deletion of adipocyte NOS3 potentiates high-fat diet-induced hypertension and vascular remodelling via chemerin.

AIMS: Obesity is an epidemic that is a critical contributor to hypertension and other cardiovascular diseases. Current paradigms suggest that endothelial nitric oxide synthase (eNOS/NOS3) in the vessel wall is the primary regulator of vascular function and blood pressure. However, recent studies have revealed the presence of eNOS/NOS3 in the adipocytes of white adipose tissues and perivascular adipose tissues (PVATs). The current understanding of the role of adipocyte NOS3 is based mainly on studies using global knockout models. The present study aimed to elucidate the functional significance of adipocyte NOS3 for vascular function and blood pressure control. METHODS AND RESULTS: We generated an adipocyte-specific NOS3 knockout mouse line using adiponectin promoter-specific Cre-induced gene inactivation. Control and adipocyte-specific NOS3 knockout (A-NOS3 KO) mice were fed a high-fat diet (HFD). Despite less weight gain, A-NOS3 KO mice exhibited a significant increase in blood pressure after HFD feeding, associated with exacerbated vascular dysfunction and remodelling. A-NOS3 KO mice also showed increased expression of signature markers of inflammation and hypoxia in the PVATs. Among the differentially expressed adipokines, we have observed an upregulation of a novel adipokine, chemerin, in A-NOS3 KO mice. Chemerin was recently reported to link obesity and vascular dysfunction. Treatment with chemerin neutralizing antibody normalized the expression of remodelling markers in the aorta segments cultured in serum from HFD-fed A-NOS3 KO mice ex vivo. CONCLUSION: These data suggest that NOS3 in adipocytes is vital in maintaining vascular homeostasis; dysfunction of adipocyte NOS3 contributes to obesity-induced vascular remodelling and hypertension.

Studies on the Effects of Hypercholesterolemia on Mouse Ophthalmic Artery Reactivity.

Atherogenic lipoproteins may impair vascular reactivity, leading to tissue damage in various organs, including the eye. This study aimed to investigate whether ophthalmic artery reactivity is affected in mice lacking the apolipoprotein E gene (ApoE-/-), a model for hypercholesterolemia and atherosclerosis. Twelve-month-old male ApoE-/- mice and age-matched wild-type controls were used to assess vascular reactivity using videomicroscopy. Moreover, the vascular mechanics, lipid content, levels of reactive oxygen species (ROS), and expression of pro-oxidant redox enzymes and the lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) were determined in vascular tissue. Unlike the aorta, the ophthalmic artery of ApoE-/- mice developed no signs of endothelial dysfunction and no signs of excessive lipid deposition. Remarkably, the levels of ROS, nicotinamide adenine dinucleotide phosphate oxidase 1 (NOX1), NOX2, NOX4, and LOX-1 were increased in the aorta but not in the ophthalmic artery of ApoE-/- mice. Our findings suggest that ApoE-/- mice develop endothelial dysfunction in the aorta by increased oxidative stress via the involvement of LOX-1, NOX1, and NOX2, whereas NOX4 may participate in media remodeling. In contrast, the ophthalmic artery appears to be resistant to chronic apolipoprotein E deficiency. A lack of LOX-1 expression/overexpression in response to increased oxidized low-density lipoprotein levels may be a possible mechanism of action.

Effects of aircraft noise cessation on blood pressure, cardio- and cerebrovascular endothelial function, oxidative stress, and inflammation in an experimental animal model.

Large epidemiological studies have shown that traffic noise promotes the development of cardiometabolic diseases. It remains to be established how long these adverse effects of noise may persist in response to a noise-off period. We investigated the effects of acute aircraft noise exposure (mean sound level of 72 dB(A) applied for 4d) on oxidative stress and inflammation mediating vascular dysfunction and increased blood pressure in male C57BL/6 J mice. 1, 2 or 4d of noise cessation after a 4d continuous noise exposure period completely normalized noise-induced endothelial dysfunction of the aorta (measured by acetylcholine-dependent relaxation) already after a 1d noise pause. Vascular oxidative stress and the increased blood pressure were partially corrected, while markers of inflammation (VCAM-1, IL-6 and leukocyte oxidative burst) showed a normalization within 4d of noise cessation. In contrast, endothelial dysfunction, oxidative stress, and inflammation of the cerebral microvessels of noise-exposed mice did not improve at all. These data demonstrate that the recovery from noise-induced damage is more complex than expected demonstrating a complete restoration of large conductance vessel function but persistent endothelial dysfunction of the microcirculation. These findings also imply that longer noise pauses are required to completely reverse noise-induced vascular dysfunction including the resistance vessels.

Perivascular Adipose Tissue Oxidative Stress in Obesity.

Perivascular adipose tissue (PVAT) adheres to most systemic blood vessels in the body. Healthy PVAT exerts anticontractile effects on blood vessels and further protects against cardiovascular and metabolic diseases. Healthy PVAT regulates vascular homeostasis via secreting an array of adipokine, hormones, and growth factors. Normally, homeostatic reactive oxygen species (ROS) in PVAT act as secondary messengers in various signalling pathways and contribute to vascular tone regulation. Excessive ROS are eliminated by the antioxidant defence system in PVAT. Oxidative stress occurs when the production of ROS exceeds the endogenous antioxidant defence, leading to a redox imbalance. Oxidative stress is a pivotal pathophysiological process in cardiovascular and metabolic complications. In obesity, PVAT becomes dysfunctional and exerts detrimental effects on the blood vessels. Therefore, redox balance in PVAT emerges as a potential pathophysiological mechanism underlying obesity-induced cardiovascular diseases. In this review, we summarise new findings describing different ROS, the major sources of ROS and antioxidant defence in PVAT, as well as potential pharmacological intervention of PVAT oxidative stress in obesity.

Models of traumatic brain injury-highlights and drawbacks.

Traumatic brain injury (TBI) is the leading cause for high morbidity and mortality rates in young adults, survivors may suffer from long-term physical, cognitive, and/or psychological disorders. Establishing better models of TBI would further our understanding of the pathophysiology of TBI and develop new potential treatments. A multitude of animal TBI models have been used to replicate the various aspects of human TBI. Although numerous experimental neuroprotective strategies were identified to be effective in animal models, a majority of strategies have failed in phase II or phase III clinical trials. This failure in clinical translation highlights the necessity of revisiting the current status of animal models of TBI and therapeutic strategies. In this review, we elucidate approaches for the generation of animal models and cell models of TBI and summarize their strengths and limitations with the aim of exploring clinically meaningful neuroprotective strategies.

Research progress on pleiotropic neuroprotective drugs for traumatic brain injury.

Traumatic brain injury (TBI) has become one of the most important causes of death and disability worldwide. A series of neuroinflammatory responses induced after TBI are key factors for persistent neuronal damage, but at the same time, such inflammatory responses can also promote debris removal and tissue repair after TBI. The concept of pleiotropic neuroprotection delves beyond the single-target treatment approach, considering the multifaceted impacts following TBI. This notion embarks deeper into the research-oriented treatment paradigm, focusing on multi-target interventions that inhibit post-TBI neuroinflammation with enhanced therapeutic efficacy. With an enriched comprehension of TBI's physiological mechanisms, this review dissects the advancements in developing pleiotropic neuroprotective pharmaceuticals to mitigate TBI. The aim is to provide insights that may contribute to the early clinical management of the condition.

Exercise ameliorates chronic inflammatory response induced by high-fat diet via Sestrin2 in an Nrf2-dependent manner.

Chronic inflammation is a major contributor to the development of metabolic disorders and is commonly seen in studies of diet-induced obesity in humans and rodents. Exercise has been shown to have anti-inflammatory properties, though the exact mechanisms are still not fully understood. Sestrins and Nrf2 are of interest to researchers as they are known to protect against inflammation and oxidative stress. In this study, we aim to explore the interconnection between Sestrin2 (SESN2) and Nrf2 and their roles in exercise benefits on chronic inflammation. Our data showed that SESN2 knockout aggravated the abnormalities of body weight, fat mass, and serum lipid that were induced by a high-fat diet (HFD), and a concomitant increase of TNF-α, IL-1ß and IL-6 in both serum and skeletal muscle. Notably, exercise was found to reverse these changes, and SESN2 was found to be necessary for exercise to reduce the inflammatory response in skeletal muscles, though not in serum. Immunoprecipitation and bioinformatics prediction experiments further revealed that SESN2 directly binds to Nrf2, indicating a protein-protein interaction between the two. Furthermore, our data demonstrated that SESN2 protein is necessary for exercise-induced effects on Nrf2 pathway in HFD-fed mice, and Nrf2 protein is necessary to enable SESN2 to reduce the inflammation caused by palmitic acid (PA)+ oleic acid (OA) treatment in vitro. Our findings indicate that exercise mitigates chronic inflammation induced by HFD through SESN2 in an Nrf2-dependent manner. Our study reveals a novel molecular mechanism whereby the SESN2/Nrf2 pathway mediates the positive impact of exercise on chronic inflammation.

Research progress of neuroinflammation-related cells in traumatic brain injury: A review.

Neuroinflammation after traumatic brain injury (TBI) is related to chronic neurodegenerative diseases and is one of the causes of acute secondary injury after TBI. Therefore, it is particularly important to clarify the role of cellular mechanisms in the neuroinflammatory response after TBI. The objective of this article is to understand the involvement of cells during the TBI inflammatory response (for instance, astrocytes, microglia, and oligodendrocytes) and shed light on the recent progress in the stimulation and interaction of granulocytes and lymphocytes, to provide a novel approach for clinical research. We searched articles in PubMed published between 1950 and 2023, using the following keywords: TBI, neuroinflammation, inflammatory cells, neuroprotection, clinical. Articles for inclusion in this paper were finalized based on their novelty, representativeness, and relevance to the main arguments of this review. We found that the neuroinflammatory response after TBI includes the activation of glial cells, the release of inflammatory mediators in the brain, and the recruitment of peripheral immune cells. These inflammatory responses not only induce secondary brain damage, but also have a role in repairing the nervous system to some extent. However, not all of the mechanisms of cell-to-cell interactions have been well studied. After TBI, clinical treatment cannot simply suppress the inflammatory response, and the inflammatory phenotype of patients' needs to be defined according to their specific conditions after injury. Clinical trials of personalized inflammation regulation therapy for specific patients should be carried out in order to improve the prognosis of patients.

Alcohol and the vasculature: a love-hate relationship?

Alcohol consumption is a leading risk factor and increases the risk of liver diseases, cancers, tuberculosis, and injuries. The relationship between alcohol use and cardiovascular risk is complex. While it is well established that heavy alcohol use and binge drinking harm cardiovascular health, the effect of light-to-moderate alcohol consumption remains controversial. Observational studies have repeatedly confirmed the U- or J-shaped relationship between alcohol consumption and cardiovascular disease risk, with the lowest risk observed in the light-to-moderate drinking group. However, the protective effect of low-level alcohol has been challenged by recent genetic epidemiological studies with Mendelian randomization. Such studies have their own limitations, and the application of this methodology in studying alcohol has been questioned. Results from the latest Global Burden of Diseases, Injuries, and Risk Factors Study suggest that the impact of alcohol consumption on health depends on the age structure and the distribution of disease burden and underlying causes in a given population. For young adults, even small amounts of alcohol cause heath loss. For older adults facing a high burden of cardiovascular diseases, light-to-moderate alcohol consumption may improve cardiovascular health outcomes. Mechanistically, all types of alcoholic beverages, including wine, spirits, and beer, have been shown to increase the levels of high-density lipoprotein cholesterol and adiponectin, and reduce the level of fibrinogen. Nonalcoholic components of wine, especially polyphenolic compounds like resveratrol, may additionally enhance endothelial nitric oxide production, and provide antioxidant and anti-inflammatory effects.

Effects of Resveratrol on Vascular Function in Retinal Ischemia-Reperfusion Injury.

Ischemia-reperfusion (I/R) events are involved in the development of various ocular pathologies, e.g., retinal artery or vein occlusion. We tested the hypothesis that resveratrol is protective against I/R injury in the murine retina. Intraocular pressure (IOP) was elevated in anaesthetized mice to 110 mm Hg for 45 min via a micropipette placed in the anterior chamber to induce ocular ischemia. In the fellow eye, which served as control, IOP was kept at a physiological level. One group received resveratrol (30 mg/kg/day p.o. once daily) starting one day before the I/R event, whereas the other group of mice received vehicle solution only. On day eight after the I/R event, mice were sacrificed and retinal wholemounts were prepared and immuno-stained using a Brn3a antibody to quantify retinal ganglion cells. Reactivity of retinal arterioles was measured in retinal vascular preparations using video microscopy. Reactive oxygen species (ROS) and nitrogen species (RNS) were quantified in ocular cryosections by dihydroethidium and anti-3-nitrotyrosine staining, respectively. Moreover, hypoxic, redox and nitric oxide synthase gene expression was quantified in retinal explants by PCR. I/R significantly diminished retinal ganglion cell number in vehicle-treated mice. Conversely, only a negligible reduction in retinal ganglion cell number was observed in resveratrol-treated mice following I/R. Endothelial function and autoregulation were markedly reduced, which was accompanied by increased ROS and RNS in retinal blood vessels of vehicle-exposed mice following I/R, whereas resveratrol preserved vascular endothelial function and autoregulation and blunted ROS and RNS formation. Moreover, resveratrol reduced I/R-induced mRNA expression for the prooxidant enzyme, nicotinamide adenine dinucleotide phosphate oxidase 2 (NOX2). Our data provide evidence that resveratrol protects from I/R-induced retinal ganglion cell loss and endothelial dysfunction in the murine retina by reducing nitro-oxidative stress possibly via suppression of NOX2 upregulation.

Dietary supplements and vascular function in hypertensive disorders of pregnancy.

Hypertensive disorders of pregnancy are complications that can lead to maternal and infant mortality and morbidity. Hypertensive disorders of pregnancy are generally defined as hypertension and may be accompanied by other end organ damages including proteinuria, maternal organ disturbances including renal insufficiency, neurological complications, thrombocytopenia, impaired liver function, or uteroplacental dysfunction such as fetal growth restriction and stillbirth. Although the causes of these hypertensive disorders of pregnancy are multifactorial and elusive, they seem to share some common vascular-related mechanisms, including diseased spiral arteries, placental ischemia, and endothelial dysfunction. Recently, preeclampsia is being considered as a vascular disorder. Unfortunately, due to the complex etiology of preeclampsia and safety concerns on drug usage during pregnancy, there is still no effective pharmacological treatments available for preeclampsia yet. An emerging area of interest in this research field is the potential beneficial effects of dietary intervention on reducing the risk of preeclampsia. Recent studies have been focused on the association between deficiencies or excesses of some nutrients and complications during pregnancy, fetal growth and development, and later risk of cardiovascular and metabolic diseases in the offspring. In this review, we discuss the involvement of placental vascular dysfunction in preeclampsia. We summarize the current understanding of the association between abnormal placentation and preeclampsia in a vascular perspective. Finally, we evaluate several studied dietary supplementations to prevent and reduce the risk of preeclampsia, targeting placental vascular development and function, leading to improved pregnancy and postnatal outcomes.

B lymphocyte-deficiency in mice promotes venous thrombosis.

Cells of the innate immune system, including monocytes and neutrophils, are key players in the process of venous thrombosis. T lymphocytes have recently been implicated in venous thrombus resolution but the role of B lymphocytes in thrombosis is unknown. The present study was conducted to address this question using a mouse model of partial ligation of the inferior vena cava. Although only a very low number of B cells was found in the venous thrombi of wild-type mice, B cell-deficient JHT mutant mice developed larger venous thrombi than the wild-type controls. Consistent with enhanced thrombogenesis, increased neutrophil counts were found in the circulating blood and in the thrombi of B cell-deficient mice. One of the mechanisms by which neutrophils contribute to venous thrombosis is the formation of neutrophil extracellular traps (NETs). In agreement, higher quantities of NETs were observed in the thrombi of B cell-deficient mice. In vitro assays showed no difference in the NET building capacity of the isolated neutrophils between B cell-deficient and wild-type mice, indicating that the enhanced NET formation in the thrombi of B cell-deficient mice is attributable to the increased number of circulating neutrophils in these animals. Furthermore, increased concentration of the clot-stabilizing macromolecule fibrinogen was detected in the plasma of B cell-deficient mice. In conclusion, B cell-deficiency in mice indirectly promotes venous thrombosis by increasing neutrophil numbers and elevating fibrinogen levels.

Intraocular Pressure-Induced Endothelial Dysfunction of Retinal Blood Vessels Is Persistent, but Does Not Trigger Retinal Ganglion Cell Loss.

Research has been conducted into vascular abnormalities in the pathogenesis of glaucoma, but conclusions remain controversial. Our aim was to test the hypothesis that retinal endothelial dysfunction induced by elevated intraocular pressure (IOP) persists after IOP normalization, further triggering retinal ganglion cell (RGC) loss. High intraocular pressure (HP) was induced in mice by episcleral vein occlusion (EVO). Retinal vascular function was measured via video microscopy in vitro. The IOP, RGC and their axons survival, levels of oxidative stress and inflammation as well as vascular pericytes coverage, were determined. EVO caused HP for two weeks, which returned to baseline afterwards. Mice with HP exhibited endothelial dysfunction in retinal arterioles, reduced density of RGC and their axons, and loss of pericytes in retinal arterioles. Notably, these values were similar to those of mice with recovered IOP (RP). Levels of oxidative stress and inflammation were increased in HP mice but went back to normal in the RP mice. Our data demonstrate that HP induces persistent endothelial dysfunction in retinal arterioles, which persists one month after RP. Oxidative stress, inflammation, and loss of pericytes appear to be involved in triggering vascular functional deficits. Our data also suggest that retinal endothelial dysfunction does not affect RGC and their axon survival.

Analysis of 16 studies in nine rodent models does not support the hypothesis that diabetic polyuria is a main reason of urinary bladder enlargement.

The urinary bladder is markedly enlarged in the type 1 diabetes mellitus model of streptozotocin-injected rats, which may contribute to the frequent diabetic uropathy. Much less data exists for models of type 2 diabetes. Diabetic polyuria has been proposed as the pathophysiological mechanism behind bladder enlargement. Therefore, we explored such a relationship across nine distinct rodent models of diabetes including seven models of type 2 diabetes/obesity by collecting data on bladder weight and blood glucose from 16 studies with 2-8 arms each; some studies included arms with various diets and/or pharmacological treatments. Data were analysed for bladder enlargement and for correlations between bladder weight on the one and glucose levels on the other hand. Our data confirm major bladder enlargement in streptozotocin rats and minor if any enlargement in fructose-fed rats, db/db mice and mice on a high-fat diet; enlargement was present in some of five not reported previously models. Bladder weight was correlated with blood glucose as a proxy for diabetic polyuria within some but not other models, but correlations were moderate to weak except for RIP-LCMV mice (r 2 of pooled data from all studies 0.0621). Insulin levels also failed to correlate to a meaningful extent. Various diets and medications (elafibranor, empagliflozin, linagliptin, semaglutide) had heterogeneous effects on bladder weight that often did not match their effects on glucose levels. We conclude that the presence and extent of bladder enlargement vary markedly across diabetes models, particularly type 2 diabetes models; our data do not support the idea that bladder enlargement is primarily driven by glucose levels/glucosuria.

Endothelial Nitric Oxide Synthase in the Perivascular Adipose Tissue.

Perivascular adipose tissue (PVAT) is a special type of ectopic fat depot that adheres to most vasculatures. PVAT has been shown to exert anticontractile effects on the blood vessels and confers protective effects against metabolic and cardiovascular diseases. PVAT plays a critical role in vascular homeostasis via secreting adipokine, hormones, and growth factors. Endothelial nitric oxide synthase (eNOS; also known as NOS3 or NOSIII) is well-known for its role in the generation of vasoprotective nitric oxide (NO). eNOS is primarily expressed, but not exclusively, in endothelial cells, while recent studies have identified its expression in both adipocytes and endothelial cells of PVAT. PVAT eNOS is an important player in the protective role of PVAT. Different studies have demonstrated that, under obesity-linked metabolic diseases, PVAT eNOS may be even more important than endothelium eNOS in obesity-induced vascular dysfunction, which may be attributed to certain PVAT eNOS-specific functions. In this review, we summarized the current understanding of eNOS expression in PVAT, its function under both physiological and pathological conditions and listed out a few pharmacological interventions of interest that target eNOS in PVAT.

Cases Abusing Brain Death Definition in Organ Procurement in China.

Organ donation after brain death has been practiced in China since 2003 in the absence of brain death legislation. Similar to international standards, China's brain death diagnostic criteria include coma, absence of brainstem reflexes, and the lack of spontaneous respiration. The Chinese criteria require that the lack of spontaneous respiration must be verified with an apnea test by disconnecting the ventilator for 8 min to provoke spontaneous respiration. However, we have found publications in Chinese medical journals, in which the donors were declared to be brain dead, yet without an apnea test. The organ procurement procedures started with initiating "intratracheal intubation for mechanical ventilation after brain death," indicating that a brain death diagnosis was not performed. The purpose of the intubation was not to resuscitate the patient but rather was directly related to facilitating the explantation of organs. Moreover, it was unmistakably stated in two of these publications that the cardiac arrest was induced in these patients without brain death determination by cold St. Thomas cardioplegic solution or other cold myocardial protection solutions. This means that the condition of these donors neither met the criteria of brain death nor that of cardiac death. In other words, the "donor organs" may well have been procured in these cases from living human beings. Thus, brain death definition is abused in China by some individuals for organ harvesting, and a systematic investigation is needed to clarify the situation of organ donation after brain death in China.

Measurement of Tetrahydrobiopterin in Animal Tissue Samples by HPLC with Electrochemical Detection-Protocol Optimization and Pitfalls.

Tetrahydrobiopterin (BH4) is an essential cofactor of all nitric oxide synthase isoforms, thus determination of BH4 levels can provide important mechanistic insight into diseases. We established a protocol for high-performance liquid chromatography/electrochemical detection (HPLC/ECD)-based determination of BH4 in tissue samples. We first determined the optimal storage and work-up conditions for authentic BH4 and its oxidation product dihydrobiopterin (BH2) under various conditions (pH, temperature, presence of antioxidants, metal chelators, and storage time). We then applied optimized protocols for detection of BH4 in tissues of septic (induced by lipopolysaccharide [LPS]) rats. BH4 standards in HCl are stabilized by addition of 1,4-dithioerythritol (DTE) and diethylenetriaminepentaacetic acid (DTPA), while HCl was sufficient for BH2 standard stabilization. Overnight storage of BH4 standard solutions at room temperature in HCl without antioxidants caused complete loss of BH4 and the formation of BH2. We further optimized the protocol to separate ascorbate and the BH4 tissue sample and found a significant increase in BH4 in the heart and kidney as well as higher BH4 levels by trend in the brain of septic rats compared to control rats. These findings correspond to reports on augmented nitric oxide and BH4 levels in both animals and patients with septic shock.

The Involvement of Sirtuin 1 Dysfunction in High-Fat Diet-Induced Vascular Dysfunction in Mice.

High-fat diet (HFD)-induced vascular impairment in mice is associated with a dysfunction of the perivascular adipose tissue (PVAT). The present study was conducted to investigate the involvement of sirtuin 1 (SIRT1). Male C57BL/6J mice were fed an HFD for 20 weeks to induce obesity. Vascular function was analyzed using a wire myograph system. In obese mice, the vasodilator response of PVAT-containing aortas to acetylcholine was reduced, although the vascular function of PVAT-free aortas remained normal. SIRT1 activity in PVAT of obese mice was reduced despite enhanced SIRT1 expression. Nicotinamide adenine dinucleotide (NAD+) levels and the NAD+/NADH ratio in the PVAT of obese mice were decreased, which was likely attributable to a downregulation of the NAD+-producing enzyme NAMPT. The reduced SIRT1 activity was associated with an enhanced acetylation of the endothelial nitric oxide synthase (eNOS) in the PVAT. Ex vivo incubation of PVAT-containing aorta from obese mice with NAD+ led to a complete normalization of vascular function. Thus, reduced SIRT1 activity due to NAD+ deficiency is involved in obesity-induced PVAT dysfunction.