Cellular recovery after prolonged warm ischaemia of the whole body.

After cessation of blood flow or similar ischaemic exposures, deleterious molecular cascades commence in mammalian cells, eventually leading to their death1,2. Yet with targeted interventions, these processes can be mitigated or reversed, even minutes or hours post mortem, as also reported in the isolated porcine brain using BrainEx technology3. To date, translating single-organ interventions to intact, whole-body applications remains hampered by circulatory and multisystem physiological challenges. Here we describe OrganEx, an adaptation of the BrainEx extracorporeal pulsatile-perfusion system and cytoprotective perfusate for porcine whole-body settings. After 1 h of warm ischaemia, OrganEx application preserved tissue integrity, decreased cell death and restored selected molecular and cellular processes across multiple vital organs. Commensurately, single-nucleus transcriptomic analysis revealed organ- and cell-type-specific gene expression patterns that are reflective of specific molecular and cellular repair processes. Our analysis comprises a comprehensive resource of cell-type-specific changes during defined ischaemic intervals and perfusion interventions spanning multiple organs, and it reveals an underappreciated potential for cellular recovery after prolonged whole-body warm ischaemia in a large mammal.

Transcriptomic taxonomy and neurogenic trajectories of adult human, macaque, and pig hippocampal and entorhinal cells.

The hippocampal-entorhinal system supports cognitive functions, has lifelong neurogenic capabilities in many species, and is selectively vulnerable to Alzheimer's disease. To investigate neurogenic potential and cellular diversity, we profiled single-nucleus transcriptomes in five hippocampal-entorhinal subregions in humans, macaques, and pigs. Integrated cross-species analysis revealed robust transcriptomic and histologic signatures of neurogenesis in the adult mouse, pig, and macaque but not humans. Doublecortin (DCX), a widely accepted marker of newly generated granule cells, was detected in diverse human neurons, but it did not define immature neuron populations. To explore species differences in cellular diversity and implications for disease, we characterized subregion-specific, transcriptomically defined cell types and transitional changes from the three-layered archicortex to the six-layered neocortex. Notably, METTL7B defined subregion-specific excitatory neurons and astrocytes in primates, associated with endoplasmic reticulum and lipid droplet proteins, including Alzheimer's disease-related proteins. This resource reveals cell-type- and species-specific properties shaping hippocampal-entorhinal neurogenesis and function.

Brain vulnerability and viability after ischaemia.

The susceptibility of the brain to ischaemic injury dramatically limits its viability following interruptions in blood flow. However, data from studies of dissociated cells, tissue specimens, isolated organs and whole bodies have brought into question the temporal limits within which the brain is capable of tolerating prolonged circulatory arrest. This Review assesses cell type-specific mechanisms of global cerebral ischaemia, and examines the circumstances in which the brain exhibits heightened resilience to injury. We suggest strategies for expanding such discoveries to fuel translational research into novel cytoprotective therapies, and describe emerging technologies and experimental concepts. By doing so, we propose a new multimodal framework to investigate brain resuscitation following extended periods of circulatory arrest.

Restoration of brain circulation and cellular functions hours post-mortem.

The brains of humans and other mammals are highly vulnerable to interruptions in blood flow and decreases in oxygen levels. Here we describe the restoration and maintenance of microcirculation and molecular and cellular functions of the intact pig brain under ex vivo normothermic conditions up to four hours post-mortem. We have developed an extracorporeal pulsatile-perfusion system and a haemoglobin-based, acellular, non-coagulative, echogenic, and cytoprotective perfusate that promotes recovery from anoxia, reduces reperfusion injury, prevents oedema, and metabolically supports the energy requirements of the brain. With this system, we observed preservation of cytoarchitecture; attenuation of cell death; and restoration of vascular dilatory and glial inflammatory responses, spontaneous synaptic activity, and active cerebral metabolism in the absence of global electrocorticographic activity. These findings demonstrate that under appropriate conditions the isolated, intact large mammalian brain possesses an underappreciated capacity for restoration of microcirculation and molecular and cellular activity after a prolonged post-mortem interval.

Chemerin Blood Levels are Associated with MRI Measured Volumes of Abdominal Adipose Tissue Compartments and Lifestyle Choices.

Obesity is a low-grade chronic inflammatory state, in which a cytokine chemerin with its immunometabolic modulation has an important role. We aimed to study in a healthy population relationships between serum chemerin levels, lifestyle choices and magnetic resonance imaging (MRI) assessed volumes of abdominal visceral (VAT) and subcutaneous (SAT) adipose tissues, which have different cytokine expression profiles. Overall, 73 healthy participants undertook lifestyle questionnaire and underwent anthropometric measurements along with MRI scanning of abdominal SAT and VAT. Furthermore, complete blood count was determined along with chemerin and fibrinogen serum levels. Regression model for prediction of chemerin serum levels was built after controlling for sex, age and anthropometric measures. Median serum chemerin was 141 (125-195) ng/mL. Serum chemerin had a moderate positive correlation with SAT and VAT volumes. The two most important predictors of chemerin levels were MRI detected SAT and thigh circumference. Independently, chemerin levels were associated with smoking, preference of salty food, and favoring flavor/simplicity of preparation over nutritional value of the food. Serum chemerin seems to be more strongly correlated with the volume of abdominal SAT than VAT, with certain lifestyle choices influencing chemerin levels independently of abdominal fat.

Transcardial gradient of adiponectin, interleukin-6 and tumor necrosis factor-α in overweight coronary artery disease patients.

BACKGROUND: Obesity is associated with coronary artery disease (CAD), where epicardial adipose tissue (EAT) express proatherogenic cytokines (i.e., interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α)) and decreases production of beneficial adiponectin. Studies on endocrine role of EAT are mostly based on assessing cytokines' mRNAs, whereas cytokine blood levels might not readily correlate. In order to get better insight into the endocrine role of thickened EAT in CAD, we assessed transcardial gradient of adiponectin, IL-6 and TNF-α. METHODS: We assessed anthropometric and ultrasound measures in cohort of fifty nondiabetic subjects (21 CAD and 29 non-CAD). Blood sampled from aortic root and coronary sinus was assayed for adiponectin, IL-6 and TNF-α, using ELISA. RESULTS: Except thicker EAT in CAD subjects, anthropometric measures were similar (overweight), with higher adiponectin in coronary sinus than in aortic root (p<0.001) in both groups. CAD subjects had lower adiponectin in aortic root (p<0.001) and higher levels of TNF-α in coronary sinus than in aortic root (p=0.05). EAT thickness positively correlated with hip circumference (p=0.038) and negatively correlated with adiponectin levels (for both p<0.05). CONCLUSIONS: Transcardial gradient of adiponectin in non-CAD and CAD overweight subjects was similar, while latter had lower systemic adiponectin level and thicker EAT. EAT with thickening reaches the threshold level of near-maximal down-regulation of adiponectin and its further thickening is not associated with continued decrease of adiponectin production. In CAD patients levels of TNF-α were higher, but IL-6 were not, and these cytokines might be flush out by lymphatic route.

Arterial tree asymmetry reduces cerebral pulsatility.

With each heartbeat, pressure wave (PW) propagates from aorta toward periphery. In cerebral circulation, at the level of circle of Willis (CW), four arteries and four PWs converge. Since the interference is an elemental property of the wave, PWs interfere at the level of CW. We hypothesize that the asymmetry of brain-supplying arteries (that join to form CW) creates phase difference between the four PWs that interfere at the level of CW and reduce downstream cerebral pulsatility. To best of our knowledge, the data about the sequence of PWs' arrival into the cerebral circulation is lacking. Evident imperfect bilateral symmetry of the vessels results with different path length of brain-supplying arteries, hence, PWs should arrive into the head at different times. The probabilistic calculation shows that asynchronous arrival is more probable than synchronous. The importance of PWs for the cerebral circulation is highlighted by the observation that barotrauma protection mechanisms are more influenced by the crest of PW (pulse pressure) than by the mean arterial pressure. In addition, an increased arterial pulsatility is associated with several brain pathologies. We created simple computational models of four converging arteries and found that asynchronous arrival of the PWs results with lower maximum pressure, slower rate of pressure amplification and lower downstream pulsatility. In analogy, the asynchronous arrival of the pressure waves into the cerebral circulation should decrease blood flow pulsatility and lower transmission of kinetic energy on arterial wall. We conclude that asynchronous arrival of PWs into the cerebral circulation influences cerebral hemodynamics and represents a physiological necessity.

Extended Eversion Carotid Endarterectomy: Computation of Hemodynamics.

BACKGROUND: Stroke prevention includes surgery for significant stenosis of internal carotid artery (ICA). Consensus on a standard approach lacks and one alternative approach is eversion carotid endarterectomy (eCEA). To overcome disadvantages of eCEA, we developed extended-eversion carotid endarterectomy (exeCEA). Aiming to investigate hemodynamics after different surgical approaches, we created computational fluid dynamics (CFD) models of exeCEA and eCEA with included progressing lumen narrowing, representation of artery restenosis at the incision line. METHODS: Blood flow velocities, volume flow rates, and wall shear stress (WSS) were established in carotid arteries from models of eCEA and exeCEA with included increasing groove (1, 1.5, 2, and 2.5 mm) at the "incision line", under input pressure of 120 and 150 mm Hg. RESULTS: For the corresponding restenosis grade, models of exeCEA had a larger orifice toward ICA, lower blood flow velocities and higher volume flow rates in ICA, with lower volume flow rates in external carotid artery. WSS values in ICA of exeCEA models were lower than in eCEA models, later reaching the thrombotic range. CONCLUSIONS: CFD showed better hemodynamic properties in exeCEA models, indicating presented approach might be better at preserving brain perfusion.

Epicardial Adipose Tissue Is Nonlinearly Related to Anthropometric Measures and Subcutaneous Adipose Tissue.

Introduction. Adipose tissue is the largest endocrine organ, composed of subcutaneous (SAT) and visceral adipose tissue (VAT), the latter being highly associated with coronary artery disease (CAD). Expansion of epicardial adipose tissue (EAT) is linked to CAD. One way of assessing the CAD risk is with low-cost anthropometric measures, although they are inaccurate and cannot discriminate between VAT and SAT. The aim of this study is to evaluate (1) the relationship between EAT thickness, SAT thickness and anthropometric measures in a cohort of patients assessed at the cardiology unit and (2) determine predictive power of anthropometric measures and EAT and SAT thickness in establishment of CAD. Methods. Anthropometric measures were obtained from 53 CAD and 42 non-CAD patients. Vascular and structural statuses were obtained with coronarography and echocardiography, as well as measurements of the EAT and SAT thickness. Results. Anthropometric measures showed moderate positive correlation with EAT and SAT thickness. Anthropometric measures and SAT follow nonlinear S curve relationship with EAT. Strong nonlinear power curve relationship was observed between EAT and SAT thinner than 10 mm. Anthropometric measures and EAT and SAT were poor predictors of CAD. Conclusion. Anthropometric measures and SAT have nonlinear relationship with EAT. EAT thickness and anthropometric measures have similar CAD predictive value.

Proton pump (H+/K+-ATPase) expression in human larynx.

OBJECTIVE: The goal of our study was to investigate and to identify the existence of proton pump in different parts of larynx. The presence of acidic content in this area is known to be connected to several laryngeal diseases. It is mostly developed by upward recurrence of acidic gastric content, but there are some signs that the acid can be produced in the larynx as well, because of the proton pump activity in laryngeal mucosa. METHODS: The study was performed on two types of specimens: (1) 50 cadaver larynges and (2) 11 surgical larynges obtained after laryngectomy. Samples were taken from supraglottis, glottis and subglottic areas and immunohistochemistry for the beta subunit of the proton pump was done. RESULTS: The presence of proton pump was proved in seromucous glands in laryngeal supraglottic area, but it was also, for the first time, found in human chondrocytes in the thyroid and epiglottic cartilage. CONCLUSION: These new findings could encourage further research that would illuminate better the etiopathogenesis not only of laryngopharyngeal reflux, but also the pathophysiology of cartilaginous disorders.

Expression of leukocyte adhesion-related glycosyltransferase genes in acute coronary syndrome patients.

INTRODUCTION: Acute coronary syndrome (ACS) is caused by destabilization and rupture of atherosclerotic plaque in the coronary artery via mechanisms affecting leukocyte signaling, rolling, adhesion, extravasation and inflammation-promoting factors. The majority of cellular communication takes place on the membrane surface that is covered with glycoproteins and glycolipids synthesized by glycosyltransferases. The aim of this study was to determine the mRNA expression of leukocyte adhesion-related glycosyltransferases in patients during the onset and the chronic phase of ACS and to compare the expression with matching subjects without coronary disease. SUBJECTS AND METHODS: The study included 26 ACS patients and 26 ACS-free matched-pair controls. Blood samples were collected at the time of hospital admittance and 8 days later. Expression analysis of six fucosyltransferases and six sialyltransferases was performed by a real-time polymerase chain reaction. RESULTS: At the time of admittance ACS subjects had lower expression levels of FUT4, ST6GalNac4, ST6Gal1 and GM3 synthase (p < 0.05) than the control subjects, and moreover, after 8 days down-regulation of FUT7 and ST6GalNac3 was also observed (p < 0.05). When compared to the initial gene expression, after treatment and stabilization of ACS subjects, FUT7, ST6GalNac2 and ST6GalNac3 were down-regulated, whereas ST6GalNac1 was up-regulated. Expression levels of FUT7, ST6GalNac1, ST6GalNac2 and ST6GalNac3 were predicted by several drugs and medical history. CONCLUSION: Expression of glycosyltransferase genes differs in ACS and control subjects. During the course of the ACS study we established further changes in gene expression levels. Medical history was predictive of gene expression levels while drugs were shown to modulate expression levels.

Function of circle of Willis.

Nearly 400 years ago, Thomas Willis described the arterial ring at the base of the brain (the circle of Willis, CW) and recognized it as a compensatory system in the case of arterial occlusion. This theory is still accepted. We present several arguments that via negativa should discard the compensatory theory. (1) Current theory is anthropocentric; it ignores other species and their analog structures. (2) Arterial pathologies are diseases of old age, appearing after gene propagation. (3) According to the current theory, evolution has foresight. (4) Its commonness among animals indicates that it is probably a convergent evolutionary structure. (5) It was observed that communicating arteries are too small for effective blood flow, and (6) missing or hypoplastic in the majority of the population. We infer that CW, under physiologic conditions, serves as a passive pressure dissipating system; without considerable blood flow, pressure is transferred from the high to low pressure end, the latter being another arterial component of CW. Pressure gradient exists because pulse wave and blood flow arrive into the skull through different cerebral arteries asynchronously, due to arterial tree asymmetry. Therefore, CW and its communicating arteries protect cerebral artery and blood-brain barrier from hemodynamic stress.

Identification of person and quantification of human DNA recovered from mosquitoes (Culicidae).

Mosquitoes (Culicidae) are widespread insects and can be important in forensic context as a source of human DNA. In order to establish the quantity of human DNA in mosquitoes' gut after different post-feeding interval and for how long after taking a bloodmeal the human donor could be identified, 174 blood-engorged mosquitoes (subfamily Anophelinae and Culicinae) were captured, kept alive and sacrificed at 8h intervals. Human DNA was amplified using forensic PCR kits (Identifiler, MiniFiler, and Quantifiler). A full DNA profiles were obtained from all Culicinae mosquitoes (74/74) up to 48 h and profiling was successful up to 88 h after a bloodmeal. Duration of post-feeding interval had a significant negative effect on the possibility of obtaining a full profile (p<0.001), and logistic regression found that the probability of obtaining the full profile is 15.5% less for every 8h increase in post-feeding interval. Culicinae mosquitoes are a suitable source of human DNA for forensic STR kits more than three days after a bloodmeal. Human DNA recovered from mosquito can be used for matching purposes and could be useful in revealing spatial and temporal relation of events that took place at the crime scene. Therefore, mosquitoes at the crime scene, dead or alive, could be a valuable piece of forensic evidence.