Neuronal dynamics direct cerebrospinal fluid perfusion and brain clearance.

The accumulation of metabolic waste is a leading cause of numerous neurological disorders, yet we still have only limited knowledge of how the brain performs self-cleansing. Here we demonstrate that neural networks synchronize individual action potentials to create large-amplitude, rhythmic and self-perpetuating ionic waves in the interstitial fluid of the brain. These waves are a plausible mechanism to explain the correlated potentiation of the glymphatic flow1,2 through the brain parenchyma. Chemogenetic flattening of these high-energy ionic waves largely impeded cerebrospinal fluid infiltration into and clearance of molecules from the brain parenchyma. Notably, synthesized waves generated through transcranial optogenetic stimulation substantially potentiated cerebrospinal fluid-to-interstitial fluid perfusion. Our study demonstrates that neurons serve as master organizers for brain clearance. This fundamental principle introduces a new theoretical framework for the functioning of macroscopic brain waves.

Circulating myeloid-derived MMP8 in stress susceptibility and depression.

Psychosocial stress has profound effects on the body, including the immune system and the brain1,2. Although a large number of pre-clinical and clinical studies have linked peripheral immune system alterations to stress-related disorders such as major depressive disorder (MDD)3, the underlying mechanisms are not well understood. Here we show that expression of a circulating myeloid cell-specific proteinase, matrix metalloproteinase 8 (MMP8), is increased in the serum of humans with MDD as well as in stress-susceptible mice following chronic social defeat stress (CSDS). In mice, we show that this increase leads to alterations in extracellular space and neurophysiological changes in the nucleus accumbens (NAc), as well as altered social behaviour. Using a combination of mass cytometry and single-cell RNA sequencing, we performed high-dimensional phenotyping of immune cells in circulation and in the brain and demonstrate that peripheral monocytes are strongly affected by stress. In stress-susceptible mice, both circulating monocytes and monocytes that traffic to the brain showed increased Mmp8 expression following chronic social defeat stress. We further demonstrate that circulating MMP8 directly infiltrates the NAc parenchyma and controls the ultrastructure of the extracellular space. Depleting MMP8 prevented stress-induced social avoidance behaviour and alterations in NAc neurophysiology and extracellular space. Collectively, these data establish a mechanism by which peripheral immune factors can affect central nervous system function and behaviour in the context of stress. Targeting specific peripheral immune cell-derived matrix metalloproteinases could constitute novel therapeutic targets for stress-related neuropsychiatric disorders.

Microdialysis and CO2 sensors detect pancreatic ischemia in a porcine model.

BACKGROUND: Pancreatic transplantation is associated with a high rate of early postoperative graft thrombosis. If a thrombosis is detected in time, a potentially graft-saving intervention can be initiated. Current postoperative monitoring lacks tools for early detection of ischemia. The aim of this study was to investigate if microdialysis and tissue pCO2 sensors detect pancreatic ischemia and whether intraparenchymal and organ surface measurements are comparable. METHODS: In 8 anaesthetized pigs, pairs of lactate monitoring microdialysis catheters and tissue pCO2 sensors were simultaneously inserted into the parenchyma and attached to the surface of the pancreas. Ischemia was induced by sequential arterial and venous occlusions of 45-minute duration, with two-hour reperfusion after each occlusion. Microdialysate was analyzed every 15 minutes. Tissue pCO2 was measured continuously. We investigated how surface and parenchymal measurements correlated and the capability of lactate and pCO2 to discriminate ischemic from non-ischemic periods. RESULTS: Ischemia was successfully induced by arterial occlusion in 8 animals and by venous occlusion in 5. During all ischemic episodes, lactate increased with a fold change of 3.2-9.5 (range) in the parenchyma and 1.7-7.6 on the surface. Tissue pCO2 increased with a fold change of 1.6-3.5 in the parenchyma and 1.3-3.0 on the surface. Systemic lactate and pCO2 remained unchanged. The area under curve (AUC) for lactate was 0.97 (95% confidence interval (CI) 0.93-1.00) for parenchymal and 0.90 (0.83-0.97) for surface (p<0.001 for both). For pCO2 the AUC was 0.93 (0.89-0.96) for parenchymal and 0.85 (0.81-0.90) for surface (p<0.001 for both). The median correlation coefficients between parenchyma and surface were 0.90 (interquartile range (IQR) 0.77-0.95) for lactate and 0.93 (0.89-0.97) for pCO2. CONCLUSIONS: Local organ monitoring with microdialysis and tissue pCO2 sensors detect pancreatic ischemia with adequate correlation between surface and parenchymal measurements. Both techniques and locations seem feasible for further development of clinical pancreas monitoring.

Assessment of hepatic function, perfusion and parenchyma attenuation with indocyanine green, ultrasound and computed tomography in a healthy rat model: Preliminary determination of baseline parameters in a healthy liver.

BACKGROUND: Defining reference intervals in experimental animal models plays a crucial role in pre-clinical studies. The hepatic parameters in healthy animals provide useful information about type and extension of hepatic damage. However, in the majority of the cases, to obtain them require an invasive techniques. Our study combines these determinations with dynamic functional test and imaging techniques to implement a non-invasive protocol for liver evaluation. The aim of the study was to determine reference intervals for hepatic function, perfusion and parenchyma attenuation with analytical and biochemical blood parameters, indocyanine green, ultrasound and computed tomography in six healthy SD rats. METHODS: Six males healthy SD rats were followed for 4 weeks. To determine hepatic function, perfusion and parenchyma attenuation analytical and biochemical blood parameters, indocyanine green, ultrasound and computed tomography were studied. Results were expressed as Means ± standard error of mean (SEM). The significance of differences was calculated by using student t-test, p < 0.05 was considered statistically significant. RESULTS: Indocyanine green clearance 5 and 10 minutes after its injection was 80.12% and 96.59%, respectively. Approximate rate of decay during the first 5 minutes after injection was 38% per minute. Hepatic perfusion evaluation with the high-frequency ultrasound was related to cardiovascular hemodynamic and renal perfusion. Portal area, hepatic artery resistance index, hepatic artery and portal peak systolic velocity and average between hepatic artery and porta was 3.41 ± 0.62 mm2, 0.57 ± 0.04 mm2/s, 693.24±102.53 mm2/s, 150.72 ± 17.80 mm2/s and 4.82 ± 0.96 mm2/s, respectively. Heart rate, cardiac output, left renal artery diammetre and renal blood flow were 331.01 ± 22.22 bpm, 75.58 ± 8.72 mL/min, 0.88 ± 0.04 mm2 and 13.65 ± 1.95 mm2/s. CT-scan hepatic average volume for each rat were 21.08±3.32, 17.57±2.76, 14.87±2.83 and 13.67±2.45 cm3 with an average attenuation coefficient of 113.51±18.08, 129,19±7.18, 141,47±1.95 y 151,67±1.2 HU. CONCLUSION: Indocyanine green and high-frequency ultrasound could be used in rats as a suitable marker of liver function. Computed tomography, through the study of raw data, help to characterize liver parenchyma, and could be a potential tool for early detection of liver parenchymal alterations and linear follow-up of patients. Further studies in rats with liver disease are necessary to verify the usefulness of these parameters.

Type-3 Hyaluronan Synthase Attenuates Tumor Cells Invasion in Human Mammary Parenchymal Tissues.

The microenvironment for tumor growth and developing metastasis should be essential. This study demonstrated that the hyaluronic acid synthase 3 (HAS3) protein and its enzymatic product hyaluronic acid (HA) encompassed in the subcutaneous extracellular matrix can attenuate the invasion of human breast tumor cells. Decreased HA levels in subcutaneous Has3-KO mouse tissues promoted orthotopic breast cancer (E0771) cell-derived allograft tumor growth. MDA-MB-231 cells premixed with higher concentration HA attenuate tumor growth in xenografted nude mice. Human patient-derived xenotransplantation (PDX) experiments found that HA selected the highly migratory breast cancer cells with CD44 expression accumulated in the tumor/stroma junction. In conclusion, HAS3 and HA were detected in the stroma breast tissues at a high level attenuates effects for induced breast cancer cell death, and inhibit the cancer cells invasion at the initial stage. However, the highly migratory cancer cells were resistant to the HA-mediated effects with unknown mechanisms.

Expression of cytokines in dairy cattle mammary gland parenchyma during chronic staphylococcal infection.

The study aim was to determine the expression of genes potentially related to chronic mastitis at the mRNA and protein levels, viz. chemokine C-C motif receptor 1 (CCR1), C-C motif chemokine ligand 2 (CCL2), C-C motif chemokine ligand 5 (CXCL5), tumor necrosis factor α (TNFα), interleukin 1ß (IL-1ß), interleukin 6 (IL-6), interleukin 8 (IL-8), interleukin 18 (IL-18), in bovine mammary gland parenchyma. The study examines the differences in expression of selected genes between cows with chronic mastitis caused by coagulase-positive (CoPS) or coagulase-negative staphylococci (CoNS) and those with healthy udders (H). Samples were collected from the udder quarters from 40 Polish Holstein-Friesian cows; 54 of these samples were chosen for analysis based on microbiological analysis of milk taken two days before slaughter. They were categorized into three groups: CoPS (N = 27), CoNS (N = 14) and H (N = 13). The RNA expression was analyzed by RT-qPCR and protein concentration by ELISA. No differences in the mRNA levels of seven genes (TNFα, IL-18, CCR1, IL-1ß, CCL2, IL-8, IL-6) and four proteins (TNFα, IL-18, CCR1, IL-1ß) were identified between the CoPS and H groups. Higher transcript levels of CXCL5 (p ≤ 0.05) gene were noted in CoPS than in H. Compared to H, higher concentrations of IL-8 and CXCL5 (p ≤ 0.05) were observed in CoPS (0.05 < p < 0.1) and CCL2 (0.05 < p < 0.1) in CoNS, while lower levels of Il-6 were found in CoPS. This may suggest that during chronic mastitis the organism stops producing pro-inflammatory cytokines, probably to protect the host tissues against their damage during prolonged infection.

Exogenous Aß seeds induce Aß depositions in the blood vessels rather than the brain parenchyma, independently of Aß strain-specific information.

Little is known about the effects of parenchymal or vascular amyloid ß peptide (Aß) deposition in the brain. We hypothesized that Aß strain-specific information defines whether Aß deposits on the brain parenchyma or blood vessels. We investigated 12 autopsied patients with different severities of Aß plaques and cerebral amyloid angiopathy (CAA), and performed a seeding study using an Alzheimer's disease (AD) mouse model in which brain homogenates derived from the autopsied patients were injected intracerebrally. Based on the predominant pathological features, we classified the autopsied patients into four groups: AD, CAA, AD + CAA, and less Aß. One year after the injection, the pathological and biochemical features of Aß in the autopsied human brains were not preserved in the human brain extract-injected mice. The CAA counts in the mice injected with all four types of human brain extracts were significantly higher than those in mice injected with PBS. Interestingly, parenchymal and vascular Aß depositions were observed in the mice that were injected with the human brain homogenate from the less Aß group. The Aß and CAA seeding activities, which had significant positive correlations with the Aß oligomer ratio in the human brain extracts, were significantly higher in the human brain homogenate from the less Aß group than in the other three groups. These results indicate that exogenous Aß seeds from different Aß pathologies induced Aß deposition in the blood vessels rather than the brain parenchyma without being influenced by Aß strain-specific information, which might be why CAA is a predominant feature of Aß pathology in iatrogenic transmission cases. Furthermore, our results suggest that iatrogenic transmission of Aß pathology might occur due to contamination of brain tissues from patients with little Aß pathology, and the development of inactivation methods for Aß seeding activity to prevent iatrogenic transmission is urgently required.

Bcl6 controls meningeal Th17-B cell interaction in murine neuroinflammation.

Ectopic lymphoid tissue containing B cells forms in the meninges at late stages of human multiple sclerosis (MS) and when neuroinflammation is induced by interleukin (IL)-17 producing T helper (Th17) cells in rodents. B cell differentiation and the subsequent release of class-switched immunoglobulins have been speculated to occur in the meninges, but the exact cellular composition and underlying mechanisms of meningeal-dominated inflammation remain unknown. Here, we performed in-depth characterization of meningeal versus parenchymal Th17-induced rodent neuroinflammation. The most pronounced cellular and transcriptional differences between these compartments was the localization of B cells exhibiting a follicular phenotype exclusively to the meninges. Correspondingly, meningeal but not parenchymal Th17 cells acquired a B cell-supporting phenotype and resided in close contact with B cells. This preferential B cell tropism for the meninges and the formation of meningeal ectopic lymphoid tissue was partially dependent on the expression of the transcription factor Bcl6 in Th17 cells that is required in other T cell lineages to induce isotype class switching in B cells. A function of Bcl6 in Th17 cells was only detected in vivo and was reflected by the induction of B cell-supporting cytokines, the appearance of follicular B cells in the meninges, and of immunoglobulin class switching in the cerebrospinal fluid. We thus identify the induction of a B cell-supporting meningeal microenvironment by Bcl6 in Th17 cells as a mechanism controlling compartment specificity in neuroinflammation.

Metabolic profiles of human brain parenchyma and glioma for rapid tissue diagnosis by targeted desorption electrospray ionization mass spectrometry.

Desorption electrospray ionization mass spectrometry (DESI-MS) is well suited for intraoperative tissue analysis since it requires little sample preparation and offers rapid and sensitive molecular diagnostics. Currently, intraoperative assessment of the tumor cell percentage of glioma biopsies can be made by measuring a single metabolite, N-acetylaspartate (NAA). The inclusion of additional biomarkers will likely improve the accuracy when distinguishing brain parenchyma from glioma by DESI-MS. To explore this possibility, mass spectra were recorded for extracts from 32 unmodified human brain samples with known pathology. Statistical analysis of data obtained from full-scan and multiple reaction monitoring (MRM) profiles identified discriminatory metabolites, namely gamma-aminobutyric acid (GABA), creatine, glutamic acid, carnitine, and hexane-1,2,3,4,5,6-hexol (abbreviated as hexol), as well as the established biomarker NAA. Brain parenchyma was readily differentiated from glioma based on these metabolites as measured both in full-scan mass spectra and by the intensities of their characteristic MRM transitions. New DESI-MS methods (5 min acquisition using full scans and MS/MS), developed to measure ion abundance ratios among these metabolites, were tested using smears of 29 brain samples. Ion abundance ratios based on signals for GABA, creatine, carnitine, and hexol all had sensitivities > 90%, specificities > 80%, and accuracies > 85%. Prospectively, the implementation of diagnostic ion abundance ratios should strengthen the discriminatory power of individual biomarkers and enhance method robustness against signal fluctuations, resulting in an improved DESI-MS method of glioma diagnosis.

EGFR and KRAS Mutations in Lung Parenchyma of Subjects With EGFR/KRAS Wild-Type Lung Adenocarcinoma.

The acquisition of driver mutations in non-tumoral cells appears to be very important during the carcinogenesis of adenocarcinoma (ADC). Recent studies suggest that cancer-related mutations may not necessarily be present only in malignant cells, but also in histologically "healthy cells". Objective: to demonstrate the presence of EGFR or KRAS mutations in non-tumoral lung cells in subjects with ADC and negative mutational status. Results: mutations in EGFR or KRAS oncogenes were identified in the normal lung in 9.7% of the subjects. Exon 21 substitution L858R in EGFR was detected in two cases while the exon 19 deletion E746-A750 in the EGFR, the G12C and G12D substitutions in the KRAS were detected once. One patient presented three different mutations in the normal lung parenchyma (EGFR_L858R, KRAS_G12C and KRAS_G12D). The negative-mutation status of the tumor and the mutations detected in the "normal lung" were confirmed using highly sensitive and specific TaqMan PCR (CAST-PCR). No differences were found in terms of progression, progression-free survival or overall survival during the 18 months follow-up. Conclusions: These results confirm the presence of driver mutations in the histologically normal lung parenchyma cells in the absence of mutations coexisting with the primary tumor.

Aging induces abnormal accumulation of Aß in extracellular vesicle and/or intraluminal membrane vesicle-rich fractions in nonhuman primate brain.

Aß metabolism in the brain is mediated by endocytosis, one part of the intracellular membrane trafficking system. We previously showed that aging attenuates the interaction of dynein with dynactin, which disrupts the endosomal/lysosomal trafficking pathway involved in Aß metabolism, resulting in intracellular accumulation of Aß. Several studies have shown that in Alzheimer's disease (AD), intraneuronal accumulation of Aß precedes extracellular Aß depositions. However, it is unclear what accounts for this transition from intracellular to extracellular depositions. Accumulating evidence suggest that autophagy has an important role in AD pathology, and we observed that autophagy-related protein levels began to decrease before amyloid plaque formation in cynomolgus monkey brains. Surprisingly, experimental induction of autophagosome formation in Neuro2a cells significantly increased intracellular Aß and decreased extracellular release of Aß, accompanied by the prominent reduction of extracellular vesicle (EV) secretion. RNAi study confirmed that EV secretion affected intracellular and extracellular Aß levels, and siRNA-induced downregulation of autophagosome formation enhanced EV secretion to ameliorate intracellular Aß accumulation induced by dynein knockdown. In aged cynomolgus monkeys, Aß levels in EV/intraluminal membrane vesicle (ILV)-rich fractions isolated from temporal lobe parenchyma were drastically increased. Moreover, EV/ILV marker proteins overlapped spatially with amyloid plaques. These findings suggest that EV would be an important carrier of Aß in brain and abnormal accumulation of Aß in EVs/ILVs may be involved in the transition of age-dependent Aß pathology.

Widespread brain parenchymal HMGB1 and NF-κB neuroinflammatory responses upon cortical spreading depolarization in familial hemiplegic migraine type 1 mice.

Neuroinflammatory changes involving neuronal HMGB1 release and astrocytic NF-κB nuclear translocation occur following cortical spreading depolarization (CSD) in wildtype (WT) mice but it is unknown to what extent this occurs in the migraine brain. We therefore investigated in familial hemiplegic migraine type 1 (FHM1) knock-in mice, which express an intrinsic hyperexcitability phenotype, the extent of neuroinflammation without and after CSD. CSD was evoked in one hemisphere by pinprick (single CSD) or topical KCl application (multiple CSDs). Neuroinflammatory (HMGB1, NF-κB) and neuronal activation (pERK) markers were investigated by immunohistochemistry in the brains of WT and FHM1 mutant mice without and after CSD. Effects of NMDA receptor antagonism on basal and CSD-induced neuroinflammatory changes were examined by, respectively, systemically administered MK801 and ifenprodil or topical MK801 application. In FHM1 mutant mice, CSD caused enhanced neuronal HMGB1 release and astrocytic NF-κB nuclear translocation in the cortex and subcortical areas that were equally high in both hemispheres. In WT mice such effects were only pronounced in the hemisphere in which CSD was induced. Neuroinflammatory responses were associated with pERK expression indicating neuronal activation. Upon CSD, contralateral cortical and striatal HMGB1 release was reduced by topical application of MK801 in the hemisphere contralateral to the one in which CSD was induced. This study reveals that neuroinflammatory activation after CSD is widespread and extends to the contralateral hemisphere, particularly in brains of FHM1 mutant mice. Effective blockade of CSD-induced neuroinflammatory responses in the contralateral hemisphere in FHM1 mice by local NMDA receptor antagonism suggests that neuronal hyperexcitability-related neuroinflammation is relevant in migraine pathophysiology, but possibly also other neurological disorders in which spreading depolarization is involved.

Types of Parenchymal Changes Diagnosed on DMSA Scans of Kidneys Affected by Different Grades of Vesicoureteral Reflux.

BACKGROUND Renal parenchymal damage and scarring usually is associated with urinary tract infection (UTI), whereas the impact of vesicoureteral reflux (VUR) on the kidneys is unclear. We aimed to compare kidneys with all grades of VUR (grades Io-V) and those without VUR by using direct radionuclide cystography, voiding cystourethrography, and findings from 99mTc-DMSA scintigraphy (DMSA scan). MATERIAL AND METHODS The present analysis included 253 renal ureteral units (RUU) from 129 children with VUR and recurrent UTI and children with a single febrile UTI associated with abnormal ultrasonographic findings. The 6 grades of VUR (Io, I, II, III, IV, and V) and 35 RUUs without VUR were divided into 4 groups: 1. Non-dilated VUR (grades Io-II); 2. Mildly dilated VUR (grade III); 3. Dilated VUR (grades IV-V); and 4. The control group. RESULTS DMSA scanning showed significant differences between the groups with non-dilated VUR, grade III VUR, grades IV-V VUR, and the control group in kidney width (χ²=30.5; P<0.001); position and shape (χ²=30.6; P<0.001); intensity of activity (χ²=38.1; P<0.001); distribution of activity (χ²=34.5; P<0.001); and existence of scars (χ²=16; P<0.001). The probability of abnormalities on DMSA scans increased with the VUR grade. However, inside the groups of dilated and non-dilated VUR we found no significant statistical differences between those characteristics. CONCLUSIONS Our results indicate that kidneys without VUR or with non-dilated lateral VUR and dilated VUR on the contralateral side represent 2 different categories of parenchymal changes.

CNS macrophages differentially rely on an intronic Csf1r enhancer for their development.

The central nervous system hosts parenchymal macrophages, known as microglia, and non-parenchymal macrophages, collectively termed border-associated macrophages (BAMs). Microglia, but not BAMs, were reported to be absent in mice lacking a conserved Csf1r enhancer: the fms-intronic regulatory element (FIRE). However, it is unknown whether FIRE deficiency also impacts BAM arrival and/or maintenance. Here, we show that macrophages in the ventricular system of the brain, including Kolmer's epiplexus macrophages, are absent in Csf1rΔFIRE/ΔFIRE mice. Stromal choroid plexus BAMs are also considerably reduced. During normal development, we demonstrate that intracerebroventricular macrophages arrive from embryonic day 10.5, and can traverse ventricular walls in embryonic slice cultures. In Csf1rΔFIRE/ΔFIRE embryos, the arrival of both primitive microglia and intracerebroventricular macrophages was eliminated, whereas the arrival of cephalic mesenchyme and stromal choroid plexus BAMs was only partially restricted. Our results provide new insights into the development and regulation of different CNS macrophage populations.

Extraparenchymal human neurocysticercosis induces autoantibodies against brain tubulin and MOG35-55 in cerebral spinal fluid.

Neurocysticercosis (NC) presents two broad clinical entities: extraparenchymal (EP-NC) and parenchymal (P-NC). Using ELISA methodology, we demonstrate autoantibodies to tubulin and the Major oligodendrocyte glycoprotein (MOG) in the CSF of most, but not all, EP-NC samples. Levels of these autoantibodies were considerably reduced or absent in the P-NC samples. There was a striking correlation between levels of anti-tubulin and anti-MOG, and the significant correlation between the levels of autoantibodies and cellularity in the CSF, suggests that stimulation of the autoantibody response may be a function of cerebral inflammation. A hypothetical model to describe the pathogenesis of EP-NC is presented.

Microparticles and Nucleosomes Are Released From Parenchymal Cells Destroyed After Injury in a Rat Model of Blunt Trauma.

We investigated the relationships between circulating procoagulants and trauma severity, including cellular destruction, and the effects of thrombin generation on procoagulants in a rat blunt trauma model. The rats were subjected to tumbling blunt trauma, where they were tumbled for 0, 250, 500, or 1000 revolutions. Creatine kinase, nucleosome, and microparticle plasma levels increased gradually with trauma severity. Strong interrelationships were observed among creatine kinase, nucleosome, and microparticle levels. Time to initiation of thrombin generation shortened with increasing trauma severity. In accordance with trauma severity, prothrombin activity decreased, but the thrombin generation ratio increased. Time to initiation of thrombin generation and the thrombin generation ratio correlated with creatine kinase levels. In an in vitro study, a homogenized muscle solution, which included massive nucleosomes and microparticles, showed accelerated thrombin generation of plasma from healthy subjects. Procoagulants, such as microparticles and nucleosomes, are released from destroyed parenchymal cells immediately after external traumatic force, activating the coagulation cascade. The procoagulants shorten the time to initiation of thrombin generation. Furthermore, although coagulation factors are consumed, the thrombin generation ratio increases.

Differential expression of receptors mediating receptor-mediated transcytosis (RMT) in brain microvessels, brain parenchyma and peripheral tissues of the mouse and the human.

Receptor-mediated transcytosis (RMT) is a principal pathway for transport of macromolecules essential for brain function across the blood-brain barrier (BBB). Antibodies or peptide ligands which bind RMT receptors are often co-opted for brain delivery of biotherapeutics. Constitutively recycling transferrin receptor (TfR) is a prototype receptor utilized to shuttle therapeutic cargos across the BBB. Several other BBB-expressed receptors have been shown to mediate transcytosis of antibodies or protein ligands including insulin receptor (INSR) and insulin-like growth factor-1 receptor (IGF1R), lipid transporters LRP1, LDLR, LRP8 and TMEM30A, solute carrier family transporter SLC3A2/CD98hc and leptin receptor (LEPR). In this study, we analyzed expression patterns of genes encoding RMT receptors in isolated brain microvessels, brain parenchyma and peripheral organs of the mouse and the human using RNA-seq approach. IGF1R, INSR and LRP8 were highly enriched in mouse brain microvessels compared to peripheral tissues. In human brain microvessels only INSR was enriched compared to either the brain or the lung. The expression levels of SLC2A1, LRP1, IGF1R, LRP8 and TFRC were significantly higher in the mouse compared to human brain microvessels. The protein expression of these receptors analyzed by Western blot and immunofluorescent staining of the brain microvessels correlated with their transcript abundance. This study provides a molecular transcriptomics map of key RMT receptors in mouse and human brain microvessels and peripheral tissues, important to translational studies of biodistribution, efficacy and safety of antibodies developed against these receptors.

Laser capture microdissection in combination with mass spectrometry: Approach to characterization of tissue-specific proteomes of Eudiplozoon nipponicum (Monogenea, Polyopisthocotylea).

Eudiplozoon nipponicum (Goto, 1891) is a hematophagous monogenean ectoparasite which inhabits the gills of the common carp (Cyprinus carpio). Heavy infestation can lead to anemia and in conjunction with secondary bacterial infections cause poor health and eventual death of the host. This study is based on an innovative approach to protein localization which has never been used in parasitology before. Using laser capture microdissection, we dissected particular areas of the parasite body without contaminating the samples by surrounding tissue and in combination with analysis by mass spectrometry obtained tissue-specific proteomes of tegument, intestine, and parenchyma of our model organism, E. nipponicum. We successfully verified the presence of certain functional proteins (e.g. cathepsin L) in tissues where their presence was expected (intestine) and confirmed that there were no traces of these proteins in other tissues (tegument and parenchyma). Additionally, we identified a total of 2,059 proteins, including 72 peptidases and 33 peptidase inhibitors. As expected, the greatest variety was found in the intestine and the lowest variety in the parenchyma. Our results are significant on two levels. Firstly, we demonstrated that one can localize all proteins in one analysis and without using laboratory animals (antibodies for immunolocalization of single proteins). Secondly, this study offers the first complex proteomic data on not only the E. nipponicum but within the whole class of Monogenea, which was from this point of view until recently neglected.

Common disbalance in the brain parenchyma of dementias: Phospholipid profile analysis between CADASIL and sporadic Alzheimer's disease.

Sporadic Alzheimer's disease (SAD) is the most common form of dementia, and cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is the most frequent hereditary ischemic small vessel disease of the brain. Relevant biomarkers or specific metabolic signatures could provide powerful tools to manage these diseases. Therefore, the main goal of this study was to compare the postmortem frontal cortex gray matter, white matter and cerebrospinal fluid (CSF) between a cognitively healthy group and CADASIL and SAD groups. We evaluated 352 individual lipids, belonging to 13 lipid classes/subclasses, using mass spectrometry, and the lipid profiles were subjected to multivariate analysis to discriminate between the dementia groups (CADASIL and SAD) and healthy controls. The main lipid molecular species showing greater discrimination by partial least squares-discriminant analysis (PLS-DA) and a higher significance multivariate correlation (sMC) index were as follows: phosphatidylserine (PS) PS(44:7) and lysophosphatidylethanolamine (LPE) LPE(18:2) in gray matter (GM); phosphatidylethanolamine (PE) PE(32:2) and phosphatidylcholine PC PC(44:6) in white matter (WM), and ether PE (ePE) ePE(38:2) and ether PC (ePC) ePC(34:3) in CSF. Common phospholipid molecular species were obtained in both dementias, such as PS(44:7) and lyso PC (LPC) LPC(22:5) in GM, PE(32:2) in WM and phosphatidic acid (PA) PA(38:5) and PC(42:7) in CFS. Our exploratory study suggests that phospholipids (PLs) involved in neurotransmission alteration, connectivity impairment and inflammation response in GM, WM and CSF are a transversal phenomenon affecting dementias such as CADASIL and SAD independent of the etiopathogenesis, thus providing a possible common prodromal phospholipidic biomarker of dementia.