Molecular Mechanisms of Hypoxic-Ischemic Encephalopathy in Newborn Piglets

Perinatal asphyxia (PA) is among the most common causes of neonatal deaths. In the survivors, PA may result in multi-organ damage including hypoxic-ischemic encephalopathy (HIE) that often leads to life long disabilities. Understanding the pathophysiological mechanisms of HIE is key to the development of neuroprotective therapies, and large animal PA/HIE models can help to bridge the translational gap between studies at the molecular/cellular level and the cotside management. Our research group has previously shown that 8- or 20-min-long PA resulted in incrementally severe HIE in newborn pigs and that neuronal-vascular injury was alleviated by molecular H2 ventilation in these models. Our purpose was to address several hypotheses related to the mechanisms of neuronal injury as well as H2 induced neuroprotection using brain samples chiefly obtained from these previous studies. First, we investigated the abundance of cyclooxygenase-2 (COX-2) immunopositive neurons in our samples, as COX-2 is known to be an important source of reactive oxygen species in the neonatal brain, however, induction of neuronal COX-2 by PA has not yet been shown in piglets. Oxidative cellular (DNA) damage was visualized with 8-hydroxy-deoxyguanozin immunohistochemistry and was correlated with neuronal COX-2 expression. Second, microglia were visualized with Iba-1 immunohistochemistry in order to detect PA induced neuroinflammatory changes in microglial morphology quantified by the so called ramification index. Third, the ratio of active (phosphorylated) and total forms of ERK and Akt kinases were determined using Western blots to assess the activity and PA induced changes of these important antiapoptotic signal transduction mechanisms. PA increased neuronal COX-2 expression neurons in several brain regions, where high percentage of COX-2 positive neurons always coincided with severe neuronal damage. In the parietal cortex, neuronal COX-2 abundance correlated both with oxidative damage and microglial activations.H2 attenuated all of these PA induced changes. Erk and Akt displayed high degree of phosphorylation in controls that was unaffected by PA in any studied region. In conclusion, PA-induced neuronal COX-2 induction, oxidative damage and neuroinflammation all contribute to neuronal injury and are reduced by H2, however, induction of antiapoptotic pathways appear to have a minor neuroprotective capacity in our HIE model

NMDA attenuates the neurovascular response to hypercapnia in the neonatal cerebral cortex

Cortical spreading depolarization (SD) involves activation of NMDA receptors and elicit neurovascular unit dysfunction. NMDA cannot trigger SD in newborns, thus its effect on neurovascular function is not confounded by other aspects of SD. The present study investigated if NMDA affected hypercapnia-induced microvascular and electrophysiological responses in the cerebral cortex of newborn pigs. Anesthetized piglets were fitted with cranial windows over the parietal cortex to study hemodynamic and electrophysiological responses to graded hypercapnia before/after topically applied NMDA assessed with laser-speckle contrast imaging and recording of local field potentials (LFP)/neuronal firing, respectively. NMDA increased cortical blood flow (CoBF), suppressed LFP power in most frequency bands but evoked a 2.5 Hz delta oscillation. The CoBF response to hypercapnia was abolished after NMDA and the hypercapnia-induced biphasic changes in delta and theta LFP power were also altered. MK-801 prevented NMDA-induced increases in CoBF and the attenuation of microvascular reactivity to hypercapnia. The neuronal nitric oxide synthase (nNOS) inhibitor (N-(4 S)-4-amino-5-[aminoethyl] aminopentyl-N'-nitroguanidin) also significantly preserved the CoBF response to hypercapnia after NMDA, although it didn't reduce NMDA-induced increases in CoBF. In conclusion, excess activation of NMDA receptors alone can elicit SD-like neurovascular unit dysfunction involving nNOS activity

A D3-vitamin-szint és a betegség súlyossága közötti kapcsolat vizsgálata herediter angioödémában

Absztrakt: Bevezetés: Az elmúlt évtized során számos közlemény látott napvilágot a D3-vitamin szérumszintje és különböző, részben immunpatomechanizmusú kórképek előfordulási gyakorisága, aktivitása közötti összefüggések elemzéséről. Célkitűzés: Korrelációt kerestünk a C1-inhibitor deficientiájában kialakuló herediter angioödémában szenvedő betegeink angioödémás rohamainak száma, lokalizációja és a felhasznált C1-inhibitor-pótlás mennyisége, valamint D-vitamin-szintjük között. Módszer: Az Országos Angioödéma Referencia Központban 2013–2014-ben gondozott 175, C1-inhibitor-deficientia következtében kialakuló herediter angioödémában szenvedő beteg közül 118 beteg D3-vitamin-szintjét határoztuk meg a téli–tavaszi (111 fő) és a nyári–őszi időszakban (105 fő). A komplement laboratóriumi eredmények és klinikai adatok az Országos Angioödéma Regiszterből és a betegnaplókból származtak. Eredmények: Betegeink mintegy 59,5%-a a téli–tavaszi időszakban, 27,6%-a a nyári–őszi időszakban, 23,5%-a pedig mindkét szezonban a D3-vitamin-hiányos csoportba (D3-vitamin-szint <20 ng/ml) tartozott. A téli–tavaszi és nyári–őszi D3-vitamin-szintek között szignifikáns különbséget találtunk (p<0,0001). A két időszakban az angioödémás rohamok akut kezelésére felhasznált C1-inhibitor-koncentrátum-ampullák száma között szignifikáns különbséget észleltünk (p = 0,01). A D3-vitamin-szint, valamint az adott időperiódusban elszenvedett rohamszám és a felhasznált C1-inhibitor-koncentrátum-ampullák száma között korrelációt egyik szezonban sem találtunk. Következtetések: Bár eddigi eredményeink alapján a herediter angioödémás betegek D3-vitamin-szintje és az elszenvedett angioödémás rohamok gyakorisága, lokalizációja között összefüggést kimutatni nem lehetett, a téli–tavaszi időszakban mégis nagyobb igény mutatkozott a rohamok kezelésére (több ampulla fogyott). Mivel betegeink körében a vártnál is gyakoribb D3-vitamin-hiányt találtunk, ez mindenképpen vitaminpótlást indokol. Orv Hetil. 2019; 160(25): 987–993. | Abstract: Introduction: In recent years, many papers analyzed the relationship between serum vitamin D3 level and the frequency and activity of various diseases at least partially attributed to immune mechanisms. Aim: We looked for correlations among the number and location of edematous episodes occurring in patients with hereditary angioedema due to C1-inhibitor deficiency (C1-INH-HAE) and the quantity of the C1-inhibitor used for supplementation as well as the vitamin D3 levels of patients. Method: We measured vitamin D3 levels in 118 of the 175 C1-INH-HAE patients of the National Angioedema Reference Center during the winter–spring (n = 111) and the summer–autumn periods (n = 105) in 2013–2014. Complement levels and clinical data were extracted from the National Angioedema Registry and from patient diaries. Results: The proportion of vitamin D3 deficient patients (serum level <20 ng/ml) was approximately 59.5% during winter–spring, 27.6% in summer–autumn, and 23.5% during both periods. There was a significant difference between vitamin D3 serum levels measured in the winter–spring or in the summer–autumn months (p<0.0001). The same applies to the number of the vials of C1-inhibitor concentrate administered as acute treatment for angioedema attacks (p = 0.01). In any season, vitamin D3 level did not correlate with the number of attacks experienced by the patients during the given period or of the vials of C1-inhibitor concentrate administered. Conclusions: We could not demonstrate a relationship between vitamin D3 level and the frequency or location of edematous episodes in HAE patients. The need for treatment (as reflected by the number of the vials administered) was higher in the winter–spring period. As vitamin D3 deficiency was more severe than expected in our patients, supplementation is clearly necessary. Orv Hetil. 2019; 160(25): 987–993

Biokémiai, molekuláris biológiai és genetikai vizsgálatok napfény okozta bőrkárosodásokban = Development of environmental health education: physical, chemical, epidemiological and dermatological aspects, with special emphasis on radiation induced damage

Munkacsoportunk évtizedek óta foglalkozik az ultraibolya fény élettani és patofiziológiai hatásaival klinikai és molekuláris szinten. Jelen pályázat futamideje alatt több photodermatologiai témájú összefoglaló közleményt és egy monográphiát publikáltunk. 3 testvéren extrém ritka hepatoerythropoeticus porphyriát diagnosztizáltunk, és a hátterében lévő compound heterozigóta mutációkat is identifikáltuk. A sporadikus porphyria cutanea tardában gyakoribb haemochromatosis mutáció és környezeti provokáló tényezők előfordulási gyakoriságáról bővítettük ismereteinket. UVB besugárzás után néhány ABC transzporter fehérje in situ, purinerg receptorok és inflammasoma alkotó elem mRNS és fehérje szintű in vitro expresszió-változását mutattuk ki keratinocita tenyészeteken. Komplettáltuk a keratinocita differenciációs teljes genom expressziós vizsgálatokat. Az irodalomban elsőként igazoltuk, hogy in vitro szintetizált módosított erszényes fotoliáz mRNS bejuttatható volt keratinocita tenyészetekbe és funkcionálisan aktív fehérje szintetizálódott róla. Tissue microarray analízis felhasználásával macrofágok, dendritikus sejtek és egy cistein tartalmú fehérje prognosztikai szerepét vetettük föl melanoma malignumban. Epidemiológiai vizsgálatunk során rámutattunk arra, hogy továbbra is fontos feladat a fényvédelem propagálása, mert 13 és 16 éves iskolás gyermekeken az anyajegyek száma magas, a gyermekek közel 40%-a napégésről, 3,6%-uk szolárium használatról számol be. | Our workgroup has investigated the physiological and pathophysiological effects of ultraviolet radiation at clinical and molecular levels for decades. Over the period of the application several photodermatological articles and one monograph were published. We diagnosed the extremely rare hepatoerythropoietic porphyria in a family, and identified compound heterozygous mutations in the background. We expanded our knowledge about the frequency of haemochromatosis mutation and environmental provoking factors in sporadic porphyria cutanea tarda. We demonstrated alterations in the expression of some ABC transport proteins in situ, and in the expression of purinerg receptors and inflammasome elements at mRNA and protein levels in vitro after UVB radiation in keratinocytes. We completed the whole-genom expression analysis related to keratinocyte differentiation. We proved for the first time that the in vitro synthesized modified marsupial photolyase mRNA can be delivered into keratinocytes, and that the translated protein is functionally active. We suggested the prognostic role of macrophages, dendritic cells and one cysteine-containing protein in melanoma malignum using tissue microarray analysis. During epidemiological studies we pointed out that propagation of light protection is an important task in the future, because the number of birthmarks is high among 13 and 16 year-old young people, and 40% of youngsters reported about sunburn and 3,6% of them use solarium

Saxagliptin Cardiotoxicity in Chronic Heart Failure: The Role of DPP4 in the Regulation of Neuropeptide Tone

Dipeptidyl-peptidase-4 (DPP4) inhibitors are novel medicines for diabetes. The SAVORTIMI-53 clinical trial revealed increased heart-failure-associated hospitalization in saxagliptin-treated patients. Although this side effect could limit therapeutic use, the mechanism of this potential cardiotoxicity is unclear. We aimed to establish a cellular platform to investigate DPP4 inhibition and the role of its neuropeptide substrates substance P (SP) and neuropeptide Y (NPY), and to determine the expression of DDP4 and its neuropeptide substrates in the human heart. Western blot, radio-, enzyme-linked immuno-, and RNA scope assays were performed to investigate the expression of DPP4 and its substrates in human hearts. Calcein-based viability measurements and scratch assays were used to test the potential toxicity of DPP4 inhibitors. Cardiac expression of DPP4 and NPY decreased in heart failure patients. In human hearts, DPP4 mRNA is detectable mainly in cardiomyocytes and endothelium. Treatment with DPP4 inhibitors alone/in combination with neuropeptides did not affect viability but in scratch assays neuropeptides decreased, while saxagliptin co-administration increased fibroblast migration in isolated neonatal rat cardiomyocyte-fibroblast co-culture. Decreased DPP4 activity takes part in the pathophysiology of end-stage heart failure. DPP4 compensates against the elevated sympathetic activity and altered neuropeptide tone. Its inhibition decreases this adaptive mechanism, thereby exacerbating myocardial damage

Characterization of Neurons Expressing the Novel Analgesic Drug Target Somatostatin Receptor 4 in Mouse and Human Brains

Somatostatin is an important mood and pain-regulating neuropeptide, which exerts analgesic, anti-inflammatory, and antidepressant effects via its Gi protein-coupled receptor subtype 4 (SST4) without endocrine actions. SST4 is suggested to be a unique novel drug target for chronic neuropathic pain, and depression, as a common comorbidity. However, its neuronal expression and cellular mechanism are poorly understood. Therefore, our goals were (i) to elucidate the expression pattern of Sstr4/SSTR4 mRNA, (ii) to characterize neurochemically, and (iii) electrophysiologically the Sstr4/SSTR4-expressing neuronal populations in the mouse and human brains. Here, we describe SST4 expression pattern in the nuclei of the mouse nociceptive and anti-nociceptive pathways as well as in human brain regions, and provide neurochemical and electrophysiological characterization of the SST4-expressing neurons. Intense or moderate SST4 expression was demonstrated predominantly in glutamatergic neurons in the major components of the pain matrix mostly also involved in mood regulation. The SST4 agonist J-2156 significantly decreased the firing rate of layer V pyramidal neurons by augmenting the depolarization-activated, non-inactivating K+ current (M-current) leading to remarkable inhibition. These are the first translational results explaining the mechanisms of action of SST4 agonists as novel analgesic and antidepressant candidates

Malignant astrocyte swelling and impaired glutamate clearance drive the expansion of injurious spreading depolarization foci

Spreading depolarizations (SDs) indicate injury progression and predict worse clinical outcome in acute brain injury. We demonstrate in rodents that acute brain swelling upon cerebral ischemia impairs astroglial glutamate clearance and increases the tissue area invaded by SD. The cytotoxic extracellular glutamate accumulation (>15 mu M) predisposes an extensive bulk of tissue (4-5 mm(2)) for a yet undescribed simultaneous depolarization (SiD). We confirm in rat brain slices exposed to osmotic stress that SiD is the pathological expansion of prior punctual SD foci (0.5-1 mm(2)), is associated with astrocyte swelling, and triggers oncotic neuron death. The blockade of astrocytic aquaporin-4 channels and Na+/K+/Cl- co-transporters, or volume-regulated anion channels mitigated slice edema, extracellular glutamate accumulation (<10 mu M) and SiD occurrence. Reversal of slice swelling by hyperosmotic mannitol counteracted glutamate accumulation and prevented SiD. In contrast, inhibition of glial metabolism or inhibition of astrocyte glutamate transporters reproduced the SiD phenotype. Finally, we show in the rodent water intoxication model of cytotoxic edema that astrocyte swelling and altered astrocyte calcium waves are central in the evolution of SiD. We discuss our results in the light of evidence for SiD in the human cortex. Our results emphasize the need of preventive osmotherapy in acute brain injury

Malignant astrocyte swelling and impaired glutamate clearance drive the expansion of injurious spreading depolarization foci

Spreading depolarizations (SDs) indicate injury progression and predict worse clinical outcome in acute brain injury. We demonstrate in rodents that acute brain swelling upon cerebral ischemia impairs astroglial glutamate clearance and increases the tissue area invaded by SD. The cytotoxic extracellular glutamate accumulation (>15 mu M) predisposes an extensive bulk of tissue (4-5 mm(2)) for a yet undescribed simultaneous depolarization (SiD). We confirm in rat brain slices exposed to osmotic stress that SiD is the pathological expansion of prior punctual SD foci (0.5-1 mm(2)), is associated with astrocyte swelling, and triggers oncotic neuron death. The blockade of astrocytic aquaporin-4 channels and Na+/K+/Cl- co-transporters, or volume-regulated anion channels mitigated slice edema, extracellular glutamate accumulation (<10 mu M) and SiD occurrence. Reversal of slice swelling by hyperosmotic mannitol counteracted glutamate accumulation and prevented SiD. In contrast, inhibition of glial metabolism or inhibition of astrocyte glutamate transporters reproduced the SiD phenotype. Finally, we show in the rodent water intoxication model of cytotoxic edema that astrocyte swelling and altered astrocyte calcium waves are central in the evolution of SiD. We discuss our results in the light of evidence for SiD in the human cortex. Our results emphasize the need of preventive osmotherapy in acute brain injury

Molecular hydrogen affords neuroprotection in a translational piglet model of hypoxic-ischemic encephalopathy

Hypoxic-ischemic encephalopathy (HIE) is the major consequence of perinatal asphyxia (PA) in term neonates. Although the newborn piglet is an accepted large animal PA/HIE model, there is no consensus on PA-induction methodology to produce clinically relevant HIE. We aimed to create and to characterize a novel PA model faithfully reproducing all features of asphyxiation including severe hypercapnia resulting in HIE, and to test whether H2 is neuroprotective in this model. Piglets were anaesthetised, artificially ventilated, and intensively monitored (electroencephalography, core temperature, O2 saturation, arterial blood pressure and blood gases). Asphyxia (20 min) was induced by ventilation with a hypoxic-hypercapnic (6%O2 - 20%CO2) gas mixture. Asphyxia-induced changes in the cortical microcirculation were assessed with laser-speckle contrast imaging and analysis. Asphyxia was followed by reventilation with air or air containing hydrogen (2.1%H2, 4 hours). After 24 hours survival, the brains were harvested for neuropathology. Our PA model was characterized by the development of severe hypoxia (pO2 = 27 +/- 4 mmHg), and combined acidosis (pH = 6.76 +/- 0.04; pCO2 = 114 +/- 11 mmHg; lactate = 12.12 +/- 0.83 mmol/L), however, cortical ischemia did not develop during the stress. Severely depressed electroencephalography (EEG), and marked neuronal injury indicated the development of HIE. H2 was neuroprotective shown both by the enhanced recovery of EEG and by the significant preservation of neurons in the cerebral cortex, hippocampus, basal ganglia, and the thalamus. H2 appeared to reduce oxidative stress shown by attenuation of 8-hydroxy-2'-deoxyguanosine immunostaining. In summary, this new PA piglet model is able to induce moderate/severe HIE, and the efficacy of hydrogen post-treatment to preserve neuronal activity/function in this PA/HIE model suggests the feasibility of this safe and inexpensive approach in the treatment of asphyxiated babies