Regional differences in expression of molecular markers during formation of the expanded subplate zone in the human fetal cerebral cortex

U doktorskoj disertaciji prikazane su regionalne citoarhitektonske razlike između čeone, tjemene, zatiljne i cingularne moždane kore u fazi ekspanzije i stvaranja subplate zone (od 13. do 15. TNZ). Pomoću imunohistokemijskih metoda na prenatalnom postmortalnom tkivu mozga čovjeka praćena je dinamika ekspresije molekularnih biljega. Rezultati su pokazali kako regionalne razlike između pojedinih izokortikalnih regija fetalne moždane kore čovjeka postaju vidljive rano tijekom prenatalnog razvoja, a postaju najistaknutije upravo tijekom razdoblja formiranja SP-a. Proces stvaranja SP-a iz dubokog dijela KP prikazan je biljegom SP neurona TBR1. Pokazano je i kako je obrazac formiranja SP-a važan kriterij za diferenciranje dorzalne izokortikalne i ventralne mezokortikalne cingularne moždane kore u ranom fetalnom razdoblju. Nadalje, karakteristike rane diferencijacije mezokortikalne cingularne moždane kore su: proširenje MZ, suženje KP i SVZ. Jedna od glavnih karakteristika mezokortikalnog dijela cingularne moždane kore je nepotpuna ekspanzija SP-a, a navedeno je prikazano pomoću biljega projekcijskih neurona. Analiza ranog razvoja cingularne moždane kore važna je radi njezine uloge u stvaranju ranih neuralnih krugova uključenih u ponašanje i emocije. Nadalje, poznavanje regionalnog razvoja bitno je za razumijevanje arealne diferencijacije i kasnije funkcionalne specifikacije moždane kore što je preduvjet za razumijevanje neurorazvojnih poremećaja.In the doctoral thesis, regional cytoarchitectonic differences between the frontal, parietal, occipital, and cingulate cortex were analyzed in the the subplate formation phase (13 to 15 PCW). Immunohistochemical methods were used on prenatal postmortem human brain tissue to analyze the molecular markers` expression pattern dynamics. The results showed that regional differences between isocortical regions of the human fetal cortex become visible early during prenatal development, and are most prominent during the SP formation period. The SP formation process is shown with the SP neuron marker TBR1. Additionally, we showed that the SP formation pattern is an important criterion for differentiating the dorsal isocortical and ventral mesocortical cingulate cortex in the early fetal period. Furthermore, the early mesocortical cingulate cortex is characterized by the widening of the MZ and the narrowing of the CP and SVZ. One of the main characteristics of the mesocortical part of the cingulate cortex is an incomplete SP expansion, and this is shown in the results using projection neuron markers. The early cingulate cortex development analysis is important because of its involvement in the formation of early neural circuits involved in behavior and emotions. Furthermore, analysis of regional differences is essential for understanding areal differentiation and later functional specification of the cerebral cortex, which is a prerequisite for understanding diverse neurodevelopmental disorders

The role of bradykinin receptor type 2 in the development of brain and retinal ischemic lesion

Usprkos velikom broju kliničkih i pretkliničkih dokaza o korelaciji ishemije mozga i mrežnice, malo je pažnje posvećeno zajedničkim patološkim mehanizmima razvoja ishemijske ozljede istih. Cilj ovog istraživanja bio je longitudinalnim in vivo praćenjem razjasniti ulogu bradikininskog receptora tipa 2 u razvoju ishemijske ozljede mozga i mrežnice te odrediti primjenjivost in vivo snimanja mrežnične vaskulature za određivanje cerebrovaskularnih promjena u mišjem modelu moždanog udara. Usporedbom Koizumi i Longa zahvata okluzije srednje moždane arterije i dugotrajnim praćenjem statusa cerebralne vaskularne perfuzije magnetskom rezonancijom pokazano je da kod Koizumi zahvata ishemiju prati kronična hipoperfuzija i ishemija mozga i mrežnice, za razliku od Longa zahvata, u kojem nakon ishemije nastupa potpuna reperfuzija koja rezultira izraženijom cerebralnom atrofijom bez znakova oštećenja mrežnice. Usporedbom miševa s onemogućenim genom za bradikininski receptor tipa 2 te njihovih kontrola nakon Koizumi zahvata, dugotrajnim praćenjem razvoja ishemije magnetskom rezonancijom, fotografijom i fluoresceinskom angiografijom očne pozadine, testovima ponašanja i mjerenjem vaskularne propusnosti pokazano je da nedostatak bradikininskog receptora tipa 2 u akutnoj fazi ishemije smanjuje oštećenje i propusnost cerebralne vaskulature, a kronično povećava atrofiju moždanog tkiva i funkcionalni deficit. U mrežnici je pokazano slično djelovanje, odnosno povećanje vaskularne propusnosti u subakutnoj fazi, te povećanje ishemijskog oštećenja u kroničnoj fazi ishemije.Despite ample clinical and preclinical evidence correlating cerebral and retinal ischemia, not enough attention has been paid to common pathological mechanisms of ischemic lesion development in these tissues. This research aimed to clarify the role of bradykinin receptor type 2 in the development of brain and retinal ischemia and to examine the applicability of in vivo retinal vascular imaging to determine cerebrovascular changes in a murine model of stroke. Comparing the Koizumi and Longa middle cerebral artery occlusion procedures by long-term cerebrovascular perfusion monitoring with magnetic resonance imaging showed that ischemia was followed by chronic hypoperfusion in both brain and retina in the Koizumi model. In contrast, complete reperfusion occurred in the Longa model, resulting in greater cerebral atrophy without retinal damage. By comparing bradykinin receptor type 2 knock-out mice and their controls after the Koizumi procedure using long-term monitoring with magnetic resonance, fundus photography, fluorescein angiography, behavioral tests and vascular permeability measurements, bradykinin receptor type 2 deficiency has been shown to reduce acute cerebral vascular permeability, while increasing cerebral atrophy and functional deficit in the chronic phase. Retinal findings showed a similar effect, increased subacute vascular permeability and increased ischemic damage in the chronic phase of ischemia

Application and effects of gene modifications mediated by a lentviral vector in the mouse brain

Unos genetskog materijala i genskih preinaka važan je pristup za proučavanje i liječenje genetskih i drugih bolesti. Lentivirusni vektori ističu se kao dominatan sustav dostave u ciljane stanične linije središnjeg živčanog sustava. Glavni cilj ovog istraživanja bio je utvrditi transdukciju neurona i astrocita za njih specifično dizajniranim lentivirusnim vektorom, procjeniti uzrokuje li dostavljen sustav shRNA utišavanje gena Spry2 te utvrditi učinak utišavanja gena Spry2 nakon izazivanja ishemijskog moždanog udara u mišjem modelu. Potvrđena je uspješna transdukcija stanica lentivirusnim vektorom te je analizom kolokalizacije pokazano da lentivirusni vektori s ovojnicom VSV-G transduciraju neurone dok Tet regulirani lentivirusni vektori sa specifičnim promotorom za astrocite i ovojnicom Mokola transduciraju astrocite. Procjenom aktivacije mikroglije i receptora TLR2 pokazano je da unos lentivirusnog vektora uzrokuje kratkotrajnu aktivaciju upalnog odgovora koja je ograničena na područje unosa. Analizom genske ekspresije potvrđeno je utišavanje gena Spry2 u injektiranim regijama mišjeg mozga. Međutim, njegova smanjena ekspresija nije pridonijela značajnom smanjenju volumena ishemijske lezije i funkcionalnom oporavku nakon ishemijskog moždanog udara. Ovo istraživanje potvrdilo je da su primjenjene strategije dizajna lentivirusnih vektora učinkovit sustav dostave koji omogućava ograničenu transdukciju stanica od interesa i uspješno utišavanje gena Spry2.The delivery of genetic material or gene modification is an important approach for the study and treatment of genetic and other diseases. Lentiviral vectors are a dominant delivery system to target cell lines of the central nervous system. The aim of this study was to determine the transduction of target cells with a specifically designed lentiviral vector for the neuron and astrocyte transduction, to assess whether the delivered shRNA system causes silencing of Spry2, and to examine the effect of Spry2 silencing after ischemic stroke induction in a mouse model. Successful cell transduction with the lentiviral vector was confirmed, and colocalization analysis confirmed that VSV-G pseudotyped lentiviral vectors transduce neurons, while Tet-regulated Mokola pseudotyped lentiviral vector with astrocyte-specific promoter transduce astrocytes. Furthermore, lentiviral vector causes a short-term activation of the inflammatory response, which was restricted near the area of injection site. Silencing of the Spry2 was confirmed in the injected regions of the mouse brain. However, downregulation of Spry2 did not reduced the volume of ischemic lesion and contribute to the functional recovery after ischemia. This study confirmed that the applied design strategies of lentiviral vector are an efficient delivery system that allows restricted transduction of cells of interest and successful Spry2 silencing

Epigenetic status and expression of SALL4 in normal and impaired testicular development

Uloga gena SALL4, koji kontrolira razvoj spermatogonija, nije dovoljno istražena u razvoju sjemenika sisavaca, a u neplodnosti čovjeka je potpuno neistražena. Cilj disertacije bio je analizirati izražaj Sall4/SALL4 u razvoju sjemenika štakora in vivo i in vitro te u ljudskim sjemenicima s različitim dijagnozama neopstruktivne azoospermije (NOA) in vivo, kao i njegov metilacijski status u in vivo uzorcima. Pirosekvenciranjem je pokazano da je SALL4/Sall4 uvijek hipometiliran u sjemeniku. qRT-PCR-om je pokazan značajno veći izražaj Sall4 mRNA u fetalnom štakorskom sjemeniku nego u postnatalnom; a u ljudskim sjemenicima sa sindromom samo Sertolijevih stanica (SCOS) SALL4 mRNA je snižena u odnosu na hipospermatogenezu (HS) i zastoj u sazrijevanju (MA). Osim u spermatogonijama, poboljšanim IHC i IF protokolima je po prvi put pokazan SALL4 u XY tjelešcu primarnih spermatocita. Također, pokazan je pojačan izražaj SALL4 u pre-pahitenskim spermatocitama i citoplazmi intersticijskih stanica u neplodnom sjemeniku u odnosu na zdravi i HS sjemenik čovjeka. Nadalje, uspostavljen je novi 3D in vitro sustav za uzgoj tkiva sjemenika štakora, a primjena siRNA snizila je izražaj Sall4 na mRNA (za 42 %) i proteinskoj razini uzrokujući smanjeni rast i narušenu histologiju. Ovi rezultati ukazuju na SALL4 kao novi potencijalni biljeg u dijagnostici NOA te podrobnije rasvjetljavaju njegovu ulogu u procesu spermatogeneze.The role of the SALL4 gene which controls the development of spermatogonia, has been insufficiently investigated in the development of the mammalian testis, and is completely unexplored in human infertility. The doctoral thesis aimed to analyze the expression of Sall4/SALL4 in the rat testis development in vivo and in vitro as well as in the human testis with a different diagnosis of nonobstructive azoospermia (NOA) and its DNA methylation status. The pyrosequencing showed that SALL4/Sall4 is always hypomethylated in the testis. The qRT-PCR has shown significantly higher expression of the Sall4 mRNA in fetal rat testis than in postnatal; while SALL4 mRNA was lower in human testis with Sertoli cell only syndrome (SCOS) than in hypospermatogenesis (HS) and maturation arrest (MA). Except in spermatogonia, improved IHC and IF protocols showed the expression of the SALL4 protein in the XY body of primary spermatocytes of human testes. Also, increased expression of SALL4 in pre-pachytene spermatocytes and cytoplasm of interstitial cells in infertile compared to healthy human testis was demonstrated. Furthermore, an original 3D in vitro system for the cultivation of rat testis tissue was established, and the application of siRNA decreased Sall4 at the mRNA (by 42 %) and protein levels, causing reduced growth and impaired histology. These results indicate SALL4 as a new potential marker in the diagnosis of NOA and shed more light on its role in the process of spermatogenesis

Proton pump inhibitors use prior to COVID-19 hospitalization is associated with higher C lostridioides difficile infection rate

Background: There are uncertainties regarding associations of prior proton pump inhibitor (PPI) use with susceptibility for COVID-19 and risks associated with SARS-CoV-2 infection. We aimed to evaluate the associations of prior PPI use with outcomes in hospitalized patients with COVID-19. Research design and methods: We have retrospectively evaluated a total of 5959 consecutively hospitalized patients with COVID-19 from a tertiary-level institution in the period 3/2020-6/2021. Associations of prior PPI use with outcomes of in-hospital mortality, mechanical ventilation, intensive care unit stay, venous thromboembolism, arterial thrombosis, major bleeding, bacteremia, and Clostridioides difficile infection (C. diff.) were evaluated in entire and case-matched cohorts. Results: Among 5959 evaluated patients, there were 1967 (33%) PPI users. In an entire cohort, prior PPI use was associated with higher in-hospital mortality and higher occurrence of C. diff. Association of prior PPI use with mortality diminished, whereas association with C. diff. persisted after multivariable adjustments. In a matched cohort, prior PPI use was associated only with higher risk of C. diff. but not other outcomes in line with multivariable analysis. Conclusions: Although prior PPI use might not have a significant impact on clinical course and mortality of SARS-CoV-2 infection, it may predispose patients to the development of complications like higher occurrence of C. diff. and thus substantially impact the course of treatment

Beyond neuromuscular activity: botulinum toxin type A exerts direct central action on spinal control of movement

Overt muscle activity and impaired spinal locomotor control hampering coordinated movement is a hallmark of spasticity and movement disorders like dystonia. While botulinum toxin A (BoNT-A) standard therapy alleviates mentioned symptoms presumably due to its peripheral neuromuscular actions alone, the aim of present study was to examine for the first time the toxin's trans-synaptic activity within central circuits that govern the skilled movement. The rat hindlimb motor pools were targeted by BoNT-A intrasciatic bilateral injection (2 U per nerve), while its trans-synaptic action on premotor inputs was blocked by intrathecal BoNT-A-neutralising antitoxin (5 i.u.). Effects of BoNT-A on coordinated and high intensity motor tasks (rotarod, beamwalk swimming), and localised muscle weakness (digit abduction, gait ability) were followed until their substantial recovery by day 56 post BoNT-A. Later, (day 62-77) the BoNT-A effects were examined in unilateral calf muscle spasm evoked by tetanus toxin (TeNT, 1.5 ng). In comparison to peripheral effect alone, combined peripheral and central trans-synaptic BoNT-A action induced a more prominent and longer impairment of different motor tasks, as well as the localised muscle weakness. After near-complete recovery of motor functions, the BoNT-A maintained the ability to reduce the experimental calf spasm evoked by tetanus toxin (TeNT 1.5 ng, day 62) without altering the monosynaptic reflex excitability. These results indicate that, in addition to muscle terminals, BoNT-A-mediated control of hyperactive muscle activity in movement disorders and spasticity may involve the spinal premotor inputs and central circuits participating in the skilled locomotor performance

Estimated plasma volume status in COVID-19 patients and its relation to comorbidities and clinical outcomes

Blood plasma is a large reservoir of circulating mediators of inflammation and its expansion has been associated with unfavorable outcomes in patients with inflammatory and cardiovascular diseases. The aim of this study was to determine clinical and prognostic value of estimated plasma volume status (ePVS) in hospitalized patients with COVID-19. We retrospectively investigated 5871 consecutive COVID-19 patient hospitalized in our tertiary-level institution in period 3/2020-6/2021. ePVS was determined using the Strauss-derived Duarte formula and was correlated with clinical characteristics and unwanted outcomes. Median ePVS was 4.77 dl/g with interquartile range 4.11-5.74. Higher ePVS was significantly associated with older age, female sex, higher comorbidity burden, worse functional status, less severe COVID-19 clinical presentation with lower severity and longer duration of symptoms, but more pronounced inflammatory profile with higher C-reactive protein, interleukin-6 and D-dimer levels (P < 0.05 for all analyses). In the multivariate regression analysis U shaped relationship of ePVS with mortality was revealed, present independently of age, sex, COVID-19 severity and comorbidity burden. In addition, higher ePVS was independently associated with higher tendency for mechanical ventilation, intensive care unit treatment, venous thromboembolism, major bleeding and bacteriemia and lower ePVS was independently associated with tendency for arterial thrombotic events. Higher ePVS, indicative of plasma volume expansion and inflammatory cytokine accumulation, may predispose respiratory deterioration and venous thromboembolism, despite less severe initial clinical presentation. Lower ePVS, indicative of hemoconcentration, may predispose arterial thrombotic events. Both may be associated with higher mortality in hospitalized COVID-19 patients

The effect of inflammation on apoptotic cell death after ischemic lesion of the mouse brain

Postishemijska upala značajan je faktor u razvoju ozljede nakon moždanog udara, a TLR2 receptor jedan je od njezinih glavnih medijatora. Glavni cilj ovog doktorata bio je odrediti utjecaj upale posredovane TLR2 receptorom na apoptozu u ishemijskom okolišu. Utjecaj TLR2 receptora istražen je na modelu ishemijskog oštećenja mišjeg mozga u životinja s normalnim (CAG-luc) i onih s onemogućenim Tlr2 genom (CAG-luc-Tlr2-/-). Oba soja ubikvitarno su izražavala luciferazni transgen. Razmjer apoptoze pratio se bioluminiscencijom zatočenim Z-DEVD-aminoluciferinom koji se cijepa i postaje bioluminiscentno aktivan nakon interakcije s pocijepanom kaspazom-3. Ishemijska ozljeda bila je uzrokovana okluzijom srednje moždane arterije, a njena progresija praćen je magnetskom rezonancom. Stanična smrt je potvrđena metodama imunofluorescencije pocijepanom kaspazom-3, protočne citometrije aneksinom-V i TUNEL esejom. Funkcionalno oštećenje izmjereno je testom neurološkog deficita. Zatočeni Z-DEVD-aminoluciferin validiran je i procijenjen kao adekvatan supstrat za praćenje apoptoze. Longitudinalno in vivo mjerenje apoptoze izazovno je te zahtijeva kvalitetnu validaciju i normalizaciju. Nije bilo ukupne razlike u apoptozi između sojeva, ali u nedostatku TLR2 receptora postojao je izraženiji mehanizam nekroze. U akutnom periodu, CAG-luc- Tlr2-/- miševi bolje su preživljavali od CAG-luc miševa, dok u kroničnom periodu nije bilo razlike u preživljenju. Ipak, magnetna rezonanca je pokazala kako su CAG-luc-Tlr2-/- miševi su u kroničnom periodu izgubili više tkiva ipsilateralne hemisfere od CAG-luc miševa. Stoga, iako smanjena upala u akutnom periodu može biti protektivna, dugoročno narušava oporavak.Postischemic inflammation is a significant contributor to ischemic injury development. The TLR2 receptor is one of its main mediators. The main aim of this thesis was to determine the effect of TLR2-mediated inflammation on apoptotic cell death in the ischemic environment. The effect of TLR2 was investigated on the ischemic injury model in animals with normal Tlr2 (CAG-luc) and those with knock-out Tlr2 gene (CAG-luc-Tlr2-/-). Both strains expressed the firefly luciferase transgene ubiquitously. The scope of apoptosis was determined by the utilization of bioluminescence imaging with caged Z-DEVD-aminoluciferin, which becomes available for the bioluminescence reaction after cleavage with activated caspase-3. Middle cerebral artery occlusion was performed to produce the ischemic injury. Its progression was followed with magnetic resonance imaging. Cell death was further confirmed using immunofluorescence with activated caspase-3, flow cytometry with annexin-V and the TUNEL assay. Functional outcomes were assessed using a neurological deficit test. Caged Z-DEVD-aminoluciferin was validated and assessed as an adequate tool for monitoring apoptosis. Longitudinal in vivo measurement of apoptosis is challenging and requires thorough validation and normalization. No total difference in apoptosis between the used strains was found. However, in the absence of the TLR2 receptor, a more pronounced mechanism of necrosis arose. In the acute period, CAG-luc-Tlr2-/- mice had better survival than CAG-luc mice. In the chronic period, there was no difference in survival between the strains. However, magnetic resonance imaging showed that CAG-luc-Tlr2-/- lost more ipsilateral hemisphere tissue than CAG-luc mice during the chronic period. Therefore, although lower levels of inflammation may be protective in the acute period post-stroke, long-term recovery is impaired