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The search for an effective and non-toxic radioprotector is ongoing. We tested a novel, natural aminothiol-based radioprotector, GL2011, that was applied 30 min, 3 h or 6 h after the exposure of male albino Wistar rats to a 6.7 Gy mild dose of gamma radiation. The molecular signatures of radioprotection were investigated with Raman microspectroscopy of brainstem tissue samples. Morphological changes and activation of astrocytes and microglia were assessed by immunohistochemistry. Global markers of neuroinflammation were followed by ELISA to monitor blood plasma levels of proinflammatory (IL-6 and TNF-α) and anti-inflammatory (IL-10) cytokines. A thirty-day follow-up determined survival of unprotected animals 37.5%. A survival increase was observed after radioprotection (75%, irrespective of the time of application). Raman spectra revealed a slightly deleterious effect of radiation on nucleic acids in surviving animals that was mitigated with the radioprotector, as GL2011 preserved the morphology of both astrocytes and microglia, with reduced microglial infiltration. Cytokine assessment revealed an immunomodulatory effect of the novel radioprotector. The overall results point out the positive effects of a single dose of GL2011 applied at different times. The molecular and cellular changes in the brainstem indicate that the radioprotector applied after radiation conferred better protection, which underlines its translation to cure radiation accidents.

Amiotrofična lateralna skleroza je smrtonosno neurodegenerativno oboljenje koje pogađa motorne i kognitivne domene centralnog nervnog sistema (CNS). Mutacije u genu za Cu, Zn-supeoksid dismutazu (SOD1) uzrokuju 20% familijalnih slučajeva ALS i izazivaju stvaranje unutarćelijskih SOD1 agregata i smanjeno vezivanje Cu i Zn, što dovodi do patoloških unutarćelijskih promena i neurodegeneracije. Istraživanja na post mortem tkivu ALS pacijenata i životinjskim modelima bolesti su istakla glijalnu aktivaciju, poremećenu homeostazu prelaznih metala, oštećenje krvno-moždane barijere (KMB) i povećanu proizvodnju reaktivnih kiseoničnih vrsta kao glavne patološke faktore u bolesti. Cilj ove studije je bio da se ispitaju navedene patološke promene u motornim (moždano stablo) i ne-motornim (hipokampus) centrima u mozgu transgenog hSOD1 G93A pacovskog modela ALS, u presimptomatskom i simptomatskom stadijumu bolesti, kako bi se detaljnije proučili ovi patofiziološki mehanizmi i identifikovale rane promene u bolesti. Prisustvo i vremenski sled aktivacije i morfoloških promena mikroglije i astrocita, kao i akumulacija i distribucija SOD1 proteina u ovim ćelijama su ispitani naprednim 3D modelovanjem i analizom kolokalizacije na slikama dobijenim konfokalnom laserskom skenirajućom mikroskopijom. Rezultati su pokazali glijalnu proliferaciju i progresivno nakupljanje SOD1 u tkivu u oba ispitivana moždana regiona hSOD1 G93A pacova, koji počinju već u presimptomatskoj fazi. U moždanom stablu ovih životinja aktivacija astrocita je bila prisutna pre početka simptoma bolesti, i bila je praćena aktivacijom mikroglije. Takođe, u moždanom stablu astrociti su pokazali progresivnu akumulaciju SOD1 proteina u ćelijskom telu i nastavcima, dok su nivoi SOD1 u mikrogliji bili smanjeni a njena distribucija ograničena na distalne ćelijske nastavke. U hipokampusu hSOD1 G93A pacova astrociti su pokazali isti reaktivni profil kao onaj uočen u moždanom stablu, dok je morfologija mikroglije bila nepromenjena, Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder affecting both the motor and cognitive domains of the central nervous system (CNS). Mutations in the Cu,Zn-superoxide dismutase (SOD1) cause 20% of familial ALS and provoke formation of intracellular SOD1 aggregates and copper and zinc unbinding, leading to pathological intracellular changes and neurodegeneration. Post mortem and animal model studies indicate glial activation, perturbed transition metal homeostasis, blood-brain barrier (BBB) disruption and elevated production of reactive oxygen species as major contributors to disease pathology. The aim of this study was to investigate the above mentioned pathological changes in motor (brainstem) and non-motor (hippocampus) brain centers of a transgenic hSOD1 G93A rat model of ALS, in presymptomatic and symptomatic stages of the disease, in order to further clarify these pathophysiological mechanisms and to identify early changes in the disease. The presence and the timeline of activation and morphological changes of microglia and astrocytes, as well as their intracellular accumulation and distribution of SOD1 were investigated by advanced 3D modeling and colocalization analysis of images obtained by confocal laser scanning microscopy. Results revealed glial proliferation and progressive tissue accumulation of SOD1 in both brain regions of hSOD1 G93A rats starting already at the presymptomatic stage. In the brainstem of these animals astrocytes activation occurred before disease symptoms onset, and was followed by activation of microglia. Additionally, in the brainstem astrocytes demonstrated progressive SOD1 accumulation in the cell body and processes, while microglial SOD1 levels were reduced and its distribution limited to distal cell processes. In the hippocampus of hSOD1 G93A rats astrocytes exhibited an identical reactive profile as the one observed in the brainstem, while microglial morphology was unchanged. In this brain region both gl

Tenascin-C (TnC) je glikoprotein prisutan u vanćelijskom matriksu (VĆM) različitih tkiva kičmenjaka tokom razvića, gde je uključen u regulaciju ćelijskog rasta, migracije i adhezije preko aktivacije različitih unutarćelijskih signalnih puteva. Ekspresija TnC je značajno smanjenja u adultnom organizmu, međutim ostaje prisutna samo u određenim perifernim tkivima, kao i u delovima centralnog nervnog sistema koji zadržavaju visok nivo plastičnosti, kao što je mali mozak, gde je uočena njegova uloga u modulaciji sinaptičkih funkcija. Obogaćena sredina (OS) oblikuje mnoge aspekte ponašanja i stoga može da posluži kao pristup za proučavanje neuronalne plastičnosti u adultnom organizmu. Izlaganje OS dovodi do brojnih promena na molekularnom i ćelijskom nivou u različitim regionima mozga uključujući mali mozak. Cilj ove studije je bio da se ispita uloga TnC u modulaciji strukturne plastičnosti malog mozga indukovane OS praćenjem distribucije perineuronalnih mreža (PNM), promene u veličini i gustini ekscitatornih i inhibitornih presinaptičkih završetaka, kao i aktivnosti glavnih enzima koji vrše razgradnju VĆM, MMP-2 i MMP-9. Takođe, ova studija je imala za cilj da ispita kako nedostatak TnC interaguje sa spoljašnjom sredinom u oblikovanju različitih domena ponašanja povezanih sa funkcijom malog mozga. U te svrhe, miševi sa nedostatkom TnC (TnC-/-) ili MMP-9 (MMP-9-/-) i odgovarajući kontrolni miševi starosti 3 nedelje su izlagani standardnim uslovima gajenja (SS) ili OS 4 ili 8 nedelja. Ova studija je pokazala da izlaganje OS u toku 8 nedelja dovodi do smanjenja intenziteta bojenja na PNM, kao i smanjenja u veličini GABAergičkih i povećanja broja i veličine glutamatergičkih sinaptičkih završetaka u kontrolnim miševima. Nasuprot tome, TnC-/- miševi su pokazali smanjen intenzitet bojenja na PNM u poređenju sa kontrolnim životinjama gajenim u SS, dok njihovo izlaganje OS nije dovelo da smanjenja, već do blagog povećanja intenziteta PNM..., Tenascin-C (TnC) is a glycoprotein present in the extracellular matrix (ECM) of a variety of vertebrate tissues during development, where it plays multiple roles in cell growth, migration and adhesion by activating diverse intracellular signaling pathways. Expression of TnC is markedly downregulated in the adulthood, persisting in a few peripheral structures and in central nervous system areas known to retain high degree of plasticity, such as the cerebellum, where it is involved in the modulation of synaptic functions. Enriched environment (EE) shapes many aspects of behavior and may, therefore, serve as a paradigm to study neuronal plasticity in the adult. Exposure to EE leads to numerous changes at the molecular and cellular levels, which target various brain regions including the cerebellum. The aim of present study was to examine the role of TnC in the modulation of cerebellar structural plasticity induced by the exposure to EE by following the appearance of perineuronal nets (PNN), changes in size and density of excitatory and inhibitory synaptic terminals, and the activity of major ECM degrading enzymes, MMP-2 and MMP-9. Furthermore, the present study aimed to examine how TnC deficiency interacts with the environment in shaping different behavioral domains associated with the cerebellum. To this end, 3-week-old mice lacking TnC (TnC-/-) or MMP-9 (MMP-9-/-) and corresponding wild-type mice were exposed to standard conditions (SC) or an enriched environment (EE) for 4 or 8 weeks. The present study shows that 8 weeks of exposure to EE leads to reduced staining of PNN, reduction in the size of GABAergic and increase in the number and size of glutamatergic synaptic terminals in wild-type mice. Conversely, TnC-/- mice showed reduced staining of PNNs compared to wild-type mice maintained under standard conditions, while exposure to EE did not further reduce, but even slightly increased PNN staining...

Aim To investigate the survival of laboratory rats after irradiation and to study the cellularity of their bone marrow and the multipotential mesenchymal stem cells (BM-MSCs) in groups treated with or without a new thiol-based radioprotector (GM2011) Methods Animals were irradiated by a Cobalt gamma source at 6.7 Gy. Treated animals were given i.p. GM2011 30 minutes before and 3 and 7 hours after irradiation. Controls consisted of sham irradiated animals without treatment and animals treated without irradiation. After 30 days post-irradiation, animals were sacrificed and bone marrow cells were prepared from isolated femurs. A colony forming unit-fibroblast (CFU-F) assay was performed to obtain the number of BM-MSCs. Results In the treated group, 87% of animals survived, compared to only 30% in the non-treated irradiated group. Irradiation induced significant changes in the bone marrow of the treated rats (total bone marrow cellularity was reduced by similar to 60% - from 63 to 28 cells x10(6)/femur and the frequency of the CFU-F per femur by similar to 70% - from 357 to 97), however GL2011 almost completely prevented the suppressive effect observed on day 30 post-irradiation (71 cells x 10(6)/femur and 230 CFU-F/femur). Conclusion Although the irradiation dosage was relatively high, GL2011 acted as a very effective new radioprotector. The recovery of the BN-MSCs and their counts support the effectiveness of the studied radioprotector.

GL2011 is a naturally occurring thiol compound and a series of thiol compounds have been proposed as radioprotectors. Radioprotective efficacy of a triple intraperitoneal dose of GL2011 of 100 mg/kg body weight of Wistar rats, 30 min prior to and 3 and 6 h following irradiation (6.7 Gy) was evaluated. Four groups of animals were used, vehicle-treated non-irradiated (VN), GL2011-treated and irradiated (GI), GL2011-treated and non-irradiated (GN) and vehicle-treated and irradiated (VI) (n = 30 per group). The radioprotective efficacy of GL2011 was determined by measuring 28-day survival and intestinal crypt cell survival. Neuroprotection in terms of behaviour was evaluated using the behavioural observational battery, open field test and elevated plus maze paradigm. An RNA microarray was carried out in order to show differences at the RNA level between VI and VN groups. Brain protein changes were identified using a gel-based proteomics method and major brain receptor complex levels were determined by blue-native gels followed by immunoblotting. 28-Day survival rate in VI was 30 %, in GI survival was 93 %, survival of VN and GN was 100 %. Jejunal crypt cell survival was significantly enhanced in GI. Protein-level changes of peroxiredoxin-5, Mn-superoxide dismutase 2, voltage-dependent anion-selective channel protein 1, septin 5 and dopamine D2 receptor complex levels were paralleling radiation damage and protection. Taken together, the findings demonstrate that GL2011 improves survival rates and jejunal crypt survival, provides partial neuroprotection at the behavioural level and modulates proteins known to be involved in protection against oxidative stress-mediated cell damage.

Aim To investigate the survival of laboratory rats after irradiation and to study the cellularity of their bone marrow and the multipotential mesenchymal stem cells (BM-MSCs) in groups treated with or without a new thiol-based radioprotector (GM2011) Methods Animals were irradiated by a Cobalt gamma source at 6.7 Gy. Treated animals were given i.p. GM2011 30 minutes before and 3 and 7 hours after irradiation. Controls consisted of sham irradiated animals without treatment and animals treated without irradiation. After 30 days post-irradiation, animals were sacrificed and bone marrow cells were prepared from isolated femurs. A colony forming unit-fibroblast (CFU-F) assay was performed to obtain the number of BM-MSCs. Results In the treated group, 87% of animals survived, compared to only 30% in the non-treated irradiated group. Irradiation induced significant changes in the bone marrow of the treated rats (total bone marrow cellularity was reduced by similar to 60% - from 63 to 28 cells x10(6)/femur and the frequency of the CFU-F per femur by similar to 70% - from 357 to 97), however GL2011 almost completely prevented the suppressive effect observed on day 30 post-irradiation (71 cells x 10(6)/femur and 230 CFU-F/femur). Conclusion Although the irradiation dosage was relatively high, GL2011 acted as a very effective new radioprotector. The recovery of the BN-MSCs and their counts support the effectiveness of the studied radioprotector.

Aim To investigate the survival of laboratory rats after irradiation and to study the cellularity of their bone marrow and the multipotential mesenchymal stem cells (BM-MSCs) in groups treated with or without a new thiol-based radioprotector (GM2011) Methods Animals were irradiated by a Cobalt gamma source at 6.7 Gy. Treated animals were given i.p. GM2011 30 minutes before and 3 and 7 hours after irradiation. Controls consisted of sham irradiated animals without treatment and animals treated without irradiation. After 30 days post-irradiation, animals were sacrificed and bone marrow cells were prepared from isolated femurs. A colony forming unit-fibroblast (CFU-F) assay was performed to obtain the number of BM-MSCs. Results In the treated group, 87% of animals survived, compared to only 30% in the non-treated irradiated group. Irradiation induced significant changes in the bone marrow of the treated rats (total bone marrow cellularity was reduced by similar to 60% - from 63 to 28 cells x10(6)/femur and the frequency of the CFU-F per femur by similar to 70% - from 357 to 97), however GL2011 almost completely prevented the suppressive effect observed on day 30 post-irradiation (71 cells x 10(6)/femur and 230 CFU-F/femur). Conclusion Although the irradiation dosage was relatively high, GL2011 acted as a very effective new radioprotector. The recovery of the BN-MSCs and their counts support the effectiveness of the studied radioprotector.

Електричне промене представљају начин комуникације између неурона и чине основу комплексних феномена у понашању као што су перцепција, сензорно- моторна интеграција, учење и памћење. Активност неурона представља колекцију различитих електричних догађаја који варирају како у трајању, тако и у простирању. Праћење електричне активности неурона у свој просторној и временској комплексности подразумева развијање метода које поседују високу темпоралну резолуцију и висок однос сигнала према шуму. Идеални метод би био у стању да прати подпражне и надпражне промене мембранског потенцијала на нивоу појединачне ћелије и са резолуцијом мањом од секунде. Примена оптичких метода за снимање електричне активности мозга представља мање деструктивну алтернативу традиционалним електрофизиолошким методама базираним на примени електрода. Oптичка снимања нуде високу просторну резолуцију омогућујући симултана снимања са различитих локација у оквиру видног поља, од нивоа појединачних ћелија и делова ћелије до нивоа нервних кола која се састоје од стотина, ако не и хиљада неурона. Током вишедеценијског развоја, почевши од раних експеримената базираних на снимању урођених сигнала који се јављају при нервној активност па све до развоја бројних волтажних боја мале молекулске масе, оптичка снимања су омогућила стицање значајнх сазнања о функционисању мозга и неуралној обради података. Ипак, и у најразвијенијој верзији овим методама недостаје просторна/временска резолуција и ћелијска специфичност која би омогућила опширне студије у будним и активним животињама. У овом раду је описан развој синтетичког протеинског конструкта који експримиран у сисарским неуронима показује промену интензитета флуоресценције у одговору на промене у мембранском потенцијалу. Развој оваквих индикатора је део великих напора који се већ дуже време улажу у области оптичког снимања а који су окренути развоју генетички кодираних волтажних индикатора. Главна предност развоја оваквих проба у односу на доступне волтажне боје м, Electrical events are the mode of communication among neurons that underly all complex behaviors including perception, sensory-motor integration, learning and memory. Neurons exhibit a collection of different transient electrical events that vary in both spatial and temporal domains. To be able to analyze these events we need methods with both high temporal resolution and large signal to noise ratio. The ideal method would be able to detect both subthreshold and suprathreshold level changes in membrane potential, in single cells (or subcellular domains) at submillisecond resolution. Use of light to record brain electrical activity represents a less invasive alternative to classical electrode-based methods and can provide high spatial resolution. Optical methods allow simultaneous recordings from different locations within a field of view, from the level of single cell and cell compartments to the level of neuronal circuits comprising of hundreds, if not thousands neurons simultaneously. Over the last couple of decades, with early experiments using intrinsic signals that arise with neuronal activity all the way to development of numerous small molecule synthetic dyes optical methods provided unique information on brain function and neuronal information processing. However, even in its most advanced form these methods lack the spatial/temporal sensitivity and cell specificity that would allow comprehensive studies of neuronal function. The present studies describe the development of synthetic protein constructs which when expressed in mammalian neurons produce change in fluorescence intensity in response to changes in membrane potential. Development of the indicators presented here is part of a greater efforts in the field of optical imaging to produce geneticallyencoded voltage indicator (GEVI). These probes would allow cell targeted expression and consequently cell type specific recordings of neuronal activity. In the last two decades efforts to produce such probes