The effect of vitamin B complexes and ribavirin on response of neuronal and glial cells and recovery of motor function after injury to the rat brain

Traumatska povreda mozga (TPM) predstavlja veliki zdravstveni problem savremenog društva jer je vodeći uzrok smrti i invaliditeta kod mlađih osoba. Uprkos znatnim istraživačkim i kliničkim naporima, još uvek ne postoji ni jedan dovoljno uspešan terapijski pristup koji utiče na sve patofiziološke procese koji prate moždanu traumu, odnosno, koji može ukloniti sve njene posledice. Stoga se uspešan tretman u borbi protiv TPM danas pokušava pronaći kombinacijom različitih terapijskih tretmana. Poznato je da je oporavak izgubljene funkcije nakon povrede mozga odraslih veoma spor i nepotpun usled nepovratnog uništenja nervnih ćelija i ograničene mogućnosti remodelovanja postojećih i formiranja novih neuronskih veza. Glavna prepreka za uspešniji oporavak posle oštećenja je nepermisivna sredina glijskog ožiljka koji se formira nakon povrede, a koji sprečava izrastanje novih nervnih vlakana i uspostavljanje novih neuronskih veza. Glijski ožiljak čine gusta mreža glijskih ćelija (astrocita i oligodendrocitnih prekursorskih ćelija) i nepropustan vanćelijski matriks (ECM). Cilj ove studije bio je da se ispita dejstvo kompleksa vitamina B (B1, B2, B3, B6 i B12) i ribavirina (RBV, purinskog nukleozidnog analoga) na oporavak motornih funkcija, posle jednostrane ablacije senzomotorne kore (ASK) prednjeg mozga pacova. Mužjacima Wistar soja, starim 2.5 meseca, urađena je ASK prema sledećim koordinatama: 2mm ispred, 4 mm iza bregme i 4 mm lateralno od središnje linije u dubinu do pojave bele mase mozga. Operisane životinje podeljene su, prema tretmanu nakon ASK, u 6 grupa: L grupa – tretirana fiziološkim rastvorom; LK1 grupa – tretirana kombinacijom vitamina B1 (33 mg/kg/dan), B6 (33 mg/kg/dan) i B12 (0,5 μg/kg/dan); LK2 grupa – tretirana kombinacijom vitamina B2 (7,5 mg/kg/dan) i B3 (500 mg/kg/dan); LK grupa – tretirana kombinacijom svih pet vitamina: B1, B2, B3, B6 i B12 u prethodno navedenim dozama; LR grupa – primala je RBV (30 mg/kg/dan); LKR grupa – tretirana kombinacijom svih 5 B vitamina i RBV u prethodno navedenim dozama. Prva injekcija B vitamina data je i.p. 15 minuta nakon ASK, a potom na svaka 24 sata tokom sledećih 14 dana, dok je RBV aplikovan od 3 dana nakon ASK, jednom dnevno do kraja eksperimenta. Uticaj primenjenih tretmana na oporavak nakon ASK praćen je testiranjem ponašanja (test prelaska preko grede - BW test), RT-PCR i Western blot analizom, kao i različitim imunohistohemijskim metodama...Traumatic brain injury (TBI) is a major health problem in modern society, being a leading cause of death and disability among young people. Despite huge basic research and clinical efforts an effective treatment for TBI has not been established yet, that will counteract all of pathophysiological processes accompanying brain trauma. Therefore, a best possible present treatment that would act on several secondary processes in TBI is based on a combination of different therapeutic approaches. A recovery of lost functions after brain injury is slow and incomplete in adults, due to the irreversible destruction of nerve cells and the limited ability of brain tissue for remodeling of existing and formation of new synaptic connections. The main obstacle for a successful recovery is existence of non-permissive glial scar, which emerges after a brain trauma preventing axonal sprouting and the establishment of new neural circuits. Glial scar consists of a dense network of glial cells (astrocytes and oligodendrocyte precursor cells) and dense extracellular matrix (ECM). The aim of this study was to explore the effects of vitamin B complex (B1, B2, B3, B6 and B12) and ribavirin (RBV, a purine nucleoside analogue) on the recovery of motor function after unilateral sensorimotor cortex ablation (SCA) of rat forebrain. Two and a half-month old male Wistar rats underwent SCA at the following coordinates: 2 mm before, 4 mm behind the bregma and 4 mm lateral to the midline, and to the depth of the white matter. After the surgery, rats were randomly divided according to treatment protocol into 6 groups: group L – treated with saline solution; LC1 group - treated with a combination of vitamin B1 (33 mg/kg/day), B6 (33 mg/kg/day) and B12 (0,5 mg/kg/day); LC2 group - treated with a combination of vitamin B2 (7.5 mg/kg/day) and B3 (500 mg/kg/day), LC group - treated with a combination of all five vitamins: B1, B2, B3, B6 and B12 in the above mentioned doses, LR group - received RBV (30 mg/kg/day); LCR group - treated with a combination of 5 B vitamins and RBV in the above doses. The first injection of vitamin B was given i.p. 15 minutes after the SCA, and then daily during next 14 days, while RBV was first administered 3 days after the SCA, once daily until the end of the experimental protocol. The effects of treatment on the recovery after SCA were followed by behavioral tests (beam walking test - BW test), while molecular changes were detected using RT-PCR, Western blot analysis and several immunohistochemical protocols..

Combined treatment with ribavirin and tiazofurin attenuates response of glial cells in experimental autoimmune encephalomyelitis

Experimental autoimmune encephalomyelitis (EAE) is an animal model of multiple sclerosis (MS), a human inflammatory and demyelinating disease. Microglia and astrocytes are glial cells of the central nervous system (CNS) that play a dual role in MS and EAE pathology. The aim of this study was to examine the effect of combined treatment with two nucleoside analogues, ribavirin and tiazofurin, on microglia and astrocytes in actively induced EAE. Therapeutic treatment with a combination of these two nucleoside analogues reduced disease severity, mononuclear cell infiltration and demyelination. The obtained histological results indicate that ribavirin and tiazofurin changed activated microglia into an inactive type and attenuated astrocyte reactivity at the end of the treatment period. Since reduction of reactive microgliosis and astrogliosis correlated with EAE suppression, the present study also suggests that the obtained beneficial effect of ribavirin and tiazofurin could be a consequence of their action inside as well as outside the CNS. [Acknowledgments. This work was supported by the Serbian Ministry of Education and Science, Project No: III41014.

Combined treatment with ribavirin and tiazofurin attenuates response of glial cells in experimental autoimmune encephalomyelitis

Experimental autoimmune encephalomyelitis (EAE) is an animal model of multiple sclerosis (MS), a human inflammatory and demyelinating disease. Microglia and astrocytes are glial cells of the central nervous system (CNS) that play a dual role in MS and EAE pathology. The aim of this study was to examine the effect of combined treatment with two nucleoside analogues, ribavirin and tiazofurin, on microglia and astrocytes in actively induced EAE. Therapeutic treatment with a combination of these two nucleoside analogues reduced disease severity, mononuclear cell infiltration and demyelination. The obtained histological results indicate that ribavirin and tiazofurin changed activated microglia into an inactive type and attenuated astrocyte reactivity at the end of the treatment period. Since reduction of reactive microgliosis and astrogliosis correlated with EAE suppression, the present study also suggests that the obtained beneficial effect of ribavirin and tiazofurin could be a consequence of their action inside as well as outside the CNS.Projekat ministarstva br. III4101

Pattern of chondroitin sulfate proteoglycan expression after ablation of the sensorimotor cortex of the neonatal and adult rat brain

The central nervous system has a limited capacity for self-repair after damage. However, the neonatal brain has agreater capacity for recovery than the adult brain. These differences in the regenerative capability depend on local environmental factors and the maturational stage of growing axons. Among molecules which have both growth-promoting and growth-inhibiting activities is the heterogeneous class of chondroitin sulfate proteoglycans (CSPGs). In this paper, we investigated the chondroitin-4 and chondroitin-6 sulfate proteoglycan expression profile after left sensorimotor cortex ablation of the neonatal and adult rat brain. Immunohistochemical analysis revealed that compared to the normal uninjured cortex, lesion provoked up regulation of CSPGs showing a different pattern of expression in the neonatal vs. the adult brain. Punctuate and membrane-bound labeling was predominate after neonatal lesion, where as heavy deposition of staining in the extracellular matrix was observed after adult lesion. Heavy deposition of CSPG immunoreactivity around the lesionsite in adult rats, in contrast to a less CSPG-rich environment in neonatal rats, indicated that enhancement of the recovery process after neonatal injury is due to amore permissive environment

Pattern of chondroitin sulfate proteoglycan expression after ablation of the sensorimotor cortex of the neonatal and adult rat brain

The central nervous system has a limited capacity for self-repair after damage. However, the neonatal brain has agreater capacity for recovery than the adult brain. These differences in the regenerative capability depend on local environmental factors and the maturational stage of growing axons. Among molecules which have both growth-promoting and growth-inhibiting activities is the heterogeneous class of chondroitin sulfate proteoglycans (CSPGs). In this paper, we investigated the chondroitin-4 and chondroitin-6 sulfate proteoglycan expression profile after left sensorimotor cortex ablation of the neonatal and adult rat brain. Immunohistochemical analysis revealed that compared to the normal uninjured cortex, lesion provoked up regulation of CSPGs showing a different pattern of expression in the neonatal vs. the adult brain. Punctuate and membrane-bound labeling was predominate after neonatal lesion, where as heavy deposition of staining in the extracellular matrix was observed after adult lesion. Heavy deposition of CSPG immunoreactivity around the lesionsite in adult rats, in contrast to a less CSPG-rich environment in neonatal rats, indicated that enhancement of the recovery process after neonatal injury is due to amore permissive environment.Centralni nervni sistem ima ograničen kapacitet za oporavak nakon povrede. Međutim, neonatalni mozak pokazuje veću sposobnost oporavka u odnosu na odrasle. Ove razlike zavise od lokalnih sredinskih faktora i stepena zrelosti aksona tokom izrastanja. U grupu molekula koji mogu da stimulišu ili inhibiraju rast aksona spada i heterogena klasa molekula označena kao hondroitin sulfatni proteoglikani (CSPG). U ovom radu ispitivanje profil ekspresije hondroitin-4 i hondroitin-6 sulfatnih proteoglikana nakonlezije leve senzomotorne kore neonatalnog i adultnog mozga pacova. Imunohistohemijska analiza pokazuje da u odnosu na normalni,nepovređeni korteks, lezija dovodi do povećanja ekspresije CSPG koji ima različiti obrazac promena u neonatalnom u odnosu na adultni mozak. Nakonlezije kod mladih, predominiraju tačkasta i membranski-vezana forma, dok kod odraslih lezija dovodi do nagomilavanja CSPG u ekstra ćelijskom matriksu. Prohodnija sredina u mozgu neonatalnih pacova koja je siromašnija CSPG, preduslov je boljeg procesa oporavka u odnosu na adulte, kod kojih se nakon povrede CSPG nagomilavaju oko mesta lezije.Projekat ministarstva br. 143005

Real-time PCR and immunocytochemical study of chondroitin sulfate proteoglycans after scratch wounding in cultured astrocytes

Background: Various in vivo and in vitro models have been described in order to elucidate the pathobiology underlying the traumatic brain injury (TBI) and test potentially suitable treatments. Since TBI is a complex disease, models differ in regard to the aspect of TBI that is being investigated. One of the used in vitro models is the scratch wound assay, first established as a reproducible, low-cost assay for the analysis of cell migration in vitro. The aim of the present study was to further investigate the relevancy of this model as a counter­part of in vivo TBI models. Methods: We have examined the astrocytic response to a mechanical injury in terms of expression of chondroitin sulfate proteoglycans (CSPGs) - phosphacan, neurocan and brevican, using real-time PCR and immunocytochemistry. Results: Our results indicate that in vitro scratch wounding alters the expression profile of examined CSPGs. Four hours after the scratch injury of the astrocytic monolayer, real-time PCR analysis revealed upregulation of mRNA levels for phosphacan (3-fold) and neurocan (2-fold), whereas brevican mRNA was downregulated (2-fold). Immunofluorescent signal for phosphacan and neurocan was more intense in astrocytes close to the injury site, while brevican was scarcely present in cultured astrocytes. Conclusions: Obtained results indicate that CSPGs are differentially expressed by astrocytes after scratch wounding, demonstrating that the scratch wound model might be suitable for investigation of astrocyte-derived response to injury.Uvod: Brojni in vivo i in vitro modeli opisani su sa ciljem da se rasvetle patobiološki procesi koji su osnova traumatske povrede mozga (TPM) i testiraju potencijalni tretmani. Imajući u vidu da je TPM kompleksno oboljenje, ovi modeli se međusobno razlikuju shodno aspektu TPM koji se ispituje. Jedan od in vitro modela je i povreda ćelijskog jednosloja grebanjem (engl. 'scratch wound' assay), isprva ustanovljen kao ponovljiv, jeftin test za analizu celijske migracije in vitro. Cilj ove studije je da se bliže ispita relevantnost ovog modela u odnosu na in vivo modele TPM. Metode: Da bi se istražio odgovor astrocita na mehaničku povredu, praćena je ekspresija odabranih hondroitin-sulfatnih proteoglikana (CSPG) - fosfakana, neurokana i brevikana, korišćenjem PCR u realnom vremenu i imunocitohemije. Rezultati: Dobijeni rezultati su pokazali da in vitro povreda astrocitnog jednosloja menja profile ekspresije ispitivanih CSPG. Četiri sata nakon povrede, primena PCR u realnom vremenu analize pokazala je povećanje nivoa iRNK za fosfakan (trostruko) i neurokan (dvostruko), dok je iRNK za brevikan bila smanjena na polovinu kontrolne vrednosti. Imunofluorescentni signal poreklom od fosfakana i neurokana je bio intenzivniji u astrocitima bližim mestu povrede, dok je signal za brevikan bio slab kako u kontrolnoj, tako i u ozleđenoj grupi. Zaključak: Dobijeni rezultati pokazuju da povreda izazvana grebanjem različito utiče na ekspresiju ispitivanih CSPG u astrocitima, sto ukazuju da ovaj model može biti pogodan za ispitivanje odgovora astrocita na povredu.Projekat ministarstva br. III 4101

Prolonged Zaleplon Treatment Increases the Expression of Proteins Involved in GABAergic and Glutamatergic Signaling in the Rat Hippocampus

Zaleplon is a positive allosteric modulator of the γ-aminobutyric acid (GABA)A receptor approved for the short-term treatment of insomnia. Previous publications on zaleplon have not addressed the proteins involved in its mechanism of action but have mostly referred to behavioral or pharmacological studies. Since both GABAergic and glutamatergic signaling have been shown to regulate wakefulness and sleep, we examined the effects of prolonged zaleplon treatment (0.625 mg/kg for 5 days) on these systems in the hippocampus of male Wistar rats. Western blot and immunohistochemical analyses showed that the upregulated components of GABAergic signaling (glutamate decarboxylase, vesicular GABA transporter, GABA, and α1 subunit of the GABAA receptor) were accompanied by increased protein levels in the glutamatergic system (vesicular glutamate transporter 1 and NR1, NR2A, and NR2B subunits of N-methyl-d-aspartate receptor). Our results, showing that zaleplon enhances GABA neurotransmission in the hippocampus, were not surprising. However, we found that treatment also increased glutamatergic signaling. This could be the result of the downregulation of adenosine A1 receptors, important modulators of the glutamatergic system. Further studies are needed to investigate the effects of the zaleplon-induced increase in hippocampal glutamatergic neurotransmission and the possible involvement of the adenosine system in zaleplon’s mechanism of action

Pattern of chondroitin sulfate proteoglycan expression after ablation of the sensorimotor cortex of the neonatal and adult rat brain

The central nervous system has a limited capacity for self-repair after damage. However, the neonatal brain has agreater capacity for recovery than the adult brain. These differences in the regenerative capability depend on local environmental factors and the maturational stage of growing axons. Among molecules which have both growth-promoting and growth-inhibiting activities is the heterogeneous class of chondroitin sulfate proteoglycans (CSPGs). In this paper, we investigated the chondroitin-4 and chondroitin-6 sulfate proteoglycan expression profile after left sensorimotor cortex ablation of the neonatal and adult rat brain. Immunohistochemical analysis revealed that compared to the normal uninjured cortex, lesion provoked up regulation of CSPGs showing a different pattern of expression in the neonatal vs. the adult brain. Punctuate and membrane-bound labeling was predominate after neonatal lesion, where as heavy deposition of staining in the extracellular matrix was observed after adult lesion. Heavy deposition of CSPG immunoreactivity around the lesionsite in adult rats, in contrast to a less CSPG-rich environment in neonatal rats, indicated that enhancement of the recovery process after neonatal injury is due to amore permissive environment.Centralni nervni sistem ima ograničen kapacitet za oporavak nakon povrede. Međutim, neonatalni mozak pokazuje veću sposobnost oporavka u odnosu na odrasle. Ove razlike zavise od lokalnih sredinskih faktora i stepena zrelosti aksona tokom izrastanja. U grupu molekula koji mogu da stimulišu ili inhibiraju rast aksona spada i heterogena klasa molekula označena kao hondroitin sulfatni proteoglikani (CSPG). U ovom radu ispitivanje profil ekspresije hondroitin-4 i hondroitin-6 sulfatnih proteoglikana nakonlezije leve senzomotorne kore neonatalnog i adultnog mozga pacova. Imunohistohemijska analiza pokazuje da u odnosu na normalni,nepovređeni korteks, lezija dovodi do povećanja ekspresije CSPG koji ima različiti obrazac promena u neonatalnom u odnosu na adultni mozak. Nakonlezije kod mladih, predominiraju tačkasta i membranski-vezana forma, dok kod odraslih lezija dovodi do nagomilavanja CSPG u ekstra ćelijskom matriksu. Prohodnija sredina u mozgu neonatalnih pacova koja je siromašnija CSPG, preduslov je boljeg procesa oporavka u odnosu na adulte, kod kojih se nakon povrede CSPG nagomilavaju oko mesta lezije.Projekat ministarstva br. 143005

Real-time PCR and immunocytochemical study of chondroitin sulfate proteoglycans after scratch wounding in cultured astrocytes

Background: Various in vivo and in vitro models have been described in order to elucidate the pathobiology underlying the traumatic brain injury (TBI) and test potentially suitable treatments. Since TBI is a complex disease, models differ in regard to the aspect of TBI that is being investigated. One of the used in vitro models is the scratch wound assay, first established as a reproducible, low-cost assay for the analysis of cell migration in vitro. The aim of the present study was to further investigate the relevancy of this model as a counter­part of in vivo TBI models. Methods: We have examined the astrocytic response to a mechanical injury in terms of expression of chondroitin sulfate proteoglycans (CSPGs) - phosphacan, neurocan and brevican, using real-time PCR and immunocytochemistry. Results: Our results indicate that in vitro scratch wounding alters the expression profile of examined CSPGs. Four hours after the scratch injury of the astrocytic monolayer, real-time PCR analysis revealed upregulation of mRNA levels for phosphacan (3-fold) and neurocan (2-fold), whereas brevican mRNA was downregulated (2-fold). Immunofluorescent signal for phosphacan and neurocan was more intense in astrocytes close to the injury site, while brevican was scarcely present in cultured astrocytes. Conclusions: Obtained results indicate that CSPGs are differentially expressed by astrocytes after scratch wounding, demonstrating that the scratch wound model might be suitable for investigation of astrocyte-derived response to injury.Uvod: Brojni in vivo i in vitro modeli opisani su sa ciljem da se rasvetle patobiološki procesi koji su osnova traumatske povrede mozga (TPM) i testiraju potencijalni tretmani. Imajući u vidu da je TPM kompleksno oboljenje, ovi modeli se međusobno razlikuju shodno aspektu TPM koji se ispituje. Jedan od in vitro modela je i povreda ćelijskog jednosloja grebanjem (engl. 'scratch wound' assay), isprva ustanovljen kao ponovljiv, jeftin test za analizu celijske migracije in vitro. Cilj ove studije je da se bliže ispita relevantnost ovog modela u odnosu na in vivo modele TPM. Metode: Da bi se istražio odgovor astrocita na mehaničku povredu, praćena je ekspresija odabranih hondroitin-sulfatnih proteoglikana (CSPG) - fosfakana, neurokana i brevikana, korišćenjem PCR u realnom vremenu i imunocitohemije. Rezultati: Dobijeni rezultati su pokazali da in vitro povreda astrocitnog jednosloja menja profile ekspresije ispitivanih CSPG. Četiri sata nakon povrede, primena PCR u realnom vremenu analize pokazala je povećanje nivoa iRNK za fosfakan (trostruko) i neurokan (dvostruko), dok je iRNK za brevikan bila smanjena na polovinu kontrolne vrednosti. Imunofluorescentni signal poreklom od fosfakana i neurokana je bio intenzivniji u astrocitima bližim mestu povrede, dok je signal za brevikan bio slab kako u kontrolnoj, tako i u ozleđenoj grupi. Zaključak: Dobijeni rezultati pokazuju da povreda izazvana grebanjem različito utiče na ekspresiju ispitivanih CSPG u astrocitima, sto ukazuju da ovaj model može biti pogodan za ispitivanje odgovora astrocita na povredu.Projekat ministarstva br. III 4101

Tiazofurin modulates lipopolysaccharide-activated microglia in vitro

Tiazofurin is a purine nucleoside analogue, with a broad spectrum of antitumoral and anti-inflammatory properties. In the present study, we have investigated the effect of tiazofurin on microglial inflammatory response to lipopolysaccharide in vitro. The cytotoxic effect of the drug was examined by sulforhodamine B assay. The Griess method was used to quantify nitrite production. Microglial morphology was assessed by measuring cell body size. Release of the pro-inflammatory cytokines, tumor necrosis factor-α, interleukin-1β, interleukin-6, and the anti-inflammatory cytokine interleukin- 10, were evaluated by enzyme-linked immunosorbent assay. Our data showed that tiazofurin decreased the number of activated microglia, lowered nitric oxide production and reduced the average cell surface of these cells. Tiazofurin reduced tumor necrosis factor-α, interleukin-6 and increased interleukin-10 secretion. Conversely, this drug promoted the release of interleukin-1β. Results obtained in this study indicate that TR displayed both anti- and pro-inflammatory modulation of activated microglia that could be relevant for its antitumor action within the central nervous system. [Projekat Ministarstva nauke Republike Srbije, br. III41014