| Sažetak | Cilj istraživanja
Cilj doktorskog rada bio je prouciti: a) stupanj oksidativnog oštecenja lipida i proteina,
potom aktivnosti antioksidativnih enzima (superoksid dismutaze, SOD i glutation
peroksidaze, GSH-Px), ekspresije razlicitih proteina (proupalnih cimbenika COX-2 i iNOS,
inhibitora NFB upalnog signalnog puta, IB, kao i endogenih neuroprotektivnih proteina,
BDNF i HSP70) i sadržaj vode u razlicitim strukturama mozga, te b) ucinke pioglitazona
(PPAR agonista) i enoksaparina (heparina niske molekularne mase) na istraživane
pokazatelje oksidativnog stresa i ekspresije gore navedenih proteina u štakora s traumatskom
ozljedom mozga (engl. traumatic brain injury, TBI).
Materijal i metode
Trauma mozga umjerene jakosti ucinjena je metodom lateralne ozljede tlakom (engl.
lateral fluid percussion injury) u podrucju lijevog parijetalnog korteksa. Razine produkata
lipidne peroksidacije, te aktivnosti SOD i GSH-Px u homogenatima tkiva, odredivane su
spektrofotometrijom. Stupanj oksidacijskog oštecenja proteina (sadržaj proteinskih
karbonilnih skupina), te razine ekspresija razlicitih proteina u tkivnim lizatima mjerene su
Western blot metodom. U dijelu istraživanja korištene su histološke i imunohistokemijske
metode. Za detekciju obrasca oštecenja tkiva korištena su bojanja hemalaun-eozin i krezil
violet. Regionalne distribucije ekspresija COX-2 i HSP70 analizirane su imunohistokemijski.
S ciljem istraživanja oksidacijskog oštecenja, upalnog odgovora, te razina ekspresija proteina
u razlicitim regijama mozga, životinje su žrtvovane 24 sata nakon indukcije TBI. Životinje
kontrolne skupine bile su podvrgnute lažnoj TBI. Dio životinja žrtvovan je 48 sati nakon TBI
s ciljem odredivanja postojanja posttraumatskog edema mozga. U dijelu pokusa, u kojima su
istraživani ucinci pioglitazona u TBI, traumatizirane životinje bile su jednokratno tretirane
navedenim glitazonom (1 ili 3 mg/kg) ili otapalom (dimetil-sulfoksid), 10 minuta nakon indukcije TBI. Štakori kontrolne skupine bili su podvrgnuti istovjetnim postupcima,
ukljucujuci aplikaciju otapala, osim što im nije bila izazvana TBI. Životinje ovog dijela
istraživanja bile su žrtvovane 24 sata nakon traume mozga. U dijelu pokusa u kojima su
istraživani ucinci enoksaparina u TBI, pokusne su životinje bile tretirane istraživanim lijekom
(1 mg/kg) ili otapalom (voda za injekcije) 1 sat nakon indukcije TBI, te s dodatnih 7 doza
primijenjenih svakih 6 sati nakon prve doze, a bile su žrtvovane 48 sati nakon izazivanja TBI.
Životinje kontrolne skupine bile su lažno ozlijedene, a umjesto enoksaparina injicirano im je
odgovarajuce otapalo.
Rezultati
TBI umjerene jakosti uzrokovala je najjace oštecenje mozga u podrucju parijetalnog
korteksa i hipokampusa štakora. U navedenim moždanim strukturama nakon TBI evidentirani
su porasti razina produkata lipidne peroksidacije i sadržaja proteinskih karbonilnih skupina, te
povišene razine ekspresija COX-2 nakon ozljede mozga. U obje navedene strukture
zabilježena je povecana aktivnost GSH-Px. Ekspresija COX-2 bila je povecana i u talamusu,
te entorinalnom korteksu ozlijedenih životinja. U hipokampusu, dodatno, bile su povecane
razine ekspresija iNOS, IB, te proBDNF, a smanjena razina mBDNF. U parijetalnom
korteksu zabilježene su i smanjene ekspresije IB, te HSP70. U entorinalnom korteksu
ekspresija proBDNF bila je povecana. U talamusu su bili pojacani i izražaji iNOS i IB.
Edem mozga uzrokovan TBI zabilježen je u ipsi- i kontralateralnom parijetalnom korteksu,
ipsilateralnom hipokampusu, te cerebelumu. Djelovanje pioglitazona i enoksaparina testirano
je u parijetalnom korteksu i hipokampusu ozlijedenih štakora. Pronadeno je da su ucinci
pioglitazona u modelu TBI ovisni o dozi lijeka i moždanoj strukturi. Sukladno navedenom,
pioglitazon može smanjiti oksidativno oštecenje lipida i proteina, povecati aktivnost GSH-Px,
smanjiti ekspresiju iNOS, te povecati izražaje proBDNF i mBDNF. Enoksaparin, u testiranim eksperimentalnim uvjetima, djelovao je neuroprotektivno u obje moždane strukture. U
parijetalnom korteksu ozlijedenih životinja povecao je aktivnost GSH-Px i ekspresiju IB, a
smanjio izražaj COX-2. U hipokampusu štakora s TBI smanjio je oksidativno oštecenje lipida
i proteina, te ekspresiju proupalnog COX-2 proteina.
Zakljucak
TBI uzrokuje jako oksidacijsko i upalno oštecenje parijetalnog korteksa i
hipokampusa štakora. U ostalim moždanim strukturama, nakon traume mozga, bilježe se
promjene pojedinih proupalnih, te endogenih neuroprotektivnih proteina. Pioglitazon i
enoksaparin mogu djelovati neuroprotektivno u modelu TBI. |
| Sažetak (engleski) | Objectives
The purpose of this PhD thesis was to study: a) the degree of lipid and protein
oxidative damage, antioxidant enzymes’ (superoxide dismutase, SOD and glutathione
peroxidase, GSH-Px) activities, the expressions of different proteins (proinflammatory factors
COX-2 and iNOS, an NFB signaling pathway inhibitor IB, and the endogenous
neuroprotective proteins, BDNF and HSP70), and water content in different brain structures,
as well as b) the effects of pioglitazone (PPAR agonist) and enoxaparin (low molecular
weight heparin) on the tested parameters of oxidative stress and the expressions of the
aforementioned proteins in rats with traumatic brain injury (TBI).
Material and Methods
Brain trauma of moderate severity was induced using the lateral fluid percussion
injury model in the area of left parietal cortex. Levels of lipid peroxidation products, as well
as the activities of SOD and GSH-Px in tissue homogenates were determined by
spectrophotometry. The degree of protein oxidative damage (protein carbonyl content), and
the levels of different proteins’ expressions were determined using the Western blot method.
In one part of the research, histological and immunohistological methods were used.
Hematoxylin-eosin and cresyl violet stainings were used for the detection of the tissue
damage extent. Regional distributions of COX-2 and HSP70 expressions were analyzed by
immunohistochemistry. With the intent to investigate oxidative damage, inflammation, the
expressions of different proteins, in various brain regions following traumatic injury, animals
were sacrificed 24 h after the TBI induction. Animals of the control group were subjected to
the sham TBI. A part of the animals were sacrificed 48 h after TBI in order to determine the
existence of posttraumatic brain edema. In the part of the research, in which the effects of
pioglitazone were investigated, injured animals were treated with single dose of the tested glitazone (1 or 3 mg/kg) or by vehicle (dimethyl sulfoxide), 10 min after the TBI induction.
Rats of the control group were handled identically, except that they were not subjected to TBI.
Animals used in this part of the research were sacrificed 24 h following TBI onset. In the part
of the research which was focused on the effects of enoxaparin in TBI, experimental animals
were treated with the tested drug (1 mg/kg) or with vehicle (water for injections) 1 h after the
TBI induction, and additionally with 7 more doses applied every 6 h after the first dose. All
rats of this part of the study were sacrificed 48 h after the TBI induction. Animals of the
control group were sham injured, and, instead of enoxaparin, they were injected with
appropriate vehicle.
Results
TBI of moderate severity caused the most prominent brain damage in the rat area of
parietal cortex and hippocampus. In the mentioned brain structures, increased levels of the
lipid peroxidation products, protein carbonyls and the COX-2 expressions after TBI were
detected. Increased GSH-Px activities in both mentioned structures were recorded. COX-2
protein expressions were also augmented in the thalamus and in the entorhinal cortex of the
injured rats. Additionally, following the brain injury, in the hippocampus, the iNOS, IB,
and proBDNF expressions were increased, while the mBDNF expression was decreased. In
the parietal cortex decreased expressions of the IB and HSP70 were detected. In the
entorhinal cortex, the proBDNF expression was increased. Furthermore, in the thalamus,
increased expressions of iNOS and IB were detected. Brain edema caused by TBI was
recorded in the ipsi- and contralateral parietal cortex, ipsilateral hippocampus, and
cerebellum. Effects of pioglitazone and enoxaparin were tested in the parietal cortex and the
hippocampus of the injured rats. It was established that the effects of pioglitazone, in the TBI
model used, were dose-, and the brain structure-related. Pioglitazone can reduce oxidative lipid and protein damage, increase the GSH-Px activity, reduce the iNOS, and increase the
proBDNF and mBDNF expressions. Enoxaparin, in the experimental conditions used, was
neuroprotective in both brain structures tested. In the parietal cortex of injured animals,
enoxaparin increased the GSH-Px activity and the IB expression, and decreased the COX-2
expression. In the hippocampus of rats with TBI, it reduced oxidative lipid and protein
damage, and also attenuated injury-induced increase in the COX-2 protein expression.
Conclusion
TBI causes prominent oxidative and inflammatory damage of the parietal cortex and
hippocampus in rats. Changes of some proinflammatory and endogenous neuroprotective
proteins were recorded in the other brain structures tested following brain trauma.
Pioglitazone and enoxaparin could be neuroprotective in experimental TBI. |
