Parameters of the brain damage and repair in different traumatic brain injury in rats : Parameters of the brain damage and repair in different regions and time points following traumatic brain injury in rats

U ovom doktorskom radu proučavani su opseg i vrijeme pojave pokazatelja oštećenja i oporavka posljedično traumatskoj ozljedi mozga (engl. traumatic brain injury, TBI) u dosad nedovoljno istraženim regijama mozga (talamus, mali mozak, rostralni dio velikog mozga). Pokusi su rađeni na odraslim Hannover-Wistar štakorima muškog spola. TBI je inducirana metodom lateralne ozljede mozga tlakom tekućine. Štakori su žrtvovani prvog, trećeg ili sedmog dana nakon TBI, a kontrolnu skupinu činile su životinje žrtvovane prvog dana nakon lažne ozljede. Uzorci mozgova proučavani su Western blot i TUNEL metodama, histološkim/imunohistološkim analizama te Fluoro-Jade B bojenjem. U talamusu ozlijeđenih životinja pronađene su progresivna degeneracija i gubitak neurona, programirana stanična smrt neovisna o aktivnoj kaspazi-3, a posredovana čimbenikom indukcije apoptoze (AIF), reaktivna astrocitoza i mikroglioza, te ograničene neuroplastične promjene tijekom prvog tjedna nakon TBI. U malom mozgu ozlijeđenih štakora, prvog dana nakon TBI, neurodegenerativne promjene zabilježene su u stražnjim režnjićima, a potom su ponovno uočene sedmog dana nakon traume u širem području ove strukture. U IX. režnjiću bio je detektiran i značajan gubitak Purkinjeovih stanica u svim istraživanim vremenskim točkama. Najjače izražena apoptoza, koja nije bila posredovana aktivacijom kaspaze-3 niti AIF-om, zabilježena je prvog dana nakon ozljede u svim slojevima kore IX. režnjića. Pojačani AIF signal pronađen je u bijeloj tvari malog mozga trećeg i sedmog dana nakon TBI. Iako su ekspresije markera astrocita i mikroglije bile smanjene u ipsilateralnoj polovici malog mozga nakon TBI, na mjestima oštećenja Purkinjeovih stanica zabilježena je njihova aktivacija. Diskretne neuroplastične promjene nakon TBI detektirane su u IX. režnjiću. U strukturama rostralnog dijela velikog mozga degeneracija i gubitak neurona nisu zabilježeni u prvom tjednu nakon TBI. U frontalnom korteksu ozlijeđenih štakora pronađene su reaktivna astrocitoza i mikroglioza, te stanice pozitivne na DCX, marker neuroblasta, a neuroplastične promjene nisu uočene. Promjene imunoreaktivnosti DCX-a detektirane su i u subventrikularnoj zoni i rostralnom migratornom putu ozlijeđenih životinja. Sumarno, rezultati ovog rada pokazali su kako TBI u štakora uzrokuje pojavu znakova oštećenja i oporavka u istraživanim moždanim strukturama u različitim vremenskim točkama tijekom prvog tjedna nakon ozljede.In this doctoral thesis regional and temporal distribution of the parameters of damage and repair following traumatic brain injury (TBI) were investigated in the rat brain structures which have not been previously studied extensively (thalamus, cerebellum, rostral part of the cerebrum). Experiments were done on adult male Hannover-Wistar rats. TBI was induced by lateral fluid percussion injury method. Rats were sacrificed on the first, third or seventh day after TBI, and the rats sacrificed on the first day after sham injury were used as the control group. Brain samples were examined by Western blot and TUNEL methods, histologic/immunohistologic analyses, and by Fluoro-Jade B staining. In the thalamus of injured animals progressive degeneration and neuronal loss, programmed cell death, which was caspase-independent, but mediated by apoptosis inducing factor (AIF), reactive astrocytosis and microgliosis as well as limited neuroplastic changes were detected during the first week after TBI. In the cerebellum of the injured rats, on the first day after TBI, neurodegenerative changes were discovered in the posterior lobules, and on the seventh day after brain trauma, diffusely through this brain structure. In the lobule IX, a significant loss of Purkinje cells was detected at all the investigated time points. Most significant apoptosis, which was caspase-3 and AIF independent, was found on the first post-injury day in all the cortical layers within the lobule IX. Enhanced AIF signal was detected in the cerebellar white matter on the third and seventh day after TBI. Even though the expressions of astrocytic and microglial markers were lower in the ipsilateral halves of the cerebellum after TBI, activation of these cells was discovered in the areas of pronounced Purkinje cells’ loss. Discrete neuroplastic changes after TBI were also detected in the lobule IX. In the structures of the rostral part of the cerebrum, degeneration and neuronal loss were not detected in the first week after TBI. In the frontal cortex of the injured animals reactive astrocytosis and microgliosis were found as well as the cells which expressed DCX, a neuroblast marker, while other neuroplastic changes were not revealed. Changes in the DCX immunoreactivity were also detected in the subventricular zone and the rostral migratory stream in the injured animals. In conclusion, results of this thesis demonstrated that TBI in the rat causes appearance of the sings of damage and repair in the investigated brain structures in different time points within the first week after the injury