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Alchajmerova bolest (AB) i Parkinsonova bolest (PB) su najčešće neurodegenerativne bolesti starenja... Alzheimer's disease (AD)and Parkinson's disease are the most common neurodegenerative diseases in elderly...

Alchajmerova bolest (AB) i Parkinsonova bolest (PB) su najčešće neurodegenerativne bolesti starenja. Neuropatologija AB i PB podrazumeva selektivan gubitak specifičnih populacija neurona, uključujući i holinergičke neurone bazalnog prednjeg mozga i moždanog stabla, kao i ushodni obrazac progresije neurodegeneracije od struktura u moždanom stablu uključenih u regulaciju REM („rapid eye movement”) spavanja ka prednjemoždanim regionima. Poremećaji ponašanja u toku REM spavanja („REM sleep behavior disorders” – RBD) veoma često ostaju neprimećeni kod AB i PB pacijenata i mogu se javiti godinama pa čak i decenijama pre motornih i kognitivnih poremećaja. Ovo ukazuje na značaj istraživanja poremećaja ponašanja u toku REM spavanja kao mogućeg ranog znaka pojave najčešćih neurodegenerativnih bolesti starenja. Predmet istraživanja ove doktorske disertacije bio je da u eksperimentalnim modelima AB i PB holinergičke neuropatologije po prvi put ispita uticaj unilateralnih i bilateralnih lezija glavnog izvora kortikalne holinergičke inervacije (nucleus basalis, NB jedro) i glavnog izvora talamo-kortikalne holinergičke inervacije (nucleus pedunculopontinus tegmentalis, PPT jedro) velikog mozga pacova na spavanje i elektroencefalografske (EEG) ritmove. U ovim eksperimentalnim modelima holinergičke neuropatologije humanih bolesti (NB i PPT lezije) unilateralne lezije su predstavljale lakše oblike ili početne stadijume neurodegeneracija, dok su bilateralne lezije predstavljale teže oblike ili kasnije stadijume neurodegeneracija, a sama progresija bolesti je praćena ukupno 5 nedelja zavisno od eksperimentalne grupe. U skladu sa osnovnom hipotezom ove doktorske disertacije, da funkcionalno različiti centralni holinergički sistemi imaju različite regulatorne funkcije u toku spavanja, koje se mogu definisati na osnovu EEG aktivnosti, postavljen je i osnovni cilj istraživanja: da se kroz analizu arhitekture spavanja, strukture prelaznih stanja i EEG mikrostrukture osnovnih faza u toku spavanja (budnost, NREM – „non rapid eye movement”, REM) u eksperimentalnim modelima funkcionalno različite holinergičke neuropatologije pronađu potencijalni EEG markeri početka, težine i progresije neurodegeneracije, sa mogućnošću dalje primene ovako senzitivnih metoda (definisanih EEG markera) u eventualnoj prevenciji i promeni terapijskog pristupa u pacijenata sa rizikom razvoja AB i PB. Mužjaci pacova Wistar soja su hronično implantirani za registrovanje spavanja. U toku operativne procedure za implantaciju elektroda za elektroencefalografiju (EEG) i elektromiografiju (EMG), korišćenjem tehnike stereotaksički navođene mikroinfuzije ibotenične kiseline (ekscitotoksina koji je generalni glutamatergički agonista), izvšene su selektivne unilateralne ili bilateralne NB ili PPT lezije. Eksperimentalni protokol registrovanja spavanja je počinjao dve nedelje nakon oporavka od operativne procedure implantacije hroničnih elektroda, sa ili bez unilateralnih ili bilateralnih NB ili PPT lezija. Registrovano je 6 sati spavanja, jednom nedeljno tokom četiri nedelje u eksperimentima NB lezija, odnosno jednom nedeljno tokom pet nedelja u eksperimentima PPT lezija, korišćenjem programa DataWave SciWorks Experimenter 8.0 (Datawave Technologies, Longmont, SAD). Nakon završetka eksperimentalnog protokola registrovanja spavanja, NB i PPT lezije su identifikovane NADPH – diaforaza histohemijskim bojenjem, a zatim i kvantifikovane u celokupnoj antero-posteriornoj dimenziji strukture korišćenjem ImageJ 1.46 programa (NIH, SAD). Analiza spavanja i EEG signala urađena je, kako u kontrolnih pacova tako i u pacova sa identifikovanim NB ili PPT lezijama, u MATLAB 6.5 programu, korišćenjem originalno razvijenog programskog paketa. Na usnimljene šestočasovne signale je najpre primenjena Furijeova transformacija, a zatim je svaka od 2160 Furijeovih epoha dužine 10 s diferencirana kao budnost, NREM ili REM faza spavanja. Pored toga, za potrebe detaljnije analize poremećaja REM faze spavanja izazvanih bilateralnom PPT lezijom, grupisanje REM epoha je na osnovu snage EMG-a dodatno razdvojeno na dva podgrupisanja REM1 (REM epohe sa većom snagom EMG-a, patološki REM) i REM2 (REM epohe sa manjom snagom EMG-a, fiziološki REM). Potom je korišćenjem EEG signala senzomotorne i motorne kore urađena topografska analiza arhitekture spavanja, strukture prelaznih stanja u toku spavanja, EEG mikrostrukture svih osnovnih faza u toku spavanja, kao i analiza kortiko-muskularih koherencija između EEG-a senzomotorne ili motorne kore velikog mozga i EMG-a mišića dorzalne vratne muskulature. Za izračunavanje grupne raspodele gustine verovatnoće relativnih amplituda svih budnost/NREM/REM/REM1/REM2 konvencionalnih EEG frekventnih opsega u toku 6 h spavanja korišćena je PDE („Probability Density Estimate”) funkcija u MATLAB 6.5 programu. U daljoj funkcionalnoj analizi poremećaja REM faze spavanja, za svaki od konvencionalnih EEG frekventnih opsega su korišćenjem „cohere” funkcije u MATLAB 6.5 izračunate REM/REM1/REM2 kortiko-muskularne koherencije između EEG-a senzomotorne ili motorne kore i EMG-a mišića dorzalne vratne muskulature. Za statističku obradu svih podataka korišćena je neparametarska jednofaktorska Kruskal-Wallis ANOVA sa post-hoc Mann-Whitney U kontrastnim testom. Bilateralna NB lezija je privremeno promenila arhitekturu spavanja 14 dana nakon lezije. Iako u slučaju fiziološke kontrole nije bilo razlika u trajanju budnosti, NREM i REM faze spavanja između senzomotorne i motorne kore, bilateralna NB lezija je u senzomotornoj kori smanjila trajanje budnosti i povećala trajanje NREM faze spavanja, dok je u motornoj kori smanjila trajanje budnosti i povećala trajanje REM faze spavanja. Istovremeno, u svim eksperimentalnim grupama, kako u fiziološkoj kontroli, tako i u unilateralnoj i bilateralnoj NB leziji, je EEG teta amplituda budnosti, NREM i REM faze spavanja bila niža u motornoj u odnosu na senzomotornu koru. Pored toga, bilateralna NB lezija je u senzomotornoj kori izazvala povećanje REM teta amplitude u trajanju od tri nedelje, dok su u motornoj kori i unilateralna i bilateralna NB lezija izazvale povećanje teta amplitude i u budnosti i u REM fazi spavanja. PPT lezija nije promenila arhitekturu spavanja, ali je značajno izmenila strukturu prelaznih stanja, kao i EEG mikrostrukturu svih osnovnih faza spavanja. Obe lezije, i unilateralna i bilateralna PPT lezija, su dugotrajno povećale broj budnost/REM i REM/budnost prelaza tokom celokunog perioda od 5 nedelja. Ova promena u strukturi prelaznih stanja je od 28. dana bila praćena smanjenjem broja NREM/REM i REM/NREM prelaza, ali samo nakon bilateralne PPT lezije, kao težeg oblika holinergičke neurodegeneracije. Unilateralna PPT lezija je izazvala povećanje EEG teta amplitude budnosti i EEG beta amplitude REM faze spavanja, a od treće nedelje nakon lezije i pad EEG delta amplitude budnosti. Bilateralna PPT lezija je izazvala povećanje EEG beta amplitude tokom svih faza spavanja, kao i povećanje EEG gama amplitude REM faze spavanja i smanjenje EEG delta amplitude budnosti i NREM faze spavanja. Poređenje efekata bilateralne NB i bilateralne PPT lezije (modeli težih, odnosno progresivnijih oblika holinergičke neurodegeneracije) na arhitekturu spavanja, strukturu prelaznih stanja i EEG mikrostrukturu svih osnovnih faza spavanja je pokazalo postojanje topografski specifičnih razlika u poremećajima nastalim kao posledica degeneracije funkcionalno različitih holinergičkih inervacija velikog mozga pacova. Za razliku od bilateralne NB lezije koja je privremeno izmenila trajanje budnosti i NREM faze spavanja u senzomotornoj kori, odnosno trajanje budnosti i REM faze spavanja u motornoj kori, bilateralna PPT lezija nije izmenila arhitekturu spavanja. Bilateralna PPT lezija je u obe kore dugotrajno (tokom 4 nedelje) povećala broj budnost/REM i REM/budnost prelaza i ovaj porast je bio praćen nekonzistentnim promenama u broju NREM/REM i REM/NREM prelaza u senzomotornoj kori, a u motornoj kori povećanjem broja ovih prelaza. Bilateralna NB lezija je u senzomotornoj kori smanjila broj NREM/REM i REM/NREM prelaza, dok je u motornoj kori povećala broj ovih prelaza tokom četiri nedelje. Promene u EEG mikrostrukturi nakon bilateralne PPT lezije bile su i u senzomotornoj i u motornoj kori izražene kao dugotrajno (tokom 4 nedelje) povećanje beta i gama amplitude budnosti, NREM i REM faze spavanja, odnosno kao smanjenje delta amplitude budnosti, ali samo u senzomotornoj kori. Nasuprot tome, bilateralna NB lezija je izazvala samo povećanje REM teta amplitude u senzomotornoj kori koje je trajalo tri nedelje. Pored promena u strukturi prelaznih stanja u toku spavanja i EEG mikrostrukturi svih osnovnih faza spavanja, bilateralna PPT lezija je dodatno uslovila izdvajanje dva funkcionalno različita REM stanja u okviru REM faze spavanja, REM1 (PPT lezijom prouzrokovan patološki REM, REM bez atonije, REM sigma koherencije) i REM2 (fiziološki REM, REM sa atonijom, REM teta koherencije), naročito u motornoj kori. Iako nije promenila arhitekturu spavanja senzomotorne kore, bilateralna PPT lezija je u motornoj kori produžila trajanje patološkog REM1 stanja, a povećala broj budnost/REM1/budnost i NREM/REM2/NREM prelaza u obe kore. U osnovi izmenjene REM EEG mikrostrukture su bile povećana beta amplituda senzomotorne kore za vreme celokupnog REM stanja i povećana teta amplituda motorne kore samo za vreme REM1 stanja. Bilateralna PPT lezija je izazvala generalizovan pad REM/REM1/REM2 beta kortiko-muskularne koherencije i dovela do izmene kontrole mišića dorzalne vratne muskulature dominantno iz senzomotorne kore. Ova doktorska disertacija je, koristeći po prvi put nov eksperimentalni model holinergičke PB neuropatologije (PPT lezija), stereotaksički navođenu mikroinfuziju kao nov metodološki pristup za selektivne lezije moždanih jedara, nov histohemijski metod identifikacije deficita holinergičkih neurona, nov pristup u kvantifikaciji neuronskog deficita, kao i nov način analize EEG signala za vreme različitih faza spavanja, po prvi put dokazala topografski specifične razlike u arhitekturi spavanja, strukturi prelaznih stanja i EEG mikrostrukturi osnovnih faza spavanja (budnost, NREM, REM), koje su nastale usled poremećaja funkcionalno različitih holinergičkih inervacija velikog mozga pacova (NB lezija u odnosu na PPT leziju). U ovom novom eksperimentalnom modelu holinergičke PB neuropatologije po prvi put su dokazani dugotrajni i veoma ozbiljni poremećaji u spavanju izraženi kao: povećanje broja budnost/REM i REM/budnost prelaza, povećana kortikalna aktivacija tokom svih osnovnih faza spavanja, izdvajanje dva različita REM stanja (pojava patološkog REM stanja pored fiziološkog REM stanja), kao i dominantna izmena kontrole mišića dorzalne vratne muskulature iz senzomotorne kore, iskazana kao pad REM/REM1/REM2 beta koherencije. Alzheimer’s disease (AD) and Parkinson’s disease (PD) are the most common neurodegenerative diseases of elderly. AD and PD neuropathology involves selective loss of specific neuronal population within the brain, including the basal forebrain and brainstem cholinergic neurons, with the ascending pattern of neurodegeneration progression from rapid eye movement (REM) sleep-related brainstem regulatory structures to the brain basal areas. REM sleep behavior disorders (RBD) very frequently go unnoticed in the AD and PD patients, and as a symptom, could precede the onset of motor and cognitive disturbances by years or even decades. This indicates the importance of RBD as a potential early marker of the neurodegenerative diseases of elderly. Using the experimental models of AD and PD cholinergic neuropathology, this PhD thesis aimed to investigate for the first time the impact of unilateral and bilateral nucleus basalis (NB – the main source of cortical cholinergic innervation) and nucleus pedunculopontinus tegmentalis (PPT – the main source of thalamo-cortical cholinergic innervation) lesions on sleep and electroencephalographic (EEG) rhythms during sleep in rat. In these experimental models of cholinergic neuropathology (NB and PPT lesions) the unilateral lesions were used as the models of mild or early stage neurodegenerations, whereas the bilateral lesions were used as the models of severe or progressed neurodegenerations, and the follow up period was up to 5 weeks depending on the experimental group. Based on the hypothesis that functionally distinct cholinergic systems have different regulatory functions during sleep, that could be defined by EEG activity, this study aimed to investigate the sleep/wake architecture, sleep/wake state-related transitions structure and EEG microstructure of three main brain states (Wake, NREM – non rapid eye movement, REM) in order to find the possible EEG markers for the onset, severity, and progression of the functionally distinct cholinergic innervations disorders. These EEG markers could be used as a sensitive and reliable methodology of early detection in the patients who are at risk to develop AD or PD, and for a potential prevention and modification of the therapeutic approach. Male Wistar rats were chronically implanted for sleep recording. During operative procedure for the electroencephalographic (EEG) and electromyographic (EMG) electrodes implantation, the selective unilateral or bilateral NB or PPT lesions were performed by stereotaxically guided microinfusion of ibotenic acid (the general glutamate agonist). Sleep recording sessions started after two weeks of recovery period. Sleep was recorded for 6 h, weekly, during 4 weeks in NB lesioned rats, and during 5 weeks in PPT lesioned rats, using DataWave SciWorks Experimenter 8.0 software (Datawave Technologies, Longmont, SAD). At the end of recording sessions the NB and PPT lesions were identified by NADPH – diaphorase histochemistry, and quantified throughout the overall NB or PPT antero-posterior dimensions, using ImageJ 1.46 software (NIH, SAD). Analysis of the signals recorded from the control rats and all rats with positively identified NB or PPT lesion was done in MATLAB 6.5 using originally developed software. Fourier analysis was applied on 6 h recordings, and each 10 s epoch was differentiated as Wake, NREM or REM state. Additionally, for detailed analysis of two distinct REM clusters, that emerged following bilateral PPT lesion, each REM epoch was further differentiated, based on the EMG power, as either REM without atonia (REM1, pathological REM), and REM with atonia (REM2, physiological REM). Further analysis included the topography of sleep/wake states architecture, sleep/wake state-related transitions structure, and EEG microstructure, as well as all REM related cortico-muscular coherences. To calculate the group probability density distribution of all conventional EEG frequency bands relative amplitudes/6 h for the Wake/NREM/REM/REM1/REM2 of each experimental group we used the Probability Density Estimate (PDE) routine within MATLAB 6.5. For the functional REM cluster differentiation the REM/REM1/REM2 cortico-muscular coherences (CMCs) were calculated separately, for each conventional EEG frequency band and each experimental group, between the EEG of sensorimotor or motor cortex and the EMG of the dorsal nuchal muscles using the MATLAB 6.5 cohere routine. All statistical analyses were done using Kruskal-Wallis ANOVA with post-hoc Mann-Whitney U two-tailed tests. Bilateral NB lesion transiently altered sleep/wake states topography 14 days following lesion. While the control rats exhibited equivalent durations of Wake, NREM and REM, as determined by sensorimotor versus motor cortex EEG, the bilateral NB lesion decreased Wake duration in both cortices, with NREM duration increased within the sensorimotor cortex, and REM duration increased within the motor cortex. At the same time, Wake, NREM and REM theta relative amplitude was lower in motor versus sensorimotor cortex in all experimental groups. In sensorimotor cortex the bilateral NB lesion increased only REM theta amplitude for three weeks, whereas in motor cortex both Wake and REM theta amplitude were transiently increased 14 days following both the unilateral and bilateral NB lesion. PPT lesion did not change the sleep/wake architecture but did change the sleep/wake state-related transitions structure and the Wake/NREM/REM EEG microstructures. Both the unilateral and bilateral PPT lesions sustainably increased Wake/REM and REM/Wake transitions during 5 weeks, followed by the decreased NREM/REM and REM/NREM transitions from 28 days only in the case of the bilateral PPT lesion, as a more severe cholinergic neurodegeneration. The unilateral PPT lesion augmented Wake theta and REM beta amplitude, and with a delay of one week it attenuated Wake delta amplitude. The bilateral PPT lesion augmented beta amplitude during all sleep states, and REM gamma amplitude, but simultaneously attenuated Wake and NREM delta amplitude. Comparison of the bilateral NB versus bilateral PPT lesion (the models of severe or progressed cholinergic neurodegenerations) effects on the sleep/wake states architecture, sleep/wake state-related transitions structure and EEG microstructures indicated the topographically specific differentiation of functionally distinct cholinergic innervation disorders. Whereas the bilateral NB lesion transiently altered Wake/NREM duration within the sensorimotor cortex, and Wake/REM duration within the motor cortex, the bilateral PPT lesion did not change the sleep/wake states distributions. Bilateral PPT lesion sustainably (during 4 weeks) increased the Wake/REM and REM/Wake transitions within the both sensorimotor and motor cortex, followed by inconsistent dysregulation of the NREM/REM and REM/NREM transitions within the sensorimotor cortex, but oppositely by their increment within the motor cortex. Bilateral NB lesion sustainably (during 4 weeks) decreased the NREM/REM and REM/NREM transitions within the sensorimotor cortex, but oppositely increased them within the motor cortex. Sleep/wake state-related EEG microstructure following the bilateral PPT lesion was expressed as the sustained (during 4 weeks) Wake/NREM/REM beta and gamma amplitude augmentations within the both sensorimotor and motor cortex, and Wake delta amplitude attenuation, but only within the sensorimotor cortex. In contrast, the bilateral NB lesion augmented only REM theta amplitude within the sensorimotor cortex during three weeks. Alongside the sleep/wake state-related transitions structure and Wake/NREM/REM EEG microstructure alteration, the bilateral PPT lesion in rats additionally potentiated the emergence of two distinct REM sleep states, REM1 (pathological REM, REM without atonia, sigma coherent REM) and REM2 (physiological REM, REM with atonia, theta coherent REM), specifically expressed within the motor cortex. Bilateral PPT lesion did not change the sleep/wake states architecture within the sensorimotor cortex, but pathologically increased the duration of REM1 within the motor cortex, alongside the increased Wake/REM1/Wake and NREM/REM2/NREM transitions within the both cortices. In addition, the augmented total REM beta amplitude within the sensorimotor cortex and REM1 theta amplitude within the motor cortex was the underlying EEG microstructure pathology. Finally, the bilateral PPT lesion dominantly induced sensorimotor cortex-dorsal nuchal muscle drive alteration, expressed throughout the REM/REM1/REM2 beta CMC decrease. Introducing the novel experimental model of PD cholinergic neuropathology (PPT lesion), the stereotaxically guided microinfusion as a novel approach for the selective lesion of the brain nuclei, the novel histochemical method for the cholinergic neuronal loss identification, and the novel methodological approach for neuronal loss quantification, as well as the novel sleep-state related EEG signal analysis methodology, this PhD thesis evidenced for the first time the topographically different expression of the sleep/wake architecture, sleep/wake state-related transitions structure and Wake/NREM/REM EEG microstructure, induced by the functionally distinct cholinergic innervation disorders in rat (NB versus PPT lesion). This study demonstrated for the first time the PPT lesion (novel experimental model of PD cholinergic neuropathology) induced sustained and more severe sleep disturbances expressed as: the Wake/REM and REM/Wake transitions increase, augmented cortical activation across all sleep/wake states, potentiation of two distinct REM clusters, and dominant sensorimotor cortex-dorsal nuchal muscle drive alteration throughout the REM/REM1/REM2 beta CMC decrease.