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UNIVERSITATEADEMEDICINIFARMACIEIULIUHAIEGANUCLUJNAPOCA

RezumatultezeideDoctorat

FACTORIIMPLICAINREGENERAREANERVOASDINSISTEMUL
NERVOSPERIFERIC

Conductortiinific:
Prof.Dr.SimonaTache

Doctorand:
AnneMarieChindri(cs.Constantin)

ClujNapoca2012
CUPRINS
ABREVIERI ................................................................................................................................................................ 12
INTRODUCERE ......................................................................................................................................................... 13
STADIULACTUALALCUNOATERII.................................................................................................................. 15
1.Fibrelenervoase ................................................................................................................................................................. 17
1.1.Consideraiigenerale.................................................................................................................................................................................17
1.1.1.Clasificareafibrelornervoase ....................................................................................................................................................18
1.1.2.Structurafibrelornervoase ........................................................................................................................................................19
1.1.2.1.Teacaconjunctiv ..............................................................................................................................................................19
1.1.2.2.TeacaSchwann ...................................................................................................................................................................19
1.1.2.3.Teacademielin.................................................................................................................................................................19
1.2.Structuranervilorperiferici.....................................................................................................................................................................20

2.Degenerescenairegenerareafibrelornervoase.................................................................................. 21
2.1.Degenerescena............................................................................................................................................................................................21
2.1.1.Degenerescenawallerian.........................................................................................................................................................21
2.1.2.Degenerescenaretrograd ........................................................................................................................................................22
2.2.Regenerarea...................................................................................................................................................................................................22
2.2.1.Factoricareinflueneazregenerareanervilorperiferici ..............................................................................................23
2.2.1.1.Factoriendogenicarestimuleazregenerareanervilorperiferici...............................................................24
Factorigenetici................................................................................................................................................................24
Factoridecretere.........................................................................................................................................................26
Factoriendogeniantioxidani..................................................................................................................................27
2.2.1.2.Factoriexogenicarestimuleazregenerareanervilorperiferici................................................................28
Factoriantioxidaniexogeni......................................................................................................................................28
Suplimentehormonale.................................................................................................................................................29
Suplimentevitaminice .................................................................................................................................................30
Medicamenteneuroprotectoare ..............................................................................................................................30
Factorifizici ......................................................................................................................................................................31
Materialeutilizatenbioinginerie...........................................................................................................................32
Efortulfizic.........................................................................................................................................................................32

CONTRIBUIAPERSONAL..................................................................................................................................................... 33
1.Obiective ............................................................................................................................................................... 35
2.Metodologiageneral ....................................................................................................................................... 37
2.1.Tehnicadeinducerealeziuniinervoase ............................................................................................................................................37
2.2.Metodefiziologiceexperimentale .........................................................................................................................................................38
2.2.1.Determinareafuncionaloriginalaindiceluisciaticfuncional...............................................................................38
2.2.2.Determinareatimpuluidelatenprintestulalgictermic............................................................................................38
2.3.Determinribiochimiceserice.................................................................................................................................................................38
2.3.1.Recoltareaprobelorbiologice.....................................................................................................................................................38
2.3.2.IndicatoriibiochimiciaibalaneiO/AO ..................................................................................................................................39
2.4.Analizahistopatologic...............................................................................................................................................................................40
2.5.Protocolulgeneral.........................................................................................................................................................................................41
2.6.Analizastatistic ............................................................................................................................................................................................41

3.Studiul1Evaluareafuncionalaregenerriinervilorperiferici .................................................. 43
3.1.Introducere ......................................................................................................................................................................................................43

3.2.Obiective...........................................................................................................................................................................................................44
3.3.Materialimetod ......................................................................................................................................................................................44
3.4.Rezultate ...........................................................................................................................................................................................................46
3.5.Discuii ...............................................................................................................................................................................................................48
3.6.Concluzii..........................................................................................................................................................................................................50

4.Studiul2Efectelemelatonineinregenerareanervuluisciatic....................................................... 51
4.1.Introducere ......................................................................................................................................................................................................51
4.2.Obiective...........................................................................................................................................................................................................52
4.3.Materialimetod ......................................................................................................................................................................................52
4.4.Rezultate ...........................................................................................................................................................................................................52
4.5.Discuii ...............................................................................................................................................................................................................58
4.6.Concluzii..........................................................................................................................................................................................................60

5.Studiul3Efectelechitosanuluinregenerareanervuluisciatic...................................................... 61

5.1.Introducere ......................................................................................................................................................................................................61
5.2.Obiective...........................................................................................................................................................................................................61
5.3.Materialimetod ......................................................................................................................................................................................62
5.4.Rezultate ...........................................................................................................................................................................................................62
5.5.Discuii ...............................................................................................................................................................................................................67
5.6.Concluzii..........................................................................................................................................................................................................68

6.Studiul4EfectelecoenzimeiQ10nregenerareanervuluisciatic ................................................. 69
6.1.Introducere ......................................................................................................................................................................................................69
6.2.Obiective...........................................................................................................................................................................................................70
6.3.Materialimetod ......................................................................................................................................................................................70
6.4.Rezultate ...........................................................................................................................................................................................................71
6.5.Discuii ...............................................................................................................................................................................................................75
6.6.Concluzii..........................................................................................................................................................................................................76

7.Studiul5Efecteleatorvastatineinregenerareanervuluisciatic................................................... 77

7.1.Introducere ......................................................................................................................................................................................................77
7.2.Obiective...........................................................................................................................................................................................................77
7.3.Materialimetod ......................................................................................................................................................................................78
7.4.Rezultate ...........................................................................................................................................................................................................78
7.5.Discuii ...............................................................................................................................................................................................................82
7.6.Concluzii..........................................................................................................................................................................................................84

8.Studiul6Efecteleefortuluifizicnregenerareanervuluisciatic.................................................... 85
8.1.Introducere ......................................................................................................................................................................................................85
8.2.Obiective...........................................................................................................................................................................................................86
8.3.Materialimetod ......................................................................................................................................................................................86
8.4.Rezultate ...........................................................................................................................................................................................................87
8.5.Discuii ...............................................................................................................................................................................................................91
8.6.Concluzii..........................................................................................................................................................................................................92

9.Discuiigenerale.................................................................................................................................................. 93

9.1.AnalizaISF ........................................................................................................................................................................................................93
9.2.Analizatimpuluidelaten .......................................................................................................................................................................94
9.3.Analizaparametrilorbiochimici .............................................................................................................................................................95
9.4.Corelaiintreparametriideterminai..................................................................................................................................................98
9.5.Analizahistopatologic...............................................................................................................................................................................99

10.Concluziigenerale ......................................................................................................................................... 101


11.Originalitateaicontribuiileinovatoarealetezei ............................................................................... 103
REFERINE................................................................................................................................................................. 105

Cuvintecheie:nervsciatic,regenerare,melatonin,chitosan,CoQ10,atorvastatin,efort

INTRODUCERE
Attnviaadezicuzi,darmaifrecventlasportivi,potaparetraumatismecareafecteazdiferii
nerviperiferici.Recuperareamorfologicifuncionalanervilorlezaiifactoriicarepotacceleraaceste
procesedevinastfelimportantepentruactivitateafizic,pentrubunstareaicalitateavieii.
ntrofibrnervoaslezat,segmentuldistal,separatdecorpulcelularneuronal,suferunproces
dedegenerare,iarncadrulsegmentuluiproximalapare,deasemenea,odegeneraretraumatic.
Factorii care stimuleaz regenerarea fibrelor nervoase periferice sunt de dou tipuri, endogeni i
exogeni.Printrefactoriiendogenisenumrfactoriigenetici iexpresialor,factoriidecretere,factorii
antioxidani endogeni. Administrarea a numeroase substane exogene (antioxidanii exogeni,

suplimentele hormonale i vitaminice, medicamentele neuroprotectoare) poate favoriza regenerarea


fibrelor nervoase periferice afectate de un traumatism. De asemenea, exist factori fizici (cmpuri
magnetice, curent electric) implicai n regenerarea nervoas periferic. Efectele neuroprotectoare i
stimulantealefactorilorexogenipotfifolositecusuccesnpracticaclinic.

CONTRIBUIAPERSONAL
Ipotezadelucru.Obiective
nncercareadeastabiliometodterapeuticeficientnmbuntirearecuperriifuncionalea
nervului periferic lezat, au fost propuse numeroase preparate i ci de administrare ale acestora.
Cercetareaefectuatsebazeazpestudiiexperimentaleiarecascopstabilireauneimetodeeficientedea
evalua recuperarea funcional dup lezarea unui nerv periferic i evidenierea rolului unor ageni cu
potenialantioxidant,nfavorizarearegenerriinervoase.
Obiectivelestudiilorexperimentaleefectuateaufosturmtoarele:
1. realizareaunuimodelexperimentaldeleziunenervoaspenervsciaticlaobolan;
2. evaluareagraduluiderecuperarefuncionalanervuluilezatprintesteadecvate;
3. evaluarea eficienei terapiei antioxidante a unor factori farmacologici i a efortului fizic
asupraregenerriifibrelornervoase;
4. efectuarea unor determinri biochimice pentru aprecierea rolului balanei
oxidani/antioxidani(O/AO)ncazulleziunilortisularenervoaseperiferice;
5. studiul modificrilor histopatologice induse de factorii regeneratori folosii, pe preparate
obinutedupsacrificareaobolanilor,lancheiereaexperimentului.
Metodologiegeneral
Studiile experimentale sau efectuat n perioada februariemai 2012, fiind studii pilot, analitice,
longitudinale, prospective, cu durata de 28 de zile. Cercetrile sau desfurat n cadrul laboratorului de
FiziologieExperimental,iardeterminrilebiochimiceaufostefectuatenLaboratorulpentruStudiulStresului
Oxidativ,dincadrulDisciplineideFiziologieaUniversitiideMediciniFarmacieIuliuHaieganu.
Sauutilizatobolanialbi,rasaWistar,masculi,nvrstde16sptmni,cugreutateade200300
grame.Loturiledeobolaniaufostoperatelanivelulmembruluiposteriordrept,lacare,dupexpunerea
nervului sciatic, a fost produs o leziune prin zdrobire a acestuia, pe un segment de 5 mm, la 11,2 cm
proximaldetrifurcaie,prinutilizareauneipensehemostaticemeninuttimpde15secunde.
Indicele sciatic funcional (ISF), corespunztor analizei funcionale a mersului, a fost calculat pe
baza amprentelor plantare, obinute prin eliberarea i deplasarea animalului ntrun tunel de sticl. Sa
utilizat o metod computerizat original, care const n fotografierea labei picioarelor posterioare
ajutorulCelestronDigitalMicroscope.
Timpul de laten la stimulul algic termic sa determinat prin utilizarea plcii nclzite Ugo Basil
7280,la53C.Sacronometrattimpuldereaciealanimaluluiaezatpesuprafaanclzit,confruntatcu
stimulultermicaplicatpesuprafaaplantar.
Probele biologice de snge au fost prelevate din sinusul retroorbitar. Sau determinat indicatorii
biochimici ai balanei O/AO, pentru stresul oxidativ malondialdehida (MDA) i proteinele carbonilate
(PC)ipentruaprareaantioxidantgruprilesulfhidriltotale(SH)iglutationul(GSH).
Pentruexaminrilehistopatologice,probeleaufostprocesateprintehnicaincluderiilaparafini
prin tehnica secionrii la ghea. Seciuni longitudinale i transversale din nerv sciatic de la fiecare
obolan,dinzonalezionataufostcolorateH&E,respectivcuOilRedOiexaminatelamicroscopuloptic
saulacelconfocal.
Protocolpentrustudiile25:latoateloturile(n=10animale/lot)saefectuatlezareanervuluisciatic
prin zdrobire la nivelul membrului posterior drept. Studiul a inclus: lotul martor sedentar, timp de 4
sptmni dup intervenie; lotul I la care sa administrat, prin injecie ip, preparatul farmacologic, 4
sptmni dup intervenie. obolanii au fost evaluai prin: determinarea ISF n ziua 1 i n ziua 28, la
ambele loturi, conform metodei descrise n studiul 1; determinarea timpului de laten la stimulul algic
termiclaambelemembreposterioareiaparametrilorbalanei O/AO;analizahistopatologicagradului
derefaceremorfologicindusdefactorulregeneratorstudiat.
Analiza statistic a utilizat testul tStudent pentru eantioane perechi i independente, analiza de
varian ANOVAFisher, testul PostHoc cu corecia Scheffe, testul MannWhitney pentru o distribuie a
datelornegaussian,cutestulKruskalWallispentrucomparaiilentre3saumaimulteloturiicoeficientul
decorelaiePearson.Pentruprezentareadateloraufostfolositetabeleigraficeledetipulboxplot.

Studiul1.Evaluareafuncionalaregenerriinervilorperiferici
Pentrustabilireamodeluluiexperimental,saefectuatisecionareaisuturanervuluisciatic.Sau
utilizat2loturia15animale,lacare,duplezareanervului sciaticprinsecionareisuturi,respectiv,

prinzdrobire,obolaniiaufostmeninuinrepaus,timpde28dezile.Saevaluatfuncionalregenerarea
nervoas periferic, bazat pe determinarea ISF i sa analizat comparativ evaluarea funcional n
cazulleziunilorprinzdrobireaiprinsecionareanervului(LotIT1nervsecionat,1sptmn;LotI
T2 nerv secionat, 2 sptmni; Lot II T1 nerv zdrobit, 1 sptmn; Lot II T2 nerv zdrobit, 2
sptmni).
Rezultate: aplicarea testului t (Student) perechi indic creterea semnificativ statistic a valorilor
ISF,attalotuluiIT2fadelotulIT1(p=0.000),ctialotuluiIIT2fadelotulIIT1(p=0.000).De
asemenea, sa observat o corelaie semnificativ statistic ntre valoarea indexului ISF la lotul I T1 i
valoareaindexuluiISFlalotulIT2(r=0.54;p<0.04),respectivntrevaloareaindexuluiISFlalotulIIT1i
valoareaindexuluiISFlalotulIIT2(r=0.991;p<0.000);obolaniicareauavutvaloricrescutealeISFla7
zile,lemeninila14zile.ValorileISFntretipuriledeleziunila14zileprezintocorelaiemedie,fapt
censeamncnivelulderegenerareesteasemntor,indiferentdetipuldeleziunefolosit.
Concluzii:1) Leziunea nervoas periferic prin zdrobire este urmat de o regenerare mai rapid,
dectceadupseciuneaisuturaterminoterminalanervului;2)Pentruevaluarearegenerriinervoase
periferice, n leziunile prin zdrobire este recomandat calculul ISF pe baza nregistrrii fotografice; 3)
Modeluldeleziuneperifericnervoasprinzdrobireanervuluisciaticsepreteazpentrustudiulefectelor
imediateaunorfactorisaupreparatecurolnregenerare.

Studiul2.Efectelemelatonineinregenerareanervuluisciatic
Sauadministratip0,01mg/kgcmelatonin.SautilizatpreparatulMellowTonin(Secom).Studiul
saefectuatconformprotocoluluidescrislametodologiegeneral.
Rezultate: comparaia prin testul tStudent pentru egalitatea mediilor (pe eantioane perechi) a
evideniat, la sfritul tratamentului, o diferen nalt semnificativ statistic ntre lotul martor i lotul
tratat cu melatonin (p=0,000), pentru valoarea ISF; dintre parametrii biochimici doar MDA a avut o
evoluie apropiat de limita semnificativitii statistice (p=0,085), indicnd o scdere a nivelului de SO.
TestulMannWhitneynuaevideniatmodificrisemnificativealetimpuluidelatenlastimululalgictermic
(p=0,6501) la nivelul ambelor membre (posterioare drept, operat i stng neoperat). Analiza
histopatologic a artat o regenerare foarte bun, cu foarte puine mononucleare care infiltreaz nervul
sciatic,mielinizareifibrenervoasedecalibruuniform.
Concluzii: 1) Administrarea de melatonin mbuntete funcia motorie n recuperarea unei
leziuninervoaseperiferice;2)ValoareaISFindicorecuperarefuncionalbunspreexcelent;3)Efectul
tratamentului cu melatonin asupra timpului de laten la stimulul algic termic este redus; 4) Aciunea
asuprabalaneisericeO/AOamelatonineideterminnprincipalscdereaSO,pebazascderiiMDA;5)
Melatonina determin o regenerare morfologic nervoas foarte bun; 6) Efectele favorabile asupra
regenerriinervoasealemelatonineisemanifestchiarindozemici;7)Efectelebeneficeantioxidante
ale administrrii de melatonin dup lezarea prin zdrobire a nervului sciatic, sunt prezente pe toat
duratastudiatarecuperriinervoaseperiferice.

Studiul3.Efectelechitosanuluinregenerareanervuluisciatic
Sau administrat ip 1,5 mg/kgc chitosan. Sa utilizat preparatul Chitosan (Solaray). Studiul sa
efectuatconformprotocoluluidescrislametodologiegeneral.
Rezultate: comparaia prin testul tStudent pentru egalitatea mediilor (pe eantioane perechi) a
evideniat, la sfritul tratamentului, o diferen nalt semnificativ statistic ntre lotul martor i lotul
tratatcuchitosan(p=0,000)pentruvalorileISFiaGSH.Timpuldelatenlastimululalgictermicavariat
semnificativ statistic la nivelul membrului posterior drept (p=0,0284) (testul MannWhitney). Analiza
histopatologicaartatprezenaunorzonedeedemcutecidemielindegradate,infiltratcunumeroase
celulemononucleare,puiniaxonivizibili,globuledemielinnceluleSchwanninmacrofage.
Concluzii:1) Administrarea de chitosan mbuntete funcia motorie n recuperarea unei leziuni
nervoaseperiferice;2)ValoareaISFindicorecuperarefuncionalbunspremedie;3)Timpuldelaten
lastimululalgictermicsemodificsemnificativduptratamentulcuchitosan,lanivelulmembruluidrept,
operat; 4) Chitosanul influeneaz balana seric O/AO i determin creterea aprrii AO, pe seama
creterii nalt semnificative a GSH; 5) Efectele morfologice asupra regenerrii nervoase ale chitosanului
suntreduse.

Studiul4.EfectelecoenzimeiQ10nregenerareanervuluisciatic
Sauadministratip0,10mg/kgccoenzimQ10.SautilizatpreparatulCoenzimaQ10(Walmark).
Studiulsaefectuatconformprotocoluluidescrislametodologiegeneral.
Rezultate: comparaia prin testul tStudent pentru egalitatea mediilor (pe eantioane perechi) a
evideniat,lasfritultratamentului,odiferennaltsemnificativstatisticntrelotulmartorilotultratatcu

CoQ10 (p=0,000) pentru valoarea GSH, o corelaie semnificativ statistic pentru MDA (p=0,019) i ISF
(p=0,013)iocorelaieapropiatdelimitasemnificativitiistatistice(p=0,058)pentruSH.Timpuldelaten
la stimulul algic termic a variat semnificativ statistic la nivelul membrului posterior drept (p=0,0073)
(testulMannWhitney).Analizahistopatologicaartatprezenaunorzonecuedemimielindegradat,
fibrenervoasedecalibruneuniform,numeroasemononucleare,proliferaredeceluleSchwann,dispariia
focalaaxonilorcuprezenamielineisubformdeglobule,odiscretreacieconjunctiv.
Concluzii:1) Administrarea de CoQ10 mbuntete funcia motorie n recuperarea unei leziuni
nervoaseperiferice;2)CoQ10areefectefavorabileasuprarecuperriifuncionalechiarindozemici;3)
ISF indic o recuperare funcional bun spre medie; 4) Tratamentul cu CoQ10 modific semnificativ
timpuldelatenlastimululalgictermic,lanivelulmembruluidrept,operat;5)Aciuneaasuprabalanei
sericeO/AOaCoQ10determinnprincipalcretereaaprriiAO,peseamaSHiGSHiscdereaSO,pe
seamaMDA;6)Histopatologic,administrareadeCoQ10determinoregenerareslablanivelulnervului
sciatic.

Studiul5.Efecteleatorvastatineinregenerareanervuluisciatic
Sauadministratip0,15mg/kgatorvastatin.SautilizatpreparatulAtorvastatin (Pfizer).Studiul
saefectuatconformprotocoluluidescrislametodologiegeneral.
Rezultate: comparaia prin testul tStudent pentru egalitatea mediilor (pe eantioane perechi) a
evideniat,lasfritultratamentului,odiferennaltsemnificativstatisticntrelotulmartorilotultratatcu
atorvastatin (p=0,000), pentru valoarea ISF i o diferen semnificativ statistic pentru valorile MDA
(p=0,005),SH(p=0,033)iGSH(p=0,012).Timpulde laten la stimulul algic termic a variat semnificativ
statistic la nivelul membrului posterior drept, operat (p=0,0452) (testul MannWhitney). Analiza
histopatologicaartatoregeneraremaiavansat,puinezonecuedemsaumielindegradat,foarterare
mononucleare, calibru uniform al fibrelor nervoase, teac de mielin uniform cu prezena axonilor n
interior.
Concluzii: 1) Administrarea de atorvastatin mbuntete funcia motorie n recuperarea unei
leziuninervoaseperiferice;2)Atorvastatinaareefectefavorabileasuprarecuperriifuncionalechiarin
doze mici; 3) ISF indic o recuperare funcional medie; 4) Tratamentul cu atorvastatin modific
semnificativ timpul de laten la stimulul algic termic, la nivelul membrului drept, operat; 5) Aciunea
atorvastatineiasuprabalaneisericeO/AOdeterminnprincipalscdereaSOicretereaaprriiAO;6)
Administrareadeatorvastatindeterminoregeneraremorfologicfoartebun.

Studiul6.Efecteleefortuluifizicnregenerareanervuluisciatic
La toate loturile sa efectuat lezarea nervului sciatic prin zdrobire la nivelul membrului posterior
drept.Studiulainclus:lotulmartorsedentar,timpde4sptmnidupintervenie;lotulIantrenatla
efort. Deoarece modificrile degenerative ating nivelul maxim la dou sptmni, dup care ncep s
regreseze,lotultestatlaefortanceputantrenamentuldinziua15,peoduratde14zile(ziua1528). Proba
denotsadesfuratntrunbazincudiminesiunilede100cmlungime/50cmlime/60cmnlime,cu
temperaturaapeimeninutla22Cinivelulapeila30cmnlime.obolaniiaufostantrenaipeparcursul
a 1 or, n 6 sesiuni a cte 5 min/sesiune, cu 5 min pauz ntre ele. Dup intervenie, obolanii au fost
evaluaiprin:determinareaISFnziua1inziua28,laambeleloturi,conformmetodeidescrisenstudiul1;
determinarea timpului de laten la stimulul algic termic la ambele membre posterioare i a parametrilor
balaneiO/AO;analizahistopatologicagraduluiderefaceremorfologicindusdeefortulfizic.
Rezultate: comparaia prin testul tStudent pentru egalitatea mediilor (pe eantioane perechi) a
evideniat,lasfritulantrenamentului,odiferenaproapedelimitasemnificativitiistatistice(p=0,053)
pentruvaloareaISF.Parametriibiochimicinusaumodificatsemnificativstatisticlalotulantrenatlaefort.
Testul MannWhitney nu a evideniat modificri semnificative ale timpului de laten la stimulul algic
termic. Analiza histopatologic a artat prezena unor zone de edem sau mielin degradat, numeroase
celule mononucleare, proliferare de celule Schwann, rare fibroblaste i celule perineuriale, creterea
cantitiidemielinidebrislipidicninteriorulfibrelornervoase,calibruneuniformalfibrelornervoase,
uneoricudispariiaaxonilor,odiscretreaciereparatoriefibroas.
Concluzii:1)Antrenamentulfizicmbunteterecuperareafuncieimotoriiladebutulprocesuluide
regenerare,dupoleziunenervoasperiferic;2)ISFindicorecuperarefuncionalbunspremedie;3)
Antrenamentulefectuatnumodificsemnificativtimpuldelatenlastimululalgictermic;4)Unexerciiu
fizicdenotpedurataa2sptmninumodificsemnificativbalanasericO/AO;5)Antrenamentulefectuat
nustimuleazrefacereamorfologicaleziuniinervoase;6)Efectelebeneficealeunuiantrenamentdenotde
14zile,dupzdrobireanervului,suntprezentenfazatardivarecuperriinervoaseperiferice;7)Exerciiul
fizicreprezintointerveniekinetoterapeutic,cuaplicareindicatdinfazadedebutaregenerriinervoase
periferice,mpreuncualtemijloaceterapeutice.

Concluziigenerale
1. Funcional, se constat pe baza creterii indicelui sciatic funcional, o recuperare motorie
dup leziunea nervoas periferic pentru toate loturile testate, mai exprimat dup administrarea de
melatonin, chitosan, dar prezent i dup tratamentul cu coenzim Q10, atorvastatin i
antrenamentullaefort.
2. Biochimic, diminuarea stresului oxidativ, pe seama scderii malondialdehidei, sa obinut
dup administrarea de coenzim Q10 i atorvastatin, iar creterea aprrii antioxidante, exprimat
prin refacerea rezervei de glutation i grupri sulfhidril, sa realizat dup administrarea de coenzim
Q10,atorvastatinichitosan.
3. Scdereastresuluialgezic,prinreducereatimpuluidelatenlatestulalgictermic,ansoit
scdereastresuluioxidativdupadministrareadecoenzimQ10,chitosaniatorvastatin.
4. Histopatologic, cea mai bun capacitate de refacere a nervului periferic lezat a fost
evideniatdupadministrareademelatoniniatorvastatin;efectelemorfologicealeadministrriide
chitosaniCoQ10nregenerareanervuluiperifericaufostmaireduse.
5. Factoriiantioxidaniutilizaiauefectefavorabilenregenerareafibrelornervoaseperiferice
lezate,celemaibuneefectefuncionale,biochimiceimorfologiceaufostobinutedupadministrarea
demelatoniniCoQ10.

Originalitateaicontribuiileinovatoarealetezei
Originalitatea lucrrii const n: cercetarea metodologic complex, unitar, din punct de vedere
histopatologic, funcional i biochimic a leziunii nervului sciatic; utilizarea unui model original pentru
studiulfuncional(indicelesciaticfuncional,timpuldelatenalgezic),caresadoveditafifuncionalia
permisobinereaderezultatecomparabilecualealtorcercettori;studiereamodificrilorhomeostaziei
redox n leziunile nervoase periferice sub influena unor ageni farmacologici neuroprotectori i cu
proprieti antioxidante (melatonina, chitosanul, coenzima Q10, atorvastatina), precum i sub influena
efortuluifizicmoderat,cafactorantioxidant.
Contribuiileinovatoaresunturmtoarele:
chitosanulafostadministratcaatareip,nusubformdederivai,ceeaceconstituieometod
noudeadministrare.Rezultateleobinuteaufostpromitoare,chiarlaadministrareandozmaimic.
CoQ10 a fost studiat pe modele de boli degenerative ale SNC, n doze mult mai mari (peste
1000x)inuamgsitdateexperimentaledespreefectulCoQ10 asupraregenerriinervoaseperiferice.
Rezultateleobinuteaufostfoartebune,chiariladozamicadministrat.
rezultatele experimentale au aplicabilitate clinic i recomand ca tratamentul chirurgical n
cazul traumatismelor nervoase periferice s fie asociat cu administrarea unor preparate
neuroprotectoare, ca strategie de regenerare i plasticitate nervoas, n recuperarea postlezional.
Aceastadeschidenoiperspectivespreelaborareaunorschemeterapeuticeeficiente,facile,carespoatfi
aplicatenmodcurent,astfelnctsbeneficiezeocategorielargdepacieni.

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UNIVERSITY OF MEDICINE AND PHARMACY IULIU HAIEGANU OF CLUJ-NAPOCA

Abstract of the doctoral thesis

Factors involved in the nerve regeneration


of the peripheral nervous system
Scientific coordinator: Prof. Dr. Simona Tache
Doctoral candidate: Anne-Marie Chindri (married Constantin)
Cluj-Napoca 2012
TABLEOFCONTENTS
ABBREVIATIONS .......................................................................................................................................................... 12
INTRODUCTION ............................................................................................................................................................ 13
CURRENTSTATEOFKNOWLEDGE .................................................................................................... 15
1.Thenervefibers ...................................................................................................................................................... 17
1.1.Generalconsiderations .................................................................................................................................................. 17
1.1.1.Classificationofnervefibers .............................................................................................................................. 18
1.1.2.Structureofnervefibers ..................................................................................................................................... 19
1.1.2.1.Connectivesheath .................................................................................................................................... 19
1.1.2.2.Schwannsheath........................................................................................................................................ 19
1.1.2.3.Myelinsheath ........................................................................................................................................... 19
1.2.Structureoftheperipheralnerve ................................................................................................................................. 20
2.Degenerationandregenerationofnervefibers ............................................................................ 21
2.1.Thedegeneration ........................................................................................................................................................... 21
2.1.1.Walleriandegeneration ...................................................................................................................................... 21
2.1.2.Retrogradedegeneration ................................................................................................................................... 22
2.2.Theregeneration............................................................................................................................................................ 22
2.2.1.Factorsinfluencingperipheralnerveregeneration .......................................................................................... 23
2.2.1.1.Endogenousfactorsthatstimulateperipheralnerveregeneration ..................................................... 24
Geneticfactors ........................................................................................................................................... 24
GrowthFactors .......................................................................................................................................... 26
Endogenousantioxidantsfactors ............................................................................................................. 27
2.2.1.2.Exogenousfactorsthatstimulateperipheralnerveregeneration ........................................................ 28
Exogenousantioxidantsfactors ............................................................................................................... 28
Hormonalsupplements ............................................................................................................................ 29
Vitaminsupplements ................................................................................................................................ 30
Neuroprotectivedrugs .............................................................................................................................. 30
Physicalfactors.......................................................................................................................................... 31
Materialsusedinbioengineering ............................................................................................................. 32
Exercise ...................................................................................................................................................... 32
PERSONALCONTRIBUTION ....................................................................................................................... 33
1.Objectives .................................................................................................................................................... 35
2.GeneralMethodology.............................................................................................................................. 37
2.1.Thetechniqueusedtoinducenerveinjury .................................................................................................................. 37
2.2.Experimentalphysiologicalmethods ........................................................................................................................... 38
2.2.1.Theoriginalfunctionaldeterminationofsciaticfunctionalindex ................................................................... 38
2.2.2.Determinationoflatencytimebypainfulheattest .......................................................................................... 38
2.3.Serumbiochemistry ....................................................................................................................................................... 38
2.3.1.Biologicalsampling ............................................................................................................................................. 38

2.3.2.BiochemicalindicatorsoftheO/AObalance ..................................................................................................... 39
2.4.Histopathologicalanalysis ............................................................................................................................................. 40
2.5.GeneralProtocol ............................................................................................................................................................ 41
2.6.Statisticalanalysis .......................................................................................................................................................... 41

3.Study1Functionalevaluationofperipheralnerveregeneration........................................ 43
3.1.Introduction ................................................................................................................................................................... 43
3.2.Objectives ....................................................................................................................................................................... 44
3.3.Materialsandmethods .................................................................................................................................................. 44
3.4.Results ............................................................................................................................................................................ 46
3.5.Discussions ..................................................................................................................................................................... 48
3.6.Conclusions .................................................................................................................................................................... 50
4.Study2Effectsofmelatonininsciaticnerveregeneration .................................................... 51
4.1.Introduction ................................................................................................................................................................... 51
4.2.Objectives ....................................................................................................................................................................... 52
4.3.Materialsandmethods .................................................................................................................................................. 52
4.4.Results ............................................................................................................................................................................ 52
4.5.Discussions ..................................................................................................................................................................... 58
4.6.Conclusions .................................................................................................................................................................... 60
5.Study3Effectsofchitosaninsciaticnerveregeneration ........................................................ 61
5.1.Introduction ................................................................................................................................................................... 61
5.2.Objectives ....................................................................................................................................................................... 61
5.3.Materialsandmethods .................................................................................................................................................. 62
5.4.Results ............................................................................................................................................................................ 62
5.5.Discussions ..................................................................................................................................................................... 67
5.6.Conclusions .................................................................................................................................................................... 68
6.Study4EffectsofcoenzymeQ10insciaticnerveregeneration............................................ 69
6.1.Introduction ................................................................................................................................................................... 69
6.2.Objectives ....................................................................................................................................................................... 70
6.3.Materialsandmethods .................................................................................................................................................. 70
6.4.Results ............................................................................................................................................................................ 71
6.5.Discussions ..................................................................................................................................................................... 75
6.6.Conclusions .................................................................................................................................................................... 76
7.Study5Effectsofatorvastatininsciaticnerveregeneration ................................................ 77
7.1.Introduction ................................................................................................................................................................... 77
7.2.Objectives ....................................................................................................................................................................... 77
7.3.Materialsandmethods .................................................................................................................................................. 78
7.4.Results ............................................................................................................................................................................ 78
7.5.Discussions ..................................................................................................................................................................... 82
7.6.Conclusions .................................................................................................................................................................... 84
8.Study6Effectsofexerciseinsciaticnerveregeneration ........................................................ 85
8.1.Introduction ................................................................................................................................................................... 85
8.2.Objectives ....................................................................................................................................................................... 86
8.3.Materialsandmethods .................................................................................................................................................. 86
8.4.Results ............................................................................................................................................................................ 87
8.5.Discussions ..................................................................................................................................................................... 91
8.6.Conclusions .................................................................................................................................................................... 92
9.GeneralDiscussion ................................................................................................................................... 93
9.1.SFIAnalysis .................................................................................................................................................................... 93
9.2.Analysisofthelatencytime........................................................................................................................................... 94
9.3.Biochemicalanalysis ...................................................................................................................................................... 95
9.4.Correlationsbetweendeterminedparameters ........................................................................................................... 98
9.5.Histopathologicalanalysis ............................................................................................................................................. 99
10.Generalconclusions ............................................................................................................................ 101
11.Originalityandinnovativecontributionsofthethesis .......................................................... 103
REFERENCES ................................................................................................................................................. 105

Keywords:sciaticnerveregeneration,melatonin,chitosan,CoQ10,atorvastatin,exercise.

INTRODUCTION
Both in everyday life, but more frequently in athletes, there can happen injuries which may be
affectingdifferentperipheralnerves.Themorphologicalandfunctionalrecoveryoftheinjurednervesand
thefactorsthatcanacceleratetheseprocessesbecomeimportantforphysicalactivityandforthewelfare
andqualityoflife.
In an injured nerve fiber, the distal segment, separated from neuronal cell body, undergoes
Walleriandegenerationandtheproximalsegmentsuffersalsoatraumaticdegeneration.
Thefactorsthatstimulateregenerationofperipheralnervefibersareoftwotypes,endogenousand
exogenous. Endogenous factors include the genetic factors and their expression, the growth factors, the
endogenous antioxidants factors. The exogenous administration of various substances (exogenous
antioxidants,hormonalandvitaminsupplements,neuroprotectivedrugs)maypromotetheregeneration
of the peripheral nerve fibers affected by trauma. There are also physical factors (magnetic fields,
electricity)involvedintheperipheralnerveregeneration.Theneuroprotectiveandstimulantseffectsof
theexogenousfactorscanbesuccessfullyusedinclinicalpractice.

PERSONALCONTRIBUTION
Workinghypothesis.Objectives
Inanattempttoestablishaneffectivetherapeuticapproachtoimprovethefunctionalrecoveryof
an injured peripheral nerve, numerous preparations and their routes of administration have been
proposed. This research is based on experimental studies and aims to establish an efficient method to
evaluatethefunctionalrecoveryafteraperipheralnerveinjuryandtohighlightthepotentialroleofsome
antioxidantagentsinpromotingthenerveregeneration.
Theobjectivesoftheexperimentalstudieswere:
1. toestablishanexperimentalmodelofthenerveinjuryintheratsciaticnerve;
2. toevaluatethedegreeoffunctionalrecoveryofinjurednervebyappropriatetests;
3. toassesstheantioxidanttherapyefficiencyofsomepharmacologicalfactorsandexerciseinthe
regenerationofnervefibers;
4. to perform biochemical measurements in order to assess the role of oxidant/antioxidant
(O/AO)balanceincaseofperipheralnervetissuedamage;
5. to study the histopathological changes induced by the regenerative factors used, on
preparationsobtainedaftersacrificingtheratsattheendoftheexperiment.

GeneralMethodology
Experimental studies were conducted from February to May 2012, and were pilot studies
analytical, longitudinal, prospective, lasting 28 days. The research was conducted in the laboratory of
ExperimentalPhysiologyandthe biochemicaldeterminations wereperformed inthe Laboratory forthe
Study of Oxidative Stress, in the Department of Physiology, University of Medicine and Pharmacy "Iuliu
Haieganu".
Weusedwhiterats,Wistar,male,aged16weeks,weighing200300grams.Thegroupsofratswere
operatedonrightposteriorlimb;aftertheexposureofsciaticnerve,wasproducedaninjurybycrushing
the nerve, in a segment of 5 mm, at 11.2 cm proximal to trifurcation, using a haemostatic clamp
maintainedfor15seconds.
The sciatic functional index (SFI), corresponding to the functional gait analysis, was calculated
basedonplantfingerprintsobtainedbyreleasingandmovingtheanimalinsideaglasstunnel.Anoriginal
computerised method was used, which consists of shooting the hind paw using Celestron Digital
Microscope.
ThelatencytimetopainfulheatstimuluswasdeterminedusingtheheatedplateUgoBasil7280,at
53 C. We registered the animal's reaction time on the heated plate, confronted to the thermal stimulus
appliedtotheplantarsurface.

The biological samples of blood were taken from the retrobulbar sinus. We determined the
biochemical indicators of the oxidant/antioxidant (O/AO) balance, for the oxidative stress (OS)
malondialdehyde (MDA) and carbonylated proteins (CP) and for the antioxidant defense (AD) total
sulfhydrylgroups(SH)andglutathione(GSH).
For the histopathological examinations, the samples were processed by paraffin inclusion
techniqueandbysectioningonicetechnique.Longitudinaland transversesectionsofsciaticnervefrom
each rat, from the lesion site, were stained H&E and Oil Red O and examined under the optical and
confocalmicroscope.
The general protocol for studies 25 was: in all groups (n = 10 animals/group) was performed
sciatic nerve injury, by crushing, at right posterior limb level. The study included: the control group
sedentary for 4 weeks after surgery, the group I who received, by ip injection, the pharmacological
preparation,4weeksaftersurgery.Ratswereevaluatedbydeterminationof:SFIonday1andday28in
both groups, as described in study 1; latency time to painful heat stimulus to both posterior limbs;
parameters of the O/AO balance and by the histopathological analysis of the morphological recovery
degreeinducedbythepreparationstudied.
ThestatisticalanalysisusedtheStudentttestforpairedandindependentsamples,theanalysisof
variance ANOVAFisher, the posthoc test with Scheffe correction, the MannWhitney test for data with
nonGaussiandistribution,theKruskalWallistestforcomparisonsbetween3orseveralgroupsandthe
Pearsoncorrelationcoefficient.Fordisplayingdatawereusedtablesandgraphssuchasboxplot.

Study1.Functionalevaluationofperipheralnerveregeneration
Inordertoestablishtheexperimentalmodel,asciaticnervesectioningandsuturingwasperformed
also.Weused2groupsof15animals,inwhich,afterthesciaticnerveinjurybysectioningandsuturing,
respectively, by crushing, the rats were kept at rest for 28 days. We asses functionally the peripheral
nerveregeneration,basedonSFIdeterminationandanalyzedcomparativelythefunctionalassessmentfor
crushingandnervesectioninginjuries(groupIT1nervecut,1week;groupIT2nervecut,2weeks;
groupIIT1nervecrushed,1week;groupIIT2nervecrushed,2weeks).
Results:thettest(Student)pairsshowedstatisticallysignificantincreasesofSFIvalues,bothingroup
IT2comparedtogroupIT1(p=0.000)andinthegroupIIT2comparedtogroupIIT1(p=0.000).Also,
there was a statistically significant correlation between the SFI index value in group I T1 and SFI index
valueingroupIT2(r=0.54,p<0.04),respectivelybetweenSFIindexvalueingroupIIT1andSFIindex
valueingroupIIT2(r=0.991,p<0.000);theratswhichhadelevatedSFIvaluesat7days,maintainthem
also at 14 days. The SFI values between the types of lesions at 14 days displayed an average correlation,
whichmeansthattheregenerationlevelissimilar,regardlessofthetypeoflesionused.
Conclusions:1)Theperipheralnerveinjurybycrushingisfollowedbyafasterrecoverythanthe
onebysectioningandendtoendnervesuture,2)Inordertoassesstheperipheralnerveregenerationin
crush injuries is recommended the SFI calculation based on photographic recording; 3) The model of
peripheral nerve injury by crushing the sciatic nerve is suitable for studying the immediate effects of
factorsandpreparationsinvolvedinregeneration.

Study2.Melatonineffectsinsciaticnerveregeneration
Weadministeredip0.01mg/kgmelatonin.WeusedthepreparationMellowTonin(SECOM).The
studywasperformedaccordingtotheprotocoldescribedinthegeneralmethodology.
Results:thecomparisonbyStudentttestfortheequalityofmeans(onsamplepairs)found,atthe
end of treatment, a statistically highly significant difference between the control group and the group
treatedwithmelatonin(p=0.000)forSFIvalue;betweenthebiochemicalparameters,onlyMDAhasfared
closetothelimitofstatisticalsignificance(p=0.085),indicatingadecreaseintheSOlevel.MannWhitney
test showed no significant change in latency time to painful heat stimulus (p=0.6501) in both limbs
(posteriors right, operated and left, unoperated). Histopathological analysis showed a very good
regeneration, with very few mononuclear cells infiltrating the sciatic nerve, myelination and uniform
diameterofnervefibers.

Conclusions: 1) The administration of melatonin improves the recovery of motor function in


peripheral nerve injuries, 2) The SFI value indicates a good to excellent functional recovery, 3) The
melatonintreatmenteffectonlatencytimetopainfulheatstimulusisreduced,4)Themelatoninactionon
serum O/AO balance determined mainly a decrease of OS, based on MDA decrease; 5) The melatonin
produce a very good morphological nerve regeneration; 6) The favorable effects of melatonin on nerve
regeneration occurs even in small doses; 7) The beneficial antioxidant effects dues to melatonin
administration, after sciatic nerve crushing injury, are present throughout the whole studied period of
peripheralnerverecovery.

Study3.Effectsofchitosaninsciaticnerveregeneration
Weadministeredip1.5mg/kgchitosan.WeusedthepreparationChitosan(Solaray).Thestudy
wasperformedaccordingtotheprotocoldescribedinthegeneralmethodology.
Results:thecomparisonbyStudentttestfortheequalityofmeans(onsamplepairs)found,atthe
end of treatment, a statistically highly significant difference between the control group and the group
treated with chitosan (p=0.000) for the SFI and GSH values. The latency time to painful heat stimulus
variedsignificantlystatisticatthelevellevelofposteriorrightlimb(p=0.0284)(MannWhitneytest).The
histopathologicalanalysisshowedthepresenceofedemaareaswithdegradedmyelinsheaths,infiltrated
with numerous mononuclear cells, few visible axons, the myelin globules inside Schwann cells and
macrophages.
Conclusions: 1) The use of chitosan improves the recovery of motor function in peripheral nerve
injuries,2)TheSFIvalueindicatesagoodtoaveragefunctionalrecovery,3)Thelatencytimetopainful
heat stimulus changes significantly after treatment with chitosan, at the level of operated right limb, 4)
The chitosan influences the serum O/AO balance and produces an increase of AD, due to a highly
significantincreaseofGSH;5)Themorphologicaleffectsofchitosaninnerveregenerationarereduced.

Study4.EffectsofcoenzymeQ10insciaticnerveregeneration
We administered ip 0.10 mg / kg coenzyme Q10. We used the preparation Coenzyme Q10
(Walmark).Thestudywasperformedaccordingtotheprotocoldescribedinthegeneralmethodology.
Results:thecomparisonbyStudentttestfortheequalityofmeans(onsamplepairs)found,attheend
of treatment, a statistically highly significant difference between the control group and the group treated
with CoQ10 (p = 0.000) for GSH value, a statistically significant correlation for MDA (p=0.019) and SFI
(p=0.013)valuesandacorrelationclosetostatisticalsignificancelimit(p=0.058)forSHvalue.Thelatency
time to painful heat stimulus varied significantly statistic at the level of posterior right limb (p=0.0073)
(MannWhitney test). The histopathological analysis showed the presence of some areas with edema and
degradedmyelin,unevencalibernervefibers,numerousmononuclearcells,proliferationofSchwanncells,
focaldisappearanceofaxonswithpresenceofmyelinglobules,adiscreteconnectivetissuereaction.
Conclusions:1)TheadministrationofCoQ10improvestherecoveryofmotorfunctioninperipheral
nerveinjuries,2)CoQ10hasfavorableeffectsonfunctionalrecoveryeveninsmalldoses,3)SFIindicates
agoodtoaveragefunctionalrecovery,4)ThetreatmentwithCoQ10significantlymodifythelatencytime
to painful heat stimulus, at the level of operated right limb, 5) The CoQ10 action on the O/AO serum
balancedeterminesmainlyanincreaseofAD,onaccountofSHandGSHandadecreaseofSO,onbehalfof
MDA,6)Histopathologically,theadministrationofCoQ10resultsinpoorregenerationofsciaticnerve.

Study5.Effectsofatorvastatininsciaticnerveregeneration
Weadministeredip0.15mg/kgatorvastatin.WeusedthepreparationAtorvastatin(Pfizer).The
studywasperformedaccordingtotheprotocoldescribedinthegeneralmethodology.
Results:thecomparisonbyStudentttestfortheequalityofmeans(onsamplepairs)found,atthe
end of treatment, a statistically highly significant difference between the control group and the group
treated with atorvastatin (p=0.000) for SFI value, a statistically significant difference for the MDA
(p=0.005), SH (p=0.033) and GSH (p=0.012) values. The latency time to painful heat stimulus varied
statisticallysignificantatthelevelofposteriorrightlimb,operated(p=0.0452)(MannWhitneytest).The

histopathological analysis showed a more advanced regeneration, few areas with edema or degraded
myelin,veryraremononuclearcells,uniformcalibernervefibers,uniformmyelinsheathconsistentwith
thepresenceofaxonswithin.
Conclusions: 1) The administration of atorvastatin improves the recovery of motor function in
peripheralnerveinjuries,2)Atorvastatinhasfavorableeffectsonfunctionalrecoveryeveninsmalldoses,
3) SFI value indicates an average functional recovery, 4) The treatment with atorvastatin significantly
modifythelatencytimetopainfulheatstimulus,atthelevel ofoperatedrightlimb,5)Theatorvastatin
action on O/AO serum balance determines mainly a decrease of OS and an increase of AD, 6) The
administrationofatorvastatincausesaverygoodmorphologicalregeneration.

Study6.Effectsofexerciseinsciaticnerveregeneration
Inallgroupswasperformedthesciaticnervedamagebycrushingthenerveattherightrearlimb.
The study included: the control group sedentary for 4 weeks after surgery, the group I trained to
swimming exercise. Because the degenerative changes touch the peak level at two weeks, after that
startingtoregress,theexercisetestedgroupbegantrainingat15thdayaftersurgery,foraperiodof14
days (day 1528). The swimming test was conducted in a 100 cm lenght/50 cm width/60 cm height
swimmingpool,watertemperaturemaintainedat22Candthewaterlevelat30cmheight.Theratswere
trained over 1 hour, in 6 sessions of 5 min/session, 5 min break between. After surgery, rats were
evaluatedbydeterminationof:SFIonday1andday28inbothgroups,asdescribedinstudy1;latency
time to painful heat stimulus to both rear limbs; serum parameters of the O/AO balance;
histopathologicalanalysisofthedegreeofexerciseinducedmorphologicalrecovery.
Results:thecomparisonbyStudentttestfortheequalityofmeans(onsamplepairs)found,atthe
end of training, a difference close to the statistical significancy level (p=0.053) for the SFI value. The
biochemicalparameterswerenotsignificantlyalteredintheexercisetrainedgroup.TheMannWhitney
testshowednosignificantchangesinlatencytimetopainfulheatstimulus.Thehistopathologicalanalysis
showed the presence of areas with edema and degraded myelin, numerous mononuclear cells,
proliferationofSchwanncells,rarefibroblastsandperineurialcells,increasedmyelinquantitiesandlipid
debrisinsidenervefibers,unevencaliberofnervefibers,sometimeswithlossofaxons,adiscretefibrous
reparativeresponse.
Conclusions: 1) The physical training improves motor function recovery at the onset of
regeneration process, after a peripheral nerve injury, 2) SFI indicates a good to average functional
recovery, 3) The conducted training does not significantly alter the latency time to the thermal painful
stimulus;4)Aswimmingexerciseduringthetwoweeksdidnotsignificantlyalterthebalanceofserum
O/AO, 5) The conducted training does notstimulatethe morphological recoveryof nerve lesion; 6) The
beneficial effects of a 14 days swimming training, after nerve crush, are present in the late phase of
peripheralnerverecovery;7)Theexerciserepresentakinetotherapeuticalintervention,withapplication
indicatedfromthefirstphaseofperipheralnerveregeneration,togetherwithothertherapeuticmeans.

Generalconclusions
1.Functionally,basedonthesciaticfunctionalindexincrease,itispresentamotorrecoveryafter
the peripheral nerve injury for all groups tested, more expressed after the administration of melatonin,
chitosan,butalsopresentafterthetreatmentwithcoenzymeQ10,atorvastatinandtrainingexercise.
2. Biochemically, the decrease of the oxidative stress, based on the decrease of malondialdehyde,
wasobtainedaftertheadministrationofcoenzymeQ10andatorvastatin,andtheincreaseofantioxidant
defense,expressedbytherestoringofglutathionereservesandsulfhydrylgroups,wasobtainedafterthe
administrationofcoenzymeQ10,atorvastatinandchitosan.
3.Thedecreaseofalgezicstress,byreductionoflatencytimetothermalpaintest,accompaniedthe
decreaseinoxidativestressaftertheadministrationofcoenzymeQ10,chitosanandatorvastatin.
4. Histopathologically, the best ability of recovering an injured peripheral nerve was evidenced
aftertheadministrationofmelatoninandatorvastatin;themorphologicaleffectsofchitosanandCoQ10
administrationinperipheralnerveregenerationwerelower.

5. The used antioxidants factors have beneficial effects in injured peripheral nerve fibers
regeneration, the best functional, biochemical and morphological effects were obtained after
administrationofmelatoninandCoQ10.

Originalityandinnovativecontributionsofthethesis
The originality of this work consists in: a complex, unitary methodological research of the sciatic
nervelesion,intermsofhistological,functionalandbiochemicalnotions;theuseofanoriginalmodelfor
the functional study (sciatic functional index, pain latency time), which proved to be functional and
allowed us to obtain comparable results with other researchers; the study of changes in redox
homeostasis in peripheral nerve lesions under the influence of neuroprotective pharmacological agents
with antioxidant properties (melatonin, chitosan, coenzyme Q10, atorvastatin) and also under the
influenceofmoderateexerciseasaantioxidantfactor.
Innovativecontributionsareasfollows:
chitosan was administered ip assuch,notderivative, which is a new methodof administration.
Theresultswerepromising,evenatlowerdoseadministration.
CoQ10hasbeenstudiedinmodelsofCNSdegenerativediseases,indosesmuchhigher(1000x)
and I have not found experimental data on the effect of CoQ10 on peripheral nerve regeneration. The
resultswereverygood,evenatthelowdoseadministered.
theseexperimentalresultshaveclinicalapplicabilityandrecommendthesurgicaltreatmenttobe
associatedwiththeuseofneuroprotectivedrugs,incaseofperipheralnerveinjuries,asabetterstrategy
toinsurenerveregenerationandplasticity,inpostlesionalrecovery.Thisopensnewperspectivesforthe
developmentofeffectivetherapeuticregimens,easy,whichcanbeappliedroutinelyinordertobenefita
widerangeofpatients.

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