Neurodegeneration und Neurogenese in organotypischen hippokampalen Schnittkulturen nach Hypoxie, Hypoglykämie [Elektronische Ressource] / von Olga Chechneva

Neurodegeneration und Neurogenese in organotypischen hippokampalen Schnittkulturen nach Hypoxie, Hypoglykämie [Elektronische Ressource] / von Olga Chechneva

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Neurodegeneration und Neurogenese in organotypischen hippokampalen Schnittkulturen nach H ypoxie/H ypoglykämieD i s s e r t a t i o nzur Erlangung des akademischen Gradesdoctor rerum naturalium(Dr. rer. nat.)genehmigt durchdie Fakultät für N aturwissenschaftender O tto-von-Guericke-Universität Magdeb urg,von Diplom-Pharmazeutin O lga Chechnevageb . am 1 .07.1 977, in Stary O skol, R ußlandGutachter: Prof. Dr. K laus G. R eymann Prof. Dr. Dr. O liver Ullrich Prof. Dr. Frank EmmrichEingereicht am: 25.1 0.2005V erteidigung am: 9.05.20061Neurodegeneration and neurogenesis in organotypic hippocampal slice cultures after oxygen and glucose deprivationD i s s e r t a t i o nT o the acquisition of the academic degreeDoctor rerum naturalium(Dr. rer. nat.)approved b yT he Faculty of N atural ScienceO tto-von-Guericke-Universität Magdeb urgfrom Master of Science in Pharmacy O lga Chechnevab orn on 1 .07.1 977, in Stary O skol, R ußlandR eviewers: Prof. Dr. K laus G. R eymann Prof. Dr. Dr. O liver Ullrich Prof. Dr. Frank EmmrichSub mitted on: 25.1 0.2005Defence on: 9.05.20062D edicatedT o my motherfor her love, care and patience3AcknowledgmentT his work was pe rformed duri ng the time from May 2002 till O ctobe r 2005 in L eibni z Ins titute for N eurobi ology, Project Group N europha rmacology he aded by Prof. Dr.

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Publié le 01 janvier 2006
Nombre de lectures 21
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Neurodegeneration und Neurogenese in organotypischen hippokampalen
Schnittkulturen nach H ypoxie/H ypoglykämie
D i s s e r t a t i o n
zur Erlangung des akademischen Grades
doctor rerum naturalium
(Dr. rer. nat.)
genehmigt durch
die Fakultät für N aturwissenschaften
der O tto-von-Guericke-Universität Magdeb urg,
von Diplom-Pharmazeutin O lga Chechneva
geb . am 1 .07.1 977, in Stary O skol, R ußland
Gutachter: Prof. Dr. K laus G. R eymann
Prof. Dr. Dr. O liver Ullrich
Prof. Dr. Frank Emmrich
Eingereicht am: 25.1 0.2005
V erteidigung am: 9.05.2006
1Neurodegeneration and neurogenesis in organotypic hippocampal slice
cultures after oxygen and glucose deprivation
D i s s e r t a t i o n
T o the acquisition of the academic degree
Doctor rerum naturalium
(Dr. rer. nat.)
approved b y
T he Faculty of N atural Science
O tto-von-Guericke-Universität Magdeb urg
from Master of Science in Pharmacy O lga Chechneva
b orn on 1 .07.1 977, in Stary O skol, R ußland
R eviewers: Prof. Dr. K laus G. R eymann
Prof. Dr. Dr. O liver Ullrich
Prof. Dr. Frank Emmrich
Sub mitted on: 25.1 0.2005
Defence on: 9.05.2006
2D edicated
T o my mother
for her love, care and patience
3Acknowledgment
T his work was pe rformed duri ng the time from May 2002 till O ctobe r 2005 in L eibni z
Ins titute for N eurobi ology, Project Group N europha rmacology he aded by Prof. Dr. K .G.
Re ymann. T he study was supported by gra dua te progra m "Biologische Grundl age n von
Erkra nkunge n des N erve nsystems", O tto-von-G ue ricke -Unive rsity, M agde burg and the
Europe an G ra nt Q L K 3-CT-2001-00407.
Here , I would like to tha nk all of the pe ople who ha ve he lpe d duri ng my studi es for
making m y e xpe rienc e i n t he l ab bot h e duc ationa l and pl easura bl e.
First of all I would like to tha nk my supe rvisor Prof. Dr. K .G. Re ymann, for giving me
the opportunity to do my PhD in his group. I sinc ere ly appre ciate his sugge stions, he lp and
support.
I would like to acknowledge the enthusiasm and critic of Prof. Dr. G. Re iser, he ad of
Gra dui ertenkolleg. I t ha nk a lso a ll s tude nts of G ra dui ertenkolleg for s upport and fri ends hip.
I am espe cially tha nkful to Dr. K . Dinke l for his exc ellent guidanc e, support and
enc oura ge ment. I would like to expre ss my large st gra titude to Dr. Fabi o Ca va liere for his
intere st in this work, inva lua bl e assistanc e for the PCR expe riments and slice pre pa ra tion,
he lpful sugge stions, advi ces and numerous discussions. I am gra teful also to Dr. M.
Stra ßburge r for he lp, s upport and re view of t he m anuscript.
M y appre ciation also goe s to Dr. Ulrich Schröde r for his kind he lp in tra nslation of
abs tra ct i n G erman a nd t o M . Roge rs for hi s corre ctions and s pe ll che ck.
M y tha nks are applied to in vitro team, espe cially Diane M undi l and Susanne von
K enne for t he ir e xc ellent t echnical assistanc e and fri endl y s upport.
I tha nk Prof. Dr. Dr. O . Ullrich for giving us possibi lity to use re al-time PCR machine
and G. W eitz for t echnical assistanc e. A lso I want t o t ha nk K. Böhm for c ell qua ntification.
I would like to tha nk for othe r technical support from Dr. K . Ba ldauf, M. T ulapurka r,
Dr. G. Zündorf, Dr. M . M artine z-S anc he z and A. L askowski, Dr. M . Ri ek as well as all othe r
4colleague s in Ins titute for Applied N euroscienc e for the good atmosphe re and the ir friendl y
coope ra tion.
I am gre atly inde bt ed to Dr. O .Y. K udri zka ya , Dr. N .Y. Frolova and Prof. E.E .
L esiovska ya , ga ve m e t he fi rst know ledge i n s cienc e and w ho gui ded m y w ay t o G ermany.
I expre ss my sinc ere tha nks to my friends D. M akhra che va -S tepoc hkina , C. Bühne mann
and A. Pivova rova for be ing m y fri ends .
I am hi ghly gra teful t o P rof. D r. H. Neumann for hi s unde rstandi ng.
5Abstract
Adul t neurogenesis plays a role in many physiological (memory formation) and
pathological (stroke, depression) processes. Increased neurogenesis in response to b rain injury
was recently reported and is considered a mechanism of regeneration after neuronal loss. T here
are evidences that numerous factors like glutamate, inflammation, and growth factors are
involved in the post-insult increase of precursors proliferation and differentiation. However the
mechanism of injury-induced neurogenesis is still poorly understood.
In the pre sent study early ne uroge ne sis in vitro in ra t orga notypic hippoc ampa l slice
culture s (OHC) was cha ra cterized and the complex interplay be tween ne urona l damage ,
microglia activa tion, cell prolife ra tion, ne uroge ne sis and the role of inflammation after
oxyge n-gl uc ose depriva tion (OGD) was inve stiga ted. In addition the effe ct of exoge nous
growth fa ctors, ba sic fibrobl ast growth fa ctor (bFGF), epidermal growth fa ctor (E GF), bra in-
derive d ne urotrophic fa ctor (BD N F) and ne rve growth fa ctor (NGF) on prolife ra tion and
ne uroge ne sis i n O HC i n t he abs enc e and pre senc e of i njury w as s tudi ed.
Proliferation was assessed b y b romodeoxyuridine (BrdU) incorporation, neurogenesis b y
BrdU-doub le lab eling with doub lecortin (DCX ) or ß-III T ub ulin, neuronal damage b y
propidium iodide uptake, microglia b y O X -42 staining, pro-inflammatory cytokines b y real-
time R T -PCR .
Massive cell proliferation was ob served in the glial envelope covering the slice. Glial
envelope was formed b y GFAP or GFAP/N estin positive astrocytes and activated microglia
during slice cultivation. T he proliferation was lower inside of the slice culture. b FGF and EGF
showed mitogenic properties in our system b y increasing the numb er of BrdU+ cells.
In addition to the dentate gyrus (D G) O HC inc lude d a second ne uroge nic zone : the
posterior pe rive ntricle (pP V ), which is a pa rt of the latera l ve ntricle wall. T his struc ture lining
the stra tum oriens containe d N estin+ pre cursors. Morphological and func tiona l diffe re nc es
6could be identified be tween DG and pP V pre cursor populations. N estin+ cells with stellar
morphology were found in the DG, while elonga ted N estin+ cells were pre sent in the pP V .
bF GF tre atment induc ed a fa st but short-lasting ne uroge nic re sponse in the DG while the pP V
showed a m ore pronounc ed and l ong l asting ne uroge nic effe ct of bF GF.
After exposure of O HC to 40 min O GD microglia activation and upregulation of IL -1 ß,
T N F- and IL -6 mR N A (2h after O GD) preceded the development of neuronal damage (6h
after O GD) that was followed b y an increase in cell proliferation (1 6h after O GD). BDN F,
EGF or N GF treatment attenuated O GD-induced proliferation. N eurogenesis was inhib ited
at 3d after O GD in b oth neurogenic zones, however the restoration of neurogenesis was
already ob served at 6d. At this time point a significant increase of newly generated neurons
was found in the pPV compared to control. N umb er of BrdU/ß-III T ub ulin+ neurons was
significantly increased in the pPV of O GD-exposed O HC b y b FGF application.
MK -801 , indomethacin or minocycline prevented the O GD-induced neuronal damage, cell
proliferation and caused a decrease of O X -42+ microglia in the damaged area. Under
control conditions MK -801 , indomethacin o r m inocycline i nduced neurogenesis i n t he p PV .
After O GD numb er of BrdU/DCX + cells was decreased in the pPV b y MK -801 treatment.
However indomethacin or minocycline did not affect O GD-induced neurogenesis in the
pPV .
In conclusion, this study is the first to show that i) two neurogenic zones, the DG and the
pPV , are present in interface O HC; ii) the DG and pPV contain neural precursors with
different neurogenic properties. High neurogenesis occur in the pPV while low
neurogenesis is present in the DG of O HC; iii) inflammation is mounted in O HC at early
time point after O GD. It is associated with activation, migration and proliferation of
microglia; iv) due to microenvironmental changes neurogenesis in b oth neurogenic zones is
inhib ited early after O GD (3d) and restored later on (6d). O GD stimulates neurogenesis in
7
athe pPV ; v) neuroprotection against O GD-induced damage in O HC b y anti-inflammatory
treatment is associated with intact neurogenesis. T aking together, these in vitro data
represent the evidences of injury-regulated neurogenesis in relation with inflammation in
O HC that could b e useful for further understanding of mechanisms of neurogenesis and
anti-inflammatory treatment strategies after cereb ral ischemia.
8Table of Contents
1. I ntroduction ......................................................................................................................... 12
1.1. N euroge ne sis i n t he CN S ............................................................................................................ 12
1.1.1. N euroge ne sis i n t he s ubve ntricular z one ................................................................ 13
1.1.2. Neuroge ne sis i n t he hi ppoc ampus .......................................................................... 13
1.1.3. S tem cells and t he ir ori gin ...................................................................................... 13
1.1.4. E xpe rimental de tection of c ell phe notype ............................................................... 14
1.1.5. F actors re gulating ne uroge ne sis .............................................................................. 15
1.1.6. G rowth fa ctors ........................................................................................................ 16
1.2. C erebr al i sche mia ........................................................................................................................ 18
1.2.1. P athophysiology of i sche mic cell de ath .................................................................. 18
1.2.2. N euroge ne sis after c ere bra l i sche mia ..................................................................... 20
1.3. Inflammation a fter cerebr al i sche mia .......................................................................................... 22
1.3.1. Mecha nism of i nflammation ................................................................................... 22
1.3.2. A nti-inflammatory m ediators and t re atment ........................................................... 23
1.3.3. Infl ammation a nd ne uroge ne sis .............................................................................. 24
1.4. Orga notypic hi ppoc ampa l s lice cultures ..................................................................................... 25
1.5. A im of t he pr oject ....................................................................................................................... 26
2. Materials an d M ethods ....................................................................................................... 28
2.1. M aterials ...................................................................................................................................... 28
2.1.1. Lab i nstruments and m aterials ................................................................................ 28
2.1.2. Che micals ................................................................................................................ 29
2.1.3. A ntibodi es ............................................................................................................... 30
2.1.4. Ce ll culture m edium and compone nts .................................................................... 31
2.1.5. Buffe rs and s olutions .............................................................................................. 31
2.1.6. P rimer s eque nc es .................................................................................................... 32
2.2. M ethods ....................................................................................................................................... 32
2.2.1. T issue pre pa ra tion ................................................................................................... 32
2.2.1.1. P re pa ra tion of orga notypic hi ppoc ampa l s lice culture s ....................................... 32
2.2.1.2. P re pa ra tion of orga notypic cortex-hi ppoc ampa l culture s ................................... 33
2.2.2. O xyge n a nd gl uc ose de priva tion ............................................................................. 33
2.2.3. E xpe rimental s etups ................................................................................................ 34
2.2.3.1. Ce ll prol ife ra tion a nd ne uroge ne sis i n O HC ........................................................ 34
2.2.3.2. G rowth fa ctor t re atment ...................................................................................... 34
2.2.3.3. Ce ll damage , m icroglia, c ell prol ife ra tion a nd pro-i nflammatory c ytokine s after
O GD .................................................................................................................................. 34
2.2.3.4. Ce ll prol ife ra tion a nd ne uroge ne sis after O GD ................................................... 35
2.2.3.5. G rowth fa ctor t re atment after O GD ..................................................................... 36
2.2.3.6. A nti-inflammatory c ompounds t re atment ............................................................ 36
2.2.4. Ce ll damage ............................................................................................................ 37
2.2.5. Im munohistoc he mistry ........................................................................................... 37
2.2.5.1. M icroglia ............................................................................................................. 37
9 2.2.5.2. P rolife ra ting c ells ................................................................................................. 38
2.2.5.3. N ewly ge ne ra ted neurons ..................................................................................... 38
2.2.5.4. O the r c ell t ype s .................................................................................................... 39
2.2.6. Is olation of RN A 40
2.2.7. Re al-time re ve rse t ra nscription pol ymera se cha in re action (re al t ime RT -PCR) ... 40
2.2.7. Ce ll qua ntification a nd s tatistical ana lysis .............................................................. 41
3. Results .................................................................................................................................. 43
3.1. Cha racterization of OHC ............................................................................................................. 43
3.1.1. Ide ntification a nd i n s itu di stribut ion of CN S cell t ype s i n O HC ........................... 43
3.1.2. D istribut ion a nd a ppe ara nc e of prol ife ra ting c ells i n O HC .................................... 44
3.1.3. Ce ll prol ife ra tion a nd grow th fa ctor t re atment ...................................................... 45
3.2. N euroge ne sis i n O HC .................................................................................................................. 47
3.2.1. Ide ntification of a s econd ne uroge nic z one i n O HC ............................................... 47
3.2.2. Q ua litative compa rison of ne uroge ne sis i n t he DG and t he pP V of O HC .............. 50
3.2.2.1. N eurons ge ne ra ted in t he DG assumed a gra nular c ell m orphology a nd s howed
mostly a punc tua ted nuc lear BrdU pa ttern ....................................................................... 50
3.2.2.2. N eurons from pP V w ere pre sent i n t he pP V and t he s tra tum ori ens and ha d
eve nly s taine d BrdU+ nuc lei ............................................................................................ 51
3.2.3. Q ua ntitative compa rison of ne uroge ne sis i n t he DG and t he pP V ......................... 53
3.2.4. T he DG and t he pP V pre cursors s howed a diffe re nt re sponse t o grow th fa ctor
tre atment ........................................................................................................................... 54
3.3. C ell damage , m icroglia activa tion a nd cell pr oliferation a fter OGD .......................................... 56
3.3.1. Ce ll damage after O GD .......................................................................................... 56
3.3.2. A ctiva tion of m icroglia after O GD ......................................................................... 58
3.3.3. Ce ll prol ife ra tion a fter O GD ................................................................................... 59
3.3.4. E ffe ct of grow th fa ctors on c ell prol ife ra tion a fter O GD ....................................... 61
3.4. Neuroge ne sis i n O HC after OGD ................................................................................................ 62
3.4.1. bF GF i nc re ases ne uroge ne sis i n t he pP V after O GD .............................................. 63
3.5. A nti-inflammatory t reatment ....................................................................................................... 65
3.5.1. A nti-inflammatory t re atment re duc es ne urona l damage after O GD ....................... 65
3.5.2. A nti-inflammatory t re atment re duc es t he num be rs of m icroglia after O GD ......... 66
3.5.3. A nti-inflammatory t re atment re duc es cell prol ife ra tion a fter O GD ...................... 67
3.5.4. A nti-inflammatory t re atment and ne uroge ne sis after O GD .................................... 68
3.5.4.1. M K -801 t re atment and ne uroge ne sis ................................................................... 68
3.5.4.2. A nti-inflammatory t re atment and ne uroge ne sis ................................................... 68
4. Discussion ............................................................................................................................. 71
4.1. G lial enve lope and pr oliferating c ells i n O HC ............................................................................ 71
4.2. I dentification of a s econd ne uroge nic z one i n O HC and m orphological differenc es of DG and
pP V pr ecursors ................................................................................................................................... 72
4.3. F unc tiona l differenc es of DG and pP V pr ecursors ..................................................................... 73
4.3.1. Low ne uroge ne sis i n t he DG compa re d to hi gh ne uroge ne sis i n t he pP V .............. 73
4.3.2. P unc tua ted pa ttern of BrdU + nuc lei of ne wly ge ne ra ted neurons i n t he DG
10