UNIVERSIDADE ESTADUAL DE CAMPINAS INSTITUTO DE BIOLOGIA
LUCIANA POLITTI CARTAROZZI
ESTUDO DOS MECANISMOS IMUNOMODULADORES EXERCIDOS PELAS CÉLULAS TRONCO MESENQUIMAIS SOBRE A REATIVIDADE DAS CÉLULAS
GLIAIS E CORRELAÇÃO COM A CAPACIDADE REGENERATIVA APÓS AXOTOMIA DE RAÍZES LOMBARES
IMMUNOMODULATORY MECHANISMS OF MESENCHYMAL STEM CELLS IN GLIAL REACTIVITY AND CORRELATION WITH REGENERATIVE CAPACITY
AFTER LUMBAR ROOT AXOTOMY
Campinas 2018
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LUCIANA POLITTI CARTAROZZI
IMMUNOMODULATORY MECHANISMS OF MESENCHYMAL STEM CELLS IN GLIAL REACTIVITY AND CORRELATION WITH REGENERATIVE CAPACITY
AFTER LUMBAR ROOT AXOTOMY
ESTUDO DOS MECANISMOS IMUNOMODULADORES EXERCIDOS PELAS CÉLULAS TRONCO MESENQUIMAIS SOBRE A REATIVIDADE DAS CÉLULAS
GLIAIS E CORRELAÇÃO COM A CAPACIDADE REGENERATIVA APÓS AXOTOMIA DE RAÍZES LOMBARES
Thesis presented to the Institute of Biology of the University of Campinas in partial fulfillment of the requirements for the degree of Doctor in Structural and Functional Biology, in Cell Biology.
Tese apresentada ao Instituto de Biologia da Universidade Estadual de Campinas como parte dos requisitos exigidos para a obtenção do Título de Doutora em Biologia Celular e Estrutural, em Biologia Celular.
Orientador: Prof. Dr. Alexandre Leite Rodrigues de Oliveira Co-orientador: Prof. Dr. Frank Kirchhoff
Campinas 2018 ESTE ARQUIVO DIGITAL CORRESPONDE À VERSÃO FINAL DA TESE DEFENDIDA PELA ALUNA LUCIANA POLITTI CARTAROZZI E ORIENTADA PELO PROF. DR. ALEXANDRE LEITE RODRIGUES DE OLIVEIRA.
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Campinas, 26 de fevereiro de 2018.
COMISSÃO EXAMINADORA
Prof. Dr. Alexandre Leite Rodrigues de Oliveira Profa. Dra. Marimélia Porcionatto
Prof. Dr. Enrico Ghizoni
Prof. Dr. Antônio Carlos Pinheiro de Oliveira Prof. Dr. Thiago Luiz de Russo
Os membros da Comissão Examinadora acima assinaram a Ata de defesa, que se encontra no processo de vida acadêmica do aluno.
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DEDICATÓRIA
Ao meu avô Roberto.
To my grandfather Roberto.
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AGRADECIMENTOS
Ao meu orientador, Prof. Alexandre, por todo suporte, ensinamentos e cuidado todos esses anos. Obrigada pelas oportunidades e pela amizade. Eu te admiro e serei sempre grata.
Ao meu co-orientador, Prof. Frank, por me receber em seu laboratório diversas vezes. Você fez possível uma grande experiência pessoal e profissional, obrigada.
A todos meus amigos e colegas do LRN, os que já passaram e os que ainda estão aqui. O laboratório é minha segunda casa desde 2007 e eu gostaria de agradecer a todos por fazerem parte disto.
Aos meus queridos amigos Matheus, Gabriela, Natália, Suzana e Aline. Nós caminhamos juntos grande parte do tempo. Nós compartilhamos sonhos e os vimos realizar. Obrigada pelas risadas, elas serão inesquecíveis.
Especialmente ao Matheus, meu melhor amigo e companheiro por todos esses anos. Por todas as vezes que você me ajudou, me fez dar risada e esteve ali por mim, sou grata.
Aos meus amigos e colegas do Lab. De Fisiologia Molecular, em Homburg. Obrigada por fazerem minha estadia divertida e com tanto aprendizado.
Especialmente Ute e Anja, pela ajuda desde o comecinho, Cai, pela ajuda com o 2P-LSM e pela doce amizade. Frank Rhode, pela ajuda com tudo no lab e Phillip, por continuar o imageamento in vivo, eu sei que não foi fácil. Obrigada!
A todos os técnicos e professores da Anatomia e do Depto. de Biologia Estrutural e Funcional.
Ao programa de pós-graduação em Biologia Celular e Estrutural. Especialmente à secretária Liliam.
Ao professor Ricardo Gazzineli e Ana Carolina Teixeira da FIOCRUZ/MG pela doação dos animais β2m knockout.
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À professora Elaine del Bel, por nos colocar em contato com o Prof. Frank e por trazer os animais transgênicos ao Brasil.
Aos meus pais, Rosangela e Humberto. Eu amo vocês, obrigada por estarem sempre comigo, mesmo quando nós não estávamos próximos.
A minha irmã, Roberta. Obrigada pelo amor e suporte durante os anos. Eu te amo e te admiro. Também ao meu cunhado, Rodrigo, pela amizade e por cuidar da Beta quando eu não pude estar por perto.
À CAPES/DAAD, pela bolsa de doutorado sanduíche (CAPES/DAAD-PROBRAL I - Nº 3294-14-5.)
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ACKNOWLEDGEMENTS
To my supervisor, professor Alexandre, for all your support, teaching, caring all these years. Thank you for the opportunities and friendship. I admire you and I will always be grateful.
To my co-supervisor, professor Frank, for welcoming me in his lab several times. You made possible a life-time scientific and personal experience, thank you.
To all my friends and colleagues from LRN, former and new ones. The lab is my second home since 2007, I want to thank all of you for being part of it.
To my dear friends Matheus, Gabriela, Natália, Suzana and Aline. We walked together most of the time. We shared dreams and we’ve seen them become true. Thank you, the laughs we had together are unforgetable.
Speacially Matheus, my best friend and partner during all these years. For all the times you helped me, made me laugh and were there for me, I am grateful.
To my friends and colleagues from Mollecular Phisiology lab, in Homburg. Thank you for making my stay fun and with so much learning.
Specially Ute and Anja, for all the help since the very beggining, Cai, for the help with 2P-LSM setup and for your sweet friendship, Frank Rhode, for all the help with everything in the lab, and Phillip, for keep running the in vivo imaging that I know it was not easy. Thank you!
To all the technicians and professors from Strutural and Functional Biology department.
To post-graduate program in Cell and Structural Biology. Specially to Liliam, the secretary.
To professor Ricardo Gazzineli and Ana Carolina Teixeira from FIOCRUZ/MG for donating β2m knockout mice.
To professor Ângela Luzo, from Hemocentro/UNICAMP, for collaboration and hMSC donation.
To professor Elaine del Bel, for putting us in contact with Prof. Frank and for bringing the transgenic mice to Brazil.
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To my parents, Rosangela and Humberto. I love you, thank you for always being with me, even when we were not close to each other.
My sister, Roberta. Thank you for all the love and support over the years. I love and admire you. Also to my brother in law, Rodrigo, for your friendship and for taking care of Beta when I couldn’t be around.
To CAPES/DAAD, for the PhD sandwich scholarship (CAPES/DAAD-PROBRAL I - Nº 3294-14-5.)
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"Science is not only a disciple of reason,
but also one of romance and passion."
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Resumo
Lesões na interface CNS/PNS são especialmente severas, levando a até 80% de degeneração neuronal nas primeiras duas semanas. Evidências recentes apontam para o envolvimento das moléculas de MHC-I na interação entre neurônios pré – e pós - sinápticos, bem como entre neurônios axotomizados e células gliais, tendo papel importante na manutenção sináptica seletiva após lesão. O presente trabalho tem por objetivo a padronização do esmagamento de raízes ventrais (VRC) em camundongos e posterior tratamento com células tronco mesenquimais humanas (hMSC), avaliando ainda a potencial interferência da ausência de MHC-I na sobrevivência neuronal, reação glial e cobertura sináptica com e sem a terapia celular. Para isto, camundongos C57BL/6J WT e β2mKO foram submetidos ao esmagamento das raízes ventrais espinais L4 – L6, tratados ou não com uma injeção intravenosa de hMSC e mantidos por 7, 14 ou 28 dias após a lesão. As análises da sobrevivência neuronal e da astrogliose mostraram padrões parecidos no que se refere ao aumento da perda neuronal e aumento da astrogliose reativa com o tempo em animais WT, sendo que a ausência de MHC-I, aumentou a susceptibilidade dos motoneurônios no período mais agudo. O tratamento com hMSC resultou na maior preservação dos motoneurônios e controle da astrogliose independente da expressão de MHC-I. A reação microglial foi mais intensa 7 dias após a lesão em animais WT, sendo reduzida no 28º dia. Na ausência de MHC-I, padrão semelhante foi detectado, porém com uma reação microglial 33% mais intensa no período agudo após a lesão. Os inputs sinápticos foram reduzidos ao redor dos neurônios axotomizados a partir de 7 dias após a lesão, sendo agudamente mais intensa nos β2mKO, alcançando uma redução de até 50% no 28º dia após a lesão. Após o tratamento com hMSC, tanto em animais WT quando nos β2mKO, aproximadamente 65% das sinapses foram mantidas. Os resultados aqui descritos, demonstram que após esmagamento de raízes ventrais em camundongos, MHC I possui um papel no controle da reação microglial aguda afetando temporariamente a perda sináptica e que o tratamento com hMSC reduziu a astrogliose reativa e reação microglial, culminando na neuroproteção de motoneurônios e manutenção da cobertura sináptica independente da expressão de MHC-I.
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Abstract
Lesions on CNS/PNS interface are especially severe, leading up to 80% of neuronal degeneration within the first two weeks. Recent data point out to the involvement of MHC-I in the interactions between pre- and post-synaptic neurons, as well as between axotomized neurons and glial cells, having an important role in selective synaptic maintenance after lesion. The present work objectives the stabilization of ventral root crush (VRC) in mice and further treatment with human mesenchymal stem cells (hMSC), evaluating the potential effect of lack of MHC-I on motoneuron survival, glial reaction, and synaptic covering, with and without cell therapy. For this purpose, C57BL/6J WT e β2mKO mice were submitted to the crush of L4 to L6 ventral roots, treated or not with one intravenous injection of hMSC and kept for 7, 14 and 28 days after injury. Analysis of motoneuron survival and astrogliosis showed similar patterns regarding the increasing loss of motoneurons and astrogliosis over time on WT animals, while the lack of MHC-I increased the motoneuron susceptibility in the acute phase. hMSC treatment resulted in higher motoneuron preservation and astrogliosis control independent of MHC-I. Microglial reaction was more intense 7 days after lesion in WT animals, becoming reduced over time. In the lack of MHC-I, an analogous pattern was detected, only with a microglial reaction 33% more intense in the acute time point after lesion. Synaptic inputs were reduced around axotomized motoneurons from the 7th day after lesion, being acutely more intense on β2mKO mice, reaching up to 50% reduction 28 days after injury. After hMSC treatment, both in WT and β2mKO mice, around 65% of synapses were maintained. Results described herein show that after ventral root crush in mice, MHC I plays a role on acute microglial reaction control, affecting temporarily synaptic loss and that hMSC treatment reduced reactive astrogliosis and microglial reaction, causing motoneuron neuroprotection and synaptic covering maintenance independent of the presence of MHC-I.
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Acronyms 2P-LSM – 2 photon laser scanning microscope AD-MSC – adipose-derived mesenchymal stem cell ALS – amyotrophic lateral sclerosis
BBB – blood brain barrier
BDNF – brain derived neurotrophic factor bFGF– basic fibroblast growth factor C1q – complement protein 1 q ChaT – Choline acil transferase CL – contralateral
CNS – central nervous system
CX3CR1 – CX3 chemokine receptor 1
DAPI – 4′,6-diamidine-2′-phenylindole dihydrochloride DNA – deoxyribonucleic acid
dpi – days post injury DRG – dorsal root ganglia
ECFP – enhanced cyan fluorescent protein EGFP – enhanced green fluorescent protein EYFP – enhanced yellow fluorescent protein FR – fluoroRuby
GABA – gamma - amino butyric acid GAD65 – glutamic acid decarboxylase 65 GDNF – glial derived neurotrophic factor GFAP – glial fibrillary acid protein
GLAST – glutamate aspartate transporter GLT-1 – glutamate transporter - 1
GM – gastrocnemius muscle GFP – green fluorescent protein
HEPES – 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid HLA - Human leukocyte antigen
hMSC – human mesenchymal stem cell
Iba-1 – ionized calcium-binding adapter molecule 1 Ig - immunoglobulin
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IL-1α – interleukin 1 α IL-10 – interleukin 10 KO – knockout
MHC-I – major histocompatibility complex class I MN – motoneuron
mRNA – messenger RNA MSC – mesenchymal stem cell MW – molecular weight
NeuN – neuronal nuclei
NMJ – neuromuscular junction PB – phosphate buffer
PBS – phosphate buffer saline PCR – polymerase chain reaction PNS – peripheral nervous system
PSD-95 – postsynaptic density protein 95 RNA – ribonucleic acid
ROI – region of interest
RT-qPCR – real time quantitative PCR siRNA – small interfering RNA
TA – tibialis anterior
TAP-1 – transporter associated with antigen processing 1 TNF – tumor necrosis factor
VRC – ventral root crush
VGluT-1 – vesicular glutamate transporter 1 WT – wildtype
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Figures list
Fig. 1 - Schematic representation of a spinal cord segment showing the dorsal root
and DRG, the ventral root and the union of both forming the spinal nerve (Drake R 2009).
Fig. 2 – The first line shows schematic representations of transverse sections from L4,
L5 and L6 spinal cord (reproduced from Paxinos (2004). Second line, the correspondent Nissl-stained sections showing the neuronal distribution through spinal cord laminae (Image credit: Allen Institute, © 2008 Allen Institute for Brain Science. Allen Spinal Cord Atlas. Available from: http://mousespinal.brain-map.org).
Fig. 3 – The molecular structure of MHC-I is illustrated in A, whereas B shows the
classical path of antigen loading and presentation on the cell surface by MHC I (reproduced from Yewdell, Reits et al. (2003))
Fig. 4 - Detailed design of mice used, treatments and time points of analysis after
lesion. There were used n = 5 mice per group each technique.
Fig. 5 – Scheme illustrating the quantification methods for each antibody.
Fig. 6 – A) scheme showing the tracer injection sites (Gastrocnemius and Tibialis
anterior muscles) in the mouse. B) shows a transversal slice of the spinal cord and unilateral motoneuron labeling 6 days after tracer injection. Astrocytes and microglia surrounding the motoneurons after tracer intramuscular injection did not show reactivity.
Fig. 7 – Surgery for window imaging opening. A shows the skin incision in the lower
part of the back of the mouse. Location of L4-L6 spinal cord segments are indicated (lumbar intumescence). In B there is a lateral incision parallel to the spine. The muscles were cleaned with a micro curette (C) and the lateral part vertebral column became evident (transverse processes indicated with arrows). After muscle cleaning, laminectomy was performed (D) to expose the ventrolateral part of spinal cord. Arrowheads show two points for paper clip holder fixation. In E, details from the imaging window can be visualized with the intact dura mater, and in F the region of interest (delimited within the dotted line circle) with ventral and dorsal roots (indicated with stars) moved to the side.
Fig. 8 – A and B show details of the mouse positioning in the angled support below
20x objective and C shows spine fixation in the angled support.
Fig. 9 – A) scheme showing the tracer application directly to sciatic nerve proximal
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motoneuron labeling 7 days after lesion and tracer application. Astrocytes and microglia surrounding the motoneurons became activated after nerve lesion.
Fig. 10 – Phase contrast micrographs from (A) panoramic view of a hMSC culture at
P11. B, higher magnitude. Scale bar = 50 μm.
Fig. 11 – Flow cytometry phenotypic analysis of hMSC. A, graph of punctual
distribution FSC (size) versus SSC (granularity) used to select the cell population of interest. B to I, average fluorescence intensity histograms versus events number (Count): B) CD 29; C) CD 90; D) CD 105; E) CD 75; F) CD 34; G) CD 45; H) HLA DR and I) HLA ABC.
Fig. 12 – Immunorreactivity anti-BDNF on human adipose derived mesenchymal stem
cells in vitro. A and D show nuclei staining by DAPI. B and E, BDNF and GDNF immunolabeling, respectively. C and F, merge images. Scale bar = 50 μm.
Fig. 13 – A) shows a transversal slice of a spinal cord after VRC showing the
preservation of the area; B) shows a detailed image from a crushed root (arrow), evidencing degenerative signs.
Fig. 14 – Neuronal survival 7, 14 and 28 days after VRC in C57BL/6J - WT mice. A)
correspond to a representative image from the contralateral lateral motor nucleus pool;
B) 7 dpi; C) 14 dpi, D) 28 dpi. E) is a schematic representation of a transversal section
of the lumbar spinal cord, highlighted the lateral motor nucleus where the counting was made (Paxinos 2004). F) Quantification of the motoneuron survival. Scale bar = 50 μm.
Fig. 15 – Neuronal survival 7, 14 and 28 days after VRC in β2mKO mice. A)
correspond to a representative image from the contralateral lateral motor nucleus pool;
B) 7 dpi; C) 14 dpi, D) 28 dpi. E) is a schematic representation of a transversal section
of the lumbar spinal cord, highlighted the lateral motor nucleus where the counting was made (Paxinos 2004). F) Quantification of the motoneuron survival. Scale bar = 50 μm.
Fig. 16 – Comparative time course of motoneuron survival in C57BL/6J - WT and KO
mice.
Fig. 17 - Neuronal survival 7, 14 and 28 days after VRC in C57BL/6J mice after hMSC
injection. A) correspond to a representative image from the contralateral lateral motor nucleus pool; B) 7 dpi; C) 14 dpi, D) 28 dpi. E) is a schematic representation of a transversal section of the lumbar spinal cord, highlighted the lateral motor nucleus where the counting was made (Paxinos 2004). F) Quantification of the motoneuron survival. Scale bar = 50 μm.
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Fig. 18 - Neuronal survival 7, 14 and 28 days after VRC in β2m KO mice, after hMSC
injection. A) corresponds to a representative image from the contralateral lateral motor nucleus pool; B) 7 dpi; C) 14 dpi, D) 28 dpi. E) is a schematic representation of a transversal section of the lumbar spinal cord, highlighted the lateral motor nucleus where the counting was made (Paxinos 2004). F) Quantification of the motoneuron survival. Scale bar = 50 μm.
Fig. 19 – Comparative time course of motoneuron survival in C57BL/6J and β2m KO
mice with or without hMSC treatment. Significance levels: * p < 0,05; ** p < 0,01; *** p < 0,001.
Fig. 20 – Anti-GFAP immunolabelling to assess astrogliosis 7, 14 and 28 days after
VRC in C57BL/6J - WT mice. A) correspond to a representative image from the contralateral lateral motor nucleus pool (insert); B) 7 dpi; C) 14 dpi, D) 28 dpi. E) Quantification of the integrated density of pixels. Scale bar = 50 μm.
Fig. 21 – Anti-GFAP immunolabelling to assess astrogliosis 7, 14 and 28 days after
VRC in B2mKO mice. A) correspond to a representative image from the contralateral lateral motor nucleus pool (insert); B) 7 dpi; C) 14 dpi, D) 28 dpi. E) Quantification of the integrated density of pixels. Scale bar = 50 μm.
Fig. 22 – Comparative time course of astogliosis in C57BL/6J and β2m KO mice. Fig. 23 – Double labelling of neuronal nuclei (NeuN; red) and astrocytes (GFAP; green)
and cellular nuclei (DAPI; blue), in the contra and ipsilateral sides of C57BL/6J - WT and β2mKO mice, 7, 14 and 28 days post injury. Scale bar = 50 µm.
Fig. 24 – Anti-GFAP immunolabelling to assess astrogliosis 7, 14 and 28 days after
VRC and hMSC treatment in C57BL/6J mice. A) correspond to a representative image from the contralateral lateral motor nucleus pool (insert); B) 7 dpi; C) 14 dpi, D) 28 dpi.
E) Quantification of the integrated density of pixels. Scale bar = 50 μm.
Fig. 25 – Anti-GFAP immunolabelling to assess astrogliosis 7, 14 and 28 days after
VRC and hMSC treatment in β2m KO mice. A) correspond to a representative image from the contralateral lateral motor nucleus pool (insert); B) 7 dpi; C) 14 dpi, D) 28 dpi.
E) Quantification of the integrated density of pixels. Scale bar = 50 μm.
Fig. 26 - Comparative time course of reactive astrogliosis in C57BL/6J and β2m KO
mice with or without hMSC treatment. Significance levels: * p < 0,05; ** p < 0,01; *** p < 0,001.
Fig. 27 – Anti-Iba-1 immunolabelling to assess microglial reaction 7, 14 and 28 days
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contralateral lateral motor nucleus pool (insert); B) 7 dpi; C) 14 dpi, D) 28 dpi. E) Quantification of the integrated density of pixels. Scale bar = 50 μm.
Fig. 28 – Anti-Iba-1 immunolabelling to assess microglial reaction 7, 14 and 28 days
after VRC in β2mKO mice. A) correspond to a representative image from the contralateral lateral motor nucleus pool (insert); B) 7 dpi; C) 14 dpi, D) 28 dpi. E) Quantification of the integrated density of pixels. Scale bar = 50
Fig. 29 – Comparative time course of microglial reaction in C57BL/6J and β2mKO
mice.
Fig. 30 – Double labelling of neuronal nuclei (NeuN; red) and microglia (Iba-1; green)
and cellular nuclei (DAPI; blue) in the contra and ipsilateral sides of β2mWT and β2mKO mice, 7, 14 and 28 days post injury. Scale bar = 50 µm.
Fig. 31 – Anti-Iba-1 immunolabelling to assess microglial reaction 7, 14 and 28 days
after VRC and hMSC injection in C57BL/6J mice. A) correspond to a representative image from the contralateral lateral motor nucleus pool (insert); B) 7 dpi; C) 14 dpi, D) 28 dpi. E) Quantification of the integrated density of pixels. Scale bar = 50 μm.
Fig. 32 – Anti-Iba-1 immunolabelling to assess microglial reaction 7, 14 and 28 days
after VRC and hMSC injection in β2m mice. A) correspond to a representative image from the contralateral lateral motor nucleus pool (insert); B) 7 dpi; C) 14 dpi, D) 28 dpi.
E) Quantification of the integrated density of pixels. Scale bar = 50 μm.
Fig. 33 - Comparative time course of microglial reaction in C57BL/6J and β2m KO
mice with or without hMSC treatment. Significance levels: * p < 0,05; ** p < 0,01; *** p < 0,001.
Fig. 34 – Anti-synaptophysin immunolabelling to assess synaptic covering 7, 14 and
28 days after VRC in C57BL/6J - WT mice. A) correspond to a representative image from the contralateral lateral motor nucleus pool (insert); B) 7 dpi; C) 14 dpi, D) 28 dpi.
E) Quantification of the integrated density of pixels. Scale bar = 50 μm.
Fig. 35 - Anti-synaptophysin immunolabelling to assess synaptic covering 7, 14 and
28 days after VRC in β2mKO mice. A) correspond to a representative image from the contralateral lateral motor nucleus pool (insert); B) 7 dpi; C) 14 dpi, D) 28 dpi. E) Quantification of the integrated density of pixels. Scale bar = 50 μm.
Fig. 36 – Comparative time course of synpatic covering in C57BL/6J and KO mice. Fig. 37 – Double labelling of neuronal nuclei (NeuN; red) and synaptic covering
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sides of C57BL/6J - WT mice and β2mKO mice, 7, 14 and 28 days post injury. Scale bar = 50 µm.
Fig. 38 - Anti-synaptophysin immunolabelling to assess synaptic covering 7, 14 and
28 days after VRC and hMSC treatment in C57BL/6J mice. A) correspond to a representative image from the contralateral lateral motor nucleus pool (insert); B) 7 dpi; C) 14 dpi, D) 28 dpi. E) Quantification of the integrated density of pixels. Scale bar = 50 μm.
Fig. 39 - Anti-synaptophysin immunolabelling to assess synaptic covering 7, 14 and
28 days after VRC and hMSC treatment in β2mKO mice. A) correspond to a representative image from the contralateral lateral motor nucleus pool (insert); B) 7 dpi; C) 14 dpi, D) 28 dpi. E) Quantification of the integrated density of pixels. Scale bar = 50 μm.
Fig. 40 - Comparative time course of synaptic covering in C57BL/6J and β2m KO mice
with or without hMSC treatment. Significance levels: * p < 0,05; ** p < 0,01; *** p < 0,001.
Fig. 41 - Anti-VGluT-1 (A to E) and anti-GAD65 (F to J) immunolabeling to assess
excitatory and inhibitory inputs, respectively, around axotomized motoneurons 28 days after VRC in C57BL/6J and β2mKO mice, with or without hMSC injection. A) correspond to a representative image from the contralateral lateral motor nucleus pool (insert); B) 7 dpi; C) 14 dpi, D) 28 dpi. E) Quantification of the integrated density of pixels. Scale bar = 50 μm.
Fig. 42 – In vivo imaging of ventral horn neurons in a triple transgenic mouse. A – D:
EGFP, ECFP, EYFP and merged images, respectively. E – H, Magnified view of the same region.
Fig. 43 – In vivo imaging of dorsal root ganglia in triple transgenic mice. A – D, from
WT. E – H, from a β2mKO.
Fig. 44 - In vivo imaging of spinal lumbar motoneurons in the double transgenic mouse
after FR intramuscular injection. A – D: EGFP, ECFP, EYFP and merged images, respectively. E – H, magnified view of the same region.
Fig. 45 – Panoramic views from β2mKO and WT 7 days after sciatic nerve transection.
Detailed images from the ventral and dorsal horn, respectively.
Fig. 46 - Panoramic views of β2mKO and WT spinal cord transverse sections 7 days
after sciatic nerve transection. Separated channels showing a microglial and astrocytic reaction. The insets show detailed images of cell morphology.
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Fig. 47 – Transverse cross sections of a fixed spinal cord showing sciatic nerve
motoneuron pool from WT and β2mKO seven days after peripheral lesion. Astrocytes (A and E: EGFAP; blue), Microglia (B and F: CXCR, green), FR-labeled motoneurons (C and G: red) and merged images (D and H). Scale bar = 40 µm.
Fig. 48 - Cross sections of a fixed spinal cord showing sciatic nerve motoneuron pool
from WT and β2mKO seven days after peripheral lesion, in a higher magnification. Astrocytes (A and E: EGFAP; blue), Microglia (B and F: CXCR, green), FR-labeled motoneurons (C and G: red) and merged images (D and H). Scale bar = 20 µm.
Fig. 49 - Cross sections of a fixed spinal cord showing contralateral sciatic nerve
motoneuron pool from WT and β2mKO. Astrocytes (A and E: GFAP; blue), Microglia (B and F: CXCR, green), FR-labeled motoneurons (C and G: red) and merged images (D and H). Scale bar = 40 µm.
Fig. 50 – Microglial cell quantification per volume (A) and the percentage in contact
with motoneurons (B).
Tables list
Table 1: Descriptive details of mouse strains used, treatments and time points of
analysis. It was used n = 5 mice per group each technique.
Table 2: Detailed description of the primary antibodies used. Table 3: Genotypes and respective experimental groups.
xxi Summary DEDICATÓRIA ... v AGRADECIMENTOS ... vi ACKNOWLEDGEMENTS ... viii Resumo ... xi Abstract ... xii Acronyms ... xiii Figures list ... xv Tables list ... xx 1. Introduction ... 23
1.1. Spinal cord anatomy and cytoarchitecture ... 23
1.2. The spinal motoneuron ... 26
1.3. Glial cells ... 27
1.4. Neural and glial reactions to injury ... 28
1.5. MHC I expression and role on CNS ... 31
1.6. Mesenchymal stem cells ... 33
1.7. Spinal cord in vivo imaging ... 35
2. Objectives ... 36
3. Materials and Methods ... 37
3.1. Surgical procedure for ventral root crush ... 37
3.2. Mice and experimental groups ... 37
3.3. Adipose-derived human mesenchymal stem cells treatment ... 39
3.4. Flow cytometry ... 39
3.5. Perfusion ... 39
3.6. Immunohistochemistry ... 40
3.7. Motoneuron survival ... 41
3.8. Transgenic and Knockout Mice strains and characterization ... 42
3.9. Tracer injection for motoneuron retrograde labeling ... 44
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3.11. 2P-LSM in vivo imaging of spinal cord ... 49
3.12. Surgical procedure for sciatic nerve transection and retrograde tracer
application ... 49
3.13. Tissue preparation for epifluorescence/confocal imaging ... 50
3.14. Microglia quantification ... 51
3.15. Statistical analysis ... 51
4. Results ... 52
Part I – Timecourse of glial reaction, synaptic changes and motoneuron survival after
VRC and hMSC treatment in C57BL/6J and β2mKO mice ... 52
4.1. Adipose-derived human mesenchymal stem cells morphological and
phenotypical characterization ... 52
4.2. Motoneuron survival following VRC ... 54
4.3. Glial reaction following VRC ... 63
4.3.1 Reactive astrogliosis ... 63
4.3.2 Microglial reaction ... 73
4.4. Synaptic covering changes following VRC ... 84
4.4.1 Excitatory and inhibitory inputs ... 93
Part II – in vivo imaging of spinal motoneurons and new strain characterization ... 95
5. Discussion ... 102 6. Concluding Remarks ... 111 7. References ... 112
8. Appendix – Published article in the Journal of Neuroscience Methods. ... 117
9. Annex ... 125
9.1 - Certificate of the Committee for Ethics in Animal Use – Institute of Biology –
CEUA/IB/UNICAMP ... 125
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1. Introduction
1.1. Spinal cord anatomy and cytoarchitecture
The spinal cord is the part of the central nervous system (CNS) that controls voluntary muscles of the limbs and trunk, and which receives sensory information from these regions. The spinal cord is cylindrical, but slightly flattened dorsoventrally and, together with its meninges (dura mater, arachnoid and pia mater) lie within the vertebral canal (Watson 2009). The spinal cord is greatly enlarged in the regions where nerves of the limbs (named brachial and lumbosacral plexuses) arise. These enlargements are called cervical and lumbosacral intumescences.
On the ventral surface of the spinal cord, there is a deep longitudinal fissure in the midline, named anterior median fissure. Its dorsal limit is formed by ventral white commissure. On the dorsal surface of the spinal cord, there is a shallow groove named posterior median sulcus. On the lateral side of the spinal cord, there is an indistinct ventrolateral sulcus and deeper dorsolateral sulcus; these correspond to the line of origin of ventral and dorsal roots, respectively (Fig. 1).
Pairs of spinal nerves arise the spinal cord and leave the vertebral column through the vertebral foramina. In rodents, each segment of spinal cord possesses about 15 dorsal and 15 ventral rootlets on each side. The dorsal roots are bundled together to form the dorsal root of a spinal nerve, and the ventral rootlets form the ventral root. Each dorsal root bears an ovoid swelling named dorsal root spinal ganglion (DRG) that consists of pseudo-unipolar neurons cell bodies and satellite cells. They give rise to a single axon which bifurcates; one branch connects to the periphery, and the other connects to the dorsal horn of spinal cord (Watson 2009).
The fundamental functional difference between ventral and dorsal roots (discovered by Magendie in 1822) is that the dorsal roots contain afferent (sensory) fibers from skin, subcutaneous and deep tissues and viscera, whereas the ventral roots contain somatic efferent (motor) fibers and presynaptic autonomic fibers (Watson 2009).
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Fig. 1 - Schematic representation of a spinal cord segment showing the dorsal root
and DRG, the ventral root and the union of both forming the spinal nerve (Drake R 2009).
In a cross-section, it is possible to notice that the spinal cord is divided into a peripheral white matter composed mostly of longitudinally running axons and glial cells and a central gray matter, made up of neuronal cell bodies, dendrites, axons and glial cells. The gray matter has horn-like projections dorsal and ventral in the white matter, with an intermediate region between them. In three dimensions, these projections represent columns that run the length of spinal cord (Paxinos 2004, Watson 2009).
Microscopic analysis of the spinal gray matter reveals a complex structure, characterized by successive layers of cells from dorsal to ventral. The description of these layers was made by (Rexed 1952, Rexed 1954), that divided the spinal gray matter into ten regions by cytoarchitecture as seen in transverse sections. The
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first nine laminae are arranged from dorsal to ventral, and the tenth is the circle of cells surrounding the central canal (Fig. 2).
Fig. 2 – The first line shows schematic representations of transverse sections from
L4, L5 and L6 spinal cord an laminae (reproduced from Paxinos (2004). Second line, the correspondent Nissl-stained sections showing the neuronal distribution through spinal cord laminae (Image credit: Allen Institute, © 2008 Allen Institute for Brain Science. Allen Spinal Cord Atlas. Available from: http://mousespinal.brain-map.org).
Briefly, laminae I - IV are the main cutaneous receptive regions. Lamina V receives afferents from the viscera, skin, and muscles, and lamina VI receives mostly proprioceptive and some cutaneous afferents. Lamina VII comprehends mostly interneurons that connect to motoneuron pools; lamina VIII contains propriospinal interneurons; lamina IX has clusters of large multipolar neurons, namely, motoneurons; lamina X receives somatic and visceral nociceptive afferents (Paxinos 2004, Watson 2009).
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1.2. The spinal motoneuron
Spinal motoneurons (MNs) are unique among the CNS neurons, by making synaptic contact with non-neuronal tissue. They constitute de final path between the CNS and skeletal muscle fibers. There are two main classes of motoneurons in the mammalian spinal cord, alfa (α) and gamma (γ) type, both cholinergic.
The α-motoneurons are large (~ 35 μm in the mouse according to Oliveira, Thams et al. (2004)) and supply extrafusal fibers of the skeletal muscles, whereas γ-motoneurons are smaller and innervate the intrafusal fibers within the muscle spindle (Ornung, Shupliakov et al. 1994, Ornung, Shupliakov et al. 1996, Watson 2009).
These motoneurons are located in the ventrolateral gray matter of spinal cord, also referred as Rexed lamina IX. They are organized into different motor nuclei (longitudinally oriented columns of MNs cell bodies). The motor nuclei are somatotopically organized: columns of motoneurons innervating proximal and distal limb muscles are located in the medial and lateral part of lamina IX, respectively, whereas MNs innervating flexor and extensor muscles are located, respectively, ventral and dorsal at lamina IX. The lateral columns are confined to the brachial and lumbosacral intumescences and innervate the upper and lower limbs, respectively, through the brachial and lumbosacral plexuses; and at these regions, the number of motoneurons is much larger as to accommodate the supply of limb muscles (Watson 2009).
First extensive analysis of the synaptology on MNs was made by Conradi (1969), in which was described that 30 – 50% of the cat motoneuron surface is covered by synaptic buttons. In the same study it was also characterized 3 types of synaptic buttons making contact to the motoneurons, based on vesicle shape and fine structure of the synaptic complex: S-type, shows spherical vesicles and asymmetric synaptic complex with a thicker post-synaptic membrane densification, containing the excitatory aminoacid glutamate; F-type, shows only flattened and/or spherical vesicles, and symmetric pre- and post-synaptic densities; and C-type, large buttons that contain spherical synaptic vesicles and are associated with a large cistern underlying the postsynaptic membrane (Ornung, Shupliakov et al. 1994, Ornung, Shupliakov et al. 1996, Ichiyama, Broman et al. 2006).
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(Ornung, Shupliakov et al. 1994, Ornung, Shupliakov et al. 1996) studied, by immunoreactivity, the distribution and fine structure of GABA, glycine and glutamate boutons on the motoneurons cell bodies in the L7 cat motor nucleus, and described that labeled boutons could be classified mainly into four groups: reactive to GABA only, to glycine only, to both GABA and glycine and to glutamate. Two-thirds of all terminals apposing to the cell bodies were classified as F-type and immunoreactive to glycine and/or GABA. Of these terminals, 2/3 were reactive to glycine only and only 2% to GABA only. Regarding to glutamate, 15 – 20% of the terminals apposed to the cell membrane were glutamatergic and classified as of the S-type.
1.3. Glial cells
In the nervous system, there are two main classes of cells: the neurons, and glial cells, or glia. The name of these cells, glia, is derived from the Greek for glue, but glia did not just hold the neurons together. In the vertebrate nervous system, the glial cells are divided into two major classes: microglia and macroglia. Microglia are the resident inflammatory cells of the CNS. In the normal brain, “resting” microglia are highly branched cells covering a 30 – 50 μm wide area in the parenchyma with no overlap with branches of nearby microglia. These branches, in contrast to the cell bodies, are highly dynamic structures, showing a continuous and high degree of extension and retraction in the “resting” state in vivo and not only after activation (Nimmerjahn, Kirchhoff et al. 2005). Through this surveillance, microglia detects diverse extracellular signals, and consequently, transduce, integrate, and respond to them to maintain brain homeostasis (Salter and Stevens 2017).
Microglia rapidly activates in response to even minor pathological changes in the CNS, and its activation is fundamental in the defense of neural parenchyma against inflammation, trauma, ischemia, neurodegeneration etc. Reactive microglia are mainly scavenger cells, but also perform functions in tissue repair and regeneration (Kreutzberg 1996).
Concerning macroglia, there are three main types: oligodendrocytes, Schwann cells, and astrocytes. Oligodendrocytes and Schwann cells form the myelin sheath, in the CNS and PNS, respectively, insulating the axons by tightly winding their membranous processes around them (Kandel 2013).
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Astrocytes comprise two major types: protoplasmic astrocytes, that are found in the gray matter; and fibrous astrocytes, that are found in the white matter. Neuroanatomical studies also indicate that both astrocyte subtypes make extensive contacts with blood vessels. Electron microscopic analyses revealed that the processes of protoplasmic astrocytes envelop synapses and that the processes of fibrous astrocytes contact nodes of Ranvier, and that both types of astrocytes form gap junctions between distal processes of neighboring astrocytes (Sofroniew and Vinters 2010). Among the functions performed by astrocytes, can be listed: increase or decrease CNS blood vessel diameter and blood flow in a coordinated manner; maintaining the fluid, ion, pH, and transmitter homeostasis in the synaptic cleft, being essential for synaptic transmission; also play essential roles in transmitter homeostasis by expressing high levels of transporters for neurotransmitters such as glutamate, GABA (γ-aminobutyric acid), and glycine that serve to clear these from the synaptic space; astrocytes can still affect directly the synaptic activity, via release of gliotransmitters; they are the principal storage sites of glycogen granules in the CNS, being important to CNS metabolism; astrocytes play also pivotal role in formation of the blood-brain barrier (BBB), that is a diffusion barrier that impedes the influx into brain parenchyma of certain molecules on the basis of polarity and size. The principal cellular constituents of the BBB are cerebral capillary endothelial cells, that form tight junctions and are surrounded by a basal lamina, perivascular pericytes, and astrocytic end feet (Araque, Parpura et al. 1999, Sofroniew and Vinters 2010, Kandel 2013).
In healthy tissue, astrocytes continually show physiological activation in the form of transient elevations in intracellular calcium ([Ca+2
i]) that represent a type of
excitability, involved in many critical dynamic astrocytic functions, including interactions with synapses and blood flow regulation (Sofroniew 2014).
Astrocytes also respond to all forms of CNS insults through a process referred to as reactive astrogliosis, which has become a pathological hallmark of CNS structural lesions (Sofroniew and Vinters 2010).
1.4. Neural and glial reactions to injury
The interaction between motoneurons and surrounding microenvironment has a critical role in its survival, functional state regulation and synaptic connectivity (Huh,
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Boulanger et al. 2000, Oliveira, Thams et al. 2004). The axotomy of spinal roots is a common incident and it is directly related to the modification in neuronal function (Rothman and Winkelstein 2007).
In this context, the brachial plexus is a critical region regarding to trauma, due to its special anatomical relationship with mobile neck and shoulder structures, making possible that traction on these structures may lesioned also the plexus nerves or roots. A Brazilian epidemiological study (Flores 2006) detected that 66% of the patients with traumatic nerve injury had brachial plexus lesion. Of these plexuses lesions, 60% were due to traction (76% of those with at least one root avulsed), 25% due to gunshots, 8.5% compression and 5.7% by penetrating wound. Most part of the trauma by traction were due to motorcycle/auto accidents.
However, both avulsion and crushing of the roots result in axotomy, yet crushing is a less severe injury since it keeps intact the Schwann cell basal membrane that surrounds nerve fibers, preserving a guidance pathway for the regenerating axon sprouts. Ventral root lesions specifically affect the motor component of the spinal nerve, and the axons from α- and γ-motoneurons are sectioned in the CNS/PNS interface, close to the cell body, increasing the lesion severity. Such proximal injuries trigger degeneration of most of the affected motoneurons (Koliatsos, Price et al. 1994, Kobayashi, Yoshizawa et al. 2004, Spejo, Carvalho et al. 2013).
Distal to the lesion takes place an event called Wallerian degeneration, that is characterized by the degeneration of axonal fragments distal to the lesion. In that context, the myelinating Schwann cells release myelin, proliferate, start producing cytokines and trophic factors and phagocyting myelin debris and degenerating axons. The soluble factors produced by Schwann cells activate resident macrophages and attract macrophages from blood circulation (Gaudet, Popovich et al. 2011). Once these macrophages reach the lesion environment, they efficiently finish the phagocytosis of myelin debris and stimulate Schwann cells migration. In a propitious environment, lesioned axons can form the growth cone and start to regenerate guided by the structures formed by the Schwann cells alignment (bands of Büngner), comprising a permissive medium for guided regenerating axon growth until the peripheral target (Gaudet, Popovich et al. 2011).
It is important to point out that not all motoneurons that survived in the first place after lesion are capable of regenerating its axons successfully. The axons must
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transpose the glial scar that is formed in the CNS/PNS interface, aiming to reach the ventral root, then regenerate through the PNS towards the target (Fraher 2000). The cell body of the lesioned neuron also undergoes several morpho-functional changes that, together, are called chromatolysis. As examples of changes, we can cite: cell body hypertrophy, nucleus displacement to the periphery, Nissl substance dissociation and expressive disturbance in the expression of structural molecules related to synaptic transmission (Zochodne 2012)
Also, as a response to peripheral injury, there are axotomy-induced retrograde synaptic responses known as “synaptic stripping”, i.e. the extensive detachment of presynaptic terminals from perikarya and dendrites of axotomized motoneurons (Blinzinger and Kreutzberg 1968, Cullheim and Thams 2007). The plasticity capacity of the nervous system ensures that a structural and functional circuitry remodeling occurs after injury, in particular, with the detachment of the excitatory boutons that were in apposition to the lesioned neuron, which leads to a metabolic change and a shift from the synaptic transmission state to a recovery state (Aldskogius, Liu et al. 1999, Linda, Shupliakov et al. 2000, Oliveira, Thams et al. 2004).
The proximal nervous lesion also results in an extensive motoneuron death (Novikov, Novikova et al. 1997, Oliveira and Langone 2000), and during the acute period after lesion, there is a significant loss of synaptic inputs, reducing or even temporally ceasing the synaptic transmission (Takata and Nagahama 1983, Delgado-Garcia, Del Pozo et al. 1988).
The axotomy rapidly activates astrocytes and microglia in the vicinity of the affected neuronal cell body.
Astrocytes respond to all forms of CNS insults such as infection, trauma, ischemia and neurodegenerative disease by a process referred to as reactive astrogliosis, which involves molecular and morphological changes that vary with severity of the insult (resulting in scar formation, in severe cases) along a graded continuum. Common features of reactive astrogliosis include up-regulation of glial fibrillary acidic protein (GFAP) and cellular hypertrophy (Sofroniew and Vinters 2010, Sofroniew 2014).
After lesion, microglia and astrocytes quickly react through structural modifications of cytoplasmatic projections that are interposed between axotomized-motoneuron membrane (post-synaptic membrane) and the retracted synaptic
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terminals (Svensson, Eriksson et al. 1993, Novikov, Novikova et al. 1997, Aldskogius, Liu et al. 1999, Oliveira and Langone 2000). The number of activated microglia in lesioned motor nuclei increases dramatically due to proliferation that lasts for about two to four days post-axotomy (Cullheim and Thams 2007).
Villacampa, Almolda et al. (2015) described in detail the microglial reaction on facial nucleus after facial nerve transection: 3 days after lesion, microglial cells changed their morphology from a ramified to an activated appearance, enlarging their cell body, retracting processes and starting to approach the motoneurons; at 7 dpi, microglia adopted a satellite position and started surrounding motoneuron soma, which was involved completely by the 14th day. At day 28, there was a
decrease in microglial reaction, but still not reaching the basal contralateral levels. It is important to emphasize that astrocytic or microglial reaction to injuries did not occur in isolation, but as part of a coordinated multicellular response to CNS insults, that includes multiple types of glia as well as neurons and nonneural cells (Sofroniew 2014).
In this context, Liddelow, Guttenplan et al. (2017) showed that A1 astrocytes (shown to be harmful; A2 astrocytes instead were shown to be protective) that, induced by microglia, gain neurotoxic function and increase motoneuron death after optical nerve crush in neonatal mice.
Understanding the signaling mechanisms among glial cells and between them and the motoneurons can enable the development of treatment strategies aiming to promote regeneration after injury (Fitch and Silver, 2008).
1.5. MHC I expression and role on CNS
Nowadays there is evidence that neurons express molecules that, originally, were assumed as immune system specific. Among them, the major histocompatibility complex of class I (MHC-I), that is a transmembrane complex of molecules (Fig. 3
A) from immunoglobulins (Ig) superfamily (Boulanger, Huh et al. 2001). MHC class
I molecules consist of a transmembrane α-chain (α1, α2, and α3) associated with a light chain, named β2-microglobulin (β2m) (Elmer and McAllister 2012).
MHC-I has a classical role in the adaptative immune response, being its primary function binding to peptides derived from intracellular proteolysis. The peptides that are processed intracellularly, are loaded to the MHC-I molecules by transporter
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associated with antigen processing 1 (TAP-1) for delivery to the cell surface; such peptides are recognized and identified by T cytotoxic lymphocytes (Fig. 3 B). In mice genetically deficient for I subunits β2m or TAP-1, the expression of MHC-I is severely impaired, since β2m is the obligatory light chain of most MHC-I molecules and TAP-1 is required as a transporter for loading peptides onto MHC-I before their transport to the cell surface (Boulanger and Shatz 2004).
Fig. 3 – The molecular structure of MHC-I is illustrated in A, whereas B shows the
classical path of antigen loading and presentation on the cell surface by MHC I (reproduced from Yewdell, Reits et al. (2003))
So far, it is known that MHC-I mRNA is expressed in neurons and glial cells in the olfactory system, cerebral cortex, striatum, hippocampus and spinal cord, both during development and in the adult. MHC I proteins are expressed on the surface of axons and dendrites and are also located both pre- and post-synaptically (Elmer and McAllister 2012)
In the healthy brain, MHC-I mRNA expression is regulated by neuronal activity (Corriveau, Huh et al. 1998, Boulanger, Huh et al. 2001) and is also related to critical periods where synaptic refinement occurs more substantially (Huh, Boulanger et al. 2000, Boulanger, Huh et al. 2001, Fourgeaud and Boulanger 2010, Needleman, Liu et al. 2010).
In the spinal cord, it was demonstrated that both MHC-I and β2m are upregulated after peripheral lesion (Linda, Hammarberg et al. 1998). Specifically, MHC class I molecules are important to the selective maintenance of inhibitory
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synaptic terminals after lesion. Oliveira, Thams et al. (2004) showed that in β2m knockout (β2mKO) or TAP-1 knockout (TAP1KO) mice, synaptic stripping after axotomy is intensified when compared to the wildtype (WT) and that preferentially the inhibitory terminals were removed.
Once glial processes are putatively involved in the detachment of pre-synaptic buttons from the lesioned motoneuron cell body, and knowing that MHC-I is important to selective maintenance of inhibitory terminals and is expressed by astrocytes, microglia and at pre- and postsynaptic terminals, it is acceptable to suggest that MHC-I signaling can be used by neurons and glia to interact both in normality and during pathological processes. Indeed, microglia and astrocytes reactivity after peripheral lesion are influenced by MHC-I modulation, also affecting the synaptic plasticity process and regenerative capacity (Zanon and Oliveira 2006, Zanon, Cartarozzi et al. 2010). However, the exact role for MHC-I neuron-glia signaling and spatiotemporal events that outline this process are still elusive.
1.6. Mesenchymal stem cells
As already cited above, it is well known that only a small percentage of motoneurons is able to successfully achieve regeneration after a proximal lesion, such as VRC, and new treatments are necessary to improve such rates.
The bone marrow contains, besides hematopoietic and endothelial stem cells, a small population of stromal cells, called mesenchymal stem cells (MSC). Minimal criteria to define human MSC is: positive expression of CD105, CD73, CD44 and CD90 and negative expression of CD45, CD34, CD14 or CD11b, CD79a or CD19 and HLA-DR surface molecules (Dominici, Le Blanc et al. 2006). Furthermore, mesenchymal stem cells exhibit plastic-adherence under standard culture conditions and are competent for in vitro differentiation into osteoblasts, chondroblasts and adipocytes. These cells can also secrete a broad spectrum of bioactive molecules that are immunomodulatory and can act in the restoration of the environment after lesion (Caplan 2007, Ladak, Olson et al. 2011).
The first and main source of MSC is the bone marrow, although they are rare cells, corresponding to 1 in 10.000 nucleated cell in the bone marrow. Luckily, it is also possible to obtain MSC from several sources as: umbilical cord blood, amniotic liquid, dental pulp and adipose tissue (Hass, Kasper et al. 2011).
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Adipose-derived mesenchymal stem cells (AD-MSC) are of great interest once the subcutaneous adipose tissue is easy to access, liposuction surgical procedure is simple and broadly performed, the cell yield is high and obtainment and isolation are simple.
Lopatina, Kalinina et al. (2011) showed that AD-MSC transplanted cells induced nerve repair and growth via BDNF production following cell xenotransplantation in mice limb re-innervation-models. Wei, Chen et al. (2009) further confirmed that the AD-MSC conditioned medium avoided neuronal apoptosis, supporting the hypothesis that AD-MSC could have therapeutic use in neurodegenerative disorders.
Local injections of MSC after ventral root avulsion or ventral root crush in rats leads to an improvement on motoneuron survival and motor function (Rodrigues Hell, Silva Costa et al. 2009, Spejo, Carvalho et al. 2013, Ribeiro, Duarte et al. 2015). Systemic injections of AD-MSC also ameliorates the clinical progression of the disease, in a murine model of Amyotrophic lateral sclerosis (ALS) (Marconi, Bonaconsa et al. 2013).
Although the exact mechanism that underlie the neuroprotection exerted by MSC are not still clear, authors hypothesize that the positive effect is mainly due to production of neurotrophic factors, that are essential for neuronal survival and maintenance, and also by the immunomodulatory potential of such cells.
So, it is important to emphasize that the beneficial effects promoted by MSC are not related to its trans-differentiation into neural phenotypes, but mainly due to secretion of molecules with anti-apoptotic, anti-inflammatory and trophic roles (Uccelli 2008, Uccelli and de Rosbo 2015).
MSc have the potential to migrate towards the lesion (Marconi, Bonaconsa et al. 2013) and evidences indicate that intercellular communication and microenvironment modulation are mediated by paracrine mechanisms, via release of soluble factors or cell-derived vesicles, that hold the capacity to merge with and transfer bioactive molecules to recipient cells both locally and systemically, directly and/or indirectly influencing the local microenvironment (Farinazzo, Turano et al. 2015).
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In this sense, AD-MSC therapy is a promising approach by providing growth factors that contribute to cell survival, lesion microenvironment modulation and axonal elongation.
1.7. Spinal cord in vivo imaging
With the advent of in vivo imaging, new studies regarding the fine changes related to neuron-glia interaction following acute lesion can potentially be carried out. In this way, novel and often unexpected findings of the behavior of cells have been described under physiological or pathological conditions (Davalos and Akassoglou 2012).
Recent studies have used advanced light microscopy approaches, such as two-photon laser scanning microscopy (2P-LSM), in combination with fluorophores/fluorescent proteins to image the CNS. 2P-LSM is an optical imaging approach that relies on non-linear infrared two-photon excitation and fluorescence emission detection (Denk, Strickler et al. 1990). This approach was initially restricted to cell culture or brain slices but has been quickly adapted to anesthetized rodents which represent an “intravital” preparation. This advance has revolutionized the field of neuroscience, allowing researchers to visualize the dynamics of cellular processes in living animals (Nayak, Zinselmeyer et al. 2012).
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2. Objectives
The main objective of this work was to establish the VRC model in mice, evaluating the time-course of events that succeed the lesion, associating them to the influence of MHC-I expression, by the use of β2m knockout mice. Further, to investigate the impact of human mesenchymal stem cell therapy in this context. Such goals were achieved by:
a) evaluation of motoneuron survival, 7, 14 and 28 days post-injury (dpi) in C57BL/6J and β2mKO mice;
b) quantification, by immunohistochemistry (integrated density of pixels), of microglial (anti-Iba-1 immunolabeling) and astroglial (anti-GFAP immunolabelling) reaction, in C57BL/6J and β2mKO mice; 7, 14 and 28 dpi;
c) quantification of synaptic covering, by anti-synaptophysin immunolabelling, in C57BL/6J and β2mKO mice; 7, 14 and 28 dpi;
d) a comparative evaluation of motoneuron survival, microglial reaction, astrogliosis and synaptic covering in C57BL/6J and β2mKO mice after VRC and hMSC treatment; 7, 14 and 28 dpi;
d) synaptic mapping (glutamatergic and GABAergic synapses), by immunolabelling, in C57BL/6J and β2mKO mice, with or without hMSC treatment; 28 dpi;
Additionally, in the lab of Prof. Dr. Frank Kirchhoff (Saarland University – Homburg, Germany) were developed the following objectives:
a) Generation of a new mouse strain: β2mKO + labeled astrocytes [TgN(GFAP -EGFP)] + labeled microglia [TgN(CX3CR1-EGFP)] + labelled neurons [TgH(Thy-1-EYFP)].
b) Retrograde tracing of sciatic nerve motoneurons with fluoro-ruby to identify lesioned neurons in vivo;
c) Set up a new 2P-LSM in vivo imaging approach that enables to reach in vivo the motoneurons deeply located in the ventral horn of lumbar intumescence (Rexed lamina IX);
d) Confocal imaging of spinal cord glial reactivity after peripheral injury both in β2mKO and β2mWT animals.
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3. Materials and Methods
3.1. Surgical procedure for ventral root crush
Mice were anesthetized with 5 mg/Kg Xylazine and 100 mg/kg Ketamine. Bepanthen (Bayer, Germany) was applied in the eyes to prevent dryness. The mice were placed in a heated bed and the hair was removed from the back. In the absence of the toe pinch withdraw, an incision parallel to the vertebral column was made at the thoracic level. The muscles were removed to expose the lower thoracic and upper lumbar vertebrae and laminectomy was performed to expose the spinal cord. After that, an incision was made in the dura mater allowing to reach ventral roots correspondent to L4, L5 and L6 spinal segments that were crushed with number 4 forceps (3 times of 10 seconds). After that, muscle and skin were sutured and mice were kept under controlled heating until completely recovered from anesthesia. A dose of painkiller (Tramadol, 5 mg/Kg) was immediately orally administered after surgery and for the following 3 days in the drinking water.
To ensure that the lesion was successfully done, in the following days after surgery the mice were behaviorally analyzed to check the correspondent paw paralysis. Moreover, the morphology of fixed spinal cords was analyzed, to check the gross anatomical preservation of the nervous tissue after lesion, as well as to detect trace elements of degeneration on the axotomized roots. Only mice fitting both these criteria were used to further analysis. The success ratio of the surgery was estimated in 80%.
3.2. Mice and experimental groups
For this study, were used 8 to 12 weeks old female wildtype C57BL/6J and β2m KO mice, distributed in the following experimental groups and techniques (Fig. 4 and Table 1):
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Fig. 4 - Design of mice used, treatment and time points of analysis after lesion.
Table 1: Descriptive details of mouse strains used, treatments and time points of
analysis. It was used n = 5 mice per group each technique.
Experimental
groups
Motoneuron survival
Immunohistochemisty (anti: GFAP, Iba-1,
synaptophysin) Synaptic mapping (anti-GAD65 and VGluT-1) 7 days 14 days 28 days 7 days 14 days 28 days 7 days 14 days 28 days C57BL/6J 5 5 3 5 5 5 5 5 5 β2mKO 5 3 3 5 5 5 5 5 5 C57BL/6J + hMSC 5 5 5 5 5 5 5 5 5 β2mKO + hMSC 5 5 5 5 5 5 5 5 5
Protocols concerning the animal use and handling were approved by the Institutional Committee for Ethics in Animal Experimentation (Committee for Ethics in Animal Use – Institute of Biology – CEUA/IB/UNICAMP, Protocol number 3336-1) and were performed in accordance with the guidelines of the Brazilian College for Animal Experimentation.
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3.3. Adipose-derived human mesenchymal stem cells treatment
Adipose-derived hMSC were obtained from liposuction adipose tissue under
donor consent and cell use approval (CAAE: 1162.0.146.000-11). The steps concerning cell collection, extraction and cultivation were done in the Cellular and Molecular Biology Lab, at Hemocentro – UNICAMP (Volpe, Santos Duarte Ada et al. 2014, Ribeiro, Duarte et al. 2015), and under supervision from Profa. Dra. Ângela Cristina Malheiros Luzo, medical director of Transfusion Service and Bank of Umbilical Cord Blood and Human Placenta.
The systemic injection of hMSC occurred via tail vein right after surgery. Mice still under anesthesia had their tails placed in 40 ºC water, for tail vein vasodilation. Were injected with an insulin syringe, 1 x 105 hMSC, between 8th and 12th passages,
resuspended in 100 μl PBS.
3.4. Flow cytometry
After hMSC purification and expansion, such cells were phenotypically characterized by flow cytometry. This procedure was developed in the Cellular and Molecular Biology Lab, at Blood Center – UNICAMP. Briefly, immunophenotypical analysis of hMSC was performed using FITC-, PE-, or PECy5 – conjugated monoclonal antibodies against positive markers (CD29, CD90, CD105 and CD73) and negative (CD45, CD34, HLA-DR and HLA-ABC) and their respective isotype control antibodies (BD Biosciences, Mountain View, CA, USA). hMSCs were resuspended at a concentration of 106 cells/ml, incubated at 4 °C for 30 min, washed
and analyzed by flow cytometry using FACS Calibur and CellQuest software (10.000 events/sample – BD Biosciences, San Jose, CA, USA).
3.5. Perfusion
Mice were perfused according to the time points described in Table 1. For that, they were anesthetized with an overdose of Xylazine and Ketamine and submitted to thoracotomy followed by transcardiac perfusion with PBS (0,1M Sodium Phosphate buffer – PB - with 0,9% NaCl; pH 7,38). Afterwards, mice designated for motoneuron survival and immunohistochemistry were perfused with a fixative solution (4% Formaldehyde in 0,1M Sodium Phosphate buffer). After fixation, lumbar intumescences were dissected out and immersed in the same fixative
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solution overnight, at 4°C, then 3 times washed with 0,1M PB and immersed in sucrose solutions (10%, 20% and 30%, 12 hours each), and finally soaked in Tissue-Tek, frozen in n-Hexane in controlled temperature (- 32°C to – 35°C) and stored at – 20°C.
3.6. Immunohistochemistry
Frozen 12 μm - thick sequential cuts were obtained in a cryostat (Microm, HM525), transferred to a gelatin covered microscopic slide and stored at -20°C until use.
For immunohistochemistry technique, the microscopic slides were left at room temperature and the cuts delimited with a hydrophobic pen. After that, slides were transferred to a humid and protected from light chamber and cuts were immersed in 0,01M PB (3 times, 5 minutes each), dried and incubated with 150 μl of blocking solution (3% Fetal Bovine Serum in 0,01M PB) for 45 minutes. Following, the different primary antibodies (Table 2) were diluted in incubation solution (1,5% Fetal Bovine Serum and 0,2% Tween in 0,1M PB) and incubated for 4 hours or overnight. After first incubation, cuts were washed with 0,01M PB and incubated with the respective secondary antibodies (cy2 anti-rabbit or cy3 anti-mouse; Jackson Immunoresearch, 1 : 500) for 45 minutes. As a nuclear marker, DAPI (4',6-diamidino-2-phenylindole) in a 1 : 1000 dilution was used. The cuts were again washed with 0,01M PB and mounted with coverslips using as medium glycerin/PB (3:1).
Table 2: Detailed description of the primary antibodies used.
Antibody Host Company Code Dilution
Anti-Iba-1 Rabbit Wako 019-19741 1 : 750
Anti-GFAP Rabbit Abcam ab7260 1 : 1500
Anti-Synaptophysin Rabbit Novus Biologicals NBP2-25170 1 : 1000
Anti-NeuN Mouse Millipore MAB377 1 : 500
Anti-VGlut-1 Rabbit Synaptic Systems 135303 1 : 1000
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Immunostainings were observed under a fluorescence microscope (Leica CTR 5500) and documented with the coupled digital camera (Leica DFC 345 FX), using specific filters according to the secondary antibodies or DAPI.
For quantification, there were selected 3 representative images from each animal of the respective experimental group. The integrated density of pixels, that represents the proteins immunolabeling, was measured in the lateral motor nucleus at anterior horn from the ipsi- and contralateral sided of the spinal cord, according to Oliveira et al. (2004), using Image J software (1.33u version, National Institutes of Health, USA).
As illustrated on Fig. 5, for GFAP and IBA-1, the hole area of the picture was quantified; for anti-synaptophysin immunolabeling, 8 small areas around each motoneuron were measured. For VGluT-1 and GAD65, one circular area around motoneurons was quantified.
Fig. 5 – Scheme illustrating the quantification methods for each antibody.
The integrated density of pixel was acquired for each animal and then calculated the media ± standard error of each experimental group.
Results were then statistically evaluated using one-way ANOVA and Bonferroni post-test. Were considered the following significance intervals: p< 0,05 (*), p<0,01 (**) and p<0,001(***).
3.7. Motoneuron survival
For motoneuron counting, the specimens were processed just as described for immunohistochemistry, until the cutting acquisition and storage of the microscopic
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slides. Transversal sections from the intumescence were then stained with 0,5% Toluidine blue for 1 minute, rinsed in water, dehydrated, diaphanized and mounted with Entellan (Merck) and coverslip, following, motoneurons from the lateral motor nucleus in the anterior horn of the spinal segments L4, L5 and L6 were counted in the ipsi- and contralateral sides every 4 slides from the specimen. In order to correct double counting, it was used Abercrombie’s formula (Abercrombie and Johnson 1946):
N = nt/(t+d)
“N” is the corrected number of neurons, “n” is the number of counted cells, “t” is the section thickness and “d” is the average diameter of the neurons. Once the neuron size difference affects the corrected number of neurons, the “d” value was calculated for each experimental group (ipsi- and contralateral).
3.8. Transgenic and Knockout Mice strains and characterization
Transgenic mice with astrocytes labelled by the cyan fluorescent protein ECFP under control of the human GFAP promoter (TgN(GFAP-ECFP)) and microglia by the green fluorescent protein EGFP controlled within the CX3CR1 gene
(TgH(CX3CR1-EGFP)) and neurons by the yellow fluorescent protein EYFP
controlled within Thy-1 promoter (TgH(Thy-1-EYFP)) were crossbreed with β2mKO mice (B6.12P2- β2mtm1Unc/J, Jackson Laboratory, USA), generating animals both
transgenic and β2mKO (and controls) used for in vivo imaging according to Table 3.
Table 3: Genotypes and respective experimental groups.
Transgenes Knockout Experimental group CX3CR1-EGFP GFAP-ECFP Thy-1-EYFP β2m Genotype xfp/wt xfp/wt xfp/wt wt WT xfp/wt xfp/wt wt wt WT – with retrograde tracer xfp/wt xfp/wt xfp/wt -/- β2mKO xfp/wt xfp/wt wt -/- β2mKO – with retrograde tracer
