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UNIVERSIDADE ESTADUAL DE CAMPINAS

INSTITUTO DE BIOLOGIA

ROGHAYEH MOZAFARAI

COMBINATION OF FIBRIN SEALANT AND BIOENGINEERED

HUMAN EMBRYONIC STEM CELLS TO IMPROVE

REGENERATION FOLLOWING SCIATIC NERVE INJURY AND

REPAIR WITH END-TO-END NEURORRHAPHY

COMBINAÇÃO DO USO DE SELANTE DE FIBRINA E

CÉLULAS TRONCO EMBRIONÁRIAS HUMANAS

TRANSGÊNICAS NA OTIMIZAÇÃO DA REGENERAÇÃO APÓS

NEURORRAFIA TÉRMINO-TERMINAL DO NERVO

ISQUIÁTICO

CAMPINAS

2017

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ROGHAYEH MOZAFARI

COMBINATION OF FIBRIN SEALANT AND BIOENGINEERED

HUMAN EMBRYONIC STEM CELLS TO IMPROVE REGENERATION

FOLLOWING SCIATIC NERVE INJURY AND REPAIR WITH END-

TO-END NEURORRHAPHY

COMBINAÇÃO DO USO DE SELANTE DE FIBRINA E CÉLULAS

TRONCO EMBRIONÁRIAS HUMANAS TRANSGÊNICAS NA

OTIMIZAÇÃO DA REGENERAÇÃO APÓS NEURORRAFIA

TÉRMINO-TERMINAL DO NERVO ISQUIÁTICO

Dissertation presented to the Institute of Biology

of the University of Campinas in partial

fulfillment of the requirements for the degree of

Master in Cellular and Structural Biology, in the

field of Cell Biology.

Dissertação apresentada ao Instituto de

Biologia da Universidade Estadual de Campinas

como parte dos requisitos exigidos para a

obtenção do Título de Mestra em Biologia

Celular e Estrutural, na área de Biologia

Celular.

Orientador: ALEXANDRE LEITE RODRIGUES DE OLIVEIRA

CAMPINAS

2017

ESTE ARQUIVO DIGITAL CORRESPONDE À VERSÃO FINAL DA DISSERTAÇÃO DEFENDIDA PELA ALUNA ROGHAYEH MOZAFARI E ORIENTADA PELO ALEXANDRE LEITE RODRIGUES DE OLIVEIRA

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Campinas, 05 de junho de 2017.

COMISSÃO EXAMINADORA

Prof. Dr. Alexandre Leite Rodrigues de Oliveira

Prof. Dr. André Schwambach Vieira

Profa. Dra. Valéria Paula Sassoli Fazan

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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Acknowledgements

First and foremost, I would like to express my sincere gratitude to my supervisor Prof.

Alexandre Leite Rodrigues de Oliveira for the continuous support of my research, for his

patience, motivation, enthusiasm, and immense knowledge. His guidance helped me in all the

time of research and writing of this thesis. I could not have imagined having a better supervisor.

I also offer my sincerest gratitude to my co-supervisor, Prof. Sergiy Kyrylenko, who supported

me throughout the thesis with his patience and knowledge. The door to Prof. Sergiy office was

always open whenever I ran into trouble or had a question about my research.

I would like to thank my exam board members: Prof. André Vieira and Prof. Valéria

Fazan for their time, interest, and helpful comments. I would also like to thank the members of

my qualification committee including Prof. Edson Pimentel, Prof. Mary Anne Heidi Dolder,

and André Luis Bombeiro.

I am thankful to my lovely friends at the Laboratory of Nerve Regeneration: André Luis

Bombeiro, Danielle Bernardes, Aline Barroso Spejo, Gabriela Bortolança Chiarotto, Luciana

Politti Cartarozzi, Mateus Vidigal de Castro, Matheus Perez, Greice Anne Rodrigues da Silva,

Simone Alves, Patrícia Ribeiro, and Júlio César Santini for their help and kindness. My thanks

go to CEMIB and CEVAP, respectively for granting the animals used in this work and granting

the fibrin sealant used in the study.

I must express my very profound gratitude to my husband Saeid for providing me with

unfailing support and continuous encouragement throughout my study and through the process

of researching and writing this thesis. This accomplishment would not have been possible

without him. Last but not the least; I would like to thank my parents, my sisters, and my one

and only loving brother for supporting me spiritually throughout my life.

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Resumo

Lesões a nervos periféricos se constituem num problema clínico global que, normalmente,

resulta em recuperação ausente ou incompleta, reduzindo a qualidade de vida de inúmeros

pacientes. O reparo de tais lesões depende de abordagem cirurgia, sendo rotineiramente

empregada a neurorrafia término-terminal, considerada padrão ouro, com os melhores

resultados clínicos. Quando o reparo direto não é possível, devido à retração dos cotos, utiliza-

se o enxerto autólogo, a partir de nervos doadores. Ao passo que as técnicas cirúrgicas têm

propiciado os melhores resultados práticos, o grau de recuperação se mostra aquém do

esperado, além de ser altamente variável. Portanto, o desenvolvimento de novas estratégias que

possam otimizar o processo regenerativo, após reparo nervoso periférico, é necessário. Neste

sentido, a terapia celular surge como promissora opção. Assim, o transplante de células tronco

embrionárias humanas (hESCs) mostra-se uma abordagem viável, tendo em vista a

pluripotência, que resulta na possibilidade de substituição de elementos celulares do nervo,

além da capacidade de se dividirem indefinidamente. Contudo, as hESCs necessitam de

constante aporte exógeno de FGF2 para manter sua capacidade de auto-renovação e

pluripotência, bem como para conduzir a diferenciação em diferentes tipos celulares. O FGF-2

exógeno pode estimular a expressão de genes fundamentais das células tronco, assim como

eliminar morte celular por suprimir genes pró-apoptóticos. O objetivo principal do presente

estudo é encontrar condições ideais para a recuperação funcional após neurorrafia término-

terminal. Para isso, postulamos que hESCs sozinhas ou combinadas com arcabouço de fibrina

possam ser empregadas para sustentar a regeneração axonal em camundongos submetidos à

neurorrafia término-terminal. Dessa forma, o nervo ciático de camundongos C57BL/6J foi

transeccionado (5mm) e, após rotação de 180º, tal segmento foi suturado simulando o enxerto

término-terminal, ligando os cotos proximal e distal. Quando previsto, foi aplicado o selante de

fibrina e/ou hESCs alteradas geneticamente para superexpressar FGF2 no local da lesão. O

estudo foi desenhado de forma a serem formados seis grupos experimentais: neurorrafia (N),

neurorrafia+selante de fibrina (N+F), neurorrafia+selante de fibrina+doxicilclina (N+F+D),

neurorrafia+selante de fibrina+hESCs selvagens (N+F+W), neurorrafia+selante de

fibrina+hESCs não induzidas (N+F+T) e neurorrafia+selante de fibrina+hESC induzidas a

produzir FGF2 (N+F+D+T). Foi avaliada a recuperação funcional da marcha utilizando-se o

sistema CatWalk, bem como o limiar nociceptivo pelo método de von-Frey. O processo

regenerativo foi estudado por imunoistoquímica, com marcadores para fibras motoras e

sensitivas, bem como para reatividade das células de Schwann. Os resultados indicaram

melhora na sensibilidade quando as hESCs foram utilizadas, na forma induzida para expressar

FGF2. A regeneração das fibras sensitivas levou à melhora do reflexo de retirada, em resposta

à estimulação da planta da pata ipsilateral à lesão, fato corroborado pela análise

imunoistoquímica. O sistema CatWalk, contudo, não mostrou diferenças significativas na

recuperação da marcha, comparando-se os grupos estudados. Conclui-se, portanto, que o

emprego de hESCs, modificadas geneticamente para superexpressar FGF2 de forma induzida,

pode ser útil como suporte adicional ao processo regenerativo, melhorando a regeneração

axonal, especialmente do componente sensitivo.

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Abstract

Peripheral nerve injury is a worldwide clinical problem that often results to incomplete or no

functional recovery, thus impairing the quality of life of many patients. To repair such injuries,

a type of surgical operation is commonly involved. The preferred surgical method for this aim

is the direct nerve repair (end-to-end neurorrhaphy) as it provides the best functional outcomes.

Where the end-to-end suturing is not possible due to large nerve gap, autologous nerve grafting

is used as the gold standard alternative. Whereas such surgical techniques have proved to lead

to better nerve regeneration, the degree of recovery is shown to be highly variable. It is thus

very important to seek complementary techniques to improve the degree of recovery. One of

the promising technique for this aim could be cell therapy. Transplantation therapy by human

embryonic stem cells (hESCs) is very appealing because they are true pluripotent that can

differentiate into specialized cell type and have the ability to self-renew indefinitely. However,

the hESCs require exogenous FGF-2 to sustain self-renewal and pluripotency, and develop the

capacity to differentiate into a large number of somatic cell types. The exogenous FGF-2 can

also stimulate the expression of stem cell genes while eliminating cell death and apoptosis

genes. The main objective of this research is to find conditions under which functional recovery

is improved after sciatic nerve neurorrhaphy. We assumed that hESC, either alone or in

combination with fibrin sealant scaffold, could be used to support regeneration in a mouse

model of sciatic nerve injury and repair via autografting with end-to-end neurorrhaphy. To

fulfill this objective, we transected 5 millimeters of the sciatic nerve of C57BL/6J mice and

inverted by 180° to simulate an injury, and then sutured the stumps. Next, we applied fibrin

sealant and/or human embryonic stem cells altered genetically to overexpress FGF2 at the site

of injury. The study was designed to include six experimental groups comprising neurorrhaphy

(N), neurorrhaphy + fibrin sealant (N+F), neurorrhaphy + fibrin sealant + doxycycline

(N+F+D), neurorrhaphy + fibrin sealant + wild type hESC (N+F+W), neurorrhaphy + fibrin

sealant + hESC off (N+F+T), and neurorrhaphy + fibrin sealant + hESC on via doxycycline

(N+F+D+T). We evaluated the recovery rate using Catwalk and von Frey tests and

immunohistochemistry analysis under a fluorescence microscope. The experiments indicated

that sensory function improved when transgenic hESCs were used. The regeneration of sensory

fibers indeed led to increased reflexes, upon stimulation of the paw ipsilateral to the lesion, as

seen by von-Frey evaluation and supported by immunohistochemistry. The Catwalk system,

however, was not successful in showing the functional recovery, likely due to its incapability

to record partial paw usage with this particular animal model. Our work indicated that the

simultaneous use of sensory test (von Frey) and Catwalk could provide a more detailed picture

of sciatic nerve recovery. Overall, our research demonstrated that transgenic embryonic stem

cells, engineered to overexpress FGF-2, could be employed to support regeneration aiming at

the recovery of both motor and sensory function.

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List of Figures

FIG. 1. AN OVERVIEW OF THE PNS. THE AXONS SURROUNDED BY MYELINATING SCHWANN CELLS ARE ENCLOSED

BY ENDONEURIUM. THE PERINEURIUM BINDS INDIVIDUAL AXONS TOGETHER TO FORM FASCICLES AND

EPINEURIUM GROUPS FASCICLES TO FORM THE NERVE CABLE (MARIEB, MITCHELL ET AL. 2013). ............... 12 FIG. 2. THE STEPS FOR OBTAINING HUMAN EMBRYONIC STEM CELLS (AFTER TERESE WINSLOW, 2006). ............... 18 FIG. 3. AUTOGRAFTING PROCEDURE IN WHICH 5 MILLIMETERS OF THE SCIATIC NERVE OF A MOUSE IS TRANSECTED,

INVERTED BY 180°, AND THEN IS SUTURED OR STITCHED TOGETHER BY NYLON SUTURE AND FIBRIN SEALANT

(~ 20X MAGNIFICATION). ............................................................................................................................. 27 FIG. 4. PHOTOGRAPHS OF HESC ACTIVATED BY DOXYCYCLINE TO OVEREXPRESS FGF-2. A) USING A PHASE-

CONTRAST FILTER. B) BY USING FLORESCENCE LIGHT. ................................................................................. 36 FIG. 5. ANTI-NEUROFILAMENT IMMUNO-STAINING OF THE CONTROL NERVES (A) AND ALL GROUPS (B TO G), 60

DAYS AFTER SURGERY. H) QUANTIFICATION OF THE INTEGRATED DENSITY OF PIXELS IN THE EXPERIMENTAL

GROUPS RELATIVE TO THE CONTROL GROUP PRESENTED IN PERCENTAGE (%) AND CALCULATED USING

IMAGEJ SOFTWARE. STATISTICALLY, THERE IS NO SIGNIFICANT DIFFERENCE BETWEEN THE EXPERIMENTAL

GROUPS. ........................................................................................................................................................ 39 FIG. 6. ANTI-CHAT IMMUNO-STAINING OF THE CONTROL NERVES (A) AND ALL GROUPS (B TO G), 60 DAYS AFTER

SURGERY. H) QUANTIFICATION OF THE INTEGRATED DENSITY OF PIXELS IN THE EXPERIMENTAL GROUPS

RELATIVE TO CONTROL GROUP (%). STATISTICALLY, THERE IS NO SIGNIFICANT DIFFERENCE BETWEEN THE

EXPERIMENTAL GROUPS. ............................................................................................................................... 40 FIG. 7. ANTI-VGLUT1 IMMUNO-STAINING OF THE CONTROL NERVES (A) AND ALL GROUPS (B TO G), 60 DAYS AFTER

SURGERY. H) QUANTIFICATION OF THE INTEGRATED DENSITY OF PIXELS IN THE EXPERIMENTAL GROUPS

RELATIVE TO CONTROL GROUP (%). STATISTICALLY, THE DIFFERENCE BETWEEN N+F VS N+F+D+T AND

N+F+D VS N+F+D+T GROUPS ARE MEANINGFUL WITH P <0.05 AND P <0.01, RESPECTIVELY. ................... 41 FIG. 8. ANTI-S100 IMMUNO-STAINING OF THE CONTROL NERVES (A) AND ALL GROUPS (B TO G), 60 DAYS AFTER

SURGERY. H) QUANTIFICATION OF THE INTEGRATED DENSITY OF PIXELS IN THE EXPERIMENTAL GROUPS

RELATIVE TO CONTROL GROUP (%).STATISTICALLY, THE DIFFERENCE BETWEEN THE FOLLOWING GROUPS ARE

MEANINGFUL: N VS N+F (P <0.05), N VS N+F+D (P<0.001), N VS N+F+W (P<0.05), AND N VS N+F+D+T

(P<0.05). THE N VS N+F+T SHOWS NO SIGNIFICANT DIFFERENCE. .............................................................. 42 FIG. 9. THE RESULTS ACHIEVED FROM CATWALK TEST SEPARATED BY GROUPS AND CALCULATED USING THE SFI

INDEX. A) NEURORRHAPHY GROUP. B) NEURORRHAPHY + FIBRIN SEALANT. C) NEURORRHAPHY + FIBRIN

SEALANT + DOXYCYCLINE. D) NEURORRHAPHY + FIBRIN SEALANT + WILD HESCS. E) NEURORRHAPHY +

FIBRIN SEALANT + TRANSGENIC CELLS. F) NEURORRHAPHY + FIBRIN SEALANT + DOXYCYCLINE + TRANSGENIC

CELLS............................................................................................................................................................ 43 FIG. 10. THE RESULTS ACHIEVED FROM CATWALK TEST SEPARATED BY DAYS AND CALCULATED USING SFI INDEX.

A) PREOPERATIVE RESULTS. B) DAY 7TH. C) DAY 14TH. D) DAY 18TH. E) DAY 22TH. F) DAY 26TH. G) DAY 30TH.

H) DAY 34TH. THE ABBREVIATIONS ARE AS TABLE 1. IN N+F+T GROUP, THE TRANSGENIC CELL IS ‘OFF’

WHEREAS IN N+F+D+T GROUP IT IS ‘ON’. THE BOX BOUNDARIES AND WHISKERS INDICATE THE MEAN ± SEM

AND ERROR BAR, RESPECTIVELY. .................................................................................................................. 45 FIG. 11. THE RESULTS ACHIEVED FROM CATWALK TEST SEPARATED BY DAYS AND CALCULATED USING SFI INDEX.

A) PREOPERATIVE RESULTS. B) DAY 38TH. C) DAY 42TH. D) DAY 46TH. E) DAY 50TH. F) DAY 54TH. G) DAY 58TH

. H) DAY 60TH.OTHER EXPLANATIONS ARE AS FIG. 10. ................................................................................. 46 FIG. 12. THE RESULTS ACHIEVED FROM VON FREY TEST DURING EIGHT WEEKS OF MEASUREMENTS SEPARATED BY

GROUPS. THE PREOPERATIVE REPRESENTS THE MEASUREMENTS BEFORE THE SURGERY (TREATMENT: P<0.05;

TIME: P<0.001). ............................................................................................................................................ 48 FIG. 13. THE RESULTS OF VON FREY TEST FOR ALL GROUPS DURING EIGHT WEEKS PERIOD. .................................. 49

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List of Tables

TABLE 1 – EXPERIMENTAL GROUPS AND PROCEDURES FOLLOWED DURING THE RESEARCH. ................................. 25

TABLE 2 – PRIMARY ANTIBODIES USED FOR IMMUNOHISTOCHEMISTRY. ............................................................... 32

TABLE 3 – THE MEAN INTENSITIES OF THE IMMUNOSTAININGS QUANTIFIED BY INTEGRATED PIXEL DENSITY FOR

CONTROL AND EXPERIMENTAL GROUPS. ....................................................................................................... 37

TABLE 4 – THE MEAN INTENSITIES OF THE IMMUNOSTAININGS QUANTIFIED BY INTEGRATED PIXEL DENSITY FOR

CONTROL AND EXPERIMENTAL GROUPS. THE NORMALIZED VALUES WERE USED TO PRODUCE THE GRAPHS

SHOWN IN FIG. 5-8. ....................................................................................................................................... 37

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Contents

ACKNOWLEDGEMENTS .................................................................................................................................. 5

RESUMO ............................................................................................................................................................... 6

ABSTRACT ........................................................................................................................................................... 7

1. INTRODUCTION ........................................................................................................................................... 11

1.1. THE NERVOUS SYSTEM ............................................................................................................................ 11 1.2. PERIPHERAL NERVE INJURY ....................................................................................................................... 13 1.3. NERVE REGENERATION (PERIPHERAL NERVE REPAIR) ................................................................................ 14 1.4. DIRECT NERVE REPAIR (NEURORRHAPHY)................................................................................................. 16 1.5. HUMAN EMBRYONIC STEM CELLS (HESC) ................................................................................................. 18 1.6. FIBROBLAST GROWTH FACTOR 2 ................................................................................................................ 20

2. JUSTIFICATION ............................................................................................................................................ 23

3. RESEARCH OBJECTIVE ............................................................................................................................. 24

3.1 GENERAL PURPOSE ...................................................................................................................................... 24 3.2 SPECIFIC OBJECTIVES .................................................................................................................................. 24

4. MATERIAL AND METHODS ...................................................................................................................... 25

4.1 ANIMALS AND SURGICAL PROCEDURES ....................................................................................................... 25 4.2 VECTOR CONSTRUCTION ............................................................................................................................. 28 4.3 MANIPULATIONS WITH STEM CELLS ............................................................................................................ 29 4.4 HUMAN ESC TRANSPLANTATION ................................................................................................................ 30 4.5 CELL CULTURE ............................................................................................................................................ 31 4.6 PREPARATION AND ADMINISTRATION OF THE FIBRIN SEALANT .................................................................. 31 4.7 IMMUNOHISTOCHEMISTRY ........................................................................................................................... 32 4.8 CATWALK TEST ........................................................................................................................................... 33 4.9 VON FREY TEST ........................................................................................................................................... 34 4.10 STATISTICAL ANALYSIS ............................................................................................................................. 35

5. RESULTS ......................................................................................................................................................... 36

5.1 EXPRESSION OF FGF-2 BY HESCS ............................................................................................................... 36 5.2 IMMUNOHISTOCHEMISTRY ........................................................................................................................... 36 5.3 MOTOR EVALUATION OF FUNCTIONAL RECOVERY VIA CATWALK .............................................................. 43 5.4 SENSORY FUNCTION EVALUATION VIA VON FREY TEST ............................................................................. 47

6. DISCUSSION................................................................................................................................................... 50

7. CONCLUSION ................................................................................................................................................ 58

REFERENCES .................................................................................................................................................... 59

ATTACHMENTS ................................................................................................................................................ 70

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1. Introduction

1.1. The Nervous System

The nervous system is didactically divided into two main parts namely Central Nervous

System (CNS) and Peripheral Nervous System (PNS). The functions of the nervous system

involve the processing of sensory information, motor control, mediation of autonomic

responses, elaboration of emotional responses, learning, and memory(Kandel, Schwartz et al.

2000).

Whereas CNS includes all parts of the brain and spinal cord, PNS is composed of

peripheral nerves and ganglia. The latter is a small group of nerve cells outside the CNS.

Neurons are specialized cells in the conduction of the nerve impulse and constitute the

fundamental unit of the nervous system. A typical neuron is composed of a body (soma),

dendrites and an axon (MACHADO 2006). Peripheral nerves contain groups of axons (nerve

fibers) that bundle together (Mescher 2009). Within a nerve, each axon is surrounded by a layer

of connective tissue called the endoneurium. The axons are bundled together into groups called

fascicles. Each fascicle is wrapped in a layer of connective tissue called the

perineurium. Ultimately, the entire nerve is wrapped in a layer of connective tissue called

epineurium (Flores, Lavernia et al. 2000) (Fig. 1).

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Fig. 1. An overview of the PNS. The axons surrounded by myelinating Schwann cells are

enclosed by endoneurium. The perineurium binds individual axons together to form fascicles

and epineurium groups fascicles to form the nerve cable (Marieb, Mitchell et al. 2013).

The cell body of the neuron is considered to be the metabolic center responsible for the

synthesis of most of the neuronal proteins and the degradation and renewal of the cellular

constituents. Dendrites, on the other hand, are responsible for receiving and generating stimuli.

The axon originates in the body of the neuron in a region named the cone of

implantation(Kandel, Schwartz et al. 2000). The length of axons are variable and in the human,

for example, varies from a few millimeters to more than one meter. The same applies to the

axons of the neurons that form the sciatic nerve. The axon is specialized in the conduction of

the electrical impulse and is responsible for the transport of substances such as neurotrophins

and growth factors. This transport is provided from the cellular body to the axonal terminals

and vice versa, which are respectively called the antegrade and retrograde axoplasmic

flow(MACHADO 2006). The cell, as a whole, receives terminals or synaptic buttons from other

neurons and forms synapses.

Axons in the PNS are surrounded by Schwann cell processes allowing close contact and

metabolic relationship. In myelinated axons, Schwann cells produce the myelin sheath around

each axon to protect and direct the nerve impulses. Every Schwann cell forms its internodal

myelin sheath, which is separated from an adjacent internode by a node of Ranvier. As a result,

Schwann cells are aligned discontinuously along the axon and are separated by nodes of Ranvier

at both the proximal and distal ends of the internode(Ide 1996).

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The PNS can be divided into two parts namely somatic and visceral. The visceral

(autonomic) PNS consists of neurons that innervate the internal organs, blood vessels, and

glands. Its sensory part carries information about the visceral function to the CNS, whereas the

motor part commands the contraction and relaxation of smooth muscles(Bear, Connors et al.

2008). The somatic PNS, on the other hand, is formed by axons that innervate the skin, joints,

and skeletal striated muscles, acting under voluntary control. Somatic motor axons command

muscle contraction under the control of motoneurons, whose cellular bodies are located in

nuclei in the ventral portion of the spinal cord(Bear, Connors et al. 2008). Somatic sensory

fibers, in turn, innervate and collect information from the skin, muscles and joints, and reach

the spinal cord via dorsal roots. The cellular bodies of these neurons are located outside the

CNS in the dorsal root ganglia(Bear, Connors et al. 2008).

After leaving the spinal cord or the sensory ganglia, the nerve fibers combine to form

peripheral spinal nerves, such as the sciatic nerve(MACHADO 2006). The sciatic nerve, which

is formed by the ventral roots of the spinal nerves namely L4, L5, L6 in the mouse, is the largest

nerve in the body and is part of the lumbosacral plexus. The sciatic nerve itself is formed by

three branches including: (i) tibial nerve, (ii) common fibular nerve, and (iii) sural

nerve(MACHADO 2006).

1.2. Peripheral Nerve Injury

Since neurons have long axons and relatively small cell bodies, most injuries in both the

CNS and the PNS involve axonal damage. Due to superficial pathways of the peripheral nerves

in some parts of the body, they are subject to various types of injuries including crushing and

transection (Sunderland 1990, Schmidt and Leach 2003). The most severe, however, is the

complete transection of a nerve called axotomy. Following an axotomy, several molecular and

cellular changes are observed at the level of the cell body, at the site of injury, and in the target

organs (Faroni, Mobasseri et al. 2015).

Initially, the axotomy divides the axon into a proximal segment, which remains attached

to the cell body, and a distal segment that, due to the lesion, loses its connection with the soma.

The cell body reaction leads to a profound response in both gene and protein expression. The

balance of these determines whether the neuron survives and attempts regeneration or results

in apoptotic death. Because the ability of protein synthesis is mostely restricted to the cell body,

axotomy gives rise to degeneration of the distal segment (Faroni, Mobasseri et al. 2015). In the

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proximal portion of the lesion, the cell body undergo a series of changes in response to injury,

the so-called chromatolysis, including: (i) swollen cell body, (ii) displacement of the nucleus to

the periphery and fragmentation of the rough endoplasmic reticulum. Metabolic changes

consisting of an overall increase in protein and RNA synthesis and changes in the gene pattern

expressed by neurons can also accompany the noted reaction (Kandel, Schwartz et al. 2000).

Typically, the injured neurons prioritize the synthesis of proteins related to axon repair and

growth. These changes are reversible if nerve regeneration is successful (Zochodne 2000).

The distal stump (distally to the lesion site) of the injured nerve undergoes a series of

molecular and cellular changes known as Wallerian degeneration, which has a crucial role in

the degeneration of the distal fragments of the injured axons. Schwann cells release myelin,

proliferate and produce cytokines and trophic factors, and participate in phagocytosis of myelin

debris and thus degenerating axons. The soluble factors produced by Schwann cells, combined

with axon debris activate resident macrophages and attract blood borne macrophages. When

the fragmented debris are removed by the combined action of Schwann cells and macrophages,

the former align to form columns called bands of Büngner. Such arrangement creates a

permissive environment rich in trophic factors, enabling guided axonal regeneration

(Sunderland 1990, Ide 1996, Bruck 1997, Faroni, Mobasseri et al. 2015).

In the target organ level, there are several obstacles for the regeneration of axon prior to

successful reinnervation. Typically, any misdirection towards the wrong target reduces

functional outcome even if there are a good number of regenerated axons. A lack of neuronal

contact in the distal stump leads to chronically denervated Schwan cells that down-regulate

growth factors and thus become unable to support axonal progression.

Similarly, the denervated target organ becomes depleted of trophic factors, resulting in

muscle fibres atrophy, leading to satellite cells apoptosis. These responses bear a significant

impact on functional recovery following proximal nerve injuries (Faroni, Mobasseri et al.

2015).

1.3. Nerve Regeneration (Peripheral nerve repair)

The PNS has far greater potential for regeneration than the CNS, mainly because the

former is associated with Schwann cells (Ren, Wang et al. 2012). Following an injury in the

PNS, Schwann cells transform into repair cells in the distal nerve stump. These cells, along with

macrophages, clean up inhibitory debris and enable the new axon sprouts to grow into the

15

degenerating nerve by the guidance of bands of Büngner. The result of these events is a healthy

environment for regeneration in the PNS(Scheib and Hoke 2013).

In the case of severe injuries, nerve regeneration commences only when Wallerian

degeneration has evolved its course. The degenerative process involves a mechanism to convert

normal nerves from a suppressed state into a state that promotes axonal growth (Salonen,

Peltonen et al. 1987, Danielsen, Kerns et al. 1995, Burnett and Zager 2004). Thus, the

regeneration process, as stated by Burnett et al. may be divided into the following anatomical

zones: “ (i) the neuronal cell body; (ii) the segment between the cell body and the injury site;

(iii) the injury site itself; (iv) the distal segment between the injury site and the end organ; and

(iv) the end organ itself “ (Burnett and Zager 2004).

The regeneration of an injured nerve may take several months. The first signs of this

process are the reversal of chromatolysis in the cell body and the centralization of the nucleus.

This signifies a fundamental shift in cell function from synaptic transmission to cellular repair.

The cell body regulates the metabolism to enable it to produce the vast amount of proteins and

lipids needed for axonal regrowth during the regeneration phase. Axoplasm, which is

responsible for the regeneration of the axon tip, arises from the proximal axon segment and cell

body. The rate of protein and lipid synthesis in the cell body affects the progress rate and the

final size of the regenerating axon (Burnett and Zager 2004).

The rate of axonal regrowth is regulated by (i) modification in the cell body, (ii) the

activity of the growth cone at the end of axon sprout, and (iii) the strength of the injured tissue

located between the cell body and end organ (Burnett and Zager 2004).

Axons that successfully enter the endoneurial tubes in the segment distal to the injury

site are more likely to reach the end organ. When the endoneurial tubes have been disrupted,

axon sprouts must first find their way into the tubes before advancing. This is achieved by

specialized growth cone at the tip of each axon, sprouting filopodia that adhere to the basal

lamina of the Schwann cell and use it as a guide. Accordingly, successful axonal regeneration

is entirely dependent on axonal sprouts making appropriate contact with Schwann cell basal

lamina in the distal nerve segment (Muir 2010). Regenerating axonal sprouts usually follow the

original Schwann cells to the denervated motor endplates to reform neuromuscular junctions.

There are chances for co-lateral sprouting to occur that result in groups of reinnervated muscle

fibers (Burnett and Zager 2004). During the regeneration, the axon’s diameter increases

16

progressively until reaching close to normal dimensions, but this growth is dependent on the

establishment of functional connections between the axon tip and the appropriate end organ

(Burnett and Zager 2004).

The transition of the nerve environment from an inhibited state to a regeneration

promoting one is likely an important event governing the onset and progression of axonal

regrowth. The degenerative process appears to involve mechanisms that convert normal nerve

from a suppressed state to one that promotes axonal growth (Salonen, Peltonen et al. 1987,

Danielsen, Kerns et al. 1995).

1.4. Direct Nerve Repair (Neurorrhaphy)

Following an injury that interrupts the nerve continuity, the primary repair is a standard

surgical procedure called “direct nerve repair” or “neurorrhaphy” (Hayat 2012). This surgical

technique can be achieved in two ways: end-to-end (ETE) repair, in which the coaptation is

performed between proximal and distal nerve stumps, and end-to-side (ETS) repair, in which

the coaptation is performed between the distal nerve stump and another healthy donor nerve

(Lundborg 1993, Hayat 2012). If direct end-to-end repair is not possible, due to retraction of

the stumps, autografting using sensory donor nerves is the gold standard approach.

Some pre-clinical studies have attempted to compare the effectiveness of ETE and ETS

techniques in the context of nerve preservation and nerve regeneration. The results have

demonstrated the superiority of the ETE neurorrhaphy over the latter in terms of recovery time

and the strength of the re-established motor force (Liao, Chen et al. 2009).

ETE neurorrhaphy repair may be performed by epineural, perineurial (Fascicular or

Funicular), and group fascicular techniques (Mafi, Hindocha et al. 2012). Conventionally,

however, the epineural repair, in which the stitches passed through the epineurium, has been

widely in use. There are several advantages to this technique including short execution time,

minimal magnification, technical easiness, not invading the intraneural contents, applicability

to both primary and secondary neural repairs, and the placement of sutures only in the outer

investing sheath (Daniel and Terzis 1977).

Whereas suturing the ends of the two nerves together can repair small defects or gaps in

the nerve (Schmidt and Leach 2003), there are cases that large lesion gaps in the peripheral

nervous system due to a defect, scar, or neuroma in continuity hinders direct repairing without

17

considerable tension. Such damages cannot be directly repaired without excessive tension and

impeding or limiting regeneration. When the gap that must regenerate across is above the

critical gap, a graft is needed to bridge the damaged ends, reconnecting the proximal and distal

stumps, and preventing the formation of a neuroma (Muir 2010). In such situations, ‘autogenous

nerve grafting’ is considered as the standard clinical treatment, particularly when there is tissue

loss and direct repair is not possible (Hayat 2012, Cartarozzi, Spejo et al. 2015). In this grafting

technique, a comparable nerve is first removed from another part of the patient's body and is

used to bridge the gap and connect the two ends of the severed nerve (Lundborg 2004, Glynn

2007). Without such grafts, these injuries will possibly never heal and can be permanently

debilitating (Muir 2010). For longer nerve gaps, however, this approach is not desired, because

tensions introduced into the nerve cable could inhibit nerve regeneration (Schmidt and Leach

2003).

There are several reasons for universal acceptance of autologous grafting in major

peripheral nerve repair. The first one is that by taking the donor nerve from within the patient's

body, there is no immune-rejection. This procedure offers a cell-rich material through which

axons can regenerate and thus has a relatively high success rate in restoring the majority of the

functionality to the damaged targets. It offers neuro-supportive architecture (that promotes

subsequent regeneration), guidance cues, neurotrophic factors, and a source for Schwann cells

(Lundborg 2004, Glynn 2007, Noaman 2012). Nerve regeneration with autografts usually

utilizes much of the graft's sheath arrangement and topology (Muir 2010).

By comparison, commercially available substances such as biodegradable polymer and

collagen-based hollow tubes have failed to match the regenerative levels of autologous nerve

grafting, mainly because they are limited to small defects and show poor functional recovery

(Faroni, Mobasseri et al. 2015). Direct nerve repair can be performed by means of fibrin glue,

or nylon suturing; however, the latter is the most common method used for this aim (Hayat

2012).

To simulate the autogenous nerve grafting in the lab, 5 millimeters of the sciatic nerve

of a mouse is transected, inverted by 180°, and then is sutured or glued (e.g. with fibrin sealant)

(Evans, Bain et al. 1991). The plan to invert the nerve of the same animal intend to minimize

the number of animals and their pain and discomfort. However, nerve repairs performed with

fibrin sealant is demonstrated to produce less inflammation, less fibrosis, better axonal

18

regeneration, and better fiber alignment in comparison with the nerve repair performed using

merely microsuturing (Pabari, Lloyd-Hughes et al. 2014).

1.5. Human Embryonic Stem Cells (hESC)

Although some surgical techniques have proved to lead to better nerve fiber

regeneration, the degree of recovery can be highly variable (Samii, Carvalho et al. 1997,

Lundborg 2002). It is thus crucial to seek for complementary techniques to improve the level

of recovery.

In recent years, stem cells have been widely investigated to be used to complement

surgery and facilitate the repair of injured peripheral nerves. The sources of these stem cells are

widespread and among them are the embryonic stem cells (ESC) routinely derived from the

inner cell mass of blastocysts (Evans and Kaufman 1981, Thomson, Itskovitz-Eldor et al. 1998,

Rahaman and Mao 2005, Ren, Wang et al. 2012). Due to the ability of ESC to self-renew

indefinitely and their pluripotency character, they have been considered as an ideal source of

cells for biomedical engineering (Weissman 2000).

Fig. 2. The steps for obtaining human embryonic stem cells (after Terese Winslow, 2006).

Although not completely clarified, the delivery of cellular components to the injured

nerve is based on the potential structural and neurotrophic additive effects exerted by paracrine,

19

differentiation and cell fusion mechanisms. Many kinds of cells including Schwann cells,

embryonic, fetal, and adult stem cells have been considered as candidates for transplantation

therapy in the peripheral nervous system (Hayat 2012).

The effectiveness of ESCs for the treatment of peripheral nerve injury and functional

recovery may lie in their ability to differentiate into Schwann cells, secrete neurotrophic factors,

promote axon regeneration, and assist in myelin formation (remyelination of axons).

Myelination, which determines both regeneration quality and functional recovery, requires the

longitudinal wrapping of Schwann cells (Ren, Wang et al. 2012). Also, these cells could be

induced to express a neural phenotype before transplantation (Hayat 2012).

Some researchers have shown that ESC transplantation can be a successful treatment.

For example, Cui et al. (Cui, Jiang et al. 2008) transplanted pre-differentiated ESC after sciatic

nerve axotomy in rats, and after three months observed that cells integrated with the host tissue

and demonstrated as expressing Schwann cell markers, suggesting that, at least part of the

remyelination process was performed by the transplanted cells. Regenerated axons were also

recorded, showing the uniform connection between the proximal and distal stumps and giving

rise to functional activity across the injury gap (Cui, Jiang et al. 2008).

However, there are many issues to be solved before the ESC can be used in nerve

therapy. Before being transplanted, these cells must be expanded invitro and differentiated, as

the undifferentiated cells can multiply in an uncontrolled manner upon injection, leading to

teratomas (Mendez-Otero, de Freitas et al. 2007). Human ESC (hESC) are traditionally

maintained in colonies on the feeder cell layers, formed for example by mitotically inactivated

mouse embryonic fibroblasts (MEF). It is nowadays accepted that the most technological way

to propagate hESC is to grow them in monolayers using Matrigel, laminin, or other extracellular

matrix components as a substrate (Kunova, Matulka et al. 2013). Various cytokines and growth

factors are used to support self-renewal of hESC in different culture systems, while the presence

of fibroblast growth factor 2 (FGF-2) is absolutely necessary (Dvorak, Dvorakova et al. 2006).

Advanced cellular bioengineering methods can provide ways to change useful

properties of the stem cells according to the objectives of the usage. This can offer opportunities

to seek for the treatment of tissues with little or no regenerative capacity including the

CNS(Kyrylenko 2012, Steward, Sridhar et al. 2013). Based on current knowledge, the engrafted

cells can perform their supportive role by two possible mechanisms: (i) Replacing the missing

20

cellular elements, not only neurons but also supporting cells, particularly the

Oligodendrocytes/Schwann cells that form myelin sheaths around axons; and (ii) Delivering

trophic factors (Pêgo, Kubinova et al. 2012). The effects of cell therapy are mediated by the

secretion of growth factors or cytokines that reduce neuronal apoptosis and inflammation and

stimulate endogenous regenerative processes as well as re-myelination and neural plasticity,

leading to the collateral sprouting of intact or damaged axons of the descending pathways

(Bradbury and McMahon 2006, Pêgo, Kubinova et al. 2012).

The application of growth factors can result in significant increase in nerve regeneration.

However, it is difficult to deliver the active growth factors over the entire duration of

regeneration in a controlled manner. Transplanted genetically modified cells, on the other hand,

offer advantages as a means of delivering a continual supply of active growth factors.

Furthermore, if the gene expression in the modified cells can be turned on (presence) and off

(absence), which is achieved by using doxycycline as the regulator for inducible gene

expression systems (Blesch, Lu et al. 2002), the expression then could be directed in a

sophisticated manner. The ability to regulate gene expression in grafted cells by genetic

engineering can enhance the in vivo efficacy of growth factor delivery by controlling axonal

entry and exit from the lesion site. By this approach, a series of growth factor expression could

lead axons to grow into a graft, switch patterns of expression, and then lead axons out of the

graft. In addition, regulated expression of growth factors at distal sites of the lesion could

enhance the exit of axons from the graft by attracting axons into the downstream regions

(Blesch, Lu et al. 2002, Schmidt and Leach 2003).

As noted, simple supplementation of hESC with doxycycline greatly improves the

viability and self-renewal capability of the cells. In practice, doxycycline enhances the

expandability of hESCs and boosts its effects for longer times. The effects of doxycycline are

mediated by direct activation of the PI3K-AKT intracellular pathway, which has been reported

to be the most crucial regulatory pathway for self-renewal of hESC (Chang, Rhee et al.). It is

worth mentioning that doxycycline has neuroprotective function in PNS(Lazzarini, Martin et

al. 2013, Santa-Cecilia, Socias et al. 2016, Gonzalez-Lizarraga, Socias et al. 2017).

1.6. Fibroblast Growth Factor 2

Fibroblast Growth Factor 2 (FGF-2, also known under the name Basic Fibroblast

Growth Factor) is a representative growth factor that has shown potentials to effects the repair

21

and regeneration of tissues. Originally, it was identified as a protein with the capability to

promote fibroblast proliferation. FGF exert multiple functions through binding into and

activation of fibroblast growth factor receptors (FGFRs), and signaling through the stimulation

of FGFRs is the RAS/MAP kinase pathway (Yun, Won et al. 2010).

FGF-2 is a member of the FGF family, which comprises up to now 23 members (Ornitz

and Itoh 2001) and encoded by a single copy gene that is alternatively translated to produce one

low (18-kDa) and four high (22-, 22.5-, 24-, and 34-kDa) molecular mass isoforms (Arnaud,

Touriol et al. 1999, Delrieu 2000).

Recent studies on the function and expression of FGF-2 and its receptors have revealed

a physiological role of these molecules in the PNS. FGF-2 and its receptors are constitutively

expressed in the dorsal root ganglia and peripheral nerve (Grothe, Meisinger et al. 2001, Grothe

and Nikkhah 2001). These molecules display an up regulation in the dorsal root ganglia and the

proximal and distal nerve stumps following peripheral nerve injury. In the ganglia, the

molecules show a chiefly neuronal expression, whereas at the lesion site of the nerve, Schwann

cells and invading macrophages represent the main cellular sources of FGF-2 and the 1–3

receptors (Grothe and Nikkhah 2001). Whereas Schwann cells are regarded as the main source

of FGF-2(Grothe, Meisinger et al. 1996, Grothe and Nikkhah 2001), the autocrine function of

FGF-2 is known to stimulate the Schwann cell proliferation 44; 45.

After sciatic nerve injury, FGF-2 is shown to increase continually for at least four weeks

in the proximal and distal nerve stump. The transcript levels of FGFR 1–3 are also upregulated

proximal and distal to the lesion site (Grothe, Meisinger et al. 1996, Meisinger and Grothe 1997,

Grothe and Nikkhah 2001). The isoforms of FGF-2 display a differential regulation in the nerve

stumps before and after a lesion (Meisinger and Grothe 1997, Grothe, Heese et al. 2000, Grothe

and Nikkhah 2001).

Results from in vivo and in vitro studies using exogenously applied FGF-2 also indicate

distinct functions for FGF-2 in spinal ganglia and at the lesion site (Grothe and Nikkhah 2001).

It commonly promotes the neurite outgrowth and vascularization of the regenerating nerve

fibers crossing the gap between proximal and distal stumps of the injured sciatic nerve

(Danielsen, Pettmann et al. 1988, Aebischer, Salessiotis et al. 1989, Jungnickel, Haase et al.

2006). Furthermore, it has been demonstrated that the use of FGF-2 to the transectioned sciatic

22

nerve can develop a neurotrophic capacity to rescue the sensory neurons from death (Otto,

Unsicker et al. 1987).

FGF-2 can also stimulate neurogenesis in animal models of neurodegenerative diseases

(Peng, Xie et al. 2008, Woodbury and Ikezu 2014), stimulate synaptic plasticity (Terlau and

Seifert 1990, Ishiyama, Saito et al. 1991), and be highly effective in promoting scarless wound

healing (Abe, Yokoyama et al. 2012). Moreover, FGF-2 is a principal constituent of hESC

growth medium and the most significant regulator of hESC self-renewal (Dvorak, Dvorakova

et al. 2006). It is now widely accepted that hESCs require exogenous FGF-2 to sustain self-

renewal and pluripotency, and develop the capacity to differentiate into a large number of

somatic cell types (Amit, Carpenter et al. 2000, Xu, Inokuma et al. 2001, Dvorak, Dvorakova

et al. 2006). It activates the FGF receptors (FGFRs) and stimulates the mitogen activated protein

kinase (MAPK) pathway in undifferentiated cells (Eiselleova, Matulka et al. 2009). Finally, the

exogenous FGF-2 can stimulate the expression of stem cell genes while eliminating cell death

and apoptosis genes.

23

2. Justification

Peripheral nerve injury as a worldwide clinical problem could impair the quality of life

of a patient and many of such injuries require surgical nerve reconstruction. This objective is

mostly achieved by autogenous nerve grafting as gold standard treatments of the peripheral

nervous system. Whereas suturing the ends of the two nerves together can repair small defects,

there are cases in which large lesion gaps in the nerve hinders direct repairing without causing

considerable tension. It is thus very important to seek complementary techniques to improve

the degree of recovery. One of the promising technique for this aim is the use of cell therapy.

Transplantation therapy by human embryonic stem cells is very appealing because they have

the ability to self-renew indefinitely and differentiate into a diverse range of specialized cell

types. The effective use of hESCs requires FGF-2 to sustain self-renewal and pluripotency and

develop the capacity to differentiate into a large number of somatic cell types. In addition, nerve

repairs performed with fibrin sealants was demonstrated to produce less inflammation, less

fibrosis, better axonal regeneration, and better fiber alignment. Accordingly, a combination of

hESCs which inducibly overexpress FGF-2 and fibrin sealants shall provide an ideal solution

for nerve regeneration in mice model following autographing with end-to-end neurorrhaphy.

24

3. Research Objective

3.1 General Purpose

The main objective of this research is to find conditions under which functional recovery

is improved after sciatic nerve neurorrhaphy. We assume that human embryonic stem cells

(hESC), either alone or in combinations with fibrin sealant scaffold can be used to support

neuronal survival and regeneration in a mice model of sciatic nerve injury and repair via

autografting with end-to-end neurorrhaphy.

3.2 Specific Objectives

Specific objectives of this project are the following: (1) To investigate how human

embryonic stem cells can be used to support functional recovery in a mice model of sciatic

nerve isograft end-to-end neurorrhaphy; (2) To examine the effects of fibrin sealant on neuronal

regeneration after sciatic nerve neurorrhaphy in mice; (3) To investigate how genetically

engineered human embryonic stem cells, which inducibly overexpress human FGF-2, can be

used to support neuronal regeneration; and (4) To examine the recovery and sensory function

of animals subjected to sciatic nerve repair combined with hESC therapy and fibrin sealant

through the walking track and Von Frey tests.

25

4. Material and Methods

4.1 Animals and Surgical Procedures

To investigate the effect of different supplementary compounds (including fibrin

sealant, doxycycline, and hESC) on the site of injury after neurorrhaphy, we designed six

groups of 8 animals each and followed the procedures described in Table 1.

Table 1 – Experimental groups and procedures followed during the research.

Tag Group Abbreviations Procedure Number

1 Neurorrhaphy alone N

Immunohistochemistry

Catwalk up to recovery

(~8weeks post surgery)

von Frey

8

2 Neurorrhaphy +

fibrin sealant N + F

Immunohistochemistry

Catwalk up to recovery

(~8weeks post surgery)

8

3

Neurorrhaphy +

fibrin sealant +

doxycycline

N + F + D

Immunohistochemistry

Catwalk up to recovery

(~8weeks post surgery)

von Frey

8

4

Neurorrhaphy +

fibrin sealant + wild

type hESC

N + F + W

Immunohistochemistry

Catwalk up to recovery

(~8weeks post surgery)

von Frey

8

5

Neurorrhaphy +

fibrin sealant +

hESC off

N + F + T

Immunohistochemistry

Catwalk up to recovery

(~8weeks post surgery)

von Frey

8

6

Neurorrhaphy +

fibrin sealant +

hESC on

(doxycycline)

N + F + D + T

Immunohistochemistry

Catwalk up to recovery

(~8weeks post surgery)

von Frey

8

26

For sciatic nerve lesion and repair, four week-old C57BL/6 male mice were obtained

from the Multidisciplinary Center for Biological Research (CEMIB), University of Campinas.

We treated them in our lab for a period of 10 days by giving vermifuge medicine for three times

with three days intervals. Both before, and after the surgery, the mice were kept with ad libitum

access to food and water under controlled light (light/dark cycle of 12 h) and temperature

conditions (i.e. 23°C) in racks specific for mice at our laboratory.

The animals were anesthetized with intraperitoneal injections of Kensol (xylasine,

Köning, Argentina; 10 mg/kg) and Vetaset (Cetamine, Fort Dodge, IA; 50 mg/kg, i.p); totaling

0.12 ml/25 g of the body weight. The left hind of the animals underwent trichotomy. Then

around 1.5 cm of the skin was incised with a scalpel. After exposing the sciatic nerve by

retracting the musculature, a 5 mm long segment of the nerve was cut out from both ends,

inverted by 180 degrees, and then inserted between the two nerve stumps. After inversion, the

nerve was repaired according to the experimental groups and was sutured under the microscope

with 9-0 nylon sutures (Fig. 3). During the surgical procedure, the first two components of the

fibrin sealant were applied and the third component was added for polymerization. For those

groups that embedded the fibrin sealant (Table 1), the cells were applied to the lesion site (6 µl)

after adding the third component. The reimplantation stability was tested by gently pulling the

nerve or by observing the clots of fibrin sealant at the site of suture under a microscope.

27

Fig. 3. Autografting procedure in which 5 millimeters of the sciatic nerve of a mouse is

transected, inverted by 180°, and then is sutured or stitched together by nylon suture and fibrin

sealant (~ 20x magnification).

We kept the mice at ambient temperature (under the warmth of a lump) and waited for

them to wake up. For groups # 3 and # 6, we gavaged 50 µl of 5 mg/ml of doxycycline instantly

after mice got conscious. Doxycycline was also included in their feed for the duration of the

experiments.

All the mice underwent surgery and then maintained in a vivarium for more than eight

weeks. During this period, we implemented a series of functionary and sensory evaluations

using Catwalk and von Frey tests (sections 4-4 and 4-5). After the predetermined survival times,

the animals were anaesthetized with an overdose of an aesthetic (a mixture of xylasine and

ketamine). The vascular system was transcardially perfused with phosphate buffer 0.1M (pH

7.4) and then perfused with 4% formaldehyde in phosphate buffer (20 ml of fixative per

animal). Their sciatic nerve was dissected out and post fixed in the same fixative solution

overnight at 4° C. They were then cryo-preserved for 24h in 10, 20, and 30% sucrose buffered

solution, respectively. Before embedding in Tissue–Tek (MilesInc., USA) and freezing at

−35°C to −40°C, we cut the nerves to yield proximal and distal parts. The longitudinal nerve

sections with 12 μm thickness, prepared by a cryostat instrument, were obtained and transferred

to gelatin-coated slides and stored at −20° C until use in immunohistochemistry studies (section

4-6).

28

All the noted experiments were conducted following the rules of ethics in animal

experimentation. We also endeavored to minimize the number of animals and their pain and

discomfort.

4.2 Vector Construction

The Phusion High Fidelity DNA Polymerase (Finnzymes/Thermo Fisher Scientific,

Vantaa, Finland) was used to amplify DNA fragments intended for cloning and/or transfections.

For constructing the hFGF-2 bacterial overexpressing vector, a PCR fragment 475 bp in length

was obtained using primers FGF-2-cds-2C, 5´-gaacatatggcagccgggagcatcac-3´ (with NdeI

recognition site, underlined), and FGF-2-cds-2D, 5´-atccctcgagctagctcttagcagacattgg-3´ (with

XhoI recognition site, underlined); and a template DNA from the ORFeome clone

OCABo5050E0522D obtained from Source BioScience (Nottingham, United Kingdom). Its

NdeI-XhoI subfragment 470 bp in length was then cloned into pET28b (Novagen/Merck

Millipore, Darmstadt, Germany) using appropriate restriction endonucleases. The resulting

vector pTe133 (GenBank accession number KX834270) was induced with IPTG in E.coli

BL21(DE3) using EnPresso growth system from BioSilta Oy (Oulu, Finland) based on enzyme-

controlled glucose autodelivery. The soluble recombinant protein was purified using Ni affinity

column. The (His)x6 tag was cleaved off by thrombin, and the resulting rFGF-2 was further

purified using heparin affinity column. The biological activity of the purified rFGF-2 was

verified using two methods: via cell proliferation assay in hESC; and then via the Erk

phosphorylation assay in hESC.

To obtain a stable hES cell line overexpressing FGF-2-GFP fusion in an inducible mode,

a Tet-On system (Clontech/Takara Bio, Mountain View, CA, USA) was used. For constructing

a vector for inducible overexpression of hFGF-2, a PCR fragments obtained with primers FGF-

2-cds-G, 5´-cagaattcatggcagccgggagcatc-3´ (with EcoRI recognition site, underlined) and FGF-

2-cds-H, 5´-tgggtaccaagctctta gcagacattgg-3 (with KpnI recognition site, underlined),

containing ORF of hFGF-2, was cloned (477 kb) into respective sites of pEGFP-N1 (Clontech),

giving rise to an intermediate vector pTe104, overexpressing hFGF-2-GFP fusion in a

constitutive (non-inducible) mode. Then, the pTe104 was used as a template to amplify the

fragment containing hFGF-2-GFP fusion ORF with the primer FGF-2-cds-G and a primer

cdsGFP-B, 5´-cgtctagattacttgtacagctcgtccatg-3´ (with XbaI recognition site, underlined). This

fragment after digestion (1,227 bp in length) was cloned into respective sites of pTRE-Tight

(Clontech), giving rise to a vector pTe106 (GenBank accession number KX844812). For stable

29

cell transfections, the DNA of the pTe106 vector was used as a template for amplifying a linear

transfection fragment 2,275 bp in length containing essential parts of the vector and omitting

the unnecessary bacterial sequences, with primers TRE-Lin-FWD, 5´-

gaagcatttatcagggttattgtctc-3´ and TRE-Lin-REV, 5´-agggagaaaggcggacaggtatc-3´.

4.3 Manipulations with Stem Cells

The hESC line CCTL12 was cultured in monolayers on Matrigel as described 38. The

karyotype of the cells was analyzed at Institut für Humangenetik und Anthropologie, Jena,

Germany. For transfection of the stem cells, we initially employed the nucleofection (Lonza,

Basel, Switzerland) and compared it to chemical transfection methods with FuGene 6, FuGene

HD (Roche, Basel, Switzerland), Lipofectamine 2,000 (Thermo Fisher Scientific, Waltham,

MA USA) and NanoJuice (Novagen/Merck Millipore, Darmstadt, Germany) reagents

according to manufacturer protocols. For the pilot experiments, cells were transfected with

pEGFP-N1 vector, and quantification of the efficiency of transfection was done by flow

cytometry with Cytomics FC500 Flow Cytometry Analyzer (Beckman Coulter, Brea, CA,

USA) 48 hrs post transfection by monitoring percentage of GFP positive cells. For this, cells

were collected by TrypLE (Invitrogen/Thermo Fisher Scientific) and resuspended in

conditioned HES medium 38 supplied with 10 µg/ml propidium iodide.

For stable selection, a concentration of G-418 and Blasticidin selection agents were pre-

determined with killing curves, such that the sensitive (wild type) cells were slowly cleared

away during two weeks. The killing curves were determined first in wide ranges and then in

narrow ranges of concentrations. The G-418 was used at 140 µg/ml and Blasticidin at 1.2 µg/ml.

The cells were plated at 20 000 cells/cm2 on Matrigel covered plates, and the selection agents

were added 24 h post plating.

The inducible overexpression in Tet-On system is regulated by rtTA transactivator,

therefore in the first round of stable selection the cells were transfected with the HindIII

linearized regulatory vector pEF1a-Tet3G (Clontech) and selected against G-418. For this, the

cells were plated at 20,000 cells/cm2 into the 24x plate. 24 h post plating the cells were

transfected with FuGene HD reagent. After additional 24h, the transfected cells were collected

with Tryple and plated into 6x plate with 2-fold serial dilutions; and the selection agent was

added 24h post plating. The medium was changed 2 times per week. Two weeks after, the

individual colonies were collected and the dissociated cells were transferred to 96x well plates.

30

The growing colonies were divided into 3 parallel 96x plates, and the individual clones

were screened for the efficiency of induction by transient transfection with pTRE-Luc vector

(Clontech) followed by induction with DOX at 1 µg/ml and determination of luciferase signal

using Bright-Glo Luciferase Assay System (Promega, Fitchburg, Wisconsin, United States).

The luciferase signal was normalized to the metabolic activity of the cells in the same well

determined by a resazurin assay. For this, the resazurin was added to each well at 5 µg/ml, and

the fluorescence was determined with 595/535 nm excitation/emission wavelength,

respectively after 4h incubation in a CO2 incubator at 37 ºC. The clones were screened for the

lowest basal expression and the highest induction rate, as well as for their ability to retain their

properties after cycles of cryopreservation.

The cells of the selected stable clone E12-1 (positive for the rtTA transactivator) was

further stably transfected with the pTe106 target vector. The transfection-selection procedure

was similarly implemented as described above. As the pTe106 (a derivative of pTRE-Tight)

vector does not possess any selection marker, its linear fragment (see above) was co-transfected

with a Blasticidin linear selection marker. The linear selection marker for Blasticidin was

obtained as a PCR fragment 1,309 bp long with primers BlastLin-A, 5´-

tataagggattttgccgatttcgg-3´ and BlastLin-B, 5´-gtatgttgtgtggaattgtgagc-3´ and DNA of vector

pcDNA6/TR (Invitrogen) as a template. The resulting double-stable clone E12-1-1

(overexpressing human FGF-2 in an inducible mode) was used in further experiments to support

neuronal survival and regeneration.

4.4 Human ESC Transplantation

Immediately following neurorrhaphy, 3×105 hES cells resuspended in 3-5 µl of DMEM

were engrafted directly at the lesion site together with the fibrin sealant matrix. To induce

overexpression of FGF-2 in the hESCs in vitro, the inducer doxycycline was added to the growth

medium at 1 µg/ml for 24-48 h. For experiments in vivo, DOX was given to animals combined

with the pelleted food (see details above). Induction was confirmed by GFP expression in the

hESCs.

31

4.5 Cell Culture

The stems cells used in this research were hESCs derived in the Masaryk University

(Dvorak, Dvorakova et al. 2006). The hESC line CCTL14 had been engineered such that the

cells inducibly overexpress human fibroblast growth factor 2 (FGF-2 ) (Kyrylenko 2012).

During the cell culture step, we first coated the plastic plates with Matrigel and produced

proper surface for the cells to grow. Then, the frozen aliquot of Matrigel (Mgl) was diluted with

DMEM on ice to reach 1/100 proportion. The whole surface of the well was covered by

Mgl/DMEM solution and finally plated by Conditioned Human Embryonic stem cell (CHES,

see below) medium. After applying the coating, we cultured the cells by following the

successive steps outlined below:

(i) the cryovial with HES cells was quickly thawed in a 37°c water bath;

(ii) the content (cells) of the cryovial was transferred to the media for washing;

(iii) the suspension was centrifuged and the cells were separated.

(iv) the cells were re-suspended and then transferred to the prepared well;

(v) the cells were cultivated in humidified incubator at 37°c with 5% CO2;

(vi) after reaching the monolayer, the cells were detached by the TrypLE enzyme, collected,

and washed.

(vii) the cells were counted and 300,000 cells were prepared for follow-up surgeries.

To prepare the CHES conditional medium, we derived mouse embryonic fibroblasts

(MEFs) from 12.5 day embryos using the standard protocols available in our Lab. The MEFs

were then frozen with the freezing medium and kept for subsequent medium preparations. Each

time it was needed, we cultured them and then added HES medium to the MEF cells to make

the CHES medium. The resultant CHES were used to culture the Human embryonic stem (HES)

cells.

4.6 Preparation and Administration of the Fibrin Sealant

Fibrin sealant (FS) derived from snake venom was supplied by the Center for the Study

of Venoms and Venomous Animals (CEVAP) of UNESP. Its constituents and instructions for

use are provided in the patents No BR1020140114327 and BR1020140114360. At the time of

usage, the components were thawed, reconstituted, and applied to the sciatic nerves (Barros,

Ferreira et al. 2009, Barbizan, Castro et al. 2013),(Barbizan, Castro et al. 2013, Gasparotto,

32

Landim-Alvarenga et al. 2014). The fibrin glue, which is composed of three separate solutions,

was homogenized immediately before being used in a final volume of 4.5 µl comprising of the

following proportion: fibrinogen (2.5 µl), calcium chloride (1µl), and thrombin-like fraction (1

µl). During the surgical procedure, the first two components were applied and the third

component was added for polymerization.

4.7 Immunohistochemistry

In order to visualize the regenerating nerves, we employed immunohistochemistry

technique. This approach enables the detailed observations of nerve regeneration mechanisms

in mice.

To accomplish this objective, the slides that were kept in a freezer at −20° C were

removed and left at room temperature for a while and then were washed with 0.1M PB. Next,

the specimens were incubated for 45 min in a 3% BSA solution. The resultant slides were

incubated with the primary antibodies reported in Table 2 overnight at 4° C. After three washes

in PB 0.1M, the respective secondary antibodies conjugated to Cy-3 (1/250, Jackson

Immunoresearch, West Grove, PA, USA) were applied and incubated for 45 minutes at room

temperature. Finally, the slides were washed and mounted with glycerol/PB (3:1) to obtain

immunostained sections.

Table 2 – Primary antibodies used for immunohistochemistry.

Antibody Supplier Host Code Concentration

Anti-S100 Dako Rabbit Z0311 1/2000

Anti-Neurofilament Milipore Rabbit Ab1989 1/2000

Anti-VGLUT1 Synaptic system Rabbit 135303 1/1000

Anti-ChAT Millipore Rabbit AB143 1/1200

The immunostained sections were observed with a fluorescence microscope (Nikon

Eclipse TS 100) using the rhodamine filter (CY3). Three representative images were captured

from normal and regenerated nerves from different experimental groups using a high-sensitivity

camera (Nikon DXM 1200F) mounted on the microscope.

For quantification purpose, each immunelabeled picture was segmented into four sub-

images to avoid null margins and then measured to obtain the integrated density of pixels using

IMAGEJ software (version 1.33u, National Institutes of Health, USA). For each animal, three

33

individual pictures from different parts of the nerve were collected. At the end, the mean

intensity ± standard error was established by averaging the results of segments and pictures for

each group. The results also were normalized against the control group (expressed in

percentage) and used to compile the bar chart of the experimental groups.

Immunohistochemistry analysis was performed aiming to quantify the following markers:

(i) Anti-choline acetyltransferase (CHAT) to label motor fibers.

(ii) Anti-neurofilament (NF) to observe regenerated axons; or analyzes the organization of

the intermediary filaments that comprise the axons of regenerated and contralateral

nerves.

(iii) Anti-VGLUT1 to label primary afferent inputs.

(iv) Anti S-100 to characterize the marker of Schwann cells.

4.8 Catwalk Test

Following the repair of peripheral nerve injury, improved behavioral outcome remains

the most important evidence for the functionality of axon regeneration. The most frequently

used behavioral test to evaluate sciatic nerve injury is the walking tract analysis from the

Catwalk XT system (www.noldus.com/animal-behavior-research/products/catwalk).

To perform this test, in a dark room, the animal is placed on a platform with a glass floor

(with 100×15×0.6 cm dimension) fitted with a fluorescent lamp that is used to record points

trodden by the mouse, and the amount of pressure exerted by his paw, which is directly

proportional to the contact area of the foot to the floor. Through the glass, the floor of this

corridor is monitored by a camera (Pulnix TM-765E CCD) equipped with a wide-angle lens.

The signal intensity will vary according to the pressure applied by the animal's paw. The higher

the pressure exerted by the animal's paw, the greater the paw contact with the floor and thus the

higher the brightness, reflected in the intensity of pixels. These signals are digitized by PC

Image-SG frame to frame (Matrix vision GmH, Oppenheime, Germany). The catwalk program

acquires, stores and analyzes videos of animals roaming the hallway.

The recorded videos were analyzed on a computer by CATWALK program. For the

calculation of motor recovery rate of the sciatic nerve, the amounts related to the distances

between the first and the fifth finger (toe spread), and between the third toe and the heel (print

34

length), both the right hind paws (normal) and left (injured) were applied to calculate the sciatic

function index (SFI) by the following formula (Pan, Cheng et al. 2007):

SFI = 118.9 ((ETS-NTS) / NTS) -51.2 ((EPL-NPL) / NPL) -7.5

Where E is the injured side, N, the normal side, TS, the "toe spread," and PL, the "print

length." For adaptation and training purposes, all animals are submitted to the test before the

sciatic nerve injury.

The Catwalk test and related calculations were performed for all the reported groups in

Table 1 in the following way: at the beginning of one week interval until 14th day followed by

four days intervals until reaching the limit of eight weeks (60 days).

4.9 Von Frey Test

Although Catwalk can serve as a standard tool for quantitative and reliable assessment

of treatment efficacy (Chen, Cheng et al. 2014), it cannot measure pain, which is an indication

of how good sensory neurons recover. To fill this gap, we considered to include electronic

pressure-meter test (von Frey) to our experiments. This test was used to quantify the mechanical

sensitivity of the feet following the surgery (Cunha, Verri Jr. et al. 2004, Shiotsuki, Yoshimi et

al. 2010).

To conduct this test, in a quiet room, mice are placed in individual Plexiglas’s box of

12×20×17 cm in dimension, whose floor consists of a mesh network with a pore size of 5 mm2,

and a non-malleable wire thickness of 1 mm. Mice remain in the boxes for 20 min before the

experiment to habituate. Mirrors are positioned 25 cm below the testing boxes for easy viewing

of the animal paws.

The experimenter (the author) was trained to apply through the mesh network, pressure

on the plantar surface of the paw at a constant increase until the mouse emit a paw withdrawal

reflex, followed by a response characterized as tremor (“flinch”) of the stimulated paw. The

stimuli were repeated until the animal shows three similar consecutive measurements (i.e. with

a difference of force less than or equal to 10%). When the paw was withdrawn, the instrument

automatically recorded the stimulus force. The maximal force applied was 8 g. The hyperalgesia

intensity is evaluated by an electronic gauge, which consists of a force transducer connected to

a digital counter and is quantified by variation of the nociceptive threshold in grams (gram-

force).

35

We measured the reflexes of the mice from groups # 1, 3, 4, 5, and 6 (Table 1) prior to

the surgery to establish a baseline or preoperative sensory function. After performing the

surgery, we repeatedly measured the same parameter for the duration of 8 weeks. However,

during this time interval, due to a problem with the instrument, we failed to measure for two

successive weeks. The three close measurements were averaged to represent the stimulus force

(g) for each mouse. The results were analyzed by the statistical procedure described in section

4-7.

4.10 Statistical Analysis

The results from all the noted experiments were presented as the mean ± standard error

of the mean (SEM) and were evaluated by the analysis of variance method. We employed the

one-way ANOVA by comparing track groups belonging to the same mouse strain over time by

assuming a significance level equal to * P <0.05; ** P <0.01; *** P <0.001. In all cases, the

ANOVA was followed by Bonferroni post-test. The resultant data were expressed as the mean

± SEM with P<0.05 being considered to be significant. All statistical analyses were performed

using the GraphPad Prism package (GraphPad Software, San Diego, CA, USA).

36

5. Results

5.1 Expression of FGF-2 by hESCs

We set a demonstration experiment in the lab to ensure that the addition of doxycycline

activates the bioengineered cells to overexpress FGF-2. We cultured the cell in a plate and after

reaching the monolayer, doxycycline was given to the medium at 1μg/ml concentration and

after 24h examined them under a microscope using phase-contrast filter and florescence light

(Fig. 4). The result clearly indicates that the cells have been activated and indeed can fulfill the

expected functions.

Fig. 4. Photographs of hESC activated by doxycycline to overexpress FGF-2. a) Using a phase-

contrast filter. b) By using florescence light.

5.2 Immunohistochemistry

Immunolabeling was performed on longitudinal sections of regenerated nerves after 60

days post injury. By using anti-neurofilament antibody (Fig. 5), we analyzed the organization

of the intermediate filaments that make up the axons of the regenerated, as well as the normal

nerves. In all groups, the nerve fibers established a parallel pattern along the axis of the nerve,

whereas in the intact (control) nerve, the fibers showed a pattern of parallel waves. Visually,

the axons have their highest density in the ‘cell-on’ group (Fig. 5g) relative to other groups

(~40% of the control group) and has the most similar pattern to control group (Fig. 5a and 5g).

Nevertheless, statistical analysis performed for this antibody showed no significant difference

between the experimental groups (Fig. 5h). The mean intensities of the immunostainings

quantified by integrated pixel density are reported in Table 3.

37

Table 3 – The mean intensities of the immunostainings quantified by integrated pixel density

for control and experimental groups.

Antibody Anti-

VGLUT1

Anti-S100 Anti-Neurofilament Anti-ChAT

Control 192.6 ± 27.40 212.8 ± 21.3 726.3 ± 49.6 383.7 ± 82.0

N 109.9 ± 11.30 392.7 ± 51.9 262.1 ± 14.6 222.7 ± 29.1

N + F 101.4 ± 14.58 214.9 ± 27.5 199.4 ± 21.9 264.3 ± 27.4

N + F + D 96.89 ± 9.256 159.9 ± 12.2 271.5 ± 37.5 260.7 ± 25.1

N + F + W 131.8 ± 9.340 192.9 ± 9.7 239 ± 23.3 259.4 ± 31.8

N + F + T 149.6 ± 6.576 260 ± 28.3 287.3 ± 35.5 275.6 ± 28.8

N + F + D +

T

189.4 ± 20.13 204.6 ± 21.2 291.4 ± 33.3 289 ± 27.9

Table 4 – The mean intensities of the immunostainings quantified by integrated pixel density

for control and experimental groups. The normalized values were used to produce the graphs

shown in Fig. 5-8.

Antibody Anti-

VGLUT1

Anti-S100 Anti-Neurofilament Anti-ChAT

N (norm) 66.19 ± 7.752 150.6

±15.67

35.18 ± 2.189 47.66 ±

5.526

N + F (norm) 57.04 ± 9.484 100.5 ±

9.04

29.51 ± 3.091 50.26 ±

7.681

N+F+D(norm) 54.36 ± 6.536 80.04 ±

6.90

38.15 ± 2.819 48.95 ±

5.090

N+F+W(norm) 73.84 ± 6.312 96.24± 4.91 35.18 ± 3.739 53.51 ±

7.145

N+F+T(norm) 81.91 ± 10.41 131.5±

16.37

39.56 ± 1.682 56.79 ±

6.056

N+F+D+T(norm) 107.3 ± 15.88 99.7 ±

12.42

39.30 ± 4.336 61.13±

5.792

The Choline acetyltransferase (ChAT) as the enzyme responsible for the biosynthesis

of acetylcholine is used currently as the most specific indicator for monitoring the functional

38

state of cholinergic neurons in the peripheral nervous systems. The Anti-ChAT is showing

intense motor axons in the control group (Fig. 6a); albeit for the experimental groups, the motor

axons are less intense. The mean intensities of the immunostainings quantified by integrated

pixel density are reported in Table 3. Despite the incremental trend from ‘N+F’ (~43%) towards

‘N+F+D+T’ group (~60%), statistical analysis performed for this antibody showed no

significant differences between the groups.

By means of Anti-VGLUT1 antibody (Fig. 7), which is a marker of sensory neurons,

we labeled the primary afferent inputs responsible for transport of Glutamate into the synoptic

vesicle. The observation with a fluorescence microscope indicated that VGLUT1 antibody is

associated with a more sensory neuron in the ‘cell-on’ (N+F+D+T) group (Fig. 7G). The mean

intensities of the immunostainings are reported in Table 3. Statistical analysis performed for

this antibody showed statistically significant differences between the experimental groups with

the ‘cell-on’ one having the highest integrated density. Based on this analysis, the ‘cell-on’

group is yielding the same level of sensory neurons as the control group, corresponding to 107%

recovery (Fig. 7h).

The anti-S100 µ-staining (Fig. 8) that is a characteristic marker of Schwann cells was

intense on N and N+F+T groups (i.e. 150% and 120%, respectively), but in the group that

incorporated transgenic cells was at the same level as the control group (i.e. 100%). The mean

intensities of the immunolabels, quantified through the integrated density of pixels are reported

in Table 3. Similar to VGLUT1 antibody, the statistical analysis shows a meaningful difference

between the experimental groups.

39

Fig. 5. Anti-Neurofilament immuno-staining of the control nerves (A) and all groups (B to G),

60 days after surgery. H) Quantification of the integrated density of pixels in the experimental

groups relative to the control group presented in percentage (%) and calculated using IMAGEJ

software. Statistically, there is no significant difference between the experimental groups.

40

Fig. 6. Anti-Chat immuno-staining of the control nerves (A) and all groups (B to G), 60 days

after surgery. H) Quantification of the integrated density of pixels in the experimental groups

relative to control group (%). Statistically, there is no significant difference between the

experimental groups.

41

Fig. 7. Anti-VGLUT1 immuno-staining of the control nerves (A) and all groups (B to G), 60

days after surgery. H) Quantification of the integrated density of pixels in the experimental

groups relative to control group (%). Statistically, the difference between N+F vs N+F+D+T

and N+F+D vs N+F+D+T groups are meaningful with P <0.05 and P <0.01, respectively.

42

Fig. 8. Anti-S100 immuno-staining of the control nerves (A) and all groups (B to G), 60 days

after surgery. H) Quantification of the integrated density of pixels in the experimental groups

relative to control group (%).Statistically, the difference between the following groups are

meaningful: N vs N+F (p <0.05), N vs N+F+D (P<0.001), N vs N+F+W (P<0.05), and N vs

N+F+D+T (P<0.05). The N vs N+F+T shows no significant difference.

43

5.3 Motor Evaluation of Functional Recovery via Catwalk

The detailed results of Catwalk test separated by days and group are summarized in Fig. 9

to 11.

Fig. 9. The results achieved from Catwalk test separated by groups and calculated using the SFI

index. a) Neurorrhaphy group. b) Neurorrhaphy + fibrin sealant. c) Neurorrhaphy + fibrin

sealant + doxycycline. d) Neurorrhaphy + fibrin sealant + wild hESCs. e) Neurorrhaphy + fibrin

sealant + transgenic cells. f) Neurorrhaphy + fibrin sealant + doxycycline + transgenic cells.

44

In all groups, in the first measurement session (7 days after the surgery), the SFI is in

the lowest level of −75 (Fig. 9 and 10b), meaning that the mice cannot use their paws at all.

However, after the second week, the changes just start. This is marked by a gradual increase in

the SFI values in most of the groups (e.g. Fig. 9a and 9d). In the case of N+F group (Fig. 9b),

the gradual increase in using the injured paw, which is indicated by a higher SFI value, drops

after 4th session (day 22) and remains constant for the remaining duration of the experiment,

whereas in the N+F+D group (Fig. 9c), this trend starts after 22 days and gradually increases

afterwards. A similar trend can be seen in the N+F+W group (Fig. 9D). In contrast, the

incremental trend in N+F+T and N+F+D+T groups (Fig. 9E and F) reverses after around 34-38

days.

In the pre-operation measurements (Fig. 10a and 11a), there are slight variations (5±2.5)

in the SFI score of all groups that seemingly is due to the intrinsic error of the

instrument/technique and also the personal walking habit of mice. Although the improvement

of injured paws starts after around two weeks (14-18 days), the most noticeable changes begin

after a month (between 34-38th days; Fig. 10H and 11B). However, the improvement is not

sustainable and fluctuates between days. The improvement also is also highly variable among

groups. The best scores belong to those groups that have incorporated hESCs (Fig. 11), whereas

the group with no additive (N) compounds or a simple one like fibrin sealant (N+F) shows no

recovery at all (Fig. 10 and 11).

We calculated the P-value for Catwalk experiment separated by groups and days. When

the one-way statistical test was performed for the results separated by days, except the 22th and

34th days in which their P-values were significant (N vs N+F, N vs N+F+D, and N vs

N+F+D+T with P<0.05 and N vs N+F+T with P<0.01 for day 22th and N+F vs N+F+T with

P<0.05 for day 34th), the groups in other days showed no significant differences. Similarly,

when the t-test was performed for the results separated by groups, only the N+F vs N+F+D

(P<0.05), N+F vs N+F+W (P<0.01), and N+F vs N+F+T (P<0.01) showed significant

differences. The general trend observed in Catwalk results, however, is the absence significant

statistical difference among the experimental groups.

45

Fig. 10. The results achieved from Catwalk test separated by days and calculated using SFI

index. a) preoperative results. b) day 7th. c) day 14th. d) day 18th. e) day 22th. f) day 26th. g)

day 30th. h) day 34th. The abbreviations are as Table 1. In N+F+T group, the transgenic cell is

‘off’ whereas in N+F+D+T group it is ‘on’. The box boundaries and whiskers indicate the mean

± SEM and error bar, respectively.

46

Fig. 11. The results achieved from Catwalk test separated by days and calculated using SFI

index. a) preoperative results. b) day 38th. c) day 42th. d) day 46th. e) day 50th. f) day 54th. g)

day 58th. h) day 60th.Other explanations are as Fig. 10.

47

5.4 Sensory Function Evaluation via Von Frey Test

The results obtained from von Frey test are shown in the Fig. 12. The pre-operative

results for healthy mice commonly vary between 5-6 g (force), which is the case for all the

groups shown in Fig. 12. Following sciatic nerve injury (first week) that leads to the loss of

sense of the paw, the stimulus force reaches to a maximum of around 8 g. After this peak, the

trend reverses and the required stimulus force decreases in several successive weeks until

reaching a minimum in week 4. In groups in which transgenic cells were incorporated, the

curves almost flatten after this minimum and show little change until the end of the period (Fig.

12D and 12E).

48

Fig. 12. The results achieved from von Frey test during eight weeks of measurements separated

by groups. The Preoperative represents the measurements before the surgery (treatment:

P<0.05; time: p<0.001).

49

Fig. 13. The results of von Frey test for all groups during eight weeks period.

50

6. Discussion

Peripheral nerve injury as a worldwide clinical problem could impair the quality of life

of a patient. In contrast to CNS, the PNS is capable of repairing and regenerating after being

injured. However, due to its structural complexity, the repair studies have been focused rather

on animal models. Such pre-clinical examinations attempt to increase the tissue regeneration

and functional recovery in the hope that eventually being translated to patients for improving

clinical outcomes. The extensively used experimental model for this aim is based on injuring

the sciatic nerve as the largest nerve trunk of mammals. This injury model provides a feasible

system for studying nerve regeneration, remyelination, and functional recovery. However, the

recovery from a severe nerve transection (i.e. autografting) is usually far more difficult and the

results are less satisfactory (Cui, Jiang et al. 2008). To improve the recovery, transected nerves

shall be surgically repaired to guide regenerating axons into the growth environment of the

distal nerve stump. Even so, surgical repair often fails to achieve remarkable functional

recovery in pre-clinical experiments. This is likely due to the decrease in the ability of neurons

to sustain axon growth, and chronic Schwann cell atrophy with subsequent failure to provide a

supportive growth environment (Bush, Tonge et al. 1996). To improve the degree of nerve

regeneration and functional recovery, cell transplantation therapy has been used with some

degree of success. In contrast to other varieties of stem cells, hESCs, have the ability to self-

renew indefinitely and differentiate into a diverse range of specialized cell types making them

an important source for transplantation therapy and biomedical engineering (Weissman 2000,

Cui, Jiang et al. 2008).

Recent studies on the function and expression of FGF-2 and its receptors have revealed

a physiological role for the molecules in the PNS. FGF-2 as a principal constituent of hESC

growth medium is the most significant regulator of the hESC self-renewal. It is now widely

accepted that the hESCs require exogenous FGF-2 to sustain self-renewal and pluripotency and

develop the capacity to differentiate into a large number of somatic cell types. In practice, since

it is difficult to deliver the active growth factors over the entire duration of regeneration in a

controlled manner, genetically modified cells are used to deliver a continual supply of active

growth factors. The gene expression in these modified cells was turned on by using doxycycline

as the regulator for inducible gene expression systems. The effects of doxycycline were

mediated by direct activation of the PI3K-AKT intracellular pathway.

51

Immunolabeling analysis is commonly used to identify the regenerated axons, Schwann cells,

and proteins associated with nerve regeneration. The technique uses an antibody (e.g. Anti-

CHAT, Anti-neurofilament, Anti-VGLUT1, Anti-S100) for fluorescently labeling a specific

biological target. When properly employed, the analysis can accurately determine the intensity

and areal fraction of the regenerated nerve.

The Immunolabeling results obtained after 60 days of cell therapy was indeed very

promising. The study of nerves by anti-neurofilament antibodies demonstrated that the axons

regenerated to some extent in all experimental groups. The highest nerve intensity and the most

uniform pattern of axons were observed in the ‘cell-on’ group. The fact that all groups showed

axon regeneration to some extent (around 40% of the control group) alongside with the absence

of inter-group statistical differences imply that ‘autografting’ has been successful in stimulating

fiber nerve regeneration. However, this antibody cannot indicate the type and accuracy of nerve

sprouting. To distinguish between them, ChAT and VGLUT-1 antibodies were employed to

label motor and sensory neurons, respectively.

Based on anti-ChAT results, the minimum number of motor axons were observed in the

N+F group meaning that fibrin sealant alone added to the site of injury offers no benefits to

nerve regeneration. Whereas the ‘cell-on’ group does not stand out as a group with the best

motor neuron regeneration, it possesses the highest recorded motor neurons (i.e. ~60%

compared to control group). The fewer number of motor neurons after sciatic nerve transection

is commonly reported in the literature (Wood, Kemp et al. 2011). In our research, statistical

analysis revealed no statistical differences between experimental groups as well as between the

‘cell-on’ and control group. This may suggest that cell therapy (hESC inducibly overexpressed

FGF-2) alone is not quite sufficient to facilitate motoneuron recovery and there is a need for

supplementary materials like neurotrophic factors to promote motoneuron regeneration. The

adding of cells, however, has been very effective in sensory neuron reinnervation. For instance,

the addition of wild and transgenic cells to the site of injury have increased the percentage of

sensory neurons labeling respectively by 73 and 82% relative to control group (For comparison,

this value for the neurorrhaphy-alone group was 66%). When the transgenic cells were activated

by doxycycline, the intensity rate dramatically increased to the same level as a control group

(~104%). This significant increase in nerve regeneration is believed to be linked to the

expression of FGF-2 growth factor, because when doxycycline solely was added to the site of

52

injury (N+F+D group), it was not very effective in stimulating the regeneration. In the

peripheral nervous system, FGF-2 modulates neuron survival, prevents the lesion-induced

death of sensory neurons, and stimulates nerve regeneration (Jungnickel, Claus et al. 2004).

The same group analyzed by S100 immunolabelling showed less activated Schwann cell and

more axon regeneration. In fact, the density of Schwann cells was identical to control group

(~100%) meaning that the combined effect of transgenic cells and fibrin sealant has been

successful in integrating cells with the host tissue and expressing Schwann cell markers. This

also suggests that the remyelination process was estimulated by the transplanted cells.

Myelination, which determines both the regeneration quality and functional recovery, requires

the secondarily developed Schwann cells by cell therapy to be wrapped longitudinal (Ren,

Wang et al. 2012).

As was marked by S100 antibody, except the neurorrhaphy group, all other groups that

incorporated fibrin sealant glue showed normal intensity of Schwann cells and thus a more

stable endoneural microenvironment. Whereas Pabari et. al. (Pabari, Lloyd-Hughes et al. 2014)

have demonstrated the constructive role of fibrin sealants in producing less inflammation, less

fibrosis, better axonal regeneration, and better fiber alignment, based on our

immunohistochemistry results, we can only confirm its crucial role in reducing inflammationan

and establishing a framework capable of retaining the grafted cells. This is corroborated by the

absence of statistical differences between the neurorrhaphy-alone group and others in the

outcomes of neurofilament immunolabeling.

In the context of behavioral patterns, in the first week after the surgery, as expected,

none of the mice were able to use their paws and thus the SFI values obtained from Catwalk

test were equal to -75. This is due to total loss of function of the nerve after neurorrhaphy and

indeed a clue for successful lesion/repair process (Félix, Pereira Lopes et al. 2013). In most of

the groups, it took about a month (26-30 days) for the mice to be able to use their paws. For

some groups that were able to use their paws, however, due to likely inflammation and relevant

pain, they stopped to use their paws altogether, so the recovery expressed by SFI was again

back to −75. The inflammation/hyperalgesia hypothesis is further supported by the results from

von-Frey test (Cunha, Verri Jr. et al. 2004). Coincidently, at fourth week, the force to stimulate

the nerve reflex reduces to their minimum of about 4gf. It is important to highlight that, in fully

denervated muscle, the highest level of post-synaptic sprouts occurs at four weeks after injury

and repair (Wood, Kemp et al. 2011). This could further explain the hypersensitivity of mice to

53

pain at this specific time. In the anti-S100 immunolabeling performed after eight weeks,

however, no such inflammation was recorded, specifically in the ‘cell-on’ group, meaning that

the likely inflammation was reduced during the time. After a sudden drop in week 4, the sensory

function of the paws were recovering, hence the force needed to stimulate the nerve reflex was

gradually coming back to normal (~ 6gf). This timing conform to the finding of anti-S100

immunolabeling.

Despite the slight initial improvement, group #2 (N+F) showed no recovery in the nerve

function. The SFI score from this group remained close to -75 after 26 days in almost all

subjects, which is evident by a flattened trend in the diagram. The fact that the results of this

group show no advantages relative to the neurorrhaphy-alone group (#1; N) implies that the

inclusion of mere fibrin sealant in the site of injury has no recovery effects. This finding indeed

is in agreement with the results of immunohistochemistry discussed above. The results from

group #1, however, are not straightforward. Despite some slight improvement in functional

recovery in the middle of the period, the SFI from this group at the final days turns back to −75.

In general, though, these two groups revealed no functional recovery.

Among the studied groups, those that included doxycycline, wild hESC, and transgenic

cells achieved better results. All these groups showed recovery to some extent from the first

month onward, though the results were not stable and were varied between measurement

sessions. Parts of the fluctuation in the SFI level could be assigned to uncertainties in instrument

function and quantification (classification) technique. The other part is linked to mice status

and habit. For instance, a stressed mouse may walk abnormally on the walkway which may lead

to variations in the results between sessions. Furthermore, those parts of the classification that

rely on experimenter (e.g. footprint detection) can be subject to variations during the time. Such

inherent variations can be easily seen in the graph of healthy mice before undergoing surgery.

Moreover, the fluctuation in the number of regenerating nerves and surviving motoneurons over

time (Ma, Omura et al. 2011) could alter the behavioral patterns of the settings.

The reversal of SFI in the N+F+T and N+F+D+T groups, however, is linked to more

painful paws arising from higher sensory nerve regeneration as was demonstrated by

immunohistochemistry studies and von-Frey test. The neuropathic pain is known to develop

after sciatic nerve injury, hence differences in SFI levels may not only reflect motor-related

impairments, but also be pain-related due to decreased weight load on the affected paw

(Medhurst, Walker et al. 2002, Deumens, Jaken et al. 2007). As was stated by Deumens. et al.

54

(Deumens, Jaken et al. 2007), “functional impairments may be purely related to pain behavior

and the behavioral effects may be a compromise between pain-related and motor-related

alterations”. Following autografting, the mice started to use their heel to contact the ground

rather than their toes. The origin of this behavior is believed to be motor-related, rather than

pain related for it began within days after injury, whereas pain sensitivity, as indicated by von-

Frey test, was only observed after a delay of about 3-4 weeks. Recovery of pain sensitivity was

also corroborated by toe pinch reflex test on awake mice. The animals displayed a blink reflex

or a withdrawal response to light paw-pinching.

Despite significant advances in the understanding of the nerve regeneration and repair

in surgical techniques, complete functional recovery of a damaged nerve seems to be scarce.

The poor functional recovery is likely due to: (i) the failure of regenerating axons to cross the

gap between proximal and distal nerve coaptation and (ii) the cross-innervation

(mismatch/dislocation) of the regenerated axons within the peripheral target sites (e.g. muscle

and tendon) (Scott 1996). In the current study, the results obtained from von-Frey sensory test

and immunohistochemistry (anti-neurofilament) denoted that axon regeneration has occurred

across the autographed gap, particularly in the ‘cell-on’ group; however, it was not as dense as

the control group. This signifies that certain numbers of axons have failed to reach end organs

and thereby the fewer regenerated axons have expressed their terminal branching capacity

(Meek, Den Dunnen et al. 1999). The consequence of this phenomenon would be the alteration

of the motor unit organization by clumping of the muscle fibers (Meek, Den Dunnen et al.

1999). Clumping may lead to changes in the force distribution profile of the muscle during

contraction and influence the mechanical input to the tendon organs (Scott, Davies et al. 1995)

and thus the feedback of walking track patterns. The other reason for muscle imbalance and a

low recovery could be the misdirection of degenerating nerve fibers in the target regions (Meek,

Den Dunnen et al. 1999) as autografting transects closely intertwined sensory and motor

neurons. A major key to functional recovery is the accurate regeneration of axons to their

original target end-organs.

Overall, the low levels of SFI in our experiment could be attributed to the following

factors: (i) the feel of pain in the injured organ; (ii) the sensitivity of the system in recording

the full footprint of paw only; (iii) the natural fluctuation in the number of regenerated motor

neurons over time, and (iv) imperfect nerve regeneration and mismatching between sensory and

motor neurons. The sensory component is very significant for correct motor coordination and

control and thus higher functional recovery (Benitez, Barbizan et al. 2014), albeit the

55

simultaneous pain arising from sensory neuron recovery can hinder the proper use of paw and

thereby leads to lower SFI score. This issue could be resolved by using painkiller during the

experiment. The second obstacle could be avoided by using the analogue walking track

technique instead of the Catwalk XT system. We speculate the hESCs have the capability to

improve and stabilize nerve regeneration. However, to witness this, the effects of the first two

factors should be eliminated primarily.

Catwalk test has been successfully used for sciatic nerve crush and spinal cord injury

models (Bozkurt, Deumens et al. 2008, Benitez, Barbizan et al. 2014). As an example, the

functional recovery of dorsal root avulsion, which is a more severe injury than autografting, has

been successfully shown via Catwalk test. However, in the case of complete sciatic nerve injury

via autografting that is the subject of this research, Catwalk was not successful in showing the

functional recovery despite clear signs of nerve regeneration given by complementary

techniques. We speculate that the animal cannot use the complete plantar surface of its paw for

walking and instead it uses the heel in the early days after surgery and thereafter the front part

of the paw including toes. The Catwalk XT system is programmed to differentiate the way the

animal uses its paw and thus the front part of the paw of the injured animal that barely carries

body weight may be considered below the detection threshold and not recorded. Whereas the

SFI yielded from the walking track analysis is not sensitive to the part of the paw carrying the

body weight (Deumens, Jaken et al. 2007). We believe this fundamental difference makes the

analogue technique more viable for nerve regeneration evaluation following end-to-end

neurorrhaphy.

To our knowledge, this is the first time that sciatic nerve regeneration has been evaluated

by combined behavioral tests namely von-Frey and Catwalk in a mouse model. The former can

provide information on sensory nerve regeneration whereas the latter (or its substitute; the

walking track analysis) gives information about motor neuron regeneration and their

aggregation translated into functional recovery.

Regarding the duration of nerve regeneration, in a rat model, some studies have been

carried on for more than 52 weeks (Meek, Den Dunnen et al. 1999). In rodent models of nerve

regeneration, “chronically denervated distal stumps already have an impaired capacity to

support axonal outgrowth by eight weeks after the injury” (Scheib and Hoke 2013). However,

in mice model, there is a critical window of approximately 35 days, in which regenerating axons

can re-establish connections with the distal muscles. Since the tissues are most permissive to

56

reinnervation during this period, any increase in the rate of axon extension could facilitate axons

to reach their targets (Ma, Omura et al. 2011). At around the same time, a local peak in the SFI

scores is noticeable in our results. On the other hand, by assuming an axon regeneration rate of

up to 3 mm/day (Brushart, Hoffman et al. 2002), and autografting as the optimal suture

technique, regenerating axons could be expected to reach the distal part of the nerve graft after

3-6 weeks of the surgery, albeit, reinnervation of muscles and improved functional recovery

would require longer times of about 12 weeks (Bozkurt, Tholl et al. 2008). Accordingly, higher

levels of SFI could be expected for our experimental groups if the studies were continued for

longer than eight weeks.

Functional recovery after complete nerve injury depends on several factors including

regrowth of axons, specific reinnervation of the target region, and maturation of nerve fibers

and reinnervated muscle fibers (Krarup, Archibald et al. 2002, Krarup, Boeckstyns et al. 2016).

Reinnervation of muscle fibers includes the establishment of neuromuscular connections and

formation of motor units as an essential element of force development and movement control.

In this phase, nerve fibers should grow from the proximal nerve stump and reconnect to muscle

fibers. Naturally, nerve fibers incorporate a small number of muscle fibers, however, during the

nerve regeneration, the muscle fibers group within the same motor unit and correspondingly,

regenerated axons incorporate additional muscle fibers. These fibers that bear the same

biochemical signatures compensate for reduced numbers of axons that succeed in reaching the

denervated muscle (Wood, Kemp et al. 2011). The fact that motor axons reinnervate (enlarge)

more than the normal number of muscle fibers causes all denervated muscle fibers to be

innervated by as few as 20-25% of the normal number of motor axons (Wood, Kemp et al.

2011). Nevertheless, reduced numbers of regenerating nerves give rise to a failure to reinnervate

all the denervated muscle fibers, thus declining the muscle contractile force. As a consequence,

poor functional recovery is recorded by catwalk experiment. In other words, the absence of

functional recovery does not necessarily mean a failure in nerve regeneration. Instead, “the

misdirection of regenerating axons (inaccurate reinnervation) could strongly affect sensory-

motor skills, which require accurate reinnervation of the proper muscles and receptors, whereas

gross functional responses are more dependent on the amount of reinnervation rather than its

accuracy” (Navarro 2016). The poor functional recovery problem is also compounded by

inaccurate (misdirection) regeneration of axons (Wood, Kemp et al. 2011). Under conditions

where the numbers of regenerated axons and reinnervated muscle fibers decline below the noted

57

level, reinnervated muscles fail to recover contractile force and thus functional recovery

remains at low levels (Wood, Kemp et al. 2011).

In the clinical situation, patients normally “reprogram” their brain to cope with an

altered input of information. Meek et. al. (Lundborg 1993, Meek, Den Dunnen et al. 1999)

argued that the reprograming of the brain can explain improvements in the SFI values of rat

model in long-term (i.e. 52 weeks) examinations. The brain reprograming is also linked to the

recovery of coordination in hind paw of rats (Bozkurt, Scheffel et al. 2011). In our mouse

model, the FGF-2 facilitates the growth of sensory neurons and thus the mice feel pain (as

indicated by von-Frey test and anti-VGLUT-1 immunolabeling). We believe if the pain is

relieved by painkiller, then due to combined effect of nerve regeneration and reprograming, one

can expect higher values for SFI after longer recovery periods.

58

7. Conclusion

A new therapeutic approach for sciatic nerve regeneration in mice model was adopted

in the present work. The approach includes the application of human embryonic stem cells

(hESCs) modified to inducibly overexpress FGF-2 to the site of injury resulted from end-to-

end neurorrhaphy. The transgenic hESCs activated by doxycycline were successful in the

regeneration of sensory function and promotion of axonal reinnervation as demonstrated by

von-Frey test and immunohistochemistry analysis. The Catwalk system, however, was not

successful in showing the functional recovery despite clear indications of nerve regeneration

provided by complementary techniques. We believe this failure results from two key factors:

(i) the incapability of Catwalk XT system to record partial usage of the paw and incomplete

footprints; and (ii) pain (hyperalgesia) in the mice’s paw arising from the regeneration of

sensory fibers. To resolve the former issues, future studies shall evaluate the behavioral function

using the analogue walking track analysis. To relieve the pain of the animals, a painkiller such

as Tramadol should be used during the analysis. Our work indicated that the simultaneous use

of sensory test (von-Frey) and walking track analysis provide a more detailed picture of sciatic

nerve recovery, and thus it shall be incorporated in future works.

Based on this study, fibrin sealant is capable of reducing inflammation and likely can

facilitate nerve fiber alignment. However, we found no evidence of its participation in axonal

regeneration in the particular experimental setup. While the mixture of this glue and transgenic

cells yielded to very promising results in regeneration of sensory function, we believe that for

higher functional recovery and better motor neuron reinnervation, these agents should be used

in combination with neurotrophic factors such as NGF, CNTF, GDNF, and BDNF (Chen, Chu

et al. 2010). Axon counting, albeit not performed in this work, should accompany the noted

settings to help evaluate the rate of regenerative success. It is worth mentioning that

reinnervation of muscles and improved functional recovery would require longer times of about

12 weeks (Bozkurt, Tholl et al. 2008). Accordingly, higher functional recovery could be

expected for similar experiments in case the studies continued for a longer time.

59

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