Histological evaluation of extracellular matrix profile ...

Histological evaluation of extracellular matrix

profile during sciatic nerve regeneration

Tobias KRETSCHMER

Verhandeling ingediend tot

het verkrijgen van de graad van

Master in de Biomedische Wetenschappen

Promotor: Prof. Dr. M. Cornelissen

Begeleiders: Dr. Victor Carriel, Charlot Philips

Vakgroep: Medische basiswetenschappen

Academiejaar 2014-2015

“De auteur en de promotor geven de toelating deze masterproef voor consultatie

beschikbaar te stellen en delen ervan te kopiëren voor persoonlijk gebruik. Elk ander

gebruik valt onder de beperkingen van het auteursrecht, in het bijzonder met betrekking

tot de verplichting uitdrukkelijk de bron te vermelden bij het aanhalen van resultaten

uit deze masterproef.”

Datum: 08/05/2015

Tobias Kretschmer Prof. Dr. M. Cornelissen

Voorwoord

Alvorens deze masterproef aan te vangen, zou ik graag een woord van dank betuigen aan enkele

mensen in het bijzonder. Deze mensen hebben het op verschillende manieren mogelijk gemaakt

om deze masterproef te realiseren:

Mijn promotor, Professor Cornelissen voor het mogelijk maken van deze masterproef, haar

kennis die zij aan mij heeft doorgegeven, haar begeleiding tijdens het laatste anderhalf jaar en

het ter beschikking stellen van haar labo en materialen. Ik ben uiterst dankbaar voor de tijd die

zij nam om mijn thesis te lezen, haar feedback en bondige gesprekken waarvan ik veel heb

kunnen leren.

Mijn begeleider, Victor Carriel die mij een groot aantal vaardigheden in het labo heeft

aangeleerd, die mij veel materiaal en tijd ter beschikking stelde en mij steeds met zijn expertise

bijstond om mijn wetenschappelijk onderzoek op een correcte manier uit te voeren. Verder ben

ik hem zeer dankbaar voor zijn feedback en begeleiding tijdens het schrijven van deze

masterproef.

Leen Pieters, de persoon die mij tips kon geven over elk onderwerp betreffende het praktische

werk in het labo. Ondanks het feit dat ze het zelf druk had, kreeg ik op elk moment en elke

vraag steeds een antwoord dat mij hielp om mijn doelen te bereiken.

De doctoraatstudenten, in het bijzonder Charlot Philips, voor haar vele tips en feedback op de

schriftelijke delen die uiteindelijk in deze thesis werden opgenomen. Zij nam de tijd om mij

grondige feedback te geven en was zo voor mij een enorme hulp tijdens het schrijven.

Ten slotte ben ik uiterst tevreden over de vele interessante en vriendelijke mensen met wie ik

in het laatste anderhalf jaar gewerkt heb op het labo. Er was altijd een vriendelijke en

aangename sfeer die zeker ook heeft bijgedragen aan mijn enthousiaste manier om deze

masterproef te realiseren.

Table of contents

Samenvatting .......................................................................................................................... 1

Abstract .................................................................................................................................... 2

1. Introduction ........................................................................................................................ 3

1.1 THE NERVOUS SYSTEM. .................................................................................................... 3

1.2 THE PERIPHERAL NERVOUS SYSTEM. .............................................................................. 3

1.2.1 Morphology of the PNS. ...................................................................................................................... 3

1.2.2 Schwann cells and myelination. .......................................................................................................... 4

1.2.3 The extracellular matrix (ECM) of the PNS....................................................................................... 5

1.2.4 Collagens in the ECM. ........................................................................................................................ 6

1.2.5 Proteoglycans in the ECM. .................................................................................................................. 6

1.2.6 Histological assessment of the PNS. ................................................................................................... 8

1.3 DEGENERATION AND REGENERATION OF THE NERVOUS SYSTEM. ................................. 8

1.4 CLINICAL ASPECTS. ........................................................................................................ 11

1.5 HYPOTHESIS AND OBJECTIVES. ..................................................................................... 11

2. Material and methods .................................................................................................... 12

2.1 EXPERIMENTAL ANIMALS. ............................................................................................. 12

2.2 TISSUE PROCESSING FOR HISTOLOGICAL ANALYSIS. .................................................... 13

2.2.1 Fixation of the tissues. ....................................................................................................................... 13

2.2.2 Paraffin embedding, sectioning and mounting. ............................................................................... 13

2.3 ROUTINE STAINING AND HISTOCHEMISTRY. ................................................................. 14

2.3.1 Hematoxylin and eosin. ..................................................................................................................... 14

2.3.2 Histochemistry with MCOLL. ........................................................................................................... 15

2.4 IMMUNOHISTOCHEMICAL ANALYSES. ........................................................................... 16

2.4.1 Antigen retrieval. ............................................................................................................................... 17

2.4.2 IHC protocols. .................................................................................................................................... 18

3. Results ............................................................................................................................... 19

3.1 H&E STAINING............................................................................................................... 19

3.2 MCOLL HISTOCHEMICAL METHOD. ............................................................................ 21

3.2.1 Myelin and collagen in autograft repaired nerves. ........................................................................... 25

3.2.2 Myelin and collagen in conduit repaired nerves. .............................................................................. 27

3.3 COLLAGEN I IHC. .......................................................................................................... 29

3.4 COLLAGEN III IHC. ....................................................................................................... 33

3.5 DECORIN IHC ................................................................................................................ 37

3.6 VERSICAN, FIBROMODULIN AND BIGLYCAN IHC. ......................................................... 41

4. Discussion and conclusion .......................................................................................... 43

Acknowledgment ................................................................................................................. 48

5. Bibliography ..................................................................................................................... 48

6. Addendum

6.1 IMMUNOHISTOCHEMISTRY – COLLAGEN I

6.2 IMMUNOHISTOCHEMISTRY – COLLAGEN III

6.3 IMMUNOHISTOCHEMISTRY – DECORIN

6.4 IMMUNOHISTOCHEMISTRY – VERSICAN

6.5 IMMUNOHISTOCHEMISTRY – FIBROMODULIN

6.6 IMMUNOHISTOCHEMISTRY – BIGLYCAN

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Samenvatting

Achtergrond: Perifere zenuwen zijn in staat om over korte afstanden terug aan elkaar te

groeien en verschillende componenten uit de extracellulaire matrix spelen hierbij volgens recent

wetenschappelijk onderzoek een belangrijke rol. Tot nu toe zijn de samenstelling, verdeling en

herschikking van deze ECM componenten tijdens zenuwregeneratie nog niet goed gekend. Het

doel van deze studie is de evaluatie van het ECM profiel tijdens de regeneratie van de nervus

ischiadicus van de rat.

Methoden: De zenuwen werden doorgesneden om een 5 mm defect te introduceren dat

vervolgens werd verbonden via een autograft of een bio-artificiële conduit. Na 20, 30 en 50

dagen werden de zenuwen verzameld. De histologische analyses werden uitgevoerd door

gebruik te maken van hematoxylin en eosine (H&E), de MCOLL histochemische methode en

immuunhistochemie (IHC) kleuringen. Door deze laatste werden de expressie en herschikking

van collageen type I en III, decorin, versican, fibromodulin en biglycan geanalyseerd.

Resultaten: Aan de hand van de H&E kleuring werd de toestand van regenererende axonen in

autograft behandelde zenuwen en van nieuw gevormde axonen in conduit behandelde zenuwen

bepaald en de progressie van axon regeneratie geëvalueerd. Door middel van de MCOLL

kleuring kon de graad van myelinisatie en de collageen herschikking in proximale, centrale en

distale zenuwuiteinden bepaald worden. De IHC kleuringen toonden aan dat collageen I en III

in het perineurium en endoneurium tot expressie kwamen en een soort omhulsel voor de

regenererende axonen vormden. Decorin kwam in nauwe associatie met het collageen tot

expressie. Versican, fibromodulin en biglycan kwamen niet tot expressie.

Conclusies: Dit onderzoek heeft nieuw inzicht geleverd in de verdeling en herschikking van

verschillende componenten van de ECM in zenuwen die op twee verschillende manieren

gerepareerd werden. Tijdens de regeneratie is er een duidelijk verschil in de herschikking van

de ECM proteïnen en het gebruik van een conduit kon deze structurele herschikking

bevorderen.

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Abstract

Background: Peripheral nerves possess the ability to regenerate over short distances after

transection and recent studies suggest that various extracellular matrix (ECM) components

contribute to the regeneration of these nerves. However, the precise composition, distribution

and reorganization of different ECM components are still largely unknown. This study aims at

evaluating the ECM profile during sciatic nerve regeneration in rat sciatic nerve (SN).

Methods: The rats were implanted with an autograft or a bioartificial conduits after a 5 mm

gap was induced in one SN. Nerve samples were harvested at 20, 30 and 50 days after

implantation. The histological analyses were carried out using H&E staining, the MCOLL

histochemical method and immunohistochemistry (IHC). By immunohistochemical means, the

expression and organization of collagen type I and III, decorin, versican, fibromodulin and

biglycan were analyzed.

Results: Based on the H&E staining the presence of regenerating axons in autograft repaired

nerves and newly-formed axons in conduit repaired nerves were observed and the progression

of axonal regeneration was evaluated. MCOLL results provided information on the degree of

myelination and collagen organization in proximal, central and distal nerve ends. The IHC

revealed collagen I and III expression in the perineurial and endoneurial space forming sheaths

around regenerating axons. Decorin was found in close association with these collagens. There

was no expression found of versican, fibromodulin and biglycan.

Conclusions: New information was obtained on the distribution and organization of several

proteins in the ECM in both types of nerve repair at different time intervals. The regeneration

process showed different characteristics regarding the ECM components’ organization and

indicated advantages for structural SN regeneration when using the conduit repair.

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1. Introduction

1.1 The nervous system. The nervous system of mammals is extremely complex from

a structural and functional point of view. It can anatomically and functionally be divided into

two different parts, i.e. the central and the peripheral nervous system. The central nervous

system (CNS) is subdivided into the brain and the spinal cord where, among other processes,

motor stimuli arise and sensory information and stimuli are processed and integrated (1). On a

macroscopic view the CNS consists of white and grey matter. The grey matter comprises mainly

unmyelinated axons which transmit the nerve stimuli, nerve cell bodies and dendrites. The

white matter comprises mainly myelinated axons and only very few cell bodies.

The supportive cells of the CNS are the glial cells, further subdivided into microglia and

macroglia. The microglia represent the immune cells of the CNS, specialized monocyte-derived

and antigen-presenting cells, which constitute a minority of all CNS cells. The macroglia cells

are the astrocytes, oligodendrocytes, ependymal cells and radial glial cells. Astrocytes are

regulatory cells and important for the neuronal environment and probably also serve as

predominant compartment of the blood-brain barrier. Oligodendrocytes perform the task of

myelin sheath production around the axons in the CNS. Ependymal cells are associated with

the cerebrospinal fluid production and secretion while forming a connection between blood and

cerebrospinal fluid in the form of a blood-cerebrospinal fluid barrier. Lastly, radial glial cells

play a role in nerve development and in the cerebellum and retina of the mature brain (2).

1.2 The peripheral nervous system. In the peripheral nervous system (PNS),

structures and cells are different and the major task of the PNS is to link the communication

between organs, tissues and the CNS (1). Peripheral nerves (PN) are found throughout the entire

body, emerging from the CNS and reaching almost every organ and all tissues. They provide

sensory, motor and autonomic innervation which is necessary for the complex functions of each

organ. In general, sensory nerve fibers originate from pseudo-unipolar neurons which are

located in the sensory ganglia, except for cranial nerve VIII and the mesencephalic root of

cranial nerve V. Motor nerve fibers have their origin in somatic and autonomic motor neurons

which are found in the CNS. Additionally, mixed nerves are the third group of nerves in the

general classification (2) (3).

1.2.1 Morphology of the PNS. PN are subdivided into two components that can be

recognized, a parenchyma and stroma. The parenchyma comprises the nerve fibers with their

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axons and Schwann cells while the stroma is made up of various connective tissue elements.

Compound nerve trunks and nerve fibers contain many nerve fascicles which are bound

together by a connective tissue layer called epineurium (3). Figure 1 gives a general schematic

overview of the structures and morphology of a PN. The epineurium is the outermost layer of

PN and delineates the nerve from the surrounding tissue. Epineurium comprises mainly

collagen and some adipose tissue and within the epineurium blood vessels are present and

fibroblasts and mast cells can be found. Surrounding the individual nerve fascicles is the

perineurium which consists of thin but dense sheath of collagen layers organized in bundles

that separate concentric layers of flattened cells. Underneath the perineurium, nerve fiber axons,

their surrounding Schwann cells, collagen fibers, fibroblasts, capillaries and few mast cells are

present in a connective tissue compartment called the endoneurium. The space which is

surrounded by the perineurium is called the endoneurial space. Perineurial cells separate the

endoneurial space from the interstitium via tight junctions. In the endoneurial space, capillaries

are present whose endothelium functions as blood-nerve barrier (3) (4).

1.2.2 Schwann cells and myelination. Axons are associated with Schwann cells which

wrap around their respective axon and produce the myelin sheath in a spiral way around an

individual axon. The myelinated portions are termed internodes and they are separated by

unmyelinated regions which display ion-channels and are termed nodes of Ranvier (3) (5).

Schwann cells thus offer support for axons by insulating them with myelin sheaths which

Figure 1: Schematic structure and components

of a peripheral nerve (58).

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basically consist of layers of phospholipid membranes. An advantage of the myelin insulation

is that signal transduction is enhanced through saltatory conduction (6).

In contrast to oligodendrocytes in the CNS, which produce myelin sheaths for more than

one axon, Schwann cells only ensheath part of one axon. However, non-myelinated axons are

also associated with Schwann cells, usually several axons enveloped as a group by a single

Schwann cell. Non-myelinated axons are usually smaller than myelinated axons and action

potential velocity is lower in the non-myelinated axons due to the fact that saltatory conduction

over nodes of Ranvier cannot occur (4).

Schwann cells derive from the neural crest which yields pluripotent cells that

differentiate under the influence of specific transcription factors. The association of Schwann

cells to developing peripheral nerves leads to their maturation in the PNS. As they maturate a

morphogenetic process called radial sorting is initiated which aims at combining Schwann cells

with large diameter and small diameter axons. The association in a 1:1 relationship of large

axon with Schwann cells will lead to myelination of the axon while small axons are not

myelinated and combine in groups around a non-myelinating Schwann cell. One of the factors

that plays a role in myelinating and non-myelinating Schwann cell development is NRG1-III

which is involved in neuregulin signaling. Furthermore, laminin which is present in the basal

lamina and interacts with certain receptors also influences myelinating Schwann cell

development (7) (8).

The contact between axons and Schwann cells is particularly important for the Schwann

cell growth and survival. Although these cells hardly divide in adult nerve tissue, cell division

is induced following nerve injury. After nerve transection, Schwann cells undergo

dedifferentiation from their myelinating state when contact to the axon is lost and turn into a

proliferative state. Due to the proliferation, the Schwann cells can remove debris together with

macrophages during Wallerian degeneration. Also, they will start to line up to form the so-

called bands of Büngner and secrete factors, such as laminin and fibronectin, to guide the

regenerating axons or axonal sprouts towards the distal nerve stump. In the absence of axon

contact and regeneration, the Schwann cells do not proliferate and the cell population decreases

by apoptosis (4) (9).

1.2.3 The extracellular matrix (ECM) of the PNS. The ECM is produced by different

cell types and provides the physical and chemical properties of different tissues. ECM

molecules are important from a structural point of view and they are even more important

6

guiding the process of regeneration of PN. The ECM molecules provide strength and structure

to tissues, have a supportive role and also have influence on cell behavior. The latter is probably

achieved through precise spatial and temporal expression of ECM molecules and

transmembrane receptor interactions (5).

1.2.4 Collagens in the ECM. In PN, the ECM is highly organized and surrounds each

nerve fiber. Furthermore, a fibrillar matrix comprising collagen-based fibrils forms part of the

ECM. Collagens are extracellular, structural proteins synthesized mostly by fibroblasts.

Schwann cells in PN also produce collagen of the types I, III, IV and V. Collagens are composed

of high amounts of hydroxyproline, proline and glycine. They typically display a triple α-helical

configuration. Collagens are found in the endoneurium as fibrils of interstitial collagen and in

the basal lamina surrounding the processes of Schwann cells and perineurial cells as non-

fibrillar elements (10) (11) (12).

Following traumatic nerve injury, fibroblasts increase their production of collagen

around the injury site and distal to it. As a result, the tensile strength of the injured nerve is

enhanced and a collagenous framework for axon orientation and sheath formation by Schwann

cells is provided. Collagen types I and III are most important in regenerating nerves since they

play a vital role in the self assembly process of fibril formation (12). In the ECM, these fibrils

form scaffolds which increase the stability of the ECM, influence cell differentiation and

migration and interact with cellular receptors (8) (13). In some cases, the production of collagen

in the regenerating nerve is excessive and can lead to scar formation. Scarring can then prohibit

axonal sprouting and a successful regeneration of the nerve (14).

1.2.5 Proteoglycans in the ECM. Several ECM macromolecules are produced by

Schwann cells and neurons including collagens, glycoproteins and proteoglycans. These ECM

molecules are involved in important cellular processes including cell proliferation, migration

and differentiation (11). Proteoglycans are heterogeneous poly-peptides existing of a core

protein coupled to one or more glycosaminoglycan (GAG) side chains. These GAG chains are

basically long, unbranched polymers of repeating disaccharide units. GAGs can be divided into

5 broad classes based on their disaccharide content, i.e. chondroitin sulphate (CS), dermatan

sulphate (DS), keratan sulphate, heparan sulphate and hyaluronan. Many proteoglycans are

secreted to become part of the extracellular matrix, or they are attached to the extracellular side

of the cell membrane (15). Chondroitin sulphate proteoglycans (CSPG) are particularly

interesting because they are expressed in both developing and mature nerves and have been

reported to inhibit neurite outgrowth (5). When enzymes that degrade CSPG were added to a

graft or the distal nerve stump, the inhibitory effects of the proteoglycans were reduced (16).

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Biglycan belongs to the group of small proteoglycans and has 2 GAG side chains either

containing CS or DS or a combination of CS and DS side chains. It is found, for instance in

fetal or young bone as CSPG or in articular cartilage as DS containing form. The precise

functions of biglycan in different tissues remain to be elucidated, but it has been suggested that

it binds transforming growth factor-β (TGF-β) in vitro, influences bone mineralization and

binds to the growth factor BMP-4 which has effects on osteoblasts (13) (17). Biglycan is also

thought to interact with collagen type I, maybe in balance with decorin, and to help assembly

the ECM (18). However, in its purified form biglycan does not bind to collagen fibrils and it is

also not found in association with classic collagen bundles in tissues. The mRNA and protein

of biglycan are expressed in several types of cells in developing human tissues (13).

Decorin is a small proteoglycan containing a single CS or DS chain attached to the core

protein. The size of decorin can differ depending on the tissue where it is found and biglycan

in the same tissue is almost always bigger than decorin. It has been shown that decorin changes

the kinetics of collagen fibril formation and that it can bind to TGF-β and thereby neutralizes

its effect on certain cells (13). Recently, it has been reported that decorin plays an important

suppressive role in acute scar formation in the CNS through inhibition of inflammatory fibrosis

by contributing to the neutralization of TGF- β1/2. Further, decorin contributes to the induction

of tissue plasminogen activator and matrix metalloproteinase in chronic lesions which leads to

fibrolysis and reduction of grown scar tissue (19).

Fibromodulin belongs to the group of keratan sulphate proteoglycan which are

expressed in different connective tissues, e.g. cartilage, skin and tendon. The structure and

amino acid sequence of fibromodulin is related to those of the proteoglycans biglycan and

decorin (13). Additionally, decorin and fibromodulin seem to be involved in the assembly and

binding of collagen in the process of fibrillogenesis (20) (21). Fibromodulin is able to bind to

collagen type I and II, and also can impede collagen fibrillation in vitro which results in the

formation of thinner fibrils (13).

Versican is a relatively large CSPG of the ECM and is released by fibroblasts. There are

different isoforms of versican expressed in various tissues, including the CNS. All of these

isoforms contain regions to interact and bind with hyaluronic acid binding domain.

Furthermore, the V3 isoform possesses the ability to bind the EGF-receptor and isoform V2 can

cause the inhibition of neurite outgrowth in CNS and PNS neurons. Through the interaction

with the ECM via specific domains, versican has a regulatory effect on cell invasion and

metastasis. Versican, together with other proteoglycans, has been reported to have inhibitory

effects on axonal regeneration and neurogenesis in the CNS after injury (13) (22). Additionally,

8

decorin and versican were found to be upregulated during mouse sciatic nerve regeneration and

it has been stated that these molecules decrease cell adhesion in different nerve cell lines (23)

(24).

1.2.6 Histological assessment of the PNS. Light microscopy is a frequently used

approach to observe and evaluate the morphological features of PN during pathophysiological

and regenerative processes. Observation of the multifarious histological appearance of cellular

and extracellular structures in PN is possible using light microscopy. However, depending on

the structure of interest and its presence and organization in the tissue, different methods of

staining can and should be applied.

Various histochemical methods allow for the identification of structures like myelin and

collagen. Toluidine blue and luxol fast blue (LFB) stain myelin with certain degree of accuracy

and specificity, but both methods also feature disadvantages. Applying toluidine blue on resin-

embedded, semi thin sections for example is more time-consuming and expensive than LFB

staining of 5 µm, paraffin-embedded sections. Furthermore, it was recently described that LFB

can be combined with the picrosirius method for simultaneous collagen staining to enhance

contrast and use less tissue sections compared to other approaches (25). Therefore, this new

method, called MCOLL method, provides a more comprehensive assessment for myelin and

collagen while being less time-consuming and less expensive.

On the other hand, several structures and molecules of the ECM are not recognized with

high accuracy or specificity through histochemical approaches. Regarding structures like ECM

proteoglycans, it seems to be more advantageous to use an immuno-histochemical approach.

Such approaches can be conducted on paraffin-embedded tissue sections of 5 µm which require

no specialized or expensive equipment (26). Immuno-histochemical protocols are usually

performed within one day, between 5 to 18 hours, depending on incubation times of the

antibodies. Furthermore, the antibodies display high potentials of specificity and accuracy

towards their respective target structure or molecule. Therefore, immunohistochemical

approaches are very useful in the assessment of histological structures which are relatively

difficult to stain using histochemical methods.

1.3 Degeneration and regeneration of the nervous system. In higher vertebrates, the

neuronal cell body can stay structurally intact for certain time and induce a regenerative

program if the neuron does not undergo cell death after transection of an axon in the CNS.

However, these regenerative efforts of the neuron are short-lived and rather unstable, ultimately

9

resulting in atrophy or degeneration of the neuron (27). Furthermore, regeneration-inhibiting

properties of glial cells in the CNS are an essential reason for sprouting of axons over only

several millimeters in immediate surroundings of the neural lesion. Oligodendrocytes, for

instance, are cells which contribute to the axonal sprout inhibition by the production and

secretion of growth-inhibiting “Nogo” molecules which are part of the mature CNS (5) (27).

After transection of a PN, the processes occurring and the involved cellular structures

are different from those in the CNS. Figure 2 schematically summarizes several of the processes

and events taking place in PN during axonal degeneration and regeneration. Morphological

changes like chromatolysis, which entails nuclear and cell body swelling, occur several hours

after nerve injury. The neurons switch from producing neurotransmission-related substances to

a growing modus where syntheses of cytoskeletal and growth-associated proteins have priority.

In the proximal part of the transected nerve, axons degenerate over a short distance back from

the site of injury in a so-called retrograde degeneration. An axon of this proximal part then

starts producing a high number of collateral and terminal sprouts starting at the tip of the

remaining axon and advancing to a distal direction. The sprouts are orientated at the basal

lamina and occur within several hours after injury. A second wave of sprouts is observed within

the following two days and earlier sprouts can degenerate before the definitive sprouts are

formed. Schwann cells play an important role in guiding these sprouts physically over the nerve

injury gap. Schwann cells form processes that provide a rate-limiting step for axonal

regeneration, rather than axonal growth itself does. Nonetheless, the space between proximal

and distal nerve stump, the interstump zone, opposes the major barrier for nerve regeneration

and success of regeneration depends specifically on chemical and cellular events taking place

at this zone (9).

Distal from the nerve transection a highly regulated process called Wallerian

degeneration occurs at the injured site (28). This process is taking place 24 to 36 hours after

nerve transection. Macrophages are responsible for removal and breakdown of myelin debris

and therefore play a vital role in Wallerian degeneration. Furthermore, macrophages secrete

neurotrophins and interact with Schwann cells to promote axonal growth and nerve repair (29)

(30). After 5 to 20 days, regenerative changes in axons can easily be detected, for example by

10

identification of the cytoskeletal proteins neurofilament, β-III tubulin and GAP-43 by

immunohistochemistry (IHC) (31).

Guiding axons during regeneration after PN damage is partly achieved by Schwann cell

basement membrane together with collagen fibers in the outer endoneurial sheath (32) (33).

Schwann cells can easily be identified in damaged nerves by the presence of an investing

basement membrane that is composed of laminin, fibronectin and other components (34).

Proliferation of Schwann cells in the endoneurial sheaths is a characteristic event which takes

place in the distal nerve stump after transection of a PN. It leads to the formation of so-called

bands of Büngner which resemble tightly packed columns of Schwann cells that aid in guiding

the regenerating axons to the periphery (35).

Regenerative changes and cellular events also take place in and are influenced by the

extracellular microenvironment (9) (10). Today, the general histological organizations of the

PN and several molecular aspects have been studied. However, there is still little known about

the expression, distribution and the role of the extracellular microenvironment in these complex

organs during regeneration.

Figure 2: Schematic representation of processes taking place during axonal degeneration and regeneration.

A. Normal peripheral nerve. B. Schwann cells comprise axon and myelin debris and have divided to form

bands of Büngner seven days after axonal injury. C. Axonal sprouts grow along the bands and proximal to

distal from the axon. D. Schwann cells are producing myelin around an axon. E. Reconnection of the axon

to its distal part with short regenerated internodes (57).

11

1.4 Clinical aspects. The frequency of PN injuries is high and can cause different

degrees of structural damage and dysfunction of the PN (36). PN are long and delicate organs

and traumatic injuries occur frequently due to their vulnerability. When a PN is transected with

a short nerve gap, the direct repair, also called neurorrhaphy, is the surgeon’s method of choice.

In certain situations however, the direct repair is not possible, for instance when a direct repair

would set the repaired nerve under too much tension. In such cases, the surgeon can make use

of autologous grafts for reconstruction without tension on the injured nerve. Such autografts

can be obtained from a donor sensory nerve, e.g. sural nerve, or a combination of veins and

muscle tissue of the patient and therefore do not cause immunogenic reactions and provide a

favorable ECM with important factors for axon repair (36). Nonetheless, autografts also feature

some major disadvantages, for example donor site morbidity. This is the result of the fact that

the graft is taken from healthy tissue leaving behind a gap which is mostly not restored to its

original state and results in scar tissue formation. Further, autograft operations require more

time and are costly (36).

Under certain circumstances the use of a tubulization technique has more advantages

than direct or autograft repair of PN. Tubulization techniques with nerve conduits (NC) have

been established to guide the sectioned nerve fiber and to speed up and increase the number and

length of regenerating axons (37). After transection of a PN, the so-called nerve guidance

conduits are sutured to the proximal and distal ends of the nerve stumps that partially lie in the

conduit. Several phases comprising different cellular and extracellular events then take place in

the course of regeneration within the NC (38) (39). NCs are advantageous, because they spare

patients additional sutures and therefore reduce episodes of neuropathies which makes them

promising alternatives to classic direct repair. However, NCs are limited to nerve gaps of 30

mm and the incorporation of biomaterials, growth factors and ECM molecules to promote

regeneration still needs experimental analysis in terms of safety and efficiency (36). Knowledge

on ECM assembly may provide a better understanding of the complex process of nerve

regeneration. In addition, the knowledge of the role of these essential ECM molecules could

help us to develop better conduits used to support repair after PN injury (40) (41).

1.5 Hypothesis and objectives. We now know that the ECM influences and guides the

formation and elongation of nerve fibers. However, complex characteristics, properties and

promising features of the ECM are not fully described and demand further research. Additional

studies on regenerative changes at the axonal level could enhance a better understanding of the

events taking place during PN regeneration (9). From a clinical point of view, more knowledge

12

on regeneration-promoting NC can improve the treatment of many patients (36) (39). However,

manipulating the microcellular environment to enhance regeneration in PN is depending on a

better understanding of the factors and their interplay in the ECM (10) (42). The topographical

information obtained with histological techniques make it one of the most efficient methods to

elucidate the ECM profile during PN regeneration (3). Figure 3 shows a typical overview

staining of a PN to identify structures and the general morphology.

Due to the lack of information regarding the ECM profile in normal and regenerated

nerves, the aim of this study is to evaluate the ECM profile during sciatic nerve regeneration

by histochemical and immunohistochemical means. The goal is also to get a better insight into

the changes in composition, distribution and reorganization of the different components of the

ECM. It is hypothesized that the expression pattern of the different ECM molecules will display

similarities to what has already been described elsewhere for the process of PN regeneration

(3) (4) (43). However, the exact association of different cellular structures and ECM molecules

in the course of regeneration within several days to weeks remains to be determined as part of

this study.

2. Material and methods

2.1 Experimental animals. All animals used in this study were obtained from the

Service of Production and Animal Experimentation, University of Granada, with the approval

of the Ethics Committee of the University of Granada, Spain. The animals were kept in a

temperature controlled environment (21 +/- 1°C) which was maintained on a 12 hours light and

dark cycle and the animals were given free access to tap water and standard rat chow. Male

Wistar rats of 13 weeks old were used and subdivided into two groups. Under general

Figure 3: Hematoxylin and eosin staining of the rat sciatic nerve.

Important structures are highlighted and mentioned in the figure by

circles and arrows.

13

anesthesia, the first group of 8 animals was implanted with a collagen type I conduit

(NeuraGen®), 8 mm in length and 2 mm in diameter, after an incision was made to produce a

gap of 5 mm in the left sciatic nerve. The second group comprised another 8 animals which

underwent autograft repair of the left sciatic nerve after a similar incision as in the first group.

The right sciatic nerve in all animals was used as control tissue. 7/0 and 4/0 polypropylene

monofilament (Premilene®) sutures were used during surgery. Each animal was housed

individually after surgery and received metamizol in the drinking water. Euthanasia was

performed using an anesthetic overdose. Nerve tissues were harvested 20, 30 or 50 days after

conduit implantation or autograft repair.

2.2 Tissue processing for histological analysis. After the experimental animals were

euthanized, part of the regenerated sciatic nerve and control nerves were removed and had to

be treated in such a way that histological analysis could be conducted. To this end, tissues were

fixed, embedded and sectioned to obtain specimens for the analysis by different histochemical

and immunohistochemical techniques.

2.2.1 Fixation of the tissues. Formalin fixation is very common under pathologists and

the processes, advantages and disadvantages of formalin fixation are known. The general

structure of cellular organelles and peptide-protein bonds are primarily preserved with

formaldehyde fixation. Only little denaturation occurs and lipids and non-protein associated

carbohydrates are not fixed. Cross-links which mask epitopes and block antibody access can

occur (44) (45). In this work 10% formalin in 0,1M phosphate buffered saline (PBS) fixation

for 8 to 12 hours was selected for half of the control and experimental tissue samples.

The fixative methacarn was used for the other half of control and experimental tissue

samples. Methacarn is a modification of Carnoy’s fixative (10 ml acetic acid, 60 ml absolute

ethanol, 30 ml chloroform) where absolute ethanol is replaced with 100% methanol (44). In this

study, tissue sections were fixed in methacarn for 3,5 hours.

2.2.2 Paraffin embedding, sectioning and mounting. The goal of embedding the

tissues after fixation is to create a tissue block that can readily be cut in sections of varying

thicknesses. Embedding of the nerve samples was achieved by using paraffin wax as embedding

medium. Since paraffin is immiscible with water, the tissues have to be dehydrated using

increasing concentrations of ethanol and finally xylene or toluene which is ultimately replaced

by paraffin.

14

For sectioning, the paraffin blocks were put into a microtome (Cut 4060, SLEE Mainz,

Germany) to produce sections of a 5 µm thickness. The sections were transferred to a water

bath of 37 – 39°C and afterwards were transferred to microscope glass slides (Klinipath, Olen,

Belgium). Slides containing sections were then put into an oven at approximately 55°C for one

hour or alternatively at approximately 30°C overnight to dry. After drying, the slides can

theoretically be stored for months or years without the loss of tissues.

Before any staining could be carried out, the slides needed to be deparaffinated using

toluene (Chem-lab, Zedelgem, Belgium) first, decreasing concentrations of alcohol

(isopropanol, 99 vol.% and ethanol, 96 vol.%, Chem-lab) afterwards and finally, distilled water

(H2Od). In our study, an automated system (Microm HMS740, Walldorf, Germany) was used

to carry out deparaffination and dehydration at the beginning or end, respectively, of each

protocol. Table 1 shows the different steps of deparaffination and dehydration with the

respective solutions and rinse times. The last step after staining sections by means of

histochemical or immunohistochemical methods was mounting which was performed using

mounting medium (Richard-Allan Scientific).

Table 1: Deparaffination and dehydration as carried out by the automated system. The bath number in

chronological order, the solution and the time of rinse in the solutions are mentioned.

Deparaffination Dehydration

Bath Solution Time (minutes) Solution Time (minutes)

#1 Toluene 5’ Tap water 4’

#2 Toluene 5’ Ethanol 96% 2’

#3 Toluene 5’ Ethanol 96% 2’

#4 Isopropanol 2’ Isopropanol 2’

#5 Isopropanol 2’ Isopropanol 2’

#6 Ethanol 96% 2’ Toluene 2’

#7 Ethanol 96% 2’ Toluene 2’

#8 Tap water 2’ Toluene 1’

#9 Distilled water 1’ -

#10 Distilled water 1’ -

2.3 Routine staining and histochemistry. The goal of a routine stain is to highlight the

most important structures of a tissue and make it possible to differentiate these structures from

one another. The general reaction taking place is based on affinities of the dye for certain types

of molecules. Generally, two types of dyes are used for a routine staining, namely basic

(cationic) dyes and acidic (anionic) dyes.

2.3.1 Hematoxylin and eosin. One of the most famous routine stainings is hematoxylin

and eosin (H&E) staining which uses hematoxylin as basophilic and eosin as eosinophilic dye

15

(46). Eosin can be combined with phloxine to produce a cytoplasmic staining that is more

clearly demonstrating different tissue components than eosin solely. H&E stainings cause

nuclei to appear blue and cytoplasm and collagen fibers to appear red, thus providing a good

contrast between nucleus and cytoplasm of the cell (47). In this study, Hematoxylin (Mayer’s

hemalum solution, VWR®) and eosin + phloxine (Thermo Scientific) staining was used on all

tissue samples as routine staining. Hematoxylin alone was also used as counterstain following

histochemical and immunohistochemical stainings to provide a better overview and contrast in

the tissue samples.

2.3.2 Histochemistry with MCOLL. The recently described MCOLL histochemical

method is based on a combination of luxol fast blue (LFB) and picrosirius histochemical

methods and achieves simultaneous staining of myelin sheaths, collagen fibers in the stroma

and morphology pattern in CNS and PNS nerve tissues. LFB utilizes a copper phthalocyanine

dye which is attracted to basic groups in lipoproteins of the myelin sheath and is well-known

for myelin staining of formalin-fixed paraffin-embedded tissue sections (25) (48). A certain

peculiarity of the LFB protocol is the differentiation step where special attention is needed.

Differentiation aims at selectively withdrawing the dye in a solvent from the tissue and the dye

is usually first lost in structure with certain permeability, for example collagen. Therefore,

during differentiation slides can be placed under a light microscope to observe the loss of dye

in collagen structures and differentiation is stopped when this process is completed. Myelin

retains the dye significantly longer during differentiation and remains clearly visible and blue

colored (49). The subsequent use of picrosirius, i.e. Sirius red F3B in a saturated solution of

picric acid, aims at staining the collagen fibers. Sirius red, a strong anionic tetrakisazo dye,

interacts with cationic molecular groups, due to hydrogen bonding, present on collagen

molecules oriented in parallel and causes an intense red color of collagen fibers (50) (51).

In this study, 0,1% LFB (Color Index C.I. 74180, BDH Chemicals) in 95% ethanol

solution with 2,5 ml of 10% acetic acid (Chem-lab) was used and incubated overnight at 56°C.

Differentiation was performed in 0,05% lithium carbonate (E. Merck, Darmstadt, Germany).

Sections were also stained in 0,2% Sirius red F3B (C.I. 35780, Sigma-Aldrich) in a saturated

solution of picric acid, i.e. picrosirius solution, for 30 minutes at room temperature. Afterwards,

a counterstain was performed in hematoxylin (Mayer’s, VWR®). A protocol with the exact

steps, including rinse and washing after sections were stained, can be found in table 2.

16

Table 2: LFB staining and picrosirius staining together with other steps in the MCOLL histochemical method

protocol.

Step Procedure, solution and timing.

1 Deparaffinate sections in toluene twice, 99% ethanol 3 times, 95% ethanol 3 times, 70% ethanol twice,

á 2 minutes, and keep in distilled water afterwards.

2 Stain sections in 0,1% LFB at 56°C for 16–24 hours (overnight).

3 Rinse in 95% ethanol, then rinse and keep in distilled water.

4 Differentiate in 0,05% lithium carbonate until gray and white matter can be distinguished.

5 Rinse in 70% ethanol for 10 seconds, two changes. Then keep slides in distilled water to stop

differentiation.

6 Stain sections in picrosirius solution at room temperature for 30 minutes.

7 Rinse in distilled water for 3 minutes, two changes.

8 Counterstain with Mayer’s hematoxylin for 1 minute and 30 seconds. Wash under tap water afterwards

for 5 minutes.

9 Dehydrate sections in 96% ethanol twice, isopropanol twice and toluene 3 times, á 2 minutes.

10 Mount sections with mounting medium.

2.4 Immunohistochemical analyses. The basis of immunohistochemical methods is the

use of an antibody which binds to antigens in the target tissue. Figure 4 shows the concept and

structures involved in an IHC staining. The antigen is the structure of interest in the staining,

e.g. collagen type-I in a PN tissue section. The visualization of the primary antibody bound to

the antigen of interest is achieved by either direct or indirect means. For example, a fluorescent

dye molecule can be coupled to the primary antibody which therefore can directly be visualized.

However, this approach has less sensitivity compared to other methods and provides only little

signal amplification. Furthermore, a fluorescence microscope is required for the evaluation of

the stained sections. Using an indirect, histochemical approach where a secondary antibody

directed against the Fc-chain of the primary antibody is used, provides more sensitivity and

amplification of the signal. Even if this approach is more prone to errors, e.g. due to non-specific

binding of the secondary antibody, the amplification is very valuable for a clear and correct

analysis (46) (52). In this study, non-labelled primary antibodies and peroxidase-conjugated

secondary antibodies were used. Alternatively, biotinylated secondary antibodies and

streptavidin horse radish peroxidase (HRP) directed against the biotin-group were used. The

17

substrate for the peroxidase was 3,3’-diaminobenzidine (DAB) which is oxidized by peroxidase

resulting in a brown color at the site of reaction.

2.4.1 Antigen retrieval. One of the major disadvantages of formalin fixation and

paraffin embedding is the fact that these methods cause inter- and intramolecular cross-linking

of antigens of interest. These changes in three-dimensional structure can yield inappropriate

antigen-antibody interactions at the cost of unsatisfactory staining results. Therefore,

procedures and specific pretreatments have been tested and developed to increase

immunoreactivity of antigens in formalin-fixed tissues.

The first attempts to increase immunoreactivity utilized proteolytic enzymes which

digest molecules to re-expose hidden antibody binding-sites. Another approach used heat near

the boiling point of water with samples in citric acid buffer of pH 6 to further forward the

development of antigen “unmasking” or retrieval techniques. Important variables in these

techniques seem to be temperature, usually 95°C, and exposure time to this temperature varying

from 10 to 60 minutes. Cooling to room temperature after pretreatment is rather slow and might

take another 20 to 30 minutes. It is now clear that several cross-linkages can be reversed while

others stay intact and proteins thusly are not denatured by the heat which shows that antigen

retrieval indeed works (52).

In this study, several antigen retrieval treatments were used depending on the antigen of

interest for the IHC. For collagen I IHC staining of formalin-fixed sections pepsin (DAKO)

pretreatment was conducted. For collagen III IHC stainings of formalin-fixed sections

Figure 4: Simplified concept of an immunohistochemical staining. The primary antibody "1"

binds to a specific antigen "Ag" and a secondary antibody "2" binds the primary antibody. The

conjugated peroxidase "P" oxidizes DAB "D" to produce a brown reaction product.

18

pretreatment was conducted with citric acid buffer (E. Merck, Darmstadt, Germany) of pH 6,1

at 95°C in a steamer (Braun®) for 25 minutes. The proteoglycans were pretreated in

chondroitinase ABC 0,2 U/ml for one hour at 37°C. Chondroitinase ABC 0,2 U/ml was

prepared using 20 µl chondroitinase 5 U/ml (C3667, Sigma-Aldrich) in 0,01% bovine serum

albumin (BSA, Roche Diagnostics GmbH, Mannheim, Germany) combined with 480 ml

solution of 50 mM Tris (Promega Corporation, USA), 60 mM sodium acetate (Merck) and

0,02% BSA.

2.4.2 IHC protocols. The IHC protocols for the different ECM components were

conducted as follows. Slides were first deparaffinated using an automated system (Microm

HMS740, Walldorf, Germany) and kept in H2Od afterwards. The tissue on the slides was

outlined with a hydrophobic, delimiting pencil (DAKO). In the following step, formalin-fixed

tissues were pretreated with the respective pretreatment in citric acid buffer and others,

depending on the target of the immune stain. After pretreatments, a washing step was performed

in PBS + 0,3% Tween20 (1 L 0,01M PBS pH 7,2 – 7,4 plus 3 ml Tween20, DAKO) twice and

consequently in pure PBS (pH 7,2 – 7,4) once, each for 3 to 5 minutes. Endogenous peroxidase

block was performed in H2O2 (VWR) in PBS 1:9 for 10 minutes. Non-specific antibody binding

sites were blocked with casein solution (Vector Laboratories®, Burlingame, CA, USA) in

H2Od, or CAS-block (Vector®), and serum (Vector®). Afterwards, primary antibodies were

incubated for different times and under different temperature conditions depending on the target

of the immune stain. After incubation, the slides were washed in PBS + 0,3% Tween20 twice

and in pure PBS once, each for 5 minutes. In table 3, the antibodies and their concentrations

together with their targets can be found.

Table 3: Antibodies used in this study, their dilution and incubation time and product code. R/T=Room

temperature.

Antibody and target Dilution and incubation time Product information

Rabbit anti-collagen I 1:500, 2 hours at R/T Acris R1038

Rabbit anti-collagen III 1:500, 1 hour at R/T Abcam ab7778

Goat anti-decorin 1:500, 1 hour at R/T R&D System AF143

Rabbit anti-versican 1:100, 1 hour at R/T Abcam ab19345

Rabbit anti-fibromodulin 1:400, 1,5 hours at R/T Larry Fisher (NIH, Bethesda, MD), LF-150

Rabbit anti-biglycan 1:100, 1 hour at R/T Abcam ab49701

Rabbit anti-goat, biotinylated 1:200, 30 min. at R/T Dako, E0466

Anti-rabbit IgG Ready-to-use kit, 30 min. at R/T MP-7401, Vector Laboratories®

Anti-mouse IgG Ready-to-use kit, 30 min. at R/T MP-7402, Vector Laboratories®

Secondary antibodies of the ready-to-use solution kits (Vector®) were incubated for 30

minutes at room temperature. These antibodies carry a peroxidase (horse radish peroxidase)

19

enzyme-conjugate which is able to react with DAB. For decorin immune staining, a different

secondary antibody system was used consisting of biotinylated rabbit anti-goat (Dako, E0466)

2nd antibody 1:200 in PBS + 0,3% Tween20 and streptavidin horse radish peroxidase (HRP,

Dako, P0397) 1:200 in PBS + 0,3% Tween20. Both antibody and HRP were incubated 30

minutes at room temperature. Following incubation of the secondary antibodies, a washing step

is performed twice in PBS + 0,3% Tween20 and once in pure PBS. The histochemistry for the

detection of antibody reaction with the structure of interest was performed using DAB substrate

solution from DAB substrate kit (SK-4100, Vector®). The DAB substrate solution was

prepared with 1 ml H2Od, 17 µl buffer stock solution, 20 µl DAB stock solution and 16 µl H2O2

solution from the kit. The reaction of DAB with the peroxidase was observed under a light

microscope and stopped by transferring the slides to H2Od when a clear reaction was observed

without background. Usually, this was the case after 2 to 4 minutes. Counterstaining was

performed with Mayer’s hematoxylin for 10 seconds and slides were subsequently washed in

tap water for 5 minutes. Lastly, slides were dehydrated in the automated system and mounting

was performed using mounting medium (Richard-Allan Scientific). Detailed protocols of each

experiment can be found in the addendum (6.1 – 6.7).

3. Results

3.1 H&E staining. This method was applied to control (healthy) nerves, autograft

repaired nerves and conduit repaired nerves. Autograft and conduit repaired nerves were

obtained at 20, 30 and 50 days after implantation of the autograft or conduit. H&E staining

showed the general organization and morphology of the PN tissues. For example, dark blue

colored cell nuclei were observed based on the reaction with hematoxylin. However, the cell

type could not be determined based on this method. Further, several acidophilic structures, e.g.

collagens, were stained in red color based on the reaction with eosin.

Twenty days after implantation of autografts and conduits, cross-sections of proximal and distal

PN tissue of both groups showed characteristics of degeneration compared to control nerves. In

the center of sections of both groups the PN regeneration was observed. The degeneration

comprised degradation of axons and extracellular structures and an increase in the number of

cells of different types, e.g. macrophages, fibroblasts and Schwann cells. Further, blood vessels

inside both types of grafts and in the surrounding tissues were observed. The tissue inside the

graft was under progressive degeneration and after 20 days signs of regeneration were only

20

observed in the proximal nerve part. Cell infiltration had occurred outside of the perineurial

space of the autograft. Autograft repaired nerves displayed a homogeneous tissue organization

with macrophages, fibroblasts and Schwann cells. Novel neural tissue was formed and in the

proximal part of the nerve stump. Regeneration of endoneurium or axons was not observed

(figure 5, A and B).

Cross-sections of conduit repaired nerves at 20 days showed a marked degeneration in

distal nerve stumps and de novo formation of nerve tissue in the center of proximal nerve

stumps. The newly-formed nerve tissue comprised cells and the formation of a novel ECM.

This period was characterized by the presence of cellular clusters and newly-formed axons.

However, close to the conduit walls it was possible to identify a loose connective tissue and

several blood vessels. In contrast, the inner part of the conduits’ lumen was characterized by a

denser ECM and abundant cells (figure 5, C and D).

In autograft repaired nerves of 30 days after implantation, cluster organization of

regenerating axons started to appear in several sections and axons were surrounded by

endoneurium. An increase in the presence of extracellular components inside of the autograft

and in the number of fascicles filled with cells, e.g. fibroblasts, in the graft wall and outside the

graft was observed. The distribution of cells and extracellular components was more

homogeneous compared to conduit repaired nerves (figure 6, A and B). After 30 days, the

conduit repaired nerves showed clusters of nerve fibers, i.e. regenerating axons lying in close

proximity to each other forming a cluster, with restored endoneurium and groups of tiny axons

associated with cells. The presence of cells and extracellular components was mainly observed

in the center of the conduit lumen forming a circular area of regeneration. Curiously, in distal

nerve stumps this area was smaller (figure 6, C and D).

The histological analysis at 50 days after implantation revealed that the tissue pattern

and organization of both studied groups was similar to the observation at 30 days after

implantation. The autograft group showed a homogeneous distribution of cells and ECM. In

addition, it was possible to observe an increase of the density of these elements in the inner part

of the grafted nerve as compared to 30 days. Nerve fibers were found organized in clusters and

each nerve fiber was immersed in a newly-formed endoneurium connective tissue (figure 7, A

and B).

In the center of conduits, a slightly smaller regeneration area was present and separated

from the conduit wall by a space of loose connective tissue of several hundred micrometers.

21

This space between conduit wall and regeneration area was almost completely free of cells and

extracellular components. In the regeneration area, perineurium and endoneurium were

observed around groups of regenerating axons associated with cells in nerve fiber clusters

(figure 7, C and D).

3.2 MCOLL histochemical method. In this study, formalin and methacarn-fixed

tissues were used for the MCOLL method and only the tissues fixed in formalin showed an

appropriate histochemical reaction for myelin, collagen and cells. Methacarn is an alcoholic

fixative which preserves several tissue elements with optimal morphology and properties.

However, due to the alcoholic nature of this solution several lipids including myelin were

completely or partially dissolved. Most information about the degree of demyelination and

remyelination was derived from longitudinal nerve sections which displayed the degree of

myelination over up to several hundred micrometers of axon length. Additionally, the typical

parallel and undulated organization of nerve fibers was clearly visible in those sections. Cross-

sections of nerves therefore gave more information about the distribution and organization of

tissue elements, collagen fibers and changes related to myelin. Cells were stained for their

nucleus by the hematoxylin counterstain though identification of the specific cell type was not

possible based on this staining. Simultaneously with the evaluation of myelin, also collagen

organization and the process of axonal regeneration were evaluated with the MCOLL

histochemical method. The contrast between myelin and collagen and the specificity and

sensibility for both of these structures were markedly improved with MCOLL compared to

H&E staining.

22

Figure 5: H&E staining results of autograft (A and B) and conduit (C and D) repaired nerves after 20 days of

regeneration. In A1 – A3 (methacarn-fixed) and B1 – B3 (formalin-fixed) sections, the cellular and extracellular

density is low and clustered nerve fibers are not visible. Note the homogeneous appearance of the graft center in

A1 and B1. The sections C1 – C3 (methacarn-fixed) show the proximal nerve stump with more pronounced

regeneration in the center of the conduit forming the so-called regeneration area. D1 – D3 (methacarn-fixed)

sections display the distal nerve stump with less intense staining and smaller regeneration area compared to C

sections. Block arrows indicate clusters of regenerating axons, small arrows indicate blood vessels. Magnification:

A1, B1, C1, D1 = 4x. A2, B2, C2, D2 = 20x. A3 – D3 = 40x.

A3 A2 A1

B1 B2 B3

C1 C2 C3

D1 D2 D3

23

A3 A2 A1

B1 B2 B3

C1 C2 C3

D1 D2 D3


regeneration. In A1 – A3 (methacarn-fixed) and B1 – B3 (formalin-fixed) sections, nerve fiber clusters and

endoneurium appear and the density of regeneration is markedly increased. Note the partial degradation of the

autograft wall in B. C1 – C3 (methacarn-fixed) display an increased regeneration area in the proximal part of the

conduit repaired nerve. Sections D1 – D3 (methacarn-fixed) show the distal nerve stump and a clearly smaller

regeneration area compared to the proximal nerve stump C. Block arrows indicate clusters of regenerating axons,

double-headed arrows indicate the autograft wall. Magnification: A1, B1, C1, D1 = 4x. A2, B2 = 20x. C2, D2,

A3 – D3 = 40x.

24

A1 A2 A3

B1

B2 B3

C1 C2 C3

D1 D2 D3


regeneration. In A1 – A3 (methacarn-fixed) endoneurium and small axons are present, but the clustered

organization is hardly visible in A3. In B1 – B3 (formalin-fixed) sections, no cluster pattern of regenerated

axons are visible. C1 – C3 (methacarn-fixed) and D1 – D3 (methacarn-fixed) sections display clusters of

regenerating axons surrounded by endoneurium. C2 shows the space between conduit wall and regeneration

area which shows no regenerating axons and only few cells. Block arrows indicate clusters of regenerating

axons, double-headed arrows indicate the autograft wall which is partially disorganized. Magnification: A1,

B1, C1, D1 = 4x. A2 – D2 = 20x. A3 – D3 = 40x.

25

3.2.1 Myelin and collagen in autograft repaired nerves. Myelin was found to be

sparsely present in the proximal nerve stumps of autograft repaired nerves at 20 days after

implantation. Further, an important decrease of myelin accompanied by Wallerian degeneration

was observed. The histochemical reaction for myelin was weaker in longitudinal sections of

autograft repaired nerves at 20 days after implantation compared control nerves. Axons were

severely degenerated and unmyelinated at different degrees in the perineurial space over the

whole length of the axon (figure 8, A).

After 30 days of implantation, autograft repaired nerves were unmyelinated over the

whole length of the axons (figure 8, B). Autograft repaired nerves of 50 days after implantation

showed a weak reaction for myelin in longitudinal sections. At this point, myelination had

occurred over parts of the regenerated axons in the proximal part of the nerve (figure 8, C).

The reorganization of collagens in the regenerating peripheral nerves was assessed by

the picrosirius reaction as part of the MCOLL method. The typical undulated and parallel

organization of collagens and nerve fibers was observed in longitudinal sections. Collagens

were mainly found associated to the epineurium and perineurium of the grafted nerve. The

endoneurial compartment was less organized and composed of a loose collagen matrix with a

weak histochemical reaction. Additionally, it was possible to identify some blood vessels with

a positive reaction for collagen at the vessel wall (figure 8, A).

After 30 days of implantation, the autograft repaired nerves showed an increase of

collagen content in comparison to 20 days after implantation. Further, the extracellular space

displayed a dense network of collagens surrounding axonal sprouts in longitudinal and cross-

sections. Thin collagen sheaths, i.e. collagen fibers oriented longitudinally and

circumferentially around axonal sprouts, were observed, but nerve regeneration was not

organized into forming nerve clusters. In addition, it was possible to identify several axonal

sprouts with irregular organization near to the perineurial layer and surrounded by collagen

fibers (figure 8, B).

In sections of 50 days after implantation, the collagen fiber pattern showed no evident

alterations compared to autograft sections of 30 days after implantation. However, the density

of other extracellular components was slightly increased and led to a more homogeneous

appearance of the regenerating nerve tissue. Organization into clusters was not observed and

reestablishment of endoneurium was found in parts of the cross-sections. In longitudinal

26

sections, collagen sheaths around regenerating axons were present with a pattern and density

that was similar to control nerves (figure 8, C).

A1 A2 A3

B1 B2 B3

C1 C2 C3

D1 D2 D3

Figure 8: Evaluation of MCOLL histochemical results of autograft repaired nerves (A, B, C) and control (healthy)

nerves (D). A1 – A3 show the autograft repaired nerves 20 days after implantation and display degeneration and

demyelination of axons. Residual myelin (circle), collagen fibers (block arrows) and blood vessels with their typical

collagen-rich vessel wall (small arrows) are present. Scale bar = 40 µm. B1 – B3 show autograft repaired nerves

after 30 days of implantation with increased collagen expression (block arrows), but absence of myelin. Scale bars

= 80 µm (B1) and 40 µm (B2 & B3). C1 – C3 show autograft repaired nerve 50 days after implantation with a

dense, regenerated collagen network and partially remyelinated axons (circles). Collagen fibers (block arrows) and

blood vessels (small arrows) are also found. Scale bars = 80 µm (C1) and 40 µm (C2 & C3). D1 – D3 show the

healthy control nerves with plenty of myelin and ordered collagen fibers. Groups of myelinated axons (circles),

collagen fibers (block arrows) and blood vessels (small arrows) are observed. Scale bars = 100 µm (D1), 40 µm

(D2) and 30 µm (D3).

27

3.2.2 Myelin and collagen in conduit repaired nerves. MCOLL staining of this group

of nerves revealed a progressive nerve regeneration but no myelination of the newly-formed

axons at 20 days after implantation. Complete absence of myelin was observed and small axonal

sprouts were found only in the central, circular regeneration area of the conduit where new

nerve tissue was formed (figure 9, A). In contrast, a proximal longitudinal sections of conduit

repaired nerves of 30 days after implantation displayed axons with a weak residual reaction for

myelin in one section. Further, axons were thicker than 20 days after implantation. The degree

of myelination in these sections was comparable to the degree of myelination in autograft

repaired nerves of 50 days after implantation, but not comparable to the control nerves (compare

figure 8 C & D to figure 9 B). Cross-sections of conduit repaired nerves showed absence of

myelin in the newly-formed nerve fibers. The same was observed in longitudinal and cross-

sections after 50 days of implantation where regenerating axons also showed an increase in

diameter compared to 20 and 30 days after implantation (figure 9).

Collagen was found in conduit repaired nerves forming fibers which appeared properly

oriented along the newly-formed axons. Further, in longitudinal sections the typical undulated

and parallel organization of collagen fibers and axons was observed. In sections of 20 days after

implantation, collagen fibers were found in the newly formed endoneurium around the axonal

sprouts. In the center of the conduits, a regeneration area was formed which showed a higher

collagen density than the space between regeneration area and the conduit wall. In this space

between regeneration area and conduit wall, collagen was present as long and thin fibers and

accompanied by cells, however without axons. Furthermore, the regeneration area showed a

denser extracellular space compared to the extracellular space found in autograft after 20 days

of implantation. A certain degree of fibrosis was observed in the regeneration area and its

surrounding. This fibrosis was characterized by an intensely stained collagen fiber deposition

(figure 9, A).

The regeneration area inside the proximal part of the conduit showed an increase in size

at 30 days after implantation. The density and organization of collagen and other extracellular

structure in the perineurial regeneration area was comparable to 20 days after implantation.

Collagen was present in endoneurium of newly-formed axons. Fibrosis was observed in the

peripheral parts of the perineurial area at a similar degree as in sections of 20 days after

implantation. Longitudinal sections at 30 days after implantation showed stronger collagen

presence around axons compared to sections after 20 days of implantation (figure 9, B).

28

After 50 days of implantation, longitudinal sections of conduit repaired nerves showed

improved organization and more collagen compared to 20 and 30 days after implantation (figure

9). The collagen fibers, endoneurium and extracellular components appeared less dense in

cross-sections after 50 days than after 30 days of implantation. However, the organization of

collagen, endoneurium and extracellular components was still comparable. Fibrosis was

observed in several sections (figure 9, C).

A1 A2 A3

B1 B2 B3

C1 C2 C3

Figure 9: Evaluation of MCOLL histochemical results of conduit repaired nerves. A1 – A3 show conduit repaired

nerves 20 days after implantation and display degeneration and demyelination of axons. Collagen fibers (block

arrows) were intensely stained and formed endoneurium and axonal sheaths. Blood vessels (small arrows) with

their typical collagen rich vessel wall were also observed. Fibrosis can be seen in A2 between block and small

arrow as red collagen stacks (squares). Scale bar = 80 µm (A1) and 40 µm (A2 & A3). B1 – B3 show conduit

repaired nerve after 30 days of implantation with increased collagen expression (block arrows) and residual myelin

(circle) in longitudinal section. Scale bars = 80 µm (B1) and 40 µm (B2 & B3). C1 – C3 show conduit repaired

nerve 50 days after implantation. Collagen fibers (block arrows), blood vessels (small arrows) and fibrosis (squares)

are observed. Scale bars = 80 µm (C1) and 40 µm (C2 & C3).

29

3.3 Collagen I IHC. Results of the collagen type I IHC staining indicated the expression

and distribution of this protein in the regenerating nerve at 20, 30 and 50 days after implantation

of autograft or conduit. In control nerves, collagen type I was observed in the endoneurium

around each nerve fiber, in the perineurium and epineurium (figure 10, A).

After 20 days of regeneration, a strong reaction for collagen I was observed in the

autograft epineurium and blood vessels. This reaction was positive with a homogeneous and

clear extracellular pattern. However, collagen type I was absent in the endoneurium layer

(figure 10, B1 & B2). In longitudinal sections, a positive reaction in the perineurium was visible

and also in endoneurial space around axons which were associated with Schwann cells and

surrounded by collagen I sheath (figure 10, B3).

After 30 days of regeneration, the reaction intensified for collagen I in the ECM inside

of the autograft. Newly-formed axons in a network of collagen I were observed, without clear

endoneurium formation or clustered organization in the distal part and with collagen I positive

endoneurium in the proximal part of the nerve. In the graft-surrounding connective tissue,

fascicular structures which comprised cells and collagen I were visible (figure 10, C).

In the autograft repaired nerves of 50 days of regeneration, regenerating axons of the

distal nerve showed no ensheathment by collagen I fibers, no clustered organization or restored

endoneurium and unorganized collagen I expression in the ECM in the graft (figure 10, D1).

However, a dense network of small axons surrounded by collagen I sheaths with an organized

collagen I expression in the ECM was observed in the proximal nerve (figure 10, D2 & D3).

Also, collagen I was abundant in the proximal longitudinal section around regenerating axons

and this section showed the typical undulated orientation of collagen I fibers and axons (figure

10, D3).

In conduit repaired nerves after 20 days of regeneration, a small regeneration area

located in the center of the conduit was observed based on the reaction for collagen I in the

perineurial space. Very few and newly-formed axons were visible which were surrounded by

collagen I fibers and organized into nerve fiber clusters. Collagen I density in the ECM was

high towards the center of the regeneration area (figure 11, A1). In longitudinal sections, the

reaction was less intense and axons surrounded by collagen I were observed in the proximal

section showing advanced axonal regeneration. In the distal section, the expression of collagen

I was lower and axonal regeneration was less advanced showing small axonal sprouts (figure

11, A3 & A4). Outside of the perineurial regeneration area and towards the conduit wall,

30

collagen I was found in longitudinally cut blood vessels and small groups of regenerating axons

(figure 11, A2). In the proximal section, a bigger perineurial space was observed and showed

axon ensheathment by collagen I fibers and organization into clusters of regenerating nerve

fibers (figure 11, A1).

In conduit repaired nerves of 30 days after implantation, the perineurial space increased

in size, filling up almost the entire lumen of the conduit. In several sections, the endoneurial

sheath around small axons was stained but hardly visible in the center of the perineurial space

and more clearly visible in the periphery of the perineurial space. However, other sections

displayed bigger axons with collagen I sheath also forming a new endoneurium (figure 11, B1

& B2). Longitudinal sections showed the undulated organization of collagen I around axonal

sprouts, but these sprouts were not visible in all of the sections. Nonetheless, collagen I was

found in the ECM forming bands to support the axonal sprouts by a network of collagens.

In conduit repaired nerves, the expression of collagen I after 50 days was homogeneous

in the regeneration area. In the distal part, axons were small and endoneurium which stained

positive for collagen I appeared weakly around those axons. However, the proximal part

showed the reaction in the endoneurium more clearly where axons were also organized into

clusters (figure 11, C1 & C2). The collagen I expression in the ECM appeared thinner and less

organized compared with sections of 30 days after implantation. In the proximal part of the

longitudinal sections, the collagen fibers around regenerating axons were organized in an

undulated orientation forming a dense ECM of collagen I. In the distal part however, this

organization of collagen I in the ECM was present to a far less extent and only small axonal

sprouts were observed. A positive reaction in the wall of the NeuraGen® collagen conduits was

also observed.

31

Figure 10: Results of immunohistochemistry for collagen type I in control (healthy) and autograft repaired nerves.

In control nerves (A1 – A3) the normal collagen I organization and ensheathment of axons in a longitudinal section

can be seen. In autograft repaired nerves after 20 days of implantation, the graft epineurium is strongly reactive for

collagen I (B1) and in the perineurial space the ECM is less reactive for collagen I (B1 & B2). Collagen I is mostly

seen around blood vessels (small arrows). The proximal longitudinal section shows collagen I around regenerating

axons forming sheaths in the endoneurial space and in the perineurium (B3, block arrow). After 30 days, the distal

section (C1) shows no clear endoneurial sheaths while the proximal part of the same nerve displays thin endoneurial

sheaths around regenerating axons (C2). In the epineurium, fascicles comprising cells and positivity for collagen I

are found (C3). Blood vessels are indicated by small arrows and perineurium positive for collagen I by block arrow.

After 50 days of implantation, in the distal section D1 no endoneurial sheath formation or axonal clusters are found

while the proximal section D2 displays individual ensheathment by collagen I of regenerating axons. Also, the

proximal longitudinal section D3 displays big, regenerated axons with individual ensheathment and collagen I

positive perineurium (block arrow). Scale bars = 80 µm (A1, B1), 40 µm (A2, B2 & B3, C1 – C3, D1 – D3) and

20 µm (A3).

A1 A2 A3

B1 B2 B3

C1

D1

C2

D2

C3

D3

32

Figure 11: Results of immunohistochemistry for collagen type I in conduit repaired nerves after 20 (A), 30 (B) and

50 (C) days of implantation. After 20 days the density of collagen I in the ECM and around axons is highest in the

center of the perineurial regeneration area (A1) and gets progressively lower towards the conduit wall (A2). Small

axons, cell nuclei and blood vessels (small arrows) are visible. In distal (A3) and proximal (A4) longitudinal

sections, the regeneration was differently advanced and shows differently sized axons and collagen I sheaths around

the axonal sprouts. Blood vessels (small arrows) in the perineurium are also found. After 30 days, the ensheathment

of axons by collagen I in the endoneurial space, axon size and collagen I organization in the ECM are more

advanced in the proximal sections B2 compared to the distal section B1. In longitudinal sections the collagen I

density varies and B4 shows restored endoneurium more clearly than B3, but regenerating axons are embedded in

a collagen I rich ECM in both sections. Blood vessels (small arrows) are also stained. 50 days after implantation,

the collagen I density in the ECM seems to decrease and it is more difficult to observe endoneurium around

regenerating axons in C1 and C2. The distal longitudinal section C3 shows less organized collagen I expression in

the ECM and around axonal sprouts while the proximal section C4 has a more dense collagen I network and

advanced regeneration of axons. The walls of the NeuraGen® collagen conduits were also positive for collagen I.

Scale bars = 40 µm.

A1

A2

A3

A4

B1

B2

B3

B4

C1

C2

C3

C4

33

3.4 Collagen III IHC. Results of this IHC staining indicated the expression and

distribution of collagen type III in the regenerating nerve at 20, 30 and 50 days after

implantation of autograft or conduit. In control nerves, collagen III was present in endoneurial

sheaths surrounding individual axons and in the wall of blood vessels. Its expression pattern

was comparable to collagen I expression in control nerves, but it appeared to be less abundant

(figure 12, D). The positive reaction in blood vessels was also observed in both groups of

repaired nerves.

Collagen III expression and distribution after 20 days of regeneration in autograft

repaired nerves was different from collagen I in several ways. There was no network of collagen

III in the ECM, but circular organized sheaths of collagen III around individual axonal sprouts

were present. In the autograft epineurium, collagen III was found in fascicles which comprised

many cells. Further, collagen III was found in the walls of blood vessels (figure 12, A). In

longitudinal nerve sections, its expression and distribution was located around axons forming

the endoneurial sheath in a similar way as collagen I (figure 12, A).

After 30 days of regeneration, an increased density and network of collagen III was

observed in autograft repaired nerves. Collagen III was found in the ECM forming a dense

network and ensheathment of individual axons was difficult to observe due to the high density

and homogeneous distribution of collagen III (figure 12, B2). However, the proximal nerve

stump displayed the ensheathment of axons by collagen III in the perineurial space. In this

section, the ECM showed lower collagen III expression and a less disperse network in the

perineurial space (figure 12, B3). The longitudinal section displayed big regenerating axons

surrounded by collagen III positive endoneurial sheath and perineurium (figure 12, B1).

In autograft repaired nerves after 50 days of regeneration, the density of collagen III was

lower in the ECM of the distal nerve section than in the distal sections of 30 days. In the

autograft of the distal section, the nerve fiber clustered orientation disappeared and also

collagen III sheaths around axons were hardly visible. In the epineurium and connective tissue,

collagen III was found in fascicles comprising cells (figure 12, C2). In the proximal sections,

collagen III was found in endoneurial sheaths around regenerating axons. Collagen III was also

found in perineurium of those sections and in blood vessel walls and fascicle in the epineurium

(figure 12, C3). In the longitudinal sections, the expression was lower in the proximal section

which also comprised axons of bigger size compared to the distal sections that comprised axons

at an earlier stage of regeneration. In proximal and distal sections collagen III was found in the

34

perineurium and perineurial space forming sheaths around regenerating axons and in the ECM

of the endoneurial space (figure 12, C1, C4 & C5).

In conduit repaired nerves at 20 days after implantation, the reaction was weaker for

collagen III than for collagen I in the perineurial space, but the expression pattern was

comparable. Collagen III was found in the endoneurial space, in several axons forming

clustered nerve fibers and in the ECM. In the distal section, the expression of collagen III in the

ECM was lower than in the proximal sections. In all sections, multiple blood vessels were

identified based on collagen III reaction in the vessel walls (figure 13, A1-A4). Collagen III

expression was lower in distal longitudinal sections of nerves, but was organized in sheaths

around regenerating axons and less abundant in the ECM surrounding these axons. Distal nerve

stump showed marked degeneration of axons and low collagen expression while the proximal

stump consisted of thicker newly-formed axons and properly oriented collagen sheaths (figure

13, A3 & A4).

Thirty days after implantation, the perineurial regeneration area in the center of the

conduit increased in size, but the expression pattern of collagen III was comparable to 20 days

after implantation. Collagen III was clearly visible in endoneurium of regenerating axons and

in the ECM (figure 13, B1 & B2), but it was less abundant in the ECM compared to collagen I.

Further, regenerating axons were packed into clusters which were positive for collagen III. In

longitudinal sections, regenerating axons were surrounded by collagen III sheaths and again in

distal and proximal sections the degree of regeneration of axons was different, i.e. more advance

in the proximal part (figure 13, B3 & B4).

In conduit repaired nerves of 50 days after implantation, the reaction for collagen III

was weak in the ECM of the perineurial space of proximal and distal sections. Collagen III was

found almost exclusively in endoneurial sheaths of axons packed in clusters (figure 13, C1 &

C2). The reaction was very weak in longitudinal sections compared to collagen I, and collagen

III was almost absent in the ECM of distal and proximal sections. However, it was still observed

around regenerating axons and the organization of the collagen III was slightly more

homogeneous than in sections of 30 days after implantation (figure 13, C3 & C4).

35

A1 A2 A3

B1 B2 B3

C1 C2 C3

C4 C5 D1

Figure 12: Results of immunohistochemistry for collagen type III in control (healthy) (D1) and autograft repaired

nerves after 20 (A), 30 (B) and 50 (C) days of implantation. After 20 days the expression of collagen III in the

perineurial ECM is low and endoneurial sheaths around axons are formed in proximal sections (A1 & A2). Collagen

III is also present in the wall of blood vessels (small arrows) and in fascicles in the autograft epineurium. In

longitudinal sections, it is found in sheaths around regenerating axons. After 30 days, the ensheathment of axons

by collagen III is more difficult to observe and collagen III is more densely present in the ECM of the perineurial

space (B2 & B3). In longitudinal sections, collagen III is found in endoneurial sheaths around axons and in the

perineurium (B1). Blood vessels (small arrows) are also stained. 50 days after implantation, endoneurial sheaths of

collagen III and nerve fiber clusters are difficult to observe in the distal section (C2), but are more clearly visible

in the proximal section (C3). The longitudinal sections show different stages of axonal regeneration and collagen

III expression and organization. The proximal C1 shows the most advanced regeneration of axons and endoneurial

sheaths of collagen III while in distal sections C4 and C5 the axons are less advanced in the regeneration process

and collagen expression is higher but less organized. Perineurium is indicated by block arrows. Scale bars = 40 µm.

36

A1

A2

A3

A4

B1

B2

B3

B4

C1

C2

C3

C4

Figure 13: Results of immunohistochemistry for collagen type III in conduit repaired nerves after 20 (A), 30 (B)

and 50 (C) days of implantation. After 20 days, the expression is low in the perineurial ECM and more pronounced

in endoneurial sheaths, blood vessel walls (small arrows) and clusters of newly-formed axons (A1 & A2). In

longitudinal sections, collagen III is found sparsely around early regenerating axons in the distal part (A3) and more

advanced regenerating axons in the proximal part (A4) forming endoneurial sheaths. In sections of 30 days after

implantation, clusters of axons surrounded by endoneurial sheaths are present and the density of collagen III

increases in the ECM (B1 & B2). Axonal regeneration and endoneurial ensheathment is also found to be more

advanced in longitudinal sections (B3 & B4). After 50 days, collagen III density in the perineurial ECM appears to

decline but nerve fiber clusters and endoneurium are still positive and visible in both cross-sections and longitudinal

sections. Scale bar = 40 µm.

37

3.5 Decorin IHC. The IHC reaction for decorin resulted in a staining pattern in healthy

control nerves, autograft and conduit repaired nerves fixed in methacarn. The expression pattern

of decorin in the ECM was analyzed in those sections of repaired nerves after 20, 30 and 50

days of implantation of autografts or conduits. In control nerves, decorin was found to be

sporadically expressed in the perineurial ECM surrounding several axons in cross-sections of

nerve and in the connective tissue surrounding the nerve (figure 14, A1).

In autograft repaired nerves of 20 days after implantation, decorin was found to be

expressed in the autograft epineurium of two sections. In the proximal section, decorin was

homogeneously expressed in the perineurial ECM around regenerating axons of various sizes

and in the wall of blood vessels. In the distal section, the ECM in the regenerating nerve tissue

was negative for decorin (figure 14, B1 & B2). In the longitudinal section of this nerve, decorin

was weakly expressed in the endoneurial ECM surrounding axons and in blood vessel walls.

Due to the reaction of decorin the typical undulated pattern of the nerve was observed (figure

14, B3).

After 30 days of regeneration, the proximal section of autograft repaired nerve showed

a positive reaction in the epineurium and in the perineurial space of the graft in the ECM.

Further, blood vessels were stained positive for decorin and the distribution of decorin was

homogeneous among the ECM (figure 14, C1, C2 & C3). In the proximal part of the nerve

section, tiny regenerating axons surrounded by sheaths and decorin in the ECM were visible

which were not observed in the distal part of this nerve sections (figure 14, C2). The distal

section of autograft repaired nerve displayed no positive reaction for decorin in the ECM of the

perineurial space (figure 14, C3). However, the epineurium and surrounding connective tissue

were positive for decorin and a small proximal part of a longitudinal section also showed

positivity around several regenerating axons (figure 14, C4).

Longitudinal sections showed positivity for decorin in the perineurial space and ECM

around regenerating axons after 50 days of regeneration. The reaction was stronger in the

proximal part of the longitudinal section which also featured a more advanced regenerative

status with bigger axons than the distal part of this nerve (figure 14, D3 & D4). In the distal

cross-sections, decorin was found to be expressed homogeneously in the perineurium and in

the ECM of the perineurial space of the graft around groups of axons organized into nerve fiber

clusters (figure 14, D1). The proximal cross-section showed less positive reaction and a more

diffuse staining pattern for decorin in the perineurium and ECM in the perineurial space of the

38

graft and nerve fiber clusters surrounded by endoneurium were sporadically stained. The

density of decorin had declined in the ECM of the proximal nerve section (figure 14, D2).

Several sections of conduit repaired nerves were evaluated after 20 days of regeneration

and decorin was found to be abundant and diffusely expressed in a central longitudinal section

around axonal sprouts (figure 15, A3). A more proximal longitudinal section showed bigger

newly-formed axons and decorin expression was much lower in the perineurial space and ECM

around those axons (figure 15, A4). Two proximal cross-sections were positive in the

perineurial space for decorin which was found in the ECM and around clusters of regenerating

axons. The presence in the ECM was more pronounced in the perineurial space where no

clusters of newly-formed axons were observed (figure 15, A1). Two distal cross-sections

showed a negative reaction for decorin, but those sections also showed axonal regeneration and

nerve fiber clusters (figure 15, A2).

After 30 days of regeneration, decorin expression was found to be markedly increased

in the periphery of the perineurial regeneration area, but not in the center where the reaction

was weak in the proximal and negative in the distal section. The ECM in the endoneurial space,

around nerve fiber clusters, comprised groups of newly-formed axons and was, together with

blood vessel walls, moderately positive for decorin (figure 15, B1 – B3). Longitudinal sections

showed a very weak staining in the endoneurial space around small axons (figure 15, B4).

After 50 days of regeneration, distal cross-sections displayed a negative reaction for

decorin in the center of the perineurial regeneration area in the conduit. However, towards the

conduit wall and at the outer edge of the regeneration area, the expression was positive in the

ECM. Nerve fiber clusters were observed in which the ECM showed positivity for decorin and

these clusters were located in the periphery of the perineurial space (figure 15, C1). Proximal

sections displayed newly-formed axons and a more homogeneous expression pattern of decorin

in the ECM over the perineurial regeneration area (figure 15, C2 & C3). In longitudinal sections,

decorin was found in the ECM of the endoneurial space and differently sized axons were

observed. The reaction was strong and homogeneous highlighting the undulated pattern of the

newly-formed axons (figure 15, C4).

39

Figure 14: Results of immunohistochemistry for decorin in control (A1) and autograft repaired nerves after 20 (B),

30 (C) and 50 (D) days of implantation. In the control nerve, decorin expression is limited to the ECM of the

perineurial space between only few axons (A1). In autograft repaired nerves after 20 days of implantation, decorin

is found in the epineurium and in the ECM of the endoneurial space (B1). The distal section is completely negative

(B2). In the longitudinal section, the reaction is very weak and decorin is observed around several regenerating

axons (B3). In sections of 30 days after implantation, the presence of decorin is markedly increased in the

perineurial space and ECM. The expression pattern is homogeneous, but regenerating axons and organized decorin

within the ECM are observed only in the proximal section (C2). The distal section is negative for decorin in the

ECM, but positive in the epineurium and connective tissue (C3). Further, decorin is expressed in the ECM

surrounding regenerating axons in the longitudinal section (C4). Cross-sections of 50 days after implantation show

decorin expression in the endoneurial space (D1 & D2), but the proximal section (D2) has a lower and non-

homogeneous reaction pattern (D2). The longitudinal section D3 shows advanced regeneration of axons and decorin

expression in the ECM. In the distal part, axons are less advanced in regeneration and smaller, but also surrounded

by decorin in the ECM (D4). Small arrows indicate blood vessels. Block arrows indicate perineurium positive for

decorin. Scale bar = 60 µm (B2) and 30 µm (A1, B1, B3, C & D).

A1 C1

C2

C3

C4

D1

D2

D3

D4

B1

B2

B3

40

A1

A2

A3

A4

B1

B2

B3

B4

C1

C2

C3

C4

Figure 15: Results of immunohistochemistry for decorin in conduit repaired nerves after 20 (A), 30 (B) and 50 (C)

days of implantation. After 20 days of regeneration, decorin is observed in the perineurial space and ECM

surrounding newly-formed axons in the proximal section (A1). The distal section is negative for decorin (A2). In

the central part of the longitudinal section, the expression was strong in the ECM surrounding axonal sprouts (A3).

The proximal part of the longitudinal section displays more advanced regeneration of axons and less decorin

expression in the ECM surrounding those axons (A4). After 30 days of regeneration, the perineurial space of the

conduits is mostly negative for decorin, but towards the borders of the perineurial space the expression increases in

the ECM (B1 & B2). Decorin is found in the ECM surrounding clusters of newly-formed axons (B3). The

longitudinal section shows very weak reaction of decorin (B4). 50 days after implantation, there is no expression

of decorin observed in the center of the regeneration area, but is observed only at the borders of the perineurial

space. Axons are packed into clusters which are surrounded by decorin positive ECM (C1 – C3). In the longitudinal

section, decorin expression is increased and it is present in the ECM around axons (C4). The transition from center

to periphery of the regeneration area is indicated by block arrows. Scale bar = 60 µm (B1, B2, C1 & C2) and 30

µm (A, B3, B4, C3 & C4).

41

3.6 Versican, fibromodulin and biglycan IHC. The IHC protocol for versican was

carried out using formalin-fixed autograft and conduit repaired nerve sections after 20, 30 and

50 days of implantation. In all sections, including control sections of healthy nerve, the reaction

was negative and no staining pattern was observed (figure 16, A1 – A4). Due to the hematoxylin

counterstain, it was possible to observe cell nuclei and blood vessels in the tissues. The

transition between autograft and conduit lumen, perineurium and epineurium and surrounding

connective tissue was visible (figure 16, A2 & A3).

Results from IHC stainings for fibromodulin in methacarn-fixed sections of autograft

and conduit repaired nerves after 20, 30 and 50 days of implantation indicated no staining

pattern in the nerve tissues. In several sections of both types of repaired nerves only the wall of

blood vessels and parts of the connective tissue showed a weak reaction (figure 16, B2 & B3).

The ECM in the regenerating nerve tissues was negative for fibromodulin. Blood vessels and

cell nuclei were found due to hematoxylin counterstain.

The IHC protocol for biglycan was performed in formalin-fixed autograft and conduit

repaired nerve sections of 20, 30 and 50 days after implantation. All sections displayed negative

reactions for biglycan and no expression was observed. The hematoxylin counterstain allowed

for the observation of cell nuclei and blood vessels based on their lumen and muscle-layered

wall (figure 16, C1 – C4).

42

Figure 16: Overview of several results of immunohistochemistry for versican (A), fibromodulin (B) and biglycan

(C). A1 and A3 display the negative reaction for versican after 20 and 50 days, respectively, after implantation of

autografts. Blood vessels (small arrows) and the perineurium (block arrows) as well as cell nuclei are observed

based on the hematoxylin counterstain. A2 and A4 show the negative reaction for versican in conduit repaired

nerves after 20 and 50 days, respectively, after implantation. In sections B2 (conduit repaired nerve, 20 days after

implantation) a slightly positive reaction for fibromodulin in the perineurium is observed (block arrow). In B3

(autograft repaired, 50 days after implantation) a slightly positive background reaction in the autograft epineurium

(block arrow) and blood vessel walls (small arrow) is observed. Autograft repaired sections of 20 and 50 days (C1

and C3, respectively) and conduit repaired sections of 20 and 50 days (C2 and C4, respectively) after implantation

show negativity for biglycan in all sections. Only blood vessels are observed based on erythrocytes reaction to DAB

(C1 & C2, small arrows). Scale bar = 60 µm.

A2

A3

A4

B1 C1

B2 C2

B3 C3

B4 C4

A1

43

4. Discussion and conclusion

The H&E stainings gave a histological overview of control, autograft and conduit

repaired nerves. It allowed us to evaluate and compare the basic morphological patterns during

sciatic nerve regeneration in rats at different time intervals, i.e. 20, 30 and 50 days after

implantation of autografts or conduits. Clusters of regenerating axons were present at an earlier

stage in time, i.e. 20 days after implantation, in conduit repaired nerves than in autograft

repaired nerves. The tissue in the autograft is first under progressive degeneration where axons

and myelin are degraded before the regenerating axons advance in the graft. The collagen-based

conduits therefore seemed to promote axonal regrowth and the formation of new nerve fiber

clusters in proximal and distal nerve stumps at an earlier stage in the regeneration process than

autografts. Further, the histochemical reaction in the ECM was more intense around those nerve

fiber clusters at 20 days after implantation in conduit repaired nerves compared to autograft

repaired nerves where the ECM density was lower due to the ongoing degeneration. However,

this difference was not observed after 30 and 50 days of implantation which indicates an equal

density and composition of ECM at those points in time.

The presence of newly-formed axons in the conduit repaired nerves after 20 days of

implantation showed that the regenerating nerve was in an advanced state. Typically,

regeneration in a hollow conduit occurs in different phases where new nerve tissue is formed

progressively. First, components with neurotrophic and supporting activities fill up the lumen

of the conduit during the first days after implantation in the fluid phase. Afterwards, a fibrin

network is formed between the proximal and distal end of the sectioned nerve and fibroblasts

along with other cells infiltrate the lumen from both nerve ends in this matrix phase. At that

time, Wallerian degeneration is taking place in the distal nerve stump where Schwann cells and

other cells recycle and remove the axonal breakdown products. This environment is important

for the Schwann cells and newly-formed axons in the second week of regeneration, the cellular

phase, where both of them migrate along the fibrin network and axons proceed from proximal

into the distal nerve end target. In the third week and later on, the axons continue to regenerate

and are myelinated by Schwann cells in the following 2 to 8 weeks in the so-called axonal phase

(38) (39). In this study, sections of conduit repaired nerves after 20 days of implantation showed

that the nerve tissue had proceeded into the axonal phase and newly-formed axons were present

in the distal nerve end.

The myelination status and collagen reorganization are important in the process of PN

regeneration and hence the MCOLL histochemical method can be conducted to determine the

44

degree of myelination and collagen expression and organization (25). The use of the MCOLL

histochemical method allowed us to observe the demyelination in autograft and absence of

myelin in conduit repaired nerves after 20 days of implantation in both types of nerve repair.

Demyelination and Wallerian degradation with loss of axon numbers and mass are typical

features of transected nerves (3). Residual myelin was observed in one autograft section which

was later degraded since all sections of 30 days after implantation of the graft were completely

negative for myelin. Also, only conduit repaired nerves, which were completely negative for

myelin after 20 days of implantation, showed myelination after 30 days of regeneration in the

proximal nerve ends. Unfortunately, there were no longitudinal sections of 50 days after

implantation of conduit repaired nerves to evaluate myelination, but in autograft repaired nerves

remyelination was observed after 50 days of implantation. The degree of myelination was still

lower than in control nerves and cross-sections of both type of nerve repair were negative for

myelin after 20, 30 and 50 days of regeneration which indicates that the myelination process

was still ongoing and far from being completed.

Collagen is an important molecule in the ECM during regeneration and guides the

axonal sprouts by forming a network along which the axons can grow (8) (12) (13). Its

expression in autograft and conduit repaired nerves was found to be linked to the degree of

axonal regeneration. 20 days after implantation of autografts or conduits, the density of the

collagen network was lower and axonal sprouts were smaller than at 50 days after implantation.

Especially autograft repaired nerves displayed a loose network of collagen in the ECM of the

perineurial space at 20 days after implantation. At this time point, collagen was found in the

perineurium and endoneurial ECM around clusters of newly-formed axons of the conduit

repaired nerves. This indicates that the conduit environment promotes the precise collagen

organization in the ECM around clusters of axons to facilitate axonal pathfinding and growth.

Further, proximal and distal sections could clearly be differentiated based on the differences in

collagen density, organization and axonal size. Although the conduit environment seemed to be

positive for collagen organization and axonal regeneration, fibrosis and scar tissue formation

which are characterized by excessive collagen deposition was observed in conduit repaired

nerve sections 20, 30 and 50 days after implantation. This phenomenon is frequently observed

in regenerating nerves and is known to influence the proper regeneration and growth of axonal

sprouts (14).

IHC stainings for two specific forms of collagen, i.e. collagen type I and III, reveal more

precisely their expression and organization in the ECM of autograft and conduit repaired nerves

45

at 20, 30 and 50 days after implantation. Collagen I and III belong to the group of fibril forming

collagens and are found in the basal lamina of myelinating Schwann cells and in the

endoneurium (10) (12) (13) (14). The results from collagen I IHC in autograft repaired nerves

indicate that the endoneurial ensheathment of regenerating axons is not restored after 50 days

in distal section, but is restored in proximal sections after 30 days and 50 days of implantation.

This collagen network which originates in the proximal nerve end and develops along the earlier

formed bands of Büngner is crucial for the axonal growth towards the distal end. Regeneration

can thus only be successful if the collagen network and ensheathment is provided for the axonal

sprouts up to the distal nerve end (16). In conduit repaired sections, axonal ensheathment by

collagen I in the endoneurium is already observed after 20 days of implantation in proximal and

distal sections. However, the reaction in the ECM is rather weak in later stages of regeneration

where individual axonal ensheathment is not observed and the highest collagen I density is

observed around clusters of newly-formed axons. It seems peculiar that collagen I is not

observed in axonal sheaths at 50 days after implantation since this ensheathment was observed

in autograft repaired nerves after 50 days and thus should also be present in conduit repaired

nerves at this time. It is possible that the antibody reaction was not optimal in those conduit

sections and the collagen was not completely stained.

Collagen III expression and organization appears to be similar to collagen I, however

collagen III was not as abundant in both types of repaired nerves as collagen I. Further, it is

observed that collagen III is present in perineurium and endoneurium of the regenerating nerves

and probably also in the basal lamina which connects myelinating Schwann cells to the axons.

The relationship of Schwann cell and the newly-formed axon is crucial for the nerve

regeneration (8) (12) and it is observed in conduit repaired sections at 20 days after

implantation. These findings indicate that the conduit repair promotes restoring the collagen III

ensheathment of newly-formed axons at an earlier point in time than autograft repair promotes

the endoneurial collagen III sheath formation.

Decorin has been reported to play a crucial role in fibril formation by interacting with

collagens (13) (53) (54). Our results suggest that decorin is expressed in the perineurial space

in close concert with collagen I and III, first in proximal nerve ends and later also in distal nerve

ends. Further, in autograft repaired nerves, decorin is not found in distal sections at 20 and 30

days after implantation while conduit repaired nerves are positive for decorin in the perineurial

and endoneurial space in both proximal and distal nerve endings after 30 days of implantation.

Additionally, it appears that advanced regeneration in conduit repaired nerves leads to lower

46

decorin expression in the endoneurial ECM and this expression equals the low decorin

expression in healthy nerves. Therefore, it seems that the conduits promote the fibril formation

of endoneurial collagen earlier than autograft repair from proximal to distal nerve ends by

increased decorin expression. This should be a factor for enhanced nerve regeneration since the

collagen fibril network is necessary and crucial factor in successful axonal pathfinding and

nerve repair (12).

The CSPG versican has been reported to be an inhibitor of axonal growth during nerve

regeneration (55). Furthermore, versican has been observed by IHC in distal nerve stumps and

in newly formed endoneurial sheaths of regenerating rat sciatic nerves (35). Another study

looked at specific isoforms of versican, namely V0/V1, and found that these isoforms were

upregulated in the proximal segment of rat sciatic nerve (56). In contrast, in the present study

it was not possible to obtain a positive staining in control or repaired nerves for versican. It is

assumed that the antibody works properly, but it may be possible that the nerve tissues were

either not treated properly or did not express versican at all or a form of the versican epitope

that is detectable with this antibody system.

Fibromodulin is expressed in connective tissues and plays a role in collagen

fibrillogenesis by binding to collagen type I and II (13) (53) (54). The positivity obtained by

the IHC for fibromodulin in our study was due to background reaction in the perineurium and

connective tissue surrounding the nerve fiber bundle. In the present study, a dense collagen I

and III network with possible fibril formation could be observed in regenerating nerves and

fibromodulin which has been reported to impede the fibril formation could be absent in those

sections to not interfere with collagen I fibrillogenesis in the ECM. However, complete absence

of fibromodulin in knockout experiments in other tissues has been shown to cause dysfunctions

in those tissues (20) suggesting that fibromodulin is needed to regulate proper collagen

fibrillogenesis at least in certain tissues. Whether fibromodulin expression is needed and in

which compartment of regenerating PN it has to be expressed for correct collagen

fibrillogenesis remains to be elucidated.

Biglycan is thought to be expressed in many tissues and to interact with collagen type I

and to work cooperatively with decorin in the process of fibrillogenesis (13) (18) (54).

However, it has also been reported that biglycan together with fibromodulin can inhibit collagen

fibrillogenesis in certain tissues (20). In this study, biglycan could not be detected by IHC

means in any compartment of the nerve sections. Since collagen type I was abundant and

47

decorin was also found, it was expected that biglycan should also be present. However, since a

dense collagen I and III network with possible fibril formation was found, biglycan could be

absent to not interfere with the ECM-collagen network in the perineurial space and

fibrillogenesis. It is rather unlikely that biglycan only has negative effects on fibril formation

and thus should be completely absent when fibril formation is observed (54). It is more likely

that in our study biglycan was not detected due to masking of the epitope or the use of protocol

with which it is not possible to detect biglycan.

Regarding the proteoglycans decorin, versican, fibromodulin and biglycan, it is difficult

to say whether they are required to be expressed and therefore detectable by IHC during sciatic

nerve regeneration and where they should be expressed in the ECM. It is clear that these

proteoglycans play important roles in the ECM of various tissues and except for versican, they

also influence collagen fibril formation in one way or the other (13) (53) (54). At least for

decorin this study shows an expression pattern in the ECM which is linked to the expression of

collagen I and III in a spatiotemporal manner and suggests that decorin is required for nerve

repair in association with collagen I and III. These perineurial and endoneurial fibril-forming

collagens which also form the ensheathment of axons are particularly important for PN

regeneration (14). The expression and distribution of these collagen types were more organized

in the perineurial and endoneurial space of conduit repaired nerves than in autograft repaired

nerves. This may further indicate that conduit repair has structural regeneration advantages over

autograft repair when this histological view is concerned.

In conclusion, the use of specific IHC protocol provided a solid base for the evaluation

though attention is needed to further improve these protocols to obtain better results in future

studies. The presence of clusters of newly-formed axons and the size of these axons showed

that conduit repair enhances the sciatic nerve regeneration compared to autograft repair within

the first 20 days after implantation. Also, myelination of the newly-formed axons occurred

earlier in conduit than in autograft repaired nerves which shows advanced regeneration after

conduit repair. This form of nerve repair also has structural advantages for collagen I and III

reorganization in the perineurial and endoneurial space and therefore axonal growth from

proximal to distal ends. Autograft repair demands more time to produce a collagen network for

axonal regeneration. Further, decorin expression and organization is enhanced by using conduit

repair and close to normal expression patterns were observed after 50 days of implantation.

This should improve collagen fibril formation and axonal regeneration more than autograft

repair. Other proteoglycans, i.e. versican, fibromodulin and biglycan were not expressed in

48

healthy or regenerating sciatic nerves and may not be required for nerve repair within the first

50 days after implantation of the autograft or conduit. Eventually, conduit nerve repair displays

advantages over autograft repair for the expression and reorganization of several important

components of the PN ECM.

Acknowledgment

The in vivo analyses and tissue collection was performed in the tissue engineering group

of the University of Granada, Spain. This study was supported by the Spanish Plan Nacional de

Investigación Científica, Desarrollo e Innovación Tecnológica (I+D+i) from the National

Ministry of Economy and Competitiveness (Instituto de Salud Carlos III), grant FIS PI14-1343

and grant IPT-2011-0742-900000 (INNPACTO program). Both grants were co-financed by

Fondo Europeo de Desarrollo Regional (FEDER), European Union.

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6. Addendum

6.1 Immunohistochemistry – Collagen I

Primary antibodies: Rabbit Anti-Collagen I (Acris R1038)

Secondary antibody: Anti-Rabbit IgG (Polymer detection kit, Vector Laboratories) (MP-

7401)

Fixation: Methacarn for 3,5 hours.

Procedure Solution Time

1. Deparaffinate and keep in H2Od 2x Xilol, 3x 99°OH, 3x 95°OH, 2x

70°OH, H2Od 10 min. C/Xilol, 5 min. C/OH

2. Rinse twice in PBS+Tween20

and once in PBS

0,1M PBS (pH7,2-7,4) + 3ml

Tween20 per liter PBS 5 min. per solution

3. Endogenous peroxidase block 3% H2O2 in PBS 10 min.


and once in PBS

0,1M PBS (pH7,2-7,4) + 3ml


5. Block non-specific antibody

binding sites

Casein block 1:9 in H2Od,

Serum 15 min. per solution

6. Incubate primary antibody Rabbit Anti-Collagen I 1:500 in

PBS+Tween20 2 hours at R/T


and once in PBS

0,1M PBS (pH7,2-7,4) + 3ml


8. Incubate secondary antibody Anti-rabbit IgG for both

Ready-to-use solutions 30 min. at R/T


and once in PBS

0,1M PBS (pH7,2-7,4) + 3ml


10. Histochemistry: DAB DAB reagent solution

Between 30 sec. to 3 min. (watch

reaction under microscope until the

reaction is intense like controls),

stop reaction with H2Od

11. Counterstain: Hematoxylin Mayer’s Hematoxylin 10 sec., put under tap-water for 5

min. afterwards

12. Dehydrate 2x Alcohol 96%, 2x isopropanol,

3x toluene 2 min. per solution

13. Mount Standard mount medium -

6.2 Immunohistochemistry – Collagen III

Primary antibodies: Rabbit Anti-Collagen III (Abcam ab7778)


7401)

Fixation: Methacarn for 3,5 hours.





and once in PBS

0,1M PBS (pH7,2-7,4) + 3ml




and once in PBS

0,1M PBS (pH7,2-7,4) + 3ml



binding sites


Serum 15 min. per solution

6. Incubate primary antibody Rabbit Anti-Collagen III 1:400 in

PBS+Tween20

Rabbit Anti-Collagen III for 1 hour

at R/T


and once in PBS

0,1M PBS (pH7,2-7,4) + 3ml


8. Incubate secondary antibody Anti-rabbit IgG

Ready-to-use solutions 30 min. at R/T


and once in PBS

0,1M PBS (pH7,2-7,4) + 3ml



Between 30 sec. to 3 min. (watch

reaction under microscope), stop

reaction with H2Od when is intense

positive


min. afterwards




6.3 Immunohistochemistry – Decorin

Primary antibodies: Goat Anti-Decorin, R&D system (AF143)

Secondary antibody: Biotinylated Rabbit Anti-Goat (); Streptavidin HRP.

Pretreatment – Chondroitinase ABC 0,2 U/ml (Sigma cat nr. C3667): 40 ml H2Od, albumin

serum bovine 0,01g, sodium acetate 0,81g, Tris base 0,3g.

Fixation: Formalin 24 hours, methacarn 3,5 hours.




2. Rinse in 37°C PBS 0,1M PBS (pH7,2-7,4) 20 min. at 37°C

3. Pretreatment with

chondroitinase ABC Chondroitinase ABC 0,2U/ml 1 hour at 37°C

4. Rinse twice in cold

PBS+Tween20 and once in PBS

0,1M PBS (pH7,2-7,4) + 3ml




and once in PBS

0,1M PBS (pH7,2-7,4) + 3ml



binding sites CAS-block solution 15 min.

8. Incubate primary antibody Goat Anti-Decorin 1:500 in

PBS+Tween20 1 hour at R/T

9. Rinse 3 times in PBS+Tween20

and once in PBS

0,1M PBS (pH7,2-7,4) + 3ml


10. Incubate secondary antibody Biotinylated rabbit anti-goat 1:200

in PBS+Tween20 30 min. at R/T


and once in PBS

0,1M PBS (pH7,2-7,4) + 3ml


12. Incubate streptavidin-

horseradish peroxidase

Streptavidin HRP 1:200 in

PBS+Tween20 30 min. at R/T


and once in PBS

0,1M PBS (pH7,2-7,4) + 3ml



Between 2 to 4 min. (watch


reaction with H2Od


min. afterwards




6.4 Immunohistochemistry – Versican

Primary antibodies: Rabbit Anti-Versican, Abcam (ab19345)


7401)



Fixation: Formalin 24 hours.









0,1M PBS (pH7,2-7,4) + 3ml




and once in PBS

0,1M PBS (pH7,2-7,4) + 3ml



binding sites CAS-block solution 15 min.

8. Incubate primary antibody Rabbit Anti-Versican 1:100 in

PBS+Tween20 1 hour at R/T


and once in PBS

0,1M PBS (pH7,2-7,4) + 3ml


10. Incubate secondary antibody Anti-rabbit IgG ready-to-use kit

(Vector) 30 min. at R/T


and once in PBS

0,1M PBS (pH7,2-7,4) + 3ml





reaction with H2Od


min. afterwards




6.5 Immunohistochemistry – Fibromodulin

Primary antibodies: Rabbit Anti-Fibromodulin, Larry Fisher (LF) 150


7401)



Fixation: Methacarn 3,5 hours.









0,1M PBS (pH7,2-7,4) + 3ml


5. Endogenous peroxidase block H2O2 1:9 in PBS 10 min.


and once in PBS

0,1M PBS (pH7,2-7,4) + 3ml



binding sites


Serum 15 min. for both blocks

8. Incubate primary antibody

Rabbit Anti-Fibromodulin 1:400

(methacarn-fixed) in

PBS+Tween20

1,5 hour at R/T


and once in PBS

0,1M PBS (pH7,2-7,4) + 3ml


10. Incubate secondary antibody Anti-rabbit IgG ready-to-use kit

(Vector) 30 min. at R/T


and once in PBS

0,1M PBS (pH7,2-7,4) + 3ml





reaction with H2Od


min. afterwards




6.6 Immunohistochemistry – Biglycan

Primary antibodies: Rabbit Anti-Biglycan abcam ab49701


7401)

Pretreatment –Citric acid buffer: 0,1g citric acid, 450 ml H2Od, adjust pH to 6,1-6,4 and fill

up to 500 ml with H2Od) for 25 min. at 95°C (steamer). Chondroitinase ABC 0,2 U/ml (Sigma

cat nr. C3667): 40 ml H2Od, albumin serum bovine 0,01g, sodium acetate 0,81g, Tris base 0,3g.

Fixation: Formalin 24 hours.




2. Pretreatment with citrate buffer

pH 6 Citrate buffer pH6 (preheated)

25 min. at 95°C (steamer), 20 min.

cool down at R/T, 5 min. in H2Od.

3. Rinse three times in PBS, last

rinse at 37°C

0,1M PBS (pH7,2-7,4) (last rinse

preheated) 5 min. per solution





0,1M PBS (pH7,2-7,4) + 3ml


6. Endogenous peroxidase block 3% H2O2 1:9 in PBS 10 min.


and once in PBS

0,1M PBS (pH7,2-7,4) + 3ml



binding sites Casein block 1:9 in H2Od 15 min.

9. Incubate primary antibody Rabbit Anti-Biglycan abcam 1:100

in PBS+Tween20

1 hour at R/T


and once in PBS

0,1M PBS (pH7,2-7,4) + 3ml


11. Incubate secondary antibody Anti-rabbit IgG Ready-to-use

solution 30 min. at R/T


and once in PBS

0,1M PBS (pH7,2-7,4) + 3ml


13. Histochemistry: DAB DAB reagent buffer solution

Between 2 – 4 min. (watch reaction

under microscope), stop reaction

with H2Od


min. afterwards




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