Evaluation of myelin sheath and collagen
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Evaluation of myelin sheath and collagen
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Article in Histochemie · December 2011
DOI: 10.1007/s00418-011-0874-3 · Source: PubMed
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Evaluation of myelin sheath and collagen
reorganization pattern in a model of
peripheral nerve regeneration using an
integrated histochemical approach
Víctor Carriel, Ingrid Garzón, Miguel
Alaminos & Antonio Campos
Histochemistry and Cell Biology
ISSN 0948-6143
Volume 136
Number 6
Histochem Cell Biol (2011) 136:709-717
DOI 10.1007/s00418-011-0874-3
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Histochem Cell Biol (2011) 136:709–717
DOI 10.1007/s00418-011-0874-3
SHORT COMMUNICATION
Evaluation of myelin sheath and collagen reorganization pattern
in a model of peripheral nerve regeneration using an integrated
histochemical approach
Vı́ctor Carriel • Ingrid Garzón • Miguel Alaminos
Antonio Campos
•
Accepted: 9 October 2011 / Published online: 29 October 2011
Ó Springer-Verlag 2011
Abstract Peripheral nerves are complex histological
structures that can be affected by a variety of conditions
with different degree of axonal degeneration and demyelination. For the study of peripheral nerve regeneration in
pathology and tissue engineering, it is necessary to evaluate
the regeneration, remyelination and extracellular matrix
reorganization of the neural tissue. Currently, different
histochemical techniques must be used in parallel, and a
correlation among their findings should be further performed. In this work, we describe a new histochemical
method for myelin and collagen fibers based on luxol fast
blue and picrosirius methods, for the evaluation of the
morphology, the myelin sheath and the collagen fiber
reorganization using a model of peripheral nerve regeneration. Whole brain, normal sciatic nerve and regenerating
peripheral nerve samples were fixed in 10% neutral buffered formalin and paraffin-embedded, for the performance
of the hematoxylin-eosin stain, the Luxol fast blue method
and the new histochemical method for myelin and collagen. The results of this technique revealed that this new
histochemical method allowed us to properly evaluate
histological patterns, and simultaneously observe the histochemical reaction for myelin sheath and collagen fibers
in normal tissue, and during the regeneration process. In
conclusion, this new method combines morphological and
histochemical properties that allowed us to determine with
high accuracy the degree of remyelination and collagen
fibers reorganization. For all these reasons, we hypothesize
V. Carriel (&) I. Garzón M. Alaminos A. Campos
Department of Histology (Tissue Engineering Group),
University of Granada, Avenida de Madrid 11,
18012 Granada, Spain
e-mail: carriel.victor@gmail.com
that this new histochemical method could be useful in
pathology and tissue engineering.
Keywords Myelin sheath Collagen fibers
Peripheral nerve regeneration Histochemistry
Tissue engineering
Introduction
Peripheral nerves are complex histological structures whose
main components are neuron axons, myelin sheaths synthesized by Schwann cells and a collagen-rich extracellular
matrix (ECM) (Mills 2007). These structures can be affected
by a variety of conditions and neuropathies with different
degrees of axonal degeneration and demyelination (Oh
2001). The study of the regeneration processes that occur
during peripheral nerve reparation is one of the goals of
current biomedical research (Chalfoun et al. 2006; Jiao et al.
2009); and assessment of peripheral nerve morphology is a
pillar in the investigation of nerve damage and regeneration
in tissue engineering (Vleggeert-Lankamp 2007). The use of
light microscopy plays an essential role in the evaluation of
morphological features of the pathologic and regenerative
processes that occur in the peripheral nerve structure. The
use of morphological, histochemical and immunohistochemical techniques provides information about pathophysiological conditions in peripheral nerves.
Light microscopy allows observing the complex histological structure of the peripheral nerve with several limitations. In many cases, the histological image that can be
observed using routine nerve staining methods is poor. For
most researchers, the gold standard in peripheral nerve
histology is toluidine blue staining of resin-embedded semi
thin sections, which allows the accurate identification of
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Histochem Cell Biol (2011) 136:709–717
most myelinated fibers (Mills 2007; Hirano 2005). However, this methodology is time-consuming, expensive and
requires special equipment such as ultramicrotomes.
Regarding the histochemical techniques, there are several
reagents that specifically stain myelin, and these techniques
have been an important part of the histopathologic examination of the nervous system (Bancroft and Gamble 2008;
Kiernan 2008; Mills 2007). The Luxol fast blue (LFB)
method described by Klüver and Barrera in 1953 is most
often used in histopathology for the evaluation of myelin in
central and peripheral nervous system. Myelin can also be
detected by immunohistochemistry and immunofluorescence using antibodies that specifically recognize the myelin
basic protein (Mills 2007; Taylor and Cote 2005).
For the study of peripheral nerve injuries and the
regeneration process that is associated to these injuries, it is
necessary to evaluate the presence of axonal sprouting,
remyelination and ECM remodeling in these tissues (Mills
2007; Oh 2001; Jiao et al. 2009). Several techniques are
available, but most of these must be performed and interpreted in parallel, and a correlation among their findings
should be further carried out. For the evaluation of these
parameters, the development of techniques that allow a
comprehensive assessment of the main histochemical
properties using a single histological slide would be useful.
In this work, we describe a new (MCOLL) histochemical method based on conventional luxol fast blue and
picrosirius histochemical methods, for the simultaneous
staining of the morphology pattern, myelin sheath and
stromal collagen fibers in different nerve tissues.
1000Ò 0.15 mg per gram of weight of the animal). Then,
10 mm of the left sciatic nerve were surgically removed
from each animal, and a commercially available type
I-collagen conduit (CC) (NeuraGenÒ) was microsurgically
implanted between both nerve ends to induce nerve
regeneration as clinically used in human patients with a
peripheral nerve lesion (Wangensteen and Kalliainen
2010). The right sciatic nerve was used as control in each
animal. After 12 weeks of the implant of the CC, the animals were euthanatized under general anesthesia and both
sciatic nerves were harvested (the normal right sciatic
nerve and the regenerating left sciatic nerve).
Materials and methods
3.
4.
Histological analysis
Normal and regenerating peripheral nerve samples were
fixed in 10% formalin in 0.1 M PBS for 8–12 h and
embedded in paraffin. Whole brains were sagittally sectioned and fixed in 10% formalin in 0.1 M PBS for 48 h
and embedded in paraffin. Samples were cut in 5 lm thick
sections for staining with conventional hematoxylin-eosin
(HE) staining, conventional LFB (Klüver and Barrera
1953; Kiernan 2008) and the new MCOLL histochemical
method described in this work.
Conventional LFB staining procedure was carried out as
follows:
1.
2.
Animal tissues
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 housed in a temperature controlled environment
(21 ± 1°C), maintained on a 12 h light/dark cycle, and
given free access to tap water and standard rat chow.
To evaluate this new histochemical method in normal
native neural tissues, we used normal brains and sciatic
nerves from 12 female Wistar rats of 12 weeks old
weighing 250–300 g.
For the evaluation of different degrees of axonal
degeneration, demyelination and remyelination process, we
used a peripheral nerve regeneration model. In this case,
animals were anesthetized by an intraperitoneal injection of
a mixture of acepromazine (Calmo-NeosanÒ 0.001 mg per
gram of weight of the animal) and ketamine (Imalgene
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5.
6.
7.
8.
9.
De-wax paraffin sections in xylene and hydrate in 100
and 95% ethanol.
Stain sections in 0.1% LFB (Color Index C.I. 74180,
BDH Chemicals) diluted in 95% ethanol with 2.5 ml
of 10% acetic acid at 56°C 16–24 h (overnight).
Rinse in 95% ethanol, and then in distilled water.
Differentiate in 0.05% lithium carbonate until gray and
white matter can be distinguished.
Rinse in 70% ethanol, two changes.
Counterstain in 0.1% aqueous solution of crystal violet
with 5 mg oxalic acid for 10 min at 56°C.
Differentiate in 70% ethanol until there is no background color.
Dehydrate in 99% ethanol (three changes).
Clear using two changes of xylene and mount using a
hydrophobic medium.
The MCOLL procedure was performed as follows:
1.
2.
3.
4.
5.
Perform the steps 1–5 as described above.
Rinse in distilled water for 5 min.
Stain sections in 0.2% sirius red F3B (C.I. 35780,
Sigma-Aldrich) in a saturated solution of picric acid
for 30 min at room temperature.
Rinse in distilled water, two changes.
Counterstain in Harris hematoxylin (Panreac, Barcelona, Spain) for 3 min.
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Histochem Cell Biol (2011) 136:709–717
6.
7.
8.
Rinse in tap water for 3–5 min.
Dehydrate in increasing concentrations of ethanol.
Clear using two changes of xylene and mount using a
hydrophobic medium.
Stained tissue sections were examined under a Nikon
Eclipse 90i light microscope, and images were captured
with a Nikon Digital Camera DXM 1200c and NIS Elements software (Nikon, Tokyo, Japan) for light microscopy. To analyze the three-dimensional collagen fiber
organization, a polarized light microscopy study was performed using an Olympus BX 51 microscope, and images
were captured with an Olympus digital camera DP70 and
DP manager software (Olympus Optical, Tokyo, Japan).
Results
Analysis of the normal tissues
In the brain sections stained with HE, we observed the
tissue pattern, which allowed us to recognize gray matter
(GM) and white matter (WM), but the limits between both
areas were imprecise and difficult to identify (Fig. 1a, e, h,
k). With the LFB method, it was possible to observe the
myelinated structures of the WM (in blue) and the metachromatic reaction of the Nissl bodies in the GM (data not
shown), but the contrast was weak, and it was difficult to
recognize all the histological structures of the central nervous system (CNS) (Fig. 1b, f, i, l).
With the new MCOLL histochemical method, an intense
histochemical reaction for myelin was observed in blue,
while the WM was clearly distinguishable from the GM
with high contrast in brain and cerebellum (Fig. 1c, g).
With this new method it was possible to accurately identify
the cortex layers based on morphological and histochemical parameters (Fig. 1d, g). The histochemical evaluation
of the cerebellum nuclei and the multiform layer of the
cerebral cortex allowed us to simultaneously observe the
soma of the neurons and individual myelinated fibers (in
blue) with high contrast. (Fig. 1j, m). Regarding the CNS
stroma, with this new histochemical method it was possible
to identify the collagen fibers network on the blood vessels
wall, the meninges (Fig. 1d, g, j) and the choroid plexus
(data not shown).
The analysis of the normal sciatic nerve with HE stain
allowed us to observe the tissue pattern with the typical
undulated and parallel organization of the nerve fibers. The
myelin sheath stain was weak and unspecific, with poor
contrast between the different histological structures
(Fig. 2a). With the LFB method it was only possible to
identify the myelin sheath of the myelinated nerve fibers
and the metachromatic granules of the mast cells (Fig. 2b).
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The analysis of the normal sciatic nerve with the new
MCOLL histochemical method allowed us to observe tissue pattern as HE stain did (Fig. 2c, d). The specific
identification of the myelin sheath (in blue) and collagen
fibers (in red) allowed us to simultaneously observe and
correlate the distribution pattern and organization of both
components with high contrast and specificity (Fig. 2c, d).
With MCOLL histochemical method it was possible to
accurately identify the Nodes of Ranvier and the organization of the collagen fibers in the connective tissue surrounding the nerve fibers and blood vessels (Fig. 2c, d).
Analysis of the nerve regeneration model
The morphologic analysis with HE stain of the CC after
12 weeks of in vivo implantation allowed us to observe the
axonal regeneration along the graft. This axonal regeneration was abundant only in the proximal anastomosis. We
observed the formation of a thick regeneration cone,
accompanied by an increased axonal sprouting (Fig. 3b–e).
It was observed that the thickness of the cone regeneration
progressively declined and we did not observe any nerve
regeneration at the distal nerve (Fig. 3f, g). In relation to
the ECM, we could identify some acidophilic components,
but it was not possible to determine and observe any
changes in the remodeling of the ECM during the process
of regeneration.
The analysis with LFB method allowed us to confirm the
presence of a myelin sheath at the proximal anastomosis
(Fig. 3h). Remyelination process was observed at the
regeneration cone and axonal sprouting (Fig. 3i–k). This
histochemical reaction progressively decreased, and was
negative at the distal anastomosis (Fig. 3l, m). However, it
was not possible to properly observe the morphologic
characteristics of the regenerating nerve, because the contrast was low and the resolution quality was poor.
The analysis with the new MCOLL histochemical
method allowed us to observe the axonal regeneration
process in the grafted CC (Fig. 3a), and correlate all the
morphologic parameters with the histochemical reaction
for myelin sheath and collagen fibers simultaneously, with
high contrast, specificity and sensibility (Fig. 3n–s).
In the proximal sciatic nerve, we observed the typical
undulated tissue pattern of the peripheral nerve with signs of
Wallerian degeneration and a significant degree of demyelination. The histochemical reaction for collagen fibers was
intense and the fibers tended to remain properly oriented
(Fig. 3n). The structural disorganization of the proximal
anastomosis, with the formation of a thick regeneration
cone, was accompanied by remyelination of the axonal
sprouting and reorganization of the collagen fibers. These
collagen fibers were oriented around the axonal sprouting
forming fascicles (Fig. 3o–q). We observed a marked
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Histochem Cell Biol (2011) 136:709–717
M
I
GM
GM
GM
II
WM
WM
WM
A
B
C
III
GM
WM
IV
E
V
F
G
VI
H
I
J
WM
K
L
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M
D
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Histochem Cell Biol (2011) 136:709–717
b Fig. 1 Morphologic and histochemical study of the normal CNS.
Sagittal section of the cerebral cortex at low magnification stained
with HE (a), the LFB histochemical method for myelin in blue (b),
and the new MCOLL method, where it is possible to distinguish the
gray matter (GM) from the white matter (WM) stained in blue with
high contrast (c). Scale bar = 500 lm. Overview of the cerebral
cortex with the MCOLL method, where it is possible to observe the
WM, and the different layers of the cerebral cortex (M meninges,
I molecular layer, II external granular layer, III external pyramidal
layer, IV internal granular layer, V internal pyramidal layer, VI
polymorphic or multiform layer). Bar = 100 lm (d). Section of
cerebellum stained with HE (e), the LFB stain (f), and the new
MCOLL method with a positive histochemical reaction for myelin in
WM and collagen fibers (arrows) at the meninges (g). Scale
bar = 100 lm. Section of the cerebellum nucleus stained with HE
(e), the LFB method (f), and the new MCOLL method where the
neuronal soma is clearly distinguished (arrowheads) between myelinated fibers stained in blue (j). Scale bar = 50 lm. Polymorphic
layer of the cerebral cortex stained with HE (k), LFB (l) and the
MCOLL method with a positive histochemical reaction for individual
myelinated nerve fibers in blue (arrows) between the bodies of the
neurons (arrowhead) with high contrast (m). Scale bar = 50 lm
process of fibrosis with disorganized collagen fibers
and demyelination process at the distal anastomosis
(Fig. 3r, s).
With the polarized light microscopy (Fig. 4a–h), we
observed the specific increase of the birefringence of the
collagen fibers and their reorganization during the peripheral nerve regeneration process. Birefringence was weak
and fibers were well organized at the proximal sciatic nerve
(Fig. 4b), but became strong and disorganized at the central
portion, with high levels of fibrosis at the distal anastomosis (Fig. 4f, h).
Discussion
Histopathologic analysis and tissue engineering of peripheral nerves should be accompanied by the application of
histological quality controls. The observation of normal
CNS, PNS and peripheral nerve regeneration using CC
with HE allowed us to evaluate the main morphologic
features and regeneration process. However, with this
method it was not possible to evaluate the integrity of the
myelin sheath and the changes that occur in the fibers of the
ECM. The identification of myelin sheaths and collagen
fibers acquires an important role in the evaluation of the
peripheral nerve in pathology and tissue engineering (Mills
2007; Jiao et al. 2009; Oh 2001). Currently, it is necessary
to carry out different specific histochemical methods for
the identification of these elements and these results should
be interpreted in parallel (Mills 2007). Recently, Rutschow
et al. (2010) described the use of both LFB and picrosirius
histological methods for the evaluation of myocytolysis
and fibrosis in myocardial tissue. However, the method
713
reported by these authors required the use of two independent staining processes using two different samples,
since both staining procedures were carried out separately.
One of the main advantages of the novel MCOLL method
is the use of one single staining process.
In paraffin-embedded tissues the LFB method is most
often used in histopathology for the study of the myelin in
CNS and PNS (Mills 2007; Klüver and Barrera 1953). In
substitution of the previously described method, several
techniques have been developed for the identification of
myelin using light and electron microscopy (Savaskan et al.
2009; Di Scipio et al. 2008; Larsen et al. 2003; Xiang et al.
2005; Schmued and Slikker 1999; McNally and Peters
1998; Tolivia et al. 1994; Schmued 1990; Schmued et al.
1982; Stilwell 1957). However, all these methodologies
only allow the specific identification of myelin, and it is not
possible to make a comprehensive assessment of the
morphologic parameters and the status of the ECM. The
osmium-tetroxide has been described for myelin sheath
impregnation in paraffin-embedded samples with different
staining contrast with promising results (Di Scipio et al.
2008). But this method provides a permanent coloration of
the myelin sheath. Therefore, it may limit the application
of other histochemical and immunohistochemical techniques in these samples.
The HE staining is commonly non-specific for many
tissue elements, due to its electropolar nature. Using this
staining, we were not able to accurately identify the myelin
sheath. In relation to the ECM, HE staining allowed us to
recognize some acidophilic elements such as the collagen
fibers. However, the staining pattern was non-specific
because all these elements became stained with similar
tones and intensities in comparison with other histological
structures.
The new MCOLL histochemical method based on conventional luxol fast blue and picrosirius histochemical
methods allowed us to evaluate the morphological features
of the nerve tissue as HE did. However, the MCOLL
method had the advantage of simultaneously identifying
the myelin sheath with the same staining pattern, specificity and accuracy of the conventional LFB method, and
the collagen fibers with the same sensibility and specificity
of picrosirius techniques, using a single analysis.
On the other hand, the analysis of the CNS, with this
MCOLL histochemical method allowed us to observe the
histological structure and analyze a specific histochemical
reaction for myelin in the white matter. The evaluation of
the CC grafted for nerve regeneration allowed us to evaluate with high contrast all major morphologic parameters,
the specific histochemical identification of the myelin
sheath in different stages of remyelination or demyelination, and the collagen fibers reorganization during the
regeneration process. In the new-formed nerve fascicles we
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714
Fig. 2 Histochemical analyses
of the normal sciatic nerve.
Longitudinal sections of the
sciatic nerve stained with HE,
where it is possible to observe
the typical undulated tissue
pattern (a). Positive
histochemical reaction for
myelin sheath and
metachromatic reaction for mast
cells (arrow) stained with the
LFB stain (b). The same
sections stained with the new
MCOLL method, where it is
possible to observe the typical
tissue pattern, the intense
histochemical reaction for
myelin sheath in blue, and the
histochemical identification of
the collagen fibers (arrows) in
red (c). The axons and the
Nodes of Ranvier are clearly
visualized (arrowheads) along
the myelinated nerve fibers;
observe the thick collagen fibers
of the epineurium stained in red
(d). Scale bar = 50 lm
Histochem Cell Biol (2011) 136:709–717
A
B
C
D
could clearly identify and evaluate the presence of axonal
sprouting and nerve regeneration. All this allowed us to
establish a more precise diagnosis of the structures that
were present in during the regeneration process in a single
sample of tissue.
The identification of collagen fibers using the MCOLL
method was specific, because it uses a strong anionic
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tetrakisazo dye called sirius red. This dye interacts with
cationic groups on the surface of the collagen molecules in
parallel, giving an intense red color to the collagen fibers in
light microscopy. In addition, this dye specifically increases the natural birefringence of collagen fibers, and allows
us to selectively identify them by polarizing microscopy.
This phenomenon is particularly induced by Picrosirius
Author's personal copy
S
M
L
R
Q
E
J
I
H
P
D
C
B
O
RC
N
A
PAN
CC
*
*
K
F
G
DAN
CENTRAL
PROXIMAL
Fig. 3 Morphologic analysis of
the collagen conduit (CC) graft.
Histochemical composition of
the entire collagen conduit graft
(a) stained with the new
MCOLL method. The
morphological study with HE
shows the nerve regeneration
process with axonal sprouting
along the graft (b–g). The
histochemical evaluation of the
CC graft with the LFB method,
shows a progressive decrease of
the myelin histochemical
reaction along the graft (h–m).
The histochemical evaluation
with the MCOLL method,
shows the regeneration process
accompanied by axonal
sprouting, a progressive
decrease of the myelin
histochemical reaction, and the
progressive increase of the
histochemical reaction for
collagen fibers stained in red
(n–s). CC collagen conduit wall,
PAN proximal nerve
anastomosis, RC regeneration
cone, DAN distal anastomosis
and distal nerve. Arrowheads
label illustrative examples of
axonal sprouting and newformed nerve fascicles. Scale
bar in panel a = 1000 lm.
Scale bar in panels
b–s = 100 lm
715
DISTAL
Histochem Cell Biol (2011) 136:709–717
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Histochem Cell Biol (2011) 136:709–717
A
B
C
D
E
F
G
H
I
Fig. 4 Light (a–f) and polarized light (g–i) microscopy images of the
CC graft stained with the new MCOLL histochemical method.
Proximal sciatic nerve (a, d, g). Proximal nerve anastomosis (b, e, h).
Distal nerve anastomosis (c, f, i). Arrows illustrative examples of
axons showing Wallerian degeneration. Arrowheads myelin sheaths.
Scale bar = 50 lm
staining (Junqueira et al. 1979; Montes and Junqueira
1991; Trau et al. 1991; Carriel et al. 2011). Interestingly,
the histochemical reaction of the myelin with LFB in this
new MCOLL method was not affected by subsequent
procedures, maintaining the sensibility and specificity, with
a considerable improvement in the contrast without background color. In addition, the histochemical reaction for
collagen fibers was not affected by treatment of the section
with high temperature, which has been previously described (Carriel et al. 2011), and provides a high contrast that
allowed us to identify the different components separately
and specifically.
Currently, there are several methods for the evaluation
of the myelin; however, a histochemical method with these
histochemical and morphologic characteristics does not
exist. Furthermore, this new MCOLL histochemical
method is carried out in about 18 h, being a specific,
simple, and inexpensive procedure for paraffin-embedded
tissue. In addition, the picrosirius and LFB solutions are
very stable at room temperature for at least 5 years, and
can be used repeatedly (Kiernan 2008).
Anyway, this is not a method that aims to replace the
conventional procedures used routinely; this new method
could be useful not only to help in a better diagnosis of the
different degrees of axonal degeneration and demyelination
in CNS and PNS injuries, but also to evaluate the
simultaneous correlation between the morphologic parameters, the myelin integrity and the collagen fibers reorganization during peripheral nerve regeneration carried out
with different protocols of tissue engineering.
In conclusion, the use of this new MCOLL histochemical method allowed us to simultaneously evaluate and
correlate the histological pattern, the specific histochemical
reaction for myelin sheath, and the histochemical reaction
for the stromal collagen fibers in a single slide with high
contrast. This method combines the properties of sensibility and specificity of the used reagents, providing a high
intensity and specificity of the histochemical reaction in the
identification of the histological structures described in this
work. For all these reasons, we hypothesize that this new
histochemical method could be useful in pathology and
tissue engineering.
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Acknowledgments The authors are grateful to Ms. Ariane Ruyffelaert from Ghent University, Belgium for revising and correcting the
English manuscript. This work was supported by grant SAS PI-135/
2007 from Junta de Andalucı́a, Spain.
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Evaluation of myelin sheath and collagen
reorganization pattern in a model of
peripheral nerve regeneration using an
integrated histochemical approach
Víctor Carriel, Ingrid Garzón, Miguel
Alaminos & Antonio Campos
Histochemistry and Cell Biology
ISSN 0948-6143
Volume 136
Number 6
Histochem Cell Biol (2011) 136:709-717
DOI 10.1007/s00418-011-0874-3
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Histochem Cell Biol (2011) 136:709–717
DOI 10.1007/s00418-011-0874-3
SHORT COMMUNICATION
Evaluation of myelin sheath and collagen reorganization pattern
in a model of peripheral nerve regeneration using an integrated
histochemical approach
Vı́ctor Carriel • Ingrid Garzón • Miguel Alaminos
Antonio Campos
•
Accepted: 9 October 2011 / Published online: 29 October 2011
Ó Springer-Verlag 2011
Abstract Peripheral nerves are complex histological
structures that can be affected by a variety of conditions
with different degree of axonal degeneration and demyelination. For the study of peripheral nerve regeneration in
pathology and tissue engineering, it is necessary to evaluate
the regeneration, remyelination and extracellular matrix
reorganization of the neural tissue. Currently, different
histochemical techniques must be used in parallel, and a
correlation among their findings should be further performed. In this work, we describe a new histochemical
method for myelin and collagen fibers based on luxol fast
blue and picrosirius methods, for the evaluation of the
morphology, the myelin sheath and the collagen fiber
reorganization using a model of peripheral nerve regeneration. Whole brain, normal sciatic nerve and regenerating
peripheral nerve samples were fixed in 10% neutral buffered formalin and paraffin-embedded, for the performance
of the hematoxylin-eosin stain, the Luxol fast blue method
and the new histochemical method for myelin and collagen. The results of this technique revealed that this new
histochemical method allowed us to properly evaluate
histological patterns, and simultaneously observe the histochemical reaction for myelin sheath and collagen fibers
in normal tissue, and during the regeneration process. In
conclusion, this new method combines morphological and
histochemical properties that allowed us to determine with
high accuracy the degree of remyelination and collagen
fibers reorganization. For all these reasons, we hypothesize
V. Carriel (&) I. Garzón M. Alaminos A. Campos
Department of Histology (Tissue Engineering Group),
University of Granada, Avenida de Madrid 11,
18012 Granada, Spain
e-mail: carriel.victor@gmail.com
that this new histochemical method could be useful in
pathology and tissue engineering.
Keywords Myelin sheath Collagen fibers
Peripheral nerve regeneration Histochemistry
Tissue engineering
Introduction
Peripheral nerves are complex histological structures whose
main components are neuron axons, myelin sheaths synthesized by Schwann cells and a collagen-rich extracellular
matrix (ECM) (Mills 2007). These structures can be affected
by a variety of conditions and neuropathies with different
degrees of axonal degeneration and demyelination (Oh
2001). The study of the regeneration processes that occur
during peripheral nerve reparation is one of the goals of
current biomedical research (Chalfoun et al. 2006; Jiao et al.
2009); and assessment of peripheral nerve morphology is a
pillar in the investigation of nerve damage and regeneration
in tissue engineering (Vleggeert-Lankamp 2007). The use of
light microscopy plays an essential role in the evaluation of
morphological features of the pathologic and regenerative
processes that occur in the peripheral nerve structure. The
use of morphological, histochemical and immunohistochemical techniques provides information about pathophysiological conditions in peripheral nerves.
Light microscopy allows observing the complex histological structure of the peripheral nerve with several limitations. In many cases, the histological image that can be
observed using routine nerve staining methods is poor. For
most researchers, the gold standard in peripheral nerve
histology is toluidine blue staining of resin-embedded semi
thin sections, which allows the accurate identification of
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Histochem Cell Biol (2011) 136:709–717
most myelinated fibers (Mills 2007; Hirano 2005). However, this methodology is time-consuming, expensive and
requires special equipment such as ultramicrotomes.
Regarding the histochemical techniques, there are several
reagents that specifically stain myelin, and these techniques
have been an important part of the histopathologic examination of the nervous system (Bancroft and Gamble 2008;
Kiernan 2008; Mills 2007). The Luxol fast blue (LFB)
method described by Klüver and Barrera in 1953 is most
often used in histopathology for the evaluation of myelin in
central and peripheral nervous system. Myelin can also be
detected by immunohistochemistry and immunofluorescence using antibodies that specifically recognize the myelin
basic protein (Mills 2007; Taylor and Cote 2005).
For the study of peripheral nerve injuries and the
regeneration process that is associated to these injuries, it is
necessary to evaluate the presence of axonal sprouting,
remyelination and ECM remodeling in these tissues (Mills
2007; Oh 2001; Jiao et al. 2009). Several techniques are
available, but most of these must be performed and interpreted in parallel, and a correlation among their findings
should be further carried out. For the evaluation of these
parameters, the development of techniques that allow a
comprehensive assessment of the main histochemical
properties using a single histological slide would be useful.
In this work, we describe a new (MCOLL) histochemical method based on conventional luxol fast blue and
picrosirius histochemical methods, for the simultaneous
staining of the morphology pattern, myelin sheath and
stromal collagen fibers in different nerve tissues.
1000Ò 0.15 mg per gram of weight of the animal). Then,
10 mm of the left sciatic nerve were surgically removed
from each animal, and a commercially available type
I-collagen conduit (CC) (NeuraGenÒ) was microsurgically
implanted between both nerve ends to induce nerve
regeneration as clinically used in human patients with a
peripheral nerve lesion (Wangensteen and Kalliainen
2010). The right sciatic nerve was used as control in each
animal. After 12 weeks of the implant of the CC, the animals were euthanatized under general anesthesia and both
sciatic nerves were harvested (the normal right sciatic
nerve and the regenerating left sciatic nerve).
Materials and methods
3.
4.
Histological analysis
Normal and regenerating peripheral nerve samples were
fixed in 10% formalin in 0.1 M PBS for 8–12 h and
embedded in paraffin. Whole brains were sagittally sectioned and fixed in 10% formalin in 0.1 M PBS for 48 h
and embedded in paraffin. Samples were cut in 5 lm thick
sections for staining with conventional hematoxylin-eosin
(HE) staining, conventional LFB (Klüver and Barrera
1953; Kiernan 2008) and the new MCOLL histochemical
method described in this work.
Conventional LFB staining procedure was carried out as
follows:
1.
2.
Animal tissues
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 housed in a temperature controlled environment
(21 ± 1°C), maintained on a 12 h light/dark cycle, and
given free access to tap water and standard rat chow.
To evaluate this new histochemical method in normal
native neural tissues, we used normal brains and sciatic
nerves from 12 female Wistar rats of 12 weeks old
weighing 250–300 g.
For the evaluation of different degrees of axonal
degeneration, demyelination and remyelination process, we
used a peripheral nerve regeneration model. In this case,
animals were anesthetized by an intraperitoneal injection of
a mixture of acepromazine (Calmo-NeosanÒ 0.001 mg per
gram of weight of the animal) and ketamine (Imalgene
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5.
6.
7.
8.
9.
De-wax paraffin sections in xylene and hydrate in 100
and 95% ethanol.
Stain sections in 0.1% LFB (Color Index C.I. 74180,
BDH Chemicals) diluted in 95% ethanol with 2.5 ml
of 10% acetic acid at 56°C 16–24 h (overnight).
Rinse in 95% ethanol, and then in distilled water.
Differentiate in 0.05% lithium carbonate until gray and
white matter can be distinguished.
Rinse in 70% ethanol, two changes.
Counterstain in 0.1% aqueous solution of crystal violet
with 5 mg oxalic acid for 10 min at 56°C.
Differentiate in 70% ethanol until there is no background color.
Dehydrate in 99% ethanol (three changes).
Clear using two changes of xylene and mount using a
hydrophobic medium.
The MCOLL procedure was performed as follows:
1.
2.
3.
4.
5.
Perform the steps 1–5 as described above.
Rinse in distilled water for 5 min.
Stain sections in 0.2% sirius red F3B (C.I. 35780,
Sigma-Aldrich) in a saturated solution of picric acid
for 30 min at room temperature.
Rinse in distilled water, two changes.
Counterstain in Harris hematoxylin (Panreac, Barcelona, Spain) for 3 min.
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Histochem Cell Biol (2011) 136:709–717
6.
7.
8.
Rinse in tap water for 3–5 min.
Dehydrate in increasing concentrations of ethanol.
Clear using two changes of xylene and mount using a
hydrophobic medium.
Stained tissue sections were examined under a Nikon
Eclipse 90i light microscope, and images were captured
with a Nikon Digital Camera DXM 1200c and NIS Elements software (Nikon, Tokyo, Japan) for light microscopy. To analyze the three-dimensional collagen fiber
organization, a polarized light microscopy study was performed using an Olympus BX 51 microscope, and images
were captured with an Olympus digital camera DP70 and
DP manager software (Olympus Optical, Tokyo, Japan).
Results
Analysis of the normal tissues
In the brain sections stained with HE, we observed the
tissue pattern, which allowed us to recognize gray matter
(GM) and white matter (WM), but the limits between both
areas were imprecise and difficult to identify (Fig. 1a, e, h,
k). With the LFB method, it was possible to observe the
myelinated structures of the WM (in blue) and the metachromatic reaction of the Nissl bodies in the GM (data not
shown), but the contrast was weak, and it was difficult to
recognize all the histological structures of the central nervous system (CNS) (Fig. 1b, f, i, l).
With the new MCOLL histochemical method, an intense
histochemical reaction for myelin was observed in blue,
while the WM was clearly distinguishable from the GM
with high contrast in brain and cerebellum (Fig. 1c, g).
With this new method it was possible to accurately identify
the cortex layers based on morphological and histochemical parameters (Fig. 1d, g). The histochemical evaluation
of the cerebellum nuclei and the multiform layer of the
cerebral cortex allowed us to simultaneously observe the
soma of the neurons and individual myelinated fibers (in
blue) with high contrast. (Fig. 1j, m). Regarding the CNS
stroma, with this new histochemical method it was possible
to identify the collagen fibers network on the blood vessels
wall, the meninges (Fig. 1d, g, j) and the choroid plexus
(data not shown).
The analysis of the normal sciatic nerve with HE stain
allowed us to observe the tissue pattern with the typical
undulated and parallel organization of the nerve fibers. The
myelin sheath stain was weak and unspecific, with poor
contrast between the different histological structures
(Fig. 2a). With the LFB method it was only possible to
identify the myelin sheath of the myelinated nerve fibers
and the metachromatic granules of the mast cells (Fig. 2b).
711
The analysis of the normal sciatic nerve with the new
MCOLL histochemical method allowed us to observe tissue pattern as HE stain did (Fig. 2c, d). The specific
identification of the myelin sheath (in blue) and collagen
fibers (in red) allowed us to simultaneously observe and
correlate the distribution pattern and organization of both
components with high contrast and specificity (Fig. 2c, d).
With MCOLL histochemical method it was possible to
accurately identify the Nodes of Ranvier and the organization of the collagen fibers in the connective tissue surrounding the nerve fibers and blood vessels (Fig. 2c, d).
Analysis of the nerve regeneration model
The morphologic analysis with HE stain of the CC after
12 weeks of in vivo implantation allowed us to observe the
axonal regeneration along the graft. This axonal regeneration was abundant only in the proximal anastomosis. We
observed the formation of a thick regeneration cone,
accompanied by an increased axonal sprouting (Fig. 3b–e).
It was observed that the thickness of the cone regeneration
progressively declined and we did not observe any nerve
regeneration at the distal nerve (Fig. 3f, g). In relation to
the ECM, we could identify some acidophilic components,
but it was not possible to determine and observe any
changes in the remodeling of the ECM during the process
of regeneration.
The analysis with LFB method allowed us to confirm the
presence of a myelin sheath at the proximal anastomosis
(Fig. 3h). Remyelination process was observed at the
regeneration cone and axonal sprouting (Fig. 3i–k). This
histochemical reaction progressively decreased, and was
negative at the distal anastomosis (Fig. 3l, m). However, it
was not possible to properly observe the morphologic
characteristics of the regenerating nerve, because the contrast was low and the resolution quality was poor.
The analysis with the new MCOLL histochemical
method allowed us to observe the axonal regeneration
process in the grafted CC (Fig. 3a), and correlate all the
morphologic parameters with the histochemical reaction
for myelin sheath and collagen fibers simultaneously, with
high contrast, specificity and sensibility (Fig. 3n–s).
In the proximal sciatic nerve, we observed the typical
undulated tissue pattern of the peripheral nerve with signs of
Wallerian degeneration and a significant degree of demyelination. The histochemical reaction for collagen fibers was
intense and the fibers tended to remain properly oriented
(Fig. 3n). The structural disorganization of the proximal
anastomosis, with the formation of a thick regeneration
cone, was accompanied by remyelination of the axonal
sprouting and reorganization of the collagen fibers. These
collagen fibers were oriented around the axonal sprouting
forming fascicles (Fig. 3o–q). We observed a marked
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Histochem Cell Biol (2011) 136:709–717
M
I
GM
GM
GM
II
WM
WM
WM
A
B
C
III
GM
WM
IV
E
V
F
G
VI
H
I
J
WM
K
L
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M
D
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Histochem Cell Biol (2011) 136:709–717
b Fig. 1 Morphologic and histochemical study of the normal CNS.
Sagittal section of the cerebral cortex at low magnification stained
with HE (a), the LFB histochemical method for myelin in blue (b),
and the new MCOLL method, where it is possible to distinguish the
gray matter (GM) from the white matter (WM) stained in blue with
high contrast (c). Scale bar = 500 lm. Overview of the cerebral
cortex with the MCOLL method, where it is possible to observe the
WM, and the different layers of the cerebral cortex (M meninges,
I molecular layer, II external granular layer, III external pyramidal
layer, IV internal granular layer, V internal pyramidal layer, VI
polymorphic or multiform layer). Bar = 100 lm (d). Section of
cerebellum stained with HE (e), the LFB stain (f), and the new
MCOLL method with a positive histochemical reaction for myelin in
WM and collagen fibers (arrows) at the meninges (g). Scale
bar = 100 lm. Section of the cerebellum nucleus stained with HE
(e), the LFB method (f), and the new MCOLL method where the
neuronal soma is clearly distinguished (arrowheads) between myelinated fibers stained in blue (j). Scale bar = 50 lm. Polymorphic
layer of the cerebral cortex stained with HE (k), LFB (l) and the
MCOLL method with a positive histochemical reaction for individual
myelinated nerve fibers in blue (arrows) between the bodies of the
neurons (arrowhead) with high contrast (m). Scale bar = 50 lm
process of fibrosis with disorganized collagen fibers
and demyelination process at the distal anastomosis
(Fig. 3r, s).
With the polarized light microscopy (Fig. 4a–h), we
observed the specific increase of the birefringence of the
collagen fibers and their reorganization during the peripheral nerve regeneration process. Birefringence was weak
and fibers were well organized at the proximal sciatic nerve
(Fig. 4b), but became strong and disorganized at the central
portion, with high levels of fibrosis at the distal anastomosis (Fig. 4f, h).
Discussion
Histopathologic analysis and tissue engineering of peripheral nerves should be accompanied by the application of
histological quality controls. The observation of normal
CNS, PNS and peripheral nerve regeneration using CC
with HE allowed us to evaluate the main morphologic
features and regeneration process. However, with this
method it was not possible to evaluate the integrity of the
myelin sheath and the changes that occur in the fibers of the
ECM. The identification of myelin sheaths and collagen
fibers acquires an important role in the evaluation of the
peripheral nerve in pathology and tissue engineering (Mills
2007; Jiao et al. 2009; Oh 2001). Currently, it is necessary
to carry out different specific histochemical methods for
the identification of these elements and these results should
be interpreted in parallel (Mills 2007). Recently, Rutschow
et al. (2010) described the use of both LFB and picrosirius
histological methods for the evaluation of myocytolysis
and fibrosis in myocardial tissue. However, the method
713
reported by these authors required the use of two independent staining processes using two different samples,
since both staining procedures were carried out separately.
One of the main advantages of the novel MCOLL method
is the use of one single staining process.
In paraffin-embedded tissues the LFB method is most
often used in histopathology for the study of the myelin in
CNS and PNS (Mills 2007; Klüver and Barrera 1953). In
substitution of the previously described method, several
techniques have been developed for the identification of
myelin using light and electron microscopy (Savaskan et al.
2009; Di Scipio et al. 2008; Larsen et al. 2003; Xiang et al.
2005; Schmued and Slikker 1999; McNally and Peters
1998; Tolivia et al. 1994; Schmued 1990; Schmued et al.
1982; Stilwell 1957). However, all these methodologies
only allow the specific identification of myelin, and it is not
possible to make a comprehensive assessment of the
morphologic parameters and the status of the ECM. The
osmium-tetroxide has been described for myelin sheath
impregnation in paraffin-embedded samples with different
staining contrast with promising results (Di Scipio et al.
2008). But this method provides a permanent coloration of
the myelin sheath. Therefore, it may limit the application
of other histochemical and immunohistochemical techniques in these samples.
The HE staining is commonly non-specific for many
tissue elements, due to its electropolar nature. Using this
staining, we were not able to accurately identify the myelin
sheath. In relation to the ECM, HE staining allowed us to
recognize some acidophilic elements such as the collagen
fibers. However, the staining pattern was non-specific
because all these elements became stained with similar
tones and intensities in comparison with other histological
structures.
The new MCOLL histochemical method based on conventional luxol fast blue and picrosirius histochemical
methods allowed us to evaluate the morphological features
of the nerve tissue as HE did. However, the MCOLL
method had the advantage of simultaneously identifying
the myelin sheath with the same staining pattern, specificity and accuracy of the conventional LFB method, and
the collagen fibers with the same sensibility and specificity
of picrosirius techniques, using a single analysis.
On the other hand, the analysis of the CNS, with this
MCOLL histochemical method allowed us to observe the
histological structure and analyze a specific histochemical
reaction for myelin in the white matter. The evaluation of
the CC grafted for nerve regeneration allowed us to evaluate with high contrast all major morphologic parameters,
the specific histochemical identification of the myelin
sheath in different stages of remyelination or demyelination, and the collagen fibers reorganization during the
regeneration process. In the new-formed nerve fascicles we
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714
Fig. 2 Histochemical analyses
of the normal sciatic nerve.
Longitudinal sections of the
sciatic nerve stained with HE,
where it is possible to observe
the typical undulated tissue
pattern (a). Positive
histochemical reaction for
myelin sheath and
metachromatic reaction for mast
cells (arrow) stained with the
LFB stain (b). The same
sections stained with the new
MCOLL method, where it is
possible to observe the typical
tissue pattern, the intense
histochemical reaction for
myelin sheath in blue, and the
histochemical identification of
the collagen fibers (arrows) in
red (c). The axons and the
Nodes of Ranvier are clearly
visualized (arrowheads) along
the myelinated nerve fibers;
observe the thick collagen fibers
of the epineurium stained in red
(d). Scale bar = 50 lm
Histochem Cell Biol (2011) 136:709–717
A
B
C
D
could clearly identify and evaluate the presence of axonal
sprouting and nerve regeneration. All this allowed us to
establish a more precise diagnosis of the structures that
were present in during the regeneration process in a single
sample of tissue.
The identification of collagen fibers using the MCOLL
method was specific, because it uses a strong anionic
123
tetrakisazo dye called sirius red. This dye interacts with
cationic groups on the surface of the collagen molecules in
parallel, giving an intense red color to the collagen fibers in
light microscopy. In addition, this dye specifically increases the natural birefringence of collagen fibers, and allows
us to selectively identify them by polarizing microscopy.
This phenomenon is particularly induced by Picrosirius
Author's personal copy
S
M
L
R
Q
E
J
I
H
P
D
C
B
O
RC
N
A
PAN
CC
*
*
K
F
G
DAN
CENTRAL
PROXIMAL
Fig. 3 Morphologic analysis of
the collagen conduit (CC) graft.
Histochemical composition of
the entire collagen conduit graft
(a) stained with the new
MCOLL method. The
morphological study with HE
shows the nerve regeneration
process with axonal sprouting
along the graft (b–g). The
histochemical evaluation of the
CC graft with the LFB method,
shows a progressive decrease of
the myelin histochemical
reaction along the graft (h–m).
The histochemical evaluation
with the MCOLL method,
shows the regeneration process
accompanied by axonal
sprouting, a progressive
decrease of the myelin
histochemical reaction, and the
progressive increase of the
histochemical reaction for
collagen fibers stained in red
(n–s). CC collagen conduit wall,
PAN proximal nerve
anastomosis, RC regeneration
cone, DAN distal anastomosis
and distal nerve. Arrowheads
label illustrative examples of
axonal sprouting and newformed nerve fascicles. Scale
bar in panel a = 1000 lm.
Scale bar in panels
b–s = 100 lm
715
DISTAL
Histochem Cell Biol (2011) 136:709–717
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Histochem Cell Biol (2011) 136:709–717
A
B
C
D
E
F
G
H
I
Fig. 4 Light (a–f) and polarized light (g–i) microscopy images of the
CC graft stained with the new MCOLL histochemical method.
Proximal sciatic nerve (a, d, g). Proximal nerve anastomosis (b, e, h).
Distal nerve anastomosis (c, f, i). Arrows illustrative examples of
axons showing Wallerian degeneration. Arrowheads myelin sheaths.
Scale bar = 50 lm
staining (Junqueira et al. 1979; Montes and Junqueira
1991; Trau et al. 1991; Carriel et al. 2011). Interestingly,
the histochemical reaction of the myelin with LFB in this
new MCOLL method was not affected by subsequent
procedures, maintaining the sensibility and specificity, with
a considerable improvement in the contrast without background color. In addition, the histochemical reaction for
collagen fibers was not affected by treatment of the section
with high temperature, which has been previously described (Carriel et al. 2011), and provides a high contrast that
allowed us to identify the different components separately
and specifically.
Currently, there are several methods for the evaluation
of the myelin; however, a histochemical method with these
histochemical and morphologic characteristics does not
exist. Furthermore, this new MCOLL histochemical
method is carried out in about 18 h, being a specific,
simple, and inexpensive procedure for paraffin-embedded
tissue. In addition, the picrosirius and LFB solutions are
very stable at room temperature for at least 5 years, and
can be used repeatedly (Kiernan 2008).
Anyway, this is not a method that aims to replace the
conventional procedures used routinely; this new method
could be useful not only to help in a better diagnosis of the
different degrees of axonal degeneration and demyelination
in CNS and PNS injuries, but also to evaluate the
simultaneous correlation between the morphologic parameters, the myelin integrity and the collagen fibers reorganization during peripheral nerve regeneration carried out
with different protocols of tissue engineering.
In conclusion, the use of this new MCOLL histochemical method allowed us to simultaneously evaluate and
correlate the histological pattern, the specific histochemical
reaction for myelin sheath, and the histochemical reaction
for the stromal collagen fibers in a single slide with high
contrast. This method combines the properties of sensibility and specificity of the used reagents, providing a high
intensity and specificity of the histochemical reaction in the
identification of the histological structures described in this
work. For all these reasons, we hypothesize that this new
histochemical method could be useful in pathology and
tissue engineering.
123
Acknowledgments The authors are grateful to Ms. Ariane Ruyffelaert from Ghent University, Belgium for revising and correcting the
English manuscript. This work was supported by grant SAS PI-135/
2007 from Junta de Andalucı́a, Spain.
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