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J. Biol. Chem., Vol. 277, Issue 33, 29881-29888, August 16, 2002
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From the Departments of
Received for publication, February 18, 2002, and in revised form, May 14, 2002
We generated double knock-out mice lacking the
GM2/GD2 and the GD3 synthase gene by mating single gene mutants,
and we analyzed the abnormal phenotypes of the mutant mice expressing
only the GM3 ganglioside. We observed a refractory skin lesion
that appeared primarily on the face of the mutant mice at 25 weeks
after birth or later. Frequent scratching of the wound sites was
observed in mutant mice with the skin injury, suggesting that it is a
triggering factor that exacerbates the injury. This was confirmed by
isolating mice in special cages for metabolic study in which the skin
injury developed more rapidly. Characteristic proliferation of nerve fibers was found in the epidermis and subepidermis at the injured sites
of the mutants, probably a result of continuous skin injury. Peripheral
nerve degeneration was observed in young mutant mice, suggesting that
reduced sensory function induced over-scratching and the resulting skin
lesion. The fact that sensory response to mechanical stimuli decreased
while that to hot stimuli increased in the mutant mice supports this
interpretation. Thus, only GM3-expressing mice displayed the
important role of gangliosides in maintaining skin integrity via
regulation of the peripheral nerves.
Acidic glycosphingolipids, gangliosides, are ubiquitously
expressed in the various tissues of vertebrates and play
important roles in the regulation of highly organized multicellular
systems (1). In particular, they are very much enriched in the nervous system and have been considered to control development, proliferation, differentiation, and maintenance of the neural tissues and cells (2,
3). Expression patterns of various ganglioside species in the nervous
system vary during development and are strictly regulated in a
spatio-temporal manner (4), suggesting that individual structures of
gangliosides contain significant implications for individual situations.
A number of studies have been performed to demonstrate the biological
function of gangliosides primarily by modifying their structures with
enzymes or using inhibitors to block some steps of synthesis (5).
Otherwise, the effects of addition of gangliosides to the culture
medium or injection into the conditioned animals have been the primary
approaches in addressing the significance of gangliosides. However,
recent success in the molecular cloning of glycosyltransferase genes
brought about an evolutional change in the experimental approaches for
carbohydrate function analysis. Availability of glycosyltransferase
genes responsible for the synthesis of gangliosides enabled us to
remodel ganglioside compositions of cells and tissues in
vivo (6, 7) and in vitro (8).
Because we isolated GM2/GD2 synthase (EC 2.4.1.92) cDNA (9) and GD3
synthase (EC 2.4.99.8) cDNA (10), we established gene knock-out
mice lines of the individual glycosyltransferases. GM2/GD2 synthase
gene knock-out mice lacking all complex gangliosides showed almost
normal morphogenesis of the nervous system and no definite abnormal
behaviors (11), although they showed nerve degeneration along with
aging (12, 13) as well as immunodeficiency (14) and male infertility
(15). GD3 synthase gene knock-out mice lacking all b-series
gangliosides1 also showed no
marked abnormal appearance in nerve tissues and exhibited almost intact
behaviors (16, 17), although regeneration of the lesioned hypoglossal
nerve was largely reduced (17).
In the present study, we established double knock-out mice of the above
described GM2/GD2 synthase gene and GD3 synthase gene by mating them to
each other, and we analyzed the abnormal phenotypes of the mutant mice
expressing only the GM3 ganglioside. Although these mutant mice might
have a serious deficit in the nervous system (16), we observed a
characteristic skin lesion that appeared mostly on the face of the
mutant mice 25 weeks after birth or later. We have investigated the
mechanisms for the development of the skin lesion and have elucidated
the reduced sensitivity of the sensory nerve based on nerve
degeneration as a primary triggering factor for the lesion.
Mice--
The mice were maintained in our laboratory. GM2/GD2
and GD3 synthase gene knock-out mice were mated, i.e.
heterozygotes of both mutants were mated, and genotypes of the
offspring were screened for the two genes as described previously (11,
17). To generate double knock-out mice efficiently, we also mated
female homozygotes of the GD3 synthase gene and male heterozygotes of
the GM2/GD2 synthase gene. Double knock-out mice with the two genes
were designated as Ho/Ho2
mutant, and those with wild type for both genes were presented as
Wd/Wd. Body weight was measured every week, and mice were observed every day.
Typing--
Genetic typing of GD3 synthase gene was performed by
polymerase chain reaction with mouse tails using primers as
follows, 5'-GCTGAGGGTACACTGACCCTGGGACATCGA-3',
5'-TCGTGCTTTACGGTATCGCCGCTCCCGATT-3', and
5'-ACTAGGGACAGACCGGCGAAATCCTTGATT-3'. The reaction was started with a
3-min cycle at 95 °C followed by 30 cycles for 1 min at 55 °C, 2 min at 72 °C, and 1 min at 95 °C. Genetic typing of the GM2/GD2
synthase gene was performed as described previously (11).
Extraction of Glycolipids--
Glycolipids were extracted as
described previously (18). Briefly, lipid fractions were extracted by
chloroform/methanol at ratios of 2:1, 1:1, and 1:2, sequentially.
Glycolipids were isolated by a Florisil column after acetylation, and
then neutral and acidic fractions were separated by DEAE-Sephadex
(A-50) column chromatography.
Isolation of Mice in Metabolic Cages to Induce Wounds--
Seven
each of Wd/Wd-type and Ho/Ho-type 25-week-old male mice were housed in
special cages usually used for metabolic study and were fed regular
chow. Daily observation was performed for 2 months to check the body
weight, generation of wounds, and frequency of skin scratching.
Scratching measurements were performed every 5 min twice a week.
Pathological Studies--
Tissue sections from the skin lesions
of the injured sites were prepared. The serial sections were divided
into three groups, i.e. the first group was stained with
hematoxylin-eosin, and the other two groups were stained with toluidine
or used for immunohistochemical study with anti-PGP9.5 antibody
directed toward the N terminus of the protein (19). The areas occupied
with nerve fibers in the epidermis were quantitatively measured in
consecutive sections, and the results were presented as an average of
the values obtained from three sections. Actual measurement of the
nerve-occupying area was performed as follows. Images were captured
using the Olympus digital camera DP-11 or classical photomicrography on film. Analysis was performed using the public domain NIH Image program
(anonymous FTP from zippy.nimh.nih.gov). These analyses were performed
by two independent persons with no preliminary information.
Electron Microscopic Study--
Skin tissues were fixed by
immersion in a fixative containing 1.5% paraformaldehyde and 1.5%
glutaraldehyde in phosphate buffer (pH 7.4) and osmicated in 1%
OsO4 solution for 1 h. Fixed tissues were dehydrated
in alcohols and embedded in Epon 812. After staining with uranylacetate
and lead citrate, ultrathin sections were prepared and observed using
the JEOL 100 CX electron microscope.
Sensitivity Test--
To analyze sensory function in the mutant
mice, we examined sensitivity to pain stimulation using the hot plate
method and with the von Frey strings test as described previously
(20).
Statistical Analysis--
Numeric data are expressed as
mean ± S.D. The number of mice used in the experiments are as
indicated in each figure. The significance of differences among groups
was determined using analysis of variance for comparison.
Generation of Double Knock-out Mice of GM2/GD2 and GD3
Synthase Genes--
The heterozygous mutants of GM2/GD2 synthase and
those of GD3 synthase were mated, resulting in nine combinations of the
genotypes. Eventually, Ho/Ho mutants were obtained at the approximate
ratio of 1:16, similar to the Wd/Wd mutants, corresponding to the
classic Mendelian rule, indicating that Ho/Ho mutants had no critical disadvantage in development and birth. They appeared somewhat normal
and could not be distinguished from their littermates at birth.
Only GM3 Remained in Double Knock-out Mice--
Brain, liver, and
skin tissues were used for the extraction of glycolipids and exhibited
dramatic changes in the compositions of gangliosides, i.e.
all complex gangliosides were deleted, and only GM3 was found in brain
extracts in the double mutants (Fig. 1B) as expected from the
synthetic pathway (Fig. 1A). In the skin extracts GM3 was a
major ganglioside, and an additional faint band of GD1a was present in
Wd/Wd skin. Otherwise, no marked change was found in the extracts of
Ho/Ho mice skin; only GM3 was detectable at similar levels during the
course (Fig. 1C).
Skin Injury Observed at 25 Weeks Old--
Approximately 35% of
the 30-week-old mutant mice (Ho/Ho) showed, more or less, skin injury
primarily on their faces as shown in Fig.
2, A and B, and the
frequency increased up to 50% as they aged to 40 weeks old. In this
situation, the injured mice showed characteristic scratching toward the
injuries on their faces and necks (Fig. 2C). The frequency
was about 90× per 5 min, whereas non-injured Ho/Ho and Wd/Wd mice
scarcely exhibited this scratching movement (Fig. 2D).
Promotion of Skin Injury by Isolation of Mice into Metabolic
Cages--
It was discovered occasionally that the skin lesions were
generated easily when mice were housed in special cages for the studies
of metabolism in which they frequently underwent trauma to the face
while taking foods through wire mesh (Fig.
3A). Therefore, we tried to
house Ho/Ho and Wd/Wd mice in the metabolic cages under an isolated
condition. Consequently, only Ho/Ho mice exhibited development of the
skin injury earlier than they did in conventional cages (Fig.
3B). Wd/Wd mice showed no injury. Ho/Ho mice undergoing the
skin injury showed a much higher frequency of scratching than Wd/Wd
mice (Fig. 3C), suggesting that this scratching might induce the skin injury and its exacerbation.
Proliferation of Peripheral Nerves in the Skin Lesion--
In the
sections of the injured skin tissues, skin erosion and granulocyte
infiltration were observed in some portions (Fig. 4A). A more characteristic
feature in these lesions was proliferated peripheral nerves as shown in
Fig. 4B. These hypertrophic nerves were stained with
toluidine blue and were found frequently in the epidermis and
subepidermis in Ho/Ho mice skin (Fig. 4, D and E)
but rarely in Wd/Wd mice (Fig. 4C). A few cells with
granules were found occasionally in Ho/Ho skin (Fig. 4D). To
identify the nerve-like structures that appeared characteristically in
the Ho/Ho mice skin, immunohistochemistry with anti-PGP9.5 antibody was
performed. As shown in Fig. 4F, a large number of nerve
fibers were found in the subepidermis and epidermis, and electron
micrographs exhibited almost normal-shaped, slightly invaginated,
myelinated, and unmyelinated nerve fibers (Fig. 4G).
Increased Area Occupied by Nerve Fibers in Ho/Ho Mice
Skin--
As shown in Fig. 4H, measurement of nerve
fiber-occupied area revealed that 6.0 ± 5.2% (as an average) of
the skin was occupied by nerve fibers in the skin of the mutants with
injury. In contrast, non-injured skin showed a minimal number of nerve
fibers similar to those in Wd/Wd mice skin (1.3% ± 1.5%).
Reduced Sensitivity to Mechanical Pain Stimuli in Ho/Ho
Mice--
To investigate the mechanisms for generation of the skin
lesion in Ho/Ho mice, we examined the sensory nerve function with two
methods using three mouse groups consisting of adolescent (20 weeks
old), adult (35 weeks old), and intermediate (25 weeks old). Ho/Ho mice
with no injury exhibited a clear reduction in sensitivity to mechanical
pain stimuli at 25 weeks old and showed exacerbation at 35 weeks old
(Fig. 5A), suggesting that
their sensitivity was reduced markedly at a certain time point after 20 weeks and that this may induce over-scratching after the first triggering event. On the other hand, sensitivity to hot stimuli as
measured with hot plates was enhanced rather significantly in Ho/Ho
mice at 35 weeks old but not at 20 weeks old (Fig. 5B). This
result suggests that the response to heat stimuli increased as a result
of the injury and subsequent nerve fiber hyperplasia.
Degenerative Changes in Trigeminal and Sciatic Nerves in Relatively
Young Ho/Ho Mutants--
In observation of the cranial
nerve (trigeminal nerve) and the peripheral nerve (sciatic nerve),
strong degenerative changes were found even in the myelinated fibers of
20-week-old Ho/Ho mice. Nerve fibers of Ho/Ho mice showed an irregular
array, swollen, beaded, and duplicate myelin appearances compared with
those of Wd/Wd mice (Fig. 6, A
and B). Degeneration in the sciatic nerves was more
prominent (Fig. 6B, bottom panel).
Width measurement of nerve fibers revealed that the diameter of fibers
including myelin sheath in homozygotes generally was increased in both
trigeminal and sciatic nerves compared with wild type (data not shown).
The width of axon and myelin sheath also was significantly increased in
homozygotes in both nerves as indicated in Fig. 6, C and
D. However, the ratio of myelin sheath/axon was almost
equivalent between homozygotes and wild type mice in both sciatic nerve
and trigeminal nerve, suggesting that nerve fibers enlarged by a whole unit (Fig. 6E).
Although the double knock-out mice containing only the GM3
ganglioside were born and grew up almost normally at a glance, they
started to die suddenly 12 weeks after birth. The accumulative survival
rates were 92.2% after 10 weeks, 77.4% after 20 weeks, and 33.2%
after 30 weeks of birth. The cause of death is not clear and may not be
due to audiogenic convulsion for these mutant mice in contrast with
those reported previously
(16).3 Namely, 12- and
18-week-old Ho/Ho mice showed no seizure when treated with various
audiogenic stimulations such as the noise of key bundles (16) or sounds
generated by a PA audiometer (Nagashima Medical Instruments Co.,
Nagoya, Japan) (21) at 200, 500, 2,000, 4,000, 8,000, 10,000, and
15,000 Hz (~80-108 decibels) (data not shown). In surviving mice,
serious skin injury was found in the significant population of Ho/Ho
mice, which never has been observed in single mutant mice,
e.g. GM2/GD2 synthase or GD3 synthase gene knock-out. Even
with the isolation in metabolic cages, the single knock-out mice as
well as the Wd/Wd mice did not show such serious injury, suggesting
that the deletion of all ganglioside structures except for GM3 induced
a novel defect in the skin. Because a definite difference in
ganglioside composition in the double mutants from that of GM2/GD2
synthase gene knock-out mice (11) is the lack of GD3, this result
indicates a significant role by at least one ganglioside (GD3). The
reason why our Ho/Ho mice showed no audiogenic seizure yet exhibited
rather prominent skin injury is not known. Genetic backgrounds of the
embryonic stem cells used are different between the two groups,
i.e. 129SvEv (TC-1) (16) and (CBA × B6)F1
(TT2) (11, 17), and seem to be a major factor for the phenotypic differences.
There were no complex gangliosides other than GM3 and a low level of
GD1 In human atopic dermatitis, scratching the itchy sites was thought to
induce serious dermatitis, and it frequently caused hypertrophy of
peripheral nerve and hyperplasia of neural fiber bundles (22-24),
leading to a high density of peripheral nerve fibers along with the
progress of the disease. This bad cycle might be one of the bases for
the refractory atopic skin inflammation (25). A similar situation may
occur in the skin of Ho/Ho mice, i.e. the reduced sensation
to mechanic pain might promote very frequent and repetitive scratching,
and the resulting inflammatory processes might induce peripheral nerve hyperplasia.
Histological analysis of the sciatic nerve and trigeminal nerve
revealed that peripheral nerves in Ho/Ho mutant mice underwent serious
degeneration more prominently than those in the single mutants as
reported previously (12).4
The reduced sensitivity to the mechanic pain observed in Ho/Ho mice
with no injury might reflect these degenerative changes.
Of course, Ho/Ho mutants should have various defects other than those
in peripheral nerves as expected from abnormal phenotypes such as
immunodysfunction and endocrinological defects in the single mutant
mice (14, 15). These factors might also be involved in the generation
of the severe skin injury. However, it seems reasonable to consider
that a defect in peripheral nerve regulation is one of the major causes
of the skin injury. Furthermore, there have been no clear studies on
the role of gangliosides in skin, although we reported previously that
transgenic mice of the GM2/GD2 synthase gene showed a rather increased
reaction to exogenous stimulation and a delay in healing (26).
Therefore, this study demonstrates for the first time an important role
of gangliosides in the regulation of skin integrity by manipulating
ganglioside synthase genes.
For a long time, gangliosides have been thought to play an important
role in the nervous system as a sort of neurotrophic factor (27, 28).
Actually, neurite extension, synaptogenesis, or regeneration of damaged
nerves was enhanced in the presence of gangliosides (29). If this is
the case, the hyperplastic nerve fibers found in Ho/Ho mice skin seem
to be a paradox. Could the deletion of all gangliosides except for GM3
result in increased neural regeneration? If so, might GM3 itself induce
and promote neurogenesis? The hypertrophic neural fibers also might be
an abnormal proliferative reaction as a result of deregulation after the deletion of complex gangliosides triggered by repetitive scratching and sustained inflammation. As yet, significant mechanisms for promotion of the proliferation of peripheral nerve fibers remain unknown. The sensitivity of neural tissues to neurotrophins, including expression levels of the neurotrophin receptors, might have changed. Neuropeptides (30, 31) or neurotrophins (32-34) from keratinocytes also may be responsible, as reported for atopic dermatitis. True molecular mechanisms remain to be investigated.
We thank T. Nakashima at the Department
of Otolaryngology, Nagoya University, for helpful discussion and
J. Tsuzuki for technical assistance.
*
This study was supported by Grants-in-aid for Scientific
Research on Priority Areas (13470021 and 12670111), Grant-in-aid for
Exploratory Research (13877022), and the Center of Excellence Research
(Grant-in-aid 10CE2006) from the Ministry of Education, Science,
Sports, and Culture of Japan. This study was also supported by a
grant-in-aid from the Mizutani Foundation.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
¶
To whom correspondence should be addressed: Dept. of
Biochemistry II, Nagoya University School of Medicine, 65 Tsurumai,
Showa-ku, Nagoya 466-0065, Japan. Tel.: 81-52-744-2070; Fax:
81-52-744-2069; E-mail: koichi@med.nagoya-u.ac.jp.
Published, JBC Papers in Press, May 22, 2002, DOI 10.1074/jbc.M201631200
1
Nomenclature of gangliosides is based on that of
Svennerholm (35).
3
M. Inoue, K. Furukawa, M. Okada, K. Furukawa, and Y. Sugiura, unpublished data.
4
Y. Sugiura, K. Furukawa, M. Okada, and K. Furukawa, unpublished data.
The abbreviations used are:
Ho/Ho, homozygous
mutant for GD3 and GM2/GD2 synthase;
Wd/Wd, wild type for GD3 and
GM2/GD2 synthase.
Refractory Skin Injury in Complex Knock-out Mice
Expressing Only the GM3 Ganglioside*
§,,,¶, and
Biochemistry II,
Anatomy II, and § Internal Medicine II, Nagoya
University School of Medicine, 65 Tsurumai, Showa-ku, Nagoya 466-0065, Japan and the ** Department of Pediatrics, Nagasaki
University School of Medicine, 1-12 Sakamoto,
Nagasaki 852-8523, Japan
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (53K):
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Fig. 1.
A synthetic pathway of gangliosides and the
effects of targeted disruption of two glycosyltransferase genes.
A, a synthetic pathway of gangliosides and ganglioside
species that would be absent from GM2/GD2 and GD3 synthase gene
knock-out mice are indicated with squares. Consequently,
structures boxed in squares are depleted in double knock-out
mice. B and C, analysis of gangliosides of brain
(B) and skin (C) of Wd/Wd and Ho/Ho mice.
B, in the left panel extracts derived from 250 µg of brain tissue from 20-week-old mice were applied. Thin layer
chromatography was performed with control gangliosides from bovine
brain (lane a), Wd/Wd (lane b), and Ho/Ho
(lane c). In the right panel brain gangliosides
from Ho/Ho mice at 10 (lane e), 20 (lane f), 30 (lane g), and 50 (lane h) weeks old were
extracted and analyzed; lane d, bovine brain gangliosides
plus GM3 as standards. C, skin gangliosides from Wd/Wd
(lane c) or Ho/Ho mice at 10 (lane d), 20 (lane e), 30 (lane f), and 50 (lane g)
weeks old were analyzed; lane a, standard mixture;
lane b, GM3. A solvent system of chloroform/methanol/water
(50:40:10) and orcinol spray for detection was used. Note that no
marked change in the GM3 level was found in either brain or skin during
the course.
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Fig. 2.
Generation of skin injury in Ho/Ho mice.
A, appearance of a 30-week-old Ho/Ho mouse with serious skin
injury on its face. Arrows indicate injured fingers.
B, time course of the generation of skin injury shown as the
ratio of mice with skin injury to the face and neck. In Ho/Ho
mice only, this emerged immensely in adulthood (25 weeks after birth).
C, example of the scratching movement observed in Ho/Ho mice
with the injury. D, frequency of scratching in 30-week-old
Ho/Ho and Wd/Wd mice. Note that only wound (+) Ho/Ho showed high
frequency.
View larger version (24K):
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Fig. 3.
Promotion of skin injury in the metabolic
study cages. A, representative examples of the injury
at 2 weeks (top panel) and 5 weeks (bottom
panel) after accommodation were shown. Arrows indicate
lesions around the eyes. B, time course of the development
of the skin injury in Ho/Ho and Wd/Wd mice housed in the metabolic
cages. The ratios of injured/whole mice examined are shown as a
function of weeks after isolation. C, frequency of
scratching as measured every 5 min. Average numbers of the movement of
three sequential counts (mean ± S.D.) are presented.
View larger version (81K):
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Fig. 4.
Histological analysis to elucidate the
peripheral nerve hypertrophy. A, erosion and marked
infiltration of inflammatory cells in epidermis of injured Ho/Ho mice
skin (hematoxylin-eosin). B, hypertrophic nerve fibers in
epidermis of injured Ho/Ho mice (hematoxylin-eosin).
Arrows indicate nerve fibers. C, toluidine blue
staining of Wd/Wd mice skin. D and E,
hypertrophic nerve fibers in Ho/Ho mice skin stained with toluidine
blue. Arrows in C, D, and E
indicate nerve fibers. Arrowheads in D indicate
cells with granules. F, proliferated nerve fibers in
epidermis in the injured lesion of Ho/Ho mice stained by an
anti-PGP-9.5 antibody; note that a number of nerve fibers were stained.
G, electron microscopic analysis of nerve fibers in the
epidermis of Ho/Ho. H, area occupied by nerve fibers in the
skin sections were measured by NIH image as described under
"Experimental Procedures." Percent areas is shown as mean ± S.D. *, p < 0.05. 30-week-old Ho/Ho and Wd/Wd mice
were used.
View larger version (25K):
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Fig. 5.
Abnormal sensory function in Ho/Ho mutant
mice. Sensitivity of sensory nerves was examined using two
approaches. A, sensitivity to mechanical pain as measured
with the von Frey test. The minimum intensity of mechanical stimuli
(g, gram) that could induce mouse reaction was determined.
B, sensitivity to hot stimuli as measured with hot plates
(53 °C). The examination was performed as described under
"Experimental Procedures" at least three times. The results from
four mice are each presented as mean ± S.D. *, p < 0.05. Note that sensitivity to the mechanical stimuli was already
reduced in 25-week-old Ho/Ho mutants.
View larger version (108K):
[in a new window]
Fig. 6.
Degeneration of peripheral nerves.
A and B, morphological changes of peripheral
nerves in 20-week-old mice were examined. Tissue sections of trigeminal
nerves (A) and sciatic nerves (B) from Wd/Wd
(top panel) and Ho/Ho (bottom panel) were stained
with toluidine blue. An arrow indicates an area with marked
nerve fiber degeneration. C and D, widths of
nerve fibers were measured (n = 100) and compared with
the width of myelin sheath (C) and axon (D). The
ratio of myelin sheath to axon in each fiber was calculated
(E). Data were analyzed with the paired Student's
t test. *, p < 0.05.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
on a thin layer chromatography of the extracted glycolipids from
Wd/Wd mice skin. This pattern was similar to that of Ho/Ho mice skin,
suggesting that the change in ganglioside composition in skin tissue
might not be a primary factor to induce the serious skin injury. The
amounts of GM3 in Ho/Ho mice showed no marked change in either brain or
skin during observation, despite the progress of skin injury. We also
focused on their movement to scratch the initially injured sites to
observe whether it induced exacerbation of the wound. Particularly in
the special cages, it was apparent that the scratching became more
frequent as the injury advanced and was tightly associated with the
development of refractory lesions.
ACKNOWLEDGEMENTS
FOOTNOTES
ABBREVIATIONS
REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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