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The Journal of Neuroscience, July 1, 2002, 22(13):5481-5491
Enhanced Rate of Nerve Regeneration and Directional Errors After
Sciatic Nerve Injury in Receptor Protein Tyrosine Phosphatase  Knock-Out Mice
Joanna
McLean1, *,
Jane
Batt2, *,
Laurie C.
Doering4,
Daniela
Rotin2, and
James R.
Bain3
1 Department of Pediatrics, Hospital for Sick Children,
University of Toronto, Toronto, Ontario, Canada, M5G 1X8,
2 Program in Cell Biology, Hospital for Sick Children, and
Department of Biochemistry and Institute of Medical Sciences,
University of Toronto, Toronto, Ontario, Canada, M5G 1X8, and
Departments of 3 Surgery and 4 Pathology and
Molecular Medicine, McMaster University, Hamilton, Ontario, Canada, L8N
3Z5
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ABSTRACT |
The receptor protein tyrosine phosphatase  (PTP) is a member
of the mammalian leukocyte common antigen-related (LAR) family. Its
expression is developmentally regulated in neuronal tissues. The
Drosophila homolog of the mammalian LAR family of
phosphatases (DLAR) controls axon guidance during
Drosophila embryogenesis. We have demonstrated
previously that mice deficient in PTP have CNS and peripheral
nervous system abnormalities. The sciatic nerve in the PTP( /)
mice demonstrates an increased number of small diameter fibers and
slower nerve conduction velocities compared with PTP(+/+) or
PTP(+/) controls. To study whether peripheral nerve regeneration
is affected by PTP activity, we assessed nerve regeneration in the
PTP(/) mouse after three standard models of sciatic nerve
injury. We report that after sciatic nerve crush injury, nerve
regeneration was significantly faster in the PTP(/) animals, as
determined by histologic, electrophysiologic, and neuromuscular
testing. After sciatic nerve transection with immediate microsurgical
repair or allografting, PTP(/) nerve fibers demonstrated errors
in directional growth compared with controls. We propose that PTP
regulates the axonal regeneration rate and guidance of regenerating fibers.
Key words:
protein tyrosine phosphatase ; axon guidance; nerve
regeneration; sciatic nerve; knock-out mouse; walking track
analysis
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INTRODUCTION |
Successful development of the
vertebrate nervous system requires the intricate timing of cellular and
molecular processes. Axons follow stereotyped patterns that, to reach a
distant target, require precise navigational instruction. This
instruction is provided by at least two classes of proteins: classic
adhesion molecules and cell surface receptors that control tyrosine
phosphorylation within the cell (Van Vactor, 1998 ; Bixby, 2000; Stoker,
2001). Cell adhesion molecules (CAMs) and substrate adhesion
molecules of many classes have been shown to play a role in axonal
fasciculation and growth (Rutishauser et al., 1978; Baldwin et al.,
1996; Krueger et al., 1996). Although mutations in these proteins
affect fasciculation, axonal trajectories are often untouched,
suggesting that adhesion molecules may stabilize, rather than direct,
neuronal growth (Van Vactor, 1998). Genetic and mutational analyses
have suggested that several protein tyrosine phosphatases (PTPs) play
an important role in axonal guidance and fasciculation (Van Vactor et
al., 1998; Bixby, 2000; Stoker, 2001).
More than 75 PTPs have been identified, with many expressed in the
nervous system and some showing neural specificity (Sahin et al., 1995;
Schaapveld et al., 1998). Members of the leukocyte common
antigen-related (LAR) mammalian subfamily of PTPs (LAR, PTP, and
PTP ), are transmembrane proteins with a cell-adhesion molecule-like
ectodomain and intracellular phosphatase domains. PTP exhibits
developmentally regulated expression (Rotin et al., 1994; Wang et al.,
1995; Schaapveld et al., 1998; Batt et al., 2002) in embryonic
neuronal, neuroendocrine, and epithelial tissues. Expression decreases
postnatally and in the adult brain is restricted to areas of high
synaptic plasticity. Chick receptor tyrosine phosphatase  (CRYP), the avian homolog, shows prominent axonal and
growth-cone localization within the embryonic chick (Stoker et al.,
1995). A loss-of-function mutant of the Drosophila homolog of LAR-PTP-PTP (DLAR) demonstrates directional guidance errors of intersegmental nerve B (ISNb) (Krueger et al., 1996). This pattern
of differential expression and the phenotype of the DLAR mutant support
a role for PTP in neuronal development.
PTP-deficient mice demonstrate abnormalities of the CNS,
peripheral nervous system (PNS), and neuroendocrine system (Elchebly et
al., 1999; Wallace et al., 1999; Batt et al., 2002). Phenotypic neurological defects in these mice include spastic movements, tremor,
abnormal limb flexion, and defective proprio-ception. Peripheral
nerve electrophysiological analysis demonstrated slower nerve
conduction velocities (NCVs) associated with an increased proportion of
slow-conducting, small-diameter myelinated fibers in the young
PTP(/) mice (Wallace et al., 1999). Normalization of some of
these parameters (NCV, nerve morphometrics, tremor, and spasticity)
with age suggested that developmental delay contributed to the
PTP(/) phenotype. Mice deficient for the other LAR family members, LAR and PTP, also demonstrate nervous system abnormalities (Uetani et al., 2000; Van Lieshout et al., 2001; Xie et al., 2001). Although no morphological differences are observed in the uninjured peripheral nerve of LAR-deficient mice, they exhibited a decreased rate
of nerve regeneration after sciatic nerve crush injury (Xie et al.,
2001). PTP expression in dorsal root ganglia (DRG) cells has been
shown to be responsive to nerve lesion and regeneration of the rat
sciatic nerve (Haworth et al., 1998). Collectively, these studies
suggest a role for the LAR family of phosphatases in developmental and
regenerative growth of the mammalian peripheral nervous system.
This study evaluates the consequences of the lack of PTP on
peripheral nerve regeneration and function using PTP knock-out mice.
We demonstrate here by means of histologic and physiologic assessment
that the peripheral nerve of the PTP(/) mouse regenerates faster
than in PTP(+/) or PTP(+/+) mice and exhibits significant directional errors, suggesting axonal pathfinding defects.
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MATERIALS AND METHODS |
Animal model. The PTP knock-out mouse was
generated and characterized as described previously (Wallace et al.,
1999). PTP(+/) animals are phenotypically indistinguishable from
wild-type animals. Three cohorts of PTP(/) can be identified:
The majority of PTP(/) animals die within 48 hr after birth
(60%). Approximately 38% live until 2-3 weeks of age and then
succumb to a wasting syndrome. Only 2.5% of PTP(/) mice survive
to adulthood, but these mice are ~25-50% smaller by weight.
Experimental design and surgical procedure. Three
standardized nerve injury models were used to evaluate peripheral nerve regeneration: sciatic nerve crush, sciatic nerve transection and microsurgical repair, and sciatic nerve allografting. For each model,
3- to 6-month-old PTP(/) animals were paired with a sibling
PTP(+/) or PTP(+/+) control. The sciatic nerves of mice of this
age have sufficient caliber to be manipulated microsurgically. The
right sciatic nerve was injured under anesthesia in each animal pair.
Nerve regeneration was subsequently assessed by histologic evaluation
of the insulted sciatic nerve and evaluation of target muscle function
(walking track analysis, electrophysiologic testing). All animal
housing and surgical interventions were performed in accordance with
the Canadian Council on Animal Care Guidelines under institutionally
reviewed animal protocols.
Sciatic nerve crush. Sciatic nerve crush injury was
performed as described by Seddon (1943). Briefly, the right hindlimb
was prepared under Halothane anesthesia (1-3%) (MTC Pharmaceuticals, Cambridge, Ontario, Canada). Using a sterile technique, a
gluteal splitting incision exposed the sciatic nerve 2 mm distal to the sciatic notch. To create the axonotmesis injury, number 5 jeweller's forceps crushed the sciatic nerve for 15 sec. The muscle
and skin were closed, and analgesic was administered (Buprenex, 1.5 mg/kg, s.c.; Reckitt and Colman Products, Richmond, VA).
Evaluation of nerve regeneration was serially performed until full
functional recovery was evident.
Sciatic nerve transection. Sciatic nerve transection injury
was performed as described by Seddon (1943). Briefly, the mice were
prepared and the sciatic nerve was exposed as described above. The
sciatic nerve was surgically divided 2 mm distal to the sciatic notch
and immediately repaired with 10-0 nylon using a standard microsurgical
technique. Because full recovery is not evident after transection and
repair, evaluation of nerve regeneration is performed at the time of
plateau of functional recovery (6 weeks after injury).
Allografts. Simultaneous inhalational Halothane (1-3%)
anesthesia (MTC Pharmaceuticals) was performed on both the
PTP(/) animal and its sibling PTP(+/) or PTP(+/+)
control. The sciatic nerves of the right hindlimb of each animal were
exposed as described above, and matching 1 cm grafts were procured. The
genotypically disparate allografts were then orthotopically repaired to
the sciatic nerve deficits via a standard microsurgical technique [i.e., the PTP(/) graft was repaired to the deficit of its sibling control and vice versa (reciprocal grafting)]. Serial histologic assessment of axonal growth through the graft by light microscopy was subsequently undertaken.
Electrophysiologic assessment. Motor nerve conduction
studies were performed both preoperatively and at experiment endpoint, as described previously (Smorto and Basmajian, 1979; Robertson et al.,
1993). Briefly, under Halothane anesthesia (MTC Pharmaceuticals) bipolar stimulating electrodes were placed proximally in the sciatic nerve at the level of the sciatic notch and distally in the posterior tibial nerve at the level of the medial malleolus. Recording electrodes were placed in the ipsilateral foot intrinsic muscles. A supramaximal square wave pulse of 0.1 msec duration was applied sequentially at the
sciatic notch and medial malleolus, and the motor latency and amplitude
of the compound motor action potential (CMAP) was recorded (Neuromax
2000 Electrophysiologic System, Excel Corp., Oakville, Canada).
The intersegmental distance between the proximal (sciatic notch) and
distal (medial malleolus) electrodes was measured and the
intersegmental NCV was calculated using standard formulas (Goodgold and
Eberstein, 1983). Direct recordings from the more proximal
gastrocnemius muscle were also performed if the CMAP was not detected
in the foot intrinsic musculature. Presence or absence of muscle
response was compared by  2 test. CMAP
amplitude and NCV comparisons between homozygotes and controls were
analyzed by one-way ANOVA and t test.
p < 0.05 was defined as significant.
Neuromuscular assessment: walking track analysis. Hindlimb
neuromuscular function was assessed by walking track analysis as described previously (Bain et al., 1989; Brown et al., 1991; Hare et
al., 1992, 1993). Briefly, nontoxic paint was applied to the hind feet
of each mouse and the animal was then placed into a walking track.
Satisfactory prints (based on consistent interprint distance and
identifiable print characteristics) were directly measured on a
digitizing tablet (Tabletworks; SummaGraphics, Cal Comp Input
Technologies Division, Scottsdale, AZ) linked to computerized planimetry software (Sigmascan; Jandel, San Rafael, CA). Foot print
length (PL), toe spread (TS), and intermediary TS (IT) (second to
fourth) were measured for control (left) and operative (right) sides of
each animal. The sciatic function index (SFI) can then be calculated as
described previously (Bain et al., 1989). Groups can be compared based
on individual footprint characteristics such as PL factor (PLF,
difference between experimental and control sides/normal print length),
toe spread factor (TSF), and IT factor (ITF) (calculated in the same
manner) (Hare et al., 1993). A one-way ANOVA evaluated differences
within each experimental group over time and between groups at
specified times. t tests were then used if justified by
ANOVA. A p value of <0.05 was considered significant.
In the rat, gait normalizes by 4 weeks after sciatic nerve crush, but
with transection/repair models, gait does not correct completely and
function plateaus by 8 weeks after injury (Hare et al., 1992).
Histological assessment. At the completion of the
functional/electrophysiologic studies, animal pairs were assigned to
either fluorescence immunostaining or light microscopy with or without quantitative cross-sectional morphological assessment.
Immunofluorescence. Neurofilament (NF) immunostaining
identifies regenerating axons after nerve injury (Sternberger and
Sternberger, 1983; Doering, 1992). The mice were sedated with
intraperitoneal chloral hydrate and underwent cardiac puncture and
perfusion fixation with 0.9% saline followed by 4% buffered
paraformaldehyde. The sciatic nerve and its terminal branches
were dissected out and cryostat sectioning of the procured nerves was
performed at the sites illustrated in Figure 3A,B. Nerves
were sampled at different locations depending on the model of nerve
injury used to obtain sections proximal, through, and distal to the
injury. Nerves were placed in 30% sucrose at 4°C for 24 hr before
cryostat sectioning. Segments were frozen by placement in a plastic
weigh boat (maintaining proximal-distal orientation) and immersion in
a beaker of 2-methylbutane cooled to -60°C with liquid
nitrogen. Samples were embedded in optimal cutting temperature compound
(TissueTech; Sakura, Torrance, CA) and 15-µm-thick
longitudinal sections were cut. Sections were transferred to
Aptex-coated slides and stored at 20°C before antibody staining.
Slides were washed in PBS for 10 min at room temperature,
blocked, and then incubated overnight at 4°C with a 1:2000 dilution of mouse monoclonal anti-neurofilament antibody (RT97 phosphorylated heavy neurofilament subunit; gift to L. Doering from J. N. Wood, University College, London, UK). Slides were washed once
with PBS before application of a goat anti-mouse IgG conjugated to fluorescein isothiocyanate or rhodamine (1:100) (Jackson
ImmunoResearch, West Grove, PA) for 3 hr at room temperature.
They were then washed three times in PBS, allowed to dry, and
coverslipped. Appropriate positive and negative controls (absence of
the primary antibody) were used. Sections were viewed subsequently with
an epifluorescence microscope to visualize the neurofilament staining.
The presence or absence of NF immunoreactivity was noted at
standardized locations (see Fig. 3A,B), and density of
staining was recorded by an observer blinded to animal group. The data
were organized in a 2 × 2 contingency table, and a
2 test was applied to determine whether
there was a statistically significant association between the presence
of NF immunostaining and mouse genotype (Lang and Secic, 1997).
p < 0.05 was considered statistically significant.
Light microscopy and quantitative morphologic analysis. Mice
were killed with intraperitoneal chloral hydrate (3%) overdose. The sciatic nerves were atraumatically excised from 5 mm proximal to
the sciatic notch to the trifurcation in the popliteal fossa and
processed. Nerves were fixed in 2.5% glutaraldehyde in a 0.025 M sodium cacodylate buffer, postfixed in 1%
osmium tetroxide (OsO4) in a 0.1 M sodium cacodylate buffer, dehydrated in
ethanol, and embedded in Epon-Analdite. Semithin sections (1 µm) were
obtained from sites illustrated in Figure 3A-C and stained
with toluidine blue in 1% sodium borate for light microscopy.
Quantitative histomorphometric assessment to determine the regenerating
nerve fiber number in the crush model was performed. Light microscopic
representative fields of the toluidine-blue stained sections were
photographed, and the images were digitized. The nerve fiber population
was analyzed based on a gray/white scale by computer-linked
morphometric software (Sigmascan). Statistical analysis was performed
using SPSS 10.0 for Windows (SPSS, Inc., Chicago, IL). ANOVA,
using a general linear model with location of assessment as the
repeated measure and genotype [PTP(/) or sibling] and time as
the group effects, was performed. An overall p value of
<0.05 was defined as significant. An experienced pathologist, blinded
to the experimental group, assessed the extent of Wallerian
degeneration present in the nerve segments, assessed the state of the
perineurium, and graded the extent of extrafascicular regeneration
(regeneration outside of the original nerve fascicle) in the
transection and allograft models as normal or increased. To determine
whether the extent of extrafascicular regeneration was significantly
different between groups in the allograft model, the data were
organized in contingency tables and analyzed by a
2 test assessing the association
between the genotype of the environment through which the fibers were
regenerating and the presence of increased extrafascicular regeneration
(Lang and Secic, 1997). A p value of <0.05 was defined as significant.
LacZ staining. PTP(/), PTP(+/), and PTP(+/+)
mice underwent sciatic nerve transection as described previously and
were killed 2 weeks after injury. The sciatic nerves in continuity with
their DRG from both the operated and contralateral control hindlimbs
were excised and fixed in a 0.1 M sodium
phosphate buffer, pH 7.9, containing 1% formaldehyde, 0.1%
glutaraldehyde, 2 mM MgCl2,
and 5 mM EGTA for 4 hr at 4°C. Nerves were
subsequently washed with four exchanges of a wash buffer (2 mM MgCl2, 0.01% deoxycholate, and 0.02% NP-40 in 0.1 M sodium
phosphate buffer, pH 7.9) at room temperature over 2 hr. The tissue was
then incubated in PBS containing 5 mM
ferricyanide, 5 mM ferrocyanide, 2 mM MgCl2 and 0.1 mg/ml
5-bromo-4-chloro-3-indolyl- -D-galactopyranoside (Roche, Hertforshire, UK) overnight at 37°C. Nerves were subsequently rinsed in 70% EtOH, embedded in paraffin, and sectioned (10 µm).
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RESULTS |
Three standardized nerve injury models were used to evaluate
peripheral nerve regeneration: sciatic nerve crush, sciatic nerve transection and microsurgical repair, and sciatic nerve allografting. PTP(+/) animals are phenotypically indistinguishable from
PTP(+/+) animals and were used interchangeably as controls
throughout the experiments.
Sciatic nerve crush
Sciatic nerve crush (axonotmesis injury) is a model of fully
reversible nerve injury (Seddon, 1943). Although the axonotmesis injury
requires a regenerative response from the proximal nerve, no
directional challenges face the regenerating fibers because they are
directed down the intact endoneurial sheath.
After crush injury, the PTP(/) mice demonstrated a more rapid
return of muscle function electrophysiologically, as evidenced by
greater amplitude of the CMAP compared with control animals (Fig.
1). The amplitude of the CMAP is directly
proportional to the number of nerve fibers innervating the muscle.
These data therefore suggest a faster rate of nerve regeneration in the
PTP(/) mice. Measurements were taken from two target muscles:
the gastrocnemius, which is 15 mm from the crush site, and the foot
intrinsics, which are ~30 mm distal to the crush. At 2 weeks after
injury, the CMAP amplitude recorded from the gastrocnemius muscle of
the PTP(/) animals was significantly greater than that of the
sibling controls (Fig. 1A), indicating that more
nerve fibers had regenerated to and reinnervated the gastrocnemius of
the PTP(/) mice. By 4 weeks after injury, the sibling controls
had "caught up" to the PTP(/) mice, with no significant
difference noted in the amplitude of the CMAP between the groups. At 6 weeks, a response became evident in the more distal musculature, the
foot intrinsics, with the CMAP amplitude being again greater in the
PTP(/) mice compared with control PTP(+/) and PTP(+/+)
mice (Fig. 1B). Therefore, both the gastrocnemius and
foot intrinsic muscles demonstrated sequentially faster reinnervation
in the PTP(/) mice, suggesting a faster rate of nerve
regeneration in the knock-out animal.
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Figure 1.
Electrophysiologic assessment of sciatic nerve
function after axonotmesis. The amplitude of the CMAP (mean ± SEM) recorded from the gastrocnemius (A) or foot
intrinsics (B) reflects and is proportional to
the degree of reinnervation of the muscle. PTP(/) mice
(n = 11) demonstrated an earlier return of
electrophysiologic response (as indicated by a greater amplitude of the
CMAP) in the gastrocnemius muscle at 2 weeks (*p < 0.05; Student's t test) and intrinsic muscles at 6 weeks (* p < 0.05; Student's
t test) compared with PTP(+/) or PTP(+/+)
sibling controls (n = 11).
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The differences noted in the rate of return of the CMAP cannot be
attributed to a size discrepancy between the experimental and control
mice. Although the adult PTP(/) mice are 25-50% smaller by
weight than their PTP(+/) or PTP(+/+) siblings, they are
essentially lean mice with no statistically significant differences in
the length of their hindlimb nerves. The sciatic nerve trifurcates
midway down the leg into the tibial, sural, and peroneal nerves. The
sciatic and a proximal branch of the tibial innervate the gastrocnemius
muscle. The distal tibial nerve terminates in and innervates the foot
intrinsic muscles. The length of the sciatic and proximal tibial
nerves innervating the gastrocnemius was 13.8 ± 1.8 mm in the
PTP(/) mice (n = 6) and 15.9 ± 2.7 mm in
the PTP(+/)/PTP(+/+) mice (n = 7)
(p > 0.05; not significant by Student's
t test). Similarly, the length of the sciatic and distal
tibial innervating the foot intrinsic muscles was 29.3 ± 3.6 mm
in the PTP(/) mice (n = 10) and 33.0 ± 4.4 mm in the PTP(+/)/PTP(+/+) mice (n = 7)
(p > 0.05; not significant by Student's
t test).
Preoperative nerve conduction studies confirmed marginally slower NCVs
and an intact CMAP in the PTP(/) mice compared with sibling
PTP(+/) or PTP(+/+) controls, as reported previously (Wallace
et al., 1999) in mice of this age group (data not shown). The slower
NCV seen in the PTP(/) group is the result of an increased
proportion of slowly conducting smaller diameter fibers (Wallace et
al., 1999), which seems to stem from developmental delay of the
PTP(/) mice. As anticipated, at the endpoint in this study, the
NCV approached the preoperative baseline in all animals, indicating
full electrophysiologic recovery (data not shown).
After crush injury, the serial walking track analyses demonstrated
significant differences in the rate of recovery of normal gait between
the PTP(/) mice and the sibling controls (Fig. 2). Hindlimb neuromuscular function can
be measured noninvasively by evaluating gait. Measurements of the
footprints from the experimental side can be serially collected to
follow nerve recovery, because characteristics of the footprint reflect
the functional muscle groups. Footprint analysis and measurement of
specific parameters are taken to determine the PLF, TSF, and ITF (Bain
et al., 1989). Formulas have also been derived and validated that
combine all relevant footprint measurements into a global index of
function (i.e., SFI) (Bain et al., 1989). Better factor scores indicate a more normal footprint and gait. Significantly better TSF and ITF
scores were measured at 1 and 1.5 weeks, respectively
(p < 0.05) in the PTP(/) mice compared
with PTP(+/) and PTP(+/+) control mice. The SFI score was also
significantly improved in the PTP(/) mice compared with controls
at 1 and 1.5 weeks after injury. After 2 weeks, there were no
significant differences noted as the PTP(+/) and PTP(+/+) mice
"catch up" to the PTP(/) mice. By 4 weeks, all test groups
had normalized their walking track parameters and had full recovery of
gait. These results suggest a faster rate of initial nerve regeneration
in the PTP(/) animal.
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Figure 2.
Walking track analysis after axonotmesis. Each
representative print characteristic is shown: A, PLF;
B, TSF; C, ITF; D, SFI
(mean ± SEM). For each factor, the value 0 represents normal
function (no difference between experimental and normal sides).
Significantly better TSF and SFI scores at 1 week
(*p < 0.05) and significantly better ITF and SFI
scores at 1.5 weeks (*p < 0.05) were noted in the
PTP(/) animals (n = 12) compared with
controls (n = 11). This is indicative of faster
regeneration of motor axons to correct motor targets. There were no
significant differences noted after 2 weeks, because the PTP(+/)
or PTP(+/+) mice catch up to the PTP(/) mice. By 4 weeks,
each factor score corrected to zero (normal function) as expected for
this injury.
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Sciatic nerves for NF fluorescent immunostaining were obtained at 0, 2, 4, and 6 weeks after axonotmesis injury. The sciatic nerves were
sectioned for analysis as illustrated in Figure
3A. Nerve sections were
examined blinded to experimental group and assessed for the presence of
NF immunofluorescence. NF immunoreactivity was evident in the distal
segment of 67% of PTP(/) mice by week 2 but was only seen in
50% of the PTP(+/) or PTP(+/+) animals
(p < 0.05 by 2
test) (Fig. 4A,B). The
earlier appearance of neurofilaments at a precisely specified distance
distal to the crush injury is evidence for a faster rate of neural
regeneration in the PTP(/) mice.
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Figure 3.
Sites of surgical intervention and histologic
assessment for the three experimental models. A, Sciatic
nerve axonotmesis (crush). The cross-hatched bar
represents the region of crush. Three millimeter longitudinal samples
at both the injury site and distally, in addition to transverse
sections at the injury site and distally, were obtained for
neurofilament immunofluorescence (NF) and light
microscopy (LM), respectively. B,
Sciatic nerve transection and repair. The suture represents the site of
transection and immediate microsurgical repair. Three millimeter
longitudinal samples at the injury site and distally, in addition to
transverse sections (proximal and distal), were obtained for NF and LM,
respectively. C, A 1-cm-long phenotypically disparate
allograft was repaired to the transection deficit in the PTP(/)
and PTP(+/) or PTP(+/+) mice. Sections for LM were obtained as
indicated by the arrows (proximal, through the graft,
and distal).
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Figure 4.
RT97 neurofilament immunostaining of crushed
nerve. Sections are 3 mm longitudinal standardized for distance from
crush site (10 mm). Adequate sections for visualization were obtained
from 10 PTP(+/)/PTP(+/+) controls and 13 PTP(/) animals.
At 2 weeks after injury, NF immunostaining (bright fluorescent
red) is evident in the PTP(/) mouse
(B) but only background staining (muted
red) is seen in the PTP(+/+) control
(A). Scale bar, 100 µm.
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After crush injury, the evaluation of the nerve cross sections by light
microscopy revealed characteristic changes of Wallerian degeneration in
the distal nerve stump and concurrent axonal regeneration (data not
shown). Quantitative evaluation of the regenerating fiber population
confirmed earlier regeneration in the PTP(/) animals compared
with PTP(+/) and PTP(+/+) controls. The regenerating fiber
number (Fig. 5) was significantly greater
at earlier stages at matched sampling sites in the PTP(/)
animals. The presence of more fibers at a standardized distance (10 mm)
from the crush site confirms a faster rate of regeneration in the
knock-out mice. Differences between experimental groups diminished by 3 weeks after injury. In all groups, as anticipated, improvement was
observed over time but did not normalize by 3 weeks.
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Figure 5.
Quantitative nerve fiber counts (mean ± SEM)
performed on semithin light-microscopy cross sections 10 mm distal to
nerve crush at matched sites (Fig. 3A). PTP(/)
animals had significantly more fibers regenerate into the distal stump
by days 7 and 14 compared with controls. By 21 d, the PTP(+/)
and PTP(+/+) controls had caught up and had an equivalent number of
fibers (*p < 0.05; n = 4 mice/group).
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Sciatic nerve transection and immediate repair
Sciatic nerve transection and microsurgical repair is a more
severe insult than axonotmesis. It is a dual challenge testing both the
regenerative capacity of the nerve and the axonal guidance system
because the endoneurial sheath has been completely disrupted. Complete
recovery does not occur after this injury, and the degree of function
regained is often extremely variable (Seddon, 1943).
After nerve transection and repair, as expected, large variance in CMAP
and walking track parameters was noted within all test groups. No
statistically significant difference was seen in the recovery of CMAP
amplitude or normal gait between the PTP(/) and PTP(+/) or
PTP(+/+) controls (data not shown).
NF immunostaining was performed on segments procured after transection
and immediate repair as illustrated in Figure 3B. After nerve transection and repair, it is common to see some fiber
misalignment. However, in the PTP(/) animals we saw a much
greater proportion of disorganized fibers at the neurorrhaphy site
(Fig. 6A) compared with
the sibling controls (Fig. 6B). The distal segment of
the nerve in the control animals had straighter and more parallel NF-positive fibers (Fig. 6D) compared with
PTP(/) mice (Fig. 6C). This suggests difficulties
with axonal pathfinding in the PTP(/) mice.
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Figure 6.
RT97 neurofilament immunostaining of longitudinal
sections through transected and repaired nerves. In PTP(/)
animals, more disoriented, transverse, and oblique fibers are evident
at the repair site (A) and distally
(C) compared with PTP(+/) or PTP(+/+)
controls (B, repair site; D, distally).
Arrows mark "end on" transverse fibers, and the
asterisk marks one of the oblique fibers. The
large black holes in A and
B are the suture sites. Scale bar, 100 µm.
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After sciatic nerve transection with immediate repair, the evaluation
of the nerve cross sections by light microscopy revealed characteristic
changes of Wallerian degeneration in the distal nerve stump and
concurrent axonal regeneration (data not shown). Several qualitative
differences between the test groups were indicative of errors in axonal
guidance in the PTP(/) mice. After transection in the normal
peripheral nerve, the large majority of axons will correctly regenerate
into the established fascicles of the distal segment, but it is typical
for a small proportion of fibers to err and regenerate outside the
fascicles (i.e., extrafascicular regeneration). In the PTP(+/)
and PTP(+/+) control animals, we noted a normal proportion of
extrafascicular fibers consistently arranged in "mini-fascicles"
within a well organized perineurium (Fig.
7B). In contrast, there was an
increase in the proportion of the extrafascicular fibers in the
PTP(/) animals and a large numbers of fibers that were obliquely
or transversely oriented (Fig. 7A). In addition, the
perineurium generally tended to be thinner and less well organized in
the PTP(/) group. Collectively, these findings suggest an
increased incidence of directional errors in the regenerating axon of
the PTP(/) mouse.
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Figure 7.
Light microscopic evaluation of sciatic nerve
cross sections from the transection model. In PTP(/) mice, more
disoriented, transverse, and oblique fibers are evident at the repair
site (A) compared with PTP(+/) or
PTP(+/+) controls (B). Arrows
mark extrafascicular fibers within mini-fascicles,
arrowheads indicate misdirected oblique fibers, the
perineurium is marked p, and an asterisk
marks a normal circumferential cross-section fiber. Scale bar, 100 µm.
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Allografts
Sciatic nerve allografting was undertaken to assess the
interaction of the regenerating axonal fibers, the Schwann cell
population, and other elements of the regenerative milieu.
Striking differences in patterns of axonal regeneration were seen in
the allografted animals. At 6 weeks after allografting, sciatic nerve
segments were obtained from sites as illustrated in Figure
3C. In the proximal nerve segment, PTP(/) mice
demonstrated extensive extrafascicular regeneration with a large number
of fibers moving retrograde (i.e., proximally) (Fig.
8D) in comparison with
PTP(+/) and PTP(+/+) controls (Fig. 8A).
These PTP(/) recipients of PTP(+/) and PTP(+/+) grafts
seemed to have some correction of their directional errors within the
graft, because sampling here revealed fewer extrafascicular fibers
(Fig. 8E). In contrast, when the control PTP(+/)
and PTP(+/+) fibers regenerated into the PTP(/) graft,
directional cues seemed to be lost, because extensive extrafascicular
regeneration was evident (Fig. 8B). Once the fibers
had completely traversed the graft and re-entered the host distal
nerve, the patterns of regeneration reversed. PTP(/) fibers
resumed an inaccurate course in the distal nerve segment, with
increased extrafascicular regeneration (Fig. 8F). However, there were a limited number of extrafascicular fibers observed
in the PTP(+/) and PTP(+/+) distal segments (Fig. 8C).
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Figure 8.
Light-microscopic evaluation of sciatic nerve
transverse sections from the allograft model. The typical histologic
pattern of regeneration in the grafted animals is shown proximal to the
graft (A, D), through the midportion of the allograft
(B, E), and distal to the graft (C,
F). Axons that grew outside the nerve (extrafascicular)
have a small likelihood of regaining contact to their original end
organ. An increased proportion of extrafascicular fibers is seen
proximally (D) and distally
(F) in the PTP(/) animal, with some
correction of growth through the PTP(+/) or PTP(+/+) allograft
(E). In contrast, the organized regeneration of
PTP(+/) and PTP(+/+) fibers proximally and distally
(A and C, respectively) is lost as the
axons traverse the PTP(/) graft (B).
Arrows indicate extrafascicular fibers.
p, Perineurium. Scale bar, 100 µm.
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The extent of extrafascicular regeneration in all microscopic sections
was graded as normal or increased to permit statistical analysis of the
data. In the PTP(+/)/PTP(+/+) mice (receiving the homozygote
allograft; n = 9), extensive extrafascicular
regeneration was seen in the proximal nerve segment of 33% of animals,
increased dramatically to 100% of animals within the graft, and was
noted in only 12.5% in the distal nerve. In contrast, in the
PTP(/) mice (receiving the heterozygote/wild-type allograft;
n = 7), extensive extrafascicular regeneration was
evident in the proximal nerve segment of 57% of animals but decreased
to 40% of animals within the graft. A
2 test assessing the association
between the genotype of the environment and the extent of
extrafascicular regeneration was statistically significant
(p = 0.0341).
Because the -galactosidase gene replaces PTP in the
knock-out cassette (Wallace et al., 1999), LacZ staining was performed to localize the pattern of PTP expression within the regenerating nerve. Our results show that blue LacZ staining is increased in the
operated nerve compared with the control contralateral unoperated nerve
(Fig. 9A,C,E). LacZ staining
was evident in the DRG (Fig. 9A), was increased throughout
the nerve on the operated side (Fig. 9C), and was
markedly intensified in the location of the advancing growth cone
within the transected nerve (Fig. 9E). Figure 9D
reveals the expression of LacZ in a cobblestone pattern around the
axons, indicating the presence of PTP in the supportive milieu of
the regenerating nerve. A higher magnification in Figure 9F
localizes LacZ staining to the Schwann cells. Unfortunately, because
of a technical limitation resulting from the mode of
construction of the knock-out cassette (where an internal ribosome
entry site immediately precedes the -galactosidase gene), we
are not able to test for the presence of PTP along the extended
axons using LacZ staining. However, Figure 9B does reveal
LacZ staining within the sensory neurons of the DRG, as has been
observed previously (Haworth et al., 1998). Thus, the regenerating
nerve exhibits enhanced expression of PTP.
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Figure 9.
LacZ staining of the operated
(O) and contralateral unoperated
(U) sciatic nerves 2 weeks after transection
injury in a PTP(/) mouse. Blue LacZ staining
indicates the pattern of PTP expression, because the PTP
knock-out cassette contains the -galactosidase gene. LacZ staining
is evident in the DRG (A; arrows) and is
markedly increased in the operated sciatic nerve
(C), particularly in the area of the advancing
growth cone (E). LacZ staining is apparent in the
sensory cell bodies of the DRG (B). Scale bar,
100 µm. D, A transverse section taken through the
proximal end of the regenerating growth cone reveals a cobblestone
pattern of LacZ staining in the supportive milieu of the axon. Scale
bar, 100 µm. F, Higher magnification localizes LacZ
staining to the Schwann cell. Scale bar, 20 µm. No LacZ staining was
evident in the PTP(+/+) controls (data not shown).
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DISCUSSION |
Peripheral nerve regeneration was significantly faster in the
PTP(/) mouse after sciatic nerve axonotmesis injury according to
all assessment techniques. In addition, the accurate navigation of the
transection site after nerve division was associated with greater
errors in the PTP(/) cohort compared with controls, and the
environment in which the regeneration occurs (heterozygote or
homozygote allograft) affects the rate of such errors.
In vitro studies have demonstrated that the state of
tyrosine phosphorylation within the neuron affects the rate of neurite outgrowth. Using confocal laser-scanning microscopy and immunoelectron microscopy, Shirasu et al. (1998) demonstrated tyrosine-phosphorylated proteins concentrated in the advancing growth cones of cultured mouse
DRG. Protein tyrosine kinase inhibitors such as genistein and
the tyrosine phosphatase inhibitor vanadate alter in vitro neurite outgrowth in cell-culture systems (Worley and Holt, 1996; Weeks
et al., 1999; Tisi et al., 2000).
The developmentally regulated expression of the LAR family PTPs
(PTP, PTP, and LAR) within the mammalian nervous system (Mizuno
et al., 1994; Sahin et al., 1995; Wang et al., 1995) supports a role
for these enzymes in nervous system growth and development. The
invertebrate LAR family PTP homologues DLAR (Drosophila) and HmLAR2 (leech) and the chicken and Xenopus CRYP (homolog
of PTP) localize to growth cone membranes and/or neurons during
periods of axonogenesis (Stoker et al., 1995; Desai et al., 1996;
Gershon et al., 1998; Johnson and Holt, 2000). This specifically
suggests a role for these enzymes in neurite extension and axonal
guidance. Indeed, Kreuger et al. (1996) have demonstrated defects in
axonal pathfinding in DLAR-deficient Drosophila. The motor
ISNb failed to exit the main intersegmental nerve trunk at a key branch
point and bypassed its target muscles in the mutant fly. In the leech, disruption of HmLAR2 function with an antibody directed against the
ectodomain (Gershon et al., 1998) or an inhibitory fusion protein
(Baker et al., 2000) resulted in decreased and aberrant intersecting
outgrowth of the neuron-like Comb cell trajectories. In avian and
Xenopus retinal explant models, CRYP has been shown to
modulate retinal axon extension (Ledig et al., 1999; Johnson et al.,
2001).
Until now, the role of PTPs in mammalian neuronal development, and
specifically axon guidance, has not been determined. The LAR knock-out
mouse demonstrates a mild neurological deficit (Van Lieshout et al.,
2001). The PTP knock-out mouse (Uetani et al., 2000) has
abnormalities of long-term potentiation, and PTP knock-out mice
(Elchebly et al., 1999; Wallace et al., 1999) demonstrate significant
developmental abnormalities of the CNS and PNS. To further investigate
the role of PTP in the mammalian PNS, the response of the sciatic
nerve to three standardized models of nerve injury and repair was
assessed. Studies evaluating nerve regeneration contribute to the
understanding of developmental mechanisms because many of the processes
responsible for neuronal development are recapitulated during
peripheral nerve regeneration (McComas, 1996).
The sciatic nerve crush model for axonotmesis injury challenges the
motor and sensory axons to regenerate to their targets; however, the
intact endoneurial sheath minimizes pathfinding requirements. After
crush injury we found an increased rate of nerve regeneration in
PTP(/) mice compared with PTP(+/) or PTP(+/+) mice, as determined by histologic, electrophysiologic, and neuromuscular testing. This work therefore assigns PTP a negative regulatory role
in axonal regenerative growth. The loss of PTP results in an
increased rate of peripheral nerve regeneration.
Using the same model of nerve injury applied to the LAR-deficient
mouse, Xie et al. (2001) found that the loss of LAR significantly decreased the rate of sciatic nerve regeneration. Therefore in mammals
the various members of the PTP LAR family seem to serve unique roles
within the PNS and affect the rate of nerve regeneration differentially. Johnson et al. (2001) reported differential effects for
the LAR family members in the developing Xenopus visual
system. Putative dominant-negative mutants (catalytically inactive
cytoplasmic domains) of LAR, PTP, and CRYP were expressed either
singly or in combination in the Xenopus retinal ganglion
cells (RGCs). Dominant-negative PTP inhibited RGC axon outgrowth,
but dominant-negative CRYP increased the rate of RGC axon outgrowth.
The LAR construct had no effect. In an embryonic avian visual system,
blocking of the CRYP-putative ligand interaction with an
anti-CRYP antibody or ectodomain fusion protein (to block the
ligand) resulted in significantly reduced axon outgrowth of the RGCs on
intact basal lamina (Ledig et al., 1999). Subsequently, Johnson et al.
(2001) proposed a model whereby the interaction of CRYP with its
ligand induces the inactivation of the CRYP phosphatase activity,
which results in the promotion of axon outgrowth by
recruiting/activating a currently unidentified signaling cascade. Our
work also shows that the loss of PTP enhances axon regenerative
growth. However, evidence for PTP ectodomain shedding (Rotin et al.,
1994; Aicher et al., 1997) and the fact that the PTP-deficient mouse
is a full-length knock-out lacking both the CAM-like ectodomain and the
intracellular phosphatase domains (Wallace et al., 1999) does not allow
us to conclude that it is specifically the downregulation of catalytic
activity that alters axon regeneration within the mammalian PNS.
We also conclude from histologic assessment of both the transection
with immediate repair and transection with allograft models that
peripheral nerve regeneration in the PTP(/) mouse shows errors
in axonal guidance. The transection injury model challenges both
regeneration and axon pathfinding because of the disruption of the
endoneurial sheath with loss of axon alignment. We noted an increased
proportion of extrafascicular regenerating axons and more fiber
misdirection in the PTP(/) mice compared with PTP(+/) or
PTP(+/+) mice. These observations are in keeping with the presence
of abnormal axon guidance in the Drosophila DLAR
loss-of-function mutant (Krueger et al., 1996) and misdirection of Comb
cell processes in the leech in which HmLAR2 is inactivated (Baker et
al., 2000).
The transection and allograft model also permitted evaluation of the
relative importance of PTP expression in the regenerating milieu on
axon guidance. PTP(+/) and PTP(+/+) controls demonstrated normal parallel axonal regeneration in the proximal and distal nerve
(where PTP is expressed within the environment) yet lost direction when traversing the PTP(/) graft (an environment lacking PTP). In contrast, the PTP(/) animals consistently demonstrated regenerating axon guidance errors that partially corrected
when the PTP(/) axons traversed the heterozygote/wild-type allografts (i.e., an environment expressing PTP). Thus, the
expression of PTP in the regenerating milieu clearly appears to be
required for regenerating axons to avoid directional errors.
LacZ staining patterns demonstrated the presence of PTP within the
Schwann cell and the supportive matrix of the regenerating nerve. The
Schwann cell is a key player in the environment of the axon and
provides a supportive, growth-promoting medium during regeneration. In
contrast, Haworth et al. (1998), using in situ hybridization, were unable to demonstrate the presence of PTP in the
neuronal support cells of the rat DRG after sciatic nerve crush. In
that model, upregulation of PTP expression occurred exclusively
within the DRG sensory neurons. The reason for the apparent discrepancy
is unclear, but it may result from different methodologies used or the
region (DRG vs regenerating growth cone) assessed.
Although the presence of PTP within the regenerating environment is
essential to correct axon pathfinding, the advancing growth cone may
not require, or can adapt to, the absence of PTP. This is suggested
by the partial correction of PTP(/) regenerating axon
trajectories within the heterozygote/wild-type allografts. The presence
of both homophilic and heterophilic interactions occurring within the
LAR family PTPs that regulate axonal outgrowth has been demonstrated.
For example, Baker et al. (2000) have shown that homophilic
interactions between HmLAR2 molecules on the growth cones of the leech
Comb cells maintain their extending trajectories in a normal parallel
pattern distant from their neighbors. PTP has been shown to be a
homophilic neurite promoting cell-adhesion molecule for CNS neurons
(Wang and Bixby, 1999; Sun et al., 2000). In contrast, heterophilic
interactions have been proposed for CRYP expressed in RGCs, with
cell- and matrix-associated ligands along the retinotectal projection
(Haj et al., 1999). One can therefore speculate that the errant fibers
in the PTP(/) mice require a heterophilic interaction to
correctly cross the transection site and enter the endoneurial
compartment of the graft or distal stump. Proper axonal guidance
necessitates the expression of PTP within the environment of the
regenerating axon, but its presence on the advancing growth cone is not
required for the select process of directional navigation. Other CAMs,
including other LAR family members, may fulfill this role on the growth cone.
Alternatively, we cannot exclude the possibility that PTP homophilic
interactions occur within the mammalian PNS. In this scenario, PTP
plays an important role within the regenerating environment, but one
could speculate that a closely related LAR family member could
substitute for the loss of PTP on the growth cone and "rescue"
the PTP-deficient axon. Definitive determination of the mechanism
involved awaits the identification of the ligand and substrate for
PTP. In Drosophila, the precise axonal responses of ISNb
to guidance cues in the environment are likely controlled by the
collaboration between the tyrosine kinase Abl and its substrate Enabled, balanced by the PTP DLAR (Wills et al., 1999). Unfortunately, the substrate(s) and ligand(s) for PTP and the signaling pathways involved to regulate its effects on neuronal growth and function are unknown.
The quiescent sciatic nerve of the PTP(/) mouse does not
demonstrate an increased proportion of extrafasicular fibers and possesses a normal well organized perineurium (Wallace et al., 1999).
This is likely attributable to the fact that during development, misdirected and improperly targeted neurites and fibers within the PNS
are pruned and therefore would not be seen in the postnatal animal.
In summary, we have demonstrated a critical role for PTP in three
in vivo models of peripheral nerve injury, repair, and regeneration. The loss of PTP results in faster peripheral nerve regeneration and significant abnormalities of axon guidance. The definitive mechanism responsible and the signaling pathways involved await identification of ligand(s) and substrate(s) for PTP.
Note added in proof. Since the acceptance of
this paper, a recent report has identified two ligands for CRYP
(PTP) (Aricesku et al., 2002).
| |
FOOTNOTES |
Received Sept. 10, 2001; revised Feb. 26, 2002; accepted April 5, 2002.
*
J.M. and J.B. contributed equally to this manuscript.
This work was supported by an Ontario Neurotrauma grant (to J.R.B.) and
a Canadian Institute of Health Research (CIHR) grant (to D.R.). D.R. is
the recipient of a CIHR Investigator Award and J.B. is supported by a
CIHR Fellowship Award.
Correspondence should be addressed to James R. Bain, 1200 Main Street
West, 4E16, Hamilton, Ontario, Canada, L8N 3Z5. E-mail: bainj{at}hhsc.ca.
| |
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Y. Shen, A. P. Tenney, S. A. Busch, K. P. Horn, F. X. Cuascut, K. Liu, Z. He, J. Silver, and J. G. Flanagan
PTP{sigma} Is a Receptor for Chondroitin Sulfate Proteoglycan, an Inhibitor of Neural Regeneration
Science,
October 23, 2009;
326(5952):
592 - 596.
[Abstract]
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J. C. Elfar, J. A. Jacobson, J. E. Puzas, R. N. Rosier, and M. J. Zuscik
Erythropoietin Accelerates Functional Recovery After Peripheral Nerve Injury
J. Bone Joint Surg. Am.,
August 1, 2008;
90(8):
1644 - 1653.
[Abstract]
[Full Text]
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S. Lee, C. Faux, J. Nixon, D. Alete, J. Chilton, M. Hawadle, and A. W. Stoker
Dimerization of Protein Tyrosine Phosphatase {sigma} Governs both Ligand Binding and Isoform Specificity
Mol. Cell. Biol.,
March 1, 2007;
27(5):
1795 - 1808.
[Abstract]
[Full Text]
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R. Siu, C. Fladd, and D. Rotin
N-Cadherin Is an In Vivo Substrate for Protein Tyrosine Phosphatase Sigma (PTP{sigma}) and Participates in PTP{sigma}-Mediated Inhibition of Axon Growth
Mol. Cell. Biol.,
January 1, 2007;
27(1):
208 - 219.
[Abstract]
[Full Text]
[PDF]
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N. Uetani, M. J. Chagnon, T. E. Kennedy, Y. Iwakura, and M. L. Tremblay
Mammalian motoneuron axon targeting requires receptor protein tyrosine phosphatases sigma and delta.
J. Neurosci.,
May 31, 2006;
26(22):
5872 - 5880.
[Abstract]
[Full Text]
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X. Wang, W. Hu, Y. Cao, J. Yao, J. Wu, and X. Gu
Dog sciatic nerve regeneration across a 30-mm defect bridged by a chitosan/PGA artificial nerve graft
Brain,
August 1, 2005;
128(8):
1897 - 1910.
[Abstract]
[Full Text]
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L. Stepanek, A. W. Stoker, E. Stoeckli, and J. L. Bixby
Receptor Tyrosine Phosphatases Guide Vertebrate Motor Axons during Development
J. Neurosci.,
April 13, 2005;
25(15):
3813 - 3823.
[Abstract]
[Full Text]
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D. G. Wansink, W. Peters, I. Schaafsma, R. P. M. Sutmuller, F. Oerlemans, G. J. Adema, B. Wieringa, C. E. E. M. van der Zee, and W. Hendriks
Mild impairment of motor nerve repair in mice lacking PTP-BL tyrosine phosphatase activity
Physiol Genomics,
September 16, 2004;
19(1):
50 - 60.
[Abstract]
[Full Text]
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J. Batt, E. Cutz, C. Fladd, and D. Rotin
Apparent normal lung architecture in protein tyrosine phosphatase-sigma -deficient mice
Am J Physiol Lung Cell Mol Physiol,
January 1, 2003;
284(1):
L214 - L223.
[Abstract]
[Full Text]
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TOP
ABSTRACT
INTRODUCTION
