Abstract
purpose. Interest in neuroprotection for optic neuropathies is, in part, based
on the assumption that retinal ganglion cells (RGCs) die, not only as a
result of direct (primary) injury, but also indirectly as a result of
negative effects from neighboring dying RGCs (secondary degeneration).
This experiment was designed to test whether secondary RGC degeneration
occurs after orbital optic nerve injury in monkeys.
methods. The superior one third of the orbital optic nerve on one side was
transected in eight cynomolgus monkeys (Macaca
fascicularis). Twelve weeks after the partial transection, the
number of RGC bodies in the superior and inferior halves of the retina
of the experimental and control eyes and the number and diameter of
axons in the optic nerve were compared by detailed histomorphometry.
Vitreous was obtained for amino acid analysis. A sham operation was
performed in three additional monkeys.
results. Transection caused loss of 55% ± 13% of RGC bodies in the superior
retina of experimental compared with fellow control eyes (mean ±
SD, t-test, P < 0.00,001, n= 7). Inferior RGCs, not directly injured by transection,
decreased by 22% ± 10% (P = 0.002). The loss of
superior optic nerve axons was 83% ± 12% (mean ± SD, t-test, P = 0.0008, n= 5) whereas, the inferior loss was 34% ± 20%
(P = 0.02, n = 5). Intravitreal
levels of glutamate and other amino acids in eyes with transected
nerves were not different from levels in control eyes 12 weeks after
injury. Fundus examination, fluorescein angiography, and histologic
evaluation confirmed that there was no vascular compromise to retinal
tissues by the transection procedure.
conclusions. This experiment suggests that primary RGC death due to optic nerve
injury is associated with secondary death of surrounding RGCs that are
not directly injured.
In the central nervous system (CNS), injury from various
primary lesions, such as ischemia and trauma, can lead to widespread
damage to neurons beyond the initial injury site.
1 2 3 4 5 6 This
phenomenon is known as secondary degeneration and can result in greater
loss of tissue than that caused by the initial disorder. Moreover, it
may continue for an extended period after termination of the primary
event. The secondary death of neighboring neurons and glia is believed
to occur by apoptosis. A variety of mechanisms for this secondary
degeneration have been proposed, including alteration of extracellular
ion concentration, release of oxygen free radicals, and high levels of
excitatory neurotransmitters.
4 5 6 7 8 9
Because the optic nerve is part of the CNS, we decided to investigate
whether secondary degeneration occurs also in the optic nerve. Damage
to the optic nerve by diseases or trauma is one of the most frequent
causes of blindness in the world. Axonal injury within the optic nerve
leads inevitably to retrograde degeneration of retinal ganglion cells
(RGCs) whose axons make up the optic nerve.
10 11 12 13
The most common optic neuropathy is glaucoma. Glaucoma is the second
leading cause of visual loss worldwide, with loss of peripheral vision
due to the death of RGCs.
14 Studies of human and
experimental glaucomatous eyes point to the optic nerve head as a major
site of injury to RGCs.
15 16 17 At this location, RGC axons
show morphologic and physiological indications of obstructed axonal
transport. This may act as a functional transection of the axon at this
site. It is not known whether there is additional secondary
degeneration of RGCs that are not primarily injured in glaucoma or in
other clinical or experimental optic neuropathies. If present,
secondary degeneration could be a substantial additive factor in
glaucoma damage, and its therapy could represent an important new
avenue of treatment. Yoles and Schwartz
1 suggest that
secondary degeneration occurs in RGCs after crush injury to the rat
optic nerve, based on the fact that some RGCs die over a protracted
period after the insult. However, in their paradigm, all axons may have
been subjected to direct insult, making separation of primary and
secondary degeneration difficult.
Experimental glaucoma models using intraocular pressure elevation
cannot be used to test for secondary degeneration, because all RGCs are
presumably exposed to the primary insult. There are, to our knowledge,
no methods of identification that distinguish primary from secondary
RGC death. To attempt to identify secondary degeneration in RGCs more
clearly, we exploited the known topographic separation of RGCs in the
primate retina and optic nerve. RGC bodies are separated into upper and
lower retinal zones, divided by a horizontal raphe. RGCs with cell
bodies quite close together above and below the raphe send their axons
into the upper and lower poles of the optic nerve, where they are
widely separated.
18 We performed partial transections
of the upper third of the intraorbital nerve in monkeys, causing
primary degeneration of upper RGCs. Our assumption was that RGCs of the
inferior retina would be unaffected unless secondary degeneration
occurred. If secondary degeneration were detected, its magnitude,
distribution, and selectivity by RGC size class could then be
estimated.
Eleven cynomolgus monkeys (Macaca fascicularis) were
included in experiments, which were approved and supervised by the
Animal Care Committee of the Johns Hopkins University School of
Medicine and adhered to the ARVO Statement for the Use of Animals in
Ophthalmic and Vision Research. Before surgery, color photographs of
the optic disc, black and white photographs of the retinal nerve fiber
layer, and fluorescein angiograms were obtained with the monkeys under
intramuscular ketamine sedation (15 mg/kg) followed by Fluothane
anesthesia (Wyeth–Ayerst, Philadelphia, PA) delivered by endotracheal
intubation.
Eight monkeys underwent partial transection of one optic nerve, and
three monkeys underwent only a unilateral sham operation. Partial optic
nerve transection was performed under Fluothane anesthesia by a
transorbital approach. The side to be operated on was chosen randomly.
In brief, a conjunctival peritomy was performed and sutures placed
under the four rectus muscles to control the position of the eye. The
pupil was dilated with 1% tropicamide eye drops. After sterile
preparation of the skin, a curved incision following the eyebrow and
lateral orbital rim was made through skin and muscle. The lateral
orbital wall was removed for approximately 5 mm with a Stryker saw and
rongeurs. The optic nerve was identified after retraction of orbital
fat with cotton pledgets. The dura of the superior nerve was focally
incised with Vannas scissors at least 5 mm posterior to the globe. A
sharp blade was used to transect the upper one third of the nerve.
Animals in the sham operation group underwent the same procedure
including the dural incision, but the optic nerve was not transected.
The retinal and choroidal circulations were inspected immediately after
transection. None of the eyes had any detectable difference between the
surgical and nonsurgical sides in blood flow or retinal color. The
muscle sutures were removed and the peritomy closed with interrupted
Vicryl sutures (Ethicon, Piscataway, NJ). The facial muscle incision
was closed with 4-0 gut, and the skin was closed with interrupted 8-0
silk sutures. The eye was dressed with antibiotic ointment.
Animals were evaluated daily for 1 week for signs of distress and pain.
None had any indication of postoperative complication. Fluorescein
angiography was repeated 2 weeks after surgery, and nerve fiber layer
and optic disc photographs were repeated 1 and 3 months after
transection.
Twelve weeks after optic nerve surgery, the animals were killed by
exsanguination under Fluothane anesthesia. The eyes were rapidly
enucleated, and a slit was made in the pars plana with a razor blade.
One milliliter of vitreous humor was aspirated from the midvitreous and
immediately frozen. Aliquots of vitreous from transection and control
eyes underwent amino acid analysis by a method identical with that used
by Dreyer et al. (Bioresource Center, Cornell University, Ithaca,
NY).
19
The anterior segment was removed, and the posterior globe was fixed by
immersion in 4% paraformaldehyde in 0.1 M phosphate buffer. After
brief fixation, the retina was separated from the choroid and optic
nerve head, and relaxing incisions were made in five to six areas to
allow flat preparation, with one incision directly on the horizontal
raphe. The vitreous was carefully removed from the retinal surface. The
retina was stained with 0.05% cresyl violet and mounted with
photoreceptors against the slide.
After 2 hours of immersion in fixative, a 1-mm-thick portion of the
optic nerve was removed 1 to 3 mm from the globe, with razor slits
marking the superior (one slit) and nasal (two slits) meridians for
orientation after sectioning. The specimens were postfixed in 1%
osmium tetroxide, dehydrated in alcohol, and embedded in epoxy resin.
One-micrometer sections were stained with 1% toluidine blue.
One observer, masked to the procedure for each eye, used the retinal
wholemounts to quantify RGC density. At ×1000 magnification and using
a camera lucida and planimeter, 60 locations with an area of 0.022
mm
2 were identified along five circles from 1 to
5 mm in radius centered on the fovea
(Fig. 1) . An equal number of locations was counted in the superior and the
inferior half of each retina. The total area sampled per retina was
estimated to be 0.5% of total retinal area. RGCs were identified by
their presence in the innermost nuclear layer and by cell and nuclear
morphology. They exhibited large, round-to-oval nuclei, frequently with
visible nucleoli and with basophilic cytoplasm
(Fig. 2) . Their diameter was most often greater than 7 μm. Glial cells and
vascular endothelium and pericytes were easily distinguished from RGCs.
It is possible that a small proportion of the cells identified were
amacrine cells present in the RGC layer, although our previous
investigations show that amacrines are rarely larger than 7 μm in
diameter.
20 21
As a second independent approach to counting RGCs, we quantified the
number of RGC axons in the optic nerve cross section by a published
method.
22 Briefly, the nerve area was determined by
outlining it on a calibrated planimeter, and the area of connective
tissues was excluded by planimetry. The nerve was divided into 16
segments by four radial and one centroperipheral division, and the area
occupied by nerve bundles was measured planimetrically in each segment.
Sixty-four areas were sampled for axon density (four per segment) with
an image analysis system (Vidas; Carl Zeiss, Thornwood, NY). The total
number of axons in each segment and in the entire nerve was calculated
by multiplying the nerve bundle area of each segment by its density of
RGC axons. This method samples approximately 4% to 5% of the total
fibers in the normal monkey optic nerve. The analytic system measures
axon diameter within the myelin sheath, providing the minimum diameter
of each axon. Those performing the optic nerve counting were also
masked as to the procedure performed on each nerve.
The estimated number of RGC bodies in the retina and axons in the nerve
was compared between the overall nerves of transected and fellow eyes,
as well as in subdivisions of retina and nerve, including superior and
inferior halves. Because all retinas were treated similarly during
preparation and because our primary data compare the right and left
eyes of each animal, we did not correct any data for shrinkage.
Statistical significance of differences was evaluated with paired t-tests, Wilcoxon rank test, and linear regression
analysis.
Nerve fiber layer photographs were normal before transection in
seven of the eight eyes with transected nerves, and in all the
sham-transected eyes. In one animal, masked review of black and white
photographs showed that there was pre-existing loss of the nerve fiber
layer in both eyes temporally. The optic nerve cross sections of both
the surgical and control eyes confirmed that central temporal areas of
both nerves had axon loss. By analogy to human clinical entities with
similar patterns of RGC loss, this animal appeared to have had RGC loss
compatible with a toxic, nutritional, demyelinative, or genetic
disorder. Because such an animal would be inappropriate as a subject in
the experiment, its data were excluded.
Nerve fiber layer photographs were inspected in a masked manner. In the
remaining 10 animals, nerve fiber layer photographs were entirely
normal before surgery. For the seven animals with transected optic
nerves, we detected loss of superior nerve fiber layer at 1 and 3
months after transection. This clinical atrophy progressed to severe
loss of superior fibers in each partially transected nerve between the
two postoperative observation points. Inferior fiber loss was also
evident in five of seven animals at 1 month after surgery and was
clearly seen in all at 3 months.
Of the 14 nerves in seven animals with transected nerves for which
retinal data are presented, two optic nerves of two animals were
unsuitable for optic nerve fiber counting because of processing errors.
Thus, the nerve data consist of five partially transected and control
nerve pairs. Although our retinal data consisted only of density
values, for the optic nerves we have estimates of the density within
segments, number of axons in a segment, region or whole nerve, as well
as the diameter distribution of axons. In seven fellow control nerves,
the estimated number of fibers in the whole nerve was 1,018,352 ±
183,706, similar to our previous estimates for this monkey
species.
22
The distribution of axonal loss in each segment of the retina in
surgically altered eyes is shown in
Figure 4A . Comparing the superior eight segments of the transected nerves with
the fellow control nerves, we found an axonal loss of 83% ± 13%
(
P = 0.0008, paired
t-test,
n = 5
animals;
Table 2 ). The inferior eight segments of the transected nerves
had 34% ± 20% fewer axons than the fellow nerves (
P = 0.02, paired
t-test,
n = 5). Cross sections of
the transected optic nerves showed that the superior nerve had total
axon loss in most areas of the upper one third, corresponding to the
surgical incision. Loss in the remainder of the nerves was subtler by
direct inspection and was diffusely distributed throughout the nerve
(Fig. 5) .
The loss in the inferior optic nerve was clearly present in four of the
five nerves, with differences from control of 36% to 50%. In one
nerve, there was no detectable loss inferiorly, compared with its
control. This nerve appears to have had the lowest axonal loss in the
superior half of the nerve. Similar to the retinal density estimates,
we found no statistically significant relationship between degree of
inferior optic nerve axon loss and the severity of superior axon loss.
However, the power to estimate such a relationship was limited by the
modest number of animals and the relatively uniform superior loss in
those affected (P > 0.05, linear regression, n= 5).
The loss in RGC density was compared with loss of optic nerve axons in
regions of the retina and optic nerve that were thought to contain the
cell bodies and the axons of the same RGCs
(Fig. 3) . In general, the
greater the RGC density loss, the more the axonal loss in the segment
of the nerve that contains the fibers of those cells of origin,
although this was statistically significant only for the superotemporal
area of the retina and its corresponding superior pole of the nerve
(
P = 0.0018, paired
t-test,
n = 5).
This study found a substantial loss of RGCs and their axons in the
inferior retina and optic nerve of monkeys whose optic nerves had only
been transected in the superior half. In the superior optic nerve, the
areas subjected to transection were almost completely devoid of axons 3
months after injury, with 97% to 99% loss in the two most peripheral
superior segments of the nerve
(Fig. 4A) . The nerve area that was
evaluated is closer to the eye than the transection site; therefore,
the measures of atrophy presented here represent the effects of
retrograde degeneration. There was a 22% loss of RGC density in the
inferior retina and a 34% loss in inferior optic nerve axons,
comprising highly significant damage. The marked histologic loss of
inferior RGCs was confirmed by masked photographic detection of nerve
fiber layer atrophy in the inferior retina. The loss of inferior RGCs
in this experiment could have occurred either from primary injury to
inferior nerve axons during the surgery or from secondary degeneration.
We will detail below the evidence that supports secondary degeneration
as the more likely cause.
Primary injury to inferior RGCs and their axons hypothetically occurred
for one or more of the following reasons: the transection extended
beyond the middle half of the optic nerve; the manipulations during
surgery primarily injured inferior RGCs or their axons; some inferior
RGC axons that passed into the superior optic nerve were cut by
transection; and transection of blood vessels in the superior nerve
decreased blood supply to the inferior nerve or cause bleeding that
could lead to axon degeneration. We are certain that extension of
transection into the inferior nerve did not occur. The monkey nerve is
3 mm in diameter in the orbit, and under direct observation with the
operating microscope, making a cut 1 mm deep into the tissue is a
straightforward procedure. Because transection leads to nearly complete
loss of all axons in the cut zone, nerves with a transection that is
too extensive would have 98% loss of axons in their inferior halves.
This was not observed in any of the specimens. The percentage loss of
axons was never greater than 70% in any individual inferior segment.
To evaluate whether inadvertent primary injury happened to inferior
RGCs, we performed a number of examinations, as well as conducting sham
operations. Clinical retinal examination and fluorescein angiography
provided no evidence that the surgery had compromised the blood supply
to the retina or choroid. If retinal ischemia had occurred during or
immediately after surgery, retinal edema would have been detected, and
nerve fiber layer loss would have occurred promptly. Neither was
observed. Histologic retinal examination showed no loss of the middle
or outer retinal layers that would have resulted from vascular
occlusion.
In eyes subjected to sham operation, some primary injury to the optic
nerve occurred, even when no direct nerve cutting was performed. In
each case, the most prominent atrophic zone in sham-transected nerves
was a wedge of peripheral loss superiorly, in the area where the
meninges had been opened, comprising much of the 19% axon loss in the
upper nerve. The retinal zones in which cell bodies were counted were
not affected at all by sham surgery. Therefore, this effect cannot be
the explanation for the decline in inferior RGCs in retinal counts in
eyes with partially transected nerves. We presume that axons were
injured during sham (and actual) transection in the far peripheral
optic nerve through the trauma of opening the meninges or by
interruption of their blood supply, which enters the nerve through
penetrating vessels in the meninges.
The diffuse loss of 14% of inferior axons is less easily explained as
direct primary trauma, although the globe was rotated and the orbital
vessels were presumably compressed during the surgery. It is clear that
the additional transection of the nerve in seven eyes produced more
than twice as much inferior RGC loss in the optic nerve as did the sham
surgery. The RGC density estimates in eyes with sham-transected nerves
showed no statistically significant decrease; however, the sampling
proportion in retinal counts was lower than in the nerve, and its
extent was limited to the central retina. Likewise, the clinical nerve
layer photographs showed no atrophy in eyes with sham-transected
nerves, expressing the mild level of damage caused by surgery without
transection.
The third manner in which primary degeneration could explain loss of
inferior RGCs in our transection experiment is random topographic
representation of RGCs in the optic nerve. Although there have been
scattered reports that axons from RGCs are not precisely ordered in the
nerve fiber layer of the retina or the optic nerve,
23 the
preponderance of evidence strongly supports an orderly
arrangement.
18 24 25 Clinical–pathologic correlations of
ocular disease with atrophy in optic nerve cross sections confirm such
regional organization.
26 If we had evaluated only RGC
density in the retina, our evidence against topographic wandering would
be much less convincing; however, we sampled optic nerve cross sections
1 to 3 mm behind the globe, with a transection that was performed at 5
to 6 mm behind the globe. For the nerve sections to have loss
of inferior axons that were misrouted to the superior nerve (and cut
there), more than 20% of inferior fibers would have to be in the
inferior nerve at 1 to 3 mm and move to the upper half of the nerve in
less than a 2-mm distance along the nerve. This appears so unlikely
that we have concluded that topographic considerations do not explain
our data.
We propose that our data indicate that much of the inferior RGC loss
after superior nerve transection was due to indirect, or secondary,
mechanisms. The magnitude of the secondary degeneration was impressive.
As many as one third of RGCs in the inferior retina died 3 months after
injury. We do not know how much damage occurred soon after the superior
nerve transection and how much might continue beyond the time of our
observations. Certainly, we observed progressive loss of the inferior
retinal nerve fiber layer between the 1-month and 3-month clinical
photographs. The death and disappearance of primarily injured RGCs
after transection is believed to be complete at 3
months.
13 However, it is reasonable to speculate that
secondary RGC degeneration could continue beyond the actual loss of the
primarily injured RGCs. Neufeld
27 detected activated
microglia in the optic nerve heads of human glaucomatous eyes. It could
be that initial RGC death begins a cascade of toxic processes mediated
by such effector cells.
It is interesting to hypothesize where the effects of primary injury
act to initiate secondary degeneration. The most obvious site would be
at the position of primary injury. In traumatized spinal cord, the
primary lesion is known to expand over time rostrally and caudally.
Crowe et al.
2 found apoptotic death of neurons and
oligodendrocytes in white matter tracts distant from the lesion site,
leading to demyelination of axons spared by initial injury. Similar
findings have been reported after transection of the optic nerve in
developing and adult rats.
28 In a variety of CNS
disorders, primary injury leads to secondary damage by creating a
hostile environment in the surrounding tissues.
29 30 31 In
the present experiment, the hostile environment may be in the orbital
optic nerve, leading to injury to axons of nontransected RGCs and
retrograde degeneration of their cell bodies. It is known that there is
a time lag of weeks between axonal injury and cell body death for
RGCs.
12 32 Our data suggested a greater proportionate loss
of optic nerve fibers than RGC bodies at 3 months after injury. It is
conceivable that this is an expression of axons that are already
injured and uncountable, whose cell bodies have not yet died. In this
regard, it would be evidence for the optic nerve as the site of
secondary degeneration. However, the differences in our retinal and
optic nerve counts may be a result of differences in methodology or
distribution of RGC loss.
It is equally likely that secondary degeneration occurs by toxic
effects of primarily dying RGC bodies in the retina. Dreyer et
al.
19 reported abnormal levels of glutamate in the
vitreous humor of experimental monkey and human eyes with glaucoma.
Perhaps the death of a large number of RGCs intraretinally disturbs the
normal mechanisms for limiting the extracellular concentration of
glutamate. Excitotoxic cell injury would result from stimulation of
N-methyl-
d-aspartate
receptors.
33 34 35 36 37 38 39 We did not find any difference in
glutamate levels in the vitreous at 3 months after injury between eyes
with partially transected nerves and control eyes although it is
possible that glutamate levels were high earlier than the time we
measured. We should stress that our experimental model was transection,
which may differ significantly from glaucoma, although both involve
optic nerve injury. More detailed sampling may be indicated at
different time points after transection. Such sampling was not
undertaken in this study to avoid the probable intraocular alterations
that would have resulted from multiple penetrations of the globe.
If the source of toxic effect for secondary degeneration is in the
retina, then greater death of RGC might be expected nearest to the
primarily dying RGCs. Our data do not provide definitive information
about this point. The regional data from the optic nerve also do not
show a clear trend for axons to die more often when they are closer to
the partial transection.
We investigated not only whether some RGCs die by secondary
degeneration, but whether particular RGCs were more likely to die. We
found no size selectivity in the superior optic nerve, but the near
total loss of axons in many superior areas made it unlikely that this
would be productive. There was, however, a significant trend for
smaller diameter axons to die preferentially in the inferior nerve. It
is intriguing that this apparent tendency toward small axon loss is
different from the susceptibility of the larger axons apparent in
human glaucoma.
40
The present result suggests that secondary degeneration can occur after
optic nerve injury. Although partial transection is different from
glaucomatous optic neuropathy, we still might consider the possibility
that secondary degeneration occurs in glaucoma. If this is true,
blocking secondary degeneration may provide additive protection in
glaucoma. Neuroprotection is a potential therapy that would intervene
in the death of neurons in novel ways.
8 9 41 42 43 44 If
secondary degeneration occurs in human optic nerve diseases, as we
believe occurred in this experimental setting, then neuroprotective
approaches might be devised to block the effector sequences that
emanate from primary injury and lead to secondary death.
In summary, we suggest that secondary degeneration may cause
substantial loss of RGCs as an indirect effect of the injury and death
of the transected RGCs. This study points to the need for further
investigations to explore the mechanisms of secondary degeneration, to
identify markers that distinguish it from primary degeneration, and to
prevent it by specific therapy.
Supported in part by US Public Health Service Grants EY02120 (HAQ) and
EY01765 (Core Facility Grant, Wilmer Institute); by the Glaucoma
Research Foundation, San Francisco, California; and by unrestricted
funding from Pharmacia & Upjohn, Kalamazoo, Michigan.
Submitted for publication August 3, 2000; revised November 22, 2000; accepted December 5, 2000.
Commercial relationships policy: N.
Corresponding author: Harry A. Quigley, Wilmer 120, Johns Hopkins Hospital, 600 North Wolfe Street, Baltimore, MD 21287.
[email protected]
Table 1. Vitreal Glutamate Levels of Eyes with Partially Transected Nerves and
the Controls
Table 1. Vitreal Glutamate Levels of Eyes with Partially Transected Nerves and
the Controls
Subject | Transected | Control |
M987 | 3.08 | 2.62 |
M988 | 3.20 | 4.38 |
M989 | 3.60 | 3.40 |
M990 | 3.40 | 3.10 |
M991 | 2.42 | 3.36 |
M992 | 1.00 | 4.84 |
M993 | 3.46 | 5.06 |
M994 | 3.22 | 3.06 |
Mean± SD | 2.92 ± 0.85 | 3.72 ± 0.9 |
Table 2. Percentage of Cells and Axons Lost in Eyes with Partially Transected
Nerves
Table 2. Percentage of Cells and Axons Lost in Eyes with Partially Transected
Nerves
Subject | RGC Body Loss | | Axonal Loss | |
| Superior | Inferior | Superior | Inferior |
987 | 62.4 | 15.1 | 68.3 | −1.5 |
988 | 50.3 | 33.0 | 100 | 50.3 |
990 | 44.7 | 6.0 | 72.3 | 37 |
991 | 69.2 | 21.2 | 88.0 | 41 |
992 | 44.7 | 29.8 | 84.5 | 43.9 |
993 | 72.0 | 32.8 | NA | NA |
994 | 39.4 | 15.1 | NA | NA |
Mean± SD | 55 ± 13 | 22 ± 10 | 83 ± 13 | 34 ± 20 |
Table 3. Percentage of Cells and Axons Lost in Eyes with Sham-Transected Nerves
Table 3. Percentage of Cells and Axons Lost in Eyes with Sham-Transected Nerves
Subject | RGC Body Loss | | Axonal Loss | |
| Superior | Inferior | Superior | Inferior |
995 | −16.9 | −23.4 | 3.7 | 7.7 |
997 | 9.8 | 3.6 | 19.5 | 12.4 |
998 | 1.0 | −1.5 | 34.4 | 24.6 |
Mean± SD | −2 ± 13 | −7 ± 14 | 19 ± 15 | 15 ± 9 |
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