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The Journal of Neuroscience, January 15, 1998, 18(2):763-778
Fetal Spinal Cord Transplants Support the Development of Target
Reaching and Coordinated Postural Adjustments after Neonatal Cervical
Spinal Cord Injury
Pamela S.
Diener and
Barbara S.
Bregman
Department of Cell Biology, Division of Neurobiology, Georgetown
University Medical Center, Washington, D.C. 20007
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ABSTRACT |
Neonatal midthoracic spinal cord injury disrupts the development of
postural reflexes and hindlimb locomotion. The recovery of rhythmical
alternating movements, such as locomotion, is enhanced in injured
animals receiving fetal spinal cord transplants. Neonatal cervical
spinal cord injury disrupts not only locomotion but also skilled
forelimb movement. The aims of this study were to determine the
consequences of cervical spinal cord injury on forelimb motor function
and to determine whether transplants of fetal spinal cord support
normal development of skilled forelimb use after this injury.
Three-day-old rats received a cervical spinal cord lesion at C3, with
or without a transplant of fetal cervical spinal cord (embryonic day
14); unoperated pups served as controls. Animals were examined daily
during the first month of life using a behavioral protocol that
assessed reflexes, postural reactions, and forelimb motor skills. They
also were trained and tested as adults to assess performance in
goal-directed reaching tasks. The onset of postural reflexes was
delayed in the lesion-only group, and goal-directed reaching and
associated postural adjustments failed to develop. The transplant group
developed reflex responses and skilled forelimb activity that resembled
normal movement patterns. Transplant animals developed both target
reaching and accompanying postural adjustments. Target reaching
requires integration of segmental, intersegmental, and supraspinal
input to propriospinal and motor neurons over many spinal cord levels.
Transplants may support the reestablishment of input onto these
neurons, permitting the development of skilled forelimb activity after
neonatal cervical spinal cord injury. The neuroanatomical
reorganization of descending and propriospinal input was examined in
the companion paper (Diener and Bregman, 1998 ).
Key words:
recovery of function; neonatal rat; reaching; postural
adjustments; transplants; development; behavior
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INTRODUCTION |
The development of hindlimb
locomotion and the coordination between forelimbs and hindlimbs relies
on the formation of interconnections between supraspinal,
propriospinal, and segmental axons, with local pattern generators for
each limb (Grillner, 1973, 1975, 1976, 1986; Andersson et al., 1978).
Midthoracic spinal cord injury damages the input to hindlimb pattern
generators and disrupts reflex development and normal hindlimb
movements for locomotion. The consequences of cervical spinal cord
injury are more severe than those after midthoracic injury. There are,
for example, greater temporal delays in the development of reflexes and
locomotion, and more abnormal movement patterns persist after cervical
injury than after thoracic injury (Bregman and Goldberger, 1983).
Forelimb deficits are also pronounced in various aspects of motor
function after high cervical spinal cord injury in the adult
(Schrimsher and Reier, 1992, 1993). After spinal cord injury in the
adult rat, for example, movement required for reaching toward a target placed on the body recovers, whereas movements required to reach for
targets placed away from the body fail to recover. To date, little is
known about (1) the development and maturation of skilled forelimb
activity such as goal-directed reaching and (2) the development of the
postural control that is essential for precise forelimb movements.
It is well established that skilled activity involves a more complex
integrative neural network than that interacting with local pattern
generators for locomotion (Grillner, 1975, 1976; Andersson et al.,
1978; Alstermark et al., 1984a,b,c, 1987c, 1990, 1991a). The
development of reaching after neonatal spinal cord injury, therefore,
may not be as extensive as the recovery of hindlimb locomotion. The
first aim of this study was to examine the development and maturation
of forelimb reaching and to determine the effect of upper cervical
spinal cord injury on this development. The second aim of this study
was to determine whether transplants have the capacity to mediate
recovery of skilled forelimb movements such as reaching or whether
recovery is restricted to rhythmic alternating movements such as
locomotion.
It is clear that transplants of fetal spinal cord tissue enhance the
development of rhythmical alternating movements after neonatal
midthoracic spinal cord injury (Kunkel-Bagden and Bregman, 1990;
Kunkel-Bagden et al., 1992; Bregman et al., 1993; Howland et al.,
1995). Transplants support the survival of immature axotomized neurons
and the regrowth of axons to spinal cord levels caudal to the injury
(Bregman and Reier, 1986; Bregman, 1987a,b; Bernstein-Goral and
Bregman, 1993; Diener and Bregman, 1994). This axonal regrowth is
associated with improved locomotor function in animals with transplants
compared with permanent deficits in locomotion in the lesion-only
animals. The effects of transplantation on the development of skilled,
goal-directed forelimb activity after neonatal cervical spinal cord
injury, however, are unknown. To assess the contribution of the
transplant to the development of reaching, a behavioral testing battery
was designed to evaluate the execution of motor skills required for
reaching in developing animals and in mature animals that had been
injured at birth. The results indicate that marked developmental delays
and abnormal compensatory motor patterns emerge in lesion-only animals
and reaching fails to develop. In contrast, skilled forelimb activity develops and mature reaching for targets on and away from the body is
established in both transplant and control animals. The greater
recovery of skilled forelimb movement is associated with greater
supraspinal input to the spinal cord in the presence of the transplants
(see companion paper, Diener and Bregman, 1998).
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MATERIALS AND METHODS |
Cervical spinal cord injury and transplantation
Timed-pregnant Sprague Dawley rats were obtained from Zivic
Miller Laboratories (Zelienople, PA) and observed two times daily for
the presence of pups. Day of birth was designated postnatal day (P) 1. Eighty-four newborn rats were prepared for this experiment. The rats
were divided randomly into three groups: control (CON, n = 13), overhemisection only (HX, n = 33), and overhemisection plus fetal transplant (HX + TP,
n = 38). On the third postnatal day, operate pups were
anesthetized by hypothermia. An incision was made along the skin
overlying the cervical vertebrae. The muscle fibers were separated to
expose the underlying vertebral column. A laminectomy of the C2 and C3
segments was performed, and the dura overlying these segments was slit
longitudinally with a no. 11 surgical blade. The right side of the C3
spinal cord segment was lesioned (overhemisection) using iridectomy
scissors. An overhemisection of the spinal cord destroys the dorsal
funiculus bilaterally and the right ventral and lateral funiculi and
the intervening gray matter unilaterally. In a separate group of
animals (HX + TP), after the spinal cord was lesioned, one or two
pieces of embryonic cervical spinal cord (1-2 mm2)
were inserted into the cavity created by the retraction of the cut ends
of the cord. The donor tissue was obtained from anesthetized (chloral
hydrate, 400 mg/kg, i.p.) timed-pregnant Sprague Dawley rats 14 d
in gestation (E14) and dissected under aseptic conditions in DMEM as
described previously (Reier et al., 1986; Bregman and McAtee, 1993).
After lesion or lesion plus transplantation, the injury site was
covered with synthetic dura (durafilm, Codman-Schurtleff, Inc.) and
covered with 0.9% saline-soaked gelfoam. The overlying muscle and skin
were sutured with 6.0 silk. The pups recovered in a warm environment
and were injected with a prophylactic dose of bicillin (Wyeth
Laboratories, Philadelphia, PA) subcutaneously and returned to their
mothers.
Behavioral testing
Rats were tested daily from birth through P30 for the
developmental series. Rats also were trained and tested as adults (>4 weeks) on various reaching and other motor tasks. The stand (109 × 37.5 × 106 cm) used for behavioral testing was constructed
with a glass top measuring 44 × 109 cm and an angled mirror
(34 × 125 cm) to permit simultaneous viewing of the lateral and
ventral aspects of the rat. Developing and mature rats were assessed
qualitatively and quantitatively for forelimb use and associated body
adjustments. Scoring criteria based on normal components of each
particular motor activity were established for each test.
Developmental and postural reflexes that were examined included
righting, chin placing, grasp reflex, and motor response to noxious
stimuli. Postural reflex testing examined both full body and individual
forelimb movements, whereas individual limb reflexes analyzed specific
components of limb function. Additionally, other forelimb skills were
used to study postural reactions, proximal control and stability, the
use of the forelimb for goal-directed movements, coordinated bilateral
forelimb movements, and distal control (tested by grasping). The use of
accurate forelimb aiming during the rhythmical alternating movement of
locomotion was evaluated qualitatively and quantitatively. At the end
of the 4 weeks of testing, randomly selected rats from each group (CON,
n = 6; HX, n = 3; HX + TP,
n = 3) were perfused, and the spinal cords and brains
were dissected. This tissue was prepared for analysis of descending and
segmental projections that may remodel after injury to the developing
spinal cord and serve as the underlying mechanism(s) for the observed
motor behaviors (Diener and Bregman, 1998). At the sixth through ninth
week of life, the remaining rats in each group were trained and tested
for forelimb use in target-directed reaching. Rats in all groups were
trained to reach at midline to remove a sticker from their heads
(simulating a grooming response) and to reach forward (grasp and
retrieve a food pellet) away from their bodies to challenge further
their balance reactions. For each test, the training period lasted 1 week, followed by 1 week of testing.
Videotape recordings
Behavioral testing for each group was recorded on videotape
approximately once a week. The animals were filmed with a Panasonic camera (WV-3260/8F) with a shutter speed of 1/1000 sec. When possible, mirrors were incorporated to allow simultaneous viewing of the rat from
lateral and ventral planes. For target reaching only, the performance
was recorded from both a lateral and anterior view. Analysis of the
videotapes occurred after all testing was completed. To analyze the
component movements used by the developing and adult rats, the
videotapes were viewed with good resolution in slow motion.
Developmental tests and scoring criteria
There are a number of motor skills that are prerequisite for the
development of target-directed reaching. These include (in sequence)
whole-body control, proximal stabilization for distal mobility, and
anticipatory postural adjustments for reaching. Scoring criteria for
individual assessments are summarized in Table
1.
Righting. The righting reflex develops postnatally
(Kunkel-Bagden et al., 1992). Righting the body requires sequential
upper and lower body movements to turn from supine to prone. Because righting requires the use of the forelimbs and the upper body, this
reflex was assessed to determine whether (1) high spinal cord injury
would delay the date of reflex development, (2) forelimb use would be
compromised, and (3) aberrant behaviors would develop. The ability to
right was tested by placing and holding the rat still in a supine
position on the glass table top. The time to turn from supine to prone
was measured with a stopwatch and recorded in seconds. The
characteristics of the movement were described according to a
five-point scale (Table 1).
Spatial orientation. Orientation of the body in space was
measured by using a board inclined to a 30° angle. Both the time to
orient the body to face upward and the motor patterns used to turn the
body around were recorded. The rat was placed with all paws on the
board, with its body and nose facing down. A five-point rating scale
(Table 1) was used to evaluate the motor components used by the rat to
achieve the upright posture.
Chin placing. The stimulus of placing the chin on a surface
is another reflex that incorporates bilateral forelimb use in the
response. To achieve light pressure on the ventral surface of the chin,
the examiner gently supported the forelimbs and lower trunk/hindlimbs
and rested the rat's chin on a smooth, horizontal surface. The timer
was started as the examiner released the support on the forelimbs. The
number of seconds required by the rat to place both paws on the surface
and the exact sequence of paw placement were recorded for each of three
trials. Because the eyes are still closed at P11-12, chin placing is
not influenced by visual input. The rats were rated according to a
three-point scale for limb placement (Table 1).
Grasping. Before active grasping and proximal control were
tested, each rat was evaluated to determine whether it had either an
immature grasp reflex or a volitional active grasp. The stimulus for
active grasping, a 15-cm-long wooden rod (2 mm in diameter) was applied
to the volar surface of the forepaw of newborn rats. Strong wrapping of
the digits around the rod as it was brushed over the palm combined with
the inability to release the rod by extending the digits was recorded
as an immature grasp reflex. If the rat could grasp and release its
grip on the rod, the grasp was recorded as volitional. To assess the
ability of the rat to grasp and maintain proximal control, the rats
were evaluated as they held onto the suspended rod. The rod was raised
above the surface to prevent the body/tail from contacting the table
top and therefore from assisting with postural support. The number of
seconds that the rat held onto the rod was recorded. Descriptions of
forelimb use were documented using two separate five-point scales, one
to evaluate forelimb use and a second to evaluate proximal limb control
(Table 1).
Grooming. Functional use of the forelimbs was evaluated
first by examining the active range of motion of each forelimb. To assess full active forelimb use in each rat, pups were placed on the
glass top of the stand, and a noxious stimulus (cold water) was applied
to the face of each rat. Each rat's forelimb and body responses were
recorded on videotape (viewing lateral and ventral sides of the
animal). The development of bilateral forelimb range of motion in
response to the stimulus was described qualitatively and
quantitatively. Each rat's face was divided into four quadrants: a
vertical line was drawn along the midline of the face, and a horizontal
line was drawn just caudal to the eyes to comprise the four quadrants.
The animals were rated to determine the functional range of motion for
each forelimb by recording the quadrant to which the forelimb reached
(Table 1) and the number of times it entered that quadrant.
Locomotion. The ability of a rat to step over obstacles
during locomotion was examined after overground locomotion developed fully. Stepping over an obstacle requires anticipatory commands both to
adjust postural support and to initiate sufficient limb flexion to
clear the obstacle (Drew, 1991). A short runway (10 × 42.5 cm)
with 1.25-cm-high obstacles placed 11.25 cm apart was used for testing.
The rat was placed at one end and walked from one end to the other
during a 2-3 min trial; its ability to step over the obstacle was
scored using a three-point scale (Table 1).
Adult tests and scoring criteria. Forelimb motor skills and
postural reactions were also assessed after the rats reached maturity (at least 4 weeks of age). Scoring criteria for individual assessments are summarized in Table 2.
Sticker removal. Removal of a sticker placed on the bridge
of a rat's nose required full range of motion of the forelimbs and at
least some grasping ability. Assessment began with placement of a
sticker (1.9 cm in diameter) on the rat's head between the nose and
eyes. After a training period of 1-2 d, the number of times and the
extent to which each forelimb reached to remove the sticker were
recorded. A rat was given a maximum of 2 min to remove each sticker
(three stickers per trial, three trials per animal). The scoring
measures were similar to those used by Schrimsher and Reier (1992).
Scoring was calculated individually for each forelimb (Table 2).
Adult target reaching. Forward reaching involves both
forelimb use and associated postural adjustments. Goal-directed
reaching into a specified region for a food pellet (target) was
analyzed in adult rats after a 48 hr fast from a solid diet. The design of the reaching apparatus was a modification of those used in other
laboratories (Whishaw and Kolb, 1988; Schrimsher and Reier, 1992,
1993). The reaching apparatus was constructed with a metal grate (1.25 cm between vertical rods) as the front wall and Plexiglas for the
remaining walls to make a box 30 × 15.6 × 20 cm. The box contained a series of 12 cubbies/shelves (2.5 × 2.5 × 2.5 cm) situated in four rows and three columns with four open shelves (2.5 × 2.5 cm) above. A space of 0.6 cm was maintained between the shelves and the grate wall. This separation between the cubby and
the grate wall ensured that the pellet could not be dragged from the
shelf and shuttled into the mouth. Instead, if the grasp was inaccurate
and the pellet fell as the limb was withdrawn from the cubby, the rat
could not retrieve it. Similarly, the floor of the apparatus was a
grate with holes of 0.6 by 0.6 cm. If the pellet was dropped as the
limb approached the mouth, the pellet would fall through the grate and
be unobtainable. In this way, being rewarded by the food was possible
only if the rat accurately maintained its grasp around the pellet. Rats
were weighed daily to verify that their weight never dropped below 80%
of baseline. Water was provided ad libitum. After the 48 hr
fast, rats received 45 mg food pellets (P.J. Noyes Company, Lancaster,
NH) during a 5 min trial. If they reached for the food pellets during
the training period, they were rewarded with their regular diet for 15-20 min; all rats received a portion of their regular diet
overnight. Testing sessions occurred after the 1 week training period,
which prepared the rats to reach into the horizontal shelves to grasp and eat the 45 mg food pellets. At the end of each test session, all
rats received their regular diet for 15-20 min.
Scoring of forelimb use was accomplished in several ways. Rats reached
into a total of 16 shelves set at four different heights as described
above. Each shelf contained four 45 mg food pellets to total 64 pellets
at the beginning of the 5 min trial. Each daily session documented both
the number of pellets that were dropped onto the surface and those that
remained in the cubby. The total was then subtracted from 64 to
determine the number of pellets consumed. Components of reaching and
postural adjustments were rated using a scale (Table 2) modified from
that described by Whishaw and colleagues (Miklyaeva et al., 1994).
The components of forelimb reaching that were evaluated included
raising the forelimb from its weight-supporting posture, neutral
positioning of the forelimb in midline with digit flexion, forward
reaching into a cubby with the forelimb moving along the midline,
extending the forelimb over the pellet that was situated in the cubby,
extending the digits over the pellet, pronating the forelimb to prepare
for the grasp, flexing the digits and grasping the pellet, lifting and
slightly supinating the forelimb to withdraw the limb, bringing the
forepaw to midline under the mouth, and finally bringing the opposite
forelimb to midline to assist in holding the pellet while eating. The
component parts of the postural reactions were scored by the same
three-point scale. The individual motor components assessed included
recording the position of the nonreaching forelimb (e.g., remained on
the ground to support the body weight), the quality of the reach (e.g., precise or multiple random attempts vs substitution of other body parts), the method of grasping and withdrawing the forelimb (e.g., grasping pellet, paw inspection before or after pellet dropped), the
lower body and hindlimb base of support (e.g., use of a stable base vs
continual repositioning to maintain balance vs losing balance), and the
coordination between the forelimbs and hindlimbs.
Lesion reconstruction
At 4 weeks to 4 months after cervical spinal cord injury with or
without transplantation, randomly selected rats were overdosed (chloral
hydrate, 1000 mg/kg, i.p.) and perfused intracardially with 0.9%
heparinized saline followed by 4% paraformaldehyde in a 0.1 M phosphate buffer, pH 7.4. Directly after perfusions, the spinal cord and brain were removed and post-fixed at room temperature for 2 hr. Tissue was cryoprotected in a graded series of sucrose solutions (10-30%) at 4°C. Spinal cord tissue was blocked and cut
in cross sections on a cryostat (16 µm sections), and a 1:5 series
was mounted on subbed slides. One slide from each series was stained
with cresyl violet to document the extent of the lesion and distinguish
transplant tissue from host spinal cord. The lesion and transplant
sites were serially reconstructed using an aus Jena microprojector to
determine the rostrocaudal and transverse extent of the lesion site and
transplant apposition. Strict lesion criteria were established to
ensure uniformity among animals used in the study. All rats included in
the behavioral analysis had a lesion that ablated the dorsal columns
bilaterally and the lateral and ventral funiculi and intervening gray
matter unilaterally (right side). Consequently, descending pathways
contributing to the development of right forelimb use were axotomized.
Inclusion in the HX + TP group also required extensive rostrocaudal and transverse apposition of the transplant to the host tissue. Table 3 lists the animals that met all of the
criteria for inclusion in the final data analysis and indicates the
behavioral tests conducted on each. All of these animals used in the
behavioral studies also underwent anatomical analysis to determine the
extent of reorganization of supraspinal and propriospinal neurons after cervical spinal cord lesions and transplants (Diener and Bregman, 1998).
Statistics
The quantitative results of behavioral tests from each
group were compared using the SPSS (Statistical Program for Social Sciences, Chicago, IL) computer program. All results are expressed as
the mean ± SD. The rating scores of each behavioral test were ordered scales and served as the dependent variables in the analysis against the animal groups, which were defined by surgical procedure (independent variable). Differences were compared across all groups using one-way ANOVA. If variability existed among the groups, specific
differences were determined by comparing the control group and each
experimental group (CON vs HX, CON vs HX + TP) or the experimental
groups against each other (HX vs HX + TP). These between-group analyses
were performed using the Bonferroni, Scheffé, and Tukey tests for
significance, with a p value of 0.05.
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RESULTS |
Lesion reconstructions
Representative cross sections from the lesion site in hemisection
and hemisection plus transplant rats included in the behavioral analysis are illustrated in Figure 1,
A and B, respectively. The motor behavior
observed within each group of animals was generally consistent. For
example, of the animals that met the lesion criteria for inclusion in
the study, in each treatment group the motor behavior of animals with
the smallest acceptable transverse lesion and the largest acceptable
transverse lesions was similar qualitatively and quantitatively. Thus,
differences observed in the motor performance between lesion-only and
lesion plus transplant animals on specific tasks could not be
attributed to differences in the transverse extent of the injury. The
experimental animals described in this section survived the surgery and
completed all behavioral testing and analysis. Table 3 lists the
animals in each group that met all lesion criteria and indicates which
behavioral analysis was performed on which animals. In each group, rats
were withheld from behavioral analysis if they survived the initial
surgery but did not survive beyond 10-14 d (HX, n = 7;
HX + TP, n = 10), did not meet the lesion criteria (HX,
n = 2; HX + TP, n = 5), or for unknown
reasons (unrelated to the injury incurred) failed to train in response
to food reward (HX, n = 3). The results reported below
are based on final sample sizes of CON, n = 13; HX,
n = 10; and HX + TP, n = 8.
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Figure 1.
Lesion or lesion plus transplant sites in rats
used for behavioral analysis. Host tissue remaining in each lesion-only
(A) or lesion plus transplant rat
(B) through the site of greatest injury extent.
The reconstructed lesion sites show a similar transverse extent of the
injury in lesion-only and lesion plus transplant animals (see Materials
and Methods for lesion criteria). In each transplant rat
(B), the transplants (TP) are well
apposed to the host cord.
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Development of postural reflexes and forelimb movement: whole-body
responses to sensory stimulation
Righting
The body-righting reflex matured by P10.6 ± 1.2 in
normal rats (Fig. 2A,
CON). Normal rats exhibited a similar motor sequence as
they developed righting responses. The supine position initially elicited random movements of all four extremities, with exaggerated flexion of the neck and abdomen. As the response matured, there was a
reduction in the time to right, and pelvic and upper body rotation
became isolated. The upper body rotated first in synchrony with the
forelimbs, and then the lower body rotated and hindpaws were placed
under the body (Fig. 2B; rating scale = 5).
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Figure 2.
Effect of neonatal cervical spinal cord
injury and transplantation on postnatal development of the righting
reflex. A, Histogram comparing average (±SD) postnatal
day at which the mature reflex develops in normal uninjured
(CON, n = 9), hemisected (HX,
n = 6), and hemisected plus transplant
(HX+TP, n = 8) rats. The righting reflex matures significantly later in HX rats as compared with CON rats
(p < 0.05). HX + TP rats demonstrate a
significant improvement compared with HX rats
(p < 0.05), because righting develops in a
time course similar to that of CON rats. B, Scatter plot
of upper body/forelimb use for righting at P11. Each
symbol represents an individual animal in the specified
group. Righting matures in CON rats by the P11, whereas HX and HX + TP
rats still exhibit immature behaviors. To initiate turning over, CON
rats use upper body rotation in advance of lower body rotation (rating
scale = 5). In their attempt to rotate the body, HX rats instead
excessively flex their pelvis and engage in other strategies to roll
over (rating scales = 2 and 3), representing a different strategy
compared with CON and HX + TP rats. HX + TP rats demonstrate emerging
abilities to rotate their bodies smoothly from supine to prone but
require multiple rotations before achieving the prone position (rating scale = 4). This represents a marked alteration in upper
body/forelimb skill development for the righting reflex compared with
CON rats.
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Cervical spinal cord hemisection delayed the development and maturation
of body righting. The time to right was increased, and abnormal
movement patterns persisted. For example, in response to being placed
supine, an exaggerated version of the normal immature response was used
and persisted for several weeks. Lesion-only rats failed to develop
isolated upper and lower body rotation. Instead, they used extreme neck
rotation or excessive flexion of their abdominal muscles resulting in
excessive elevation of the pelvis from the surface. Emerging abilities
to roll over were impeded by limited right forelimb movement. By
P10-11 (mean day of maturation of body righting for normal rats),
significant impairments in motor control were evident in lesion-only
rats as compared with the CON group (Fig. 2B; HX
rating scale = 2.3 ± 0.52 vs CON rating scale = 5.0;
p < 0.05). By the third week of life, isolated rotation was still minimal (Fig. 2A; P14.8 ± 2.9, significantly different from CON; p < 0.05). This
abnormal motor pattern was used consistently by all lesion-only rats
and was evident in those with the smallest and largest transverse
extent of lesion. Although the response was aberrant, it was considered
mature, because there was little subsequent improvement in motor
performance for body righting.
The presence of a transplant at the cervical lesion site accelerated
the time of development of the righting reflex toward normal and
permitted the establishment of normal movement patterns. Transplant
animals initially used random motor patterns in righting. The righting
response matured during the second week, at which time all transplant
rats incorporated upper body rotation, forelimb external rotation, paw
placement on the surface, and pelvic rotation to turn over. This
sequence of righting movements in HX + TP animals resembled normal
motor patterns (Fig. 2A; CON P10.6 ± 1.2 vs HX + TP P13 ± 2.9; p > 0.05), although the
maturation of righting was delayed slightly. The quality of the body
movements used by transplant rats to right was reduced significantly
compared with normal rats (Fig. 2B; CON rating
scale = 5 vs HX + TP rating scale = 4; p < 0.05), but was significantly better when compared with lesion alone (HX
rating scale = 2.3 ± 0.52 vs HX + TP rating scale = 4;
p < 0.05).
Spatial orientation
Normal rats repositioned themselves to face upward when they were
inverted on a board inclined to a 30° angle. Initial placement on the
board elicited immediate attempts to achieve postural stability. Once
secure in their stance, rats easily transferred body weight from one
limb to another to coordinate reciprocal smooth repositioning of all
limbs to turn 90° (i.e., placing their body perpendicular to the
angle of incline). Once facing upward, they ascended to the top edge of
the board using an alternating, rhythmical locomotor pattern. Normal
development of this response occurred by P11 (Fig. 3; CON rating scale = 5).
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Figure 3.
Effect of neonatal cervical spinal cord injury on
the ability to orient the body upright during the second postnatal
week. Each symbol represents an individual animal in the
specified group. As normal uninjured rats mature, they turn their
bodies on an inclined board to orient their head upward by first moving
their forelimbs and then realigning their body with their hindlimbs. HX rats develop alternative strategies for failed
development of isolated forelimb movements and respond with movement
substitutions to accomplish the goal. These aberrant movements are
different from the pattern used by both CON and
HX+TP rats. HX + TP rats, although delayed, develop the
ability to turn themselves around on an inclined board, using a
developmental sequence similar to that of normal rats rather than the
substitutions used by HX rats.
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Lesion-only rats were unable to support their body weight on all four
extremities when they were placed on the angled board. Instead, during
the first 14 postnatal days, they firmly pushed their nose into the
board and widened their hindlimb base of support to maintain a static
posture. Transferring of body weight was impaired in this group;
consequently, any attempt to reorient the body by repositioning the
limbs was unsuccessful, and the rats rolled off of the board. From the
third to fourth postnatal weeks, all HX rats substituted novel motor
patterns for the normal motor sequence used to turn upright. For
example, five of six HX rats laterally flexed their trunk and swiftly
pivoted their hindlimbs around their stationary upper body, making
final postural adjustments with the left forelimb. This motor pattern
was the reverse of that used by normal rats (Fig. 3; CON rating
scale = 5 vs HX rating scale = 2.5 ± 1.0;
p < 0.05). Use of right forelimb to assist in turning
or in final body placement was minimal or absent in all HX rats.
Initially in the lesion plus transplant animals, attempts to reposition
any limb on the board caused these rats to roll off of the board. By
the end of the first postnatal week, however, they were able to turn on
the inclined board by sequencing left forelimb lateral stepping with
subsequent hindlimb movements, which suggests the development of
abilities to shift body weight to maintain balance. During the second
week, seven of eight rats realigned their body initially through minor
positional changes (e.g., lateral stepping) followed by moving the
right forelimb forward, laterally flexing the trunk, and stepping with
both hindlimbs. Forelimb and upper body movements preceded hindlimb and
lower body movements, similar to the normal pattern (Fig. 3; CON rating scale = 5 vs HX + TP rating scale = 4.1 ± 0.64;
p > 0.05). In response to loss of balance, transplant
rats reacted automatically to stabilize themselves by widening their
hindlimb base of support and abruptly extending all limbs. In contrast,
lesion-only animals lost control and rolled off of the board. This is
one example of movement patterns used by the HX + TP group that were
significantly better than those used by the HX group (Fig. 3; HX + TP
rating scale = 4.1 ± 0.64 vs HX rating scale = 2.5 ± 1.0; p < 0.05).
Development of postural reflexes and forelimb movements: motor
skills that are prerequisite for target-directed reaching
Chin placing
Chin-placing responses are absent at birth in normal rats.
During the first postnatal week in the normal rat, only small, random,
bilateral forelimb movements were elicited in response to stimulation
of the ventral surface of the chin. Chin placing developed during the
second postnatal week when, in response to stimulation of the ventral
surface of the chin, one or both forelimbs were placed reciprocally or
simultaneously onto the surface (Fig. 4A, CON).
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Figure 4.
Effect of neonatal cervical spinal cord injury on
the development of chin placing. A, Comparison of the
day of maturation of the reflex (x-axis = DAYS POSTNATAL). Both HX and
HX+TP rats develop the paw-placing response to stimuli
to the chin later than CON rats. The delay in maturation
of the response is more pronounced in HX rats
(p < 0.05) than in HX + TP rats.
B, Comparison of the development of the chin-placing
reflex in normal (CON) and lesion with
(HX+TP) or without (HX)
transplantation. Each open symbol represents the
response of all animals in the specified group. Symbols with the
center marked represent 50% of the animals. CON rats place
both forelimbs on the surface during the second week of life in
response to a tactile stimulus to the chin (forelimb use = 2). The
onset of the response is delayed in HX rats, and the mature response
never develops. Instead, the mature response of HX rats is to place the
left forelimb on the surface, with occasional random unproductive right forelimb movements (forelimb use = 1), which is a markedly different strategy from that used by CON and HX + TP rats. HX + TP rats follow a similar sequence of development as
compared with CON, but do so with a slight developmental delay. By 2 weeks postnatal, however, all transplant rats place both forepaws on
the surface in response to the stimulus.
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The typical time course for the development of chin placing in the
three groups is illustrated in Figure 4B. Lesion-only
rats responded to stimulation on their chin with an exaggerated version of the normal immature response (Fig. 4B; HX,
forelimb use = 1.0; open squares at P6-11;
p < 0.05). For example, the rats struggled, rotated
their heads, and infrequently and inconsistently moved their left
forelimbs randomly. By P11, although there was a temporal delay between
application of the stimulus and the motor response, the frequency of
the random left forelimb movements increased and occasionally the limb
was placed on the surface. By the third postnatal week, all HX rats
immediately placed their left forelimbs onto the surface in response to
the stimulus, indicating partial maturation of the reflex (Fig.
4A; HX P14.5 ± 1.0; p < 0.05;
and Fig. 4B). Lesion-only rats never developed a
bilateral forelimb response to stimulation under the chin and therefore
never achieved the mature response.
Transplantation after neonatal spinal cord injury prevented the delay
in the development of chin placing. In the first week of life, the
reaction of HX + TP rats was similar to that of normals; they responded
to the stimulus with head rotation, lateral trunk flexion to either
side, and random movements of both forelimbs (Fig.
4B, open triangles; P6-8). By P13, the
response matured (Fig. 4A; 13.2 ± 3.4), and one
or both forelimbs were placed on the surface in addition to the chin
(Fig. 4B; HX + TP; forelimb use = 1.5 ± 0.53). Placing in the HX + TP group was characterized by a momentary
delay between application of the stimulus and limb placement,
distinguishing the response from that in normal animals. Unlike HX
animals, however, all of the HX + TP rats were able to respond with
both forelimbs (HX rating scale = 1.0 vs HX + TP rating scale = 1.5; p > 0.05).
Grasping and proximal control
A mild obligatory grasp reflex that was elicited in all rats at
birth became integrated in normal rats during the first postnatal week
when active grasping developed (P6). The strength of active grasp was
measured functionally by evaluating rats during strenuous activities
that incorporated grasping with proximal control. Proximal control,
also a prerequisite for target-directed reaching, was achieved through
active use of the muscles surrounding the shoulder joint and associated
stabilizing movements of the upper and lower body. The development of
proximal stability was measured by observing the rats supporting their
body weight (chin-up) when grasping a horizontal rod. Normal rats
exhibited proximal control of the upper body by the beginning of the
second week of life (Fig. 5A, CON). During the second week of life, muscle
strength, proximal stability, and control over the body improved,
enabling the rat not only to sustain a static chin-up but also to
reposition the forelimbs along the rod (Fig. 5A, open
circles; rating scale = 5.0). Over the next 2 weeks, normal
animals narrowed their base of support on the rod, sustained their grip
for increasing periods of time, and transferred to the opposite side of
the rod.
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Figure 5.
Effect of neonatal cervical spinal cord injury on
the development of proximal control during static grasping and dynamic
forelimb use. A, Proximal control for achieving and
maintaining a chin-up at 2 weeks postnatal. Rating scores range from 1 to 5: 1 indicates the most immature and aberrant response, 5 represents
maturation of the response, and the intermediate scores represent
progressive development of skills for this task. Each
symbol represents an individual animal in the specified
group. CON rats use proximal muscle control to achieve and maintain a
chin-up. HX rats never maintain a chin-up and instead achieve various
intermediate levels of control, often using their obligatory right
grasp reflex to sustain their grip. Mostly, when HX rats attempt to
support their body weight, they excessively flex their pelvis, swinging
their hindlimbs up onto the pole. Even with this excessive effort, they do not maintain their grasp and soon drop to the surface (rating scale = 1). Although the HX + TP rats initially drop to the
surface, unlike the HX rats they eventually develop the motor
components for the mature response. Most HX + TP rats use both
forelimbs to hold onto the pole and use proximal control to achieve a
chin-up (rating scale = 4), although others use varying degrees of
normal behavior. B, Forelimb use to groom the face. Each
symbol represents the typical response of the animals in
the specified group. Filled symbols indicate mature
behavior. Open symbols indicate immature behavior.
Partially filled symbols indicate emerging mature
behaviors. Unlike CON rats (n = 9), HX rats
(n = 6) never develop mature use of the forelimbs for grooming. Immature responses (e.g., backing away from a noxious stimulus) are used initially, and later emerging mature behaviors (e.g., only the left forelimb bats at the stimulus) develop in HX rats. Although HX + TP rats (n = 8)
demonstrate a prolonged period during which they exhibit immature
behaviors, they all develop the motor skills required for maturation of
the grooming response.
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Neonatal cervical spinal cord overhemisection disrupted the development
of volitional grasping, proximal control, and isolated use of muscles
around the shoulder (Fig. 5A, HX). During the second and third postnatal weeks, three of five HX rats used an obligatory grasp reflex to wrap their digits around the rod (Fig.
A; rating scale = 1.0), they but
lacked the proximal control to pull to the chin-up posture. Instead,
their attempts to contract their muscles resulted in movement
substitutions including excessive abdominal flexion with the hindlimbs
swinging up to grip the rod, which consequently
inverted the rats (Fig. 5A, open squares; rating scale = 1.0). At this 2 week period, the two other rats in the group
demonstrated emerging capabilities to execute the motor sequences that
are necessary to accomplish a chin-up position (2 weeks) (Fig.
5A, open squares; rating scale = 3.0 and 4.0). In
contrast to normal developing rats, the base of support used by HX rats
was wide (approximately the width of their shoulders), and the
forelimbs were not repositioned on the rod, indicating weakness in
flexor musculature and a predominant grasp reflex (Fig. 5A;
significantly different from CON, p < 0.05). By
maturity (P31), HX rats typically either used a moderately wide base of support on the rod or released one forelimb, sustaining their grasp
with the other and alternating between hanging and momentarily pulling
to a chin-up.
HX + TP rats developed the ability to achieve and maintain the chin-up
posture, although the emergence of mature skills was delayed and varied
within the group. Differences in motor performance in the HX + TP
animals could not be attributed to differences in the transverse extent
of the lesion. All HX + TP rats grasped and hung from the pole until
proximal control improved and they could pull to a chin-up (Fig.
5A, 2 weeks postnatal, open triangles; n = 4 at rating scale = 4.0; significantly
different from CON, p < 0.05). At 2 weeks postnatal,
two HX + TP rats were more precocious than others in the group: they
developed full proximal control to achieve a chin-up (Fig. 5A,
open triangles; at rating scale = 5.0). Although the
performance of all HX + TP rats resembled normals by the fourth week of
life, there were differences in the strategy for accomplishing the
task. For example, although the left forelimb maintained its original
grasp, the base of support on the rod widened because the right
forelimb slid laterally. As proximal control developed over the third
postnatal week, the rat could return the right forelimb to a neutral
position or could adjust total body position to evenly redistribute
(recenter) body weight between the forelimbs. Similar to normal rats,
by P31 HX + TP rats narrowed their base of support and repositioned
both forepaws laterally along the rod.
Grooming
A noxious (cold) stimulus was applied to the bridge of each rat's
nose to evaluate active range of motion and postural stability requisite for functional skills such as grooming. In normal rats, forelimb responses to remove the stimulus were not developed at birth.
Instead, the initial responses included a combination of retraction of
the head and body or pivotal movements with the forelimbs to escape
from the stimulus. These motor patterns were categorized as immature
(Fig. 5B, open symbols). The use of one forelimb was
observed occasionally and was recorded as partially mature (Fig.
5B, partially opened circle at P4). By P6, the
response matured and a bilateral swiping motion was used to displace
the stimulus (Fig. 5B, filled circles from P6 to
adulthood). The quality and accuracy of the swiping motion improved
during the second and third week of life and developed into a grooming
pattern (i.e., forelimbs move through quadrants I-II or III-IV; see
Materials and Methods).
Lesion-only rats failed to develop mature bilateral forelimb movements
in response to the stimulus and instead substituted immature avoidance
patterns and compensatory strategies. Forelimb swiping movements were
not used; instead, immature behaviors such as head retraction,
pivoting, or walking away from the noxious stimulus persisted (Fig.
5B, open squares). By P22, HX rats used only the left
forelimb to swipe at the stimulus (Fig. 5B; partial mature
response). Right forelimb adduction failed to develop. HX rats never
developed bilateral forelimb use in midline (Fig. 5B, no
completely filled squares).
The mature bilateral response developed in transplant rats (Fig.
5B, filled triangles), although the onset and maturation of
the response was delayed compared with normals (CON maturation at P6 vs
HX + TP maturation at P18). The immature response pattern was present
during the first week (Fig. 5B, open triangles).
A partial mature response developed during the second postnatal week
(Fig. 5B, partially filled triangle representing
left forelimb used for batting and swiping at the stimulus). By P18,
all transplant rats consistently used both forelimbs (reciprocally or
symmetrically) to groom their faces. Most transplant rats lacked full
supination and therefore could only rub their faces with the medial
side of their paws. The frequency and quality of movement improved during the fourth postnatal week. In HX + TP rats, right and left forelimb movements into quadrants I and II (see Materials and Methods)
was 72 and 98%, respectively, of normal forelimb movement. This
represents a dramatic improvement when compared with the lesion-only
animals, who do not use the right forelimb at all and use the left
forelimb at only 37% of the frequency of normal rats.
Stepping over obstacles
Qualitative assessments indicated that overground locomotion
developed in all rats by the end of the second week of life. Normal
rats walked earlier and with better form than the lesion or lesion plus
transplant animals (Bregman and Goldberger, 1983; Kunkel-Bagden and
Bregman, 1990; Kunkel-Bagden et al., 1992; Diener, unpublished data).
During the third and fourth week of life, the ability of each rat to
anticipate and step over obstacles placed in its path was evaluated.
The ability to step over the obstacle requires anticipatory postural
adjustments, proximal and distal muscle control, and accurate aiming of
the lifting limb. These forelimb skills and postural reactions are also
prerequisites for smooth, coordinated reaching. Normal animals walked
easily overground, anticipating and clearing each obstacle with all
four extremities (Fig. 6; CON
rating scale = 3.0). Lesion-only rats walked overground but
demonstrated significant deficits as compared with CON in anticipating
and stepping over obstacles (Fig. 6; HX rating scale = 1.0; p < 0.05). For example, HX rats did not lift
their right forelimb voluntarily over the obstacle. Instead, the
forward movement of the body caused the right forelimb to contact the
obstacle. After contact, the limb was placed reflexively over the
obstacle in a pattern resembling forelimb proprioceptive placing. This
movement also resembled a dragging motion that ended with either the
ventral or dorsal surface of the paw on the runway. Both hindlimbs and
the left forelimb of HX rats usually anticipated and cleared the
obstacle. Initially, HX + TP rats dragged the right forelimb over the
obstacle but soon developed the ability to anticipate and step over it.
This response was a significant improvement compared with the response
of the lesion-only animals (Fig. 6; HX rating scale = 1.0 vs HX + TP rating scale = 2.3 ± 0.5;
p < 0.05). Secondary to reduced forelimb range for
shoulder flexion during the swing phase, most (four of six) of
transplant rats brushed the tip of their right forepaws on the top edge
of the obstacle as the forelimb stepped over. The inability to lift the
right forelimb completely over the barrier distinguished transplant rats from normals, although the difference was not significant.
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Figure 6.
Effect of neonatal cervical spinal cord injury on
the acquisition of precise right forelimb placement during locomotion.
CON (n = 3); HX
(n = 4); HX+TP
(n = 6). Rating scale ranges from 1 to 3: 3 represents mature behavior and 1-2 are degrees of aberrant movements.
Each symbol represents an individual animal in the specified group. CON rats step over obstacles in their path during overground locomotion. CON rats anticipate the obstacle and raise their
forepaws to completely clear the height of the obstacle during
swing-through. HX rats demonstrate significant deficits compared with
both CON and HX + TP rats: they fail to anticipate the obstacles and
instead drag their forelimbs and place them over the obstacle. In
contrast, most HX + TP rats anticipate the obstacle but fail to
sufficiently clear it during the swing phase, contacting the distal tip
of their paw to the upper edge of the obstacle.
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Forelimb use in adult rats: target-directed reaching and associated
postural adjustments in adult animals
Sticker removal
Forelimb movements used to remove a sticker from the head were
similar to grooming motions. Normal rats swiftly reached for, grasped,
and abruptly pulled the sticker from the head using either or both
forelimbs. Balance was maintained by shifting body weight backward
toward the midtrunk (Figs. 7, 8).
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Figure 7.
Comparison of representative responses for sticker
removal in each group of rats. A, CON
rats use a bilateral approach to remove the sticker, with both forepaws
gripping and pulling the sticker from the nose. Simultaneously, they
maintain a stable base with both hindlimbs slightly externally rotated.
B, C, Two different rats representing the typical
response of the HX group. Each uses aberrant strategies
when attempting to dislodge the sticker. To maintain balance, the right
forelimb (arrows) moves forward and across midline while
the left forelimb approaches the sticker. The photographs also depict
the usual posture of the rats in the HX group (i.e., slightly contorted
body position with the hindlimbs asymmetrically placed and the upper
body frequently rotated in opposition to the lower body). D,
E, Two representative HX + TP
rats demonstrating the response consistently observed in this group.
Similar to CON rats, the HX + TP rats use both forelimbs to reach for
the sticker while maintaining a stable base of support.
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Lesion-only rats exhibited significant deficits in sticker removal
bilaterally (Figs. 7 B,C, 8,
CONTRALATERAL rating scale = 3.8 ± 0.45 and
IPSILATERAL rating scale = 1.2 ± 0.84;
p < 0.05). Only the left forelimb was raised to the
height of the sticker (Fig. 7B,C). Grasping failed to
develop; the radial and dorsal surface of the left forepaw rubbed at
the sticker, loosening but never removing it (Figs. 7B,C, 8,
CONTRALATERAL rating scale = 3.8). Deficits in balance
were evident during this task. When they raised both forelimbs to the
sticker, HX rats often fell to one side. To compensate for the
compromised balance reactions, postural control was acquired through
the use of a three-point stance (not pictured) or by altering the
hindlimb base of support and counterbalancing with the right extremity
moving across the midline while the left forelimb was raised to the
sticker (Fig. 7B,C).
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Figure 8.
Effect of neonatal cervical spinal cord injury on
reaching for targets on the body. Rating scales range from 0 to 6: 6 represents mature behavior (i.e., full range of motion), 0 represents
abnormal behavior (i.e., little to no movement), and intermediate
numbers reflect degrees of active range of motion of each forelimb,
analyzed individually. Each symbol represents the
specified forelimb of an individual animal. CON rats
quickly and consistently use either or both forelimbs to remove a
sticker placed between the eyes. HX rats
(n = 5) demonstrate marked deficits in target
reaching compared with CON rats and HX+TP rats. Their
best response is to bring their left (contralateral) forelimb to touch
the sticker. The right (ipsilateral) forelimb, at best, moves up to the
level of the sticker, but the limb remains lateral and fails to contact the sticker. The HX + TP rats (n = 6) use either or
both forelimbs to remove the sticker. Because they require multiple
attempts to free the stickers from their faces, the HX + TP rats
exhibit obvious deficits in forelimb motor control as related to speed and dexterity when compared with CON animals.
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Transplant rats removed the sticker but lacked the speed, smooth
coordination, and powerful grasp of normals (Fig. 8, HX+TP, CONTRALATERAL rating scale = 5.0 and
IPSILATERAL rating scale = 4 ± 1.1;
p < 0.05), yet they were significantly better than HX
animals (p < 0.05). In contrast to normal rats,
HX + TP rats required multiple reaching attempts to remove the sticker.
Because grasping was impaired bilaterally, much of the sticker was
dislodged initially from the face by repetitive rubbing before it was
grasped and removed (Fig. 7D, E). Although the movement
pattern was similar, HX + TP rats used their left forelimb more than
their right (compare scores on Fig. 8, CONTRALATERAL rating
scale = 5.0 and IPSILATERAL rating scale = 4 ± 1.1). Similar to CON rats, HX + TP rats shifted their body weight
backward to maintain balance during reaching [compare rats in Fig.
7A
(CON),D,E
(HX + TP)]. These results indicate that after
cervical spinal cord lesions, transplants not only mediate recovery of
rhythmic alternating movements such as those in locomotion
(Kunkel-Bagden and Bregman, 1990; Bregman et al., 1993), but they also
mediate recovery of skilled forelimb movements.
Target reaching
Normal rats used appropriate postural reactions when using either
forelimb to reach for a target at a distance from the body. To lift one
forelimb from its resting posture in stance, the rat transferred its
weight to the opposite forelimb as well as the lower trunk/pelvis. They
then actively extended their nonweighted forelimb through a vertical
grate to reach a series of horizontal shelves (wells) containing 45 mg
food pellets. The reaching limb adducted and externally rotated as it
raised (Fig. 9A) and then extended (Fig. 9B) into the well. The forelimb pronated and
the digits extended over the pellet, anticipating pellet retrieval (Fig. 9B). Next, the digits of the forelimb grasped the
pellet, supinated slightly, and withdrew from the shelf (Fig.
9C) The forelimb further supinated as it approached the
mouth, and the contralateral forelimb mirrored its position to assist
in holding the pellet under the mouth to eat (Fig. 9D).
Normal animals reached more accurately toward a target and consumed
more pellets than either experimental group (see Fig.
11A,B) and never used compensatory strategies (Fig.
10, CON). Normal
animals did not exhibit a dominant limb for reaching (Fig.
11).
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Figure 9.
Motor sequences used to reach targets away from
the body. A-D (serial images), Normal rats sniff at the
target (A), transfer body weight to free a
forelimb to reach into a shelf (B), and grasp a
pellet (C). After grasping the pellet, the limb
withdraws, and the rats sit back on their haunches, bringing both
forelimbs to midline to eat the pellet (D).
E, F (serial images), Hemisected rats substitute for
failed development of target reaching by using their tongues
(arrow) to obtain a pellet from the shelf
(E). After obtaining the pellet
(E), they fail to bring their paws to midline to
assist in holding and eating the pellet (F).
G-I (serial images), Hemisected plus transplant rats
sniff at the pellet and initiate the weight shift to free the reaching
forelimb (G). They reach through the grate for
the pellet but use a qualitatively different pattern of forelimb and
digit extension (H) as compared with
normal rats. Transplantation reestablishes postural support, reaching, and bilateral forelimb movements to midline to assist with eating the
retrieved pellet (I).
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Figure 10.
Effect of neonatal cervical spinal cord injury on
target reaching. CON rats (n = 4)
consistently use either forelimb to retrieve food pellets from
horizontal shelves and never engage in compensatory movements.
Conversely, HX rats (n = 5)
consistently failed to develop both reaching and coordinated lower body
responses and compensated by using tongue protraction.
HX+TP rats (n = 6) use a combination
of patterns, but more consistently use forelimbs to grasp a
pellet.
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Figure 11.
Effect of cervical spinal cord injury on pellet
consumption and associated postural adjustments. A, Each
group score is based on the preferred method of each individual animal
for pellet retrieval. The score represents the mean number (±SD) of
pellets each rat consumed per minute. HX rats ate
significantly fewer pellets than both CON and
HX+TP rats, which indicates obvious deficits in motor control for eating. B, Both CON and HX + TP rats
typically use their forepaws to grasp pellets, whereas lesion-only rats
primarily use their tongues. When the results shown in A
and B are combined, it appears that the transplant rats
must reach more frequently to consume the same number of pellets as
normal rats. In other words, because of aiming deficits in the
transplant animals, they must reach repetitively before actually
contacting the pellet. T, Tongue; L, left
forepaw; R, right forepaw.
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Skilled reaching and associated postural adjustments failed to develop
in lesion-only rats. Occasionally these rats fell to one side either
while obtaining food or returning to stance after food retrieval. Food
was obtained by substituting tongue extension for absent reaching
skills (Figs. 9E,F, 10, bar at 0%). Furthermore, once the
pellet was on the tongue and ready to be eaten, impaired bilateral
forelimb use was pronounced; the right paw failed to approach midline
to assist in holding the pellet (Fig. 9F). This movement pattern frequently resulted in awkward posturing, including body contortions and frequent loss of balance (Fig.
12). The use of compensatory strategies
limited the range over which HX rats could accumulate pellets;
consequently, significantly fewer pellets were taken and consumed as
compared with normal and transplant rats (Fig. 11A,B;
p < 0.05).
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Figure 12.
Comparison of the use of postural adjustments in
concert with pellet retrieval. Rating scale ranges from 1 to 3: 1 indicates abnormal behavior and 3 indicates mature, normal movements.
Each symbol represents an individual animal in the specified group. CON rats consistently demonstrate postural adjustments
while reaching and eating. HX rats do not reach and
therefore fail to make subsequent postural adjustments. They also lack
appropriate postural adjustments to maintain balance while retrieving
pellets with their tongue. Impaired motor control in each HX animal
frequently results in a loss of balance when they move away from their
center of gravity. HX+TP rats usually adjust their
posture to maintain balance when reaching forward for a food pellet.
This represents marked improvements as compared with HX animals in the
ability to coordinate postural responses with forelimb use.
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Although qualitatively different from normal rats, target reaching
developed in HX + TP rats (Fig. 10; score = 84%). Despite bilateral forelimb deficits, HX + TP rats preferred to use their left
limbs for reaching. Only when the pellets rested on the very outer edge
of a shelf did the CON and HX + TP rats use their tongues for pellet
retrieval (Fig. 11). Typically, one forelimb (left more than right)
entered the shelf (Fig. 9G) to retrieve the pellet (Fig.
9H) while the opposite forelimb remained on the
ground (Fig. 9G) or on the edge of a shelf for support (Fig.
9H). Forelimb pronation was occasionally excessive
compared with that of normal animals and was accompanied by extreme
digit extension over the pellet. Aiming of the forelimb was not always
smooth or directed toward any particular pellet or shelf; often the
left forelimb was inserted and withdrawn repetitively before a
successful grasp was achieved (significant improvement over HX animals;
p < 0.05). Occasionally after the rat grasped with the
right forepaw, the pellet dropped from the paw as the limb was
withdrawn from the shelf. After successful withdrawal of the grasped
pellet, both forelimbs came to midline under the mouth to hold the
pellet to eat (Fig. 9I). The HX + TP rats usually
shifted their weight backward and relied on other postural adjustments
associated with reaching in a manner similar to that of normal animals
(Fig. 12). For example, HX + TP rats used the nonreaching forelimb on a
shelf to maintain balance while the opposite forelimb reached for and
grasped a pellet. HX + TP rats resembled normals in their approach to
reach for, grasp, withdraw, and ingest the pellet (compare Fig. 9,
A-D with G-I). Despite some qualitative
differences in reaching styles, HX + TP rats used their forelimbs to
grasp significantly more pellets than HX rats in a time frame more
consistent with but not identical to that of normals (Fig.
11A,B). Thus, transplants supported the development
and maturation of skilled forelimb function in goal-directed reaching
to targets in space as well as on the body. The qualitative differences
in execution of the reaching movements in HX + TP animals as compared
with control animals suggest that there may be differences in the
descending supraspinal input controlling reaching movements (Diener and
Bregman, 1998).
Summary
Neonatal cervical spinal cord overhemisection retarded the onset
of forelimb motor control and resulted in the production of abnormal
motor patterns. Lesion-only rats also failed to develop forelimb
goal-directed movements or associated postural adjustments and instead
used compensatory behaviors. In contrast, after neonatal spinal cord
lesion plus transplants, there was a short temporal delay in the
development of some postural reflexes compared with normal rats, but
the forelimb motor patterns developed and resembled those used by
normal rats. Transplant rats developed both forelimb use for
target-directed reaching and appropriate associated postural adjustments to sustain balance during movement. The greater behavioral recovery in the presence of a transplant was accompanied by anatomical reorganization resulting in greater supraspinal input to spinal cord
levels caudal to the lesion (Diener and Bregman, 1998).
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DISCUSSION |
Although the recovery of skilled forelimb movement has been
examined in the adult rat after various injuries, including spinal cord
contusion (Schrimsher and Reier, 1992, 1993), cortical ablations (Castro, 1972a,b; Whishaw and Kolb, 1988; Jones and Shallert, 1992,
1994), striatal or nigrostriatal lesions (Whishaw et al., 1986;
Miklyaeva et al., 1994), and globus pallidus lesions (Schneider and
Olazabal, 1984), this is the first study to explore the development of
forelimb reaching and postural ad |
Top
Abstract
Introduction
