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Departments of 1Anatomy and Neurobiology, and 2Physical Therapy, Virginia Commonwealth University, Medical College of Virginia, Richmond, Virginia 23298-0709
Submitted 13 September 2002; accepted in final form 24 March 2003
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONCONCLUSIONACKNOWLEDGMENTSREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONCONCLUSIONACKNOWLEDGMENTSREFERENCES |
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) and/or
orotracheal intubation (Bier et al.
1993) demonstrate impaired suckling abilities at term. Delays in
the developmental progression of feeding behavior can result in prolonged
hospital stays (Schanler et al.
1999). Ultimately, children who required neonatal intensive care
exhibit an increased frequency of motor speech disorders (Jennische and Sedin
1998,
1999). These outcomes may be
due, in part, to a disruption of the normal development of the hypoglossal
motor system under the influence of altered suckling behavior.
Several animal models have been used to investigate skeletal muscle, in the
developing animal, under the influence of a reduced load or altered activity
level. These models include hindlimb suspension
(Asmussen and Soukup 1991;
Asmussen et al. 1989;
Elder and McComas 1987;
Huckstorf et al. 2000;
Mozdziak et al. 2000; Ohira et al.
2001; Saitoh et al.
1999; Walton et al.
1992), spaceflight (Adams et
al. 2000), or antagonist muscle removal
(Lowrie et al. 1989) to study
the soleus and restricting animals to a soft diet to study the masseter
(Kiliaridis et al. 1988;
Maeda et al. 1987;
Miyata et al. 1993). In
general, a reduction in load or activity level produces a decrease in muscle
fiber size and a shift toward faster muscle fiber types. These adaptations are
more substantial than those seen in adult skeletal muscles
(Asmussen and Soukup 1991;
Asmussen et al. 1989;
Saitoh et al. 1999), and it is
unclear whether they are reversible or permanent (Mozdziak et al. 2000;
Ohira et al. 2001). However,
the acquisition of a normal adult locomotion pattern appears dependent on
hindlimb loading during a critical time period in neonatal rats
(Walton et al. 1992).
Like the soleus and masseter muscles, the tongue is composed of skeletal
muscle tissue. However, despite its obvious functional importance, the
developing tongue musculature has been infrequently studied. Newborn mammals
obtain all of their nutrients through nutritive suckling. It is just one of
several vital processes including respiration, chewing, swallowing, and
vocalization that require normal tongue muscle function
(Lowe 1981;
Sokoloff 2000). Nutritive
suckling involves a protrusion of the tip of the tongue against the nipple,
followed by a retraction to exert a milking or stroking movement from below
(Pieper 1963). Electromyographic and kinematic studies have confirmed tongue
muscle activity during nutritive suckling
(Thexton et al. 1998). As
mammals mature, suckling gives way to chewing; in rats, this transition occurs
between birth and postnatal day 30 (Maeda
et al. 1987). During the same time period, the rat genioglossus
muscle (a tongue protruder) shifts from a developmental to adult fast myosin
heavy chain (MHC) isoform composition
(Brozanski et al. 1993).
Additionally, hypoglossal motoneurons are undergoing significant morphological
and neurophysiological changes (Berger et
al. 1996). No previous studies have investigated the morphological
or physiological characteristics of the developing tongue retractor
musculature.
This study's purpose was to examine the influence of an altered activity
level, via artificial rearing, on the contractile properties, MHC phenotypes
and muscle fiber sizes of the developing rat tongue retractor musculature.
Artificially reared rat pups were fed via a gastric cannula, eliminating
nutritive suckling behavior. This alteration in activity level interferes with
the developmental progression of feeding behavior. Rat pups artificially
reared from postnatal day 1 until postnatal day 18 took twice as long as
dam-reared pups to eat commercial rat chow
(Hall 1975). We hypothesized
that the tongue retractor musculature would develop abnormally under the
influence of an altered activity level.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONCONCLUSIONACKNOWLEDGMENTSREFERENCES |
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Sprague-Dawley rat dams (Harlan Sprague-Dawley, Madison, WI) and their litters of pups (postnatal day 2 on arrival to our facility) were used in the experiments. Litter sizes were maintained at 10 rat pups per dam. Litters were housed in regular cages in light- and temperature-controlled quarters with commercial rat chow and water provided ad libitum.
Litters were allocated into three groups: 1) dam-reared; 2) sham-operated, rat pups that underwent the cannulation procedure on postnatal day 4, had their cannulas cut and sealed, and were dam-reared; and 3) artificially reared, rat pups artificially reared from postnatal day 4 to postnatal day 14. On postnatal day 14, rat pups from each group were subdivided into short-term and long-term groups. Rat pups assigned to the short-term groups immediately underwent study. Rat pups assigned to the long-term groups were dam-reared until postnatal day 21, weaned, and housed in regular cages in light- and temperature-controlled quarters with commercial rat chow and water provided ad libitum. On postnatal day 42, rat pups in the long-term groups underwent study.
All six groups had a sample size of 10 rat pups obtained from two to three different litters. At the conclusion of each study, rat pups were killed using an intraperitoneal injection of Euthasol (150 mg/kg pentobarbital with phenytoin). All experimental procedures complied with the United States Public Health Service Policy on Humane Care and Use of Laboratory Animals and were approved by the Virginia Commonwealth University Institutional Animal Care and Use Committee.
Artificial rearing
The artificial rearing techniques used were developed by Hall
(1975) and derived from a
review written by Patel et al.
(1994). On postnatal day 4,
anesthetized rat pups were cannulated intragastrically. The cannulas of rat
pups to be artificially reared were connected via polyethylene tubing to
disposable syringes mounted on multi-syringe pumps (Harvard Apparatus,
Holliston, MA). The disposable syringes were filled with a rat milk-substitute
formula developed by Hiremagalur et al.
(1993). The rat
milk-substitute formula had a macronutrient composition similar to that in
rat's milk (Table 1),
containing 1.56 kcal/ml. The rat pups were fed at a rate of 0.45 kcal/g body
weight per day, based on average dam-reared rat pups body weights. The
feedings were delivered in 12 equal allotments (23–25 min in a 2-h
cycle) over a 24-h period.
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The rat pups were housed individually in Styrofoam cups filled with bedding material and floated in a temperature-controlled water bath (heated to 38–40°C to maintain the temperature inside the cup at 30–32°C). Daily maintenance included weighing the rat pups, stimulating them to urinate and defecate, and flushing their cannulas. Additionally, the rat milk-substitute formula was renewed and the rate of delivery was adjusted. On postnatal day 14, cannulas of artificially reared rat pups assigned to the long-term group were cut and sealed and the rat pups returned to a dam.
Physiology surgery and measurement of contractile properties
Prior to surgery, rats were anesthetized with an initial intraperitoneal injection of urethane (1.3 g/kg), followed by subsequent doses (300 mg/kg) as needed to maintain deep anesthesia (absence of a withdrawal response to paw pinch). The respiratory rate was continuously monitored and body temperature was maintained between 38 and 40°C using a heating pad. A ventral approach was used to expose the whole hypoglossal nerve with its medial and lateral branches. On one side, the nerve to the geniohyoid muscle and the medial branch of the hypoglossal nerve was excised. The lateral branch of the hypoglossal nerve innervates the tongue retractor musculature, which includes the styloglossus, hyoglossus, inferior longitudinal, and superior longitudinal muscles. The hypoglossal nerve trunk was freed from surrounding tissue. Special care was taken to not disrupt the blood supply and innervation zone of the musculature. The animal was secured in a supine position, and a silk suture was placed into the tip of the tongue.
The tongue was attached to a semiconductor strain gauge (Pixie Model 8101,
ENDEVCO; see Goldberg et al.
1976; Lennerstrand
1974) via the silk suture. The strain gauge has a compliance of 2
µm/g, a resonant frequency of 2 kHz, and is capable of linearly resolving
forces from <1 mg to 100 g. The tongue was aligned with the strain gauge
and pulled slightly out of the mouth to record tongue retraction forces. This
applied stretch (preload) was set at the tension that produced the maximal
isometric tension in response to supramaximal lateral branch stimulation.
A bipolar silver/silver chloride hook electrode was placed around the hypoglossal nerve trunk, proximal to its bifurcation into medial and lateral branches. Recall that the medial branch had been excised, confining nerve conduction to the lateral branch only. Mineral oil was applied to keep the tissues moist and warm and prevent current spread during stimulation.
Twitch characteristics were determined in response to 0.1-ms rectangular
pulses (300–500 µA) delivered 1/s through the lateral branch of the
hypoglossal nerve. Twitch tension, contraction time, and half-decay time were
averaged over 10 trials. The twitch measurements preceded tetanic stimulation
to avoid potentiation. Tetanic forces and fusion frequency were assessed using
200-ms trains at stimulation rates ranging from 20 to 160 Hz in 10-Hz
increments. Fatigability was measured in response to 500-ms trains, at
stimulation rates of 50 Hz (short-term groups) or 90 Hz (long-term groups),
delivered 1/s for 2 min. A stimulation frequency of 50 Hz was chosen because
it approximated the fusion frequency of the postnatal day 14 rat tongue
(unpublished results). A stimulation frequency of 90 Hz was chosen because it
approximated the fusion frequency of the adult rat styloglossus muscle
(Sutlive et al. 1999). The
fatigue index (FI), which is the ratio of tension remaining after 2 min of
stimulation to the tension generated by the initial train, was used to express
muscle fatigability (Burke et al.
1973).
A Master-8 digital, programmable stimulator (A.M.P.I., Jerusalem, Israel) and stimulus isolation unit were used to provide electrical stimulation. Data were visualized during the experiment on a Tektronix 2221 digital oscilloscope (Tektronix, Beaverton, OR) and simultaneously recorded on a Vetter 420-H FM magnetic tape recorder (Vetter Corp., Rebersburg, PA) for subsequent analysis.
Muscle removal and preparation
After measurement of the contractile properties, styloglossus and biceps brachii (long head) muscles were surgically isolated and removed bilaterally. The styloglossus, unlike the other tongue retractor muscles (hyoglossus, inferior longitudinal, and superior longitudinal), is easily isolated surgically. The biceps brachii (long head) is a forearm flexor that served as a control for the possible systemic effects of artificial rearing.
The muscles used for MHC analysis were frozen in liquid nitrogen and stored
at –70°C. Frozen muscles were lyophilized, minced with scissors, and
homogenized with a pellet pestle in ice-cold extraction buffer (0.3 M NaCl,
0.15 M Na2HPO4, 10 mM EDTA, pH 6.5)
(Zhang et al. 1997). The
solution was agitated and stirred at 4°C for 60 min and centrifuged
(10,000g) for 10 min. The total protein concentrations of the
supernatants were determined using the Bio-Rad protein assay for microtiter
plates (Bio Rad Laboratories, Hercules, CA), based on the Bradford dye-binding
procedure (Bradford 1976). The
supernatants were diluted to 0.25 mg/ml in extraction buffer and stored at
–70°C.
The muscles used for muscle fiber size determination were placed in tissue embedding medium, frozen in liquid nitrogen-cooled isopentane, and stored at –70°C. The frozen muscle blocks were trimmed, and serial 10-µm sections were cut with a croyostat kept at –20°C. The unfixed frozen sections were thaw-mounted on gelatin-coated slides.
Electrophoresis of MHCs
MHC isoforms were separated using a sodium dodecyl sulfate-polyacrylamide
gel electrophoresis (SDS-PAGE) technique
(Adams et al. 1999;
Talmadge and Roy 1993). This
specific technique enables the separation of the two developmental MHC
isoforms from the four adult MHC isoforms typically expressed in rodent
skeletal muscles.
Gel slabs (0.75 mm thick) consisted of a 13.5-cm 8% separating gel and a
4-cm 4% stacking gel. All gels were made from the same stock solutions and all
chemicals were of electrophoresis grade. A 2x Laemmli sample buffer
(Laemmli 1970) was added to
the muscle samples to yield a final protein concentration of 0.125 mg/ml.
Samples were boiled for 5 min to denature the protein. Each lane on a gel was
loaded with 20 µl of a muscle sample. Tris-glycine-SDS running buffers
cooled to 4°C were used. Electrophoresis was performed using a vertical
slab gel unit (Protean II xi Cell, Bio Rad Laboratories) run at 275 V for 30 h
at 4°C.
Separating gels were silver stained using the Silver Stain Plus Kit (Bio Rad Laboratories). Images of silver-stained gels were obtained using an AGFA Duoscan HiD scanner (AGFA Corp., Ridgefield Park, NJ). Relative amounts of MHC isoforms were determined using Gel-Pro Analyzer (Media Cybernetics, Silver Spring, MD), image analysis software.
Using this electrophoretic protocol, the migration pattern of adult rat MHC
isoforms has been clearly established (Bar
and Pette 1988; Talmadge and
Roy 1993; Termin et al.
1989). Adult rat MHC isoforms separate in the following order from
top to bottom of the gel: IIa, IIx, IIb, and I
. Adams et al.
(1999,
2000) have determined, based on
Western blot analysis, the migration pattern of the developmental rat MHC
isoforms. Embryonic MHCs demonstrate the slowest migration, appearing above
the adult MHCIIa isoform. Neonatal MHCs demonstrate an intermediate migration,
appearing between the adult MHCIIx and MHCIIb isoforms. In this study,
identification of the individual MHC isoform bands was achieved using a rat
skeletal muscle standard that contained all six MHC isoforms. The rat skeletal
muscle standard was formulated from a mixture of adult and neonatal rat
gastrocnemius, plantaris, and soleus muscles prepared as described (see
Muscle removal and preparation).
Muscle fiber sizes
Styloglossus muscles from three animals in each of the dam-reared and
artificially reared short-term groups were used to determine muscle fiber
sizes. Immunohistochemistry was performed using a MHCIIa + IIx (Developmental
Studies Hybridoma Bank, University of Iowa, Iowa City, IA) monoclonal
antibody. Muscle sections were placed in 10 mM phosphate buffered saline (PBS,
pH 7.5). Nonspecific binding was blocked by a 20-min incubation period in 10%
normal blocking serum. The sections were incubated with diluted (1:50) primary
antibody for 1 h, washed with 10 mM PBS, and incubated with diluted (1:1000)
biotinylated secondary antibody for 30 min. After a wash in 10 mM PBS, the
sections were reacted with Vectastain Elite ABC Reagent (Vector Laboratories,
Burlingame, CA) for 30 min, followed by another wash in 10 mM PBS. A
diaminobenzadine solution was used for visualization. The stained sections
were dehydrated in ascending alcohols, cleared in xylene, and mounted in
permount. An Image-ProPlus image analysis system with an Olympus BH-2
Microscope and a Cool Snap-Pro digital camera (Media Cybernetics, Silver
Spring, MD) was used to determine mean least diameter
(Dubowitz 1985) and mean
cross-sectional area (CSA) of 200 randomly selected muscle fibers (stained and
unstained) from each muscle.
Statistical analysis
Descriptive statistics were calculated for each parameter. Outliers, those values greater or less than 2 SD were removed from the data set. Less than 10% of the samples showed outliers, and these appeared to reflect methodological problems such as poor fixation or an inappropriate maximal tetanic tension setting for the tongues muscles. Differences in contractile properties (twitch tension, twitch contraction time, twitch half-decay time, maximum tetanic tension, fusion frequency, fatigue index) and myosin heavy chain isoform profiles between groups (artificially reared, dam-reared, sham-operated), at each time period (short-term and long-term), were determined by one-factor ANOVA. If an overall significant F value was obtained, Tukey's HSD post hoc analysis was used to isolate statistically significant means. Independent Student's t-tests were used to test for significant differences in muscle fiber sizes (mean least diameter and mean CSA) between dam-reared and artificially reared short-term groups. All tests were carried out using SPSS statistical software. Statistical significance was set at P < 0.05. Values are expressed as means ± SD, except where otherwise noted.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONCONCLUSIONACKNOWLEDGMENTSREFERENCES |
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Our artificial rearing system allowed us to simultaneously rear ≤20 rat
pups per experimental period (postnatal day 4 to postnatal day 14). Two
experimental periods each yielded a success rate of 55%. The majority of rat
pups were lost due to "bloat," a condition characterized by
abdominal distension (Tonkiss et al.
1985).
Body weight
Figure 1 shows the growth curves of dam-reared and artifi-cially reared rat pups from postnatal day 4 to postnatal day 14; initial body weights were not significantly different. Body weights of both groups were significantly higher at the conclusion of the experimental period, indicating that all rat pups grew (P < 0.001). At the time of death, postnatal day 14 artificially reared rat pups (21.45 ± 2.72 g) weighed 22% less than age-matched sham-operated (27.58 ± 3.47 g) and damreared rat pups (27.37 ± 4.24 g; P < 0.05). Postnatal day 42 artificially reared rats (126 ± 17 g) weighed 10.5% less than age-matched sham-operated (140 ± 20 g) and dam-reared rats (144 ± 17 g), although these differences were not significant. No significant differences were found between dam-reared and sham-operated animals in either the short-term or long-term groups.
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Contractile properties
The contractile properties of the rat tongue retractor musculature are summarized in Table 2. In terms of controls, no significant differences were found in any of the contractile properties between dam-reared and sham-operated animals in either the short-term or long-term groups.
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SHORT-TERM GROUPS. Figure 2 illustrates the twitch, tetanic, and fatigue characteristics of a dam-reared and artificially reared rat pup at postnatal day 14. The mean preload or stretch applied to the muscle to produce the maximal isometric tension was not significantly different between the three groups. Despite their smaller body weights, artificially reared rat pups mean twitch and tetanic tensions were similar to sham-operated and dam-reared rat pups. The mean twitch contraction time was not significantly different between the three groups.
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However, postnatal day 14 artificially reared rat pups demonstrated significantly longer twitch half-decay times and lower fusion frequencies than dam-reared rat pups (P < 0.05). Comparable differences existed between artificially reared and sham-operated rat pups, although they were not significant. Moreover, artificially reared rat pups mean fatigue index was significantly less than both sham-operated and dam-reared rat pups (P < 0.001).
LONG-TERM GROUPS. Figure 3 illustrates the twitch, tetanic, and fatigue characteristics of a dam-reared and artificially reared rat pup at postnatal day 42. No significant differences were found in any of the contractile properties between the three groups.
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MHC isoform composition
The MHC phenotypes of the rat styloglossus muscle are summarized in Table 3. In terms of the controls, no significant differences were found in MHC isoform composition between dam-reared and sham-operated animals in either the short-term or long-term groups.
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SHORT-TERM GROUPS—STYLOGLOSSUS MUSCLES. No significant
differences were found in MHC isoform composition between the three groups. At
postnatal day 14, the styloglossus in all groups was composed of MHCIIa >
MHCneonatal > MHCIIx/d > MHCIIb. MHC embryonic was not detected.
However, MHC neonatal comprised approximately 31–32% of the total MHC
complement, indicating that the muscle was in an undifferentiated state
relative to its postnatal day 42 phenotype. One artificially reared rat pup
expressed a small amount of the MHCI isoform.
LONG-TERM GROUPS—STYLOGLOSSUS MUSCLES. At postnatal day
42, the styloglossus in all groups was composed of MHC IIx/d > MHCIIa >
MHCIIb; MHCneonatal was no longer expressed. Artificially reared rats mean
MHCIIx/d isoform expression was similar to sham-operated and dam-reared rats.
However, artificially reared rats demonstrated a significant increase in
MHCIIa expression and decrease in MHCIIb expression compared with
sham-operated and dam-reared rats (P < 0.05). No rats in any group
expressed the MHCI isoform.
SHORT-TERM AND LONG-TERM GROUPS—BICEPS BRACHII MUSCLES. MHC isoform composition of five biceps brachii (long head) muscles from each group was determined to identify possible systemic effects of artificial rearing. No signifi-cant differences were found between dam-reared, sham-operated, and artificially reared animals in either the short-term or long-term groups (data not shown).
Muscle fiber size
Mean muscle fiber size was determined in styloglossus muscles from three animals in each the dam-reared and artificially reared short-term groups. The mean least diameter of muscle fibers from dam-reared rat pups was 16.97 ± 1.38 µm, whereas that from artificially reared rat pups was 16.06 ± 0.82 µm. The mean CSA of muscle fibers from dam-reared rat pups was 425.20 ± 20.85 µm2, whereas that from artificially reared rat pups was 384.60 ± 54.57 µm2. There was no significant muscle fiber size difference between the postnatal day 14 styloglossus muscles from dam-reared and artificially reared rat pups.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONCONCLUSIONACKNOWLEDGMENTSREFERENCES |
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). There is also a progressive fall in hypoglossal motoneuron
input resistance due to a decrease in specific membrane resistivity
(Viana et al. 1994), while
electronic coupling exists between genioglossus motoneurons (prior to
postnatal day 10 only) (Mazza et al.
1992), perhaps to synchronize activity within the motoneuron pool
to coordinate early suckling behavior
(Berger et al. 1996). Also, the
genioglossus muscle shifts from a developmental to adult fast MHC isoform
composition (Brozanski et al.
1993). This impressive maturation of the hypoglossal motor system
occurs as the rat is transitioning from a suckling to chewing feeding motor
pattern (Maeda et al.
1987).
While the importance of studying the impact of various motor tasks (e.g.,
suckling) on hypoglossal motor system development has been recognized
(Berger et al. 1996), this is
the first study of its kind. The artificial rearing model used here attempts
to mimic that of human infants necessarily deprived of normal suckling.
Artificial rearing does not alter activity by traumatizing the muscle,
restricting joint movement, or disrupting the central or peripheral nervous
systems; it simply eliminates nutritive suckling behavior.
It has previously been shown that artificial rearing from postnatal day 4
to postnatal day 14 results in differences in the gustatory afferent terminal
fields of the nucleus of the solitary tract into adulthood
(Lasiter and Diaz 1992). In
response to orochemical stimulation, the nucleus of the solitary tract sends
input to the hypoglossal nucleus
(Wilson-Pauwels et al. 1988).
Therefore the same time period of artificial rearing was used in these initial
attempts to examine the effects of an altered activity level on the developing
rat hypoglossal motor system.
Artificial rearing from postnatal day 4 to postnatal day 14 appeared to affect the normal postnatal development of the tongue retractor musculature. The short-term effects of artifi-cial rearing were an increase in twitch half-decay time, decrease in fusion frequency, and a marked decrease in fatigue resistance. The long-term effects were an increase in MHCIIa expression and a decrease in MHCIIb expression. In all parameters studied, sham-operated animals were similar to damreared animals, eliminating the possibility that the cannulation procedure itself influenced tongue muscle development. Only the results pertaining to the dam-reared and artificially reared groups will be discussed.
Body weight
Postnatal day 14 artificially reared rat pups weighed significantly less
than dam-reared rat pups. The lower body weights were likely due to attempts
to treat bloat by reducing the rate of milk infusion. According to Tonkiss et
al. (1987), rat pups can
recover from bloat if the rate of milk infusion is greatly reduced, but this
results in decreased body weight gain. Human infants on enteral feeding suffer
from a similar condition known as necrotizing enterocolitis
(Diaz et al. 1980).
Contractile properties
At birth, future "slow" and "fast" muscles exhibit
slow isometric contractile speeds (Kernell
1998). During the postnatal period, slow muscles become fast and
then slow, while fast muscles become faster
(Kernell 1998). Differences in
isometric twitch contraction time between muscles have been attributed to
varying calcium kinetics rather than myosin ATPase activity
(Brody 1976). Hindlimb
suspension of the postnatal rat produced a decrease in the twitch contraction
time and half-relaxation time in the slow twitch soleus muscle
(Asmussen et al. 1989;
Elder and McComas 1987).
Denervation of the developing fast twitch rabbit tibialis anterior muscle
impeded the normal increase of Ca2+ATPase and decreased
calsequestrin (Leberer et al.
1986), changes that are functionally related to slower contraction
times (Burke 1981). Similarly,
artificial rearing from postnatal day 4 to postnatal day 14 resulted in an
overall, but modest, slowing of contractile speed in the fast twitch tongue
retractor musculature.
To our knowledge, no previous studies have investigated the effects of a
reduced load or altered activity level on fatigue resistance of developing
skeletal muscle. While ultrasound imaging has confirmed gross protrusive and
retrusive movements of the tongue during human infant nutritive suckling, it
also revealed that the lateral portions of the tongue (styloglossus and
hyoglossus muscles) serve primarily a sustained stabilizing function (Bosma et
al. 1990). The removal of nutritive suckling resulted in a substantial
decrease in fatigue resistance of the tongue retractor musculature. According
to Faulkner et al. (1972),
relative inactivity easily reduces the oxidative capacity of skeletal muscles,
which is associated with a decrease in fatigue resistance
(Burke 1981).
A 1-mo resumption of function was adequate for recovery of the contractile speed and fatigue characteristics of the tongue retractor musculature affected by artificial rearing from postnatal day 4 to postnatal day 14. In a previous study, immobilization of the adult rat hindlimb musculature shortened the isometric twitch duration of the soleus and lengthened the isometric twitch duration of the extensor digitorum longus; in both muscles, the contractile speed characteristics recovered in 14 days (Witzman et al. 1982).
MHC isoform composition
The short-term artificially reared group showed no signifi-cant differences in MHC phenotype of the styloglossus muscle compared with the dam-reared group. However, the long-term artificially reared group showed an increase in MHCIIa isoform expression and a decrease in MHCIIb isoform expression of the styloglossus muscle compared with the dam-reared group. A 1-mo resumption of function altered the MHC phenotype of the styloglossus.
While artificial rearing did not initially alter the MHC phenotype of the
styloglossus, it should be recalled that MHC neonatal still comprises about
32% of the muscle at 14 days. Perhaps the survival period used was not long
enough to reveal MHC differences in the styloglossus muscle. However, 14 days
may have been enough to alter the development of other masticatory muscles,
impacting subsequent function. Suckling involves coordinated movements between
the mandible, hyoid, and tongue (Bosma et al. 1990). Electromyographic
activity has been recorded in the rat masseter, anterior digastric,
sternohyoideus, and genioglossus muscles during nutritive suckling
(Westneat and Hall 1992).
While the effects of artificial rearing on these muscles have not been
examined, alteration in mastication behavior has been shown to alter the
development of the rat masseter (Maeda et
al. 1981) and craniofacial growth
(Kiliaridis et al. 1988).
Perhaps the change in masticatory function after artificial rearing results
in a relative increase of endurance activity for the styloglossus muscle and
thus led to the MHC phenotype shift from MHCIIb to MHCIIa. Resistance training
induces an apparent down-regulation of the MHCIIb isoform with a corresponding
up-regulation of the MHCIIa isoform (Booth
and Baldwin 1996). The altered MHC phenotype of the styloglossus
muscle did not appear to be enough to impact the contractile properties of the
tongue retractor musculature.
The biceps brachii (long head) served as a control muscle to distinguish the "altered activity" effects from those resulting from the stresses of artificial rearing (e.g., social isolation) and slight undernourishment. The myosin heavy chain phenotypes of the postnatal day 14 and postnatal day 42 biceps brachii (long head) muscles were not significantly different between the dam-reared and artificially reared groups. These findings suggest that the stresses of artificial rearing and level of undernourishment did not have a systemic impact on the MHC phenotypes of skeletal muscles.
Muscle fiber size
Despite their smaller body weights, the mean muscle fiber size of postnatal day 14 styloglossus muscles from artificially reared rat pups was similar to that of dam-reared rat pups. These data compare favorably with the contractile tension data in that artificial rearing also did not impact contractile tensions.
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CONCLUSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONCONCLUSIONACKNOWLEDGMENTSREFERENCES |
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). Hindlimb suspension not only reduces the load placed on the
soleus, it places the foot in plantarflexion so that the muscle is in a
shortened resting position relative to normal
(Thomason and Booth 1990).
Differing from hindlimb suspension, artificial rearing does not alter the
resting position of the tongue musculature. The soft diet model replaces a
forceful activity (e.g., biting hard food), with a nonforceful activity (e.g.,
lapping) (Miyata et al. 1993);
the masseter muscle is one of the main muscles activated during biting and
chewing food (Kiliaridis and Shyu
1988). Perhaps artificial rearing does not significantly reduce
the load or force production of the tongue retractor musculature, but rather
alters its endurance activity level.
While the tongue is similar to other skeletal muscles in that its MHC
phenotype is undifferentiated at birth
(Brozanski et al. 1993), the
time course for the postnatal myosin isoform transition is species and muscle
specific (Pette and Staron
1997). The rate at which a muscle decreases developmental myosin
isoform expression and increases adult myosin isoform expression occurs in the
following order in the rat: diaphragm > soleus > tongue > masseter
(d'Albis et al. 1989, 1991).
d'Albis et al. (1991)
suggested that the differences in transition time courses are in part related
to a muscle's function. At postnatal day 14, MHC neonatal still comprised
approximately 32% of the total MHC isoform composition in the rat styloglossus
muscle. It is possible that our time period of artificial rearing was not
initiated early enough, sustained long enough, or encompassed the
developmental age during which it would have the most dramatic effects on the
tongue musculature. Additionally, further effects of artificial rearing on the
tongue retractor musculature may exist, yet were undetected due to small
sample sizes.
Regardless, our results provide evidence to support artificial rearing as a
useful model to alter the activity level of the tongue. The experiments
reported herein are a first crucial step toward understanding the influence of
a motor task (e.g., suckling) on hypoglossal motor system development. Future
investigations in this laboratory are aimed at investigating the effects of
artificial rearing on the developing genioglossus muscle and hypoglossal
motoneuron compartmentalization (Aldes
1995). Since the exertion of pressure on the nipple by the tongue,
a protrusive movement, is characteristic of nutritive suckling
(Sameroff 1973), artificial
rearing may have a greater impact on the genioglossus. Different time periods
of artificial rearing will be utilized to further determine if critical
periods for hypoglossal motor system development exist.
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ACKNOWLEDGMENTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONCONCLUSIONACKNOWLEDGMENTSREFERENCES |
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This work was supported by National Institute of Deafness and Other Communication Disorders Grant 5 RO1 DC-02008.
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FOOTNOTES |
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Address for reprint requests: S. J. Goldberg, Virginia Commonwealth Univ., Medical College of Virginia, Dept. of Anatomy and Neurobiology, P.O. Box 980709, 1101 E. Marshall St., Richmond, VA 23298-0709 (E-mail: sgoldber{at}hsc.vcu.edu).
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