Objectives/Hypothesis: Regenerative properties of age-associated changes in the intrinsic laryngeal muscles following injury are unclear. The purpose of this study was to investigate the regenerative properties of the thyroarytenoid (TA) muscle in an aging rat model. The hypothesis was that following myotoxic injury, old animals would exhibit a decrease in mitotic activities of muscle satellite cells when compared with younger rats, suggesting reduced regenerative potential in the aging rat TA muscle. Study Design: Animal group comparison. Methods: Regeneration responses following injury to the TA muscle were examined in 18 young adult, middle-aged, and old Fischer 344/Brown Norway rats. TA muscle fiber cross-sectional area (CSA), satellite cell mitosis (number/fiber), and regeneration index (CSA injured side/CSA noninjured side) were measured and compared across age groups. Results: Young adult animals had a significantly higher regeneration index than the middle-aged and old groups. Within the lateral region of the TA muscle (LTA), the regeneration index was significantly higher in the young adult animals than in the middle-aged and old animals. The regeneration index of the medial TA was significantly higher than the LTA across all age groups. Conclusions: The regenerative capacity of the TA muscle is impaired with increasing age. Laryngoscope, 2012

Satellite cells are the myogenic cells lying between the myofiber sarcolemma and basal lamina. The objective of this study was to determine the expression patterns of MyoD, myogenin, and Pax7 within the satellite cell population in the growing rat soleus and extensor digitorum longus (EDL) muscles. Secondly, the expression of the myogenic markers was also studied within the interstitial cell compartment and myonuclei. It was discovered that the soleus contained a higher number of Pax7, MyoD, or myogenin-positive nuclei compared with the EDL. Similarly, myogenin was expressed at a lower level in the myonuclei of the soleus compared with the EDL, and myogenin was expressed at a higher level in the interstitial compartment of the soleus compared with the EDL. When interstitial nuclei, myonuclei, and double-labeled nuclei were used in the estimate of the satellite cell population, it was discovered that approximately of 13% of the myofibers in a transverse section of the soleus muscle and 4.1% of EDL myofibers exhibit a labeled satellite cell nucleus. Overall, results from this study suggest that expression patterns of these markers vary predictably among muscles with different growth dynamics and phenotypic characteristics.

The biomechanical and regenerative responses in rabbit tibialis anterior muscle following a single eccentric stretch injury were investigated 1, 3 and 7 days after traumatization. The stretch injury produced a transient biomechanical effect. On day 1, the load at failure was decreased in the injured muscles compared with the uninjured controls (P<0.05), but on days 3 and 7 significant biomechanical differences were no longer recorded between the injured and control muscles. In immunohistochemical analyses, no overt myonecrosis or connective tissue disruption was observed in any of the stretch-injured muscles. Yet satellite cells were activated to incorporate a thymidine analogue, bromodeoxyuridine indicating mitotic activity. They did not, however, produce muscle-specific proteins, i.e., they did not differentiate further. Furthermore, the intact appearing but mildly injured myofibers of the stretched muscles expressed both neonatal myosin and vimentin near the myotendinous junctions. Collectively, these results suggest that a single mild eccentric stretch of skeletal muscle which does not cause gross structural alterations results in a short-term decrease in tensile load and induces transient proliferation of satellite cells and fibroblasts together with expression of primitive proteins in myofibers.

This paper presents the final report on Skeletal Muscle Growth and Repair During Microgravity. This report covers the period of July 1, 1998 through June 30, 2002.

The hindlimb-unloading model was used to study the ability of muscle injured in a weightless environment to recover after reloading. Satellite cell mitotic activity and DNA unit size were determined in injured and intact soleus muscles from hindlimb-unloaded and age-matched weight-bearing rats at the conclusion of 28 days of hindlimb unloading, 2 wk after reloading, and 9 wk after reloading. The body weights of hindlimb-unloaded rats were significantly ( P < 0.05) less than those of weight-bearing rats at the conclusion of hindlimb unloading, but they were the same ( P > 0.05) as those of weight-bearing rats 2 and 9 wk after reloading. The soleus muscle weight, soleus muscle weight-to-body weight ratio, myofiber diameter, number of nuclei per millimeter, and DNA unit size were significantly ( P< 0.05) smaller for the injured soleus muscles from hindlimb-unloaded rats than for the soleus muscles from weight-bearing rats at each recovery time. Satellite cell mitotic activity was significantly ( P < 0.05) higher in the injured soleus muscles from hindlimb-unloaded rats than from weight-bearing rats 2 wk after reloading, but it was the same ( P > 0.05) as in the injured soleus muscles from weight-bearing rats 9 wk after reloading. The injured soleus muscles from hindlimb-unloaded rats failed to achieve weight-bearing muscle size 9 wk after reloading, because incomplete compensation for the decrease in myonuclear accretion and DNA unit size expansion occurred during the unloading period.

The satellite cell population in postnatal skeletal muscle is heterogeneous because individual satellite cells isolated from a single muscle have differing abilities to proliferate under the same in vitro conditions. Telomeres are structures found at the ends of all eukaryotic chromosomes that are characterized by repetitive DNA sequences, and they are important in determining cellular proliferation potential. The relationship between satellite cell proliferative heterogeneity and telomeric DNA was examined by digesting genomic DNA from large-colony-forming and small-colony-forming turkey satellite cell clones with HinfI, separating the restriction fragments on an agarose gel, and hybridizing the gels with an oligonucleotide probe specific for telomeric DNA. Turkey satellite cells generated telomeric restriction fragments up to approximately 180 kB. The large-colony-forming satellite cell clones had a larger proportion (P<0.05) of total telomeric restriction fragments below 33 kB than the small-colony-forming satellite cell clones. However, telomerase expression was detected in cultures from large-colony-forming and small-colony-forming turkey satellite cells suggesting that the differences in telomeric restriction fragments may not be related to the differences in in vitro proliferative behavior and that telomerase may contribute to the high in vitro growth capacity of turkey satellite cells.

Nutritional status influences muscle growth and athletic performance, but little is known about the effect of nutritional supplements, such as creatine, on satellite cell mitotic activity. The purpose of this study was to examine the effect of oral creatine supplementation on muscle growth, compensatory hypertrophy, and satellite cell mitotic activity. Compensatory hypertrophy was induced in the rat plantaris muscle by removing the soleus and gastrocnemius muscles. Immediately following surgery, a group of six rats was provided with elevated levels of creatine monohydrate in their diet. Another group of six rats was maintained as a non-supplemented control group. Twelve days following surgery, all rats were implanted with mini-osmotic pumps containing the thymidine analog 5-bromo-2'-deoxyuridine (BrdU) to label mitotically active satellite cells. Four weeks after the initial surgery the rats were killed, plantaris muscles were removed and weighed. Subsequently, BrdU-labeled and non-BrdU-labeled nuclei were identified on enzymatically isolated myofiber segments. Muscle mass and myofiber diameters were larger (P < 0.05) in the muscles that underwent compensatory hypertrophy compared to the control muscles, but there were no differences between muscles from creatine-supplemented and non-creatine-supplemented rats. Similarly, compensatory hypertrophy resulted in an increased (P < 0.05) number of BrdU-labeled myofiber nuclei, but creatine supplementation in combination with compensatory hypertrophy resulted in a higher (P < 0.05) number of BrdU-labeled myofiber nuclei compared to compensatory hypertrophy without creatine supplementation. Thus, creatine supplementation in combination with an increased functional load results in increased satellite cell mitotic activity.

5-Bromo-2'-deoxyuridne (BrdU) and H-3-thymidine label mitotically active cells, but they do not adequately mark the progeny of dividing cells for long term study. An alternative method is to label cells using the replication-defective CXL retroviral vector, which carries the lacZ gene encoding beta-galactosidase; however. the ability of the CXL retroviral vector to pulse-label mitotically active cells selectively is not known. Cultures of proliferating muscle cells were simultaneously incubated with the CXL retrovirus and BrdU (10 mu M) for 2 hr. After removing the retrovirus containing medium. the cells were maintained for an additional 24 hr in vitro before they were stained to detect beta-galactosidase and BrdU simultaneously. More than 95% of beta-galactosidase positive cells were also BrdU positive suggesting that the majority of beta-galactosidase positive cells were in the S-phase of the cell cycle at the time of CXL retroviral administration. Therefore, the CXL retroviral vector is an appropriate pulse marker for dividing cells, and it is useful when it is desirable to know the fate of the progeny of a particular cell following a mitotic event.

The role of satellite cells and DNA unit size in determining muscle size was examined by inhibiting postnatal skeletal muscle development by using hindlimb suspension. Satellite cell mitotic activity and DNA unit size were determined in the soleus muscles from hindlimb-suspended and age-matched weight-bearing rats before the initiation of hindlimb suspension, at the conclusion of a 28-day hindlimb-suspension period, 2 wk after reloading, and 9 wk after reloading. The body weights of hindlimb-suspended rats were significantly (P < 0.05) less than those of weight-bearing rats at the conclusion of hindlimb suspension, but they were the same (P > 0. 05) as those of weight-bearing rats 9 wk after reloading. The soleus muscle weight, soleus muscle weight-to-body weight ratio, myofiber diameter, nuclei per millimeter, and DNA unit size for the hindlimb-suspended rats were significantly (P < 0.05) smaller than for the weight-bearing rats at all recovery times. Satellite cell mitotic activity was significantly (P < 0.05) higher in the soleus muscles from hindlimb-suspended rats 2 wk after reloading, but it was the same (P > 0.05) as in weight-bearing rats 9 wk after reloading. Juvenile soleus muscles failed to achieve normal muscle size 9 wk after reloading because there was incomplete compensation for the hindlimb-suspension-induced interruptions in myonuclear accretion and DNA unit size expansion.

BACKGROUND 5-Bromo-2'-deoxyuridine (BrdU) is a powerful compound to study the mitotic activity of a cell. Most techniques that identify BrdU-labeled cells require conditions that kill the cells. However, the fluorescence intensity of the membrane-permeable Hoechst dyes is reduced by the incorporation of BrdU into DNA, allowing the separation of viable BrdU positive (BrdU+) cells from viable BrdU negative (BrdU-) cells. METHODS Cultures of proliferating cells were supplemented with BrdU for 48 h and other cultures of proliferating cells were maintained without BrdU. Mixtures of viable BrdU+ and viable BrdU- cells from the two proliferating cultures were stained with Hoechst 33342. The viable BrdU+ and BrdU- cells were sorted into different fractions from a mixture of BrdU+ and BrdU- cells based on Hoechst fluorescence intensity and the ability to exclude the vital dye, propidium iodide. Subsequently, samples from the original mixture, the sorted BrdU+ cell population, and the sorted BrdU- cell population were immunostained using an anti-BrdU monoclonal antibody and evaluated using flow cytometry. RESULTS Two mixtures consisting of approximately 55% and 69% BrdU+ cells were sorted into fractions consisting of greater than 93% BrdU+ cells and 92% BrdU- cells. The separated cell populations were maintained in vitro after sorting to demonstrate their viability. CONCLUSIONS Hoechst fluorescence intensity in combination with cell sorting is an effective tool to separate viable BrdU+ from viable BrdU- cells for further study. The separated cell populations were maintained in vitro after sorting to demonstrate their viability.

BACKGROUND:Myogenin expression is associated with a slow myofiber phenotype, and MyoD expression is associated with a fast myofiber phenotype. Hindlimb suspension (HS) will induce muscular atrophy, and a transition from a slow to fast myofiber phenotype in the rat soleus.HYPOTHESIS:Hindlimb suspension will induce myofiber atrophy, and a slow to fast myofiber type transition with corresponding changes in myogenin and MyoD expression.METHODS:Myofiber phenotype was evaluated by electrophoretically separating the myosin heavy chain isoforms. Myogenin expression was evaluated by Northern analysis, while MyoD expression was evaluated by Northern analysis and semiquantitative RT-PCR.RESULT:After 28 d of hindlimb suspension, there was significantly (p < 0.05) less myosin heavy chain Type IIA, and more (p < 0.05) myosin heavy chain Type IIX in the soleus muscles of hindlimb suspended rats compared with soleus muscles from weight-bearing (WB) rats. Although there was a shift to a faster myosin heavy chain phenotype in soleus muscles from hindlimb suspended rats, there was no change in myogenin expression, and MyoD expression was undetectable by Northern analysis. Semi-quantitative RT-PCR revealed an up-regulation of MyoD expression following 14 d of hindlimb suspension.CONCLUSIONS:Myogenin expression levels do not change during the slow to fast myofiber phenotypic transition that occurs during hindlimb suspension; MyoD expression appears to increase at the same time as the phenotypic transition. Thus, MyoD expression or the Myogenin: MyoD mRNA ratio may be important in the phenotypic transition. Neither myogenin nor MyoD appear to play a critical role in the muscular atrophy that occurs during weightlessness.

Nitric oxide (NO) is an inter- and intracellular messenger involved in a variety of physiologic and pathophysiologic conditions. The effect of two NO donors, sodium nitroprusside (SNP) and S-nitroso-N-acetylpenicillamine (SNAP) and their effect on myoblast proliferation was examined. Both donors stimulated an increase in myoblast cell number over a range (1–10 µM) of donor concentrations. However, 50 µM SNAP inhibited myoblast proliferation. Cell numbers from cultures treated with degraded 10 µM SNAP were equivalent to the control. Therefore, it appears NO can stimulate as well as inhibit myoblast proliferation.

The gross and microscopic anatomy of the rat larynx was studied with particular attention to myology and neuromuscular structures to further validate it as a model to evaluate morphologic and functional changes induced by botulinum injection, A laryngeal alar cartilage (LAIC), alar cricoarytenoid (ACA) muscle, and a superior cricoarytenoid muscle (SCA) were identified as anatomic-structures not previously described. Two portions (medial and lateral) of the thyroarytenoid muscle (TA) were distinguished, The function of the ACA was suggested to be similar to the aryepiglottis muscle in humans and the function of the SCA was suggested to be similar to the human interarytenoid muscle, The predominant pattern of motor endplate (MEP) distribution in rat laryngeal muscles (posterior cricoarytenoid, lateral cricoarytenoid, cricothyroid, and SCA) was to have MEPs concentrated mostly at the midbelly of muscle where they were distributed throughout the cross-sectional area of the midbelly. The TA and ACA differed from this pattern, The lateral TA had MEPs concentrated at the anterior third of its belly and those of the medial TA were located at the midbelly. Motor endplates in the ACA were located mostly at the posterior portion of muscle, Muscle fiber-typing showed subtle differences between the intrinsic laryngeal muscles, Fast fibers were predominant in the rat laryngeal muscles, This study supports the expanded use of rats in studies of laryngeal neuromuscular function and disease in humans.

805 Sodium nitroprusside (SNP), a nitric oxide (NO·) generator has recently been shown to enhance myoblast proliferation in vitro suggesting that NO· could have the potential to enhance muscle fiber regeneration following muscle strain injury. However, SNP is not considered an ideal NO· generator because it releases NO· sporadically and produces cyanide. The intent of this study was to compare the myoproliferative effects of SNP to those of S-nitroso-N-acetylpenicllamine (SNAP), another NO· generator which does not produce cyanide or other detrimental byproducts. Primary cell cultures were obtained from the pectoralis thoracicus of four, three week old turkey embryos. The myoblasts were isolated following enzymatic liberation and Percol density gradient centrifugation. The cells were allowed to attach to gelatin coated plates for 48 hours in plating media. After the attachment period, a random sample of cells (n=4) was counted with a Coulter Counter. Growth media as well as growth media with SNP, SNAP and degraded SNAP (8-10 hrs) were introduced to random populations of cells and changed every 48 hours thereafter. Cells were counted at 72, 96, 120, and 144 hours after introduction of growth media (n=4 for each treatment and each time). SNP stimulated myoblast proliferation significantly (p<0.01) at 0.01mM, 0.005mM and 0.001mM with maximal cell growth at 0.005mM (p<0.01). SNAP significantly (p<0.01) stimulated myoblast proliferation at 0.01mM and 0.005mM with maximal stimulation at 0.01mM (p<0.01). Optimal concentrations of SNAP and SNP stimulated myoblast proliferation equally. Furthermore, cell growth with the degraded 0.01mM SNAP was equivalent to control. Therefore, this study shows that both SNP and SNAP stimulate myoblast proliferation. Because SNAP enhances myoblast proliferation without the detrimental production of cyanide it may be the generator of choice for stimulating in vitro myoblast proliferation.

The relationship between myogenin or MyoD expression and hypertrophy of the rat soleus produced either by clenbuterol and 3,3', 5-triiodo-L-thyronine (CT) treatment or by surgical overload was examined. Mature female rats were subjected to surgical overload of the right soleus with the left soleus serving as a control. Another group received the same surgical treatment but were administered CT. Soleus muscles were harvested 4 wk after surgical overload and weighed. Myosin heavy chain isoforms were separated by using polyacrylamide gel electrophoresis while myogenin and MyoD expression were evaluated by Northern analysis. CT and functional overload increased soleus muscle weight. CT treatment induced the appearance of the fast type IIX myosin heavy chain isoform, depressed myogenin expression, and induced MyoD expression. However, functional overload did not alter myogenin or MyoD expression in CT-treated or non-CT-treated rats. Thus pharmacologically and surgically induced hypertrophy have differing effects on myogenin and MyoD expression, because their levels were associated with changes in myosin heavy chain composition (especially type IIX) rather than changes in muscle mass.

Exposure of juvenile skeletal muscle to a weightless environment reduces growth and satellite cell mitotic activity. However, the effect of a weightless environment on the satellite cell population during muscle repair remains unknown. Muscle injury was induced in rat soleus muscles using the myotoxic snake venom, notexin. Rats were placed into hindlimb-suspended or weightbearing groups for 10 days following injury. Cellular proliferation during regeneration was evaluated using 5-bromo-2′-deoxyuridine (BrdU) immunohistochemistry and image analysis. Hindlimb suspension reduced (P<0.05) regenerated muscle mass, regenerated myofiber diameter, uninjured muscle mass, and uninjured myofiber diameter compared to weightbearing rats. Hindlimb suspension reduced (P<0.05) BrdU labeling in uninjured soleus muscles compared to weightbearing muscles. However, hindlimb suspension did not abolish muscle regeneration because myofibers formed in the injured soleus muscles of hindlimb-suspended rats, and BrdU labeling was equivalent (P>0.10) on myofiber segments isolated from the soleus muscles of hindlimb-suspended and weightbearing rats following injury. Thus, hindlimb suspension (weightlessness) does not suppress satellite cell mitotic activity in regenerating muscles before myofiber formation, but reduces growth of the newly formed myofibers.

OBJECTIVES:To characterize the acute and chronic cellular effects of botulinum toxin (BT) injection into rat laryngeal muscles. A complete characterization of these effects is important because patients with focal dystonias of the head and neck are commonly treated with BT injection. Further, potential muscular changes in the larynx must be carefully delineated owing to the critical phonatory and airway protective functions of these muscles. STUDY DESIGN:The acute and chronic cellular effects of BT injection were studied using 5'-bromo 2'-deoxyuridine (BrdU) following single and repeated BT injection into rat laryngeal muscles. BrdU is incorporated into mitotically active nuclei such that changes in cell proliferative behavior following BT injection can be monitored. RESULTS:Increased mitotic activity was detected in the tissue samples studied following BT injection. Differences in the times of the peak distribution of BrdU-labeled cells in each laryngeal muscle were observed. This may be related to the diffusion effects of BT. Prolonged muscle fiber changes, including splitting, were also observed as the result of repeated BT injection. CONCLUSIONS:The results of this study suggest that BT may induce a proliferative response in muscle tissue.

This pilot study was designed to determine if the interval between repeated botulinum toxin injections influenced physiologic and histologic effects on laryngeal muscles in a rat model. The physiologic measurements included digitized videomicroscopic recording of vocal fold movement and electromyography. The histologic measurements included muscle fiber size and digitized optical density of laryngeal muscles after glycogen depletion by electrical stimulation. The results demonstrated that the effect of timing of the second injection was strongly correlated to laryngeal changes. Most notable were results in the subjects that underwent injections 6 weeks apart. We hypothesize that these findings might be related to terminal axonal sprouting with reinnervation. The results from this study help confirm and expand the validity of using the rat laryngeal model to understand the effect of botulinum toxin. Moreover, we believe that the data might be extrapolated to prove useful in predicting human responses to botulinum toxin treatment for functional dystonias such as spasmodic dysphonia.