Effect of Fascia Implantation and Controlled Release of Basic Fibroblast Growth Factor for Muscle Atrophy in Rat Laryngeal Paralysis.

OBJECTIVE: To improve lateral thyroarytenoid (TA) muscle atrophy after laryngeal paralysis, reconstruction of the vascular network of the atrophied muscle is necessary. We therefore evaluated whether the controlled release of basic fibroblast growth factor (bFGF) with autologous fascia implantation could affect vascular reconstruction in the lateral TA muscle. STUDY DESIGN: Animal experiment. SETTING: Laboratory. METHODS: Unilateral laryngeal paralysis was induced in 20 rats. The rats were implanted with autologous fascia and a gelatin hydrogel sheet with or without 1 µg of bFGF (fascia and bFGF + fascia groups; n = 5 each) and with only a gelatin hydrogel sheet with bFGF (bFGF group: n = 5). Another group remained untreated (n = 5) at 4 months after paralysis. At 3 months since transplantation, intra- and intergroup comparisons of the muscle volumes and total area of blood vessels in the lateral TA muscle were performed. RESULTS: When compared with the untreated group, the bFGF + fascia group showed a significant increase in muscle volume (P =.0008) and vascular area (P =.0002) in the lateral TA muscle, whereas the other 2 treated groups demonstrated an insufficient effect. CONCLUSION: bFGF + fascia implantation showed histologic improvement in severe laryngeal paralysis. We demonstrated that the decrease in lateral TA muscle mass after paralysis might be countered by the reconstruction of the vascular network. Our findings indicate that hypovascular and denervated areas of the laryngeal muscle can be regenerated by the implantation of growth factors and scaffolds with surgical stress. LEVEL OF EVIDENCE: 5.

Microglial SIRPα regulates the emergence of CD11c+ microglia and demyelination damage in white matter.

A characteristic subset of microglia expressing CD11c appears in response to brain damage. However, the functional role of CD11c+ microglia, as well as the mechanism of its induction, are poorly understood. Here we report that the genetic ablation of signal regulatory protein α (SIRPα), a membrane protein, induced the emergence of CD11c+ microglia in the brain white matter. Mice lacking CD47, a physiological ligand of SIRPα, and microglia-specific SIRPα-knockout mice exhibited the same phenotype, suggesting that an interaction between microglial SIRPα and CD47 on neighbouring cells suppressed the emergence of CD11c+ microglia. A lack of SIRPα did not cause detectable damage to the white matter, but resulted in the increased expression of genes whose expression is characteristic of the repair phase after demyelination. In addition, cuprizone-induced demyelination was alleviated by the microglia-specific ablation of SIRPα. Thus, microglial SIRPα suppresses the induction of CD11c+ microglia that have the potential to accelerate the repair of damaged white matter.

Evaluation of Autologous Fascia Implantation With Controlled Release of Fibroblast Growth Factor for Recurrent Laryngeal Nerve Paralysis Due to Long-term Denervation.

OBJECTIVES: Paralyzed tissue due to long-term denervation is resistant to many treatments because it induces irreversible histological changes and disorders of deglutition or phonation. We sought to determine the effect of autologous transplantation of fascia into the vocal fold (ATFV) with controlled release of basic fibroblast growth factor (bFGF) on long-term unilateral vocal fold paralysis (UVFP). METHODS: Unilateral recurrent laryngeal nerve (RLN) section was performed on 20 rats. Five rats were implanted with autologous fascia only (fascia group), and 10 rats were implanted with autologous fascia and a gelatin hydrogel sheet with 1 µg (1 µg bFGF + fascia group) or 0.1 µg (0.1 µg bFGF + fascia group) of bFGF 4 months after RLN section. We evaluated the normalized glottal gap and laryngeal volume and histological changes 3 months after implantation. RESULTS: The normalized glottal gap was significantly reduced in the 3 fascia implantation groups. Normalized laryngeal volume, fat volume, and lateral thyroarytenoid muscle volume were significantly increased in the 2 fascia implantation with bFGF groups. CONCLUSIONS: The ATFV with controlled release of bFGF repaired the glottal gap and laryngeal volume after RLN section and may reduce the occurrence of aspiration and hoarseness. We speculate that this treatment improves laryngeal function in long-term RLN denervation.

Fascia implantation with fibroblast growth factor on vocal fold paralysis.

PURPOSE: The purpose of this prospective study was to determine the effect of autologous transplantation of fascia into the vocal fold (ATFV) with controlled release of basic fibroblast growth factor (bFGF) on unilateral vocal fold paralysis (UVFP) in a rat model. MATERIALS AND METHODS: Unilateral recurrent laryngeal nerve (RLN) section was performed on 15 rats. Ten rats received an autologous fascia implant and gelatin hydrogel with or without bFGF (1 µg) to their larynxes (fascia only, "fascia group"; bFGF + fascia, "fascia + bFGF group"), while the rest underwent RLN transection ("RLN section group"). Four months later, evaluation of the laryngeal glottal gap and histological analysis were performed. RESULTS: The glottal gap was significantly reduced in the fascia + bFGF group, and fat volume increased significantly relative to the RLN section. The volume of the remaining fascia in the bFGF + fascia group was significantly greater than that of the fascia group. CONCLUSIONS: ATFV with controlled release of bFGF may compensate for diminished laryngeal volume in UVFP by reducing resorption of the implanted fascia and increasing fat volume. Our findings suggest that this modality may represent an attractive option for treating UVFP.

Association between osteopontin promoter variants and diastolic dysfunction in hypertensive heart in the Japanese population.

Heart failure with preserved ejection fraction is recently highlighted as a major health problem, and diastolic dysfunction associated with hypertension has a dominant role in the development of heart failure with preserved ejection fraction. Osteopontin (OPN) is a secreted phosphoprotein, which mediates fibrosis. In animal models, OPN is upregulated in response to pressure overload and is thought to be involved in systolic dysfunction. However, the functional role of OPN in diastolic dysfunction is unknown. The guanine base insertion polymorphism at -156 position of the OPN promoter is postulated to upregulate the transcription of OPN in human. To investigate whether -156del/G polymorphism of OPN promoter is associated with diastolic dysfunction in hypertensive hearts, the patients with hypertension have been genotyped for variants of -156del/G polymorphism by genomic sequencing. Diastolic function of the left ventricle was estimated as the ratio of early to atrial filling (E/A ratio), obtained by pulsed-Doppler derived transmitral flow in echocardiographic analysis. The patients with -156G allele displayed lower E/A ratio compared with those with -156del/del genotype, suggesting exacerbated diastolic function. Notably, in case of the population with diabetes mellitus, the patients with -156G allele showed significant association with lower E/A ratio, compared with -156del/-156del patients. Multiple linear regression analysis revealed that prevalence of -156G allele was an independent factor for lowering E/A ratio. The -156del/G genetic variants of OPN promoter were associated with decreased E/A ratio in hypertensive patients. These results suggest that OPN has a functional role in the development of diastolic dysfunction in hypertensive hearts.

Identification of a royal jelly glycoprotein that carries unique complex-type N-glycans harboring the T-antigen (Galß1-3GalNAc) unit.

In this study, we identified a royal jelly glycoprotein (RJG) that carries a unique complex-type N-glycans harboring the T-antigen (Galß1-3GalNAc) unit. The amino acid sequence of the tryptic glycopeptide harboring the T-antigen unit was G-E-S-L-X-K (X might be glycosylated Asn), confirmed in the major royal jelly glycoprotein 1 (MRJP1), which is also expressed in the mushroom body of the honeybee brain.

[Follow-up of dysphagia in the elderly--clinical survey of deglutition disorder at a private ENT office].

In a one-year-plus follow-up study in 17 of 26 cases involving apparent liquid aspiration during videoendoscopic screening, subjects were instructed in swallowing using video images and in choosing food, utensils, and posture. The amount of sputum decreased in 10 case (59%), and sputum sticking in the throat disappeared in 4 (18%). Body weight also increased in 4 (24%). Choking coughs noted in 11 during initial videoendoscopy disappeared in 2 (18%). We thus, concluded that detecting dysphagia symptoms early and providing follow-up could help prevent aspiration pneumonia.

[Screening test is for dysphagia in the elderly].

In screening tests for dysphagia conducted using videoendoscopy in 81 subjects over 75-years-old apparent liquid aspiration was found in 26 (32%). In 2 of these 26 saliva aspiration was observed. Logistic analysis of aspiration versus clinical symptoms, including endoscopic findings, showed a significant correlation between aspiration and longer time required for meals, bending forward during swallowing, choking during meals, hypoesthesia of the larynx, delayed swallowing reflex and difficulty in spitting. Results were considered clinically significant although they did not appear to fully agree with those of previous studies. The usefulness of videoendoscopy was confirmed.

Differences in age-related alterations in muscle contraction properties in rat tongue and hindlimb.

PURPOSE: Because of differences in muscle architecture and biomechanics, the purpose of this study was to determine whether muscle contractile properties of rat hindlimb and tongue were differentially affected by aging. METHOD: Deep peroneal and hypoglossal nerves were stimulated in 6 young and 7 old Fischer 344-Brown Norway rats to allow recording of muscle contractile properties of tongue and extensor digitorum longus (EDL) muscle in the hindlimb. In the same animals, the following measurements were made: (a) twitch contraction time (CT; in milliseconds), (b) half decay time (HDT; in milliseconds), (c) maximum twitch force (in grams), (d) tetanic force, and (e) fatigue index determined from repetitive stimulation of the muscles. RESULTS: No significant differences were observed in young versus old groups in retrusive tongue forces, whereas a significant (p < .05) decrement in EDL tetanic forces was found in old rats. Slower CT in old rats was observed only in the tongue. Old and young groups were not significantly different in fatigue index or HDT for tongue or EDL. CONCLUSIONS: Old animals generated equivalent maximum tongue forces with stimulation, but they were slower in achieving these forces than young animals. Limb and cranial muscles were not affected equally by aging. As such, information derived from limb muscle studies may not easily generalize to the cranial motor system.

Effect of aging on blood flow in rat larynx.

OBJECTIVE/HYPOTHESIS: Age-associated muscular changes and fatigue have been shown to affect phonatory function. Reductions in blood flow with aging could translate to reductions in oxidative capacity within laryngeal muscles and increased fatigability. We tested the hypothesis that there would be increased capillary red blood cell (RBC) velocity and a reduction of capillary density in the thyroarytenoid (TA) muscle of senescent rats. STUDY DESIGN/METHODS: Ten male Fisher 344/Brown Norway rats in two age groups were used: young adult (9 mo) and old (28-30 mo). Sixteen additional young and old rats were used in a fluorescent microsphere experiment that examined blood flow rates before and after a surgical manipulation. With use of a specially equipped intravital microscope, in vivo measurements of capillary geometry and flow were obtained, including RBC velocity, capillary density, tortuosity, and number of branch points. RESULTS: There was an age-related reduction in capillary surface area as evidenced by reduced lineal density of capillaries. In addition, reduced RBC transit time was suggested by the reduction in branch points found with age. There was no change in RBC velocity with aging. The surgical method used to expose the TA muscle for blood flow recordings did not significantly affect resultant blood flow measurements. CONCLUSIONS: We developed a method to evaluate in vivo laryngeal microvasculature. We found age-related changes in microvascular geometry within the TA muscle of the rat that could affect blood flow to this critical muscle of phonation and airway protection. These microvascular changes could contribute to age-related laryngeal dysfunction.

Effect of aging on tongue protrusion forces in rats.

The purpose of this study was to ascertain the effect of aging on muscle contractile properties associated with tongue protrusion in a rat model. Fischer 344/Brown Norway hybrid rats, ten young (9 months old) and ten old (32 months old), were used to measure protrusive contractile properties. Results showed a significant reduction in tetanic forces in the old animals. The following measures of muscle contraction were not different between age groups: mean twitch contraction force, twitch contraction time, twitch contraction half-decay time, and a calculated measure of fatigability. In conclusion, aging influenced protrusive tongue muscle contractions in a rat model such that tetanic forces were reduced. The reduction of tetanus force may parallel findings in human subjects relative to isometric tongue force generation and may be associated with age-related disorders of swallowing.

Long-term result of the new endoscopic vocal fold medialization surgical technique for laryngeal palsy.

OBJECTIVE: The conventional surgical method for a case of unilateral laryngeal nerve paralysis with large glottal gap requires an external cervical incision. In the present study, we developed an endoscopic technique of vocal fold medialization that can make the external incision unnecessary. This procedure of autologous transplantation of fascia into the vocal fold (ATFV) was developed for the successful treatment of unilateral laryngeal nerve paralysis. However, the method seemed to be effective only for patients with a relatively mild glottal gap. STUDY DESIGN AND METHODS: In the present study, we modified the method of medialization using the ATFV technique to obtain effective closure of a large glottal gap. To overcome this difficulty, an attempt was made to extend the site of transplantation more posteriorly so as to adduct the vocal process of the arytenoid cartilage in the body of the vocal fold. RESULTS: This new technique was applied to eight cases of patients with unilateral laryngeal paralysis with severe dysphonia. None of the patients showed any evidence of falling off of the graft. Elongation of the maximum phonation time and a decrease in airflow rate during phonation were obtained with improvement in voice quality in all patients 1 year after the surgery. CONCLUSIONS: This method, with its less invasive approach, proved to be useful for the treatment of large glottal gap due to unilateral laryngeal nerve paralysis.

Endoscopic vocal cord medialization: a new surgical technique without neck incision for laryngeal palsy.

Different types of surgery have been reported for glottal insufficiency due to unilateral vocal cord paralysis. We recently developed a new surgical technique of fascia implantation known as vocal cord medialization. This new method achieves a wide glottal gap during phonation, without the use of an external approach. This new technique is described in detail herein, together with the satisfactory results we obtained in six cases of unilateral vocal cord paralysis with a wide glottal gap during phonation. We recommend this technique in cases of unilateral vocal cord paralysis in which both a satisfactory clinical result and an aesthetically pleasing cosmetic result are required.

Discoordination of laryngeal and respiratory movements in aged rats.

Voice and swallowing actions require the coordination of multiple motor systems, and this coordination may be impaired with aging. Although recent work has reported impairments in age-related laryngeal kinematics in rats, the temporal relationship of laryngeal excursions to the respiratory cycle is unknown. The goal of this study was to assess laryngeal-respiratory coordination by examining temporal interrelationships between change in laryngeal aperture and chest wall movement during quiet breathing in a rat model. Glottal images were recorded, digitized, and synchronized with respiratory signals, and temporal features were measured. In the young animals, glottal opening began before the onset of inspiration, and glottal and respiratory cycles were phasic and stereotypic. In old animals, however, inspiration often began during the glottal closing phase, and both respiratory signals were asymmetric. Discoordination of laryngeal and respiratory motor actions associated with aging may be caused by a generalized decline in sensorimotor cranial functions and may contribute to age-related swallowing and communication impairment.

Effect of deficits in laryngeal sensation on laryngeal muscle biochemistry.

Swallowing deficits in elderly people are significant clinical problems and may be associated with impaired pharyngolaryngeal sensation. However, the extent to which sensory innervation affects the motor system is unclear. Our purpose was to examine differences in biochemical properties of laryngeal muscles following sensory nerve ablation. We used sodium dodecyl sulfate-polyacrylamide gel electrophoresis to evaluate laryngeal muscles of young and old Fischer 344/Brown Norway rats, and rats that underwent sensory ablation via bilateral section of the superior laryngeal nerve, internal branch (SLNi), or mixed sensory-motor nerve ablation via left-sided recurrent laryngeal nerve (RLN) section. In lateral thyroarytenoid muscle, a reduction was found in the proportion of the most rapidly contracting myosin heavy chain isoform (type 2B) with SLNi section, RLN section, and aging. Section of the SLNi did not alter the proportion of any myosin heavy chain isoform within the lateral cricoarytenoid or posterior cricoarytenoid muscles, but RLN section resulted in a reduction in the proportion of type 2B. Accordingly, alteration in biochemical properties of the lateral thyroarytenoid muscle alone was demonstrated following sensory ablation. We conclude that sensory changes may affect properties of laryngeal muscles, and may thus have an impact on motor control during critical functions, such as airway protection during swallowing.

Regeneration of aged vocal folds with basic fibroblast growth factor in a rat model: a preliminary report.

Aged vocal folds have been reported to have dense collagen deposition and decreased hyaluronic acid (HA) in the lamina propria. These characteristics are thought to contribute to vocal problems that occur with age (presbyphonia). To restore better viscoelasticity to aged vocal folds, an intervention that might increase HA and decrease collagen production from aged vocal fold fibroblasts would appear to be a potentially useful approach. Our previous in vitro study has revealed that basic fibroblast growth factor (bFGF) consistently stimulates HA production and decreases collagen production from aged rat vocal fold fibroblasts. The present in vivo study examined the effects of intracordal injection of bFGF into aged rats' vocal folds in terms of restoration of HA and collagen distribution in the lamina propria. We injected bFGF transorally into the lamina propria of (unilateral) vocal folds. The injection was repeated 4 times weekly, and rats were painlessly sacrificed 1 week, 1 month, and 2 months after the final injection. Histologic examination revealed that bFGF significantly increased the HA content of the lamina propria up to 2 months, but showed no effect on collagen, even after 2 months. Because it might take longer for excessive collagen to be degraded, further studies are necessary to clarify the long-term effect on collagen. A drug delivery system for bFGF also needs to be developed to maximize its effect in the future. The present study suggested at least a positive effect of bFGF in restoring the HA content in the aged vocal fold lamina propria.

New estimation method for highly sensitive quantitation of human immunodeficiency virus type 1 DNA and its application.

A new estimation method for quantitation of HIV-1 DNA was established by introducing a pre-quantitation polymerase chain reaction (PCR) before conventional real-time PCR. Two alternative methods for estimating the copy number can be used: the first method utilizes the rate of beta2-microglobulin (beta2M) gene amplification during the pre-quantitation PCR, and the second utilizes a calibration curve of the crossing point of real-time PCR versus the standard HIV-1-plasmid concentration. These methods could be used to reproducibly and accurately detect a provirus density down to five copies/10(6) cells (for methods 1 and 2, inter-assay CV=17 and 16% and accuracy=81 and 92%, respectively). The levels of HIV-1 DNA could be measurable using as little as 100 microl of whole blood or buffy coat cells. Using a combination of a conventional and highly sensitive methods, we found that the amount of HIV-1 DNA ranged from 2 to 5960 copies/10(6) cells (median of 830 copies/10(6) cells) in CD4-positive T lymphocytes isolated from 30 patients responding well to highly active antiretroviral therapy (HAART). Thus, the highly sensitive method developed in this study allows estimation of the HIV-1 reservoirs in peripheral CD4-positive T lymphocytes of patients responding well to HAART.

Growth factor therapy for vocal fold scarring in a canine model.

Vocal fold scarring remains a therapeutic challenge. Hepatocyte growth factor (HGF) has strong antifibrotic activity and has proved to have therapeutic potential in restoration of scar tissues such as liver cirrhosis and lung fibrosis. The present study aimed to clarify the effects of HGF injection into scarred vocal folds in a canine model. Canine vocal folds were stripped unilaterally and treated with intracordal injection of saline solution (sham group), HGF (HGF group), or HGF with cultured autologous normal vocal fold fibroblasts (Fb/HGF group) 1 month after injury. The larynges were harvested 6 months after the initial injury and then subjected to vibratory and histologic examination. The results of vibratory examinations in the excised larynx setup revealed that phonation threshold pressure significantly increased and vocal efficiency was significantly reduced in all treated groups as compared to normal data obtained from normal canine larynges. However, the HGF group presented much better results than both the sham and Fb/HGF groups in terms of mucosal wave amplitude and incidence of vocal fold bowing, glottal incompetence, and phase asymmetry. The histologic data indicated a significant increase of collagen in both the sham and Fb/HGF groups, while normal levels of collagen were found in the HGF group. Tissue contraction of the lamina propria was also observed in both the sham and Fb/HGF groups, but was barely detectable in the HGF group. Although the HGF-treated vocal folds appeared to require more driving forces for vibration, HGF might prevent excessive collagen deposition and tissue contraction and thus reduce the effects of scarring on the vibratory properties of the vocal folds. From these data it is concluded that HGF has considerable potential in the treatment of vocal fold scarring.

Delayed HIV-1 infection of CD4+ T lymphocytes from therapy-naïve patients demonstrated by quantification of HIV-1 DNA copy numbers.

Measuring the amount of HIV-1 DNA in infected cells is important to estimate the size of the viral reservoir in patients. However, the clinical impact of the intracellular viral DNA level remains unclear. The present study examines the clinical significance of the HIV-1 DNA level in peripheral CD4+ T lymphocytes from 21 therapy-naïve patients. HIV-1 DNA levels in purified peripheral CD4+ T lymphocytes were measured by the real-time PCR method using the Roche LightCycler system that can detect 200 copies/10(6) cells. We detected intracellular HIV-1 DNA in 15 (71.4%) of 21 patients at levels ranging from 270 to 98,120 copies/10(6) CD4+ cells, with a median of 2,220 copies/10(6) cells. We also found HIV-1 DNA that was below the detection limit in the remaining 6 patients, although 8,800-150,000 copies/ml of HIV-1 RNA were detected in plasma. Circular HIV-1 DNA was not detected in 5 of 6 cases, suggesting that reverse transcription in CD4+ T lymphocytes of these cases was not active. Thus, delayed HIV-1 infection of CD4+ T lymphocytes was demonstrated in these patients. The level of HIV-1 DNA in peripheral CD4+ T lymphocytes indicates the clinical status of therapy-naïve patients.

Prevalence of infection and genotypes of GBV-C/HGV among homosexual men.

Since the discovery of GB virus-C (GBV-C) and hepatitis G virus (HGV), many studies have been performed. These viruses are now known to be parenterally, as well as sexually transmitted. A phylogenetic analysis also revealed that GBV-C has five major genotypes: type 1 predominates in West Africa, type 2 in Europe and the United States, type 3 in parts of Asia, type 4 in Southeast Asia, and type 5 in South Africa. Despite the number of reports so far, there have been few large-scale surveys of homosexual men to determine the prevalence of the GBV-C/HGV infections. We examined the levels of GBV-C/HGV viremia in 297 homosexual men who attended the Nagoya Lesbian and Gay Revolution held in Nagoya, Japan. Reverse transcription-polymerase chain reaction (RT-PCR)/nested PCR of the GBV-C/HGV 5 ' -non-coding region (NCR), and base sequence analyses showed that the infection rate was 12.5%, and genotypes in this population were classified into type 2 (32%) and type 3 (68%). None were classified as types 1, 4, or 5 in this study. Our results indicate that the GBV-C/HGV type 2 seen mainly in Europe and the US is spreading widely in Japan, especially in the Nagoya district.