James B. Kobler

TBX1 is responsible for cardiovascular defects in velo-cardio-facial/DiGeorge syndrome

Velo-cardio-facial syndrome (VCFS)/DiGeorge syndrome (DGS) is a human disorder characterized by a number of phenotypic features including cardiovascular defects. Most VCFS/DGS patients are hemizygous for a 1.5-3.0 Mb region of 22q11. To investigate the etiology of this disorder, we used a cre-loxP strategy to generate mice that are hemizygous for a 1.5 Mb deletion corresponding to that on 22q11. These mice exhibit significant perinatal lethality and have conotruncal and parathyroid defects. The conotruncal defects can be partially rescued by a human BAC containing the TBX1 gene. Mice heterozygous for a null mutation in Tbx1 develop conotruncal defects. These results together with the expression patterns of Tbx1 suggest a major role for this gene in the molecular etiology of VCFS/DGS.

Recurrent Laryngeal Nerve Identification and Assessment during Thyroid Surgery: Laryngeal Palpation

Electrical identification and monitoring of the recurrent laryngeal nerve (RLN) has been proposed as an adjunct to standard visual identification of the nerve during thyroid and parathyroid surgery. This study was undertaken to assess laryngeal palpation as an intraoperative technique for identifying and assessing the RLN during surgery and to investigate the relation between laryngeal palpation and associated laryngeal electromyographic (EMG) activity. The postcricoid region of the larynx during surgery was palpated through the posterior hypopharyngeal wall to sense posterior cricoarytenoid muscle contraction in response to ipsilateral RLN stimulation (i.e., the “laryngeal twitch response.”) Laryngeal palpation was performed in a series of 449 consecutive thyroid and parathyroid surgeries with 586 RLNs at risk. All patients underwent preoperative and postoperative laryngoscopy to assess vocal cord mobility. In a subset of patients, laryngeal palpation and simultaneous laryngeal EMG recordings were compared during intraoperative RLN stimulation. In this series, there was no permanent RLN paralysis. There was one case of temporary RLN paralysis secondary to neural stretch that resolved 6 weeks postoperatively (temporary paralysis rate: 0.2% of patients, 0.2% of nerves at risk). Intraoperative laryngeal palpation of the laryngeal twitch response reliably correlated with normal postoperative vocal cord function. Loss of the laryngeal twitch response occurred in the single case of temporary paralysis in the setting of an anatomically intact nerve. Laryngeal palpation correlated well with simultaneous laryngeal EMG activity. There were no palpation-induced laryngeal injuries or laryngeal edema. There were also no RLN injuries due to repetitive neural stimulation. Intraoperative laryngeal palpation during RLN stimulation is a safe, reliable method for neural monitoring that can assist in RLN identification and assessment during thyroid and parathyroid surgery. Most importantly, it provides important prognostic information regarding ipsilateral vocal cord function at the completion of the initial side of the thyroid or parathyroid surgery. Intraoperative laryngeal palpation allows the surgeon to stage contralateral surgery if RLN damage is diagnosed, thereby avoiding the potential for bilateral vocal cord paralysis. We believe that laryngeal palpation is useful as an adjunct to formal EMG monitoring during thyroid and parathyroid surgery.

Incorporation of a matrix metalloproteinase-sensitive substrate into self-assembling peptides - a model for biofunctional scaffolds.

Controlling and guiding cell behavior requires scaffolding materials capable of programming the three-dimensional (3-D) extracellular environment. In this study, we devised a new self-assembling peptide template for synthesizing nanofibrous hydrogels containing cell-responsive ligands. In particular, the insertion of a matrix metalloproteinase-2 (MMP-2) labile hexapeptide into the self-assembling building blocks of arginine-alanine-aspartate-alanine (RADA) was investigated. A series of peptides, varied by the position of the MMP-2 hexapeptide substrate and the length of RADA blocks, were prepared by parallel synthesis. Their self-assembling capabilities were characterized and compared by circular dichroism spectroscopy and dynamical mechanical analysis. Among all the different insertion patterns, the sequence comprising a centrically positioned MMP-2 substrate was flanked with three RADA units on each side self-assembled into a hydrogel matrix, with mechanical properties and nanofiber morphology comparable to the native material built with (RADA)(4) alone. Exposure of the new gel to MMP-2 resulted in peptide cleavage, as confirmed by mass spectroscopy, and a decrease in surface hardness, as detected by nanoindentor, indicating that the enzyme mediated degradation was localized to the gel surface. The new design can be used for introducing biological functions into self-assembling peptides to create scaffolding materials with potential applications in areas such as tissue engineering and regenerative medicine.

The recruitment order of stapedius motoneurons in the acoustic reflex varies with sound laterality.

In many muscles, motor units are recruited in a fixed order with increasing strength of muscular contraction. We show that for the stapedius muscle of the cat, vastly different recruitment orders can be obtained, depending on which ear is acoustically stimulated. The data support the idea that the distribution of inputs to a motoneuron pool can be inhomogeneous and a significant factor in determining recruitment order.

Quantitative and Comparative Studies of the Vocal Fold Extracellular Matrix II: Collagen

Objectives: This study examines the collagen content and turnover in the midmembranous vocal fold laminae propriae (LPs) of humans, dogs, pigs, and ferrets. Methods: The LP collagen levels were assessed by quantifying tissue hydroxyproline. Quantitative histology allowed evaluation of the collagen content in specific LP regions. Several collagen types and Two markers of collagen turnover were examined immunohistochemically. Results: Collagen made up 43.4% ± 2.6% of human LP total protein (TP), with men having approximately 30% higher collagen content than women (p > .024). The collagen levels in pigs (52.6% ± 1.9% of TP) and ferrets (29.8% ± 3.7% of TP), but not that in dogs (45.3% ± 1.2% of TP), varied significantly from that in humans (pigs, p > .016; ferrets, p > .011). Quantitative histology indicated marked interspecies differences in total collagen distribution. Collagen types I, III, and IV were detected in the LP, and spatially complex staining patterns were observed for the Two markers of collagen turnover studied. Conclusions: The collagen content of the human LP is approximately 60% to 70% of that of human dermis. Although canine LP collagen levels are most similar to those of humans, quantitative histology indicates that the collagen distribution of the human LP is best matched by the porcine LP. Collagen types I and III seem to be the dominant LP collagens. Spatial variations in collagen turnover appear to exist that may contribute to normal LP physiology.

Quantitative and comparative studies of the vocal fold extracellular matrix. I: Elastic fibers and hyaluronic acid.

Objectives: This study examines the elastic fiber and hyaluronic acid (HA) content of the midmembranous vocal fold laminae propriae (LPs) of humans, dogs, pigs, and ferrets. Methods: Lamina propria elastin was quantified by measuring the amino acid desmosine, and HA was measured by an enzyme-linked immunosorbent assay-based technique. Quantitative histology was used to evaluate elastin and HA levels in specific LP regions. The distributions of fibrillin-1, a primary microfibrillar component of elastic fibers, and of tropoelastin, an indicator of elastin synthesis, were immunohistochemically analyzed. Results: Elastin and HA constituted 8.5% ± 2.1% and 0.82% ± 0.11% of human LP, respectively, relative to tissue total protein. Although the mean LP desmosine levels were similar across species, the mean HA levels in canine (p < 3.1 × 10−5), porcine (p < 1.5 × 10−5), and ferret (p < 6.6 × 10−4) LPs were 3 to 4 times higher than that in humans. Marked interspecies differences in elastin, fibrillin-1, tropoelastin, and HA distributions were observed histologically. Conclusions: The elastin content of the human LP is roughly twice that of the dermis, whereas the HA content of the human LP is similar to that of the dermis. Although all species had similar levels of desmosine, histologic evaluation indicates that the porcine elastin distribution is most similar to that of the human LP. Fibrillin-1 staining suggests that stress in the human LP may be particularly high in the superior superficial layer, and tropoelastin staining indicates that the rate of LP elastin turnover may vary spatially.

Echo intensity compensation by echolocating bats.

When mounted on a swinging pendulum, mustache bats, Pteronotus p. parnellii, emit ultrasonic pulses as they move toward and away from fixed targets. During forward swings they systematically decrease the intensity of their emitted pulses and during backward swings they increase the intensity. In this way, echo strength is continuously adjusted and apparently optimized for signal analysis. We have called this behavior echo intensity compensation. Pteronotus simultaneously Doppler and echo intensity compensate during forward swings of the pendulum but during backward swings they only echo intensity compensate. Pteronotus can regulate the intensity of both the constant frequency and frequency modulated components of their pulses; this regulation is independent of vestibular cues, pulse repetition rates, pulse durations and pulse-echo intervals.

Imaging the mucosa of the human vocal fold with optical coherence tomography.

Objectives: Discerning the layered microstructure of the vocal folds is critical for effective phonomicrosurgery. Optical coherence tomography (OCT), a noncontact, noninvasive technology that provides cross-sectional images by means of backscattered light, offers the potential for delineating these layers in vivo. Methods: The glottal mucosa of 3 human cadaver larynges was imaged with conventional OCT and polarization-sensitive OCT (PS-OCT). Images were obtained through the epithelium and lamina propria. Results: Although the superficial layer of the lamina propria appeared quite homogeneous, the outer surface of the superficial lamina propria was correlated with an increase in backscatter with OCT. The superficial lamina propria and vocal ligament were correlated with a marked increase in tissue birefringence with PS-OCT. Conclusions: This preliminary study demonstrates the capacity of OCT and PS-OCT for visualizing the layered microstructure of the vocal fold mucosa. We believe that these imaging techniques will have applications in the exploration of solutions to vocal fold scarring and in imaging vocal fold disorders in the clinic and operating room.

Clinical Ultrafast Laser Surgery: Recent Advances and Future Directions

Ultrafast pulsed lasers can be used to achieve remarkable precision during surgical ablation. Through nonlinear interactions with tissue, ultrafast lasers can provide a largely non-thermal mechanism of ablation and a unique ability to create targeted damage within bulk tissue. These advantages have made ultrafast lasers the ideal surgical tool for various novel applications in ophthalmology. Clinical adoption of ultrafast lasers in other surgical applications remains limited in part due to the lack of a means for fiber delivery of ultrafast laser pulses as a flexible, hand-held surgical endoscope. This review provides an overview of the recent advances in bringing this unique surgical tool into the clinic. We discuss fundamental mechanisms and limitations of ultrafast laser ablation, novel techniques for overcoming these limitations, the current state of clinical applications, and conclude with our recent efforts in developing fiber-coupled probes for flexible ultrafast laser surgery and imaging.

Midmembranous vocal fold lamina propria proteoglycans across selected species.

Objectives: We examined the proteoglycan (PG) and associated sulfated glycosaminoglycan (GAG) content of the midmembranous vocal fold lamina propria (LP) of humans, dogs, pigs, and ferrets. Methods: The LP PG levels were assessed indirectly by quantifying the associated sulfated GAGs, and immunohistochemical analyses of specific PGs and/or GAGs (PGs/GAGs) were conducted. Results: Sulfated GAGs constituted approximately (average ± SEM) 14.7 ±2.1 μg per milligram of tissue total protein in the human LP — similar to levels in canine, porcine, and ferret LPs (p > .05). Immunohistochemical analysis identified versican, chondroitin 4- and 6-sulfate, and heparan sulfate in the LP extracellular matrix — PGs/GAGs previously believed to be localized only intracellularly and in the basement membrane. Observations of PG/GAG staining patterns resulted in identification of microstructurally based subdivisions of canine, porcine, and ferret LPs. Conclusions: The sulfated GAG concentration in human LP was similar to that of dermis. In contrast to the interspecies similarity in LP sulfated GAG levels, immunohistochemical analysis indicated notable interspecies differences in specific PG/GAG distributions. Moreover, spatial variations in the presence of several PGs/GAGs were observed — variations that may be integral in maintaining normal LP physiology. Finally, the noted canine, porcine, and ferret LP subdivisions may yield insight into the adaptation of LP microstructure to the phonatory needs of each species.