Tissue engineered muscle implantation for tongue reconstruction: a preliminary report

OBJECTIVES/HYPOTHESIS: Because current tongue reconstructive methods introduce adynamic, variably sensate tissue into the mouth, the critical functions of the tongue in articulation and deglutition may be compromised. The objective of this work was to introduce a combination of myoblasts and scaffolding material into rat hemiglossectomy defects and to examine the extent of neomuscle formation in the reconstructed area, under the hypothesis that the presence of myoblasts leads to formation of new muscle. STUDY DESIGN: Randomized, prospective animal study. METHODS: Myoblasts were harvested from neonatal Lewis rats, and a growth factor enriched collagen gel was prepared. Syngeneic adult animals received either hemiglossectomy alone or reconstruction with one of four experimental reconstructive preparations: collagen gel alone, collagen gel with suspended myoblasts, the gel-cell combination in undifferentiated muscle construct form by way of tissue culture for 7 days in a preformed mold, or differentiated constructs, cultured in myoblast fusion medium. After 6 or 16 weeks, animal weight gain was recorded, animals were killed, and the tongues harvested. The tissue was examined histologically, and quality of the muscular regenerate was rated on a scale according to predefined criteria. RESULTS: Animals in all groups gained weight appropriately. In groups receiving hemiglossectomy alone or acellular (gel only) reconstruction, there was significant scarring and lack of neomuscle formation. In groups receiving myoblast transplantation, either by way of gel suspension or in the form of undifferentiated or differentiated constructs, muscle quality was superior to controls. CONCLUSIONS: Myoblast transplantation into hemiglossectomy defects appears to lead to new muscle formation and does not inhibit normal weight gain in animals after tongue implantation.     

The resistance to changing guidelines--what are the challenges and how to meet them

Bridging the gap between scientific evidence and its practical application is of the utmost importance in improving the quality of care and increasing patient safety. Guidelines based on evidenced-based medicine (EBM) have led to improved performances and better outcomes. However, even though scientific data are available, resistance to adopting evidence-based guidelines is still enormous. Significant barriers hinder the introduction of best medical practice into the daily clinical routine. The barriers to implementing change are complex, multifunctional, and influenced on many levels by various interests both inside and outside the health-care system. Political, organizational, financial, cultural and scientific interests are regarded as being as important as the perception of patients and health workers. Strategies need to be planned which take account of the multidimensional character of quality of care and incorporate it at the various levels. The conclusion, therefore, is that we need to combine methods and tools to tailor our interventions to the patient's needs.     

Imaging of the calf vocal fold with high-frequency ultrasound

Objectives/Hypothesis: High‐frequency ultrasound imaging offers the potential for assisting in the diagnosis and treatment of vocal fold pathology if it allows aspects of vocal fold microstructure to be visualized noninvasively. The objective of this study was to assess the ability of high‐frequency ultrasound to image vocal fold anatomy and injected biomaterials. Study Design: The vocal folds of two excised calf larynges were imaged ex vivo and compared with corresponding histological sections. Methods: High‐frequency ultrasound imaging was performed under saline submersion using 40 and 50 MHz transducers, and corresponding cryostat cross‐sections were stained with H&E, Trichome, and Verhoeff's Van Gieson stains. Results: The epithelial surface, lamina propria, and underlying muscle were easily identified with the high‐frequency ultrasound as verified with histological sections representing each imaged region. The arytenoid cartilage vocal process can also be clearly distinguished from the surrounding tissue, as can the full extent of injected biomaterials within the superficial lamina propria. Useful ultrasound resolution was obtained to depths of at least 10 mm within the tissue with the 40 MHz transducer. Conclusions: This preliminary study demonstrates the capability of high‐frequency ultrasound to image the layered anatomy of the calf vocal fold and to discern materials injected into the superficial lamina propria, indicating that this technology holds a strong potential for use in phonosurgery.     

Predicting clinical efficacy of photoangiolytic and cutting/ablating lasers using the chick chorioallantoic membrane model: implications for endoscopic voice surgery

Objectives/Hypothesis: The optimal balance between a laser's clinical efficacy and collateral thermal damage is the major determinant for selection of a particular laser in endolaryngeal surgery. The chick chorioallantoic membrane (CAM) simulates the microvasculature of the human vocal fold and is, therefore, useful for testing effects of laser settings, mode of delivery, active cooling, and wavelength. Such information is essential for optimizing the effectiveness of lasers in treating laryngeal pathology while preserving vocal function. Study Design and Methods: The thermal and coagulative effects of four lasers (585 nm PDL, 532 nm KTP, 2.01ìm Thulium, 10.6ìm CO₂) were quantified at selected (and clinically relevant) energy settings before and after tissue cooling using the CAM model. Measures included imaging real‐time vascular reactions in the CAM model (i.e., vessel coagulation and/or rupture), and post‐procedure histologic analysis of CAM tissue. In each experiment, laser energy was applied to the CAM in a controlled manner. Cooling was done using a dermatological cold‐air device, and temperatures were measured with a thermistor. Lasers tested included the photoangiolytic pulsed‐dye (PDL) and KTP, as well as the ablative/cutting CO₂ and thulium lasers. The vessel rupture/coagulation and thermal effects of various energy‐delivery parameters on the CAM, with and without cooling, were assessed. After removal of the CAM, specimens were stained as whole‐mounts, photographed at 4X magnification, and evaluated by two independent, blinded surgeon reviewers. The efficacy of increased pulse‐width (KTP laser) on treating larger vessels (>0.5 mm) and the effects of extravasated blood on photoangiolysis were also evaluated. Results: Photoangiolytic lasers: Vessel coagulation/rupture rates showed that the PDL caused more frequent vessel rupture than the KTP laser. For both lasers, cooling the CAM by ～20°C resulted in 30% ‐ 60% reduction in the thermal‐damage zone (P < .05). Cooling reduced the efficacy of coagulation with the PDL but not with the KTP laser. The clinically observed phenomenon that laser heating of extravasated blood increases thermal damage and decreases efficacy of coagulation was clearly evident in the CAM model. Ablative lasers: The thermal‐damage zone of the CO₂ laser (0.3 mm spot size) was not significantly different with or without cooling (0.32 mm² and 0.34 mm², respectively) (P = .30). However, when the spot size was defocused to 1 mm, the thermal‐damage zone was over 2× greater when the tissue was not cooled (0.74 mm² vs 0.35 mm²) (P < .002). The thermal‐damage zone of the Thulium laser was reduced by an average of 58% for the three power settings tested when the CAM was air‐cooled (P < .05). Conclusions: The CAM was an excellent model for studying the effects of photoangiolytic lasers, for which optimal pulse‐widths exist for vessel coagulation. Smaller vessels coagulated reliably at pulse widths >15 msec, and larger vessels required pulse widths >35 msec for optimal coagulation. Cooling the target tissue decreased the thermal‐damage zone created by photoangiolytic lasers. While cooling had no effect on the efficacy of coagulation with longer pulse widths (KTP), tissue cooling decreased the coagulation rate at shorter pulse widths (PDL). The thermal effects of cutting/ablating lasers can be reduced with cooling, but the CAM was not a good model with which to study coagulation/rupture rates in cutting/ablating lasers.     

Comparison of a flexible laryngoscope with calibrated sizing function to intraoperative measurements

OBJECTIVES: The objectives were to assess the clinical performance and accuracy of a prototype fiberoptic transnasal laryngeal endoscope with an auxiliary optical system that allows images to be spatially calibrated. METHODS: A novel fiberoptic endoscope was developed that projects green laser beams across the field of view from a separate optical channel. According to the location of the spots in the field of view, the images can be calibrated with a software routine. To assess its performance, we compared measurements of 14 lesions imaged with the calibrated endoscope and during microlaryngoscopy, where a calibration instrument was placed next to the lesions. Four clinicians measured lesion length, width, and area from the collected images. RESULTS: The calibrated endoscope performed as well as current flexible fiberoptic laryngoscopes in terms of image quality and patient comfort. For lesions with well-defined borders, the error ranged from 14% to 23% for length, from 20% to 30% for width, and from 33% to 50% for area across observers. Factors contributing to larger errors in some subjects were identified. CONCLUSIONS: The calibrated endoscope is capable of providing useful sizing information for laryngeal structures, and these measures correspond quite well to more direct measurements in the operating room. Objective sizing of laryngeal lesions is complicated by subjective judgments of lesion boundaries, which can be indistinct in many cases.     

Histologischer Gehirnbefund nach Insulinkoma

Zusammenfassung An einem Fall einer nach der Methode vonSakel insulinshockbehandelten schizophrenen Patientin, die in einem schweren von epileptiformen Krämpfen begleiteten Koma starb, wurden diffuse Veränderungen im Bereiche des ZNS. gefunden, teils in leichter, teils in schwerer Erkrankung der Zellen, geringer gliöser Reaktion und in geringen Gefäßveränderungen bestehend, wie sie bei schweren Vergiftungen vorkommen können. Darüber hinaus zeigten sich Auflösungen des Zellkerns, bis zur Vakuolenbildung führend, die in der bisherigen Literatur wenig Beachtung fanden und die wir mit der Darreichung hoher Insulindosen in Verbindung brachten. Ferner wurde daraus auf eine direkte Zellwirkung als einer Komponente der cerebralen Insulinwirkung geschlossen. Ein Befund, der die Einwirkung des Insulins auf dem Umwege über das Gefäßsystem wahrscheinlich machen würde, konnte nicht mit Deutlichkeit erhoben werden, was diesen Mechanismus jedoch nicht ausschließt.     

Separation of presynaptic Cav2 and Cav1 channel function in synaptic vesicle exo- and endocytosis by the membrane anchored Ca2+ pump PMCA

Synaptic vesicle (SV) release, recycling, and plastic changes of release probability co-occur side by side within nerve terminals and rely on local Ca²⁺ signals with different temporal and spatial profiles. The mechanisms that guarantee separate regulation of these vital presynaptic functions during action potential (AP)–triggered presynaptic Ca²⁺ entry remain unclear. Combining Drosophila genetics with electrophysiology and imaging reveals the localization of two different voltage-gated calcium channels at the presynaptic terminals of glutamatergic neuromuscular synapses (the Drosophila Ca_v2 homolog, Dmca1A or cacophony, and the Ca_v1 homolog, Dmca1D) but with spatial and functional separation. Ca_v2 within active zones is required for AP-triggered neurotransmitter release. By contrast, Ca_v1 localizes predominantly around active zones and contributes substantially to AP-evoked Ca²⁺ influx but has a small impact on release. Instead, L-type calcium currents through Ca_v1 fine-tune short-term plasticity and facilitate SV recycling. Separate control of SV exo- and endocytosis by AP-triggered presynaptic Ca²⁺ influx through different channels demands efficient measures to protect the neurotransmitter release machinery against Ca_v1-mediated Ca²⁺ influx. We show that the plasma membrane Ca²⁺ ATPase (PMCA) resides in between active zones and isolates Ca_v2-triggered release from Ca_v1-mediated dynamic regulation of recycling and short-term plasticity, two processes which Ca_v2 may also contribute to. As L-type Ca_v1 channels also localize next to PQ-type Ca_v2 channels within axon terminals of some central mammalian synapses, we propose that Ca_v2, Ca_v1, and PMCA act as a conserved functional triad that enables separate control of SV release and recycling rates in presynaptic terminals.

Neuronal network function critically depends on the tight control of synaptic vesicle (SV) release probability at chemical synapses over wide ranges of activity regimes. At the same time, synaptic gain remains adjustable to render network function flexible. To maintain synapse function over time, SV recycling rates must be matched to vastly different activity patterns and synaptic gains. While SV release and recycling as well as their plasticity-related adjustments all include Ca²⁺-dependent steps, they operate in parallel but on different time scales. A tight spatial and temporal coordination of presynaptic Ca²⁺ signals and their effectors is thus needed for both the induction of changes in synaptic strength and the maintenance of robust synapse function. However, the mechanisms that effectively separate Ca²⁺ signals in time and space (e.g., through different voltage-gated calcium channels [VGCCs]) to allocate these to different presynaptic functions are not well understood.SV release probability depends on the sensitivity of the vesicular Ca²⁺ sensor and the positioning of VGCCs inside active zones (AZs) (1). Various mechanisms that can tune release probability by modulating their precise localization or kinetic properties have been uncovered (2–4). Irrespective of such modulation, efficient Ca²⁺-triggered SV release through presynaptic VGCCs (mainly Ca_v2.1 and Ca_v2.2 in vertebrates) remains spatially restricted to a few hundred nanometers due to the limited abundance and brief opening of the channels and the presence of endogenous Ca²⁺ buffers (5, 6). It is thus conceivable that Ca²⁺ signals originating within presynaptic terminals but outside AZs are engaged to tune SV recycling and plastic changes according to changes in activity.Apart from the need for fast activating and inactivating Ca_v2 channels for SV release, other types of VGCCs have been implicated in presynaptic plasticity. In GABAergic synapses, pharmacological blockade of Ca_v1 channels does not affect AP-induced SV release but converts posttetanic potentiation into synaptic depression (7). In hippocampal CA3 mossy fiber boutons (8–10) or in synapses of the lateral amygdala (11), Ca_V2.3 and Ca_V1.2 channels are required for presynaptic long-term plasticity but are unable to trigger SV release (9, 11).Differential functions of Ca_v2 and Ca_v1 channels in neurotransmitter release versus other Ca²⁺-dependent presynaptic processes can hardly be explained just by different coupling distances to SVs, since there are also situations where loose coupling is predominant (4, 10). Moreover, compared with Ca_v2.1 and Ca_v2.2, Ca_v1 channels display higher conductances (12), suggesting that additional mechanisms are required to allocate Ca_V1-related Ca²⁺ signals to specific presynaptic functions while avoiding interference with SV release. SV recycling also includes regulation by presynaptic Ca²⁺ signals but operates mostly at different subsynaptic sites and at slower time scales than Ca²⁺-triggered SV release (13–15). We hypothesize that activity-dependent regulation of SV recycling employs Ca_v1-dependent Ca²⁺ entry and that active mechanisms exist to regulate the relative contributions of Ca_v2 and Ca_v1 channels to SV release versus recycling. We address these hypotheses at the Drosophila larval neuromuscular junction (NMJ), an established model for glutamatergic synapse function (16–18).