Voice Disorder in Cystic Fibrosis Patients

<div><p>Cystic fibrosis is a common autosomal recessive disorder with drastic respiratory symptoms, including shortness of breath and chronic cough. While most of cystic fibrosis treatment is dedicated to mitigating the effects of respiratory dysfunction, the potential effects of this disease on vocal parameters have not been systematically studied. We hypothesized that cystic fibrosis patients, given their characteristic respiratory disorders, would also present dysphonic symptoms. Given that voice disorders can severely impair quality of life, the identification of a potential cystic fibrosis-related dysphonia could be of great value for the clinical evaluation and treatment of this disease. We tested our hypothesis by measuring vocal parameters, using both objective physical measures and the GRBAS subjective evaluation method, in male and female cystic fibrosis patients undergoing conventional treatment and compared them to age and sex matched controls. We found that cystic fibrosis patients had a significantly lower vocal intensity and harmonic to noise ratio, as well as increased levels of jitter and shimmer. In addition, cystic fibrosis patients also showed higher scores of roughness, breathiness and asthenia, as well as a significantly altered general grade of dysphonia. When we segregated the results according to sex, we observed that, as a group, only female cystic fibrosis patients had significantly lower values of harmonic to noise ratio and an abnormal general grade of dysphonia in relation to matched controls, suggesting that cystic fibrosis exerts a more pronounced effect on vocal parameters of women in relation to men. Overall, the dysphonic characteristics of CF patients can be explained by dysfunctions in vocal fold movement and partial upper airway obstruction, potentially caused by the accumulation of mucus and chronic cough characteristic of CF symptomatology. Our results show that CF patients exhibit significant dysphonia and suggest they may potentially benefit from voice therapy as a parallel treatment strategy.</p></div

A New Low Cost Wide-Field Illumination Method for Photooxidation of Intracellular Fluorescent Markers

<div><p>Analyzing cell morphology is crucial in the fields of cell biology and neuroscience. One of the main methods for evaluating cell morphology is by using intracellular fluorescent markers, including various commercially available dyes and genetically encoded fluorescent proteins. These markers can be used as free radical sources in photooxidation reactions, which in the presence of diaminobenzidine (DAB) forms an opaque and electron-dense precipitate that remains localized within the cellular and organelle membranes. This method confers many methodological advantages for the investigator, including absence of photo-bleaching, high visual contrast and the possibility of correlating optical imaging with electron microscopy. However, current photooxidation techniques require the continuous use of fluorescent or confocal microscopes, which wastes valuable mercury lamp lifetime and limits the conversion process to a few cells at a time. We developed a low cost optical apparatus for performing photooxidation reactions and propose a new procedure that solves these methodological restrictions. Our “photooxidizer” consists of a high power light emitting diode (LED) associated with a custom aluminum and acrylic case and a microchip-controlled current source. We demonstrate the efficacy of our method by converting intracellular DiI in samples of developing rat neocortex and post-mortem human retina. DiI crystals were inserted in the tissue and allowed to diffuse for 20 days. The samples were then processed with the new photooxidation technique and analyzed under optical microscopy. The results show that our protocols can unveil the fine morphology of neurons in detail. Cellular structures such as axons, dendrites and spine-like appendages were well defined. In addition to its low cost, simplicity and reliability, our method precludes the use of microscope lamps for photooxidation and allows the processing of many labeled cells simultaneously in relatively large tissue samples with high efficacy.</p> </div

Representative voice recordings and spectrograms of female patients with CF (red) and healthy controls (blue).

<p>A: Sound recordings of/a/vowel phonations from a female control subject (A1) and a female patient with CF (A2); note the markedly reduced amplitude range of the signal from the CF patient in relation to the control. B: Amplified views of the recordings presented in A1 and A2; note the irregularity of the voice signal from the CF patient in relation to the control. C: Spectrogram (frequency domain) representations of the recordings presented in A1 and A2; note the higher level of background noise and low formant segregation in the CF patient in relation to the healthy control.</p

Conflit de juridictions, commerce électronique et consommateur en Europe

<p>Horizontal lines and error bars represent median±IQR for all variables. Subjects were pooled independently of their sex. <b>A:</b> Values of F₀ for each group. <b>B:</b> Values of intensity for each group; note the significant reduction in intensity in the CF group. <b>C:</b> Values of jitter for each group; note the significant increase in jitter in the CF group. <b>D:</b> Values of shimmer for each group; note the significant increase in shimmer in the CF group. <b>E:</b> Values of HNR for each group; note the significant reduction in this variable in the CF group. ***  = <i>P</i><0.0001.</p

Schematics and function of the photooxidizer.

<p>A: Schematic drawing of the PCB. 1: Voltage regulator microchip; 2: Input/output connectors; 3: Resistors; 4: Red low power LED (indicates power feed). B: Schematic drawing of the internal components of the photooxidizer. This panel represents a vertical transection of the apparatus. 1: Green high power LED (promotes photooxidation); 2: LED chamber heat sink; 3: PCB; 4:On/off switch; 5: Power feed connector; 6: Red low power LED (indicates power feed); 7: Voltage regulator microchip; 8: PCB heat sink. C: Radiance spectrum of the green high power LED used for DiI photoconversion. The orange dot represents the peak absorbance wavelength of DiI, confirming the appropriateness of the used LED.</p

Representative photomicrographs of human retinal neurons and axons stained by the new photooxidation method.

<p>Cells were processed and observed in intact post-mortem human retinas. A and B: High magnification (100× objective lens) photomicrographs of DAB-stained putative horizontal retinal neurons. Note the clear delineation of the cell body (blue arrows) and dendritic tufts (green arrows). Scale bars correspond to 10 µm. C: Low magnification (40× objective lens) photomicrograph of two DAB-stained retinal cells (putatively horizontal cells). Scale bar corresponds to 20 µm. D: Low magnification (40× objective lens) photomicrograph of a DAB stained axon terminal in a sample of human retina. Note that even fine structures, such as axonal knobs (arrows), are clearly stained. Scale bar corresponds to 20 µm.</p

Comparison between the new photooxidizer illumination method and conventional mercury arc lamp microscope photooxidation.

*<p>With a 10× objective lens and a 528–553 nm wavelength light filter.</p

Representative photomicrograph of a Cajal-Retzius cell stained by the new photooxidation method.

<p>A: Low magnification (40× objective lens) photomicrograph of the DAB-stained Cajal-Retzius Cell obtained from a transverse section of developing rat neocortex. Note that the cell is stained over its entire volume, with no variations in staining intensity. The red arrow points at the cell's axon. The blue arrow indicates the large horizontal dendrite that defines the Cajal-Retzius cell type. The orange arrow points at the cell's soma Scale bar corresponds to 20 µm. B: High magnification (100× objective lens) photomicrograph of the insert indicated in A (dotted line square). Note the successful staining of dendritic shafts and spine-like appendages (green arrows), highlighting the high degree of detail and acuity of the staining. Scale bar corresponds to 10 µm.</p

Photooxidizer apparatus and tissue chamber.

<p>Scale bars correspond to 10 mm. A: Front view of the apparatus in an offline configuration. B: Rear view of the apparatus. The machine is connected to an outlet, but the switch is turned off. C: View of the photooxidizer with the connected tissue chamber. D: View of the photooxidizer with the connected tissue chamber while the apparatus is turned on. Note that the chamber fits over the LED light beam. E: Disassembled tissue chamber, including the acrylic ring, the glass lid and the aluminum ring with nylon mesh used for holding the tissue in place.</p

Representative photographs of the maximum illumination fields of the photooxidizer apparatus and of a conventional fluorescent microscope (Eclipse 80i microscope; Nikon, Japan).

<p>Illumination fields are defined by the region of the tissue were DAB was successfully photooxidized into an opaque polymer. The labeled structures include cell bodies, dendrites and axons of human retinal neurons. Observe that the geometry of the fields approximates that of a perfect circle. Also note that the illumination field area of the photooxidizer (42 mm²) is over 7.5 times larger than that of the microscope (5.5 mm²). This confirms the advantage of the new method for the processing large tissue samples. A: Very low magnification (1.6× objective lens) photographs of DAB-stained neurons in a human retina photooxidized using our new illumination method. The region of tissue that was successfully photooxidized can be fitted within a circle with a radius of approximately 3.66 mm and area of 42 mm². Scale bar corresponds to 1 mm. B: Low magnification (4× objective lens) photographs of DAB-stained neurons in a human retina photooxidized using a conventional fluorescent microscope. The region of tissue that was successfully photooxidized can be fitted within a circle with a radius of approximately 1.32 mm and area of 5.5 mm². Scale bar corresponds to 500 µm.</p