Spontaneous Calcium Oscillations through Differentiation: A Calcium Imaging Analysis of Rat Cochlear Nucleus Neural Stem Cells.

Causal therapies for the auditory-pathway and inner-ear diseases are still not yet available for clinical application. Regenerative medicine approaches are discussed and examined as possible therapy options. Neural stem cells could play a role in the regeneration of the auditory pathway. In recent years, neural stem and progenitor cells have been identified in the cochlear nucleus, the second nucleus of the auditory pathway. The current investigation aimed to analyze cell maturation concerning cellular calcium activity. Cochlear nuclei from PND9 CD rats were microscopically dissected and propagated as neurospheres in free-floating cultures in stem-cell medium (Neurobasal, B27, GlutaMAX, EGF, bFGF). After 30 days, the dissociation and plating of these cells took place under withdrawal of the growth factors and the addition of retinoic acid, which induces neural cell differentiation. Calcium imaging analysis with BAPTA-1/Oregon Green was carried out at different times during the differentiation phase. In addition, the influence of different voltage-dependent calcium channels was analyzed through the targeted application of inhibitors of the L-, N-, R- and T-type calcium channels. For this purpose, comparative examinations were performed on CN NSCs, and primary CN neurons. As the cells differentiated, a significant increase in spontaneous neuronal calcium activity was demonstrated. In the differentiation stage, specific frequencies of the spontaneous calcium oscillations were measured in different regions of the individual cells. Initially, the highest frequency of spontaneous calcium oscillations was ascertainable in the maturing somata. Over time, these were overtaken by calcium oscillations in the axons and dendrites. Additionally, in the area of the growth cones, an increasing activity was determined. By inhibiting voltage-dependent calcium channels, their expression and function in the differentiation process were confirmed. A comparable pattern of maturation of these channels was found in CN NSCs and primary CN neurons. The present results show that neural stem cells of the rat cochlear nucleus differentiated not only morphologically but also functionally. Spontaneous calcium activities are of great relevance in terms of neurogenesis and integration into existing neuronal structures. These functional aspects of neurogenesis within the auditory pathway could serve as future targets for the exogenous control of neuronal regeneration.

Different Neurogenic Potential in the Subnuclei of the Postnatal Rat Cochlear Nucleus.

In patients suffering from hearing loss, the reduced or absent neural input induces morphological changes in the cochlear nucleus (CN). Neural stem cells have recently been identified in this first auditory relay. Afferent nerve signals and their impact on the immanent neural stem and progenitor cells already impinge upon the survival of early postnatal cells within the CN. This auditory brainstem nucleus consists of three different subnuclei: the anteroventral cochlear nucleus (AVCN), the posteroventral cochlear nucleus (PVCN), and the dorsal cochlear nucleus (DCN). Since these subdivisions differ ontogenetically and physiologically, the question arose whether regional differences exist in the neurogenic niche. CN from postnatal day nine Sprague-Dawley rats were microscopically dissected into their subnuclei and cultivated in vitro as free-floating cell cultures and as whole-mount organ cultures. In addition to cell quantifications, immunocytological and immunohistological studies of the propagated cells and organ preparations were performed. The PVCN part showed the highest mitotic potential, while the AVCN and DCN had comparable activity. Specific stem cell markers and the ability to differentiate into cells of the neural lineage were detected in all three compartments. The present study shows that in all subnuclei of rat CN, there is a postnatal neural stem cell niche, which, however, differs significantly in its potential. The results can be explained by the origin from different regions in the rhombic lip, the species, and the various analysis techniques applied. In conclusion, the presented results provide further insight into the neurogenic potential of the CN, which may prove beneficial for the development of new regenerative strategies for hearing loss.

Button batteries and typical swallowed foreign bodies can be differentiated in high-resolution X-Rays.

BACKGROUND: Button battery ingestions have emerged as an increasing medical issue in recent years, especially for children. The frequent use of these energy sources in small appliances and toys is responsible for their ubiquitous occurrence in households. In addition to other possible foreign bodies, button batteries are particularly dangerous as they can cause severe complications in the aerodigestive tract. OBJECTIVE: The study aimed for a detailed analysis of specific radiographic identifiers of button batteries and similarly configured potential esophageal foreign bodies in high-resolution X-ray scans. METHODS: A selection of potentially hazardous button batteries - in cases of ingestion (CR2032) or aspiration (LR44, LR1130) - was made. Other clinically relevant radio-opaque objects of similar size and shape have been selected accordingly. High-resolution X-ray scans (33.3 lp/mm) were made using an in vitro model in porcine esophageal preparations. A systematic, comparative analysis was carried out with the digital radiographic images. FINDINGS: In the study described, the selected foreign bodies were scanned at distinct angles in high image quality. Using button batteries, radiographically details of their internal structures were shown that have not yet been described. The known markers, as well as new detailed characteristics, were found in the experimental setting. The differentiation from other typical esophageal foreign bodies was possible by analyzing four relevant identification features: the edge properties, internal structures, the size, and differences in 0°/90° scans. CONCLUSIONS: The study results reveal that specific features of potential esophageal foreign bodies can be identified by improved radiographic resolution, contrast, and dynamics. Thus, the diagnostic reliability in distinguishing 'high-risk foreign bodies' from less dangerous ones could be increased. In the future, button batteries could be detected more reliably with intelligent digital image analysis and enhanced radiographic technology. This may further support clinical triage algorithms and help reduce medical complications in cases of foreign body ingestions.

Severe tracheobronchial harm due to lithium button battery aspiration: An in vitro study of the pathomechanism and injury pattern.

BACKGROUND: Button battery incidents have become a rising medical issue in recent years, especially for infants. The increasing number of these cases can be explained by the expanding use of objects of everyday life and toys. As a result, button batteries in many households are ubiquitous in different states of charge. The extremely long shelf-life and the increasing energy densities of lithium button batteries boost the potential medical complications of accidental swallowing. OBJECTIVE: The study aimed to analyze the pathophysiology of damage to tracheobronchial structures by button batteries aspiration over time. METHODS: CR2032 and CR927 lithium button batteries (3.2/3.0 V) were exposed to porcine trachea preparations intraluminal at 37 °C in intervals up to 36 h. Measurements were made of the voltage curve, the discharge current, and the resulting pH values around the electrodes. The effects on tissue were examined using macroscopic time-lapse images and microscopic pictures of sections of the fixed specimens over time. FINDINGS: The examinations showed a tissue electrolysis reaction directly after the beginning of battery exposure, which led to an immediate coagulation impairment of the respiratory epithelium. Over time, a strongly alkaline environment was established around the batteries. The resulting tissue colliquation caused profound tissue damage beyond the basal membrane of the mucosa, affecting the tracheobronchial cartilage after only 4 h of exposure time. After 12 h, there was significant necrosis of the annular ligaments of the trachea and the peribronchial pulmonary tissue. After completion of the experimental exposure time of 36 h, there was still a sufficient residual voltage on all button batteries of the experiments. CONCLUSIONS: Besides accidental ingestion, the aspiration of button batteries is a life-threatening situation. The partial or complete acute airway obstruction in the trachea or the bronchi initially is the leading symptom, as with any foreign body aspiration. However, the results of the investigations show that even after a short exposure time, relevant tissue damage can be caused by the electrolysis reaction of the battery. After 12 h, a profound destruction of cartilage, connective tissue, and smooth muscles was observed in vitro, which may cause significant consequential damage in vivo. These findings reveal the need for rapid diagnosis and immediate foreign body removal after any battery ingestion. Moreover, the results show how relevant prevention of these accidents is, and that future safety modifications of these types of battery by the manufacturers would be appropriate.