Romuald Wróblewski

Ionic environment of cochlear hair cells

The scala media of the adult cochlea in mammals comprises a morphologically closed compartment sealed with tight junctions of the intermediate to tight types. The unique ionic composition of endolymph is maintained by the stria vascularis through active reabsorption of sodium and active secretion of potassium against ionic gradients. The subtectorial space is only a partially closed compartment which communicates with the endolymph via holes in the tectorial membrane at its outer insertion to the organ of Corti. Hardestys membrane divides the subtectorial space into two compartments: one facing the surfaces of inner hair cells and one facing the surfaces of outer hair cells. In the study of comparative anatomy, hair cells, e.g. in the lizard, basilar papilla are of two types: those covered with a tectorial membrane and those being free-standing lacking the tectorial membrane. The ionic environment of the hair cell surface seems to be the same, independent of whether covered with a tectorial membrane or not. The tectorial membrane itself is semipermeable to ions in the endolymphatic space. Only the surface structures of the hair cell with the sensory hairs facing the subtectorial space are exposed to the high concentration of potassium, whereas the remaining parts of the hair cell are surrounded by a fluid having a more normal extracellular type of ionic composition (cortilymph/perilymph). During embryonic development the ionic composition of endolymph develops in parallel with the morphologic maturation of the stria vascularis. A completely mature composition of endolymph is reached before any electrophysiological potentials in the cochlea can be elicited. The sensory hair surface of hair cells has reached a mature morphology prior to the maturation of endolymph. In several species the tectorial membrane is morphologically only partially mature when the increase of the potassium concentration of endolymph starts. Drugs primarily affecting the stria vascularis causing a transient change of the ionic composition of endolymph result in a transient dysfunction of inner ear potentials. If the ionic changes persist for longer time, morphological changes can occur in both the stria vascularis and the hair cells of the organ of Corti. Whether such changes are primarily caused by the ototoxic drug itself or by changes in the ionic composition of endolymph has to be explored further.

Human and experimental lens repair and calcification

Lens repair and calcification have been studied in an experimental rabbit model of anterior segment necrosis. Findings were compared with those in a human senile cataractous lens with subcapsular calcification. Rabbit lenses subjected to anterior segment ischemia underwent a repair process similar to that observed in perforating lens injuries. Cellular response included the formation of fibroblast-like cells that covered epithelial defects of the anterior pole. These observations suggest that the lens epithelium can transform into fibroblast-like cells. The calcification process was a non-cell-induced, and the observed mineral was probably nucleating on organic molecules. Elemental analysis demonstrated that crystals contained calcium and phosphorus with a ratio of 2:1. The mineral was probably hydroxyapatite. Since morphological findings in rabbit lenses closely resemble those of the studied cataractous human lens, the rabbit model appears to simulate one type of lens calcification in senile cataract.

In situ elemental analysis and visualization in cryofixed nervous tissues: X-ray microanalytical investigations of embryological and mature brain, inner ear, photoreceptors, muscle and muscle spindles. Comparison of preparation methods for analysis and visualization at cellular and subcellular levels

For meaningful X‐ray microanalysis (XRMA) in biology and medicine, the development of preparative and quantitative methods has been necessary. The methods need to preserve close to in vivo distribution of diffusible ions with at the same time reasonable morphological preservation of the tissue.

Why low temperature embedding for X-ray microanalytical investigations?

Freeze‐drying followed by infiltration with resin and polymerization by UV light at low temperatures and under constant vacuum conditions is an alternative tissue preparation technique for microprobe analysis. Embedding is carried out with the nonpolar low‐temperature embedding resin (Lowicryl HM20) which allows infiltration and polymerization at temperatures down to −50°C. Sections of low temperature embedded material can be cut dry at −60°C or at room temperature. Sectioning at low temperatures is an alternative for preparations that are difficult to cut at room temperature.

A new type of striated muscle in mammalian body: Morphological, histochemical, and x-ray microanalytical observations of stapedius muscle in guinea pig

The guinea pig stapedial muscle presented an uncommon ultrastructural organization compared with its skeletal muscles or the stapedial muscles from other species. In the majority of muscle fibers the cross-sectional area was small (5–20 μm φ) and the nuclei were centrally located. Golgi complexes and centrioles were frequently found near the nuclei. The mitochondria had a dense matrix and contained a large number of dense granules, adjacent to the internal membranes. Nerves were found among the muscle fibers and had end plates with excessive numbers of vesicles. Capillaries appeared in a small number. Degenerating and split muscle fibers were common. The muscle fiber composition based on its stainability for myofibrillar ATPase exhibited the presence of type I and type II fibers, the latter with subtypes A, B, and C. Elemental analysis performed on unfixed cryosections revealed an extremely low potassium and high calcium and sodium concentrations. The highest concentrations of calcium and phosphorus were found in the mitochondrial areas. The quadriceps muscle of guinea pig was analyzed simultaneously and its elemental composition was similar to that found in ordinary striated skeletal muscles from rat and man.

Energy dispersive X-ray microanalysis of individual yestibular hair cells

SummaryMicroprobe analysis was performed at the cellular and subcellular levels of type I and type II vestibular hair cells. In principle the same types of elemental histograms were found in the two types of hair cells studied. High concentrations of Cl and K were detected in stereocilia, whereas calcium was found when analyzing stereocilia and the supranuclear cytoplasm.

Simultaneous investigations of elemental changes in individual cells of the stria vascularis and in endolymph.

SummaryUsing the microprobe for energy dispersive X-ray microanalysis, the elemental compositions of both the individual cells of the stria vascularis and of the endolymph were followed simultaneously under normal conditions and after the administration of 120 mg/kg ethacrynic acid (EA). Marginal cells and intermediate cells showed reversible increases in potassium and decreases in sodium concentrations. Shifts in the ionic composition of endolymph occurred later than after elemental changes in the strial cells. The present results indicate that the marginal and the intermediate cells are the primary target for EA-induced ototoxicity. However, generalized toxic effects of EA are also indicated, with a general leakage of different elements occurring during the 30–60 min period after EA administration.

Microprobe Analysis of Cryofixed, Chemically Untreated Freeze-Dried Cochlear Hair Cells

Elemental (energy-dispersive X-ray) microanalysis was performed of cryofixed, chemically untreated freeze-dried cochlear hair cells of 1-month-old CBA/CBA mice. This technique is very difficult to carry out and time consuming, requiring a large number of specimens to be sectioned, quite at random, in order to obtain absolutely perpendicular section planes. Microprobe determination was performed in different parts of outer and inner hair cells. In addition to the normal intracellular elemental content a high calcium concentration was found at the base of all hair cells. Lower calcium levels were evident in the entire cytoplasm, including the cuticular plate and stereocilia. Technically it was feasible to analyze individual stereocilia.