Development of a 3D printing process of bolus using BolusCM material for radiotherapy with electrons.

This work presents the optimized parameters of 3D printing for print bolus using BolusCM material. Printing parameters were selected of the homogeneity and absence of air gaps. The dosimetric features of printed bolus were measured with a plane-parallel ionization chamber and EBT3 radiochromic film. Measured features were compared with those estimated with Monte Carlo methods. BolusCM shows good characteristics to be used as bolus material in radiotherapy with electrons, where the printing process allows personalizing the bolus in function of patient characteristics. The material low-cost, the 3D printing and the dosimetric features are few of the advantages of using BolusCM material in radiotherapy with electrons in skin cancer treatment.

Bolus 3D printing for radiotherapy with conventional PLA, ABS and TPU filaments: Theoretical-experimental study.

A theoretical-experimental study of the interaction of electron beams with 3 filaments conventionally used for 3D printing is presented in this paper. Pieces of polylactic acid (PLA), acrylonitrile butadiene styrene (ABS) and thermoplastic polyurethane (TPU) are studied using Monte Carlo simulation with Geant4 and experimental measurements with plane-parallel ionization chambers and radiochromic films. Using different printing parameters and computed tomography, the presence of air gaps and the uniformity in the bolus density made with the different materials are evaluated. The main parameters in the Percentage Depth Dose (PDDs) are determined, the manufacturing process is standardized and the printing profiles are generated for each of the materials in order to obtain uniform attenuation characteristics in the pieces and improve adaptation to irregular anatomical areas.

Characterization of a novel material to be used as bolus in radiotherapy with electrons.

Features of new material to be used as bolus in external radiotherapy were determined and their performance were evaluated. The characterization was carried out using Monte Carlo methods with the Geant4 code where the Percentage Depth Dose (PDD) due to electrons was estimated. In the Monte Carlo model the linear accelerator head was included. Calculated results were experimentally validated with measurements made for 6, 9, 12 and 16 MeV electron beams. The key characteristics of the implemented material were identified, guaranteeing a low cost bolus, easy to be customized and to be used in clinical applications. In comparison with commercial materials the new materials are superior from the cost to the effectiveness of their use in clinical treatments.

Assessment of the effectiveness of attenuation of Pb aprons by using TLD dosimetry and Monte Carlo calculations.

We developed an experimental set-up by using a continuous emission X-ray (Pantak DXT-3000) and three types of Pb aprons, with thicknesses of 0.25, 0.5, and 0.75mm, coated with Mylar fiber on their surface. Aprons were placed at a distance of 2.5m from the focus. Aluminum filtration was performed at the beam output to reproduce the qualities of narrow beams, N40 (Eeffective =33keV), N80 (Eeffective =65keV), and N100 (Eeffective =83keV), according to the ISO standard 4037 (1-3). Each apron was fixed with 10 thermoluminescent dosimeters (TLDs) over its surface, five dosimeters before and five dosimeters after irradiation with X-rays. Dosimeter readings were noted, and the attenuation coefficients for each effective energy were calculated. To confirm the method of effective energy of ISO-4037 and evaluate the effectiveness of aprons according to the energy range required for different medical practices, a Monte Carlo simulation using GEANT4 code was performed. Thus, the fluence and the absorbed dose in each of the dosimeters were determined, and then the coefficients of linear attenuation were calculated and compared with the experimental data and with those reported by the National Institute of Standards and Technology. Results were consistent between theoretical calculations and experimental measures. This work will serve to make assessments for other personalized radiation protectors made of Pb.

Golgi-Colonnier method: correlation of the degree of chromium reduction and pH change with quality of staining.

We examined the role of chromium reduction in the Golgi-Colonnier method, correlating the quality of neuronal impregnation with the levels of hexavalent (CrVI) and trivalent (CrIII) chromium in the tissue and in the chromation fluid (CF). The concentrations of both chromium species were assessed by measuring spectrophotometrically the CrVI before and after oxidizing the sample and by calculating the ratio of CrVI to total chromium (chromium ratio, CrR). The CrR was almost identical in the tissue and the CF, decreasing exponentially during chromation due to a progressive consumption of CrVI to form CrIII. Satisfactory cell impregnation was obtained only when the CrR was 0.45-0.7, regardless of other factors. The CrR values could be accurately predicted by the pH increase of the CF; this increase has proven to be a most reliable criterion to decide the endpoint of the chromation process. The dependence of cell staining on the [CrIII], together with the well-known ability of this species to bridge proteins, suggests that the key event for cell impregnation is the cross-linking of neuronal proteins by CrIII polymers.

Pilocarpine-induced changes in the saccharide composition of the tectorial membrane and interdental cells of the organ of Corti: a study with gold-labeled lectins.

The glycoconjugates in the cytoplasm of inner ear interdental cells and those constituting the limbal tectorial membrane were identified by a post-embedding cytochemical method using low-temperature embedding in Lowicryl K4M and labeling with biotinylated lectins, goat anti-biotin antibody, rabbit anti-goat antibody, and gold-labeled protein A in control animals, and after the systemic injection of pilocarpine. The lectins used were ConA, PHA-E, PSA, RCA, SBA, Succ-WGA, UEA, and WGA. In control animals, a semiquantitive analysis of gold particles showed that Succ-WGA produced the strongest labeling on the tectorial membrane, followed by SBA, ConA, WGA, RCA, PHA-E, and PSA. The lowest values were obtained with UEA. The cytoplasm of the interdental cells was also labeled with all the lectins, but the number of particles/microns2 was lower than on the tectorial membrane. The concentration of gold particles on the limbal tectorial membrane in pilocarpine-treated animals was higher than in control animals for some lectins (RCA, PSA, UEA) but lower for others (WGA, SBA, PHA-E, Succ-WGA). The changes in the labeling pattern of the cytoplasm of the interdental cells paralleled those in the tectorial membrane. These results demonstrate that the saccharide composition of the limbal tectorial membrane can be modified by systemic injection of pilocarpine. This action may take place through a change in either the secretion rate or the amount of some glycoconjugates by the interdental cells.

A reliable method for Golgi staining of retina and brain slices.

Although the classical Golgi method is a powerful means for structural analysis of the brain, it is generally considered to be an unpredictable technique making anatomists wary of using it. Often, even when successful staining has occurred, deposits of silver chromate crystals on the surface of the tissue obscure examination. This paper describes a simple procedure for Golgi impregnation of retina and brain slices so that good, even staining is obtained and crystal formation is avoided. The most outstanding feature of the method is the consistency of results. This consistency is due to two factors: (1) the accurate determination of the optimal chromation by measuring the rise of pH in the solutions and (2) the uniform penetration of dichromate and silver nitrate to the specimen by using a freely hanging, sandwiching technique. We suggest that the method described here can be applied to other parts of the nervous system and will be a reliable way to identify and better classify new cell types.

The effect of hypothyroidism on the development of the glycogen content of organ of Corti's hair cells.

The density of glycogen particles in organ of Corti's sensory cells was measured to determine the effect of congenital hypothyroidism upon the normal development of this energy source. This density in both normal and hypothyroid inner hair cells remains in low values from birth to adulthood. On the other hand, that of normal outer hair cells undergoes a great increase between the 10th and the 20th postnatal days, coinciding with the maturation of both the efferent innervation of these cells and the tuning properties of the auditory receptor. The glycogen stores of the hypothyroid outer hair cells do not show any significant increase from birth to adulthood. This latter fact suggests that the congenital hypothyroidism restrains the development of an important energy source of outer hair cells, most surely disturbing the physiological processes relying on glycogen metabolism.

Lectin staining of saccharides in the normal and hypothyroid developing organ of Corti.

Lectin staining has been used to detect mono- and oligosaccharides in normal and hypothyroid developing organs of Corti in the rat. Eight developmental stages were studied (1, 5, 8, 10, 15, 20, 50 and 60 days after birth). Congenital hypothyroidism was induced by oral administration of propylthyouracil to pregnant rats. Labelling of the tectorial membrane with 3 lectins, Ulex europaeus agglutinin-I (UEA-I), Lens culinaris agglutinin (LCA) and Ricinus communis agglutinin-I (RCA-I) showed no significant differences between normal and hypothyroid animals. Staining with peanut agglutinin (PNA) showed that the hypothyroid adult tectorial membrane (but not the normal one) possesses the disaccharide galactose + N-acetyl galactosamine. Phaseolus vulgaris agglutinin-L (PHA-L) labels the whole tectorial membrane in both groups of animals, but the staining is more intense in the hypothyroid one for a narrow band of oligosaccharide located just between the tectorial membrane and the underlying organ of Kölliker. Both soybean agglutinin (SBA) and succinylated wheat germ agglutinin (WGA) stain the tectorial membrane as well as the cytoplasm of the cells constituting the inner portion of the organ of Kölliker; this latter feature disappears in the normal animals about the 8th postnatal day, but it is abnormally preserved until the 60th postnatal day in the hypothyroid ones. In the adult hypothyroid animals, 3 of the lectins (LCA, PHA-L and WGA) stain extracellular conglomerates located under the synaptic pole of the outer hair cells.

Specific staining of nonpyramidal cell populations of the cerebral cortex by lectin cytochemistry on semithin sections.

The pattern of lectin labeling in the cerebral cortex of the cat was studied using semithin sections. The labeling produced by some lectins (Concanavalin A, Lens culinaris, Phaseolus vulgaris-L, Phaseolus vulgaris-E, Pisum sativum, wheat germ agglutinin, and succynilated-wheat germ) appeared inside every neuron as small cytoplasmic granules, probably corresponding to cisterns of endoplasmic reticulum and/or the Golgi complex. Lectins with affinity for alpha-mannosyl residues (Pisum sativum, Lens culinaris, and Concanavalin A) stained the cell surface of a subset of cortical neurons. The labeled cells were round or polygonal, medium to large neurons present in layers II-VI, exhibiting the morphological features of nonpyramidal cells. Previous lectin studies of perineuronal nets have shown that these extracellular specializations contain N-acetylgalactosamine and N-acetylglucosamine. Our results show that mannose is also a component of perineuronal nets and that lectins specific for alpha-mannose can be used as tools for the cytochemical detection of a separate class of cortical neurons, which have not yet been fully characterized. In addition, some lectins (Bandeiraea simplicifolia, Concanavalin A, Lens culinaris, Phaseolus vulgaris-L, Phaseolus vulgaris-E, Pisum sativum, and succynilated-wheat germ agglutinin) specifically labeled a population of a type of microglia-related cells known as perivascular cells. The data presented here report for the first time the selective staining of perivascular cells and further support the hypothesis that they are different from typical microglial cells.

Tannic acid staining of the cell coat of the organ of Corti.

Tannic acid staining of the cell coat of the organ of Corti shows a deep asymmetry between the endo- and the perilymphatic surfaces, the former being 6 times thicker. This fact may be related to a barrier mechanism against potassium-induced cell damage. Horizontal cross-links between stereocilia were heavily stained, but vertical ones were not preserved, thus suggesting they are not glycoconjugates.

Regional specialization of the cell coat in the hair cells of the organ of Corti.

The anionic sulfated groups of the cell coat glycoconjugates were studied in sensory and supporting cells of the organ of Corti with the colloidal iron hydroxide technique of Mowry, modified by Seno et al. [(1985) Histochemistry 82, 307-312]. This technique specifically stains the apical (endolymphatic) surface of both inner and outer hair cells. As this is the area at which the influx of potassium into the sensory cells takes place, the accumulation of negative charges in this location may be a mechanism to concentrate cations (mainly potassium) close to this portion of the hair cell membrane.

Two different secretion mechanisms in the inner ear's interdental cells.

Fixation with a mixture of tannic acid and glutaraldehyde reveals a darkly stained substance in the intercellular clefts between juxtapposed interdental cells, where it is included by pynocytotic vesicles. Over the interdental cells, similar-sized vesicles (35-45 nm) are present in clear spaces of the amorphous layer of the limbal portion of the tectorial membrane. Some images suggest that they may be secreted through small disruptions of the membrane of the interdental cells' microvilli. In addition, a TA-unstained, amorphous material is present inside both basal ducts and apical cavities of the interdental cells, thus suggesting another secretory route from the basal region of the interdental cells towards either the endolymph, or the tectorial membrane. These two secretion processes coexist in a single interdental complex, and the two secretion products may be involved in the turnover of the adult tectorial membrane and/or the secretion of some component of the endolymph.

Pilocarpine elicits the interdental cell secretory activity of the inner ear.

Subcutaneous injection of pilocarpine in guinea pigs resulted in the following ultrastructural changes: 1) the apical cavities of the interdental cells were filled with a substance indistinguishable from the overlying amorphous layer of the TM; 2) a great number of spherical structures appeared over the limbal portion of the tectorial membrane. In TEM photomicrographs these structures displayed the same appearance as the amorphous layer of the TM and were usually continuous to it; 3) the number of holes that decorate the upper surface of the limbal portion of the TM was dramatically increased and it was found that they connect the endolymphatic space to the apical cavities of the interdental cells; 4) there was an increase in the number of the small extracellular vesicles found in the clear spaces of the tectorial membrane. These facts suggest that pilocarpine stimulates the secretion of the interdental cells, confirming the existence of the secretory processes previously described (Prieto et al., 1990). These findings can be related to the turnover of the TM in the adult animal and, perhaps, to the secretion of some organic compound to the endolymph. We postulate that the actions of pilocarpine on the interdental cells are most probably mediated by the activation of muscarinic acetylcholine receptors in these cells.

Subsurface material in outer hair cells.

Tannic acid stains a homogenous material inside the outer hair cells of the organ of Corti of the guinea pig. This material is always placed between the plasma membrane and the first layer of subsurface cisterns, but only in those areas along the lateral surface of the outer hair cell lining the spaces of Nuel. The possibility that this material is related to some particular function of outer hair cell lateral face is discussed.

Localization of anionic sulfate groups in the tectorial membrane.

Colloidal iron hydroxide (CIH) staining demonstrates the existence of anionic sulfate groups of glycoconjugates associated with several constituents of the tectorial membrane (TM). In the adult animal, labelling in the main body of the TM appears as long, electron-dense patches surrounding type A fibrils which show alternating stained and unstained zones. On the other hand, labelling of the fibrils of the matrix of the TM appears as single, CIH particles with no special arrangement. Some of the structurally distinct regions of the TM are also labelled (limbal zone, Hensen's stripe and inner portions of the cover net), while others are not (marginal band and outer portions of the cover net). Staining of type A fibrils in the major TM is already present in newborn animals; while, both the outermost region of the TM closest to the cells of the organ of Kölliker and the minor TM are not labelled. The implications of these distributions of sulfated glycoconjugates for the electrochemical properties of the TM are discussed.

The effects of kainic acid on the cochlear ganglion of the rat.

The effects of locally applied kainic acid on cells and fibers in the rat cochlea were examined in a quantitative and ultrastructural study. Doses of 5 nM per microliter of artificial perilymph destroyed part of the spiral ganglion type I cell population, with no ototoxic effects on cochlear hair cells or supporting cells. Type II cells also appeared unaffected. A quantitative evaluation of the cell loss with the 5 nM dosage showed that 34% of spiral ganglion neurons were lost 10 days after treatment. Doses of 20 nM per microliters and 40 nM per microliters did not result in increasing neuronal loss. This differential toxicity could reflect the presence of a sub-population of spiral ganglion cells with an increased number of KA receptors.

Development of the tectal cells in the mouse cochlea.

Tectal cells appear at birth in the outer part of the developing organ of Corti. At first they are attached to the basilar membrane, but later they ascend through the auditory epithelium. During the 1st postnatal week (coinciding with the development of the minor tectorial membrane), the newly formed tectal cells show several cytological characteristics suggesting increased metabolic and secretory activities, which include: (1) a large Golgi complex, (2) abundant amorphous material inside the cisterns of rough endoplasmic reticulum, and (3) dense granules inside the mitochondrial matrix. All these features gradually disappear, and by the 14th postnatal day the tectal cells show a dark cytoplasm and few and short microvilli. In addition, tectal cells were stained selectively by some lectins. These findings suggest that tectal cells may participate in the secretion of some components of the minor tectorial membrane, different from those produced by Deiters' cells, Hensen's cells and pillar cells.

Surgical trauma to the cochlea results in reversible damage to spiral ganglion type I neurons.

Type I neurons are reversibly damaged when the cochlear bony wall is opened. The reversibility is indicated by the absence of neuronal loss, as demonstrated by quantification of the spiral ganglion neuronal population. Reversible damages included ultrastructural signs of excessive ion and water influx into the type I neuron cytoplasm, whose functional implications must be investigated in the future.

Reversible damage to the nerve fibres in the organ of Corti after surgical opening of the cochlea in the rat.

After minimal opening of the cochlear bony wall, the efferent and the outer spiral fibres showed no changes; inner radial fibres (afferents to inner hair cells) were highly sensitive to this mild trauma, appearing swollen and empty of cytoplasmic content. Available data suggest that this may be due to alterations in the cochlear micromechanical environment, related to the surgical manipulation. The swellings were reversible, although the normal structure had not completely recovered until one month after the manipulation.