Age estimation based on different molecular clocks in several tissues and a multivariate approach: an explorative study.

Several molecular modifications accumulate in the human organism with increasing age. Some of these "molecular clocks" in DNA and in proteins open up promising approaches for the development of methods for forensic age estimation. A natural limitation of these methods arises from the fact that the chronological age is determined only indirectly by analyzing defined molecular changes that occur during aging. These changes are not linked exclusively to the expired life span but may be influenced significantly by intrinsic and extrinsic factors in the complex process of individual aging. We tested the hypothesis that a combined use of different molecular clocks in different tissues results in more precise age estimates because this approach addresses the complex aging processes in a more comprehensive way. Two molecular clocks (accumulation of D-aspartic acid (D-Asp), accumulation of pentosidine (PEN)) in two different tissues (annulus fibrosus of intervertebral discs and elastic cartilage of the epiglottis) were analyzed in 95 cases, and uni- and multivariate models for age estimation were generated. The more parameters were included in the models for age estimation, the smaller the mean absolute errors (MAE) became. While the MAEs were 7.5-11.0 years in univariate models, a multivariate model based on the two protein clocks in the two tissues resulted in a MAE of 4.0 years. These results support our hypothesis. The tested approach of a combined analysis of different molecular clocks analyzed in different tissues opens up new possibilities in postmortem age estimation. In a next step, we will add the epigenetic clock (DNA methylation) to our protein clocks (PEN, D-Asp) and expand our set of tissues.

Estimation of age at death based on aspartic acid racemization in elastic cartilage of the epiglottis.

Age estimation based on aspartic acid racemization (AAR) has been applied successfully to various tissues. For routine uses, AAR is analyzed in dentine. For cases in which teeth are unavailable, analyzing AAR in purified elastin has been shown to be an alternative method. The suitability of elastic cartilage from the epiglottis as an elastin source for age estimation based on AAR was tested. A total of 65 tissue samples (cartilage) of epiglottis and 45 samples of elastin purified from the elastic cartilage of epiglottis samples were analyzed. While the D-aspartic acid content of total tissue samples increased with age only slowly, its increase with age in purified elastin samples was similar to that in purified elastin from other tissues. The relationship between the D-aspartic acid content and age was shown to be close enough for age estimation based on AAR in purified elastin from the elastic cartilage of the epiglottis, provided a sufficient quality of elastin purification. Age estimation based on AAR in purified elastin from the epiglottis might serve as a valuable alternative in cases in which other tissues (e.g., teeth) are unavailable.

Morphology and chemical characteristics of subepithelial laminar nerve endings in the rat epiglottic mucosa.

The laminar nerve endings are distributed in the laryngeal mucosa, and described as sensory receptors evoked by laryngeal pressure changes. The present study aimed to determine detailed morphological characteristics of the laryngeal laminar endings of the rat. Immunohistochemistry for Na(+)-K(+)-ATPase, α(3) subunit, showed that laminar endings were distributed in the entire laryngeal surface of the epiglottis. The parent axons of the endings were thick in diameter, and they were branched and continued to the endings. In some cases, several endings from different parent axons fused into a large complex structure of 500 µm in width. The laminar endings were also immunoreactive for vesicular glutamate transporter 1 (vGLUT1) and vGLUT2, but not for P2X(3) purinoceptor. Around the laminar endings, terminal Schwann cells with immunoreactivity for S-100 protein were closely associated with axon terminals. Use of scanning electron microscopy with alkaline maceration method showed that the terminal Schwann cells consisted of a rounded perinuclear region and lamellar cytoplasmic processes. Ultrastructurally, axon terminals with numerous mitochondria were partly covered with Schwann cell sheath, and some terminals intruded into the epithelial layer. Clear vesicles of 50 nm in diameter were also observed especially in small cytoplasmic processes of 400 nm to 1 µm in size. The results in the present study suggested that the laminar endings in epiglottic mucosa have morphological characteristics of slowly adapting mechanoreceptors and contribute to sensation of laryngeal pressure via mucosal tension.

[Differences between epiglottis and cricoid cartilages. Histochemical study with lectins in hamsters]. / Diferencias entre el cartílago de epiglotis y el de cricoides. Estudio histoquímico con lectinas en el hámster.

An histochemistry study with lectins was performed in the epiglottis and cricoid cartilages of 4 Syrian golden hamsters (Mesocricetus auratus). The results show that there are differences in glycoconjugates between both cartilages. In the epiglottic matrix there was no reactivity to HPA, PNA and DBA lectins. However the cricoid cartilage showed no reactivity to SBA. Neither of cartilage matrixes were reactive to LTA or Con-A.

Hyaluronan localization in the rabbit larynx.

BACKGROUND: The larynx is a complex organ composed of different connective tissue elements. So far, the extracellular matrix of the larynx has not been thoroughly described. Hyaluronan is a matrix polysaccharide with physicochemical effects and biological cell functions in soft connective tissues. METHODS: The histochemical distribution of hyaluronan (hyaluronic acid, hyaluronate) was studied in tissue sections from various levels of the rabbit larynx by means of a hyaluronan-binding protein and avidin biotin peroxidase staining. Microwave-aided fixation was used to retain the extracellular location of hyaluronan. RESULTS: Hyaluronan accumulated chiefly in the subepithelial lamina propria and in the connective tissue enclosing striated muscle fibres of the thyroarytenoid muscle and vocalis muscle. This localization contrasted sharply with the weak staining for hyaluronan in muscles external to the thyroid cartilage. Intensive staining for hyaluronan was found in perivascular and periglandular connective tissue, as in the vacuoles of the hyaline cartilage of the thyroid, cricoid and arytenoid cartilages, and to a lesser extent in the lacunae of the chondrocytes and in the perichondrium of the elastic cartilage of the epiglottis. CONCLUSIONS: Hyaluronan was heterogenously distributed in the rabbit larynx. It was abundant in intrinsic laryngeal muscles performing small, precise, and rapid movements and in the subepithelium at the glottic level, where it may facilitate mucosal movements. The abundant hyaluronan in the subglottic region may be involved in the control of vascular leakage and edema formation.

Morphology, histochemistry, and differentiation of the cat's epiglottic cartilage: a supporting organ composed of elastic cartilage, fibrous cartilage, myxoid tissue, and fat tissue.

BACKGROUND: In carnivores, the supporting organ of the epiglottis is usually called "epiglottic cartilage" (EC) although it is composed of elastic cartilage and unilocular fat storing cells. We studied the cat's EC in order to decide whether these fat storing cells are true adipocytes or fat storing (dedifferentiated) chondrocytes. METHODS: ECs were studied in cat embryos at gestation days 40 and 60, in newborn, postnatal, and adult cats. We used classical staining methods, immunohistochemistry, and transmission electron microscopy to identify the different kinds of tissues contributing to the EC and to follow their differentiation. RESULTS: The cat's EC was defined by a layer of coarse collagen fibers representing a tunica albuginea. This tunica covered irregularly formed and irregularly sized areas of elastic cartilage, fibrous cartilage, myxoid tissue, and lobules of unilocular fat cells. All these tissue showed regular morphology. Adipocytes were provided with continuous basal laminae and fat lobules were well supplied with capillaries. Alcianophilia of ground substance was observed in all tissue components but was strongest in elastic cartilage. Most islets of elastic cartilage adhered to the tunica albuginea of the EC at one surface and were connected to the opposite surface by coarse strands of connective tissue traversing the organ. Intercalated areas of fibrous cartilage contained fuchsinophilic collagen bundles. Myxoid tissue was characterized by stellate cells in alcianophilic ground substance with intermingled fuchsinophilic bundles. All kinds of supporting tissues combined with each other without clear demarcation. Immunohistochemistry revealed strong reactivity for S-100 of chondrocytes, myxoid cells, and fat cells. Chondrocytes and myxoid cells also stained for glial fibrillary acidic protein, neurofilament protein 200, and neuron specific enolase. During development, condensation of mesenchymal cells indicated the blastema of the EC at gestation day 40. At day 60, delicate collagen fibrils indicated the future tunica albuginea, faint alcianophilia was noted in the ground substance, and multilocular fat cells were scattered throughout the blastema. At birth, alcianophilia was moderate and multilocular fat cells were numerous. Three weeks after birth, single and grouped unilocular fat cells were seen, alcianophilia of ground substance was prominent, and former blastema cells presented as ramified myxoid cells. Eight weeks after birth, the EC primarily consisted of myxoid tissue, but the first islets of cartilage were seen in the center of myxoid areas. Unilocular fat cells already formed lobules. CONCLUSIONS: These results show that in the cat EC a) differentiation of adipocytes precedes differentiation of all the other tissue components, and b) differentiation of myxoid tissue precedes differentiation of cartilage. It is concluded that myxoid tissue may serve as a precursor of fibrous and elastic cartilage.

Neoplastic infiltration of laryngeal cartilages: histocytochemical study.

The relationship between cartilage and invading neoplastic cells was studied in 32 cases of laryngeal cancer by histological and cytochemical methods. Cartilage invasion was present in 12 cases, 10 of which were in proximity or in contact with areas of calcification and ossification. It was significantly correlated only to tobacco consumption (P less than .05) and, in regard to glottic tumors, to tumor diameter greater than 3 cm (P less than .01). Histologically, neoplastic invasion in cartilage was massive in 2 cases, occurred in areas of ossification in 4, between cartilage and bone in 4, and in epiglottic cartilage in 2. In 3 of the cases with bone invasion, there was also new bone formation. Hyaline cartilage and bone resorption was due to tartrate-resistant acid phosphatase (TRAP)-positive giant cells; in epiglottic cartilage only mononuclear cells were present, some of which were TRAP-positive. These results show that neoplastic cells can promote not only resorption and formation of bone, but also resorption of cartilage, which is considered resistant to neoplastic invasion. The different types of resorbing cells in contact with hyaline cartilage and bone in laryngeal cancer, and elastic cartilage in epiglottic cancer, suggest that the structure of the tissue being resorbed can influence the type of resorbing cells.