Sources of stress and worry in the development of stress-related mental health problems: A longitudinal investigation from early- to mid-adolescence.

BACKGROUND AND OBJECTIVES: Stress and stress-related mental health complaints are common and increasing among adolescents, especially girls. Identifying typical sources of stress as well as central intervention targets is an important effort in the development of effective prevention and treatment protocols. This study investigated worry as potential mediator in the development of mental health problems in response to common stressors in adolescence. We also examined to what sources adolescents ascribe their stress over the years from the 7th through the 9th grade. DESIGN: Prospective cohort study. METHODS: Self-reported subjective stressor load, worry, anxiety and depressive symptoms were assessed in a sample of Swedish 7th graders (N = 1137; 46% girls, mean age 13.2) with follow-up assessments one and two years later. RESULTS: School was the most common source of stress across all time-points, with girls reporting considerable more stress than boys. Worry mediated the relationship between overall stressor load and depressive symptoms and anxiety over time and was not moderated by gender. CONCLUSIONS: Worry may be an important target in stress prevention and efforts to prevent stress-related problems would benefit from focusing on early adolescence as especially school stress is already relatively common in grade 7.

Expression of EGFR, HER2, HER3, and HER4 in metastatic squamous cell carcinomas of the oral cavity and base of tongue.

The expressions of all four receptors in the epidermal growth factor receptor family, EGFR. HER2, HER3, and HER4 were evaluated by immunohistochemistry in 19 cases of metastatic squamous cell carcinoma of the oral cavity and base of tongue. EGFR had a similar and high expression in both primary tumours and the corresponding metastases, while the expression in normal epithelium was lower in most cases. HER2 was not expressed to the same extent as EGFR. However, when HER2 was well expressed, it was in most cases expressed to the same extent and intensity in the primary tumours, metastases, and normal epithelium. The expression of HER3 and HER4 varied and was mainly cytoplasmic in all cases studied. No overexpression of HER3 and HER4 in tumours was seen as compared to normal epithelium. In order to further investigate the distribution of HER3, two HER3 expressing cell lines originating from tongue cancer were analysed in vitro, using radiolabelled anti-HER3 antibodies directed to the extracellular domains of the receptor. The results indicated that HER3 was not present in measurable amounts in the cellular membrane. There is a need for improved diagnostics and therapy for the studied type of tumours, e.g. using radiolabelled antibodies or ligands, and EGFR seemed suitable as target since the expression was high, membrane associated and similar in the primary tumours and the corresponding metastases.

Glycoprotein tumour markers in head and neck neoplasms--a consecutive study on CA-50, CA 19-9, and CEA.

Serum levels of three glycoprotein tumour antigens (carcino-embryonic antigen, CEA; cancer-associated antigen 50, CA-50; gastrointestinal cancer-associated antigen, CA 19-9) were determined on 125 consecutive patients with tumours of the head and neck region. Elevated CEA values (greater than 5 units/ml) were found in 13/70 squamous cell carcinomas, 3/21 benign and 4/18 malignant salivary gland neoplasms. Elevated CA-50 values (greater than 17 units/ml) were found in 19/70 squamous cell carcinomas, 6/18 malignant and 1/21 benign salivary neoplasms. CA 19-9 displayed higher values (greater than 37 units/ml) in 9/68 squamous cell carcinomas, 4/18 malignant and none of 21 benign salivary gland tumours. Combination of CEA and CA-50 analyses increased the proportion of elevated values to 30/70 in squamous cell carcinomas and 10/18 in salivary gland malignancies. In squamous cell carcinomas no correlation between staging or grading and serum levels was detected for any of the markers. Among malignant salivary gland tumours, CA-50 displayed enhanced serum values in 4/6 mucoepidermoid carcinomas. The mean values for CA-50 and CA 19-9 serum levels were significantly higher for malignant salivary gland neoplasms compared to benign tumours. There was a close correlation between CA-50 and CA 19-9 serum levels. Although, the results suggest that at present none of the tumour markers tested have a place alone in the routine examination of patients with tumours affecting the head and neck region, further studies on salivary gland neoplasms and combinations of the tumour markers are justified.

Appearance and distribution of two Ca2+-binding proteins during development of the cochlea in the musk shrew.

In the developing cochlea of the musk shrew, Suncus murinus, the localization of two Ca2+-binding protein, calbindin and calmodulin, which are thought to play different roles in the nervous system, was examined during gestational and postpartum periods. Calbindin is thought to play a Ca2+ buffering role, while calmodulin activates other proteins. Cochleae from the musk shrews sacrificed from gestational day (GD) 15 to postnatal day (PP) 9 and as adults, were immunohistochemically analyzed. The localization and order of appearance of calmodulin in sensorineural elements were similar to those of calbindin, except for timing of appearance. Calmodulin-staining was recognized first in the spiral ganglion neurons on GD21, followed by the inner hair cells (IHCs) on GD23 and outer hair cells (OHCs) on GD26, while calbindin immunoreactivity in the spiral ganglion neurons on GD19, the IHCs on GD21 and the OHCs on GD23. In hair cells, during development, immunostaining of calbindin and calmodulin was initially seen in the cytoplasm, followed by the cuticular plate. Cytoplasmic staining then decreased in mature hair cells. Non-sensorineural components also showed positivity for both calbindin and calmodulin. The lateral wall of the cochlear duct was positive for calbindin, while the stria vascularis was positive for calmodulin. Immunoreactivity for calbindin was present earlier than that of calmodulin in sensorineural elements, suggesting that in the developing cochlea, calbindin and calmodulin have different functions and that Ca2+ buffering capacity, which is regulated by Ca2+ buffer proteins, such as calbindin, may be required before trigger proteins, such as calmodulin, function.

Cytopathogenic effects of atoxyl, an ototoxic compound, on human diploid fibroblasts in vitro.

Atoxyl, an arsenic compound, may cause degeneration in vivo of the inner ear including cells of the stria vascularis and hair cells. The mechanism behind the cytotoxic effect is not known. The effects of atoxyl at the subcellular level were investigated in this study using human diploid embryonic lung fibroblasts in monolayer cultures as an in vitro model system. Atoxyl caused a subtle but significant increase in the permeability of the fibroblast plasma membrane, as measured by release of a low molecular weight cytoplasmic marker (alpha-amino isobutyric acid). At higher concentrations or after longer incubation times, protein synthesis was impaired. This effect occurred in parallel with alterations in the cellular morphology as viewed by light microscopy. In the final stages of atoxyl intoxication the cells released also a higher molecular weight marker (nucleotide), indicating a further increased membrane permeability following the primary damage. It is concluded that atoxyl exerts a dual effect on the human fibroblasts, namely on membrane permeability and protein synthesis. Although the concentrations used were higher than those exerting the ototoxic effects in vivo, the prolonged exposure times to low concentrations obtained in whole animals may very well compensate for this fact. The effects observed in the in vitro fibroblast model system may thus be relevant to the mechanism of action of atoxyl during induction of ototoxic effects in vivo.

Specific pathology of the stria vascularis in postnatal progressive genetic inner ear disorder.

After reaching normal morphological maturation the stria vascularis of the Shaker-2 (Sh-2) mouse postnatally underwent a progressive degeneration, especially of the intermediate cell layer. Considerable changes were present on the 3rd week after birth. The marginal cells became atrophic and showed a reduction in the number and extent of the cellular extensions towards the intermediate cell layer. The intermediate cells disintegrated but were present 9 weeks after birth. The basal cells showed a normal morphology. Specific inclusion bodies were identified in the marginal cells from the 3rd week on. Occasionally such inclusions occurred in marginal cells. In the Shaker-1 (Sh-1) a flattening of intermediate cells occurred but otherwise no pathological changes were found 6--9 weeks after birth.

Elemental analysis of the early postnatal tectorial membrane.

The analytical electron microscope has revealed that the elemental composition of the early postnatal tectorial membrane is principally the same before and after the development of endolymph. The elemental composition of the tectorial membrane does not give absolute information on the permeability of the membrane to ions such as potassium and chlorine from the endolymphatic space. Such a permeability is, however, likely when comparing the relative peak intensities of chlorine and potassium before and after the development of endolymph.

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. Hardesty's 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.

Cellular localization of adenylate cyclase in the developing and mature inner ear of the mouse.

The localization of adenylate cyclase in the developing and mature inner ear of the CBA/CBA mouse' in in Shaker-1 and Shaker-2 mutants and in organ culture was determined by a histochemical procedure with adenylyl imidodiphosphate as substrate and Sr2+ as capture ion. Enzymatic activity was associated with cell membranes in stria vascularis and Reissner's membrane but could not be demonstrated in other cochlear structures. In stria vascularis, adenylate cyclase was found on the contraluminal infoldings of marginal cells but not on their luminal surface, while Reissner's membrane showed activity on its endolymphatic surface. Activity was also associated with utricular dark cells. Essentially the same localization but differing in quantitative aspects was observed during development, in organ culture and in the mutant strains. The speculative role of adenylate cyclase in cochlear ion and fluid balance is discussed.

Adenylate cyclase activity in the fetal and the early postnatal inner ear of the mouse.

The adenylate cyclase activity was analyzed in fetal, early postnatal and adult inner ears of the CBA/CBA mouse and also in approximately one month old inner ears from Shaker -1 and Shaker -2 mice. A comparison was made with the maturation of potassium levels in endolymph as investigated with the X-ray energy dispersive technique. Adenylate cyclase activity in the developing normal inner ear shows two significant periods of increases: from the 16th to the 19th gestational day in both the cochlear and vestibular parts of the labyrinth, and from birth to day 6 after birth in the lateral wall tissues of the scala media. During the first period the anatomical boundaries of the secretory epithelia are developing. The postnatal rise in adenylate cyclase activity correlates with the morphological maturation of stria vascularis at the cellular and subcellular levels and the rise in potassium content of endolymph. The rise of enzyme activity in the cochlear during the maturation of endolymph supports a link between adenylate cyclase and the control of inner ear fluids. Adenylate cyclase activity in stria vascularis/spiral ligament of Shaker -1 and Shaker -2 mice were at normal levels and correlated better with the rather normal morphology of the tissues than the abnormal composition of endolymph in these mutants.

Lipopolysaccharide-induced expression of inducible nitric oxide synthase in the guinea pig organ of Corti.

The purpose of the investigation was to ascertain whether inoculation of bacterial lipopolysaccharide (LPS) into the cochlea of the guinea pig could elicit formation of inducible nitric oxide synthase (iNOS). Immunohistochemical study revealed that immunoreactivity to iNOS was seen below outer hair cells representing nerve fibers and synaptic nerve endings. iNOS-staining could also be observed in phalangeal dendrites of Deiter's cells pointing to the cuticular membrane, Hensen's cells and on stria vascularis 48 h after inoculation with LPS. Immunohistochemical investigation with a specific anti-nitrotyrosine antibody also revealed intense immunoreactivity identical to that of iNOS, suggesting formation of peroxynitrite in the organ of Corti by the reaction of NO with O(2)(-). On the basis of these findings, it can be concluded that NO together with O(2)(-), which form the more reactive peroxynitrite, are the most important pathogenic agents in LPS-induced damage of cochlea in the guinea pig.

Age-dependent disruption of basal lamina and extracellular matrix formation in L-proline analog treated otic explants.

L-azetidine-2-carboxylic acid, LACA, a naturally occurring vegetable imino acid, can be incorporated into mammal proteins instead of proline. This incorporation has an especially inhibitive effect on collagen secretion. Exposure of embryonic mouse inner ear explants to LACA causes dysmorphogenesis and retarded differentiation, reduces the number of collagen fibrils in the perilymphatic spaces and capsules, and gives rise to a dose-dependent derangement of the basal lamina. In control specimens, both in vivo and in vitro, the inner ear epithelia had a dense contiguous basal lamina overlying a well-developed network of collagen fibrils. When the inner ears were exposed to LACA at a concentration of 150 micrograms per ml of medium, there was a loss of the collagen network and gaps appeared in the basal lamina. At exposure to 300 micrograms LACA/ml, scarcely any collagen fibrils were present and the basal lamina was disrupted in many areas, especially beneath the sensory epithelia.

Morphological and electrophysiological study of the inner ear and the central auditory pathways following whole body fetal irradiation.

Pregnant CBA/CBA mice were whole body irradiated with 2 Gy on the 13th or 16th day of gestation, respectively. The exposed fetuses were raised to an age of 21 postnatal days. Auditory brainstem recordings of threshold levels showed a considerable elevation independent of if irradiation had been performed on either the 13th gestational day or the 16th gestational day. In exposed animals a latency difference occurs in the peaks that increases from peak 1 to peak 5, measuring in peak 5 up to 1.16 ms. Also the peak-to-peak length of waves 1-5 increases in irradiated animals. Scanning electron microscopy of the cochleae showed varying degrees of stereociliary derangement of both outer and inner hair cells, particularly in cochleae where irradiation had been performed on the 13th gestational day, but not loss of hair cells. Light microscopic analysis of auditory brainstem nuclei revealed normal conditions except that in inner ears exposed on the 16th gestational day the flocculus was fused to the lateral surface of the anterior ventral cochlear nucleus. It is concluded that the elevated threshold levels in irradiated animals are most likely due to pathological changes in the peripheral receptor organ whereas the increased latencies and the increased peak-to-peak length likewise reflect functional changes in the brainstem auditory nuclei.

Effects of cisplatin and thiosulfate upon auditory brainstem responses of guinea pigs.

Two side effects which limit the use of cisplatin in cancer chemotherapy are severe nephrotoxicity and ototoxicity. The concurrent administration of sodium thiosulfate with cisplatin reportedly protects from cisplatin nephrotoxicity, however, protection from ototoxicity has not been documented. The purpose of this study was to examine the efficacy of using thiosulfate to ameliorate the ototoxic effects of cisplatin. Toward this end, the effects of cisplatin alone, cisplatin administered concurrently with sodium thiosulfate (CIS/THIO), and sodium thiosulfate alone on the auditory brainstem response (ABR) of guinea pigs were compared. ABR waveforms, comparing latencies, amplitudes and response thresholds, were monitored before, immediately after, and 30 days post treatment. Sodium thiosulfate administered with cisplatin (CIS/THIO) consistently protected animals from hearing loss and surprisingly yielded significant increases in amplitude when compared to baseline and saline controls. However, ABRs of CIS/THIO animals returned toward baseline values after 30 days.

Postnatal maturation of cochlear sensory hairs in the mouse.

The surface morphology of inner hair cells in the cochlea of the mouse is fully developed at birth. At this time a regular pattern of elongated microvilli covers the surfaces of outer hair cells at all levels of the cochlea. The sensory hair on outer hair cells undergo a morphological maturation during the first days postnatally. A mature surface morphology is reached 5-7 days after birth. There are two gradients in hair cell maturation in the cochlea: (1) basal to apical, and (2) from inner hair cells to the 3rd row of outer hair cells. Polarization of sensory hairs on an outer hair cell, i.e. polar differentiation of the sensory hairs in the radial direction, occurs as a stepwise increase in length of those facing the stria vascularis.

Embryogenesis of the mammalian inner ear. III. Formation of the tectorial membrane of the CBA/CBA mouse in vivo and in vitro.

The development of the tectorial membrane in the basal coil of the cochlea has started already in the 15th gestational day inner ear and has reached a considerable thickness and maturation at birth. The development of the tectorial membrane occurs synchronously in in vivo labyrinths and the in vitro material cultured to an age corresponding to birth. At least during this part of the development the formation of the tectorial membrane is independent of the specific composition of endolymph. In the in vivo material a secretory maximum was reached on the 18th gestational day, whereafter the secretory activity was low, especially after birth. In the in vitro specimens, however, a rather constant secretion of material occurred also post partum, which indicates a lack of control mechanisms during in vitro conditions. A complete maturation of the tectorial membrane did not occur in vitro. When passing the point of time corresponding to birth, in the in vitro inner ear explants the gross structure of the tectorial membrane is only slightly changed. In vivo a mature configuration of the tectorial membrane is observed on the 14th DAB (day after birth).

Structural changes in the round window membrane following exposure to Escherichia coli lipopolysaccharide and hydrocortisone.

The present study focused on structural changes of the round window membrane (RWM) from agents that evoke transient or permanent impairment of the auditory brainstem response when applied into the RW niche. Escherichia coli (E. coli) lipopolysaccharide (LPS) in sterile water (SW) and a 2% suspension of hydrocortisone (CORT), micronized in SW, were instilled into the round window (RW) niche of Sprague-Dawley rats. The morphology of the RWM was analyzed 3 to 21 days after instillation of either substance. Both substances caused minor structural alterations at the light microscopic level. The RWM showed a slight thickening and an invasion of inflammatory cells. At the ultrastructural level, the CORT-treated specimens showed an increased epithelial height and numerous microvilli, whereas the epithelium of the LPS-treated specimens was extended and contained few microvilli resembling those in the normal RWM. We postulate that the RWM may undergo dynamic structural changes when exposed to various agents. The structural alterations per se can influence the passage of substances from the middle ear to the inner ear.

Principles in embryonic development and differentiation of vestibular hair cells.

The early development and maturation of vestibular hair cells in the CBA/CBA mouse were analyzed at the ultrastructural level with conventional transmission electron microscopy and freeze fracturing. Cells differentiating into future hair cells pass their terminal mitosis close to the otocyst lumen. The earliest morphologic sign of differentiation into future hair cells was the arrangement of microvilli in a regular fashion. Hair cell cytodifferentiation occurred with a gradient from the hair cell surface to the base. In parallel with the maturation of sensory hairs a structural intracellular rebuilding occurred: the number of polyribosomes and amount of rough endoplasmic reticulum decreased, the nucleus moved basally, and mitochondria accumulated in the supranuclear region. Both tight junctions and gap junctions occurred initially on developing hair cells. From the sixteenth gestational day on, gap junctions disappeared, indicating an uncoupling phenomenon. Afferent nerve terminals developed before efferent nerve endings. Maturation of innervation occurred, for the most part, postnatally, and after that hair cells reached mature morphology.

Auditory epithelial migration. III: An immunohistologic study using anti-BrdU antibody on tympanic membrane in mouse.

A localization pattern of epidermal cells on the tympanic membrane (TM) and their migratory patterns were studied in mice, by means of immunohistologic technique using an anti-bromodeoxyuridine (BrdU) antibody. The BrdU was instilled intraperitoneally and the animals were painlessly sacrificed between 1 hour and 10 days after the injection. An immunostaining technique using anti-BrdU antibodies was applied on whole mount TM tissues. One hour after injection, BrdU-labeled cells were found in the handle of the malleus (HM) region and in the annular region of the pars tensa of the TM. Some labeled cells were observed in the intermediate region of the upper half of the superior quadrant, but no labeled cells were found in the remaining part of the intermediate region. Labeled cells were also evident in the pars flaccida without any particular pattern of distribution. As time elapsed after the injection, the labeled cells first appearing in the HM region had migrated laterally and inferiorly from the HM toward the annulus, while those in the annular region had considerably decreased in number. Results of the present study are the following: 1) the proliferation center of epidermal cells in the pars tensa is located in two different areas, i.e., the HM region and annular region, 2) newly generated cells in the HM region migrated from the HM region toward the annular region, whereas those in the annular region migrate from the the annular region to the external auditory canal, and 3) no specific generation center is located in the pars flaccida. On the basis of these results, we discuss the relationship between the site of the proliferation center of epidermal cells and their migratory patterns.

Elemental composition of the developing inner ear.

The elemental composition of the inner ear fluid-filled compartments has been analyzed using the x-ray energy dispersive technique (CBA mouse). Special attention has been focused on the maturation of endolymph. A few days before and after birth the relative peak intensity of potassium (RK) in the vestibular endolymphatic compartment was slightly surpassing that in the cochlear part of the labyrinth. From the fourth day after birth (DAB) a rapid increase occurred concerning the RK. The highest RK was found in the endolymphatic space in the basal part of the cochlea followed by that in the vestibular endolymph. The lowest RK was measured in endolymph of the apical part of the cochlea. These obvious differences were abandoned already on the sixth DAB. A mature composition of endolymph was reached on the eighth DAB. The present technique does not allow analyses of differences between cochlear and vestibular endolymph with regard to minimal shifts.