Biomedical subjects
Publications and source records attributed to M Anniko.
The 2 day post partum mouse Crista ampullaris was cultured "in vitro" for 7 days. The differentiation into secretory and sensory areas was preserved. The ultrastructure of the dark cells of the secretory region was well maintained. The hair cell morphologically was considerably changed though the hair cells themselves survived 7 days in culture.
The histopathological features of the inner ears in a case of deafness in an old English sheepdog reveal a cochleo-saccular (Scheibe) type of degeneration. However, also the hair cells in the vestibular part of the labyrinth were reduced in number though there appeared no clinical signs or symptoms of vestibular dysfunction. The present case is the so far only known old English sheepdog with a hearing loss reported in Sweden.
To investigate which fixative is most suitable for the preservation of cochlear hair cells three main groups of fixatives were used: 1. 3% glutar aldehyde in various buffers (osmolality altered). 2. Glutar aldehyde/paraform aldehyde combination (ad modum Karnovsky). 3. Osmic acid solutions (OsO4) in veronal acetate buffer according to Rhodin (1954). The 2% osmic acid fixative (350--370mosm/kg) resulted in an excellent ultrastructural preservation; an acceptable result was obtained with a 3% glutar aldehyde fixative in 0.1 M sodium phosphate buffer, 600--700 mosm/kg and the Karnovsky's fixative.
Post-mortem change (autolysis) of the sensory and the secretory epithelia was studied by means of the light and the electron microscope. The first ultrastructurally recognizable sign of autolysis occurred 10-15 min after death in the mitochondria of the hair cells and their adjacent nerve endings. The morphology of hair cells was occasionally well preserved 30 min post-mortem, but after 90 min or more autolytic changes were invariably severe. The secretory epithelium, however, was considerably less affected by autolysis than the hair cells during the first 5 h after death.
Whether an active accumulation of ototoxic substances or their metabolites occurs in the cochlea, or whether there exists a delayed elimination of these products from the endo-and perilymphatic fluids, following a passive equilibrium between the blood and the body tissues, is still a subject for discussion. To investigate the cochlear distribution of an ototoxic substance and its elimination rate, compared to other organs in the body, atoxyl, with known ototoxic effects (Anniko and Wersäll, 1975a, b; Anniko, 1976a, b) was used as a test substance. The neutron activation analysis technique was used to measure the atoxyl concentration in various parts of the body (cochlea, blood, muscle, kidney and cortical femoral bone) after various lengths of time following a subcutaneous injection of 100 mg/kg b.w. of atoxyl. Atoxyl was retained in the cochlea for a long period and a delayed elimination occurred from the inner ear. The excretion of atoxyl was likely to appear via the kidneys.
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.
The arsenical compound atoxyl has been used in labyrinthine research because of its documented effects both on cells engaged in an active transport of ions (secretory epithelia) and sensory epithelia (cochlear hair cells and vestibular sensory cells type I and type II.) In the present experiments both the kidney and the structures of the inner ear were studied. The renal tubules were damaged by exposure to atoxyl. Tubular casts, haemorrhages (both in the tubules and in the renal calices) and tubular epithelial cell necrosis occurred. A common denominator in the ototoxic and the nephrotoxic actions of acute atoxyl poisoning might be a disturbance of the very active ion transport systems of the cells involved.
Atoxyl-induced damage to the inner and the outer hair cells in the guinea pig cochlea starts in the apical coil and spreads successively downward toward the round window. Vesicular degeneration of both the inner and the outer hair cells and the cell organelles occurs. The degeneration is probably caused by an alteration in ionic composition of the endolymph resulting from primary changes of the stria vascularis (vesiculation and rejection of degenerated marginal and intermediate cells) as reported by Anniko and Wersäll (1975). However, a direct action of atoxyl on the sensory cells of the organ of Corti cannot be excluded.
In a retrospective study of 58 patients from all over Sweden, treated for small squamous cell carcinoma of the mobile tongue (T1N0M0), different prognostic factors were evaluated. A partial glossectomy was performed as primary treatment in all patients. The cancer recurred in 28 (38%) of 58 patients. One of 7 patients with only local recurrence, and 9 of 14 with recurrence in the neck died of their disease. The relative survival rate was 77%. Of the prognostic factors tested, tumor thickness was found to be the most significant. In patients with tumor thickness was found to be the most significant. In patients with tumor thickness exceeding 10 mm, recurrences occurred in 58% (P less than 0.0048), and 87% (P less than 0.0164) died of their cancer. This represents a statistically significant higher degree when compared with tumor thickness of less than 10 mm. Elective treatment of the neck nodes is recommended in epidermoid cancer of the mobile tongue when tumor thickness exceeds 10 mm, even in stage I tumors (i.e., the surface extension diameter is smaller than 20 mm at most).
The distribution of alpha-actinin, vinculin, alpha-spectrin, beta-spectrin and fibronectin was analyzed in 14- to 21-week-old fetal human inner ears using immunofluorescence microscopy. Staining for alpha-actinin was fairly evenly distributed at the epithelial surfaces of all five vestibular organs, whereas in the cochlea it was mainly at the surface of the receding greater epithelial ridge and in some foci apically at the lesser epithelial ridge. Fluorescence for vinculin was observed mainly at the surface of vestibular organs, but was lacking in the LER. Intense fluorescence for alpha-spectrin was found at the apical surface of individual cells of the cristae and maculae. Antibodies against beta-spectrin mainly stained the endothelial cells of blood vessels, but faint staining of the epithelial cell surfaces of the vestibular organs was also detected. The fluorescence pattern of the actin-associated proteins is indicative of structural differences between cochlear and vestibular hair cells. Fibronectin was identified only between mesenchymal cells and its functional importance in the mature inner ear epithelia can be discounted.
The ototoxic potentials of netilmicin and gentamicin were compared in three models: the standard in-vivo animal model (guinea pig), organ culture of the embryonic inner ear (mouse), and perilymphatic perfusions (guinea pig). Toxicity was assessed by light and electron microscopy of hair cells or by measurement of cochlear microphonic potentials. In vivo, netilmicin did not have any significant morphologic effect on hair cells under conditions (dose X duration) where gentamicin caused excessive hair cell loss. In organ culture, concentrations of netilmicin had to be at least tenfold higher than those of gentamicin in order to produce comparable damage. In direct applications of the drugs to the cochlea by perilymphatic perfusion, similar effects on cochlear microphonics were seen when netilmicin concentrations were more than threefold higher than those of gentamicin. It is suggested that netilmicin ototoxicity observed in vivo is low because of lesser penetration into the inner ear and lower intrinsic toxicity.
The development of the cupula cristae ampullaris in the mouse starts on the 14th gestational day and occurs mainly during the 15th and 16th gestational days. Subsequently the secretory activity of the epithelial cells diminishes. The cupula is formed from the underlying epithelium when there are only a small number of differentiated hair cells present. Secretion of cytoplasmic material occurs mainly from supporting cells. The surface of the epithelium at a location corresponding to the planum semilunatum in the adult organ has a thick layer of microvilli and demonstrates a particularly high secretory activity on the 15th gestational day. After the 16th gestational day there are few morphologic signs of secretion. The cupula also develops in inner ear anlages that have been explanted at the otocyst stage on the 12th or 13th gestational day and cultured for eight days in vitro.
The development and maturation of tight junctions in secretory epithelia of the inner ear, i.e., the stria vascularis in the cochlea and the dark cell epithelium around the vestibular organs, have been analyzed with the freeze-fracture technique. Inner ears were followed from the otocyst stage to the mature stage (mouse; gestational age 20 to 21 days). Before epithelial cells differentiate into specific tissues, tight junctions have a loose network of sealing elements with zero to four sealing strands. During and after cytodifferentiation, the numbers of strands increase both in the marginal cells of the stria vascularis and in the dark cells around the vestibular organs. Large intra- and intercellular variations in the numbers of sealing strands of the tight junction occur prior to birth in both types of secretory epithelia. In the dark cells the mature structure of the tight junction with five to eight sealing elements is reached at birth. Maturation of marginal cell junctions occurs during the first few days postnatally. Thus, the tight junctions are morphologically mature before the development of the high potassium concentration in the endolymph.
The ultrastructure of aging vestibular hair cells of the guinea pig can be abnormal even though they appear morphologically normal by light microscopy. In only a few of the hair cells studied did nonspecific changes occur. In general, the age-related changes involved specific structures in the hair cells: aggregations of lipofuscin pigments, multivesiculated bodies, disintegration of the cuticular plate, and rod-shaped inclusions from the cuticle area into the hair cell. Furthermore, sensory hairs are generally present when the cuticular plate shows an advanced disintegration. Nerve calyces can be ultrastructurally changed without morphologic changes in the adjacent hair cell. Type I hair cells show age-related morphologic changes more often than do type II hair cells.
Cytodifferentiation of a limited number of cochlear hair cells in the mouse starts on the 14th gestational day. One day later, the number of identified hair cells increases considerably. Cytodifferentiation apparently occurs in a gradient from the hair cell surface to the base. First, the irregular microvilli covering the future hair cell surfaces begin to show a regular pattern but are of the same thickness and length as microvilli on supporting cells. Second, a polarization of sensory hairs occurs with a stepwise increase in stereocilia length in the different rows toward the kinocilium. Finally, the cuticle is formed, giving an anchorage for sensory hair rootlets in the hair cell. At birth some hair cells can be found with immature surface morphologic features, e.g., stereocilia of the same length on the entire hair cell surface and lack of a cuticular plate. The onset of hair cell differentiation takes place without morphologic contact with ingrowing nerve fibers.
The early embryonic development of the inner ear anlage, from the otic placode stage to formation of the otocyst, was documented in CBA/CBA mice by scanning electron microscopy. The otic placode is identified at the eight- to ten-somite stage, as revealed by the surface morphology of the developing ectoderm. The cells in the otic depression are considerably smaller than those of the adjacent ectodermal surface. Rapid invagination of the otic anlage occurs during the ninth gestational day (approximately eight to 20 somites). In most specimens from the tenth gestational day, the otic vesicle has closed toward the ectodermal surface. The surface of the otocyst on the 13th gestational day shows considerable specialization, with regional differences of the surface structure.
Thirty-two CBA/CBA mice were irradiated in utero on the 12th, 13th, or 16th gestational day with doses of 0.5, 1, and 2 Gy, respectively (1 Gy = 100 rads). One month after birth, the inner ears were examined by light microscopy and transmission and scanning electron microscopy. Otoconia with defective shapes was identified frequently. The strict hexagonal shape of normal otoconia seldom developed and, in exposed animals, had often been replaced with rounded, oval, or elongated shapes. The otoconial substructure was disarrayed, and fusion of two or three otoconia occurred. Degenerating otoconia appeared in the intercellular space of the dark-cell epithelium. Fetal gross structures of otoconia persisted into maturity.
The tumor tissue from 62 consecutive patients with pituitary adenomas was analyzed with regard to ploidy and percentage of cells in different phases of the cell cycle by use of flow-cytofluorometry. In six of 62 cases, two tumor-cell populations were identified; therefore the study material comprised 68 cell lines. Approximately half of the cell lines were diploid (33 of 68, or 49 per cent); five of 68 (7 per cent) were hypodiploid, and 30 of 68 (44 per cent) were hyperdiploid-aneuploid. A low occurrence of aneuploid cell lines appeared in hormonally nonsecreting tumors (22 per cent). An aneuploid DNA pattern was predominantly found in prolactinomas (70 per cent). In acromegaly, tumors secreting growth hormone had only an aneuploid DNA pattern in 41 per cent of the cases, whereas 67 per cent of the tumors with concomitant secretion of growth hormone and prolactin were aneuploid. The latter group also comprised the largest number of adenomas with two cell lines and all but one hypodiploid tumor. Most tumors with an aggressive clinical course were either aneuploid or diploid but with a high percentage of proliferating cells.