A ferritin-containing cell type in the stria vascularis of the mouse inner ear.

This report describes a new cell type within the stria vascularis of the mouse inner ear. The cell is similar ultrastructurally to the classically described intermediate cell. However, it can be distinguished by the presence of dense vacuoles, presumably lysosomes, within which can be visualized electron dense particles resembling ferritin molecules. In addition, the ferritin-like particles are present throughout the cytoplasm and occasionally within the endoplasmic reticulum. These cells characteristically abut capillary basal lamina. Electron probe analysis of the dense vacuoles revealed the presence of iron. It is suggested that these cells may sequester iron released from dying erythrocytes in the strial capillary system, whereupon the iron is conserved through ferritin synthesis.

DNA content, mitotic activity, and incorporation of tritiated thymidine in the developing inner ear of the rat.

The rat inner ear is ectodermally derived from a region adjacent to the developing hindbrain. Beginning on day 8 of a 22-day gestational period, This zone of ectoderm first forms the otic placode, then the otocyst, and ultimately the definitive membranous labyrinth. This report provides an estimation of total DNA content of the developing inner ear, and hence an estimation of the total number of cells that comprise the inner ear at each developmental stage. The incorporation of 3H-thymidine indicates that most cells of the inner ear undergo DNA synthetic activity during gestational days 13 to 15. Radioautographic observations indicate a zone of DNA synthetic activity at the base of the outpocketing cochlear duct during early development. At the later stages of development, DNA synthesis is restricted to the cristae ampullares of the semicircular canals and the maculae of the utricle and the saccule. In contradistinction to the findings of other investigators, the statoacoustic ganglion complex undergoes terminal mitosis during gestational days 17 and 18. The gestational period between days 13 and 15 may prove to be a critical stage in normal otic development. The normal values of total DNA content and the number of cells that comprise the inner ear during development, established by these methods, can be compared with pathologic inner ears to provide quantitative means of assessing the damage in malformed inner ears. These values also form the baseline for future experimental studies of inner ear development.

Effects of fluorodeoxyuridine on the developing inner ear of the rat.

The inner ear in rats develops from the surface ectoderm on day 8 of a 22-day gestational period. Labeled thymidine incorporation studies have indicated that in the developing inner ear most of the cells undergo terminal mitosis between gestational days 13 and 15. During this period the developing inner ear would be particularly vulnerable to environmental hazards. To test this hypothesis, pregnant rats were given a single intraperitoneal injection of 5-fluoro-2'-deoxyuridine (FUdR), an antimitotic substance, on gestational days 12 to 16. The rats also received one injection of 3H-thymidine 1 h prior to the removal of the fetuses. The animals were killed after various time intervals following the treatment, and the otocysts or inner ears were prepared for morphologic observations and biochemical assays. The cells in the inner ear of rats exposed to FUdR exhibited pyknotic nuclei and chromatolytic degeneration, and they eventually died. By 4 h after the administration of FUdR, pyknotic nuclei were seen in the antiluminal zone of the otic epithelium, and there was a substantial decrease in the number of the otic cells. This decline in cell number was seen until 24 h after treatment. However, the inner ears from the fetuses exposed to FUdR during gestational days 12--15 showed complete recovery from the toxic effects of the drug when examined on day 21 of gestation. The phenomenon of programmed cell death observed in the developing inner ear of the rat indicates that more cells are produced during the earlier stages of development than are required for the definitive adult structures. This phenomenon may represent an important protective feature. The redundant production of cells perhaps allows the developing otocysts to respond to an environmental stress by subtotal destruction of cells from the pool of undifferentiated cells, resulting in relatively fewer congenital anomalies of the inner ear.

Apparent immunologic nonidentity of human placental and endometrial 17beta-estradiol dehydrogenase.

Immunohistochemical techniques were used to search for the presence of 17beta-estradiol dehydrogenase activity in human endometrial and placental tissues, with the use of antibodies raised against highly purified human placental 17beta-estradiol dehydrogenase. Sensitivity and specificity of the antibodies were documented by radioimmunoassay and immunodiffusion on cellulose acetate. Although staining was consistently demonstrated in the syncytiotrophoblast layer of term placentas, in both cytoplasm and nuclei, no immunohistochemical reaction was observed in endometrial samples. These results support the contention that placental 17beta-estradiol dehydrogenase is immunologically dissimilar from the endometrial enzyme.

In vitro hippocampal uptake of tritiated serotonin (3H-5HT): A morphological, biochemical, and pharmacological approach to specificity.

The in vitro uptake of tritiated serotonin (3H-5HT) into hippocampal slices was measured in Ringer's solution (37 degrees C) containing pargyline, ascorbic acid, and dextrose. The specific uptake of 3H-5HT rose asymptotically as the 3H-5HT molarity was increased from 5 x 10(-10) to 1.5 x 10(-6) M. Linear regression analysis gave a Km value for the specific uptake of 1.4 x 10(-7) M. The nonspecific binding (NSB) was the amount of 3H-5HT retained by the slices following incubation in a medium with a very large excess of unlabeled 5-HT added to dilute the specific uptake of 3H-5HT. This NSB increased with increasing molarity of 3H-5HT, and was linearly related to 3H-5HT concentrations between 5 x 10(-9) and 1.5 x 10(-6) M. The ratio of specific uptake to NSB was highest at 5 x 10(-8) M (2.75) and lowest at 1.5 x 10(-6) M of 3H-5HT (0.54). Competition studies with noradrenaline, desipramine (a noradrenergic uptake blocker), fluoxetine (a 5-HT uptake blocker), and tryptophan confirmed the specificity of the 3H-5HT uptake mechanism. Radioautographic studies of in vitro incubated hippocampal slices showed silver grain aggregates at 3H-5HT specific uptake sites. Addition of an excess of unlabeled 5-HT to the slices, or the use of hippocampi from 5,7-dihydroxytryptamine intracerebral microinjected rats (5 microgram/400 nl into the fornix-fimbria and the cingulum bundle, 6 day survival) caused a dramatic decrease in these aggregates. The distribution of hippocampal 5-HT axons and terminals, inferred from the pattern of silver grain aggregates, is more widespread than previously described. 5-HT varicosities were clearly seen in all layers of Ammon's horn, dentate gyrus, and the subicular cortex. Innervation routes were seen to the stratum radiatum and stratum lacunosum from stratum oriens in Ammon's horn, and to the polymorphic layer of the dentate gyrus from the subicular cortex and from the fimbria. Semiquantitation of the occurrence of silver grain aggregates was done in the various hippocampal regions. The highest density in Ammon's horn was 119.5 boutons/10,000 micron2, in the dentate gyrus it was 67.4 boutons/10,000 micron2, and in the subicular cortex it was 79.2 boutons/10,000 micron2. These results are consistent with previous quantitative results.

An evaluation of normal strial capillary transport using the electron-opaque tracers ferritin and iron dextran.

Enzymatic tracer techniques to study normal and pathologic strial capillary transport pose various problems. The use of electron opaque tracers can circumvent many of these problems. Iron dextran (mol. diam 20--70 A) and ferritin (mol. diam 110 A) were injected intravenously and the mice sacrificed at intervals of 1/2, 1, 2, 5, and 24 h. The iron dextran results were unusual in that from 1/2 to 5 h after administration the tracer was present within the cytoplasmic matrix of endothelia, but by 24 h it had been cleared out. No transendothelial exchange was noted. The ferritin results were in conflict and previous results using horse-radish peroxidase. Transport of ferritin was minimal regardless of time sacrificed. No more than a few molecules were scattered about the capillary basal limina. Those molecules transported across capillaries were apparently delivered by means of the micropinocytotic system. The results suggest a blood-strial barrier similar to the blood-thymic and blood-myenteric barriers. Experimental as well as control animals exhibited strial light cells which contained ferritin-like particles within their cytoplasmic matrices. These light cells are probably reticuloendothelial type cells. Ferritin may be useful to gauge strial capillary transport alterations associated with auditory pathologies.

Intercellular junctions in the differentiating rat otocyst.

Intercellular junctions between cells of the rat otocyst on the 12th day of gestation were studied using lanthanum tracer and freeze-fracture techniques. At the luminal surface, the intercellular space is closed by a series of tight junctions. Gap junctions are also present between cells both within and below the luminal junctional complex. The presence of tight and gap junctions at this early stage in the differentiating otocyst is probably essential for the development of a normally functioning adult ear.

Transformation of DBA/2 mouse fetal liver cells infected in vitro by the anemic strain of Friend leukemia virus.

Fetal liver cells of DBA/2 mice were infected with the anemic strain of Friend leukemia virus (FLV-A), which has no spleen focus-forming virus (SFFV) activity. The infected cells were grown in medium with or without erythropoietin. Transformed lines were isolated only from the infected cultures that had been treated with erythropoietin at the time of their initiation. The properties of three permanent cell lines in serial passage for over 2 years are described. Each has an aneuploid karyotype. Only the immature hematopoietic cells of the first line have metacentric chromosomes. They grow in suspension, as do the erythroleukemic lines derived from leukemic spleens of FLV-infected mice, and clone on agar. They produce tumors resembling reticulum cell sarcomas upon subcutaneous inoculation into syngeneic hosts. Stimulation of differentiation induced after treatment with dimethyl sulfoxide identifies the cells of the first line as being erythroid in origin. The two other lines are adherent and epithelioid in appearance. These lines may have originated from the nonhematopoietic cells present in fetal liver. No tumors were produced after the subcutaneous inoculation of 10(6) cells. All three lines synthesize virus. The virus is attenuated for leukemogenicity and has no SFFV activity. The transforming event appears to be specific, because fetal liver cells from C57BL/6 mice, which are resistant to the induction of leukemia by FLV, were not affected by the virus. Malignant transformation of erythroid cells by FLV-A in vitro confirms the in vivo findings that SFFV may not be a necessary prerequisite for the induction of erythroleukemia in susceptible hosts.

Detection of H2-b and Thy-1.2 surface antigens on the differentiating murine otocyst using Nomarski optics.

The 12th postcoital day otocyst appears as a hollow cellular ball of pseudostratified columnar epithelium that has entered into its inital stages of differentiation and organogenesis. H-2b antigen was demonstrated on the ectodermally derived epithelial cells of the otocysts and on the mesodermally derived cells of the surrounding mesenchyme. Thy-1.2 antigen was detected in the mesenchymal cells, but not on the epithelial cells of the otocyst. The use of Nomarski optics as a new method for detecting cell surface staining that would otherwise be undetected by bright field optics was demonstrated.

Observations on cell lines derived from a patient with Hodgkin's disease.

Permanent cell lines have been established from a spleen nodule and lymph node of a male Hodgkin's disease (HD) patient whose father has the same disease. Th in vitro growth pattern morphological and cytogenetic characteristics of these lines maintained continuously for over 2 years are described. The cultures contain a population of mixed cell types that grow in suspension. Between 5 and 10% of the cells have surface immunoglobulins M and D. B-cell alloantigens are also detectable. While the cultures are predominantly lymphoid, some of the large cells, by light and electron microscopy, resemble the Reed-Sternberg and Hodgkin's cells of the original biopsies. Although the cells maintain the human diploid karyotype, they are heterotransplantable in nude mice. After 14 months of culture, chromosome rearrangement and losses, commonly seen in leukemic bone marrow, occurred. Close to 100% of the cells are Epstein-Barr nuclear antigen positive, but they lack Epstein-Barr viral (EBV) capsid antigen and EBV-induced early antigen. Nucleic acid hybridization tests indicated that there were no more than two EBV genome equivalents per cell. Tests with HD sera free of anti-EBV were negative. Electron microscope examination of the cells revealed the presence of intracellular as well as extracellular rare pleomorphic particles ranging from 400 to 1200 A. The nature of these particles, which increased in number after the cultures were treated with halogenated pyrimidines but not with dimethyl sulfoxide, remains questionable. The cultures derived from the mouse-passaged HD cells, however, had reverse transcriptase activity and readily identifiable type C particles which were probably of murine origin. These cultures have some unique features that make them useful in studying the perplexing pathological entity of HD.

The evaluation of middle ear meningiomas using computerized axial tomography.

Meningiomas localized within the middle ear and mastoid which have no intracranial component are rare tumors. Three patients with meningiomas which were thought to involve only the middle ear were treated surgically, were followed for two to six years and were thought to have been cured. However, when computerized axial tomography became clinically available, these patients were examined using this radiographic technique. Unexpectedly, all three patients were found to have a large intracranial meningiomatous extension. A review of the literature reveals only 13 primary meningiomas; these case reports are summarized. It was noted that definitive tests for an intracranial extension of the meningioma were performed in only 2 of the 13 cases. Therefore, in view of our recent clinical experience, it is recommended that a patient presenting with a middle ear meningioma should have a computerized axial tomographic examination of the head to detect the possible presence of a clinical silent intracranial involvement.

Tympanic neurectomy and chorda tympanectomy for the control of drooling.

Seventeen patients suffered from drooling that either occurred as a sequelae of extensive head and neck cancer resections or was due to neurological disorders. In these patients, a tympanic neurectomy and/or chorda tympanectomy was performed in an attempt to eliminate the drooling. The conditions in five of 12 (41%) patients with head and neck cancer were improved following such surgery. Two of four children with cerebral palsy initially had a good result. However, the long-term follow-up of the patients demonstrated that the drooling recurred. An additional patient suffering from bulbar weakness and drooling owing to a cerevrobascular accident had less problems with salivary secretions. The results were relatively disappointing; there are several possible explanations for this.

Evaluation of tympanic neurectomy and chorda tympanectomy surgery.

Thirty patients were treated by tympanic neurectomy, chorda tympanectomy, or both for a variety of conditions. Out of six patients with gustatory sweating treated by tympanic neurectomy, two patients were relieved of symptoms, two were improved, and two remained unchanged. In five cases of benign recurrent painful parotid swelling, only two patients noted improvement in symptoms. Seventeen patients suffered from drooling. Out of 12 postresection head and neck patients, 5 (41%) were improved following such surgery. Two of four cerebral palsy children initially had a good result. However, the long term follow-up of the patients demonstrated that the drooling recurred. An additional patient who suffered from drooling caused by bulbar weakness following a cerebrovascular accident had fewer problems with salivary secretions postoperatively. The pertinent anatomy and pathophysiology is outlined. The possible reasons for the relatively disappointing results achieved are discussed.

Comparative histological study of mini pig skin after chemical peel and dermabrasion.

Although individual studies of the histological changes after chemical peel and dermabrasion have been reported, there has not been a comparative study of these modalities in the same animal mode. Using the mini pig, whose skin most closely resembles human skin, a study was performed of histological changes 24 hours to 16 weeks after dermabrasion and chemical peel. A considerable increase in thickness of the new collagen layer was noted after chemical peel in contrast to that seen in skin after dermabrasion. The skin surface after chemical peel appeared smoother after 16 weeks, suggesting that chemical peel may be a more effective modality for treatment of the fine wrinkles of aging skin.

Ultrastructural development of the early rat otocyst.

The ultrastructural development and differentiation of cells forming the rat otocyst were studied from the 9th to the 13th postcoital day (PCD). The earliest stage investigated was a simple ovoid structure with a connecting stalk to the surface ectoderm. A process of programmed cellular death involving surface ectoderm, connecting stalk, and lateral otocyst wall rapidly detached the otocyst. The cells forming the otocyst were roughly columnar, the organelles were polarized; mitochondria occurred in greatest number basally and in the supranuclear area; Golgi membranes when present were supranuclear. Luminal cells had many microvilli and cilia of various lengths were detected. The shorter, incompletely formed cilia terminated in small knob-like blebs. With each day the otocysts became more complicated and the endolymphatic duct made its appearance as an evagination of the otocyst. Many more cells were seen to have cilia in various stages of development, and by the 12th PCD possibly each cell of the main otocystic cavity had a kinocilium. Growth of the otocyst due to mitosis occurred to a great extent from a single ventromedial center. Cells in mitosis, although seen at other sites, were in greatest abundance in this area; cellular involution apparently was a related function. Together the process of over-production and programmed cellular involution of supranumerary cells not lost to other causes (e.g., environmental) may represent an evolutionary advantage.

Scanning electron microscopic appearance of rat otocyst of the twelfth postcoital day: elaboration of a method.

A method for removal, fixation, microdissection, and drying of early rat otocyst for examination by the scanning electron microscope is elaborated. Tissues were dissected, fixed as for conventional transmission electron microscopy and dried by critical point evaporation using amylacetate as the transitional fluid and carbon dioxide as the pressure head. Otocysts were either dissected at the time of initial fixation, or subsequent to drying. The otocyst of the 12th postcoital day was used as a model system in this preliminary report. Critical point drying retained the overall configuration and the fine ultrastructural detail of the otocyst. The interior otocystic surface was visualized and cilia bearing cells of the luminal surface were identified. Most if not all of these cells had a comspicuous, but short kinocillum which terminated in an ovoid bulb. The scanning electron microscopic appearance was correlated to the transmission electron microscopic image seen in the second paper in this Supplement.

An improved method for processing early rat otocysts for the electron microscope.

Various methods of dissection, fixation, osmication, sectioning and staining were tested in order to develop an acceptable technique for preparing 9, 10, 11, and 12-day-old rat otocysts for electron microscopic study. The general problems associated with embryonic tissue-difficult handling, high water content and poor stainability are discussed, and concrete methods of preparation which significantly decrease these difficulties are proposed. The specific fixation and sectioning requirements of rat otocyst are also described in the elaboration of a method which will be used in subsequent studies of the organogenesis of rodent ear.