Concentration of nitric oxide metabolites in middle ear effusion.

Free radicals such as nitric oxide (NO) seem to be important in the pathogenesis of otitis media with effusion (OME). NO can be quantitated by measuring its metabolites, nitrate (NO(3)(-)) and nitrite (NO(2)(-)). The purpose of this study is to determine the concentrations of NO in human middle ear effusion (MEE). Samples of human MEE were collected at the time of myringotomy and tympanostomy tube insertions. The type of MEE was classified as serous (SOM), mucoid (MOM) or purulent (POM) at the time of surgery. Samples of MEE were assayed for NO metabolites (nitrate and nitrite) with colorimetric assay (Griess method). Concentrations of NO metabolites were highest in MOM followed by SOM and POM. This study suggests that NO is present in human MEE and may play an important role in the pathogenesis of OME.

Effects of common topical otic preparations on the morphology of isolated cochlear outer hair cells.

Otic drops are commonly used not only for otitis externa but also for otorrhea in the presence of tympanostomy tube or tympanic membrane perforation. Many studies have demonstrated the ototoxicity of common otic preparations such as Cortisporin otic drops. Recent studies have suggested the use of fluoroquinolone antibiotic drops as an alternative owing to their excellent antimicrobial coverage and no ototoxic effect. The purpose of this study was to assess the relative ototoxicity of four common otic preparations by direct exposure to isolated cochlear outer hair cells (OHCs). OHCs from adult chinchilla cochlea were exposed to standard bathing solution (control), Cortisporin, Cipro HC, Ciloxan, and Floxin. The cells were observed using an inverted microscope, and the images recorded in digital still-frame and video, and analyzed on the Image Pro-Plus 3.0 program. As measured by time to cell death and change in morphology of OHCs, Cortisporin was most toxic to OHCs. Among the fluoroquinolone drops, Floxin was more toxic than Ciloxan or Cipro HC.

Determination of ototoxicity of common otic drops using isolated cochlear outer hair cells.

OBJECTIVES: Otic drops are commonly used not only for otitis externa, but also for otorrhea in the presence of tympanostomy tubes or tympanic membrane perforations. Many studies have demonstrated the ototoxicity of common otic preparations such as Cortisporin otic drops (Monarch Pharmaceuticals, Bristol, TN). The purpose of this study was to assess the relative ototoxicity of common otic preparations by direct exposure to isolated cochlear outer hair cells (OHCs). METHODS: OHCs from adult chinchilla cochlea were exposed to standard bathing solution (control), acetic acid, Acetasol HC (Alpharma USPD Inc., Baltimore, MD), Gentacidin (CIBA Vision Ophthalmics, Atlanta, GA), and Tobradex (Alcon, Fort Worth, TX). The cells were observed using an inverted microscope, and the images were recorded in digital still-frame and video, and analyzed on the Image Pro-Plus 3.0 program (Media Cybernetics, Silver Spring, MD). RESULTS AND CONCLUSIONS: As measured by time to cell death and change in morphology of OHCs, acetic acid with or without hydrocortisone was most toxic to OHCs. Cortisporin was more cytotoxic than gentamicin and Tobradex.

Feasibility of acoustic distortion product testing in newborns.

Acoustic distortion products (ADP) have been used as a neonatal screen for newborn, full-term infants resident on maternity wards. ADPs were measured across frequency with f2 (the higher frequency tone) between 1 and 8 kHz and the f1 (the low frequency tone) determined by f2/1.225. Testing was attempted on 77 infants. Of these, 13% were untestable, 69% were tested in both ears and 18% had one ear tested; 48% gave data from both ears and 25% gave data from one ear. If results from both ears were required to pass the test, 52% would fail. Of the 67 babies that could be tested, 27 out of 120 ears gave no result: a failure rate of 22.5%. It was often not possible to record ADP across all the frequencies tested, but only four babies out of the 31 who were tested at each frequency bilaterally showed no ADP at any frequency. Older babies (4-6 days old) gave distortion at more frequencies than younger babies (0-3 days old). ADP were most easily recorded with f2 at 3 and 4 kHz. Lower incidence of ADP at frequencies below 3 kHz could be explained by greater levels of background noise. Left or right ears and method of delivery did not affect the ability to record ADP, but, unexpectedly, boys showed a higher incidence of ADP (though not higher level) than girls. Behavioural testing of the infants was carried out at 7 months of age without knowledge of the previous ADP test results. It yielded three referrals (out of 52 who could be followed up), one of whom was re-tested and passed. The two remaining infants were among the four who gave no distortion bilaterally, indicating accurate prediction by the ADP measure. It was concluded that ADP measurement alone at birth would lead to an unnecessarily large number of referrals for further investigation. This high failure rate may be reduced by measurement methods designed to avoid or cancel infant-generated noise, and by sound-treated facilities, but the lack of co-operation by the infant coupled with limited test time poses a more serious problem.

Covalent linkage between proteins of the inter-alpha-inhibitor family and hyaluronic acid is mediated by a factor produced by granulosa cells.

The direct interaction of hyaluronic acid (HA) and proteins of the inter-alpha-inhibitor family plays a critical role in organization and stabilization of the expanding cumulus extracellular matrix (cECM) following an ovulatory stimulus. Despite similarities in the morphology of cumulus oocyte complexes (COCs) expanding in vivo and in vitro, we find that the cECM of COCs which expand within intact follicles are more elastic and resistant to shear stress than the cECM of those stabilized in vitro. Western blot analysis shows that only the heavy chains of inter-alpha-inhibitor are incorporated into the cECM and appears to be covalently linked to HA after stabilization in vivo while intact inter-alpha-inhibitor is bound to the HA-enriched cECM by a non-covalent mechanism in in vitro stabilized COCs. However, purified pre-alpha-inhibitor and HA can form covalent linkage in the presence of granulosa cells or with granulosa cell-conditioned medium. In addition, COCs resistance to shear stress is also enhanced by coincubation with granulosa cells. Upon formation of the apparent covalent linkage between heavy chains and HA in culture medium, the light chain (bikunin) is concomitantly released into the medium as a complex with chondroitin sulfate moieties of inter-alpha-inhibitor supporting the possibility that HA may replace the chondroitin sulfate linkage to the heavy chains. We speculate that a factor(s) secreted by granulosa cells within the follicle may catalyze a transesterification reaction resulting in an exchange of chondroitin sulfate with HA at the heavy chain/chondroitin sulfate junction followed by release of chondroitin sulfate-bikunin into the follicular fluid. It is also possible that the consequent further stabilization of the cECM through the covalent interaction of HA and heavy chains of inter-alpha-inhibitor may play an important role in the process of ovulation.

Gonadotropin-stimulated regulation of blood-follicle barrier is mediated by nitric oxide.

Although initially described over 30 years ago, the blood-follicle barrier has remained a biological enigma. In this study, we characterize the blood-follicle barrier with respect to its regulation of intrafollicular inter-alpha-trypsin inhibitor protein (I alpha I) influx after an ovulatory stimulus. We have found that I alpha I is localized within the ovarian vasculature but is excluded from the follicular compartment until an ovulatory stimulus is given. Within minutes after an ovulatory dose of human chorionic gonadotropin, I alpha I is localized within the follicular fluid of responding follicles where this protein becomes associated with and stabilizes the newly synthesized hyaluronic acid-rich cumulus extracellular matrix. Analysis of this process has shown that intravenous injection of sodium nitroprusside or excess substrate for nitric oxide synthase, L-arginine, mimics the effect of gonadotropic hormones on the influx of I alpha I into the follicular compartment of preovulatory follicles. Moreover, intravenous injection of specific nitric oxide synthase inhibitors, NG-nitro-L-arginine methyl ester and NG-nitro-L-arginine, inhibits gonadotropin-mediated intrafollicular influx of I alpha I and also inhibits ovulation in the mouse.

Steroid hormone regulation of rat myometrial gap junction formation: effects on cx43 levels and trafficking.

The formation of myometrial gap junctions coincides with onset of labor in many mammalian species, including humans. The assembly of gap junction protein into functional gap junction plaques is a final step in a cascade that begins with estrogen-dependent expression of the connexin43 (cx43) gene and continues with synthesis of cx43 in the rough endoplasmic reticulum (RER) and transport to the Golgi, followed by its trafficking to the plasma membrane and its assembly into functional gap junctions. Moreover, in several models of preterm labor in rats, precocious synthesis, trafficking, and assembly of cx43 follow an increase in the estrogen:progesterone ratio. The actions of these steroids on cx43 expression, gap junction formation, and labor led us to consider whether or not the cascade of cx43 expression and gap junction assembly typical of preterm and term labor would be disrupted by manipulations that inhibit labor through experimental reduction of the estrogen:progesterone ratio. Ovariectomized and non-ovariectomized pregnant rats were treated with minimal doses of progesterone or the anti-estrogenic compound ICI 182780 over a time course sufficient to inhibit labor. We found that cx43-positive gap junction formation was prevented in all animals treated with ICI 182780 or progesterone but that the mechanism by which this disruption occurred was different in anti-estrogen- and progesterone-treated animals. We found that ICI 182780 significantly inhibited the typical rise in myometrial cx43 concentrations normally observed just before labor. In contrast, it was surprising to find that significant cx43 was synthesized in myometrium of progesterone-treated intact and ovariectomized animals even though labor was inhibited. However, we found that the trafficking of myometrial cx43 from the Golgi and assembly into gap junctions at the plasma membrane were suppressed in these progesterone-treated animals, providing further support for the hypothesis that it is not synthesis of cx43 per se but trafficking of cx43 to the plasma membrane and its assembly into gap junctions that are required for effective synchronized myometrial contractions typical of labor.

Acoustic distortion products (ADP) from the ears of term infants and young adults using low stimulus levels.

It has been shown previously that relatively low stimulus levels (L1 = 55, L2 = 40 dB SPL) elicit recordable levels of ADP from healthy adult ears (Gaskill and Brown, 1990). We have used the same stimulus levels in healthy term infants within 6 days of birth. The results from the 38 infants who provided data from both ears (from a total of 66 attempted) are reported here. The data were compared with those from 12 adults aged 20 to 30 years with normal audiometric thresholds. ADP was measured across frequency with f2 (the high frequency tone) between 1 and 8 kHz (infants) and 0.5 kHz and 8 kHz (adults) and f1 (the low frequency tone) determined by f2/1.225. ADP was also measured at a range of different stimulus frequency separations. The mean level of ADP was higher in infant than in adult ears but this difference was not significant. The f2/f1 ratio of approximately 1.2 which has been widely adopted for use in adults is also an appropriate frequency separation for term infants.

Sequential effects of follicle-stimulating hormone and luteinizing hormone on mouse cumulus expansion in vitro.

LH and FSH play critical roles in the development and function of the cumulus oophorus of the mammalian follicle, but the significance of cumulus LH receptors and the nature of the functional interaction between LH and FSH in relation to the process of cumulus expansion are not well understood. Controversy may arise from contradictory results of cumulus LH receptor assays, possibly as a consequence of relative sensitivities of the assays utilized, limited availability of tissue of specific developmental stages, or limitations on the access of radiolabeled ligands to cumulus LH receptors in the intact follicle. It has also been difficult to understand the basis for the apparent difference in gonadotropin requirements for cumulus expansion in vitro and in vivo; FSH seems to be required for expansion in vitro and LH initiates expansion in vivo. In this study, we show that expansion of mouse cumulus-oocyte complexes (COCs) in vitro occurs to a degree comparable to that seen in vivo when COCs are challenged with highly purified FSH (hpFSH) and highly purified LH (hpLH) together or when treatment with hpFSH precedes treatment with LH. Saturation radioreceptor assays revealed that FSH up-regulates cumulus cell LH receptors in the absence of LH and that LH may stimulate expansion upon binding to these acutely formed receptors. A model of cumulus expansion describing effects of both FSH and LH in vivo is proposed.

Functional significance of cumulus expansion in the mouse: roles for the preovulatory synthesis of hyaluronic acid within the cumulus mass.

Gonadotropin-stimulated expansion of the mouse cumulus oocyte complex (COC) in vitro, measured with a quantitative videographic method, is comparable to that observed to occur in vivo when medium is supplemented with porcine follicle stimulating hormone (pFSH), 10% fetal bovine serum (FBS), and 2.5 mM glucosamine or optimal concentrations of glutamine and glucose. In the absence of glucosamine, the volumetric expansion of COCs in vitro is never more than 25% of that occurring in its presence. The addition of 6-diazo-5-oxo-1-norleucine (DON), an inhibitor of glucosamine synthesis to medium supplemented with glutamine and glucose, completely inhibits cumulus expansion in vitro. This system was utilized to examine the relationship between cumulus expansion and fertilization rates, and the maintenance of fertilizability in culture. Successful fertilization (as determined by development to the 2-cell stage) was correlated with the quantity and quality of the expanded cumulus mass, and conversely, the spontaneous loss or mechanical removal of the cumulus was correlated with a loss of fertilizability following additional incubation in culture medium. In addition, the i.p. injection of DON inhibited cumulus expansion within the intact follicle and suppressed ovulation.

Hyaluronic acid synthesis and gap junction endocytosis are necessary for normal expansion of the cumulus mass.

The application of a quantitative videographic technique has provided an opportunity to compare the quantitative volumetric expansion of cultured oocyte complexes (COCs) to quantitative changes in gap junction down-regulation and hyaluronic acid synthesis and to investigate the effects of physiological agents that influence these processes. Results of these experiments support the idea that the down-regulation of cumulus gap junctions is required for the initial phase of cumulus cell disaggregation and confirm earlier reports that hyaluronic acid synthesis plays a major role in additional expansion of the cumulus. These studies also provide evidence that the degree of expansion observed in culture lacking substrates of hyaluronic synthesis is significantly attentuated when compared with expansion occurring in vivo and that the failure of cultured complexes to expand maximally can be overcome by the addition of substrates of hyaluronic acid synthesis to the culture medium.

Arachidonic acid in uterine phospholipid during early pregnancy and following hormone treatment.

Evidence that prostaglandins are involved in intercellular communication during blastocyst implantation suggested that development and loss of uterine sensitivity to deciduogenic stimuli during early pregnancy might depend upon changes in uterine capacity to mobilize arachidonic acid from phospholipid. We measured levels of arachidonic acid in lipid fractions on Day 6 of pregnancy in uterine segments containing implantation sites, in uterine segments between implantation sites, and in luminal epithelial cells after a deciduogenic stimulus. Arachidonic acid in uterine phospholipid was depleted at implantation sites. With an intrauterine deciduogenic stimulus of hormonally primed ovariectomized rat uteri, the arachidonic acid content of the luminal epithelium decreased. When the fatty acid composition of the luminal epithelium was examined during pseudopregnancy and after progestin-estrogen treatment, however, no changes in arachidonic acid composition and content were observed. These data suggest that during blastocyst implantation, luminal epithelial cells at implantation sites mobilize arachidonic acid from phospholipid for prostaglandin synthesis, but that uterine sensitivity and the capacity to synthesize prostaglandins in response to the blastocyst does not depend upon changes in arachidonic acid levels in uterine phospholipid.

Comparison of human and sheep chorion laeve permeability to glucose, beta-hydroxybutyrate, and glycerol.

Significant permeability of in vitro human chorion laeve to glucose, glycerol, and beta-hydroxybutyrate (betaOH butyrate) and in vitro sheep chorion leave to glycerol and betaOH butyrate was measured. Diffusion across these tissues of glycerol and betaOH butyrate corresponded to the relative molecular size, charge, and lipid solubility of these compounds. The simple diffusion of glucose across human chorion leave was somewhat reduced by metabolic conversion of this compound during the transfer process. Demonstration of in vitro human placental tissue permeability to ketoacids and glycerol suggests that these compounds cross to the fetus when elevated in the maternal diabetic state.

Changes in amniotic fluid glucose, beta-hydroxy-butyrate, glycerol, and lactate concentration in diabetic pregnancy.

Amniotic fluid glucose, beta OH butyrate, glycerol, and lactate concentrations were measured in 75 samples collected in the third trimester of pregnancy from 50 diabetic patients, all but four of whom required insulin. Increases in maternal fasting plasma sugar were accompanied by corresponding increases in amniotic fluid glucose and on occasion increases in amniotic fluid beta OH butyrate. These data correspond to previous reports of placental glucose transfer and in addition, provide statistically significant evidence of placental betaOH butyrate transfer since the hyperglycemic, hyperinsulinemic fetus of a diabetic mother would be a poor primary source for ketogenesis. Relatively poor correlation of elevated fluid levels of these solutes to fetal outcome probably reflects a low incidence of maternal hyperglycemia, ketogenesis. Relatively poor correlation of elevated fluid levels of these solutes to fetal outcome probably reflects a low incidence of maternal hyperglycemia, ketoacidosis, and over-all reduced neonatal morbidity-mortality rates in this group of metabolically well-controlled, predominantly insulin-requiring diabetic patients managed in a regional high-risk perinatal center.

Neurohistochemical evidence supporting an absence of adrenergic and cholinergic innervation in the human placenta and umbilical cord.

Neurohistochemical techniques were used to determine the distribution of adrenergic and cholinergic nerves in the human placenta and the umbilical cord adjacent to the chorion. These morphologic studies demonstrate the absence of neural elements in these sites. The significance of these findings to the placental innervation controversy is discussed.