Jiri Prazma

Immunohistochemical characterizationof nitric oxide synthase activity in squamous cell carcinoma of the head and neck

Abstract This study was designed to investigate the presence of nitric oxide in human squamous cell carcinoma of the head and neck. We localized the activity of nitric oxide synthase in these tumors through immunohistochemical analysis using antibodies to L -citrulline (a byproduct of nitric oxide synthase), to inducible nitric oxide synthase, and to constitutive nitric oxide synthase. We found presence of inducible enzyme in squamous cells throughout these tumors, with the highest intensity staining occurring directly around keratin pearls. Our findings suggest that inducible nitric oxide synthase activity is present in squamous cell carcinomas of the head and neck, leading us to conclude that inducible nitric oxide synthase may play a significant role in tumor growth. (OTOLARYNGOL HEAD NECK SURG 1995;113:541-9.)

Insulin-dependent diabetic microangiopathy in the inner ear

Thickening of the basement membrane in capillaries is implicated in the microangiopathic complications of diabetes mellitus. This study was designed to evaluate microangiopathic changes of the inner ear associated with insulin‐dependent diabetes mellitus (IDDM) and concurrent moderate‐intensity noise exposure.

GASTROESOPHAGEAL REFLUX AND EUSTACHIAN TUBE DYSFUNCTION IN AN ANIMAL MODEL

Objective To explore the possible relationship between gastroesophageal reflux and eustachian tube dysfunction in an animal model.

Nitric oxide synthase is an active enzyme in the spiral ganglion cells of the rat cochlea

Nitric oxide (NO) mediates the effects of the excitatory amino acids in the central nervous system. Excitatory amino acids, in particular L-glutamate, are thought to be the neurotransmitter(s) present at the cochlear hair cell-afferent nerve synapse. To our knowledge, no studies to date have documented the presence of NO in the cochlea nor attempted to elucidate the role of NO in hearing. Rat cochlea frozen sections were examined for the presence of nitric oxide synthase (NOS) by NADPH diaphorase histochemistry. Vibratome sections of rat cochlea were examined by immunocytochemistry with an antibody to citrulline, an indication of NOS activity. Spiral ganglion cells in the rat cochlea were positive by NADPH diaphorase histochemistry and by anti-citrulline immunocytochemistry. These results indicate that NOS is present and that the enzyme actively produces nitric oxide in the spiral ganglion cells of the rat cochlea. Given our current understanding of neurotransmission in the cochlea, it is reasonable to postulate that the actions of NO in cochlear neuronal tissue are similar to the actions of NO in the CNS and that NO acts as a neurotransmitter/neuromodulator in the cochlea. In addition, because NO has been implicated as a mediator of excitotoxicity in the CNS, NO may play a role in neurotoxicity in the cochlea.

Interaction Between Hypertension and Diabetes Mellitus in the Pathogenesis of Sensorineural Hearing Loss

The purpose of this study is to support the hypothesis that diabetic end‐organ damage of the cochlea is augmented in the setting of hypertension. A historical perspective reviewing the effects of diabetes and hypertension as causative factors in the development of sensorineural hearing loss, as well as the basic epidemiology and pathophysiology of the renal and vascular effects of diabetes and hypertension, is presented. The results of audiologic findings in insulin‐dependent diabetic patients, both normotensive and hypertensive, were analyzed and correlated with the results of animal studies to support the hypothesis that sensorineural hearing loss in patients and cochlear hair cell loss in animal studies result from the effects of hypertension in conjunction with insulin‐dependent diabetes mellitus.

Immunohistochemical Localization of Nitric Oxide Synthase Activity in Upper Respiratory Epithelium

Nitric oxide (NO) production in the respiratory epithelium of the upper airways has recently been described. To better delineate the role of epithelial NO, the authors of this study attempted to identify the cell type responsible for the production of NO in rat tracheal epithelium and human nasal epithelium. They localized the activity of NO through immunohistochemical analysis with an antibody to L‐citrulline, a marker for activity of the L‐arginine‐dependent nitric oxide synthase (NOS) pathway. Using anti‐inducible NOS (iNOS) and anti‐constitutive NOS (cNOS) antibodies, they also attempted to identify the specific NO isotypes that were present.

The ototoxicity of 3,3′-iminodipropionitrile: Functional and morphological evidence of cochlear damage

Previous reports have suggested that IDPN may be ototoxic (Wolff et al., 1977; Crofton and Knight, 1991). The purpose of this research was to investigate the ototoxicity of IDPN using behavioral, physiological and morphological approaches. Three groups of adult rats were exposed to IDPN (0-400 mg/kg/day) for three consecutive days. In the first group, at 9-10 weeks post-exposure, thresholds for hearing of 5.3- and 38-kHz filtered clicks were measured electrophysiologically and brainstem auditory evoked responses (BAERs) were also recorded to a suprathreshold broadband click stimulus. A second set of animals was tested at 9 weeks for behavioral hearing thresholds (0.5- to 40-kHz tones) and at 11-12 weeks post-exposure for BAER thresholds (5- to 80-kHz filtered clicks). A third group of animals was exposed (as above), and killed at 12-14 weeks post-exposure for histological assessment. Kanamycin sulfate was used as a positive control for high-frequency selective hearing loss. Surface preparations of the organ of Corti were prepared in order to assess hair cells, and mid-modiolar sections of the cochlea were used to examine Rosenthals canal and the stria vascularis. Functional data demonstrate a broad-spectrum hearing loss ranging from 0.5 kHz (30 dB deficit) to 80 kHz (40 dB deficit), as compared to a hearing deficit in kanamycin-exposed animals that was only apparent at frequencies greater than 5 kHz. Surface preparations revealed IDPN-induced hair cell loss in all turns of the organ of Corti, with a basal-to-apical gradient (more damage in the basal turns) at the lower dosages. At higher dosages there was complete destruction of the organ of Corti. There was also a dosage-related loss of spiral ganglion cells in all turns of the cochlea, again with a basal-to-apical gradient at the lower dosages. These data demonstrate that IDPN exposure in the rat results in extensive hearing loss and loss of neural structures in the cochlea.

Transient Inflammation and Dysfunction of the Eustachian Tube Secondary to Multiple Exposures of Simulated Gastroesophageal Refluxant

Gastroesophageal reflux is a common problem in the newborn and preschool periods. Recent research suggests that it may be related to eustachian tube dysfunction and otitis media with effusion. The purpose of this experiment was to investigate the relationship between simulated gastroesophageal reflux and eustachian tube dysfunction. Rat middle ears were repeatedly exposed (transtympanically) to pepsin in hydrochloric acid or to phosphate-buffered saline solution. Their eustachian tube function was evaluated by assessing passive opening and passive closing pressures, and active clearance of positive and negative pressure. The passive pressure function tests showed variable results. The rats exposed to pepsin in hydrochloric acid had an impaired ability to clear positive and negative pressure from the middle ear as compared to the rats exposed only to phosphate-buffered saline solution. The results demonstrate that multiple middle ear exposures to pepsin in hydrochloric acid leads to eustachian tube dysfunction in rats.

Nitric oxide mediates mucin secretion in endotoxin-induced otitis media with effusion*

The mechanisms that regulate mucin release in chronic otitis media with effusion, a leading cause of hearing loss in children, remain largely unknown. We developed an animal model using Sprague-Dawley rats to determine the factors responsible for mucin production in chronic otitis media with effusion. N-nitro-L-arginine methyl ester (L-NAME), a competitive inhibitor of nitric oxide synthase, was used to investigate the role of nitric oxide in mucin secretion by the middle ear epithelium. All rats underwent eustachian tube obstruction. In the first set of rats, the middle ear was then injected transtympanically with 35 microl of either 300 mOsm Krebs-Ringer bicarbonate buffer (control group) or 1 mg/ml lipopolysaccharide in Krebs-Ringer (experimental group 1). In a second set of rats, the middle ear space was injected with lipopolysaccharide and then infused at a continuous rate for 7 days with either Krebs-Ringer (experimental group 2) or 1 mmol/L L-NAME in Krebs-Ringer (experimental group 3) through an osmotic infusion pump. After 7 days the volume of effusion and the quantity of mucin collected were significantly greater in lipopolysaccharide-exposed ears than in controls. In addition, antimucin immunostaining demonstrated mucous cell hyperplasia in response to lipopolysaccharide. The lipopolysaccharide-induced production of mucin and mucous cell hyperplasia was inhibited in ears treated with lipopolysaccharide and L-NAME. These results suggest that nitric oxide is a mediator in the pathway of mucin secretion in chronic otitis media with effusion.

Nitric Oxide: A Mediator of Endotoxin‐Induced Middle Ear Effusions

Using a rat model, the authors investigated the role of nitric oxide (NO) in endotoxin‐induced middle ear effusion (MEE). After the eustachian tube was obstructed, the middle ear was transtympanically injected with 35μL of either 1 mg/mL lipopolysaccharide (LPS) or LPS and 1 mmol/L N‐nitro‐L‐arginine methyl ester (L‐NAME), a competitive inhibitor of NO synthase. Over the next 6 hours, the fluid within the middle ear was collected every 2 hours, and the quantity of albumin in the fluid, an index of vascular leakage, was determined using enzyme‐linked immunosorbent assay.