Hyperbaric oxygen upregulates cochlear constitutive nitric oxide synthase

Abstract

Background: Hyperbaric oxygen therapy (HBOT) is a known adjuvant for treating ischemia-related inner ear diseases. Controversies still exist in the role of HBOT in cochlear diseases. Few studies to date have investigated the cellular changes that occur in inner ears after HBOT. Nitric oxide, which is synthesized by nitric oxide synthase (NOS), is an important signaling molecule in cochlear physiology and pathology. Here we investigated the effects of hyperbaric oxygen on eardrum morphology, cochlear function and expression of NOS isoforms in cochlear substructures after repetitive HBOT in guinea pigs.

Results: Minor changes in the eardrum were observed after repetitive HBOT, which did not result in a significant hearing threshold shift by tone burst auditory brainstem responses. A differential effect of HBOT on the expression of NOS isoforms was identified. Upregulation of constitutive NOS (nNOS and eNOS) was found in the substructures of the cochlea after HBOT, but inducible NOS was not found in normal or HBOT animals, as shown by immunohistochemistry. There was no obvious DNA fragmentation present in this HBOT animal model.

Conclusions: The present evidence indicates that the customary HBOT protocol may increase constitutive NOS expression but such upregulation did not cause cell death in the treated cochlea. The cochlear morphology and auditory function are consequently not changed through the protocol.

Figure 1

Morphology of eardrum after HBOT. Otoscopic view of the eardrums in guinea pigs. A. normal eardrum; B. Image of the eardrum with moderate hemorrhage (arrow) after 20 sessions of HBOT.

Figure 2

Auditory level. Box plots of the hearing level in the control, normobaric air (NBA) group and the experimental, hyperbaric oxygen (HBO) treatment group before (_pre) and 4 weeks after (_4w) the treatment sessions. There were no significant intra-group changes or inter-group differences in the hearing levels before and 4 weeks after the start of the treatment. X-axis indicates the different frequencies for auditory measurement, from 1 k to 8 k Hz. The dots represent the outliers.

Figure 3

Immunohistochemistry of NOS subtypes in cochlea. Immunohistochemistry of cochlear nNOS (A,B), eNOS (C,D) and iNOS (E,F) expression in the control NBA and HBOT groups. (G) Spleen tissue was used as a positive control for iNOS. (H) Schematic drawing depicting a cross-section through cochlear canals and its related sub-structures. (A,B) nNOS immunoreactivity was present in the spiral ganglion, stria vascularis, organ of Corti, limbus and modiolar nerve bundles in both groups but enhanced immunoreactivity was present in the HBOT group. (C,D) eNOS immunoreactivity was found in the modiolar blood vessels, stria vascularis, organ of Corti, limbus and spiral ganglion in both groups with more enhanced immunoreactivity in the HBOT group. iNOS did not exhibit immunoreactivity in the control NBA (E) and HBOT (F) groups, as compared with the positive control of iNOS expression in the spleen tissue (G). Scale bar, 50 μm.

Figure 4

Morphometric analysis of nNOS and eNOS. Box plots of the optical densities from morphometric measurements of nNOS (A) and eNOS (B) in the control NBA (in white box) and experimental HBO treatment group (in dash box). The expression of nNOS was significantly enhanced in nerve fiber bundles after HBOT. The expression of eNOS was significantly enhanced in the limbus, nerve fiber bundles and modiolar blood vessels after HBOT. The dots represent the outliers. OD, optical densities; SL, spiral ligament; SV, stria vascularis; OG, organ of Corti.

Figure 5

Confocal microscopic view of eNOS and nNOS after HBOT. Immunofluorescence labeling of eNOS (red) and nNOS (green) in the spiral ganglion (A-C), cochlear modiolus (D-F) and stria vascularis (G-I) of the cochlea after repetitive HBOT. Co-expression of eNOS and nNOS occurred in the spiral ganglion (C) and stria vascularis (I). In the cochlear modiolus, nNOS was expressed in the modiolar nerve fibers, and eNOS was expressed along the modiolar blood vessel. Scale bar, 20 μm.

Figure 6

TUNEL assay of cochlea after HBOT. TUNEL assay of cochleae after HBOT. Significant DNA fragmentation was shown in the brain tissue with penetrating injury (A-C); however, no evidence of DNA damage was identified in the cochlear tissue after HBOT (D-F).