Berberine exerts antioxidant effects via protection of spiral ganglion cells against cytomegalovirus-induced apoptosis.

Cytomegalovirus (CMV) is the leading cause of sensorineural hearing loss (SNHL) in children because of its damage to the cochlea and spiral ganglion cells. Therefore, it has become a top priority to devise new methods to effectively protect spiral ganglion cells from damage. Berberine (BBR) has gained attention for its vast beneficial biological effects through immunomodulation, and its anti-inflammatory and anti-apoptosis properties. However, the effect of BBR on spiral ganglion cells and molecular mechanisms are still unclear. This study aims to investigate whether BBR has an anti-apoptosis effect in CMV-induced apoptosis in cultured spiral ganglion cells and explore the possible mechanism. In this study, TUNEL and MTT assays significantly demonstrated that low doses of BBR did not promote cell apoptosis and they also inhibited the CMV-induced cultured spiral ganglion cell apoptosis. Immunofluorescence and Western blot assays indicated that the anti-apoptosis effect of BBR was related to Nox3. Mitochondrial calcium and Western blot assays revealed that NMDAR1 mediated this anti-apoptosis effect. Our results demonstrated that BBR exerted an anti-apoptosis effect against CMV in cultured spiral ganglion cells, and the mechanism is related to NMDAR1/Nox3-mediated mitochondrial reactive oxygen species (ROS) generation.

A Cell Culture Model of Latent and Lytic Herpes Simplex Virus Type 1 Infection in Spiral Ganglion.

PURPOSE OF THE STUDY: Reactivation of latent herpes simplex virus type 1 (HSV-1) in spiral ganglion neurons (SGNs) is supposed to be one of the causes of idiopathic sudden sensorineural hearing loss. This study aims to establish a cell culture model of latent and lytic HSV-1 infection in spiral ganglia. PROCEDURES: In the presence of acyclovir, primary cultures of SGNs were latently infected with HSV-1 expressing green fluorescent protein. Four days later, these cells were treated with trichostatin A (TSA), a known chemical reactivator of HSV-1. TCID50 was used to measure the titers of virus in cultures on Vero cells. RNA from cultures was detected for the presence of transcripts of ICP27 and latency-associated transcript (LAT) using reverse transcription polymerase chain reaction. RESULTS: There is no detectable infectious HSV-1 in latently infected cultures, whereas they could be observed in both lytically infected and latently infected/TSA-treated cultures. LAT was the only detectable transcript during latent infection, whereas lytic ICP27 transcript was detected in lytically infected and latently infected/TSA-treated cultures. CONCLUSION: Cultured SGNs can be both latently and lytically infected with HSV-1. Furthermore, latently infected SGNs can be reactivated using TSA, yielding infectious virus.

[Distributions of cells infected by AD5-EGFP infused in different ways in guinea pig's cochlear].

OBJECTIVE: To explore the distribution of cells infected by AD5-EGFP infused in different ways in the cochlea of guinea pig. METHOD: AD5-EGFP was infused into the endolymphatic system through a hole on the lateral wall of the scala media or into the extralymphatic system through the round window membrane respectively. The infected cochlear cells confirmed by expression of EGFP were examined on the whole mount or cryostat sections. RESULT: In the cochleae in which AD5-EGFP was infused into the extralymphatic system through the round window membrane, expression of EFGP could be found in the type I, IV and V fibrocyte of the stria vascularis, superlimbal cells of the spiral lip, cells in Ressenal membrane, spiral ganglion neurons in Rosenthal hole and cells lining the inner wall of scala vestibular and scala tympani, indicating that these cells were infected by adenovirus. None of the inner, outer hair or supporting cells was found to be infected in these cochleae. In the cochleae in which AD5-EGFP was infused into the endolymphatic system through a hole on the lateral wall of scala media, expression of EFGP could be found in supporting cells in the organ of Corti and lining cells of the scala media. CONCLUSION: Adenovirus5 is a good and effective vector for delivering genes into cells in guinea pig's cochlea. The scope of infected cells will be very different when the vector is applied to the cochlea through different infusion ways. No cells in the endolymphatic system would be infected if the vector is infused into the extralymphatic system.

Ménière's disease is a viral neuropathy.

Morphological and clinical evidence supports a viral neuropathy in Ménière's disease (MD). Quantitative examination of 11 sectioned temporal bones (TBs) from 8 patients with a history of MD revealed a significant loss of vestibular ganglion cells in both the endolymph hydropic (EH) and non-EH ears. Transmission electron microscopy of vestibular ganglion cells excised from a patient with MD revealed viral particles enclosed in transport vesicles. Antiviral treatment controlled vertigo in 73 of 86 patients with vestibular neuronitis (85%) and 32 of 35 patients with MD (91%).

Highly variable distribution of HSV-1-specific DNA in human geniculate, vestibular and spiral ganglia.

Viral reactivation in temporal ganglia is the suspected cause of Bell's palsy, vestibular neuritis and sudden hearing loss. Since the distribution of latent herpes simplex type 1 (HSV-1) in geniculate, vestibular and spiral ganglia of individual human temporal bones could have implications for the explanation of isolated as well as combined disorders of these three cranial nerves, we examined these ganglia in 18 human temporal bones of adults by nested polymerase chain reaction. In all of the temporal bones HSV-1 specific DNA was detected: 10/18 (56%) of the geniculate, 11/18 (61%) of the vestibular and 9/18 (50%) of the spiral ganglia samples were positive. All combinations of positive and negative ganglia were found in individual temporal bones at roughly equal frequencies. These data support a viral etiology of all three conditions, especially their occasional combinations.

Defining responsiveness of avian cochlear neurons to brain-derived neurotrophic factor and nerve growth factor by HSV-1-mediated gene transfer.

The importance of individual members of the neurotrophin gene family for avian inner ear development is not clearly defined. Here we address the role of two neurotrophins, brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF), for innervation of the chicken cochlea. We have used defective herpes simplex virus type 1 (HSV-1) vectors, or amplicons, to express these neurotrophins in dissociated cultures of cochlear neurons. HSV-1-mediated expression of BDNF promotes neuronal survival similar to the maximal level seen by exogenously added BDNF and exceeds its potency to produce neurite outgrowth. In contrast, cochlear neurons transduced with an amplicon producing bioactive NGF show no response. These results confirm BDNF as an important mediator of neurotrophin signaling inside avian cochlear neurons. However, these neurons can be rendered NGF-responsive by transducing them with the high-affinity receptor for NGF, TrkA. This study underlines the usefulness of amplicons to study and modify neurotrophin signaling inside neurons.

Detection of viral DNA in vestibular ganglia tissue from patients with Menière's disease.

OBJECTIVE: The main goal of this study was to examine the vestibular ganglia from 11 patients with intractable classic Menière's disease (MD) for the presence or absence of DNA from three neurotropic viruses (herpes simplex virus, cytomegalovirus, and varicella zoster virus) using exquisitely sensitive molecular biologic techniques. STUDY DESIGN: This was a prospective controlled study with vestibular ganglia from patients with MD and from patients with small vestibular schwannomas undergoing resection. Polymerase chain reaction was used for viral DNA detection from the ganglia along with known positive and negative polymerase chain reaction control subjects. SETTING: The study was performed in an academic tertiary referral center. PATIENTS: Patients for inclusion had medically uncontrolled MD, including documented fluctuating sensorineural hearing loss, episodic vertigo, and tinnitus who elected to undergo vestibular nerve section. Control patients were undergoing vestibular schwannoma removal. INTERVENTIONS: The intervention was vestibular nerve section with removal of vestibular ganglion. MAIN OUTCOME MEASURES: The presence or absence of viral DNA (herpes simplex virus, cytomegalovirus, and varicella zoster virus) in vestibular ganglion tissues detected by polymerase chain reaction. RESULTS: No viral DNA was detected in the vestibular ganglia of patients with MD (p = 0.028) nor in the control group. The likelihood of a type II or beta type error was < 10%. CONCLUSIONS: In patients with MD requiring surgical intervention, infection with herpes simplex virus, cytomegalovirus, or varicella zoster virus of the vestibular ganglia does not appear to play a major role in the pathoetiology of the disease.

Transfection of neonatal rat cochlear cells in vitro with an adenovirus vector.

A recombinant adenovirus vector containing a beta-galactosidase reporter gene was used to transfect neonatal rat organ of Corti or spiral ganglion explants in vitro. Infection at appropriate titers (10(6)-10(7) pfu/ml) transduced virtually all cells in the cultures after 72 hr. However, spiral ganglion neurons and cells in the inner hair cell regions of the organ of Corti showed the highest levels of expression. Viral titers that produced high levels of beta-galactosidase expression did not appear to damage the cultures, and did not inhibit neurite outgrowth from spiral ganglion cells. However, higher titers (10(8)-10(9) pfu/ml) clearly diminished explant viability and inhibited neurite extension. The results demonstrate that cochlear cells can be transfected successfully with an adenovirus vector, at viral titers which do not induce obvious signs of cellular damage or dysfunction.

Detection of latent varicella-zoster virus infection in human vestibular and spiral ganglia.

Varicella-zoster virus (VZV) becomes latent in the sensory ganglia after primary infection and VZV DNA has been found in human trigeminal, thoracic, and geniculate ganglia. In this study, human vestibular and spiral ganglia, which do not received innervation from the skin, were examined for VZV DNA using the polymerase chain reaction. VZV DNA was detected in 2 of 10 (20%) vestibular ganglia and in 2 of 10 (20%) spiral ganglia from five adults. VZV DNA was undetectable in either type of ganglion from a newborn and from two of the five adults. These two adults were VZV seronegative. The results indicate that VZV becomes latent in several types of sensory ganglion after primary infection and suggest the possibility that reactivation of the virus from the vestibular and spiral ganglia may cause disorders in the labyrinth.