Elena Koulich

NF-κB is involved in the survival of cerebellar granule neurons: association of Iκβ phosphorylation with cell survival: NF-κB in neuronal survival

The NF‐κB transcription factor consists of dimeric complexes belonging to the Rel family, which include p50, p52, p65 (RelA), RelB and c‐Rel. NF‐κB activity is tightly controlled by IκB proteins which bind to NF‐κB preventing its translocation to the nucleus. Activation of NF‐κB is most often mediated by IκB degradation, which permits NF‐κB to enter the nucleus. We investigated the role of NF‐κB in the survival of cerebellar granule neurons. We found that survival of these neurons in high potassium medium is blocked by three separate inhibitors of NF‐κB activity: SN‐50, N‐tosyl‐l‐phenylalanine chloromethyl ketone and pyrrolidinedithiocarbamate, indicating that NF‐κB is required for neuronal survival. Gel‐shift assays reveal three complexes that bind to the NF‐κB binding site in high potassium medium. Switching these cultures to low potassium medium, a stimulus that leads to apoptotic death, causes a reduction in the level of the largest complex, which contains p65. Overexpression of p65 by transfection inhibits low potassium‐induced apoptosis, whereas overexpression of IκBα promotes apoptosis even in high potassium medium. Surprisingly, however, neither the level of endogenous p65 nor that of IκBα and IκBβ is altered by low potassium treatment. Similarly, no changes are seen in the nuclear or cytoplasmic levels of p50, p52, RelB and c‐Rel. Phosphorylation of p65, which can lead to its activation, is unchanged. Phosphorylation of IκBβ is, however, reduced by low potassium treatment. Besides being necessary for high potassium‐mediated neuronal survival, NF‐κB is also involved in the survival‐promoting effects of IGF‐1 and cAMP as judged by the ability of SN‐50 to inhibit the actions of these survival factors and the ability of these factors to inhibit the low potassium‐induced alterations in the DNA‐binding activity of NF‐κB. Taken together, our results show that NF‐κB may represent a point of convergence in the signaling pathways activated by different survival factors and that uncommon mechanisms might be involved in NF‐κB‐mediated survival of cerebellar granule neurons.

Apoptosis in cerebellar granule neurons is associated with reduced interaction between CREB-binding protein and NF-κB

Cerebellar granule neurons undergo apoptosis when switched from medium containing depolarizing levels of potassium (high K+ medium, HK) to medium containing low K+ (LK). NF‐κB, a ubiquitously expressed transcription factor, is involved in the survival‐promoting effects of HK. However, neither the expression nor the intracellular localization of the five NF‐κB proteins, or of IκB‐α and IκB‐β, are altered in neurons primed to undergo apoptosis by LK, suggesting that uncommon mechanisms regulate NF‐κB activity in granule neurons. In this study, we show that p65 interacts with the transcriptional co‐activator, CREB‐binding protein (CBP), in healthy neurons. The decrease in NF‐κB transcriptional activity caused by LK treatment is accompanied by a reduction in the interaction between p65 and CBP, an alteration that is accompanied by hyperphosporylation of CBP. LK‐induced CBP hyperphosphorylation can be mimicked by inhibitors of protein phosphatase (PP) 2A and PP2A‐like phosphatases such as okadaic acid and cantharidin, which also causes a reduction in p65–CBP association. In addition, treatment with these inhibitors induces cell death. Treatment with high concentrations of the broad‐spectrum kinase inhibitor staurosporine prevents LK‐mediated CBP hyperphosphorylation and inhibits cell death. In vitro kinase assays using glutathione‐S‐transferase (GST)‐CBP fusion proteins map the LK‐regulated site of phosphorylation to a region spanning residues 1662–1840 of CBP. Our results are consistent with possibility that LK‐induced apoptosis is triggered by CBP hyperphosphorylation, an alteration that causes the dissociation of CBP and NF‐κB.

Effects of cochlear drilling with Piezosurgery Medical device in rats.

Drilling on the otic capsule for cochleostomy should be less traumatic to the cochlea with the Piezosurgery Medical device (PZ) than with a standard diamond drill (DD). “Soft” cochleostomy is used for preservation of residual hearing in cochlear implant patients. PZ drilling can be used for accurate cochleostomy placement with minimal soft‐tissue damage and may be superior for atraumatic drilling on the cochlea, as compared with a DD. This study compared inner ear effects after drilling the rat otic capsule with the PZ versus the DD.

Comparison of systemic and otic administration of ofloxacin

To assess the feasibility of delivering ofloxacin across the intact tympanic membrane; to compare middle ear bioavailability of ofloxacin after otic and systemic administrations; to determine distribution of otically delivered ofloxacin to other tissues.

Ototoxicity of topical azithromycin solutions in the guinea pig

OBJECTIVE To investigate possible ototoxic effects of topical azithromycin (AZ) in the guinea pig. DESIGN A prospective, controlled animal study. SETTING The University of Texas Southwestern Medical Center at Dallas. PARTICIPANTS Twenty-three pigmented guinea pigs were given single, unilateral middle ear applications of a solution containing 3% (n = 3), 2% (n = 5), 1% (n = 5), or 0.5% (n = 5) AZ or saline (n = 5). The contralateral ear served as the untreated control. MAIN OUTCOME MEASURES The animals were observed for behavioral changes for 2 weeks and then humanely killed. The ears were processed for anatomical evaluation. Morphologic changes were analyzed by quantitation of middle ear changes and cochlear inner and outer hair cell loss. Statistical analysis was performed to examine effects by dose. RESULTS Analysis revealed extensive middle and inner ear changes associated with all formulations of AZ. Moderate correlation was found between the extent of middle ear changes and AZ concentration (r(2) = 0.59), whereas a strong correlation was seen between inner ear damage and AZ concentration (r(2) = 0.94). Both inner and outer hair cells were affected, with inner hair cell damage consistently greater than outer hair cell damage. CONCLUSIONS The results of this study demonstrate that ototopical AZ can cause middle ear changes and significant hair cell loss in the guinea pig. This finding, together with previous clinical reports, indicates that topical AZ should be used with caution in the clinical setting.

Ephrins and Ephs in cochlear innervation and implications for advancing cochlear implant function

Determine if the neuronal pathfinding cues resulting from Eph/ephrin interaction in the inner ear play a role in establishing the tonotopic innervation of the cochlea.

Ototopic applications of povidone iodine/dexamethasone in the rat

Hypothesis Repeated applications of low-concentration povidone iodine (PI) combined with dexamethasone (Dex) through a tympanic membrane ventilation tube will not cause ototoxic changes in the rat. Background Otitis externa (OE) and acute otitis media (AOM) are 2 of the most common otologic disorders requiring outpatient antibiotic treatment. The development of topical treatments that are easy to administer would help to limit systemic exposure to antibiotics in these patients. Topical formulations containing Dex and low-dose PI were designed to provide both antimicrobial and anti-inflammatory effects for the treatment of OE and AOM. Treatment with PI alone has shown mixed results in studies designed to determine PI. Low concentrations of PI combined with Dex should yield less ototoxicity while maintaining effectiveness. Methods We performed tympanostomies on rats, inserting a ventilation tube to administer 1% or 2% PI, plus 0.1% Dex over a period of 7 days. Hearing was accessed via auditory brainstem response (ABR) testing over the duration of the study and histologic analysis was performed 15 days after the initial application to determine the effect of administration of PI/Dex on middle and inner ear structures. Conclusion The preparations used in the present investigation were formulated to allow repeated applications to both the external and middle ear, without risk to hearing or equilibrium. Neither of the PI/Dex formulations tested caused pathologic changes in the ear that significantly affected equilibrium, hearing function or morphology.