The reporter mouse used in the present experiments permitted identification of individual cells in which NF-κB was activated, regardless of which the five forms of NF-κB was involved. The results show that the ability to determine which cells in complex tissues employ NF-κB in response to different stressors offers opportunities to get a more realistic picture of the circumstances under which it operates. Remarkably few cochlear cell classes showed activation in response to noise trauma or systemic inflammatory challenge. Although both classes of challenge were found to activate NF-κB, each stimulus activated predominantly one cochlear cell type with a characteristic and unexpected anatomical pattern. The levels of noise used here are known to induce sensory cell damage and hearing loss, but not sensory cell death (Wang et al., 2002
), but neither sensory cells nor the ganglion cells that innervate them showed any evidence of NF-κB activation in response to noise exposures. Somewhat unexpectedly, only fibrocytes were activated by noise exposures. The nature of the stress that provoked this activation is not clear because little is known about these cells’ functions. The sensory epithelium moves only nanometers in response to acoustic stimulation (Harris, 1968
), so it is unlikely that type I fibrocytes located remotely from the basilar membrane would be mechanically stressed by sound. Further, the present results would not have been predicted by previous work on acoustic trauma or cochlear inflammation, much of which implicates reactive oxygen species in cochlear epithelial cells’ responses to acoustic trauma (Henderson et al., 2006
). The finding of activation exclusively in connective tissue cells casts a new light upon the inner ear’s responses to acoustic stress and to systemic inflammatory stresses. It also raises questions about what stress pathways may be involved in sensory cell responses to noise-induced stress. NF-κB is generally assumed to be present in all cells and is known to directly or indirectly affect expression of hundreds of genes (Pahl, 1999
; Ahn and Aggarwal, 2005
; Ghosh et al., 1998
), so its importance in biology is difficult to overstate. Although it is known to play a central role in many stress responses (Mercurio and Manning, 1999
), it is clear that it does not participate in all stress responses. The lack of NF-κB activation in cochlear epithelial cells by traumatic noise exposure may reflect the fact that noise trauma seldom occurs in nature and was therefore most likely not a significant source of stress while the mammalian ear was evolving. The negative results with epithelial cells and ganglion cells emphasizes how little is known about the nature of the stress responsive pathways in various inner ear cell classes. On the other hand, the findings concerning connective tissue cells’ involvement in noise-induced and inflammatory responses in the cochlea affords opportunities for extending this research to learn more about these poorly understood cells and their functional roles in hearing.
In the present report an NF-κB reporter mouse is employed to visualize which cochlear cells showed activation of NF-κB following exposure to traumatizing noise or following administration of inflammatory agents. Immunolocalization of nuclear NF-κB confirmed the results of noise-induced activation in the reporter mice. Likewise, the present finding of systemic LPS administration inducing NF-κB activation in type II fibrocytes was confirmed by a previous report of immunostaining of nuclear p65 (a form of NF-κB) in those cells following LPS administration (Adams, 2002
). The power of the present reporter mouse approach is that activation of all forms of NF-κB are reported and that the reporter is retained within activated cells for at least one day post activation so that it is not necessary to do an exhaustive time series in order to capture the brief period during which NF-κB is in the nucleus. Given the breath of the NF-κB forms reported by the assay and the relative lack of temporal constraints on the post stress time for detecting activation, the limited number of cell types that were found to be activated was remarkable.
There have been a number of reports of NF- B activation in the cochlea following stresses, including noise exposure (Ramkumar et al., 2004
; Masuda et al., 2006
; Tahera et al., 2006a
; Tahera et al., 2006b
; Nagashima et al., 2007
; Selivanova et al., 2007
; Miyao et al., 2008
), administration of ototoxic drugs (Watanabe et al., 2002
; Jiang et al., 2005
;So et al., 2008; Chung et al., 2008), and inflammatory challenges (Moon et al., 2007
; Miyao et al., 2008
). Some of these did not include reports of which cell classes showed NF- B activation. Of those that did, agreements with the present results are mixed. Masuda et al., (2006)
used immunostaining for P65 and P50 to identify NF- B activated cells in the cochlea following noise exposure. As in the present study, they found no noise-induced nuclear translocation indicative of NF- B activation in ganglion cells or in sensory cells. Rather, activated cells were exclusively within the spiral ligament and stria vascularis. The principal difference between those results and the present findings appears to be that they found noise-induced activation of type II fibrocytes (their Figure 7). This apparent difference from the present results may be due the fact that they used a noise intensity that was more than two orders of magnitude greater than was used in the present study, and/or due to their use of C57/Bl6J mice, which are more vulnerable to noise damage than CBA/CaJ mice used in the present study.
In contrast with Masuda et al., 2006
, and with the present results, Tahera et al. (2006a
found abundant nuclear NF- B localization of ganglion cells, even in mice with no noise stress. This unlikely finding may have resulted from the use of a non-specific NF- B antibody or some other error in immunostaining. Likewise, the report that NF- B was still present in the nuclei of ganglion cells and epithelial cells 8 days following stress (Miyao et al., 2008
) needs confirmation and an explanation. Although we never found NF- B activation in sensory cells or ganglion cells, we cannot rule out the possibility that such activation could occur if much higher intensity noise had been utilized. We limited noise exposure levels to those that have been shown to permanently damage hair cells without destroying them so as to exclude effects that may be associated with tissue repair that would be induced by physical disruption of the tissue, such as occurs when extreme noise levels are employed. Consequently, our results can not be taken as strong evidence that sensory cells and ganglion cells never activate NF- B following noise trauma, but if it occurs, it appears to do so it happens at extreme levels of trauma. In any case, the relatively low levels of noise required to activate connective tissue cells shows that these cells either have much lower thresholds for noise-induced stress or that they utilize different tactics for dealing with the stresses.
Exposure of mice to low level noise has been shown to protect the ears from subsequent traumatizing noise exposure (Yoshida and Liberman, 2000
). Activation of type I fibrocytes by noise stimulation suggest that these cells may play a role in protection of the ear from noise damage. How this might be achieved is not immediately clear, but two possibilities suggest themselves. Activation of NF- B in type I fibrocytes could result in up-regulation of inflammatory cytokines and/or nitric oxide (Ichimiya et al., 2000; Hashimoto et al., 2005
), both of which are known to regulate gap junctional permeability. A combination of TNF and a purinergic agonist has been shown to modulate gap junctional connectivity and connexin 26 immunostaining in the trigeminal ganglion (Damodaram et al., 2009) and in immortalized mouse hepatocytes (Temme et al., 1998
). Increased nitric oxide synthase has been reported to be correlated with decreased connexin 26 levels (Pitre et al., 2001
). Decreasing gap junctional permeability in type I fibrocytes would be expected to deprive the stria vascularis of K+
ions, which should lead to decreasing endolymphatic potential. Decreasing endolymphatic potential would produce a transient decrease in cochlear sensitivity to sound and thereby decrease its vulnerability to noise-induced trauma. The obvious limitation of this hypothesis is that it remains to be demonstrated that nitric oxide or inflammatory cytokines actually affect permeability of gap junctions within the cochlea. Another possibility is that NF- B activation could result in type I fibrocytes secreting signaling compounds such as inflammatory cytokines that affect cochlear epithelial cells in a paracrine fashion. Such signaling of nearby root cells, for example, could ultimately result in signals being transmitted to sensory cells or to their adjacent epithelial cells via gap junctions (Kikuchi et al., 1995
) or by purinergic signaling (Gale et al., 2004
). How such signals might lead to protection of sensory cells from acoustic trauma remains a matter of conjecture. Both of these possibilities are based on the premise that type I fibrocytes are sensitive to excessive acoustic stimulation, perhaps by sensing K+
flux and/or other associated ion changes through the tissue. Their location between the primary site of K+
uptake from perilymph (type II fibrocytes) and the stria vascularis has them situated in a key site for controlling ion input to the stria (Kikuchi et al., 2000
; Wangemann, 2006
). Clearly, much work will be needed to test these and other possibilities.
Loss of function of type II fibrocytes would likely have a drastic impact upon hearing due to their critical roles in K+
ion uptake from perilymph. Transgenic mice with degenerated type II fibrocytes have hearing threshold elevations (Delprat et al., 2005
). The finding that these cells may be selectively stressed by systemic inflammation raises the possibility that their vulnerability to systemically administered inflammatory stress may underlie two poorly understood forms of hearing loss, sudden hearing loss and immune-mediated hearing loss. Both types of hearing loss are usually unilateral, like the response of type II fibrocytes to systemic inflammatory challenge reported here. Both types of hearing loss are responsive to treatment with anti-inflammatory steroids (Chen et al., 2003
; Ruchenstein, 2004
). The fact that steroids are potent blockers of NF-κB activation suggests that, if the activation of NF-κB in type II fibrocytes by systemic inflammatory agents can result in a loss of function of these cells, this activation could be blocked or reversed by steroid treatment (Auphan et al., 1995
; Scheinman et al., 1995
). It remains to be determined what effects inflammatory stresses may have upon type II fibrocytes but the present results show that among cochlear cells these cells are selectively vulnerable to such stresses and that this effect can be blocked by steroid administration. Clearly systemic inflammations do not routinely cause unilateral hearing loss, but the present findings suggest that if a second stress were compounded with the largely unilateral stress upon type II fibrocytes like that shown in the present results, the result could compromise hearing in that ear.
The present results provide important insight into previously unrecognized signaling pathways activated in response to excessive noise, a health hazard of increasing clinical importance (Nelson et al., 2005
). Understanding how the cochlea protects itself from acoustic trauma is of paramount importance, given the epidemic proportions of noise-induced hearing loss (Nelson et al., 2005
; Catlin, 1986
; Kurmis and Apps, 2007
). Uncovering two previously unrecognized cell specific stress-related responses within the ear by use of the NF-κB reporter mouse demonstrates the power of the present approach for identifying individual cells that are stress responsive in vivo
. There is every reason to believe that similar insights could be gained by this approach in a wide variety of other tissues.