Migration of cochlear lateral wall cells
Introduction
Our prior studies of the alteration of energy metabolism during the onset of hearing in neonatal rats demonstrated a direct correlation between the levels of the M-type subunit of 6-phosphofructo-1-kinase (PFK) in cochlear cells (Armour et al., 2001) and endocochlear potential (EP) (Rybak et al., 1992) as well as increased 2-deoxy-D-glucose (2-DG) incorporation (Canlon and Anniko, 1987). The high levels and unique kinetic and regulatory properties of the M-type subunit of PFK (Dunaway, 1983) agree with earlier studies suggesting a high degree of cochlear energy production. While examining cochlear sections during these studies and examining electron micrographs, it was observed that some of the marginal stria vascularis cell layer appeared to exhibit characteristics of apoptosis. In particular, the marginal cells displayed blebbing or bulging into the endolymph. Also, morphologically abnormal nuclei in marginal strial cells were occasionally observed on electron micrographs (Rybak et al., 1991).
Apoptosis in the mammalian cochlea during embryogenesis and in response to ototrauma is well accepted (Fekete et al., 1997, Nishizaki et al., 1998, Nikolic et al., 2000). However, in adult mammals there are positive and negative reports related to the occurrence of apoptosis. Apoptosis in the early neonatal but not adult cochlea was observed predominately in areas of remodeling, cavitation, and differentiation. (Nikolic et al., 2000). Also, little signs of apoptosis or cell division in the inner ear from adult human cadaver temporal bone were observed (Jokay et al., 1998). In the senescence-accelerated mouse, apoptosis, as evaluated by the TdT-mediated dUTP-biotin nick-end-labeling (TUNEL) assay method, was detected in the inner and outer hair cells, pillar cells, Deiters’ cells, the marginal, intermediate, and basal cells of the stria vascularis, and Reissner’s membrane (Usami et al., 1997). A subsequent investigation using BALB/c mice evaluated whether the fixation method or the interval between death and initiation of fixation influenced results of the TUNEL assay in the inner ear (Nishizaki et al., 1999). It was found that irrespective of the fixation method or the interval between death and fixation, the extent of apoptosis in marginal stria vascularis cells in the adult mouse was greater than any other structure in the cochlea (Nishizaki et al., 1999). It was speculated that the high metabolic rate of the stria vascularis could lead to autolysis and increased DNA fragmentation and that not all of the TUNEL-positive cells reflected apoptosis (Nishizaki et al., 1999).
Realizing the potential interpretation concerns and controversy, apoptosis and cell division were each investigated by two independent techniques, TUNEL assay and Hoechst 33342 fluorescence staining were employed to reveal apoptotic cells in the cochlear lateral wall cells of the Fischer 344 rat, i.e. stria vascularis, spiral prominence, and spiral ligament. Cell division was detected by immunohistochemical detection of 5-bromo-2′-deoxyuridine (BrdU) incorporation and proliferating cell nuclear antigen (PCNA), which is the DNA polymerase delta-associated protein (van Dierendonck et al., 1991, Wijsman et al., 1992). Further, to reduce ambiguities, a double labeling immunohistochemical technique was developed and used to measure the incidence of cell death (TUNEL assay) and division (BrdU pulse) in the same cochlear sections. With the exception of marginal strial cells, cell division was present in the lateral wall cells. To measure the role of cell movement in marginal cell replacement, a pulse-chase paradigm using a pulse of BrdU and a chase of deoxythymidine (dT) was employed. These data are consistent with marginal strial cells replacement occurring by migration of underlying cochlear cells.
Section snippets
Handling of animals
Sixty day old male rats of the Fisher 344 strain (Harlan/Sprague Dawley, Inc., Indianapolis, IN, USA) were purchased directly and used after 72 h of acclimation in the animal quarters. Purina laboratory rat chow and tap water was available ad libitum. Ambient noise was minimal, and lighting was appropriate for nocturnal animals.
Processing of cochlea
Temporal bones were rapidly removed, and each temporal bone was handled simultaneously and individually. The oval and round windows were opened, and a small hole was
Results
As mentioned in Section 1, it was suspected that the marginal stria vascularis cells were apoptotic. More definitive evidence of cell death in cochlear sections was sought by examining markers of apoptosis by immunohistochemical methods. Specifically, the presence of condensed chromatin which is produced during apoptosis was detected using the Hoechst fluorescence stain (Fig. 1A). Also, the presence of nuclear DNA fragmentation was detected employing the TUNEL assay (Fig. 1B). As shown in Fig.
Discussion
This study supports the following hypotheses: (1) marginal strial cells undergo a high degree of cell death and undetectable proliferation; and (2) the marginal cell layer is continuously maintained by an influx of cells of unidentified origin. The occurrence of significant apoptosis in the senescence-accelerated mouse stria vascularis has been previously reported (Usami et al., 1997). Subsequently it was argued that the high incidence of TUNEL-positive strial cells could be related, in part,
Acknowledgements
This study was supported by Research Grant DC-00321 from the National Institutes on Deafness and Other Communicative Disorders, National Institutes of Health. The authors wish to thank Jennifer Parrish for her technical contributions and Steve Verhulst, Ph.D., Division of Statistics and Research Consulting for conducting the statistical analyses.
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