Hearing loss is a significant problem in the United States today. Nearly 35 million Americans suffer from measurable hearing impairment and related speech disorders. Hearing loss affects approximately 17 in every 1,000 children under the age 18. The incidence increases with age: approximately 314 in 1,000 people over age 65 have a hearing loss and 40 to 50 percent of people 75 and older have a hearing loss1
. Hearing loss affects more people than epilepsy, multiple sclerosis, spinal injury, stroke, Huntington’s, and Parkinson’s diseases combined2
, and although it is rarely life-threatening, it has a huge financial impact on our economy and lifestyle. Two million Americans are completely deaf and two-to-three out of every thousand children born are severely to profoundly deaf, half of those due to hereditary causes1
. The primary cause of these hearing impairments is thought to be damage to the sensory cells, supporting cells and neurons in the cochlea, and is referred to clinically as sensorineural or “nerve” deafness, as opposed to conductive hearing loss. These hearing deficits can be caused by administration of ototoxic drugs, exposure to intense work-related or recreational noise, genetic mutations, or as a consequence of the aging process. The loss of hair cells in the mammalian cochlea leads to permanent hearing loss because these cells are generated only during embryonic development3
and must last throughout a person’s lifetime. However, it was recently discovered that birds are able to rapidly and repeatedly produce new hair cells and supporting cells in their cochleae following hair cell damage which leads to a significant recovery of hearing4,5,6
The primary mechanism for regeneration in the bird cochlea is the proliferation of supporting cells in the damaged region of the sensory epithelium that results in the generation of new hair cells and supporting cells. In the normal bird cochlea, both the hair cells and supporting cells are post-mitotic and remain in a state of quiescence known in the cell cycle field as G07
. But once the dying hair cells are ejected from the epithelium, the adjacent supporting cells are stimulated to leave quiescence, re-enter the cell cycle (the G1
phase), double their DNA content (the DNA synthesis, or S phase), generate proteins needed to divide (G2
phase), and finally split into two identical daughter cells (the Mitosis, or M phase). In the avian cochlea, the daughter cells will go on to differentiate into new hair cells or supporting cells replacing those that were lost. At the time of our original regeneration discovery in the bird, there was morphological evidence that new stereociliary bundles were appearing in the region of hair cell loss within 4-6 days after the trauma8
. However, we were unsure as to whether this was from the repair of surviving hair cells or the generation of new ones.
In order to test whether new hair cells were being produced by cell divisions, we needed to label the tissues with markers for evidence of the production of new cells. At that time, the standard technique was to inject radioactive (tritiated, or 3H) thymidine, one of the four nucleotides required to duplicate DNA. Tritiated thymidine is detected by the radioactive decay of the 3H tag, however, this weak decay can only travel through tissues over a short distance. Thus, the method for detecting tritiated thymidine is to slice the tissue into thin (1-30 um) sections, cover the sections with a photographic emulsion, expose the tissues in the dark for several hours to days in order to detect exposed silver grains in the overlying emulsion. While this is a very powerful technique, it has several drawbacks, such as dealing with radioactive isotopes, having to section tissues, and the fact that the signal is only detected in the overlying emulsion, not in the tissue itself. Plus, the necessity of excluding extraneous light that would ruin the emulsion during the exposure can be frustrating.
In the mid-1980′s a new technique was developed for detecting DNA synthesis using a non-radioactive analog of thymidine, 5-bromo-2′-deoxyuridine (BrdU). This analog is incorporated into DNA just as readily as thymidine during S phase and can be detected with a monoclonal antibody that binds directly to the BrdU molecule within the DNA9
. The BrdU technique to label mitotically active cells is quite beneficial, as one can sidestep the significant dangers, regulations, and permitting associated with the radioactive isotope tritiated thymidine. Moreover, since the antibody binds directly to the DNA within the cell, it does not require sectioning and overlaying with a photographic emulsion. The detecting signal can be a stain such as diaminobenzidine (DAB), for brightfield histological analysis or a fluorescent probe attached to a secondary antibody or even directly to the primary antibody for immunofluorescent detection. Finally, detection of the signal within the tissue, rather than in an overlying emulsion, and penetration of the antibody label through thick sections of tissue or even whole-mount preparation of tissues like the cochlear sensory epithelium, enables three dimensional localization of the labeled nuclei with tools such as the confocal laser scanning microscope or computer-aided 3D reconstruction programs coupled with standard light microscopy. While these benefits of BrdU as a method for detecting proliferating cells offer several major advantages, there are also a few significant drawbacks. Detection of BrdU incorporated into the DNA requires harsh denaturation techniques to give the primary antibody access to the BrdU molecule. This harsh denaturation also tends to affect many protein epitopes and significantly hinders the ability of many standard antibodies to detect their target proteins. Moreover, the requirement of a monoclonal antibody to detect the BrdU limits the ability to detect other proteins of interest within the tissue that are normally also labeled with monoclonal antibodies.
A new technique for detecting DNA synthesis in proliferating cells in vivo
and in vitro
has been developed within the last two years10
. The thymidine analog 5-ethynyl-2′-deoxyuridine (EdU) has a terminal alkyne group replacing a methyl group at the 5 position of the pyrimidine ring and can be readily incorporated into DNA during synthesis. The incorporated EdU molecule can be detected by a reaction of the terminal alkyne group with fluorescent azides, in a Cu(I)-catalyzed [3 + 2] cycloaddition “click” chemistry (Click-iT™, Invitrogen/Molecular Probes, Carlsbad)10
. Because the Click-iT™ reagents are significantly smaller than antibody molecules, they can penetrate much more easily through tissues and the incorporated EdU can be detected without the necessity of DNA denaturation. This leads to a greater sensitivity of detection and retains the availability of other proteins for double-labeling using standard immunocytochemistry. In this study, we have examined the ability of EdU incorporation and detection by Click-iT™ chemistry to identify proliferating supporting cells in the regenerating chick cochlea following gentamicin injection. We have shown that EdU is as sensitive, if not more so, than traditional BrdU labeling with less background labeling and that several antibodies to other proteins involved in regeneration can be used in conjunction with EdU.