Article

Comparison of activated caspase detection methods in the gentamicin-treated chick cochlea

Laboratory for Cellular and Molecular Hearing Research, Department of Otolaryngology, Evans 637, Boston University Medical Center, 715 Albany Street, Boston, MA 02118, USA. <>
Hearing Research (Impact Factor: 2.85). 07/2008; 240(1-2):1-11. DOI: 10.1016/j.heares.2008.03.003
Source: PubMed

ABSTRACT Aminoglycoside antibiotics induce caspase-dependent apoptotic death in cochlear hair cells. Apoptosis, a regulated form of cell death, can be induced by many stressors, which activate signaling pathways that result in the controlled dismantling of the affected cell. The caspase family of proteases is activated in the apoptotic signaling pathway and is responsible for cellular destruction. The initiator caspase-9 and the effector caspase-3 are both activated in chick cochlear hair cells following aminoglycoside exposure. We have analyzed caspase activation in the avian cochlea during gentamicin-induced hair cell death to compare two different methods of caspase detection: caspase antibodies and CaspaTag kits. Caspase antibodies bind to the cleaved activated form of caspase-9 or caspase-3 in specific locations in fixed tissue. CaspaTag is a fluorescent inhibitor that binds to a reactive cysteine residue on the large subunit of the caspase heterodimer in unfixed tissue. To induce cochlear hair cell loss, 1-2 week-old chickens received a single injection of gentamicin (300 mg/kg). Chicks were sacrificed 24, 30, 42, 48, 72, or 96 h after injection. Cochleae were dissected and labeled for activated caspase-9 or caspase-3 using either caspase-directed antibodies or CaspaTag kits. Ears were co-labeled with either phalloidin or myosin VI to visualize hair cells and to determine the progression of cochlear damage. The timing of caspase activation was similar for both assays; however, caspase-9 and caspase-3 antibodies labeled only those cells currently undergoing apoptotic cell death. Conversely, CaspaTag-labeled all the cells that have undergone apoptotic cell death and ejection from the sensory epithelium, in addition to those that are currently in the cell death process. This makes CaspaTag ideal for showing an overall pattern or level of cell death over a period of time, while caspase antibodies provide a snapshot of cell death at a specific time point.

Download full-text

Full-text

Available from: Christina Kaiser Marko, Jan 03, 2014
0 Followers
 · 
83 Views
  • Source
    • "To address this issue it was necessary to compare the patterns of hair cell loss after streptomycin exposure with and without prior anti-miR181a transfection. BPs were therefore transfected with either anti- miR181a or a non-targeting miRNA and then cultured for 48 hours with 78 µM streptomycin, before fixation and labeling for the apoptosis marker activated caspase-3 (Kaiser et al., 2008), the hair cell marker myosin VI, and the major hair bundle component actin (Figure 2). Streptomycin treatment causes complete hair cell loss at the high frequency segment of the BP (Figure 2). "
    [Show abstract] [Hide abstract]
    ABSTRACT: Specialized sensory-transducing hair cells regenerate in response to injury in non-mammalian vertebrates such as birds and fish but not in mammals. Previous work has shown that overexpression of microRNA181a (miR181a) in cultured chicken basilar papillae, the avian counterpart of the cochlea, is sufficient to stimulate proliferation with production of new hair cells. The present study investigates the role of miR181a in hair cell regeneration after injury in explants of chicken auditory epithelia. Basilar papillae were explanted from 0-day-old chickens and transfected with either anti-miR181a, which knocks down endogenous miR181a, or a non-targeting miRNA and cultured with streptomycin to eliminate all hair cells from the epithelium. Labeling with BrdU was used to quantify proliferation. Explants exposed to streptomycin and transfected with anti-miR181a had significantly fewer BrdU positive cells than basilar papillae treated with streptomycin and transfected with a non-targeting miRNA. Activated caspase-3 and myosin VI labeling were used to show that the pattern of hair cell death and loss, respectively, were not affected by anti-miR181a transfection. MiR181a downregulation therefore seems to dimish the proliferative component of hair cell regeneration rather than prevent hair cell death following ototoxic injury.
    Neuroscience Letters 02/2011; 493(1-2):44-8. DOI:10.1016/j.neulet.2011.02.017 · 2.06 Impact Factor
  • Source
    • "Primary antibodies were a rabbit polyclonal pan-TLE antiserum (# 4681, Cell Signaling Technology, Danvers, MA) used at a dilution of 1:200, a rabbit polyclonal antibody against the active fragment of cleaved caspase-3 (# 9661S, Cell Signaling Technology Inc.) at a dilution of 1:200, a goat polyclonal OMP antibody (544-10001, Wako Chemicals USA, Richmond, VA) at a dilution of 1:500, a rabbit polyclonal antibodies against phosphorylated histone-3 and phosphorylated CREB (#06-570 and #06-519, Millipore, Billerica, MA) at dilution of 1:200 and 1:1,000, respectively, a rabbit polyclonal antibody against Crocc supplied by Dr. Tiansen Li (Harvard University) at a dilution of 1:5,000, a mouse monoclonal against ASCL1 (MAB2567, R & D Systems, Minneapolis, MN), a rabbit polyclonal against ADCY3 (#sc-588, Santa Cruz Biotechnology, Sant Cruz, CA) at a dilution of 1:200, and a mouse monoclonal antibody against tyrosine hydroxylase (# ab11, Abcam, Cambridge, MA) at a dilution of 1:200. The specificities of these antibodies are documented in publication (Blount et al., 2008; Buiakova et al., 1996; Cheong et al., 2003; Hendzel et al., 1997; Hu et al., 2000; Huang et al., 2007; Kaiser et al., 2008; Rodriguez-Gil and Greer, 2008; Yang et al., 2005; Yang et al., 2002), or via data made available on-line in the case of the pan-TLE antiserum (http://www.cellsignal.com/products/4681.html). A Cy3-conjugated donkey anti-rabbit secondary antibody and a donkey anti-goat antibody (#711-165-152, #705-165-147, Jackson ImmunoReseach) were used at a dilution of 1:1000. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Uncx (Phd1, Chx4) is a paired homeobox transcription factor gene. It and its probable functional partners, Tle co-repressors, were expressed by neurally-fated basal progenitor cells and olfactory sensory neurons of the olfactory epithelium. Uncx expression was rare in olfactory epithelia of Ascl1(-/-) mice, but common in Neurog1(-/-) mice. In Uncx(-/-) mice olfactory progenitor cell proliferation, progenitor cell number, olfactory sensory neuron survival, and Umodl1 and Kcnc4 mRNAs were reduced. Evidence of sensory neuron activity and functional connections to the olfactory bulb argue that decreased neuronal survival was not due to loss of trophic support or activity-dependent mechanisms. These data suggest that UNCX acts downstream of neural determination factors to broadly control transcriptional mechanisms used by neural progenitor cells to specify neural phenotypes.
    Molecular and Cellular Neuroscience 12/2010; 45(4):398-407. DOI:10.1016/j.mcn.2010.07.013 · 3.73 Impact Factor
  • Source
    • "It does so on western blots only when apoptosis-initiating proteases such as caspase-8 and caspase-9 are active, and it does not detect the 17kDa fragment if these enzymes are inhibited (Cheong et al., 2003). Its ability to label cells in tissue sections is directly correlated with both the detection of the active 17kDa fragment in western blots and the presence of conditions known to cause apoptosis (Cheong et al., 2003;Hu et al., 2000;Kaiser et al., 2008). To confirm the ability of this antiserum to bind its antigen, we used a 10-fold molar excess of the antigen to block immunoreactivity (#1050, Cell Signaling Technology, Inc.), and as a control used a 10-fold molar excess of an irrelevant peptide antigen (KNNLKECGLY, #sc-262 P, "
    [Show abstract] [Hide abstract]
    ABSTRACT: More than any other neuron, olfactory sensory neurons are exposed to environmental insults. Surprisingly, their only documented response to damaging stress is apoptosis and subsequent replacement by new neurons. However, they expressed unfolded protein response genes, a transcriptionally regulated defense mechanism activated by many types of insults. The unfolded protein response transcripts Xbp1, spliced Xbp1, Chop (Ddit3), and BiP (Hspa5) were decreased when external access of stressors was reduced by blocking a nostril (naris occlusion). These transcripts and Nrf2 (Nfe2l2) were increased by systemic application of tunicamycin or the selective olfactotoxic chemical methimazole. Methimazole's effects overcame naris occlusion, and the unfolded protein response was independent of odor-evoked neuronal activity. Chemical stress is therefore a major and chronic activator of the unfolded protein response in olfactory sensory neurons. Stress-dependent repression of the antiapoptotic gene Bcl2 was absent, however, suggesting a mechanism for disconnecting the UPR from apoptosis and tolerating a chronic unfolded protein response. Environmental stressors also affect both the sustentacular cells that support the neurons and the respiratory epithelia, because naris occlusion decreased expression of the xenobiotic chemical transformation enzyme Cyp2a5 in sustentacular cells, and both naris occlusion and methimazole altered the abundance of the antibacterial lectin Reg3g in respiratory epithelia.
    The Journal of Comparative Neurology 05/2010; 518(10):1825-36. DOI:10.1002/cne.22305 · 3.51 Impact Factor
Show more