The Experts below are selected from a list of 17898 Experts worldwide ranked by ideXlab platform
Edwin W Rubel - One of the best experts on this subject based on the ideXlab platform.
-
response of mechanosensory hair cells of the zebrafish lateral line to aminoglycosides reveals distinct cell death pathways
Hearing Research, 2009Co-Authors: Kelly N Owens, Edwin W Rubel, Allison B Coffin, Lisa S Hong, Keri O Bennett, David W RaibleAbstract:Abstract We report a series of experiments investigating the kinetics of hair cell loss in lateral line neuromasts of zebrafish larvae following exposure to aminoglycoside antibiotics. Comparisons of the rate of hair cell loss and the differential effects of acute versus chronic exposure to gentamicin and Neomycin revealed markedly different results. Neomycin induced rapid and dramatic concentration-dependent hair cell loss that is essentially complete within 90 min, regardless of concentration or exposure time. Gentamicin-induced loss of half of the hair cells within 90 min and substantial additional loss, which was prolonged and cumulative over exposure times up to at least 24 h. Small molecules and genetic mutations that inhibit Neomycin-induced hair cell loss were ineffective against prolonged gentamicin exposure supporting the hypothesis that these two drugs are revealing at least two cellular pathways. The mechanosensory channel blocker amiloride blocked both Neomycin and gentamicin-induced hair cell death acutely and chronically indicating that these aminoglycosides share a common entry route. Further tests with additional aminoglycosides revealed a spectrum of differential responses to acute and chronic exposure. The distinctions between the times of action of these aminoglycosides indicate that these drugs induce multiple cell death pathways.
-
notch signaling regulates the extent of hair cell regeneration in the zebrafish lateral line
The Journal of Neuroscience, 2008Co-Authors: Eva Y, Edwin W Rubel, David W RaibleAbstract:Mechanosensory hair cells within the zebrafish lateral line spontaneously regenerate after aminoglycoside-induced death. Exposure of 5-d-old larvae to 400 μm Neomycin for 1 h results in death of almost all lateral line hair cells. Regeneration of new hair cells is observed by 24 h after Neomycin treatment with nearly complete replacement by 72 h. Using bromodeoxyuridine incorporation, we show that the majority of new hair cells are generated from a transient increase in support cell proliferation that occurs between 12 and 21 h after Neomycin damage. Additional observations reveal two distinct subsets of proliferating support cells within the neuromasts that differ in position, morphology, and temporal pattern of proliferation in response to Neomycin exposure. We hypothesize that proliferative hair cell progenitors are located centrally within the neuromasts, whereas peripheral support cells may have a separate function. Expression of Notch signaling pathway members notch3 , deltaA , and atoh1a transcripts are all upregulated within the first 24 h after Neomycin treatment, during the time of maximum proliferation of support cells and hair cell progenitor formation. Treatment with a γ-secretase inhibitor results in excess regenerated hair cells by 48 h after Neomycin-induced death but has no effect without previous damage. Excess hair cells result from increased support cell proliferation. These results suggest a model where Notch signaling limits the number of hair cells produced during regeneration by regulating support cell proliferation.
-
ultrastructural analysis of aminoglycoside induced hair cell death in the zebrafish lateral line reveals an early mitochondrial response
The Journal of Comparative Neurology, 2007Co-Authors: Kelly N Owens, Edwin W Rubel, Glen Macdonald, David W Raible, Dale E Cunningham, Remy PujolAbstract:Loss of the mechanosensory hair cells in the auditory and vestibular organs leads to hearing and balance deficits. To investigate initial, in vivo events in aminoglycoside-induced hair cell damage, we examined hair cells from the lateral line of the zebrafish, Danio rerio. The mechanosensory lateral line is located externally on the animal and therefore allows direct manipulation and observation of hair cells. Labeling with vital dyes revealed a rapid response of hair cells to the aminoglycoside Neomycin. Similarly, ultrastructural analysis revealed structural alteration among hair cells within 15 minutes of Neomycin exposure. Animals exposed to a low, 25-μM concentration of Neomycin exhibited hair cells with swollen mitochondria, but little other damage. Animals treated with higher concentrations of Neomycin (50–200 μM) had more severe and heterogeneous cellular changes, as well as fewer hair cells. Both necrotic-like and apoptotic-like cellular damage were observed. Quantitation of the types of alterations observed indicated that mitochondrial defects appear earlier and more predominantly than other structural alterations. In vivo monitoring demonstrated that mitochondrial potential decreased following Neomycin treatment. These results indicate that perturbation of the mitochondrion is an early, central event in aminoglycoside-induced damage. J. Comp. Neurol. 502:522–543, 2007. © 2007 Wiley-Liss, Inc.
-
jnk signaling in Neomycin induced vestibular hair cell death
Hearing Research, 2006Co-Authors: Kazuma Sugahara, Edwin W Rubel, Lisa L CunninghamAbstract:Mechanosensory hair cells are susceptible to apoptotic death in response to exposure to ototoxic drugs, including aminoglycoside antibiotics. The c-Jun n-terminal kinase (JNK) is a stress-activated protein kinase that can promote apoptotic cell death in a variety of systems. Inhibition of the JNK signaling pathway can prevent aminoglycoside-induced death of cochlear and vestibular sensory hair cells. We used an in vitro preparation of utricles from adult mice to examine the role of JNK activation in aminoglycoside-induced hair cell death. CEP-11004 was used as an indirect inhibitor of JNK signaling. Immunohistochemistry showed that both JNK and its downstream target c-Jun are phosphorylated in hair cells of utricles exposed to Neomycin. CEP-11004 inhibited Neomycin-induced phosphorylation of both JNK and c-Jun. CEP-11004 inhibited hair cell death in utricles exposed to moderate doses of Neomycin. However, the results were not uniform across the dose-response function; CEP-11004 did not inhibit hair cell death in utricles exposed to high-dose Neomycin. The CEP-11004-induced protective effect was not due to inhibition of PKC or p38, since neither Chelerythrine nor SB203580 could mimic the protective effect of CEP-11004. In addition, inhibition of JNK inhibited the activation of caspase-9 in hair cells. These results indicate that JNK plays an important role in Neomycin-induced vestibular hair cell death and caspase-9 activation.
-
Neomycin induced hair cell death and rapid regeneration in the lateral line of zebrafish danio rerio
Jaro-journal of The Association for Research in Otolaryngology, 2003Co-Authors: Julie A Harris, Lisa L Cunningham, Alan G Cheng, Glen Macdonald, David W Raible, Edwin W RubelAbstract:Mechanoreceptive hair cells are extremely sensitive to aminoglycoside antibiotics, including Neomycin. Hair cell survival was assessed in larval wild-type zebrafish lateral line neuromasts 4 h after initial exposure to a range of Neomycin concentrations for 1 h. Each of the lateral line neuromasts was scored in live fish for the presence or absence of hair cells using the fluorescent vital dye DASPEI to selectively label hair cells. All neuromasts were devoid of DASPEI-labeled hair cells 4 h after 500 µM Neomycin exposure. Vital DASPEI staining was proportional to the number of hair cells per neuromast identified in fixed larvae using immunocytochemistry for acetylated tubulin and phalloidin labeling. The time course of hair cell regeneration in the lateral line neuromasts was also analyzed following Neomycin-induced damage. Regenerated hair cells were first observed using live DASPEI staining 12 and 24 h following Neomycin treatment. The potential role of proliferation in regenerating hair cells was analyzed. A 1 h pulse-fix protocol using bromodeoxyuridine (BrdU) incorporation was used to identify S-phase cells in neuromasts. BrdU incorporation in Neomycin-damaged neuromasts did not differ from control neuromasts 4 h after drug exposure but was dramatically upregulated after 12 h. The proliferative cells identified during a 1 h period at 12 h after Neomycin treatment were able to give rise to new hair cells by 24–48 h after drug treatment. The results presented here provide a standardized preparation for studying and identifying genes that influence vertebrate hair cell death, survival, and regeneration following ototoxic insults.
David W Raible - One of the best experts on this subject based on the ideXlab platform.
-
response of mechanosensory hair cells of the zebrafish lateral line to aminoglycosides reveals distinct cell death pathways
Hearing Research, 2009Co-Authors: Kelly N Owens, Edwin W Rubel, Allison B Coffin, Lisa S Hong, Keri O Bennett, David W RaibleAbstract:Abstract We report a series of experiments investigating the kinetics of hair cell loss in lateral line neuromasts of zebrafish larvae following exposure to aminoglycoside antibiotics. Comparisons of the rate of hair cell loss and the differential effects of acute versus chronic exposure to gentamicin and Neomycin revealed markedly different results. Neomycin induced rapid and dramatic concentration-dependent hair cell loss that is essentially complete within 90 min, regardless of concentration or exposure time. Gentamicin-induced loss of half of the hair cells within 90 min and substantial additional loss, which was prolonged and cumulative over exposure times up to at least 24 h. Small molecules and genetic mutations that inhibit Neomycin-induced hair cell loss were ineffective against prolonged gentamicin exposure supporting the hypothesis that these two drugs are revealing at least two cellular pathways. The mechanosensory channel blocker amiloride blocked both Neomycin and gentamicin-induced hair cell death acutely and chronically indicating that these aminoglycosides share a common entry route. Further tests with additional aminoglycosides revealed a spectrum of differential responses to acute and chronic exposure. The distinctions between the times of action of these aminoglycosides indicate that these drugs induce multiple cell death pathways.
-
notch signaling regulates the extent of hair cell regeneration in the zebrafish lateral line
The Journal of Neuroscience, 2008Co-Authors: Eva Y, Edwin W Rubel, David W RaibleAbstract:Mechanosensory hair cells within the zebrafish lateral line spontaneously regenerate after aminoglycoside-induced death. Exposure of 5-d-old larvae to 400 μm Neomycin for 1 h results in death of almost all lateral line hair cells. Regeneration of new hair cells is observed by 24 h after Neomycin treatment with nearly complete replacement by 72 h. Using bromodeoxyuridine incorporation, we show that the majority of new hair cells are generated from a transient increase in support cell proliferation that occurs between 12 and 21 h after Neomycin damage. Additional observations reveal two distinct subsets of proliferating support cells within the neuromasts that differ in position, morphology, and temporal pattern of proliferation in response to Neomycin exposure. We hypothesize that proliferative hair cell progenitors are located centrally within the neuromasts, whereas peripheral support cells may have a separate function. Expression of Notch signaling pathway members notch3 , deltaA , and atoh1a transcripts are all upregulated within the first 24 h after Neomycin treatment, during the time of maximum proliferation of support cells and hair cell progenitor formation. Treatment with a γ-secretase inhibitor results in excess regenerated hair cells by 48 h after Neomycin-induced death but has no effect without previous damage. Excess hair cells result from increased support cell proliferation. These results suggest a model where Notch signaling limits the number of hair cells produced during regeneration by regulating support cell proliferation.
-
ultrastructural analysis of aminoglycoside induced hair cell death in the zebrafish lateral line reveals an early mitochondrial response
The Journal of Comparative Neurology, 2007Co-Authors: Kelly N Owens, Edwin W Rubel, Glen Macdonald, David W Raible, Dale E Cunningham, Remy PujolAbstract:Loss of the mechanosensory hair cells in the auditory and vestibular organs leads to hearing and balance deficits. To investigate initial, in vivo events in aminoglycoside-induced hair cell damage, we examined hair cells from the lateral line of the zebrafish, Danio rerio. The mechanosensory lateral line is located externally on the animal and therefore allows direct manipulation and observation of hair cells. Labeling with vital dyes revealed a rapid response of hair cells to the aminoglycoside Neomycin. Similarly, ultrastructural analysis revealed structural alteration among hair cells within 15 minutes of Neomycin exposure. Animals exposed to a low, 25-μM concentration of Neomycin exhibited hair cells with swollen mitochondria, but little other damage. Animals treated with higher concentrations of Neomycin (50–200 μM) had more severe and heterogeneous cellular changes, as well as fewer hair cells. Both necrotic-like and apoptotic-like cellular damage were observed. Quantitation of the types of alterations observed indicated that mitochondrial defects appear earlier and more predominantly than other structural alterations. In vivo monitoring demonstrated that mitochondrial potential decreased following Neomycin treatment. These results indicate that perturbation of the mitochondrion is an early, central event in aminoglycoside-induced damage. J. Comp. Neurol. 502:522–543, 2007. © 2007 Wiley-Liss, Inc.
-
Neomycin induced hair cell death and rapid regeneration in the lateral line of zebrafish danio rerio
Jaro-journal of The Association for Research in Otolaryngology, 2003Co-Authors: Julie A Harris, Lisa L Cunningham, Alan G Cheng, Glen Macdonald, David W Raible, Edwin W RubelAbstract:Mechanoreceptive hair cells are extremely sensitive to aminoglycoside antibiotics, including Neomycin. Hair cell survival was assessed in larval wild-type zebrafish lateral line neuromasts 4 h after initial exposure to a range of Neomycin concentrations for 1 h. Each of the lateral line neuromasts was scored in live fish for the presence or absence of hair cells using the fluorescent vital dye DASPEI to selectively label hair cells. All neuromasts were devoid of DASPEI-labeled hair cells 4 h after 500 µM Neomycin exposure. Vital DASPEI staining was proportional to the number of hair cells per neuromast identified in fixed larvae using immunocytochemistry for acetylated tubulin and phalloidin labeling. The time course of hair cell regeneration in the lateral line neuromasts was also analyzed following Neomycin-induced damage. Regenerated hair cells were first observed using live DASPEI staining 12 and 24 h following Neomycin treatment. The potential role of proliferation in regenerating hair cells was analyzed. A 1 h pulse-fix protocol using bromodeoxyuridine (BrdU) incorporation was used to identify S-phase cells in neuromasts. BrdU incorporation in Neomycin-damaged neuromasts did not differ from control neuromasts 4 h after drug exposure but was dramatically upregulated after 12 h. The proliferative cells identified during a 1 h period at 12 h after Neomycin treatment were able to give rise to new hair cells by 24–48 h after drug treatment. The results presented here provide a standardized preparation for studying and identifying genes that influence vertebrate hair cell death, survival, and regeneration following ototoxic insults.
Dev P Arya - One of the best experts on this subject based on the ideXlab platform.
-
targeting c myc g quadruplex dual recognition by aminosugar bisbenzimidazoles with varying linker lengths
Molecules, 2013Co-Authors: Nihar Ranjan, Dev P AryaAbstract:G-quadruplexes are therapeutically important biological targets. In this report, we present biophysical studies of Neomycin-Hoechst 33258 conjugates binding to a G-quadruplex derived from the C-myc promoter sequence. Our studies indicate that conjugation of Neomycin to a G-quadruplex binder, Hoechst 33258, enhances its binding. The enhancement in G-quadruplex binding of these conjugates varies with the length and composition of the linkers joining the Neomycin and Hoechst 33258 units.
-
recognition of hiv tar rna using Neomycin benzimidazole conjugates
Bioorganic & Medicinal Chemistry Letters, 2013Co-Authors: Nihar Ranjan, Sunil Kumar, Deyun Wang, Daniel H. Appella, Derrick Watkins, Dev P AryaAbstract:Abstract Synthesis of a novel class of compounds and their biophysical studies with TAR-RNA are presented. The synthesis of these compounds was achieved by conjugating Neomycin, an aminoglycoside, with benzimidazoles modeled from a B-DNA minor groove binder, Hoechst 33258. The Neomycin–benzimidazole conjugates have varying linkers that connect the benzimidazole and Neomycin units. The linkers of varying length (5–23 atoms) in these conjugates contain one to three triazole units. The UV thermal denaturation experiments showed that the conjugates resulted in greater stabilization of the TAR-RNA than either Neomycin or benzimidazole used in the synthesis of conjugates. These results were corroborated by the FID displacement and tat-TAR inhibition assays. The binding of ligands to the TAR-RNA is affected by the length and composition of the linker. Our results show that increasing the number of triazole groups and the linker length in these compounds have diminishing effect on the binding to TAR-RNA. Compounds that have shorter linker length and fewer triazole units in the linker displayed increased affinity towards the TAR RNA.
-
Click Dimers To Target HIV TAR RNA Conformation
2012Co-Authors: Sunil Kumar, Patrick Kellish, Edward W. Robinson, Deyun Wang, Daniel H. Appella, Dev P AryaAbstract:A series of Neomycin dimers have been synthesized using “click chemistry” with varying functionality and length in the linker region to target the human immunodeficiency virus type 1 (HIV-1) TAR RNA region of the HIV virus. The TAR (Trans-Activation Responsive) RNA region, a 59 bp stem–loop structure located at the 5′-end of all nascent viral transcripts, interacts with its target, a key regulatory protein, Tat, and necessitates the replication of HIV-1. Neomycin, an aminosugar, has been shown to exhibit multiple binding sites on TAR RNA. This observation prompted us to design and synthesize a library of triazole-linked Neomycin dimers using click chemistry. The binding between Neomycin dimers and TAR RNA was characterized using spectroscopic techniques, including FID (fluorescent intercalator displacement), a FRET (fluorescence resonance energy transfer) competitive assay, circular dichroism (CD), and UV thermal denaturation. UV thermal denaturation studies demonstrate that binding of Neomycin dimers increases the melting temperature (Tm) of the HIV TAR RNA up to 10 °C. Ethidium bromide displacement (FID) and a FRET competition assay revealed nanomolar binding affinity between Neomycin dimers and HIV TAR RNA, while in case of Neomycin, only weak binding was detected. More importantly, most of the dimers exhibited lower IC50 values toward HIV TAR RNA, when compared to the fluorescent Tat peptide, and show increased selectivity over mutant TAR RNA. Cytopathic effects investigated using MT-2 cells indicate a number of the dimers with high affinity toward TAR show promising anti-HIV activity
-
sequence specific targeting of rna with an oligonucleotide Neomycin conjugate
Bioconjugate Chemistry, 2007Co-Authors: I Charles, Dev P AryaAbstract:The synthesis of Neomycin covalently attached at the C5-position of 2′-deoxyuridine is reported. The synthesis outlined allows for incorporation of an aminoglycoside (Neomycin) at any given site in an oligonucleotide (ODN) where a thymidine (or uridine) is present. Incorporation of this modified base into an oligonucleotide, which is complementary to a seven-bases-long R-sarcin loop RNA sequence, leads to enhanced duplex hybridization. The increase in Tm for this duplex (¢Tm ) 6 °C) suggests a favorable interaction of Neomycin within the duplex groove. CD spectroscopy shows that the modified duplex adopts an A-type confirmation. ITC measurements indicate the additive effects of ODN and Neomycin binding to the RNA target (Ka ) 4.5 10 7 M -1 ). The enhanced stability of the hybrid duplex from this Neomycin-ODN conjugate originates primarily from the enthalpic contribution of Neomycin {¢¢Hobs )- 7.21 kcal/mol (¢HNeomycin conjugated - ¢H nonconjugated)} binding to the hybrid duplex. The short linker length allows for selective stabilization of the hybrid duplex over the hybrid triplex. The results described here open up new avenues in the design and synthesis of nucleo-aminoglycoside-conjugates (N-Ag-C) where the inclusion of any number of aminoglycoside (Neomycin) molecules per oligonucleotide can be accomplished.
-
aminoglycoside nucleic acid interactions remarkable stabilization of dna and rna triple helices by Neomycin
Journal of the American Chemical Society, 2001Co-Authors: Dev P Arya, R L Coffee, Bert Willis, A I AbramovitchAbstract:The stabilization of poly(dA).2poly(dT) triplex, a 22-base DNA triplex, and poly(rA).2poly(rU) triple helix by Neomycin is reported. The melting temperatures, the association and dissociation kinetic parameters, and activation energies (E(on) and E(off)) for the poly(dA).2poly(dT) triplex in the presence of aminoglycosides and other triplex binding ligands were determined by UV thermal analysis. Our results indicate that: (i) Neomycin stabilizes DNA triple helices, and the double helical structures composed of poly(dA).poly(dT) are virtually unaffected. (ii) Neomycin is the most active and triplex-selective stabilization agent among all aminoglycosides, previously studied minor groove binders, and polycations. Its selectivity (DeltaT(m3-->2) vs DeltaT(m2)(-->)(1)) exceeds most intercalating drugs that bind to triple helices. (iii) Neomycin selectively stabilizes DeltaT(m3)(-->)(2) for a mixed 22-base DNA triplex containing C and T bases in the pyrimidine strand. (iv) The rate constants of formation of triplex (k(on)) are significantly enhanced upon increasing molar ratios of Neomycin, making triplex association rates closer to duplex association rates. (v) E(on) values become more negative upon increasing concentration of aminoglycosides (paromomycin and Neomycin). E(off) values do not show any change for most aminoglycosides except Neomycin. (vi) Aminoglycosides can effectively stabilize RNA [poly(rA).2poly(rU)] triplex, with Neomycin[being one of the most active ligands discovered to date (second only to ellipticine). (vii) The stabilization effect of aminoglycosides on triple helices is parallel to their toxic behavior, suggesting a possible role of intramolecular triple helix (H-DNA) stabilization by the aminoglycosides.
Remy Pujol - One of the best experts on this subject based on the ideXlab platform.
-
ultrastructural analysis of aminoglycoside induced hair cell death in the zebrafish lateral line reveals an early mitochondrial response
The Journal of Comparative Neurology, 2007Co-Authors: Kelly N Owens, Edwin W Rubel, Glen Macdonald, David W Raible, Dale E Cunningham, Remy PujolAbstract:Loss of the mechanosensory hair cells in the auditory and vestibular organs leads to hearing and balance deficits. To investigate initial, in vivo events in aminoglycoside-induced hair cell damage, we examined hair cells from the lateral line of the zebrafish, Danio rerio. The mechanosensory lateral line is located externally on the animal and therefore allows direct manipulation and observation of hair cells. Labeling with vital dyes revealed a rapid response of hair cells to the aminoglycoside Neomycin. Similarly, ultrastructural analysis revealed structural alteration among hair cells within 15 minutes of Neomycin exposure. Animals exposed to a low, 25-μM concentration of Neomycin exhibited hair cells with swollen mitochondria, but little other damage. Animals treated with higher concentrations of Neomycin (50–200 μM) had more severe and heterogeneous cellular changes, as well as fewer hair cells. Both necrotic-like and apoptotic-like cellular damage were observed. Quantitation of the types of alterations observed indicated that mitochondrial defects appear earlier and more predominantly than other structural alterations. In vivo monitoring demonstrated that mitochondrial potential decreased following Neomycin treatment. These results indicate that perturbation of the mitochondrion is an early, central event in aminoglycoside-induced damage. J. Comp. Neurol. 502:522–543, 2007. © 2007 Wiley-Liss, Inc.
Lisa L Cunningham - One of the best experts on this subject based on the ideXlab platform.
-
jnk signaling in Neomycin induced vestibular hair cell death
Hearing Research, 2006Co-Authors: Kazuma Sugahara, Edwin W Rubel, Lisa L CunninghamAbstract:Mechanosensory hair cells are susceptible to apoptotic death in response to exposure to ototoxic drugs, including aminoglycoside antibiotics. The c-Jun n-terminal kinase (JNK) is a stress-activated protein kinase that can promote apoptotic cell death in a variety of systems. Inhibition of the JNK signaling pathway can prevent aminoglycoside-induced death of cochlear and vestibular sensory hair cells. We used an in vitro preparation of utricles from adult mice to examine the role of JNK activation in aminoglycoside-induced hair cell death. CEP-11004 was used as an indirect inhibitor of JNK signaling. Immunohistochemistry showed that both JNK and its downstream target c-Jun are phosphorylated in hair cells of utricles exposed to Neomycin. CEP-11004 inhibited Neomycin-induced phosphorylation of both JNK and c-Jun. CEP-11004 inhibited hair cell death in utricles exposed to moderate doses of Neomycin. However, the results were not uniform across the dose-response function; CEP-11004 did not inhibit hair cell death in utricles exposed to high-dose Neomycin. The CEP-11004-induced protective effect was not due to inhibition of PKC or p38, since neither Chelerythrine nor SB203580 could mimic the protective effect of CEP-11004. In addition, inhibition of JNK inhibited the activation of caspase-9 in hair cells. These results indicate that JNK plays an important role in Neomycin-induced vestibular hair cell death and caspase-9 activation.
-
Neomycin induced hair cell death and rapid regeneration in the lateral line of zebrafish danio rerio
Jaro-journal of The Association for Research in Otolaryngology, 2003Co-Authors: Julie A Harris, Lisa L Cunningham, Alan G Cheng, Glen Macdonald, David W Raible, Edwin W RubelAbstract:Mechanoreceptive hair cells are extremely sensitive to aminoglycoside antibiotics, including Neomycin. Hair cell survival was assessed in larval wild-type zebrafish lateral line neuromasts 4 h after initial exposure to a range of Neomycin concentrations for 1 h. Each of the lateral line neuromasts was scored in live fish for the presence or absence of hair cells using the fluorescent vital dye DASPEI to selectively label hair cells. All neuromasts were devoid of DASPEI-labeled hair cells 4 h after 500 µM Neomycin exposure. Vital DASPEI staining was proportional to the number of hair cells per neuromast identified in fixed larvae using immunocytochemistry for acetylated tubulin and phalloidin labeling. The time course of hair cell regeneration in the lateral line neuromasts was also analyzed following Neomycin-induced damage. Regenerated hair cells were first observed using live DASPEI staining 12 and 24 h following Neomycin treatment. The potential role of proliferation in regenerating hair cells was analyzed. A 1 h pulse-fix protocol using bromodeoxyuridine (BrdU) incorporation was used to identify S-phase cells in neuromasts. BrdU incorporation in Neomycin-damaged neuromasts did not differ from control neuromasts 4 h after drug exposure but was dramatically upregulated after 12 h. The proliferative cells identified during a 1 h period at 12 h after Neomycin treatment were able to give rise to new hair cells by 24–48 h after drug treatment. The results presented here provide a standardized preparation for studying and identifying genes that influence vertebrate hair cell death, survival, and regeneration following ototoxic insults.
-
caspase activation in hair cells of the mouse utricle exposed to Neomycin
The Journal of Neuroscience, 2002Co-Authors: Lisa L Cunningham, Alan G Cheng, Edwin W RubelAbstract:Aminoglycoside exposure results in the apoptotic destruction of auditory and vestibular hair cells. This ototoxic hair cell death is prevented by broad-spectrum caspase inhibition. We have used in situ substrate detection, immunohistochemistry, and specific caspase inhibitors to determine which caspases are activated in the hair cells of the adult mouse utricle in response to Neomycin exposure in vitro. In addition, we have examined the hierarchy of caspase activation. Our data indicate that both upstream caspase-8 and upstream caspase-9, as well as downstream caspase-3 are activated in hair cells exposed to Neomycin. The inhibition of caspase-9-like activity provided significant protection of hair cells exposed to Neomycin, whereas the inhibition of caspase-8-like activity was not effective in preventing Neomycin-induced hair cell death. In addition, caspase-9 inhibition prevented the activation of downstream caspase-3, whereas the inhibition of caspase-8 did not. These data indicate that caspase-9 is the primary upstream caspase mediating Neomycin-induced hair cell death in this preparation.
