Blanca 1 Jesse Blanca CD 652 Audiology Marcia Raggio, Ph.

D San Francisco State University Friday, November 25, 2011

Preventing Hair Cell Death with Hsp70 Aminoglycosides are types of antibiotics used to treat (Gram-negative; proteobacteria, E Coli, Salmonella, Shingella, Legionella) aggressive viral, bacterial infections and diseases. The Food and Drug Administration approves of nine aminoglycosides (gentamicin, tobramycin, amikacin, streptomycin, neomycin, paromomycin, netlimicin, spectinomycin and Kanamycin) for clinical use in the United States. Aminoglycosides are inexpensive to produce and have a high efficacy rate, which makes this type of antibiotic very popular around the world. Unfortunately the side effects are drastic that tends to be organ specific, all aminoglycosides are ototoxic that can also be cochleotoxic IHC/OHC target specific others are vestibulotoxic hair-cell target specific and all are to a varying degree nephrotoxic (toxic to kidney). Significant hearing loss and vestibular disturbances occurs for nearly 15-20% of patients (Forge and Schacht 2000) who receive aminoglycosides (variance occurs with dosage/s and length of treatment). Researchers have found that Heat-Shocked-Protein (Hsp) can help mitigate the effects of ototoxicity (Cell Stress and Chaperones, journal no. 12192). Mechanosensory hair cells found in the auditory and vestibular areas of the inner ear are affected by aminoglycosides toxicity by inducing a kind of apoptopic cell death (programmed cell death, PCD) at a molecular level. Mammalian hair cells are non-regenerative making damage terminal. Aminoglycoside induced apoptotic cell death has been isolated to two specific apoptotic proteins, c-Jun N-terminal (JNK) and caspase activation (cystein-aspartic proteases). JNK is activated by the aminoglycosides ability to generate within the inner ear Reactive Oxygen Species (ROS), which are chemically-reactive molecules that play a role in cell signaling and homeostasis. In the case of apoptotic hair-cell death ROS is triggered to such high levels sufficient to create an oxidative stress cell environment (Tabuchi, Nishimura, Nakamagoe, Hayashi, Nakayama, Hara). The two types of hair cells (IHC, OHC) in the cochlea have different sensitivity to aminoglycoside as demonstrated by the types of damages that occur in hearing. The most susceptible to aminoglycosides are the outer-hair-cells (OHC) and even in the most severe cases inner-hair-cells (IHC) survivability remains high. It is also known that aminoglycosides affects the OHC spatial sensitivity, more in the basal turn than the apical turn. Suggesting that high frequency to mid frequencies are the most effected in hearing loss.

Blanca 2 Researchers from the Medical University of South Carolina and UC San Diego published a paper titled Hsp 70 Inhibits Aminoglycoside-induced Hearing Loss and Cochlear Hair Cell Death (Cell Stress Society International January 15 2009). The research paper details the result of an experiment with the potential of alleviating the ototoxic side effects of aminoglycosides by using over-expressed heat-shocked proteins. Mona Taleb, Carlene S. Brandon, Lisa L. Cunningham (Department of Pathology and Laboratory Medicine, Medical University of South Carolina) Fu-Shing Lee, Kelly C. Harris, (Department of Otolaryngology-Head and Neck Surgery, Medical University of South Carolina) and Wolfgang H. Dillmann, (Department of Endocrinology, School of Medicine UCSD) conducted lab experiments that inhibited JNK and Caspase from inducing hair-cell deaths. The induction of over-expressed (upregulated over 250 times at the mRNA level) heat shock proteins (Hsp) during lab experiments had significant results in lab rats. Hsp70 protein being the most promising in the study and would be the principle in the experiment. Heat shocked proteins (Hsps) are a class of proteins found in virtually all living organism, in humans and in bacteria. Often coined as, molecular chaperones Hsps are primal instrument of cellular protection (cytoprotective) that is expressed during a barrage of stresses. Hsps responds to hyperthermia, oxygen radicals, heavy metals, ethanol, amino acid analogues, which externally can be manifested as infections, inflammation, rigorous physical activity, hunger, hypoxia, water deprivation among others, consequently Hsp is also called the stress protein. Hsps are commonly named after their molecular weight, Hsp60, Hsp70, etc. The 70 measured in kilodaltons or amu (unified atomic mass unit), 1 unit being 1.660538921(73) 1027 kg. What the study did is use Hsp70 OE protein in reinforcing (Hsp70 OverExpressed, more mRNA than normal on a specific protein, a common biotech technique) the hair cells in both the auditory and vestibular system. Thereby creating a molecular-protein buffer for the toxic effect of the aminoglycoside in vivo. Method of the Experiment The experimental design involved carefully bred mice, twenty-two to be exact, 10 with over-expressed (OE) Hsp70 protein and twelve wild-type mice, using both gender. Distributed randomly, at only 4 weeks old the mice underwent auditorybrainstem response (ABR) threshold test. The test was used to establish threshold levels for both the control substance and the actual aminoglycoside to be used. The result of ototoxicity is often threshold-shift (TS or STS Standard Threshold Shift OSHA avg loss of 10dB or more at 2000, 3000, or 4000 Hz in either ear), a process in which when certain hair-cells of a particular frequency dies, then hair cells of other frequencies move over to the vacant space. The mice were placed on a warming pad during ABR, using platinum subcutaneous needle electrodes the responses were recorded on the TDT System 3, a Neurophysiology Workstation. Inserting the non-inverting electrode on the vertex midline and over the mastoid of the right ear then a common electrode was placed

Blanca 3 subcutaneously on the left upper leg. A tone burst of 3ms duration on a sampling rate of 50kHz with a 1 ms rise/fall times delivered via a free-field loudspeaker at 4, 8, 11.3, 16, 22.4 and 32 kHz was done at a rate of 31/s. The mice ear was 2.5 cm from the loudspeaker and at 0 azimuth. A Medusa 4-channel (TDT RA4PA) preamplifier obtained the responses. Filtering was done at 60Hz for the notch, 3kHz for the low pass and 300 Hz for the high pass. 1st recording was at 95-105 dB SPL with a 10dB step graduation until no response was recordable. Again from 0dB to a gradual increase of 5dB till no recordable response was seen. After which the threshold responses was given to a certified audiologist for evaluation. Then the aminoglycoside antibiotic Kanamycin or saline was administered regimentally in vivo for 14 days. Kanamycin was selected for this experiment because it was shown to be more cochleotoxic than vetibulotoxic (Aran, Taylor et. al 1995). Kanamycin sulfate powder was dissolved in a sterile saline with a final concentration of 45mg/ml with an experimental dosage target of 700mg/kg. Administered at 0.02 ml/g per body weight of the mice twice per day. All Hsp70 over-expressing (n=5) mice and wild-type mice (n= 6) received the same Kanamycin target dose of 700mg/kg . The control group, (n=5) Hsp70 over-expressing mice and wild type mice received subcutaneous injections of saline. After which the mice were euthanized and tissue samples were collected for immunochemistry examination. Preparation for examination required the disassembly of the vestibular and auditory system of each mouse. Including the removal of the utricles and the stapes from the oval window. The cochleas were perfuse-in ice-cold para-formaldehyde at about 4% concentration through a pinhole opening on the apical end of the cochlea. The dissected cochlea and exposed hair cells were labeled immunohistochemically. A Zeiss Axioplan 2 (high resolution microscope with a digital monochrome camera) was used in the fluorescent microscopy as well as the hair cell counts. Statistical analysis was done under one-way or two-way analysis of variance (ANOVA) via the SYSTAT 8.0 software. Results The mice injected with saline both the Hsp70 OE and Wild-Type had no significant hearing loss. Saline mice where tested at 4kHz: F 1,17 = 0.25 and p> 0.05; 8 kHz: F 1,17 = 0.01 and p > 0.05; 11.3 kHz: F1,17 = 0.07 and p >0.05; 16kHz: F1,17 = 0.07 and p> 0.05; 22.4 kHz:F1,17 = 0.36 and P>0.05; 32 kHz: F 1,17 = 0.00 and P > 0.05. While the WildType mice with Kanamycin had severe hearing loss as the

Figure 1

Blanca 4 chart (figure 1) indicates, tested at 4kHz: F 1,17 = 8.39 and p< 0.05; 8 kHz: F 1,17 = 11.83 and p < 0.05; 11.3 kHz: F1,17 = 9.41 and p <0.05; 16kHz: F1,17 = 19.89 and p< 0.05; 22.4 kHz:F1,17 = 4.75 and P<0.05; 32 kHz: F 1,17 = 5.11 and P < 0.05;. Hsp70 OE with Kanamycin had some threshold shift but a significant amount of the hair cells survived, testing at; 4kHz: F 1,17 = 8.39 and p< 0.05; 8 kHz: F 1,17 = 11.84 and p < 0.05; 11.3 kHz: F1,17 = 9.41 and p <0.05; 16kHz: F1,17 = 19.89 and p< 0.05; 22.4 kHz:F1,17 = 4.75 and P<0.05; 32 kHz: F 1,17 = 5.11 and P < 0.05;. Wild-type mice with Kanamycin had drastic threshold shift significantly at higher frequencies. At 22.4 kHz range, three Wild-Type Kamamycin mice were hearing impaired and all five had hearing loss at 32kHz. Contrasted to Hsp70 overexpressed mice that had Kanamycin resulted in a dramatically lower loss in threshold shift ( Hsp70 OE Kanamycin). Aminoglycosides specific effect on OHC can be seen on figure 2, slide Base-K-Kanamycin-Wild-Type shows no OHC that survived the experiment. Slide Middle-G-Kanamycin-Wild-Type also shows significant OHC death on the middle frequency range.

Figure 2

Blanca 5 The future of Hsp70 Heat shocked protein induction shows promise in the survival of hair cells during the presence of aminoglycoside in the system. The utricles of the mouse with overexpressed Hsp70 were also protected from apoptotic hair-cell death. The paper postulates that, The protective effect of Hsp 70 against aminoglycoside-induced hair cell death was observed at a range of neomycin concentrations, suggesting that induction of Hsp70 may hold the potential to be developed into a relevant clinical cotherapy for patients receiving aminoglycoside antibiotics (M. Taleb et al) suggesting that further study could lead to a viable agent against ototoxicity both in cochleotoxic and vetibulotoxic effect. The use of aminoglycoside type of antibiotic is often used as a last resort. In the case of Tobramycin its either death or the ototoxic side effect. Newborns with a severe fever are often administered a two-month regimen of Gentamicin in conjunction with Ampacilin. Staphylococcus Aureus (Staph Infection) is an aggressive bacterium is also treated with Gentamicin. Further discussion on the efficacy of aminoglycoside within the medical industry persistently leans on the basic equation of hearing loss vs. death. Fortunately a multi faceted approach to mitigating otoxicity and hair-cell death is being studied and experimented. From the potential of stem cells re-generating dead or weakened hair-cells to Nsp70 OE future clinical viability. Even the need to evolved aminoglycosides due to some drug-resistant bacteria fostered creation of semisynthetic derivatives named 3rd generation aminoglycosides (Davis, J. E ). Hearing loss throughout the lifespan of any individual warrants serious research consideration. With current biotechnology and medical research the boundary of hearing is no longer relegated on the periphery. Reinforcement of the IHC/OHC with HSP shows promise to an otherwise one-way compromise of life or hearing loss.

Blanca 6 Source: Taleb, M. , Brandon, C. , Lee, F. , Harris, K. , Dillmann, W. , et al. (2009). Hsp70 inhibits aminoglycoside-induced hearing loss and cochlear hair cell death. Cell Stress & Chaperones, 14(4), 427-437. References: Pandit, S R. (2011). Functional effects of adult human olfactory stem cells on earlyonset sensorineural hearing loss. Stem cells, 29(4), 670. Bremer, H. , de Groot, J. , Versnel, H. , & Klis, S. (2011). Combined administration of Kanamycin and furosemide does not result in loss of vestibular function in guinea pigs. Audiology & Neuro-Otology, 17(1), 25-38. Tabuchi, K. , Nishimura, B. , Nakamagoe, M. , Hayashi, K. , Nakayama, M. , et al. (2011). Ototoxicity: Mechanisms of cochlear impairment and its prevention. Current Medicinal Chemistry, 18(31), 4866-4871. Julian E. Davies Reviews of Infectious Diseases , Vol. 5, Supplement 2. A Clinical Perspective of Antibiotic Therapy: Aminoglycosides vs. Broad-Spectrum -Lactams (May - Jun., 1983), pp. S261-S267 (Resistance to Aminoglycosides: Mechanisms and Frequency [with Discussion] ) Published by: Oxford University Press Article Stable URL: http://0-www.jstor.org.opac.sfsu.edu/stable/4453102