Overview
Nuclear lamin
molecules
DNA
Non-muscle myosin-II
molecules
Dr. O’neil W. Guthrie is the Director of the Cell
and Molecular Pathology Laboratory (CMPL). The
CMPL is broadly focused on defining the cellular
and molecular bases of diseases as a necessary
prerequisite to the development of biomedical
therapies. To this end, work in the CMPL is
identifying and manipulating endogenous
molecular mechanisms that mediate
cellular/tissue injury and repair. The laboratory
employs multiple strategies that encompasses
molecular biology, biochemistry, genetics,
epigenetics, exploratory pharmacology,
electrophysiology, microscopy, histopathology &
cytology and design-based stereology.
RESEARCH PROGRAMS
BIOMEDICINE DISCOVERY PROGRAM
The CMPL is interested in developing biomedical approaches to prevent or treat
intractable conditions such as hearing loss. Therefore, two independent approaches
are currently under research and development. The newest approach is based on the
engineering of specific molecular constructs that will increase the capacity of cells to
repair themselves after injury (further information is restricted at this time). The
other approach employs specific low molecular weight chemotypes called carboxy
alkyl esters (CAEs) to enhance recovery of sensory and neural functions following
injury. This research is important because it suggests (for the first time) that CAEs
may be used as a novel approach to prevent or treat hearing loss. The goals of this
line of research are to 1) determine the underlying molecular mechanisms that
facilitate cellular repair, 2) optimize the treatment regimen to achieve maximal repair,
3) identify other molecules that have the capacity to augment cellular repair, and 4)
conduct clinical trials to determine efficacy among patients.
What are CAEs and how do they work? CAEs are phytochemicals found in healthy
foods and beverages such as fruits, vegetables, coffee and tea. They provide direct
trophic support to cells. However, they can function through systemic metabolism.
For instance, CAEs are involved in systemic biochemical reactions that increase
bioavailability of essential nutrients. One proposed reaction that is currently under
investigation is illustrated.
OOH
HO C
OH
1
5
4
OH
1
3
Quinic acid
O
OH
OH
OH
O
(CAE)
HO
OOH
HO C
O
4
OH
H2 O
2Pi
Caffeic acid
COO
1
5
4
3
OH
NAD
ATP
PEP
OH
esterase
5
OH
A
OOH
HO C
B
H
3
OH
CH2
OH
OH Quinic acid
O
Chorismate
C
OH
Shikimate biosynthetic
pathway of the
gatrointestinal microflora
COO-
COO+
+
H3 N
C
H
CH2
synthesis of aromatic amino acids
COOH
H3 N
C
CH2
H
COO +
H3 N
C
CH2
C
HC
N
H
DNA
repair
antioxidation
NAD + ATP
synthesis
inflammatory and
immune responses
Trp
OH
Phe
Tyr
H
A) The metabolism of a CAE by enteric microbiota increases host DNA
repair capacity, antioxidation, NAD/ATP synthesis and regulates
inflammation and immune responses. B) Production of aromatic amino
acids (Trp, Phe and Tyr) by the shikimate biosynthetic pathway of the
gastrointestinal microflora. Abbreviations: NAD, nicotinamine adenine
dinucleotide; ATP, adenosine triphosphate; 2Pi, two-inorganic
phosphates; PEP, phosphoenolpyruvate; Trp, tryptophan; Phe,
Phenylalanine; Tyr, tyrosine.
CELLULAR REPAIR PROGRAM
Green = cell borders marked by the F-actin molecule
Blue and Red = DNA molecules coiled into polytene chromosomes
Excessive noise exposure, certain medications and the aging process can all lead to progressive
degeneration of a large portion of sensory neurons; however, a smaller population of sensory neurons
consistently survive. We are pursuing the hypothesis that differences in survival capacity between
neurons are dependent on the neurons’ ability to effectively mobilize a protective DNA damage
response. Noise exposure and cancer chemotherapies can independently damage DNA within
peripheral neurons leading to the loss of neural functions (e.g., hearing loss). We have shown that
only a small population of peripheral neurons are able to defend themselves by mobilizing DNA repair
pathways to repair damaged genes. However, the larger population of neurons do not express DNA
repair proteins or fail to appropriately coordinate DNA repair pathways. We suspect that these
neurons lack the repertoire necessary to effectively defend their genome beyond basal demands and
are therefore a major mediator of sensory impairments.
We are employing a range of molecular, epigenetic and pharmacologic strategies to increase the
survival of neurons by improving their capacity to defend/repair their genome. This work is relevant
to the development of biomedical approaches aimed at rescuing neurons from cell death processes.
NEUROAUDIOLOGY PROGRAM
The neuroaudiology research program is focused on delineating the underlying
neural substrates that drive specific types of auditory dysfunctions, such as
tinnitus. Tinnitus is the abnormal perception of a sound that has no external
source. The sounds heard by tinnitus suffers can be a ringing, buzzing, chirping,
roaring and/or a large variety of other types of sounds. The sound may be
intermittent or constant and may localize to the right ear, left ear, either ears or
neither ears but instead perceived in the head. The intensity of the tinnitus sound
can be so loud that patients report interference with everyday listening. The effect
of tinnitus can be so great that patients often contemplate suicide. Fifty million
Americans suffer with tinnitus and it is the most prevalent service-connected
disability among veterans (exceeding PTSD and TBI). The Veterans Administration
(VA) spent over $1.1 billion in 2010 to compensate veterans for tinnitus and is
projected to exceed $2.26 billion (ATA). Interestingly, public and private funding
for research focused on tinnitus treatment is only $10 million (ATA).
Our research is focused on an unorthodox line of thinking regarding the auditory
system and tinnitus. For instance, we are pursuing the notion that some forms of
neuronal dysfunctions that drive tinnitus may also lower the threshold of
susceptibility and/or increase severity of other neuropathologic conditions.
Supporting evidence for this notion comes from observations that 1) specific
lesions to primary auditory neurons alter excitatory and inhibitory neural circuits
in the brainstem and cortex. 2) Alterations to excitatory and inhibitory neural
circuits in the brainstem and cortex are common to tinnitus and other
neuropathologic conditions.
BRAIN INJURY PROGRAM
This research is a collaboration with the Wright-Patterson Air Force Base in Ohio.
Epidemiologic evidence suggest an increased risk of auditory impairments among Air Force personnel
exposed to Jet fuel. Therefore, the current work aims to determine whether jet fuel exposure affects the
peripheral and/or central auditory nervous system as a necessary prerequisite to the development of
novel biomedical therapies. The research has already led to several important results. First, we
discovered that low level jet fuel exposure induced a central auditory dysfunction that manifested as
impaired brainstem encoding of stimulus intensity. Second, this central auditory dysfunction was
exacerbated by background noise exposure. Third, the brainstem impairment was dominant among
neurons that are responsive to high levels of acoustic stimulation. These findings could represent
important and major shifts in the theoretical framework that governs current understanding of jet fuel
and/or noise induced ototoxicity. From a clinical perspective the results indicate that jet fuel+noise
exposure have consequences to brainstem function that may be more wide-spread and insidious than
what was previously known. Therefore, it is possible that a large population of military personnel who
are suffering with the effects of jet fuel or jet fuel+noise exposure may be misidentified because they
would exhibit normal auditory sensitivity (normal hearing thresholds) but harbor a "hidden" brainstem
dysfunction. Such brainstem dysfunctions may be associated with a large variety of clinical conditions
such as depression, anxiety, sleep disorders, post-traumatic stress, tinnitus (ringing in the ear),
hyperacusis (hypersensitivity to sounds), diploacusis (miss perception of pitch) and impaired speech
perception (in ability to understand speech sounds).
Brain Injury Program (continued):
2f1 - f2 (dB SPL)
40
A
B
30
R3
20
10
0
-10
R2
Control
Noise
Fuel
Fuel+Noise
Noise floor
R1
Nerve fibers
-20
2
4
8
16
32
64
f2 (kHz)
C
D
Control
Gain
1 µV
1 ms
Fuel+Noise
Brain Injury Program (continued):
Panel A shows the pre-synaptic sensory function of a group of animals exposed to jet
fuel+noise. In addition to this group (Fuel+Noise), there are groups that were exposed to only
noise and only jet fuel. There is also a control group which was unexposed. Note that the presynaptic function between the groups are similar. Panel B is a representative
photomicrograph which reveals that the pre-synaptic sensory cells and post-synaptic
peripheral nerve fibers were undamaged by the exposures. Panels C & D, are brainstem
recordings that illustrate that the jet fuel+noise exposures altered brain activity (p < 0.05).
Taken together, the data suggest that exposure to jet fuel combined with background noise
may preserve hearing sensitivity but impairs brain functions. All panels were taken from
Guthrie et al., 2014, J Toxicol Environ Health A.,77,261-280.
Data Presentations at Scientific Meetings
Differential DNA Damage Response Among Auditory Sensory Neurons Following Ototrauma
O'neil Guthrie Ph.D1,2, Terry Fleck MD2, Helen Xu MD1,2
Linda Veterans Affairs Hospital, 2Department of Otolaryngology – Head & Neck Surgery, School of Medicine, Loma Linda University Medical Center
1Loma
A
Introduction
-15
2
4
8
16
f2
32
25
E
0.4
0.0
Noise
XPA
**
Control
Noise
0.6
0.2
0.2
0.0
0.0
APEX
MIDDLE
APEX
MIDDLE
F
BASE
CSA
*
BASE
A
5
APEX
MIDDLE
BASE
2
4
8
16
32
64
200
D
E
nuclear
nucleoplasm
150
20
10
10
Control
Noise
1.5
nuclear translocation
Neurons/120 µm2
30
Control
Noise
0
0.4
Control
Noise
0.5
0.4
**
0.0
Noise
**
**
0.4
**
*
*
*
**
*
0.3
*
0.3
**
0.3
0.2
0.2
0.2
0.1
0.1
0.1
Control
0.0
Noise
0.5
Control
0.0
Noise
0.5
0.4
**
Control
middle
Noise
0.5
*
*
0.4
**
**
**
*
**
*
0.4
*
**
*
**
0.3
0.3
0.2
0.2
0.2
0.1
0.1
0.1
**
base
Control
0.0
Noise
Control
0.0
Noise
Control
Noise
Figure 5. Spatial remodeling of subcellular distribution patterns. Neurons in the apical cochlear coil exhibited an
increase in both nuclear and cytoplasmic retention of CSA which was accompanied by significant nuclear exclusion of
XPA. Neurons in the middle coil of the cochlea exhibited significant nuclear exclusion of both CSA and XPA. These
neurons also showed an increase in cytoplasmic retension of CSA coupled with a significant reduction of cytosplamic
XPC. Neurons in the basal cochlear coil exhibited significant nuclear retention of XPC and reduction of cytoplasmic and
nuclear XPA, [* = p < 0.05; ** = p < 0.01].
B
A
C
γH2Ax
80
0
2
4
5
7
9 11 12 14
Distance (µm)
60
F
250
Apex
Middle
Base
1.5
control
150
100
50
nucleoplasm
0
2
4
5
7
Apex
9 11 12 14
Distance (µm)
Middle
Base
1.0
0.5
Middle
60
20
195
60
apex
20
0
80
D
40
210
225
240
255
0
195
80
E
60
210
225
240
255
240
255
F
base
middle
40
40
20
20
0
0
210
225
240
255
195
210
225
gray level
Figure 7. Noise induced γ-H2Ax. (A) Photomicrograph of γ-H2Ax immunoreactivity within the spiral ganglia. (B)
Enlargement of the area outlined in (A) showing dense nuclear reactions. Note the punctus reaction products that line the
circumference of the nucleus. (C) Morphometric analysis of the reaction products with and without noise exposure. Note
that the noise exposure induced an increase in γ-H2Ax (see arrows). Panel C is the overall frequency distribution of the
density of the reaction products under control (dotted line) and noise (solid line) conditions. Panels D-F is the data for the
three different cochlear coils. In panels C-F, the ordinate axis is the frequency of occurrence in percent and the abscissa is
the measured gray level. The step tablet above panel C shows the direction of the gray levels where 0 = black (heavy
staining) and 255 = white (no staining). Scale bar in panel A = 10 µm and panel B = 20 µm.
XPA
CSA
0.0
Conclusions


Base
Figure 2. Preservation of spiral ganglion neurons. (A) Representative photomicrograph of H&E stained spiral
ganglion neurons. (B) The total number of neurons from the control or noise groups is not significantly different (p >
0.05) at 1-day post noise exposure. Furthermore, neuronal populations at the apical, middle and basal coils are similar
(p > 0.05) between the control and noise groups (C). These combined data indicates that subsequent observations were
not due to missing neurons.
Noise
Control
0.5
XPC
Figure 5. Noise induced dys-synchrony. Under control conditions, spiral ganglion neurons exhibit a general response of the following order:
Apex = XPA > CSA + XPC; Middle = XPA + CSA > XPC; Base = XPA + CSA > XPC. However, after noise stress the general response is
characterized by: Apex = CSA > XPC > XPA; Middle = XPC > CSA + XPA; Base = XPC > CSA + XPA.
Apex
80
C
40
200
noise
1.0
0.0
0
Control
0.5
50
0
C
20
0.0
Noise
**
100
0
(kHz)
B
30
Control
0.5
0.0
apex
**
250
Figure 4. Representative example of subcellular distribution patterns. (A) Photomicrograph of a field of neurons exhibiting cytoplasmic
expression. (B) Enlargement of the area outlined in (A) showing that reaction products were predominantly localized in the cytoplasm. (C)
Representative 1-pixel wide linescan demonstrating that cytoplasmic reactive neurons exhibit a specific morphologic profile where
chromogen intensity in the nucleoplasm (~4-10 µm) is minimal compared to the cytoplasm. The y-axis in panels C and F are inverted gray
(g) level (1/g). (D) Photomicrograph of a field of neurons exhibiting nuclear expression. (E) Enlargement of the area outlined in (D) showing
that reaction products were predominantly localized in the nucleus. (F) Representative 1-pixel wide linescan revealing that nuclear reactive
neurons exhibit a specific morphological profile where chromogen intensity in the nucleoplasm (~4-10 µm) is maximal compared to the
cytoplasm.
40
0.1
0.0
H&E
40
0.2
0.1
**
B
cytoplasmic
L1/L2 (65/55)
f2
50
0.2
Linescan Profiles
(kHz)
50
**
0.1
0.0
**
**
0.3
*
0.2
0.3
0.4
*
Figure 1. Loss of cochlear function from the noise (8 kHz OBN @105 dB SPL /4 h) exposure. The levels of 2f 1-f2
DPOAE as a function of f2 frequency driven with primary levels (L) at 65/55 dB SPL are shown for both the control
and noise treated groups. The gray bar in panel B represents the frequency bandwidth of the damaging noise. Note
that the nose exposure induced a significant (p < 0.05) loss of sensitivity. The gray solid line in each panel is the noise
floor. Error bars are standard errors of the means.
A
Proportion immunoreactive neurons
Proportion reactive neurons
Control
-5
-25
64
XPC
0.2
0.0
15
-15
L1/L2 (65/55)
0.2
0.6
D
0.4
**
0.3
*
Frequency (%)
5
-5
0.6
Intensity (g-1)
2f1 - f2 (dB SPL)
15
-25
Control (n=12)
Noise (n=11)
Noise Floor
35
25
C
*
0.4
*
**
0.3
negative control
0.4
CSA
0.5
*
0.4
**
*
0.6
XPA
0.5
Cytoplasmic
Nuclear
0.4
Figure 3. Heterogeneity among spiral ganglion neurons. (A) No immunoreactivity for NER proteins are found in the spiral ganglia after the
immunohistochemistry procedure with antibodies omitted. (B) A representative photomicrograph of a field of negative (arrows) and
positive immunoreactive neurons and satellite cells (*), scale bars = 20 µm. (C) A small (~40%) cohort of neurons are immunoreactive
under normal conditions and a similar cohort appears to be reactive following (1-day post) noise stress. (D) This noise stress did not cause a
significant change in the proportion of neurons that are immunoreactive for XPC, however there was a reduction in the proportion of XPA
reactive neurons at the middle and basal coils (E). Additionally, the proportion of CSA reactive neurons showed a significant reduction in
the basal coil (F), [* = p < 0.05; ** = p < 0.01].
1-day post-noise exposure
45
Control (n=12)
Noise (n=11)
Noise Floor
35
2f1 - f2 (dB SPL)
B
Baseline
45
XPC
0.5
*
Intensity (g-1)
A
B
*
Noise exposure has been shown to precipitate free radical induced DNA damage products in the cochlea. Free
radical DNA damage can result in mutated gene fragments that alter cellular functions and/or induce progressive cell death.
The nucleotide excision repair (NER) pathway is particularly adapted to protecting both active and inactive genes through
genetically distinct subpathways that require a Cockayne syndrome-A (CSA) E3-ubiquitin ligase complex
(CSA/DDB1/Cullin-4A/ROC1/Rbx1/COP9) and the xeroderma pigmentosum-C (XPC) heterotrimeric
(XPC/nHR23B/centrin2) preincision complex. The rate limiting factor in both pathways is the C4-type zinc-finger (Cys105X2-Cys108 -X17 -Cys126 -X2 -Cys129 ) scaffolding protein XPA, which positions and activates nucleases for excision of damaged
gene fragments. Noise is known to induce progressive degeneration of spiral ganglion neurons, however a subpopulation of
neurons consistently remain viable. This difference in viability might be mediated through NER. Therefore, it was posited
that noise exposure would induce differential DNA damage responses among spiral ganglion neurons. Male Long-Evans rats
were exposed to an 8 kHz octave band of noise at 105 dB SPL for 4 hours. Distortion product otoacoustic emissions were
recorded before and after noise exposure and the animals were sacrificed via transcardial perfusion for temporal bone
harvesting, immunohistochemistry and quantification of intracellular protein distribution. The results revealed that the
majority (~60%) of spiral ganglion neurons do not express NER protein complexes as a defense against noise exposure
which may help to explain selective neurodegeneration. However, under normal conditions a cohort of neurons (~40%)
exhibited either cytoplasmic or nuclear localization of NER factors. After noise exposure the overall number of reactive
neurons stayed the same but there was significant (p < 0.01) cytoplamic and nuclear redistribution of the various NER
factors. This noise effects was accompanied by phosphorylation of histone H2Ax, indicating genomic stress. Translocation
of NER factors from the cytoplasm to the nucleus was dependent on the location of neurons along the cochlear spiral. For
instance, neurons at the apical coil exhibited significant (p < 0.01) nuclear translocation of the CSA complex while neurons
at the basal coil revealed significant (p < 0.05) nuclear translocation of the XPC complex. This spatial difference in nuclear
translocation between CSA and XPC complexes suggests a difference in genome defense repertoire between apical and basal
spiral ganglion neurons. Furthermore, noise exposure depleted XPA from the nucleus regardless of location along the
cochlear spiral. These findings provide a novel mechanism for interpreting noise induced primary neuropathy and provide a
basis for noise induced mutagenesis.
Acknowledgments
This work was supported by a CDA-2 (C7600-W) Award from the Rehabilitation Research and Development Service of the
Office of Research and Development United States Department of Veterans Affairs. The Loma Linda Veterans Affairs Hospital
provided the facilities for conducting the experiments.

NER is one of the most potent molecular mechanisms for determining resistance to cell death.
The majority of spiral ganglion neurons do not express NER proteins with or without noise
stress. This is significant because spiral ganglion neurons are hyper-susceptible to cell death
from noise exposure, ototoxic xenobiotics and/or microbial infections.
A small cohort of spiral ganglion neurons express NER proteins and mobilized these proteins in
response to noise exposure. This is significant because in humans and animals a small
proportion of spiral ganglion neurons persistently survive a wide variety of stressors. This
capacity to survival may be driven by NER.
DNA Repair Adjuvant Therapy Regenerates Neural Sensitivity
When Administered After Noise Trauma
1Loma
O’neil Guthrie 1,2, Jinwei Hu2, Helen Xu2
Linda Veterans Affairs Medical Center, 2Loma Linda University Medical Center
Background
Previous research has demonstrated the
existence of an inducible DNA repair
pathway within spiral ganglion neurons 1,2.
After noise exposure or cisplatin intoxication,
there is active transcription and intracellular
translocation of DNA repair enzymes within
a distinct population of neurons. Recent
experiments have shown that carboxy alkyl
esters (CAEs) that have been standardized
to increase intracellular DNA repair can
augment the capacity of spiral ganglion
neurons to mobilize DNA repair enzymes 3.
However, it is unclear whether such
mobilization of DNA repair enzymes would
restore neural sensitivity after trauma.
Therefore in this study, we investigated
whether or not adjuvant therapy with CAEs
that begins after noise trauma could restore
neural sensitivity.
Methods
Long-Evans rats were used as subjects and
they were randomly divided into four
experimental groups: vehicle-control, noise,
CAE and noise+CAE. Hair cell and neural
sensitivity were monitored with distortion
product otoacoustic emissions (DPOAE) and
auditory brainstem responses (ABR).
Reference
1.
Guthrie et al, Hear Res 239, 79-91, 2008;
2.
Guthrie and Xu, Hear Res 294, 21-30, 2012;
3.
Guthrie, Int J Neurosci 122, 757-766, 2001
Fig. 3A, The average ratio of click ABR thresholds at baseline and 1 day post-exposure.
The control groups exhibited a ratio of ~1.0 which indicates that there were no differences
in threshold between time points. Both the noise-only and the CAE+noise groups had a
ratio of 0.4, indicating the acoustic overexposure induced similar loss of function. B. The
average ratio of click ABR thresholds at 1 day and 1 week post-exposure. A ratio that is
greater than 1.0 indicates that the threshold at 1 week has improved from that at baseline. C
click ABR threshold shift at 1 month relative to baseline threshold for each experimental
group. CAE+noise group exhibited greater recovery in ABR threshold than the noise-only
group.
Fig.1 DPOAE are robust within the time period of the study and elevated from the noise
floor in the control and CAE treated rats. However, the acoustic overexposure depleted
DPOAE levels into the noise floor and this depletion persisted over the duration of the
study (1 month), which replicated in the noise+CAE group. N = 6-7 rats per group and
the noise was a 5.6-11.3 kHz noise band @105 dB/4 hours.
Fig. 4A DPOAE thresholds at 1 month after noise exposure. Thresholds for the noise groups
(noise and CAE+noise) are similarly elevated above the control groups. 4B ABR thresholds at 1
month after noise exposure. The thresholds for the control groups are similar, there is a clear
difference in thresholds between the noise and CAE+noise groups. The CAE+noise group has
significantly better thresholds than that of the noise group.
Fig. 2A Both noise groups (noise-only and the CAE+noise group) exhibited significant
threshold elevations. 2B, ABR threshold shifts relative to the threshold of the control
group. Note that the noise-only and the CAE+noise groups exhibited similar loss at 1 day
post-noise exposure but the CAE+noise group showed better threshold recovery.
Acknowledgements
This work was supported by a CDA-2 (C7600-W) Award from the Rehabilitation Research and Development Service of the Office of
Research and Development United States Department of Veterans Affairs. The Department of Otolaryngology and Head & Neck Surgery
in the School of Medicine at Loma Linda University Medical Center also provided support.
Conclusion
1. These results suggest that DNA repair adjuvant therapy
may rescue neural functions when administered after injury.
2. This raises the possibility that treatment for NIHL may
involve the use of hearing aids to address the permanent
loss of audibility from OHC dysfunction combined with DNA
repair therapy to preserve neural sensitivity.
Neuronal Coupling of Cell Surface Receptor with DNA Repair Enzyme
O’neil W. Guthrie1,2, Daniel Kwon2, Helen Xu2
1Research
2Department of
Service-151, Loma Linda Veterans Affairs Medical Center, Loma Linda, CA 92357, USA.
Otolaryngology and Head & Neck Surgery, School of Medicine, Loma Linda University Medical Center, Loma Linda, CA 92354, USA.
Introduction
Selected populations of spiral ganglion neurons have
been shown to mobilize DNA repair enzymes1. We
conducted experiments to determine whether or not
such neurons express the epidermal growth factor
receptor (EGFR). Gene and enzyme expression of the
EGFR and the xeroderma pigmentosum-A (XPA) DNA
repair factor were determined. Immunofluorescence
assays were used to localize neurons expressing both
EGFR and XPA. This work was then confirmed with
double-immunohistochemical reactions. Rosenthals’
canal served as the reference space in these
experiments and design-based stereology was
employed. The results revealed that three populations
of spiral ganglion neurons could be distinguished. For
instance, there was a population that only expressed
XPA and these neurons were predominantly clustered in
the superior-medial quadrant of Rosenthal’s canal.
There was a second population that only expressed the
EGFR and these neurons were predominantly
distributed throughout the inferior and superior-lateral
quadrants of Rosenthal’s canal. The third and most
abundant population, were neurons that expressed
both the EGFR and the XPA enzyme. These neurons
were distributed throughout the four quadrants of
Rosenthal’s canal. The combined results provide the
basis for future experiments designed to manipulate
the EGFR in order to regulate DNA repair capacity and
regenerate gene function after DNA damage.
G
I
Fig I: Qualitative model of the data, showing distribution
of the three population of neurons. Note the even
distribution of neurons co-expressing EGFR and XPA.
Conclusion
Fig A, B: PCR and Western blot showing
expression of both EGFR and XPA.
Fig G: The 4 quadrants of Rosenthal’s canal and the
corresponding stereologic quantification of EGFR and
XPA; SM = superior medial; SL = superior lateral; IM =
inferior medial and IL = inferior lateral
H
Subjects and Methods
Six Long-Evans rats served as subjects in this study.
Gene expression was measured with end-point
polymerase chain reaction assays and related enzyme
expression was measured with Western blots. After
temporal bone harvesting, immunofluorescence
confirmed by immunohistochemistry was used to
localize expression of the EGFR and XPA enzyme.
Design-based stereology employing the physical
dissector approach was used for quantification of firstorder stereologic parameters.
Fig C-F: Immunofluorescence (C-E) and
immunohistochemical reactions (F) showing the
immunoreactivity of distinct populations of neurons.
Fig H: Breakdown of the three neuronal populations in
each quadrant.
Neurons are terminally differentiated cells
and are known to have poor DNA repair
defenses2. Cochlear neurons easily die after
noise stress or injury from ototoxic exposure
and with aging3 which might relate to the
lack of DNA repair enzymes seen in these
neurons1. However previous research has
discovered a small population of cochlear
neurons that express DNA repair enzymes1.
This population of neurons may be more
protected compared to the larger population
of cochlear neurons. The current study
further characterized this population of
neurons by revealing that they express both
EGFR as well as DNA repair enzymes which is
a signature finding in cells that are resistant
to cell death (i.e. cancer cells). This finding is
a basis for future studies into the survival
capacity of spiral ganglion neurons.
References:
1. Guthrie and Xu. Noise exposure potentiates the subcellular
distribution of nucleotide excision repair proteins within spiral
ganglion neurons. Hearing Research. 294 (2012) 21e30
2. Nouspikel, T., Hanawalt, P.C., 2002. DNA repair in terminally
differentiated cells. DNA Repair (Amst.) 1, 59e75.
3. Lin, H.W., Furman, A.C., Kujawa, S.G., Liberman, M.C., 2011.
Primary neural degeneration in the Guinea pig cochlea after
reversible noise-induced threshold shift. J. Assoc. Res. Otolaryngol.
12, 605e616.
Subtoxic Levels of Jet Fuel Result in Abnormal Auditory Function
O’neil W. Guthriea,b, Daniel Kwonb, Helen Xub, Pedro A. Ortizc, David R Mattie d
aResearch Service-151, Loma Linda Veterans Affairs Medical Center, Loma Linda, CA
of Otolaryngology-Head and Neck Surgery, Loma Linda University Medical Center, Loma Linda, CA
cNaval Medical Research Unit - Dayton, Wright-Patterson, Air Force Base, OH.
dMolecular Bioeffects Branch, Bioeffects Division, 711 Human Performance Wing, Human Effectiveness Directorate, Air Force Research Laboratory (711 HPW/RHDJ), Wright Patterson AFB, OH.
bDept.
Introduction
Individuals who work within and around aircrafts are at
risk for developing hearing loss. This is supported by studies that demonstrated
that pilots, aircrew, aircraft technicians and mechanics have high (32-47%)
prevalence rates of hearing loss when compared to the general adult population
(15-20%). It is accepted that noise over-exposure is a prominent factor in the
development of the hearing loss. In addition, preliminary epidemiologic analyses on
aircraft maintenance personnel concluded that jet propulsion fuels (JP-8 or JP-4)
may interact with noise to further induce hearing loss. Recent animal studies
support this conclusion by revealing that exposure to JP-8 combined with noise may
result in the loss of pre-neural cochlear sensitivity as well as loss of cochlear hair
cells. This study investigated whether or not sub-ototoxic exposure to fuel,
combined with a non-damaging dose of noise could interact to induce auditory
impairment [Support for this research was obtained from the U.S. Air Force Surgeon
General (SG5I) and managed through 711 HPW/RHDJ, Henry Jackson Foundation
Subjects
and Methods
for Military Medicine, Loma Linda
VA Medical
Center, and Navy work unit number
Eighty pigmented Long-Evans rats served as subjects in
61062].
the present study. Baseline screening of disortion product otoacoustic emission
(DPOAE) levels was conducted on each animal to ensure normal DPOAE levels as
determined by normative values publisehd by our laboratory. Animals were then
randomized into 4 experimental groups, where each group consisted of 10 males
and 10 females. There was a control (n = 20), a noise-only (n = 20), a jet fuel-only (n
= 20) and a jet fuel+noise group (n = 20). Jet fuel animals were exposed to sub-toxic
JP-8 for 6 hours per day, five days per week for four weeks. Noise exposure animals
were exposed to non-damaging levels (85 dB) of noise for 6 hours per day, five days
per week for four weeks. DPOAE, Auditory Brainstem Response (ABR) and
Cytocochleograms were conducted on all rats.
Statistical analyses were conducted with Prism 5, version 5.03 (GraphPad Software,
Inc., La Jolla, CA. USA). ABR click thresholds were treated with one factor analysis
of variance (ANOVA). The DPOAE, threshold shift, rate-level and gain-level data
were treated with a split-plot ANOVA followed by Bonferroni pair-wise contrasts and
Dunnett's post hoc testing. F-tests wereResults
conducted to determine differences
between slopes.
Peripheral pre-neural function:
DPOAE and cytocochloegrams revealed that fuel and noise exposures did not
induce a pre-neural impairment. The sensitivity of the outer hair cells from the
noise, fuel and fuel+noise groups is similar to that of control . Almost all outer hair
cells, as well as inner hair cells and innervating nerve fibers were present (Fig.1).
Peripheral neural function:
Analysis of auditory electrophysiologic recordings revealed no detectable
peripheral nerve impairment (wave I). There was no significant differences in click
thresholds between groups. This was confirmed with frequency specific threshold
shift analysis (Fig 2).
Brainstem response function:
Auditory brainstem response analysis demonstrated central auditory dysfunction
with fuel and fuel+noise exposures. Amplification of peripheral neurotransmission
was disrupted by the noise exposure as demonstrated by abnormal morphologies
of evoked response recordings in the brainstem component in the fuel+noise
exposed rats (Fig 3). Also, the fuel and fuel+noise groups exhibited a blunted
response in the normal linear growth function of simulus intensity versus evoked
response rate, suggesting compromise of brainstem encoding of stimulus intensity
TEMPLATE DESIGN © 2008
www.PosterPresentations.com
Discussion
Individual noise and jet fuel exposure dosages used in the current
study were deliberately chosen to be sub-ototoxic. This was done to determine whether or not subototoxic levels of each exposure could become ototoxic when combined (jet fuel+noise). The
results revealed peripheral auditory function was not markedly effected but there was the presence
of central auditory dysfunction. This indicates that central auditory processing dysfunction may be
part of the ototoxic profile of jet fuel exposure. This line of thinking is supported by recent studies
showing that military personnel exposed to jet fuel as well as gas station attendants demonstrate
Central Auditory Processing Dysfunction (CAPD) such as poor auditory frequency discrimination
and impaired ability to recognize speech in background listening situations despite normal hearing
thresholds.
From an epidemiologic perspective, the results imply that jet fuel
exposure may have consequences to auditory function that could be more wide-spread and
insidious than what was previously known. It is possible that a large population of military and
civilian personnel who are suffering with the effects of jet fuel exposure may be misidentified
because they might exhibit normal auditory sensitivity (normal hearing thresholds) but harbor a
"hidden" brainstem dysfunction. Such brainstem dysfunctions may be associated with auditory
and/or non-auditory symptoms. Therefore, the current study provides a basis for further research
focused on jet fuel induced CAPD..
References
Carrillo-de-la-Pena, M. T. 2001. One-year test-retest reliability of auditory evoked potentials (AEPs) to tones of increasing intensity.
Psychophysiology 38:417-424
Fechter, L. D., J. W. Fisher, G. D. Chapman, V. P. Mokashi, P. A. Ortiz, J. E. Reboulet, J. E. Stubbs, A. M. Lear, S. M. McInturf, S. L. Prues, C.
A. Gearhart, S. Fulton, and D. R. Mattie. 2012. Subchronic JP-8 jet fuel exposure enhances vulnerability to noise-induced hearing loss in
rats. J Toxicol Environ Health A 75: 299-317.
Fechter, L. D., C. A. Gearhart, and S. Fulton. 2010. Ototoxic potential of JP-8 and a Fischer-Tropsch synthetic jet fuel following subacute
inhalation exposure in rats. Toxicol Sci 116 :239-248.
Fechter, L. D., C. Gearhart, S. Fulton, J. Campbell, J. Fisher, K. Na, D. Cocker, A. Nelson-Miller, P. Moon, and B. Pouyatos. 2007. JP-8 jet
fel can promote auditory impairment resulting from subsequent noise exposure in rats. Toxicol Sci 98 :510-525.
Guest, M., M. Boggess, J. Attia, C. D'Este, A. Brown, R. Gibson, M. Tavener, I. Gardner, W. Harrex, K. Horsley, and J. Ross. 2010. Hearing
impairment in F-111 maintenance workers: The study of health outcomes in aircraft maintenance personnel (SHOAMP) general health and
medical study. Am J Ind Med 53:1159-1169
Kaufman, L. R., G. K. LeMasters, D. M. Olsen, and P. Succop. 2005. Effects of concurrent noise and jet fuel loss. exposure on hearing J Occup
Environ Med 47:212-218.
Quevedo, S., Tochetto, T., Siqueira, M.A., Machado, M.S. 2012. Auditory brainstem response in gas station attendants. Braz J
Otorhinolaryngol 78:63-68.
Genetic/transgenic conditional expression of nonmuscle myosin-II in auditory neurons:
Head domain regulates assembly of the α-helical coiled-coil tail
1Loma
O’neil Guthrie 1,2, Jinwei Hu2, Helen Xu2
Linda Veterans Affairs Medical Center, 2Loma Linda University Medical Center
Background
Nonmuscle myosin-II (MyoII) is an actin-binding protein that
is involved in several motor functions including growth cone
motility and neural migration, outgrowth and retraction. The
molecule can be functionally divided into a head-domain
and a α-helical coiled-coil tail. Self-assembly of the MyoII αhelical coiled-coil tail with other MyoII molecules form
bipolar filaments that power cell movements. This selfassembly of the tail is intrinsically regulated by
intramolecular interactions of the tail with the head-domain
as demonstrated by in vitro experiments1. However, there is
no in vivo evidence for this regulatory mechanism.
Methods
In this study, we further define whether the head-domain of
MyoII regulates in vivo self-assembly of the α-helical coiledcoil tail in auditory neurons. A GAL4-UAS gene expression
system was used to selectively express zip/MyoII fulllength, zip/MyoII tail with and without isoleucine-glutamine
(IQ) motifs in Drosophila melanogaster auditory (Johnston’s
organ) sensory neurons. The N-terminus of each construct
was fused to green fluorescent protein (GFP) to follow the
distributions of transgene expression.
Figure 1. Sound evoked neural responses. The MyoII molecule was
cloned and the head domain was removed to generate a headless
(MyoII-tail) protein that harbors only the tail. This construct was
genetically targeted to neurons, particularly auditory neurons. Note
that conditional expression of the MyoII-tail showed reduced auditory
nerve activity which suggest a potential hearing loss .
Figure 2. Elav-GAL4 drives Elav-GFP expression in the auditory neurons of
Johnston’s organ. Panel A shows a field of DAPI stained (blue) DNA from cells in
Johnston’s organ. DNA from both neuronal and non-neuronal cells is stained. The
neuron specific Elav promoter was used to drive GAL4 transcriptional regulation of
UAS-GFP-elav (Panel A') in auditory neurons of Johnston’s organ. Panel A'' is a
merger of panels A and A'. Panel A''' reveals the location of the neurons relative to
the F-actin enriched (phalloidin stained) scolopale rods (red).
Figure 3. Full-length zip/MyoII
may exhibit rod or globular
conformations in auditory
neurons of Johnston’s organ.
Panels A- A' reveals major
sensory structures such as the
actin enriched scolopale rods
(red) and bipolar auditory
neurons (green). The neuron
specific Elav promoter was used
to drive GAL4 transcriptional
regulation of full-length UASGFP-zip/MyoII. Panels A''- A'''
are enlarged from panel A and
they reveal the two dominant
conformations of GFP-zip/MyoII
in the neurons. Note that
zip/MyoII may assemble as rods
(panel A'') or globular oligomers
(panel A''' ).
Figure 5. The tail domain of zip/MyoII assembles as rods in sensory
neurons from living whole-mount preparations of Johnston’s organ. Panels
A-A''' are optical sections through living tissue. The neuron specific Elav
promoter was used to drive GAL4 transcriptional regulation of UAS-GFPzip/MyoII-tail domain in sensory neurons of Johnston’s organ. Panel A'''' is
a Z-stack of the entire neuronal tissue of Johston’s organ.
Figure 6. The tail+IQ domain of zip/MyoII diffuses throughout the
cytoplasm of neuronal cell bodies and axons from fixed and living wholemount preparations of Johnston’s organ. Panels A-A''' shows neuronal
expression of the tail+IQ domain in neurons from fixed whole-mount
preparations of Johnston’s organ. The neuron specific Elav promoter was
used to drive GAL4 transcriptional regulation of UAS-GFP-zip/MyoII-tail+IQ
domain. Panels B-B' are 3 µm optical sections through living tissue.
Figure 4. The tail domain of zip/MyoII assembles as rods in sensory neurons of
Johnston’s organ. Panel A shows neuronal expression of the tail domain. The
neuron specific Elav promoter was used to drive GAL4 transcriptional regulation
of UAS-GFP-zip/MyoII-tail domain in sensory neurons of Johnston’s organ. Panel
A' reveals dendrites projecting into the actin enriched (phalloidin stained red)
scolopale rods. Panels A''- A''' are enlarged from panels A and A'. Note the
elongated conformation of the tail domain. Scale bar =5 µm.
Conclusion
1. Head-domain is critical for modulating the selfassembly of the tail in vivo.
2. These findings may help to explain why human
mutations in the head domain result in severe
diseased phenotypes.
Reference:
1. Jung H.S, et al . Proc Natl Acad Sci U S A, 2008, 105: 6022-6026.
Remodeling the intracellular distribution of DNA repair enzymes in spiral ganglion neurons
in response to noise stress and otoprotective therapy
1Loma
O’neil Guthrie 1,2, Jinwei Hu2, Helen Xu2
Linda Veterans Affairs Medical Center, 2Loma Linda University Medical Center
Background
Intracellular stress gradients drive the spatial distribution of
DNA repair enzymes 1,2. In this study, we investigated if
spiral ganglion neurons could remodel intracellular
distributions of DNA repair enzymes in response to stress
and otoprotective therapy.
Methods
The intracellular locations of DNA excision repair enzymes
were determined within spiral ganglion neurons under
normal conditions, after noise exposure, and following
otoprotective treatment with carboxy alkyl esters (CAEs).
Line scans were used to record immunoreactivity within the
soma of each neuron to objectively profile the subcellular
distribution of the enzymes.
Figure 1. Positive and
negative controls. (A)
positive control cells. (BD) negative control.
Scales bar=10 µm.
Figure 3. Allocation of repair proteins across subcellular compartments
from the control group. Each subcellular compartment is enriched with at
least one repair protein. The XPC protein is predominantly localized in the
cytoplasm while XPA is primarily diffused throughout the cytoplasm and
nucleus. Additionally, the CSA protein exhibits a preference for
cytoplasmic and perinuclear loci.
Figure 7. The effect of the experimental conditions on the distribution patterns.
The experimental conditions did not significantly change the distribution patterns
for the XPC protein. However, there were significant changes for the XPA and
CSA proteins. The experimental conditions were control (N = 5), noise (N = 5),
CAE (N = 3) and CAE+noise (N = 3). Each plot displays the mean ± SEM.
Figure 4. Re-allocation of repair proteins to the cytoplasmic
compartment from the noise exposed group. Note that all the proteins
exhibited preferential localization in the cytoplasm.
Figure 8. Neuronal threshold shifts. Compound action potentials were
recorded from each group at 4 weeks after the noise exposure. Note that the
CAE treatment was neuroprotective.
Figure 5. Allocation of repair proteins across subcellular compartments from
the CAE treated group. The CAE treatment apparently equalized the
distribution of the proteins across patterns. Statistical analyses reveal that
no one localization pattern is significantly different than the other patterns for
a given protein.
Conclusion
1. The current study revealed that spiral ganglion
neurons exhibit multiple compartmentalizing modes for
excision repair enzymes and these modes exhibit
plasticity following noise stress and otoprotective
treatment.
2. Regulation of intracellular gradients of DNA excision
repair enzymes may represent a novel approach to
preserving neural function following stress.
Reference
1.Griffiths LM, et al . Mol Cell Biol. 2009;29:794-807.
2.Guthrie OW..Anticancer Res. 2008;2637-2640.
Figure 2. Representative examples of the intracellular distribution
patterns of Neurons. (A-C) Diffuse expression. (D-F) Cytoplasmic
expression. (G-I) Both nuclear and diffuse expression. (J-L) Perinuclear
expression. The y-axis in panels C, F, I and L are inverted gray (g) levels
(1/g). The scale bar (20 µm) in panel A applied to panels D, G and J.
The scale bar (10 µm) in panel B applied to panels E, H, and K.
Figure 6. Mixed allocation of repair proteins across subcellular
compartments from the CAE+noise treated group. CAE+noise equalized
the intracellular distribution of the XPC and XPA proteins. Statistical
analyses reveal that no one localization pattern is significantly different than
the other patterns. However, the CSA protein exhibited a preference for
cytoplasmic and perinuclear loci.
Acknowledgements
This work was supported by a CDA-2 (C7600-W)
Award from the Rehabilitation Research and Development Service of the
Office of Research and Development United States Department of Veterans
Affairs. The Department of Otolaryngology and Head & Neck Surgery in the
School of Medicine at Loma Linda University Medical Center also provided
support.
The α-Helical Coiled-Coil Tail of Nonmuscle Myosin-II Mediate Interactions with the Nucleus
O'neil Guthrie Ph.D1,2, Terry Fleck MD2, Helen Xu MD1,2
1Loma
Linda Veterans Affairs Hospital, 2Department of Otolaryngology – Head & Neck Surgery, School of Medicine, Loma Linda University Medical Center
A
B
C
A
B
C
Introduction
Human genetic mutations (MYH9 and MYH14 related
diseases) that affect the nonmuscle myosin-II (MyoII) molecular motor result in syndromic and
nonsyndromic forms of sensorineural hearing loss but the underlying mechanisms are unresolved.
We posit that one potential mechanism to account for MYH9/14 related diseases (e.g., hereditary
macrothrombocytopenias) might be the interaction between MyoII and the cell nucleus. As a first
approximation, we conducted genetic/transgenic conditional expression experiments to determine
whether or not MyoII associates with the nucleus of cells in metazoan tissue. The giant cells within
salivary gland organs from 3rd instar Drosophila melanogaster larvae were evaluated in living and
fixed preparations. A UAS-Gal4 conditional expression system was used to drive gene expression
of MyoII specifically within salivary gland organs. A GFP-MyoII protein trap line, which uses the
endogenous MyoII promoter to control expression of full-length GFP-MyoII was also employed.
Additionally, epitope immunoreactivity was used to localize endogenous MyoII proteins. The
combined results demonstrate that the MyoII molecule may interact with the nucleus. For instance,
the molecule formed oligomerized (filament-like) conformations on the cytoplasmic side of the
nuclear lamin. The α-helical coiled-coil tail of the MyoII molecule was necessary and sufficient to
mediate all nuclear interactions including perinuclear localization and oligomerization. These
interactions affected the morphology of the nucleus and the nuclear associations of filamentous
actin and actin-binding proteins. The data provide direct evidence for a nuclear association of
MyoII within metazoan tissue and suggest that the interaction between MyoII and the nucleus
might contribute to MYH9/14 related diseases.
head domain
α-helical coiled-coil tail domain
Wild-type
full-length
ELC
G
A
A1
B
B1
DNA
Lamin
zip/MyoII
D
E
F
D
G
G'
G''
G
E
F
G'
DNA
0 μm
3 μm
6 μm
Figure 3. Oligomerized perinuclear pools zip/MyoII in a whole-mount
salivary gland organ. In all panels green is GFP-zip/MyoII, red or
blue is DNA and the cell junction protein, moesin is in white. Panel A
is a Z-section through a whole-mount salivary gland. Panels B-F are
stacked Z-sections. Panels B-C provides a 3D view of zip/MyoII and
DNA. Panels D-F reveal the spatial orientation of DNA and/or GFPzip/MyoII within the salivary gland in general and specific cells in
particular. Panels G-G'' are 0-6 μm sections through the nucleus
outlined in panel A.
Figure 4. zip/MyoII exhibits cytoplasmic perinuclear localization.
Panels A-F are serial Z-zections through a single nucleus. Note that
GFP-zip/MyoII is localized on the cytoplasmic side of the nuclear
lamin (red). Panels G-G' are 3D reconstructions of panels B-F. Scale
bar (10 µm) in panel C applies to all panels.
A
RLC
B
Figure 7. Perinuclear zip/MyoII co-localize with some actin-binding proteins. Panel A shows
that spectrin (red, antibody stain) is localized around the nucleus of salivary gland cells. Panel
A1 shows the same salivary gland with co-localization of spectrin (red) and GFP-zip/MyoII.
Note that the yellow loci are areas of strong co-localization. Panel B shows that moesin (red,
antibody stain) is localized at the periphery of the salivary gland. Panel B1 shows no colocalization between GFP-zip/MyoII and moesin.
C
A
B
neck + tail
tail
GFP-Tail fragment
Figure 1. Schematic molecular structure of MyoII. MyoII consists of a hexameric holo-complex of two myosin
heavy chains and two pairs of light chains. The N-terminal of the heavy chain comprises the MyoII head and
consists of an SH3 domain and the motor domain which is the chemomechanical force producer and exhibits both
ATPase and actin binding activities. The C-terminal of the head (called the neck domain) consists of an extended
α-helix with isoleucine-glutamine or IQ motifs that bind to an essential light chain (ELC) and regulatory light chain
(RLC). Both light chains (LC) stabilize the IQ motifs of the neck and stiffen the lever arm during force generation.
The tail structure functions to dimerize the heavy chains through the formation of an α-helical coiled-coil and also
includes a short, C-terminal non-helical tail piece.
A
A'
GFP-Head+neck fragment
GFP-Neck+tail fragment
Figure 5. zip/MyoII tail domain regulates the assembly of perinuclear oligomers. Panels A-C are photomicrographs of
living whole-mount salivary gland organs. Note that perinuclear oligomers are observed with expression of the tail
and neck+tail domains and not the head+neck domain.
neck+tail
DAPI
lamin
C
A
D
B
A''
tail
actin
GFP-zip/MyoII
DNA
DAPI
B
zip/MyoII antibody staining
B'
B''
zip/MyoII protein trap
DAPI
C
merge
C'
C
D
Conclusion
merge
C''
merge
Figure 6. zip/MyoII co-localize with perinuclear actin. Panel A is phalloidin stained actin in whole mount salivary
gland. The arrow reveals projections from both perinuclear and cell border actin pools. Panel B is the same salivary
gland showing perinuclear localization of GFP-zip/MyoII. Panel C is the merger of panels A and B. Note that the
yellow loci reveal strong perinuclear co-localization. Panel D is the Z-stack profile of the organ.
DAPI
zip/MyoII driven by Gal 4
merge
Figure 2. Perinuclear localization of endogenous and transgenic zip/MyoII in single cells from whole-mount
salivary gland organs. Endogenous (antibody stained) zip/MyoII (panels A-A") exhibit a prominent perinuclear
localization. However, GFP-zip/MyoII protein trap (panels B-B") exhibit a modest perinuclear localization. Overexpression of GFP-zip/MyoII with the salivary gland specific Gal4 driver (panels C-C") result in prominent
perinuclear localization. Arrows point to the perinuclear clusters of zip/MyoII.
Figure 8. Headless myosin-II molecules may affect the morphology of the nucleus. Panel A-B
shows that the morphology of the nucleus is maintained when the neck is attached to the tail.
However, panels C-D reveals that removing the neck alters the morphology of the nucleus.
The inset in panel D shows the percent of nuclei with altered morphology as a function of the
various MyoII transgenic constructs. Abbreviation: F, full-length; H, head; N+T, neck+tail; T,
tail.
Acknowledgments
The authors would like to thank Professor Dan Kiehart for helpful comments and support. The lamin Dm0(ADL67.10) monoclonal
antibody developed by Paul A. Fisher was obtained from the Developmental Studies Hybridoma Bank and developed under the auspices of
the NICHD and maintained by the University of Iowa, Department of Biology, Iowa City, IA 52242. The work was supported by the
Hargitt Cell Biology Research Award.
Previous cell culture observations were further bolstered in this
study in which we clearly revealed perinuclear pools of MyoII in living and fixed preparations of
Drosophila salivary gland organs. MyoII in cooperation with filamentous actin is known to
generate force at the nucleus during disperate conditions such as mitosis, cellular locomotion and
cell lineage determination. In this study MyoII affected the morphology of the nucleus and the
nuclear associations of filamentous actin and actin-binding proteins. The data provide direct
evidence for a nuclear association of MyoII within metazoan tissue and suggest that the interaction
between MyoII and the nucleus might contribute to MYH9/14 related diseases.
AUTOMETALLOGRAPHICAL AMPLIFICATION OF INTRACELLULAR ANTI-CANCER PLATINUM MOLECULES
O’neil W. Guthrie1, Carey Balaban2
Science and Disorders, University of Pittsburgh, Forbes Tower, Pittsburgh, PA, United States,
2Otolaryngology and Neurobiology, University of Pittsburgh, 203 Lothrop, Pittsburgh, PA, United States
1,2Communication
ABSTRACT
METHODS
This study utilized autometallography (Danscher et at, Neuroscience 105, 941947, 2001) to localize platinum in tissue. Platinum molecules from the drug
cisplatin were amplified by reducing silver ions to silver atoms while chelating
platinum bound to intracellular ligands. This redox activity allowed for detection of
platinum embedded in black silver deposits at the light microscopy level. Kidneys
were harvested from a Sprague-Dawley rat that was treated with 13 mg/kg of
cisplatin i.p., euthanized 24 hours later and perfused transcardially with formalin.
Control kidneys were harvested from an untreated, formalin-perfused SpragueDawley rat. Both control and experimental tissues were post-fixed with 10%
buffered formalin and cryoprotected in PBS + 30% sucrose. Sections were then
rinsed in Na acetate buffer before being immersed in a physical developer (AgNO3,
Na acetate trihydrate, glacial acetic acid, cetylpyridinium Cl, Triton X-100, Na
tungstate and Ascorbic acid). Silver deposits were fixed in potassium thiosulfate
and sections were dehydrated and cover slipped. Autometallography silver
deposits were observed in the nuclei of the proximal tubule cells of the kidney from
the rat receiving systemic treatment of cisplatin. In vitro exposure of kidney tissues
to 5 mM of cisplatin for 1 hour, by contrast, resulted in heavy staining of the renal
cortex and less staining in the renal medulla. These findings suggest that
autometallography may be a useful method for studying cisplatin uptake and
distribution at the cellular level. [Supported by NIH-NIDCD (F31) DC05757-01 and
the Eye & Ear Institute Foundation. The authors wish to thank Dr. Richard Salvi for
providing tissue treated in vitro and Dr. Len Rybak for providing the in vivo treated
materials.]
Kidneys were harvested from a Sprague-Dawley rat that was treated with 13
mg/kg of cisplatin i.p., euthanized 24 hours later and perfused transcardially with
formalin. Control kidneys were harvested from an untreated, formalin-perfused
Sprague-Dawley rat. Both control and experimental tissues were post-fixed with 10%
buffered formalin. Both tissues were sulfidated in 1% (NH4)2S in 70% ethanol, pH 9,
at 22 C for 15 min. The tissues were then treated with 10% formic acid for seven
days at 22° C and neutralized with 5% Na2SO4 for 24 hours at 22° C and then
cryoprotected in PBS + 30% sucrose. Frozen sections of both tissues were cut on a
sliding microtome (Microm HM440E) at 20 mm. The tissue sections were then ready
for autometallography (see table 1 for autometallographic solutions).
INTRODUCTION
Cisplatin is an effective, widely used, broad-spectrum anticancer xenobiotic
(Gordon & Hollander, 1993; Weiss & Christian, 1993).
Its dose-limiting side
effects are nephrotoxicity and ototoxicity. Therefore, knowledge of the cellular and
subcellular binding sites of cisplatin is necessary to elucidate loci of toxic effects as
well as aid in designing targeted prophylactic approaches. Thus, there is a need to
develop sensitive techniques to identify trace amounts of the drug in the kidney
and the cochlea. When cisplatin (cis-diamminedichloroplatinum II, (NH3)2PtCl2)
enters a cell the dichloride ligands are dispelled, which renders the remaining
platinum molecule highly reactive. This reactive platinum molecule acts as a
potent electrophile, which reacts with nucleophilic ligands on nucleic acids
(Kartalou & Essigmann, 2001). The approach in this study is based upon the
observation that platinum bound to intracellular ligands may be chemically
converted to a heterogeneous catalyst by reaction 1 (Zdolsek, 1993):
Pt-ligand + S2-
PtS + apo-ligand
(1)
This
initial
reaction
facilitates
the
process
of
autometallography.
Autometallography is the most sensitive cytochemical process for identifying the
topographical distribution of catalytic metals in cells. Metallo-catalytic atoms or
molecules within a cell may serve as a heterogeneous catalyst for the reduction of
silver ions to metallic silver atoms. This redox activity leads to the identification of
the catalytic metal, which allows for detection with electron and light microscopy.
Autometallography has been used to identify endogenous trace metals such as,
Cu2+, Fe2+, Zn2+, Co2+ and Ni2+ (Danscher, 1981). Additionally, nonessential and
toxic metals such as, Hg2+, Cd2+, Pb2+, As2+, Bi2+, Tl2+, Au2+, and Ag2+ can be
identified with autometallography (Danscher, 1981). In this study we have
2+
developed aAgsensitive
platinum-sulfide autometallography (PtSAMG) procedure that
2+
allowed for Ag
the detection and amplification of intracellular anti-cancer platinum
Ag°
moleculese in the
nucleus of kidney cells
(see 1.
figure
1).
Figure
Principles
of Autometallography:
An
-
-
Ag°
Ag2+
PtS
-
ee-
Ag°
t1
Ag°
eAg2+
-
t2
Ag2+
Ag2+ + e2-
Ago
insoluble crystal lattice is formed from the
electrovalent bonding of a metal (Pt) to a non-metal
(S2-), which is able to conduct electrons (e-). An
electrophile (Ag+) is reduced by a nucleophile (-) to
form a metallic product (Ag°). This redox activity
speeds up when both reagents come in contact with
the metallic crystal lattice. The reagents chellates the
initial crystal lattice, which generates a metallic shell
of Ag° which grows with time, allowing for detection at
the electron and light microscopy levels, t1= beginning
time; t2= anytime post t1.
RESULTS
Panel A
Table 1
Autometallography Chemicals
Solution (sol.)
C2H3O2Na.3H2O
g
*GAA
AgNO3
C21H38NCl
•TX
Na2WO4.2H2O
C6H8O6
K2S2O3
dH2O
A
112.5 g
B
-
18.75 ml
2.75 g
1.0 g
2.5 ml
12.5 g
1872.5 ml
250 ml
*GAA (Glacial Acetic Acid),
•TX
(Triton
X-100)
2000 ml
C
-
D
-
E
-
F
20
0.3 g
300 ml
20 ml
1980 ml
20 g
2000 ml
-
Panel B
Panel C
Black silver deposits show the localization of putative catalytic platinum molecules in the kidney.
Panel A, is a photomicrograph at an intermediate depth within the renal cortex of a control kidney
from a Sprague-Dawley rat. This section was processed with the autometallographic procedure.
There are no black silver deposits. The same region of the kidney from a rat treated with cisplatin
(13 mg/kg, i.p.) is shown in panels B and C. Note the prominent autometallographic black silver
deposits in the nuclei of the proximal tubule cells and the lack of deposits within the renal corpuscle.
This staining pattern was restricted to the middle third of the cortex. The nuclear localization is
consistent with the known formation of cisplatin-DNA adducts (Kartalou & Essigmann, 2001). The
localization of binding to proximal tubule cells in the middle third of the cortex suggests a selective
affinity of these cells for cisplatin, which is consistent with the selective damage to proximal tubule
cells after cisplatin treatment (Townsend, Deng, Zhang, Lapus & Hanigan, 2003).
CONCLUSION
 We have presented a cytochemical approach to localize platinum molecules in
cells.
All chemicals were obtained from Sigma Chemical Company, St. Louis, MO.
 Autometallography may be a useful method for demonstrating cisplatin uptake
and distribution at the cellular level.
 This method should be applicable to localize platinum in the inner ear.
REFERENCES
Autometallography Development
Sequence
}
free floating
sections
sol. F
2 min
}
physical developer
sol. A, B & C
1 or 4 min
}
Danscher, G. (1981). Histochemical demonstration of heavy metals: A revised version of the
sulphide silver method suitable for both light and electron microscopy. Histochemistry 71, 1-16.
}
sol. D
10 min
}
sol. E
30 sec
Gordon, M., & Hollander, S. (1993). Review of platinum anticancer compounds. Journal of Medicine,
24, 209-265.
Kartalou, M., & Essigmann, J. M. (2001). Recognition of cisplatin adducts by cellular proteins.
Mutation Research, 478, 1-21.
Townsend, D. M., Deng, M., Zhang, L., Lapus, M. G., & Hanigan, M. H. (2003). Metabolism of
cisplatin to a nephrotoxin in proximal tubule cells. Journal of the American Society of Nephrology,
14, 1-10.
Autometallographic Technique for Visualizing Platinum in the Kidney
Weiss, R. B., & Christian, M. C. (1993). New cisplatin analogues in development. Drugs, 46, 360377.
Step 1:
Kidney from an animal treated with cisplatin is sulfidated in vitro with (NH4)2S at pH 9
for 15 minutes. An insoluble platinum-sulfide reaction product is formed from the
electrovalent bonding of platinum to sulfur, which is able to conduct electrons.
Zdolsek, J. M., Roberg, K., & Brunk, U. T. (1993). Visualization of iron in cultured macrophages: A
cytochemical light and electron microscopic study using autometallography. Free Radical Biology
and Medicine, 15, 1-11.
Step 2:
Silver ions in solution A, are reduced by ascorbic acid (solution C) buffered by
sodium tungstate (solution B) to form metallic silver. This redox activity speeds up
when silver ions and ascorbic acid come in contact with tissue containing platinumsulfide reaction products (step 1).
Silver ions once reduced by ascorbic acid
chelates the initial platinum-sulfide reaction product, which generates black silver
deposits. The silver deposits grow with incubation time, allowing for detection at the
light microscopic level.
ACKNOWLEDGEMENT
This work was supported by NIH-NIDCD (F31) DC05757-01 and the Eye & Ear
Institute Foundation. The authors wish to thank Dr. Len Rybak for providing the in
vivo treated materials. The authors would also like to thank Drs. Catherine Palmer
and John Durrant for helpful discussions regarding this project.
Cisplatin Induces Cytoplamic to Nuclear Translocation of Nucleotide Excision Repair
Factors Among a Proportion of Spiral Ganglion Neurons
1, Department
O’neil W. Guthrie1, Carey Balaban2
of Biology, Developmental, Cell and Molecular Biology Group, Duke University, French Science Research Center, Durham, NC.
2, Otolaryngology and Neurobiology, University of Pittsburgh, 203 Lothrop, Pittsburgh, PA.
INTRODUCTION
Compared to other cell-types, neurons are poor at repairing damaged DNA among silent genes
(global genome)[1]. The underlying mechanisms that result in poor neuronal repair of their
global genome is unknown. Therefore, we evaluated the response of spiral ganglion neurons to
sub-toxic cisplatin treatment cycles in order to better understand the mechanisms that underlie
poor global genomic repair among neurons. Cisplatin is a potent anticancer drug that induces
DNA damage in the cochlea among various cell types and human temporal bones reveal that
cisplatin chemotherapy kills spiral ganglion neurons [2, 3]. Cancer research has revealed that
cell survival from cisplatin chemotherapy is dependent on DNA nucleotide excision repair (NER)
pathways [4]. One pathway is called global genomic-NER (GG-NER) and only repairs DNA
damage among silent genes. The rate-limiting step in this pathway is DNA damage identification
and verification by the proteins xeroderma pigmentosum complementation group C and A (XPC
& XPA). The other pathway is called transcription coupled-NER (TC-NER) and only repairs DNA
damage among active genes. The rate-limiting step in this pathway is accomplished by RNA
polymerase II and XPA. Unlike RNA polymerase II, XPC and XPA have no known cellular
function beyond DNA repair. Therefore, we suspected that cisplatin chemotherapy may
stimulate the expression of XPC and XPA among spiral ganglion neurons.
METHODS
Sub-toxic cisplatin treatment cycles of 2 mg/kg, i.p., per day results in cisplatin DNA adducts
among various cell types in the guinea pig cochlea [2] and stimulates antioxidant responses in
the rat cochlea [5]. Therefore, we employed a 2 x 3 x 2 factorial design, consisting of two
treatment conditions (cisplatin and saline treatment), three survival times (days 5, 19 and 22 of
cisplatin treatment) and two analysis methods (quantitative RT-PCR and immunohistochemistry).
The rats were treated with two cycles of cisplatin [5], each cycle consisting of four days of
treatment (1 mg/kg, i.p., twice daily) separated by 10 days of rest. Each animal received daily
hydration therapy consisting of 10 ml of saline administered subcutaneously. Survival times
were 24 hours after each treatment cycle (days 5 and 19) and four days (recovery period) after
the second treatment cycle (day 22). A total of 30 animals were treated with cisplatin, 10 were
sacrificed at each of the three survival times for analyses. Another 30 animals received sterile
physiological saline (vehicle) instead of cisplatin.
Figure 4. XPC and XPA are expressed among cochlear hair
cells with or without cisplatin treatment. A-B, are radial views of
the organ of Corti showing XPC expression after cisplatin
treatment (B) compared to saline treatment (A). C-D, are
longitudinal views of the outer hair cells showing XPA
expression after cisplatin treatment (D) compared to saline
treatment (C). ihc, inner hair cell; ohc, outer hair cells, DC,
Dieter’s cell; ip, inner pillar cells; op, outer pillar cells; sv, spiral
vesicle. A-D are representative of all days sampled (days 5, 19
& 22), scale bars = 10 μm.
Figure 5. Cisplatin treatment increases XPC and XPA immunoreactivity
in the stria vascularis and spiral ligament. All cell types in the stria
vascularis and spiral ligament seem to be immunoreactive after either
saline (A, B, E, F) or cisplatin (C, D, G, H) treatment. Note that cisplatin
treatment results in heavier immunoreactivity compared to saline
treatment. B, D, F and H (scale bars = 10 μm) are enlargements of the
outlined areas in A, C, E and G (scale bars = 50 μm) respectively. A-H
are representative of all days sampled (days 5, 19 & 22). RM,
Reissner’s membrane; Stv, stria vascularis; SLg, spiral ligament; O,
osteocytes; SMC, strial marginal cells; SIC, strial intermediate cells;
SBC, strial basal cells; I, type I fibrocytes; II, type II fibrocytes; III, type III
fibrocytes.
Figure 6. Cisplatin treatment increases XPA and XPC
immunoreactivity in the spiral limbus. Spiral limbus
fibrocytes (SLFs) and interdental cells (IDCs) exhibit light
XPA (A-D) and XPC (E-F) immunoreactivity after saline
treatment (A, B & E). SLF and IDC exhibit heavy XPA and
XPC immunoreactivity after cisplatin treatment (C, D & F).
No immunoreactivity is found in the negative control (G).
RM, Reissner’s membrane; OC, organ of Corti; SL, spiral
limbus; Stv, stria vascularis; SLg, spiral ligament. A-G are
representative of all days sampled (days 5, 19 & 22).
Scale bars A & C = 50 μm, scale bars for B, D, E & F = 10
μm and the scale bar for G = 100 μm.
Figure 10. Cytoplasmic to nuclear translocation of
XPA.
XPC mRNA level x 10-9
140
cisplatin
control
A
120
100
80
60
40
20
0
180
saline
Weight in grams
175
Saline
Cisplatin
170
cisplatin
Day 5
165
saline
cisplatin
Day 19
saline
cisplatin
Day 22
140
XPA mRNA level x 10-7
160
155
150
145
140
135
130
125
B
120
100
80
60
Figure 11. Total proportion of XPC and XPA immunoreactive ganglion
cells.
40
20
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22
0
saline
Days
cisplatin
Day 5
saline
cisplatin
Day 19
saline
cisplatin
Day 22
Figure 1. Weight loss as a function of cisplatin treatment cycles.
A
15
10
5
0
-5
Day 5
Day 19
Day 22
-10
OAE amplitude shift (dB)
ABR threshold shift (dB)
Figure 3. Cisplatin treatment increases cochlear XPC and
XPA mRNA levels (2 ─∆Ct: y-axis). (A) XPC mRNA level for
saline (n = 15) and cisplatin (n = 15) treated groups after days
5 (n = 5), 19 ( n = 5) and 22 (n = 5). (B) XPA mRNA level for
saline (n = 15) and cisplatin (n = 15) treated groups after days
5 (n = 5), 19 (n = 5) and 22 (n = 5). Each bar represents
mean ± SE for triplicate runs.
25
25
20
Figure 9. Cytoplasmic to nuclear translocation of XPC
20
B
15
10
5
0
-5
-10
-15
-15
4
8
16
1
2
3
4
5
6
7
8
Frequency (kHz)
Figure 2. Cisplatin induced ABR threshold shifts and OAE amplitude shifts.
Figure 8. Differential translocation of XPC (A-C) and XPA (D-F) among spiral ganglion neurons
after cisplatin treatment. After the first treatment cycle (day 5) both XPC (A) and XPA (D) are
localized in the nucleus of a proportion of spiral ganglion neurons (see arrows). After the
second treatment cycle (day 19) XPA (E) remains localized in the nucleus while XPC (B) is now
localized in the cytoplasm. After four days of recovery from the second treatment cycle (day
22) both XPC (C) and XPA (F) are predominantly localized in the cytoplasm. Interestingly,
satellite cells (see arrow heads) are more immunnoreactive for XPA than for XPC and remained
immunoreactive on all days sampled (D-F). Bars = 10 μm.
Figure 7. Representative example of cytoplasmic to nuclear translocation among spiral ganglion neurons
due to cisplatin treatment. (A) Negative control showing no immunoreactivity. (B) Enlarged area in (A)
showing cell bodies of spiral ganglion neurons. (C) Schematic representation of a bipolar spiral ganglion
neuron from (A). (D) XPC immunoreactivity is present under normal conditions (saline treatment) among
some spiral ganglion neurons. (E) Enlarged area outlined in (D) showing predominant cytoplasmic
localization of XPC. (F) Schematic representation of a bipolar spiral ganglion neuron from (E). (G) XPC
immunoreactivity remained present after cisplatin treatment among spiral ganglion cells, except the
immunoreactivity is now predominantly localized in the nucleus (H), which suggest translocation to the
nucleus due to cisplatin treatment. (I) Schematic representation of a bipolar spiral ganglion neuron from
(G). Scale bar (50 μm) in (A) applies to D and G as well. Scale bar (10 μm) in (B) applies to E and H as
well.
CONCLUSIONS:
•Sub-toxic cisplatin treatment activates the NER cell survival
pathway in the cochlea.
•The time courses of nuclear translocation of XPC & XPA in spiral
ganglion neurons vary along an apical to basal gradient.
•Nuclear translocation of XPC occurs after the initial but not the
second cisplatin treatment cycle in spiral ganglion cells. This
finding suggests a global genomic NER response to only the first
cycle of cisplatin exposure.
•Nuclear translocation of XPA after both cycles of treatments
suggests a transcription-coupled NER response to the second cycle
of exposure.
References:
1.
Nouspikel, T. & Hanawalt, P. C. (2000). Molecular and Cellular Biology, 20, 1562-1570.
2.
van Ruijven, M. W. M., de Groot, J. C. M. J., Hendriksen, F. & Smoorenburg, G. F. (2005). Hearing Research 203, 112-121.
3.
Strauss, M., Towfighi, J., Lord, S., Lipton, A., Harvey, H. A., & Brown, B., (1983). Laryngoscope 93, 1554-1559.
4.
Wu, X., Fan, W., Xu, S. & Zhou, Y. (2003) Clinical Cancer Research, 9, 5874-5879.
5.
Minami, S. B., Sha, S. H. & Schacht, J. (2004). Hearing Research, 198, 137-143.
Acknowledgements: NIH-NIDCD (F31), Eye and Ear Institute Foundation, Pennsylvania State K. Leroy Irvis Award, SHRS Research Development
Fund
Factors that Contribute To Tinnitus Distress in A Veteran Population
Jinwei Hu, MD1; Jane Xu, BS1; Matthew Streelman, DDS2; Helen Xu, MD1; O’neil Guthrie, Ph.D1,3
1 Department of Otolaryngology-Head and Neck Surgery, Loma Linda University Medical Center,
2 Loma Linda University School of Medicine, 3Jerry L. Pettis VA Medical Center
Objective: Neuronal dysfunctions that manifest as tinnitus are
known to alter neuronal circuits in the brainstem and cortex,
which are common to several comorbid conditions. This study
is focused on the relations between tinnitus and
anxiety/depression.
Table 2A: Correlations between the degree of tinnitus and depression
Pearson
Correlation
Significance
(2-tailed)
Total THI score *
Depression
0.205
0.044
Study design: retrospective case review.
Functional THI score *
Depression
0.429
0.000
Setting: Veterans Affairs Medical Center (VAMC) Tinnitus
Clinic.
Emotional THI score *
Depression
0.331
0.001
Catastrophic THI score *
Depression
0.271
0.007
Patients: Ninety-one male veterans who reported subjective
tinnitus were enrolled in a VAMC Tinnitus Clinic.
Table 3: Tinnitus characteristics and duration
Interventions: Case history, audiometric thresholds and selfassessment of tinnitus handicap were examined in all patients.
Main outcome measures: Scores from the Tinnitus Handicap
Inventory (THI) was used to assess tinnitus severity. ICD-9
codes for depression and anxiety were used to determine their
prevalence among the patients. Pure tone averages (PTA) were
used to assess hearing status.
Table1: The median age, standard deviations, and age ranges of veterans with tinnitus
with/without anxiety and depression (n=91).
Age
parameters
Anxiety
No anxiety
Depression
No
depression
Both
Median
62.5
61
64
65
65
SD
10.04
11.40
10.18
11.76
10.18
Range
31-73
51-80
31-79
31-84
39-79
Figure 1. The mean PTAs (500, 1000, 2000Hz) showed no differences among the
five groups or between ears (p > 0.05). The average hearing loss was around 38
dB HL among all the groups
Figure 2: Tinnitus severity presented with total THI (A) and Functional (B), Emotional (C) and
Catastrophic subscales (D) among tinnitus patients with and without anxiety and depression.
Table 2A: Correlations between the degree of tinnitus and anxiety
Pearson
Correlation
Significance
(2-tailed)
Total THI score * Anxiety
0.401
0.000
Functional THI score *
Anxiety
0.474
0.000
Emotional THI score *
Anxiety
0.473
0.000
Catastrophic THI score *
Anxiety
0.279
0.006
Tinnitus
characteristics
x/n (%)
Anxiety
No anxiety
Depression
No
depression
Both
Intermittent
n=32
28/32
(87.5)
4/32
(12.5)
18/32
(56.2)
14/32
(43.7)
18/32
(56.2)
Persistent
n=47
37/47
(78.7)
10/47
(21.3)
31/47
(65.9)
16/47
(34.0)
30/47
(63.8)
Bilateral
n=71
59/71
(83.0)
12/71
(16.9)
46/71
(64.8)
25/71
(13.2)
45/71
(63.4)
Unilateral
n=6
6/6
(100.0)
0/6
(0)
3/6
(50.0)
3/6
(50.0)
3/6
(50.0)
More than 10
years
n=33
30/33
(90.9)
3/33
(9.0)
20/33
(60.6)
13/33
(39.4)
20/33
(60.6)
Less than 10
years n=16
12/16
(75.0)
4/16
(25.0)
7/16
(43.7)
9/16
(56.2)
7/16
(43.7)
Conclusions: The majority of patients with tinnitus exhibited a
diagnosis of anxiety and depression. These patients suffer with
more severe tinnitus than tinnitus patients without anxiety and
depression. The data support the need for multidisciplinary
intervention of veterans who suffer with tinnitus.
Acknowledgements ?? The Department of Otolaryngology and Head & Neck
Surgery in the School of Medicine at Loma Linda University Medical Center provided
support.
Regeneration of Pre-synaptic Sensory Functions May Not Restore Post-Synaptic Neurotransmission
Eric Mendonsaa, Jinwei Hub, Helen Xub, O’neil W. Guthriea,b,
aResearch
Service-151, Loma Linda Veterans Affairs Medical Center, Loma Linda, CA 92357, USA.
bDepartment
of Otolaryngology and Head & Neck Surgery, School of Medicine, Loma Linda University Medical Center, Loma Linda, CA 92354, USA.
Some instances of individual
subject cochlear microphonics
and summating potentials at
low and high input stimuli
show levels of sensory
recovery 1 month after noise
exposure. Rat 14 CMs and Rat
19 SPs displayed to the right.

Subjects: Long-Evans rat model

Treatment: An octave band of noise centered at 8 kHz at 105 dB SPL for 4
hours.

Techniques:

Pre-synaptic

Distortion Product Otoacoustic Emission (DPOAE)

Cochlear Microphonic (CM)

Summating Potential (SP)

Post-synaptic

Auditory Brainstem Response (ABR) waves I-IV

Measurements of pre and post synaptic cochlear functions were taken at
baseline and 1 day, 1 week, and 1 month after noise exposure
Experimental data illustrates a decoupling between pre-synaptic
sensory function and post-synaptic neurotransmission after
damaging levels of noise exposure. DPOAE responses from noise
treated subjects exhibit detectable levels of sensory function
recovery. A similar (although not as dramatic) effect can be
observed in the pre-synaptic cochlear microphonic and summating
potential data in which experimental subjects realize recovery at
both low and high input stimuli. Post-synaptic neurotransmission,
on the other hand, displayed a lack of recovery as illustrated by the
negative trend from ABR waves I to IV. Overall, the data suggests
that the restoration of pre-synaptic sensory functionality does not
correlate with the degeneration of neural impulses from the
peripheral to central nervous system. Consequently, further
experimentation requires investigation of possible molecular
mechanisms.