Download Hair cell regeneration Part 2 - Academy of Doctors of Audiology

Hair cell regeneration Part 2
Brenda M. Ryals, Ph.D.
JJames M
Madison
di
U
University
i
it
Robert Dooling, Ph.D.
University of Maryland
Academy of Doctors of Audiology
November 4-6, 2010
With special appreciation to Dr. Robert
Dooling, University of Maryland
Co-investigator – Functional aspects of
Hair cell Regeneration
Work supported by NIDCD grants DC01245 DC1372,
DC00198 and DC00353 and The Comparative and
Evolutionary Biology of Hearing Training Grant (DC00046)
And to Dr. Beth Brittan-Powell, University
of Maryland for all of the ABR results
Work supported by NIDCD grants DC01245 DC1372,
DC00198 and DC00353 and The Comparative and
Evolutionary Biology of Hearing Training Grant (DC00046)
1
Will Hair Cell Regeneration Cure
Hearing Loss?
• Will
regenerated hair cells result
in the return of normal hearing
(audiogram), complex auditory
perception (speech
understanding)?
Will Hair Cell Regeneration Cure
Hearing Loss?
•Does the onset and etiology of
hearing loss matter?
•Pre-lingual/post-lingual
•Congenital/genetic
•Acquired
Birds offer a unique animal model of complex
auditory perception, vocal learning and vocal
production
2
Birds offer a unique animal model of complex
auditory perception, vocal learning and vocal
production
AND..they regenerate hair cells
Effect of Early Deafening on Warble Song
Normal Budgerigar
Deafened Budgerigar
Effect of Adult Deafening on Contact Calls
Heaton, et al, 1999
3
Vocal Learning in Budgerigars
Farabaugh, et al, 1994
Birds naturally regenerate
and re-innervate hair
cells after acquired injury
Birds naturally use
hearing to modify vocal
production
What does the world
sound like after hair cell
regeneration? Does it
make a difference to vocal
production?
4
Hearing Sensitivity Recovers to near normal within 8
weeks
125
10
20
30
250
Frequency in Hz
500
1000
X
X
X
2000
4000
8000
X = pre-injection
X X X
X X
X
X
X
40
X = 2 weeks post
injection
X
X
X= 8 weeks post
injection
50
X
70
X
Absolute Threshold dB SPL (re 20 pa)
60
80
90
100
80
Injections
70
60
50
40
30
PTS of 23 dB
20
10
0
0
5
10
15
20
25
30
Days
Dooling, Ryals and Manabe 1997
Hearing Resolution returns to
normal even sooner
A
30
F (Hz)
1.0 kHz
2.86 kHz
20
10
0
Pre
4-6
8-10
12+
Pre
4-6
8-10
12+
Weeks
B
6
1.0 kHz
2.86 kHz
4
I (dB)
Hearing Level in dB SPL
0
2
0
Pre
4-6
8-10
12+
Pre
4-6
8-10
12+
Weeks
Dooling, Ryals and Manabe 1997
5
Does Hair Cell Regeneration Cure Hearing Loss? Yes.
Behavioral audiograms show 40-50dB recovery of
sensitivity for even the most severely damaged regions.
Difference limens for frequency and intensity return to
normal.
Human
pigeon
What does
Wh
d
the brain
have to do
with
anything?
What is the effect of regeneration on
perception and production of relevant,
complex acoustical stimuli?
6
Perceptual Maps of Budgerigar Contact Calls
Before Kanamycin
(VAF = 79%)
28-Day Recovery
(VAF = 78%)
Dimension 2
0.50
D
D'
0.25
B
B'
DB'B
D
D'
A'
E E'
0.00
C'
C
-0.25
E'E
A'
A
-0.50
-0.50
-0.25
0.00
0.25
C'C
0.50
-0.50
-0.25
0.00
0.25
0.50
Dimension 1
Recovery was back to pre-injection levels by 23 weeks
Dooling, Ryals, Dent and
Reid, 2006
7
Will Hair Cell Regeneration Cure Hearing
Loss?
When the papilla first becomes
repopulated, the world sounds
different. Absolute sensitivity and
simple
p discrimination recovers within
weeks of regeneration while more
complex recognition tasks may take
several months to recover.
Full regeneration, maturation and
re-innervation of hair cells occurs
well before full recovery of function.
Vocal production in budgerigars
Kazu Manabe, Michael Osmanski, Ashwin Plachikkat, Manjit Sahota
Training birds to produce specific
vocalizations by operant conditioning
Relattive Similarity
Effect of Kanamycin (200mg/kg/day) on Vocal Precision
1.0
0.9
0.8
0.7
Injections
0.6
0
5
10
15
20
25
Days
8
Relative Similarity
1.1
1.0
0.9
0.8
0.7
0.6
0.5
A
Injections
0.0
-5
0
5
10
15
20
25
15
20
25
30
Days
Sequence Stereotyp
py
1.1
10
1.0
0.9
Injections
0.8
0.7
0.6
0.5
B
0.0
-5
0
5
10
30
Days
Woolley and Rubel
2002
Can regenerated hair cells support normal
vocal production?
 The precision of vocal production
is only temporarily disrupted and
only
y when hearing
g loss is the most
severe
Does the onset and etiology of
hearing loss matter?
Belgian Waterslager
Canaries – a model of prelingual, hereditary deafness
i conjunction
in
j
i with
i h ongoing hair cell regeneration
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Canary Domestication
•Initial domestication
over 500 years ago by
Spanish monks
•Breeding by miners in
Tyrol and the Hartz
mountains
•Developed first song
strain: Hartz Rollers
Canary Song Strains
• Dozens of canary strains bred for color, plumage style,
conformation
• Several strains bred specifically for song: German
(Hartz) Rollers, Belgian Waterslagers, Spanish
Timbrados, Russian Singers, American Singers,
Borders.
Absolute Threshold in dB SPL (re 20Pa)
• Earliest known reference to Belgian Waterslager is
1713, near Malinois Belgium, possible strain bottleneck
around turn of the century
90
Behavioral Audiograms
80
Belgian Waterslager
70
Canary
60
50
40
30
20
10
0.1
Non-Belgian Waterslager
Canary
1
10
Frequency (kHz)
10
What is the Inheritance Pattern?
Absolute Threshold in dB SPL (re 20Pa)
Auditory Sensitivity in Hybrid Canaries
90
Belgian Waterslager
Canary
80
70
Symbols = F1 Hybrids
60
50
40
30
20
10
Non-Belgian Waterslager
Canary
0.1
1
10
Frequency (kHz)
Okanoya, et al, 1987
Canary ABR Thresholds
Threshold in dB SPL(re 20 uPa)
110
100
90
Waterslagers
80
70
60
Non-Waterslagers
50
Only Waterslager Z's (n=6)
40
At least 1 Border Z (n=11)
30
20
500
1000
1500 2000
2860
4000
5700
8000
Frequency (Hz)
Does the onset and etiology of hearing loss
matter?
BWS have genetic inner
ear abnormality (Wright et al
2004)
-non-syndromic,
non syndromic
recessive, rapidly
progressive, sex-linked
1
V)
8
4
2
0
-4
Amplitude (
-8
0
2
4
6
8
10
Time (ms)
stimulus reaches bird's ear
(.88 ms delay)
Brittan-Powell, Dooling and Ryals 2008
11
Can BWS canaries regenerate hair cells?
27 hours after BrdU injection
136 hours after BrdU injection
Gleich, et al, 1997
After 10 days of tritiated thymidine injections
Yes, regeneration rate in
BWS canaries ~ 6
cells/day
Ryals, et al
1999
Not only are new hair cells being “born”
every day, but other hair cells are dying
every day.
Wilkins et al 2001
Does the onset and etiology of hearing loss
matter?
So hair cell abnormalities increase and cells begin
dying soon after hatching (~20 days).
ABR thresholds in BWS never achieve normal
sensitivity
12
Does this constant turn-over of
hair cells affect the auditory
nerve? The cochlear nucleus?
•Number of afferent synapses are
reduced from 4-5/HC to ~2/HC in
BWS
•Efferent synapses are similar in
BWS and normal canary ~1-2 per
HC
There is a corresponding
•There
average 12% loss of neural
fibers – BUT the loss of HC in
adults is 30% or more
•Cell size but not number is
reduced in BWS at first order
nuclei
Kubke et al 2002; Gleich Dooling
and Ryals 2001; Ryals and Dooling
2008
•Could we “cure” hearing loss in
BWS by stimulating the
remaining precurser cells
(supporting cells) to divide and
repopulate the epithelium?
13
Threshold sh
hift (dB)
Recovery from Kanamycin (200mg/kg/day)
50
1000 Hz
40
BWS canaries (n=4)
Normal canaries (n=4)
Pre-injection threshold
2000 Hz
2860 Hz
4000 Hz
50
40
30
30
20
20
10
10
0
0
-10
-10
2
4
6
8 10 12
2
4
6
8 10 12
2
4
6
8 10 12
2
4
6
8 10 12
Weeks following injections
What have we learned from birds?
•Verifying the influence of regenerated hair cells
on hearing
•We
We must measure more than the pure tone audiogram if
we want to know what regeneration means to hearing
•We see two time courses of recovery – Perception of
complex vocal calls returns much more slowly than
simple auditory sensitivity. What is the role of
experience/training?
What have we learned from birds?
– Onset and Etiology of Hearing Loss
– Genetic bases of Hearing Loss
•Small songbirds such as budgerigars and canaries are
essentially “deaf” in the first two weeks post-hatch. This
offers a “window” for studies of the influence of
regeneration on early onset, “prelingual” deafness.
•Because these are songbirds, we may also be able to
gain some insight into the influence of regeneration on
early vocal learning
14
What have we learned from birds?
•Issues of Candidacy
– Onset and Etiology of Hearing Loss
– Genetic bases of Hearing Loss
•At least for the Belgian Waterslager Canary, genetic inner
ear abnormalities at the level of the hair cell do not
preclude the hair cell regeneration
•Stimulation of increased hair cell regeneration, in this
case of genetic abnormality, results in improved – but
not normal- auditory sensitivity
With appreciation to my Co-Author Dr.
Robert Dooling and to students and
collaborators:
Auditory Research Lab James Madison University
Edwina Westbrook; Tracy Tucker; Vince Hottinger; Ericka Mills;
Kristin Wehman; Rhonda Snorrason; WC Sheets; Kelly Moran,
Tracy Tucker, Deb Leap, Katie Roper, Heather Bennett
Laboratory of Comparative Psychoacoustics University
of Maryland
Beth Brittan-Powell; Micheal Dent; Otto Gleich;Tracy Kidd; George
Klump; Melonie Mavilia; Kazuo Okanoya; Thomas Park;Joelle
Presson; Richard Sleboda; Heather Wilkins; Elena Sanovich,
Timothy Wright
Work supported by NIDCD grants DC01245 DC1372,
DC00198 and DC00353 and The Comparative and
Evolutionary Biology of Hearing Training Grant (DC00046)
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