Preliminary Study of Apoptotic Genetics in the Rat Cochlea

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Preliminary Study of Apoptotic Genetics in the
Rat Cochlea

• Syed Ahsan, MD
• Thomas Van De Water, PhD
• Thomas Balkany, MD

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Introduction

• There are two distinct modes of cell death
• Necrosis - Apoptosis
• Apoptosis is active, necrosis is passive
• Apoptosis is morphologically distinct
• Often the two are coexistent

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Extrinsic Cell Death Pathway
Intrinsic Cell Death Pathway
ROS
HNE
MAPK/JNK
Active caspase-8
(low concentration)
BID
(high concentration)
IAPs
TRV, 2003
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Introduction (Contd)

• Apoptosis plays an important role in the
  development of the cochlea up to P3.
• OHC loss after aminoglycosides occurs via
  apoptosis.
• Apoptosis has also been shown to be the driving
  force leading to progression of OHC death in
  chinchillas exposed to noise. (Hu et al, 2002,
  Hearing Research).

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Introduction (Contd)

• OHC loss may occur through multiple simultaneous
  apoptotic events
• Hypothesis mechanical trauma (CI) ?oxidative
  stress (ROS) leading to activation of the stress
  kinase pathway ? ?caspase 3,7,9 ?Apoptosis
• Understanding the genetics of apoptosis can lead
  to development of apoptotic inhibitors (caspase
  In, JNK In, Hypothermia) in improving function in
  CI patients as well as preserving hearing

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MAPK/JNK Signaling Pathway
Oxidative Stress
MEKK 1-3, MEKK 4, TAK 1,
ASK 1, Tpl-2
DLK MLK 3
3x Kinases
MKK 4, MKK 7
2x Kinases
JNK 1, JNK 2, JNK 3
1x Kinases
ATF-2, Elk-1,c-Jun, p53, DPC4, NFAT4a
APOPTOSIS
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Objectives

• To develop a model of CI in which to study the
  effects of CI and to develop methods to improve
  function after CI.
• Identify the baseline expression of early MAP
  kinase pathway genes (MKK4, MKK7, JNK1, JNK2,
  JNK3) in young rats.
• To determine if hypothermia decreases expression
  of MAP kinase pathway genes.
• Determine if direct mechanical trauma to the
  cochlea leads to different expression of
  apoptotic genes compared to controls.

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Materials and Methods

• Animal subjects and lesioning.
• Fisher 344 strain P3-7 rats.
• One group of rats were anesthetized with
  hypothermia (ice) for 20 minutes prior to surgery
  and before sacrifice (P3, P5, P7).
• Another group were anesthetized using ketamine
  prior to surgery and prior to sacrifice to
  control for effects of hypothermia.
• Surgery was performed by making an incision
  behind one ear and dissecting till the bullae was
  exposed.
• A 28 gauge needle was used to pass through the
  bullae and blindly injure the cochlea. Animals
  were allowed to recover prior to sacrifice.

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Rat Bulla
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Materials and Methods

• Total RNA extraction.
• Cochlear tissue disrupted with M/P, Needle and
  syringe .
• Quiagen Rneasy Mini system.
• Primers for rat MKK4, MKK7, JNK1, JNK2, JNK3 were
  designed from published sequences.

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Materials and Method

• RT-PCR.
• Invitrogen 1st strand system RT- using Oligo dT
  for mRNA.
• PCR 6 different primers.
• Eppendorf Master Mix.
• 35 cycles.
• Temp. optimized.
• 2 agarose gel used to ID PCR products.
• Visulaized by UV light following EtBr staining
  (5?g/ml).

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Results P3-4 rat cochlea apoptotic gene
expression. Exp vs. Control
MKK 7 ?
MKK4 ?
28 gauge needle injury to P3-4 rat cochlea, 4hr
later sacrificed hypothermia before injury and
prior to sacrifice used to anesthesia. JNK 1,
MKK4, MKK7 found in control and experimental.
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P5-7 rat cochlea apoptotic gene expression. Exp
vs. control

• 28 ga needle injury to cochlea
• hypothermia before injury and before
  sacrificing
• Expression of both MKK7 and JNK2 in exp and
  control rats

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P7 rat cochlea apoptotic gene expression Exp
vs. control

• Needle jab injury to cochlea
• Hypothermia prior to injury and before
  sacrificing
• MKK7 and JNK2 in exp and control

MKK7 ?
?S29
JNK 2 ?
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P4 rat cochlea apoptotic gene expression No
hypothermia controls
?S29
MKK7 ?
?JNK1
MKK4?
20 rats used. No hypothermia. expression of
S29, JNK1, MKK4, MKK7
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Discussion

• Baseline expression of MAP kinase genes.
• JNK1, MKK4 expressed only in very young (ltP4).
• JNK2 expressed in ?P5.
• MKK7 expressed at all ages examined (P3-7).
• Effect of hypothermia on MAP kinase gene
  expression.
• No effect noted (JNK1, MKK4, MKK7).

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Discussion

• No difference was noted in apoptotic pathway gene
  expression after needle injury to the cochlea.
• Apoptotic gene expression continues after P3
  stage (new finding).
• Current Project Insertion of electrode into
  adult rat basal turn of cochlea.