Enhanced Expression And Activity of NAD(P)H Oxidase in Mouse - - PowerPoint PPT Presentation
Enhanced Expression And Activity of NAD(P)H Oxidase in Mouse Periaqueductal Gray Tissue During Morphine Antinociceptive Tolerance Department of Pharmacology & Toxicology Virginia Commonwealth University Emily C. Wright Background:
Department of Pharmacology & Toxicology Virginia Commonwealth University
Emily C. Wright
Area surrounding
cerebral aqueduct in brain stem levels 9 and 10
Contains receptors
for opiate peptides which can eliminate the perception of pain
Pain reduction takes place when opiates
Antinociceptive tolerance may result from
Morphine causes increase in intracellular
Role of NAD(P)H Oxidase in Morphine Induced Tolerance
NAD(P)H NAD(P) H
+ H
2O
2
e extracellular cytoplasm membrane P22 P67 P47 Morphine (
NA
+
H
+ +
P22 P67 NO (
._
ONOO -
Rac
gP91
R
analgesia
NAD(P)H NAD(P) H
+ H
2O
2
e extracellular cytoplasm membrane P22 P67 P47 Morphine (
NA
+
H
+ +
P22 P67 NO (
._
ONOO -
Rac
gP91
R
analgesia
Is NAD(P)H oxidase (subunits p47 and NOX-2) present
in the PAG?
(process used to localize proteins in cells of tissue sections)
β -actin gp91phox
M V M V PAG Cortex 58kDa 45kDa PAG Cortex 0.0 0.6 1.2 1.8 2.4 3.0 Vehicle Morphine
gp91phox protein expression
(Ratio to β−action)
47kDa 45kDa M M V V PAG Cortex
p47phox β-actin
0.0 0.6 1.2 1.8 2.4 3.0 Vehicle Morphine PAG Cortex
p47phox protein expression (Ratio to β − actin)
PAG 1 2 3 4 5 Vehicle Morphine Cortex
Expression of gp91phox mRNA (Tn )
PAG 5 10 15 20 25 Vehicle Morphine Cortex
Expression of p47phox mRNA (Tn)
3 groups of mice: naïve, placebo pellet,
Performed a two-day immunohisto-
Qualitatively analyzed results by taking
Figure 1: Expression of the p47 antigen in the periaqueductal gray and cortex of placebo pellet mouse brain tissue. A) 400X magnification. B) 1000X magnification.
PAG Cortex p47 Expression
A
Negative Control
B
p47 Expression Negative Control PAG Cortex
Figure 2: Expression of the NOX-2 antigen in the periaqueductal gray and cortex of placebo pellet mouse brain tissue. A) 400X magnification. B) 1000X magnification.
PAG Cortex
NOX-2 Expression
A
Negative Control
B
Negative Control PAG Cortex
NOX-2 Expression
NAD(P)H oxidase is present in the PAG of
Perform ESR to detect the levels of
Perform HPLC to assess the functioning of
Figure 3: Expression of the NOX-1 antigen in the cortex and medulla of rat kidney tissue. A) 400X magnification. B) 1000X magnification
Cortex Medulla
NOX-1 Expression
A
Negative Control
B
Negative Control Cortex Medulla
NOX-1 Expression
Figure 4: Expression of the NOX-1 antigen in the cortex and medulla of mouse kidney tissue. A) 400X magnification. B) 1000X magnification
B
Negative Control Cortex Medulla
NOX-1 Expression
Cortex Medulla
NOX-1 Expression
A
Negative Control
Figure 5: Expression of the NOX-2 antigen in the cortex and medulla of rat kidney tissue. A) 400X magnification. B) 1000X magnification
Cortex Medulla
NOX-2 Expression
A
Negative Control
B
Negative Control Cortex Medulla
NOX-2 Expression
Figure 6: Expression of the NOX-2 antigen in the cortex and medulla of mouse kidney tissue. A) 400X magnification. B) 1000X magnification
B
Negative Control Cortex Medulla
NOX-2 Expression
Cortex Medulla
NOX-2 Expression
A
Negative Control
Figure 7: Expression of the NOX-3 antigen in the cortex and medulla of rat kidney tissue. A) 400X magnification. B) 1000X magnification
Cortex Medulla
NOX-3 Expression
A
Negative Control
B
Negative Control Cortex Medulla
NOX-3 Expression
Figure 8: Expression of the NOX-3 antigen in the cortex and medulla of mouse kidney tissue. A) 400X magnification. B) 1000X magnification
B
Negative Control Cortex Medulla
NOX-3 Expression
Cortex Medulla
NOX-3 Expression
A
Negative Control
Figure 9: Expression of the NOX-4 antigen in the cortex and medulla of rat kidney tissue. A) 400X magnification. B) 1000X magnification
Cortex Medulla
NOX-4 Expression
A
Negative Control
B
Negative Control Cortex Medulla
NOX-4 Expression
Figure 10: Expression of the NOX-4 antigen in the cortex and medulla of mouse kidney tissue. A) 400X magnification. B) 1000X magnification
B
Negative Control Cortex Medulla
NOX-4 Expression
Cortex Medulla
NOX-4 Expression
A
Negative Control
There are some differences between rat
Positive controls for NOX-3 and NOX-4
Labs of Dr. Li and Dr. Dewey Program for Summer Research
isolated from mice chronically treated with morphine. (2005).
Mouse Periaqueductal Gray Neurons During Morphine Antinociceptive
http://www.neuroanatomy.wisc.edu/virtualbrain/BrainStem/24PAG.html. (2006).