84% of the population will suffer from low back pain at some point in their lives, and 39% of the cases may be related to pain originating from the intervertebral disc (IVD) also known as discogenic pain. Age or injury can cause progressive degeneration of the IVD, and this can allow innervation by nociceptive (pain-sensing) neurons from the dorsal root ganglion (DRG) into the inner IVD.6 Long-term exposure to pain-inducing stimuli can cause nociceptor sensitization, manifested as increased pain sensation in response to non-painful or painful stimuli.7 Sensitization results in the nociceptors responding more strongly to a stimulus, and the activation energy required for response is reduced.8 During IVD degeneration, inflammation and oxidative stress create excess reactive oxygen species (ROS) .9  ROS such as superoxide may play a role in nociceptive signaling and lead to nociceptor sensitization.8 ROS are involved in the chronic pain that results from nerve injury or inflammation in many contexts and may be involved in discogenic pain as well.10 The focus of this study is to determine if long-term oxidative stress causes a change in the DRG nociceptive neuron phenotype related to sensitization. This would increase the expression of pain-related ion channels and lower the neuronal activation threshold.
1. DePalma et al. (2011) 2. Balague et al. (2012) 3. Zhang et al. (2010) 4. Humzah et al. (1988) 5. Ohtori et al. (2015) 6. Yang et al. (2018) 7. Woolf et al. (2010) 8. Chung et al. (2004) 9. Nasto et al. (2013) 10. Yowtak et al. (2011

Redwine, Adan

Romereim, Sarah M

Wachs, Rebecca A.

English

DigitalCommons@University of Nebraska

University of Nebraska - Lincoln DigitalCommons@University of Nebraska - Lincoln UCARE Research Products UCARE: Undergraduate Creative Activities & Research Experiences Spring 4-14-2020 Quantification of Phenotypic Change Resulting in Sensitization of Primary Sensory Neurons due to Oxidative Stress Adan Redwine University of Nebraska - Lincoln, aredwine@huskers.unl.edu Sarah M. Romereim University of Nebraska - Lincoln, sromereim2@unl.edu Rebecca A. Wachs University of Nebraska - Lincoln, rebecca.wachs@unl.edu Follow this and additional works at: https://digitalcommons.unl.edu/ucareresearch  Part of the Biomedical Engineering and Bioengineering Commons Redwine, Adan; Romereim, Sarah M.; and Wachs, Rebecca A., "Quantification of Phenotypic Change Resulting in Sensitization of Primary Sensory Neurons due to Oxidative Stress" (2020). UCARE Research Products. 180. https://digitalcommons.unl.edu/ucareresearch/180 This Poster is brought to you for free and open access by the UCARE: Undergraduate Creative Activities & Research Experiences at DigitalCommons@University of Nebraska - Lincoln. It has been accepted for inclusion in UCARE Research Products by an authorized administrator of DigitalCommons@University of Nebraska - Lincoln. Quantification of Phenotypic Change Resulting in Sensitization of Primary Sensory Neurons due to Oxidative StressRedwine, Adan L; Romereim, Sarah M; Wachs, Rebecca ADepartment of Biological Systems Engineering, University of Nebraska - LincolnMOTIVATION AND SIGNIFICANCE METHODS RESULTSACKNOWLEDGEMENTSDISCUSSION AND FUTURE WORKRNA Isolation1. Flash frozen DRG samples homogenized and lysed using TRI Reagent. 2. Chloroform used to separate sample into three phases. A red organic phase (containing protein), an interphase (containing DNA), and an aqueous phase (containing RNA). (Figure 2)3. Aqueous phase transferred to another vial. 4. RNA phase then precipitated into RNA pellet using isopropanol. (Figure 2)5. RNA pellet was washed and resuspended innuclease-free water for NanoDrop analysis.• Current ROS treatments cause some oxidative stress in DRGexplants but not enough.• We will move forward with optimizing additional analysis methodssuch as qPCR (Figure 4), Western blotting, and live calciumimagining.• Future work will focus on increasing oxidative stress levels in DRGsto desirable levels while keeping the cells alive.• Once oxidative stress inducing methods are established it will thenbe possible to determine if long-term oxidative stress causes aphenotypic change in DRG sensory neuron phenotype.DRG Harvest1. DRGs dissected from sacrificed neonatal or adult rats.2. Surrounding tissue removed and neuronal projections (neurites)trimmed.a) Some DRGs flash frozen and stored at -80°C for subsequent RNAisolation.b) Some DRGs and embedded in hydrogel for culture (see below).DRG Culture1. DRGs embedded in hydrogels consisting of type I collagen, laminin,and methacrylated hyaluronic acid (MAHA).2. Cultured in 300 µL complete DRG media (Neurobasal media with10% FBS, 1% Glutamax, 1% pen/strep, 2% B27, and 0.01% NGF).3. ½ media changed every other day.4. DRGs cultured for six days to allow neurite growth.5. Imaged on day six using bright-field microscopy. (Figure 1, scale bar 500 µm) RNA Isolation The wavelength absorbance ratios measured by NanoDropA260/A280  & A260/A230 indicate whether RNA quality is desirable to use for qPCR analysis. (Table 1 & Figure 5)• c1–3• When healthy, the IVD is mainly aneural and absorbs compressionloads for spine motion and flexibility.4,5• Age or injury can cause progressive degeneration of the IVD, andthis can allow innervation by nociceptive (pain-sensing) neuronsfrom the dorsal root ganglion (DRG) into the inner IVD.6• Long-term exposure to pain-inducing stimuli can cause nociceptorsensitization, manifested as increased pain sensation in response tonon-painful or painful stimuli.7• Sensitization results in the nociceptors responding more strongly toa stimulus, and the activation energy required for response isreduced.8• During IVD degeneration, inflammation and oxidative stress createexcess reactive oxygen species (ROS) .9• ROS such as superoxide may play a role in nociceptive signaling andlead to nociceptor sensitization.8• ROS are involved in the chronic pain that results from nerve injuryor inflammation in many contexts, and may be involved indiscogenic pain as well.10• The focus of this study is to determine if long-term oxidative stresscauses a change in the DRG nociceptive neuron phenotype relatedto sensitization. This would increase the expression of pain-relatedion channels and lower the neuronal activation threshold.METHODSREFERENCES1. DePalma et al. (2011)2. Balague et al. (2012)3. Zhang et al. (2010)4. Humzah et al. (1988)5. Ohtori et al. (2015)6. Yang et al. (2018)7. Woolf et al. (2010)8. Chung et al. (2004)9. Nasto et al. (2013)10. Yowtak et al. (2011) Figure 1. – Bright-field image of DRG after six days of culture in hydrogel. ROS Induction1. DRGs embedded in hydrogels and cultured for 4 days in normal media.2. DRGs were treated 3 times every 3 hours for 3 days with the ROS hydrogen peroxide (H2O2 ) and the intracellular antioxidant inhibitor L-BSO to induce oxidative stress or cultured in normal media as a control.● 100 µM H2O2 ● 100 µM H2O2 + 10 µM L-BSO● 200 µM H2O2 ● 200 µM H2O2 + 10 µM L-BSO3. Separately, oxidative stress induction by culturing in different concentrations of oxygen was performed after 3 days of culture in normal media in normoxia. Normoxia culture is 21% O2, 5% CO2. Hypoxia is 3.5% O2, 5% CO2.● Normoxia  ● Hypoxia for 30 min every 3 hrs for 3 days, otherwise normoxia● Hypoxia     ● Normoxia for 30 min every 3 hrs for 3 days, otherwise hypoxiaAnalysis:1. ROS quantification using the fluorescence probe dihydroethidium (DHE).i. DRGs incubated in 20 µM DHE.ii. DRG gels embedded in cryo-medium and flash frozen in liquid nitrogen.iii. Frozen DRG blocks sectioned onto slides and immediately imaged on confocal microscope to detect fluorescence from ROS-reacted DHE.iv. Sections stained with DAPI for 3 minutes followed by re-imaging on the confocal to visualize cell nuclei.2. DRG gels fixed, cryoembedded, sectioned, immunofluorescence for oxidative stress markers, and fluorescence intensity quantified using Image J (NIH):● 8-hydroxy-2'-deoxyguanosine (8-OHdG; RNA/DNA damage)● 4-hydroxylnonenal (lipid damage) ● Carboxymethyl lysine (protein damage)Figure 2.a) RNA phase separationb) RNA aqueous phase c) RNA pellet a b cCellsRNA + Genomic DNARNAcDNA + RNATRI Reagent DNase Superscript III qPCRqPCR DesignRNA Isolation and cDNA SynthesisSelect genes of interestDesign PrimersOrder PrimersDesign Plate LayoutRun qPCR PlateSample IDNucleic Acid Conc. (ng/µl)260/280 260/2301 151.5 1.86 1.382 217.3 1.89 23 144.4 1.8 2.09ROS Induction• 200 µM H2O2 + 10 µM L-BSO showed a significant difference influorescence intensity of the RNA/DNA damage marker 8-OHdG.• Slight increase in 8-OHdG fluorescence in hypoxia-to-normoxiacondition.Table 1. NanoDrop results from RNA isolationFigure 5. NanoDrop results from RNA isolation• Thank you to UCARE UNL for supporting and allowing me the opportunity to conduct this research.Figure 3. Overview of steps to prepare for qPCR Figure 4. Overview of qPCR plate preparation.UCARE(1)(2)(3)• An A260/A280 of 1.8-2.1 indicates highly purified RNA, and an A260/A230near 2.0 is ideal, therefore samples 2 and 3 favorable for subsequent cDNA synthesis and qPCR analysis. (Figure 3)

Quantification of Phenotypic Change Resulting in Sensitization of Primary Sensory Neurons due to Oxidative Stress

UNL | Libraries

University of Nebraska - Lincoln 
DigitalCommons@University of Nebraska - Lincoln 
UCARE Research Products UCARE: Undergraduate Creative Activities & Research Experiences 
Spring 4-14-2020 
Quantification of Phenotypic Change Resulting in Sensitization of 
Primary Sensory Neurons due to Oxidative Stress 
Adan Redwine 
University of Nebraska - Lincoln, aredwine@huskers.unl.edu 
Sarah M. Romereim 
University of Nebraska - Lincoln, sromereim2@unl.edu 
Rebecca A. Wachs 
University of Nebraska - Lincoln, rebecca.wachs@unl.edu 
Follow this and additional works at: https://digitalcommons.unl.edu/ucareresearch 
 Part of the Biomedical Engineering and Bioengineering Commons 
Redwine, Adan; Romereim, Sarah M.; and Wachs, Rebecca A., "Quantification of Phenotypic Change 
Resulting in Sensitization of Primary Sensory Neurons due to Oxidative Stress" (2020). UCARE Research 
Products. 180. 
https://digitalcommons.unl.edu/ucareresearch/180 
This Poster is brought to you for free and open access by the UCARE: Undergraduate Creative Activities & Research 
Experiences at DigitalCommons@University of Nebraska - Lincoln. It has been accepted for inclusion in UCARE 
Research Products by an authorized administrator of DigitalCommons@University of Nebraska - Lincoln. 
Quantification of Phenotypic Change Resulting in Sensitization of 
Primary Sensory Neurons due to Oxidative Stress
Redwine, Adan L; Romereim, Sarah M; Wachs, Rebecca A
Department of Biological Systems Engineering, University of Nebraska - Lincoln
MOTIVATION AND SIGNIFICANCE METHODS RESULTS
ACKNOWLEDGEMENTS
DISCUSSION AND FUTURE WORK
RNA Isolation
1. Flash frozen DRG samples homogenized and lysed using TRI Reagent. 
2. Chloroform used to separate sample into three phases. A red organic phase 
(containing protein), an interphase (containing DNA), 
and an aqueous phase (containing RNA). (Figure 2)
3. Aqueous phase transferred to another vial. 
4. RNA phase then precipitated into RNA pellet 
using isopropanol. (Figure 2)
5. RNA pellet was washed and resuspended in
nuclease-free water for NanoDrop analysis.
• Current ROS treatments cause some oxidative stress in DRG
explants but not enough.
• We will move forward with optimizing additional analysis methods
such as qPCR (Figure 4), Western blotting, and live calcium
imagining.
• Future work will focus on increasing oxidative stress levels in DRGs
to desirable levels while keeping the cells alive.
• Once oxidative stress inducing methods are established it will then
be possible to determine if long-term oxidative stress causes a
phenotypic change in DRG sensory neuron phenotype.
DRG Harvest
1. DRGs dissected from sacrificed neonatal or adult rats.
2. Surrounding tissue removed and neuronal projections (neurites)
trimmed.
a) Some DRGs flash frozen and stored at -80°C for subsequent RNA
isolation.
b) Some DRGs and embedded in hydrogel for culture (see below).
DRG Culture
1. DRGs embedded in hydrogels consisting of type I collagen, laminin,
and methacrylated hyaluronic acid (MAHA).
2. Cultured in 300 µL complete DRG media (Neurobasal media with
10% FBS, 1% Glutamax, 1% pen/strep, 2% B27, and 0.01% NGF).
3. ½ media changed every other day.
4. DRGs cultured for six days to allow neurite growth.
5. Imaged on day six using bright-field microscopy. 
(Figure 1, scale bar 500 µm) 
RNA Isolation 
The wavelength absorbance 
ratios measured by NanoDrop
A260/A280  & A260/A230 indicate 
whether RNA quality is desirable 
to use for qPCR analysis. 
(Table 1 & Figure 5)
• c1–3
• When healthy, the IVD is mainly aneural and absorbs compression
loads for spine motion and flexibility.4,5
• Age or injury can cause progressive degeneration of the IVD, and
this can allow innervation by nociceptive (pain-sensing) neurons
from the dorsal root ganglion (DRG) into the inner IVD.6
• Long-term exposure to pain-inducing stimuli can cause nociceptor
sensitization, manifested as increased pain sensation in response to
non-painful or painful stimuli.7
• Sensitization results in the nociceptors responding more strongly to
a stimulus, and the activation energy required for response is
reduced.8
• During IVD degeneration, inflammation and oxidative stress create
excess reactive oxygen species (ROS) .9
• ROS such as superoxide may play a role in nociceptive signaling and
lead to nociceptor sensitization.8
• ROS are involved in the chronic pain that results from nerve injury
or inflammation in many contexts, and may be involved in
discogenic pain as well.10
• The focus of this study is to determine if long-term oxidative stress
causes a change in the DRG nociceptive neuron phenotype related
to sensitization. This would increase the expression of pain-related
ion channels and lower the neuronal activation threshold.
METHODS
REFERENCES
1. DePalma et al. (2011)
2. Balague et al. (2012)
3. Zhang et al. (2010)
4. Humzah et al. (1988)
5. Ohtori et al. (2015)
6. Yang et al. (2018)
7. Woolf et al. (2010)
8. Chung et al. (2004)
9. Nasto et al. (2013)
10. Yowtak et al. (2011) 
Figure 1. – Bright-field image of DRG 
after six days of culture in hydrogel. 
ROS Induction
1. DRGs embedded in hydrogels and cultured for 4 days in normal media.
2. DRGs were treated 3 times every 3 hours for 3 days with the ROS hydrogen 
peroxide (H2O2 ) and the intracellular antioxidant inhibitor L-BSO to induce 
oxidative stress or cultured in normal media as a control.
● 100 µM H2O2 ● 100 µM H2O2 + 10 µM L-BSO
● 200 µM H2O2 ● 200 µM H2O2 + 10 µM L-BSO
3. Separately, oxidative stress induction by culturing in different concentrations 
of oxygen was performed after 3 days of culture in normal media in 
normoxia. Normoxia culture is 21% O2, 5% CO2. Hypoxia is 3.5% O2, 5% CO2.
● Normoxia  ● Hypoxia for 30 min every 3 hrs for 3 days, otherwise normoxia
● Hypoxia     ● Normoxia for 30 min every 3 hrs for 3 days, otherwise hypoxia
Analysis:
1. ROS quantification using the fluorescence probe dihydroethidium (DHE).
i. DRGs incubated in 20 µM DHE.
ii. DRG gels embedded in cryo-medium and flash frozen in liquid nitrogen.
iii. Frozen DRG blocks sectioned onto slides and immediately imaged on 
confocal microscope to detect fluorescence from ROS-reacted DHE.
iv. Sections stained with DAPI for 3 minutes followed by re-imaging on the 
confocal to visualize cell nuclei.
2. DRG gels fixed, cryoembedded, sectioned, immunofluorescence for oxidative 
stress markers, and fluorescence intensity quantified using Image J (NIH):
● 8-hydroxy-2'-deoxyguanosine (8-OHdG; RNA/DNA damage)
● 4-hydroxylnonenal (lipid damage) ● Carboxymethyl lysine (protein damage)
Figure 2.
a) RNA phase separation
b) RNA aqueous phase c) RNA pellet 
a b c
Cells
RNA + Genomic 
DNA
RNA
cDNA 
+ RNA
TRI Reagent DNase Superscript III qPCR
qPCR Design
RNA Isolation and cDNA Synthesis
Select genes 
of interest
Design 
Primers
Order 
Primers
Design Plate 
Layout
Run qPCR 
Plate
Sample ID
Nucleic Acid 
Conc. (ng/µl)
260/280 260/230
1 151.5 1.86 1.38
2 217.3 1.89 2
3 144.4 1.8 2.09
ROS Induction
• 200 µM H2O2 + 10 µM L-BSO showed a significant difference in
fluorescence intensity of the RNA/DNA damage marker 8-OHdG.
• Slight increase in 8-OHdG fluorescence in hypoxia-to-normoxia
condition.
Table 1. NanoDrop results from RNA isolation
Figure 5. NanoDrop results from RNA isolation
• Thank you to UCARE UNL for supporting and allowing me the 
opportunity to conduct this research.
Figure 3. Overview of steps to prepare for qPCR 
Figure 4. Overview of qPCR plate preparation.
UCARE
(1)
(2)
(3)
• An A260/A280 of 1.8-2.1 indicates highly purified RNA, and an A260/A230
near 2.0 is ideal, therefore samples 2 and 3 favorable for subsequent 
cDNA synthesis and qPCR analysis. (Figure 3)


https://digitalcommons.unl.edu/cgi/viewcontent.cgi?article=1195&context=ucareresearch

Quantification of Phenotypic Change Resulting in Sensitization of Primary Sensory Neurons due to Oxidative Stress

Abstract

Similar works

Full text

Available Versions

DigitalCommons@University of Nebraska

UNL | Libraries