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Expression of DNA methyltransferases in adult dorsal root ganglia is cell-type specific and up regulated in a rodent model of neuropathic pain.

Pollema-Mays SL, Centeno MV, Apkarian AV, Martina M - Front Cell Neurosci (2014)

Bottom Line: Real-time RT PCR analysis revealed robust and time-dependent changes in DNMT transcript expression in ipsilateral DRGs from spared nerve injury (SNI) but not sham rats.Interestingly, DNMT3b transcript showed a robust upregulation that appeared already 1 week after surgery and persisted at 4 weeks (our endpoint); in contrast, DNMT1 and DNMT3a transcripts showed only moderate upregulation that was transient and did not appear until the second week.We suggest that DNMT regulation in adult DRGs may be a contributor to the pain phenotype and merits further study.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology, Northwestern University Feinberg School of Medicine Chicago, IL, USA.

ABSTRACT
Neuropathic pain is associated with hyperexcitability and intrinsic firing of dorsal root ganglia (DRG) neurons. These phenotypical changes can be long lasting, potentially spanning the entire life of animal models, and depend on altered expression of numerous proteins, including many ion channels. Yet, how DRGs maintain long-term changes in protein expression in neuropathic conditions remains unclear. DNA methylation is a well-known mechanism of epigenetic control of gene expression and is achieved by the action of three enzymes: DNA methyltransferase (DNMT) 1, 3a, and 3b, which have been studied primarily during development. We first performed immunohistochemical analysis to assess whether these enzymes are expressed in adult rat DRGs (L4-5) and found that DNMT1 is expressed in both glia and neurons, DNMT3a is preferentially expressed in glia and DNMT3b is preferentially expressed in neurons. A rat model of neuropathic pain was then used to determine whether nerve injury may induce epigenetic changes in DRGs at multiple time points after pain onset. Real-time RT PCR analysis revealed robust and time-dependent changes in DNMT transcript expression in ipsilateral DRGs from spared nerve injury (SNI) but not sham rats. Interestingly, DNMT3b transcript showed a robust upregulation that appeared already 1 week after surgery and persisted at 4 weeks (our endpoint); in contrast, DNMT1 and DNMT3a transcripts showed only moderate upregulation that was transient and did not appear until the second week. We suggest that DNMT regulation in adult DRGs may be a contributor to the pain phenotype and merits further study.

No MeSH data available.


Related in: MedlinePlus

Von Frey measurements in SNI and sham rats. The spared nerve injury (SNI) model of neuropathic pain was used. The left or ipsilateral nerve was injured while the right or contralateral nerve was intact. Tactile sensitivity of the spared region of the operated paws was measured from the withdrawal responses to mechanical stimulation with von Frey filaments. 50% response thresholds were calculated according to Chaplan et al. (1994). Data are shown by dividing the contralateral/right paw over the ipsilateral/left paw, at 1, 2, and 4 weeks post SNI or sham surgery. All animals that underwent SNI surgery developed significant tactile allodynia in the ipsilateral/left paw that persisted until they were sacrificed, while sham rats showed no changes, *p < 0.01. n = 8 SNI and sham for 1 week, 4 for 2 weeks, and 6 for 4 weeks. The left foot von Frey thresholds for sham-operated and SNI rats, respectively, were 3.93 ± 0.58 g and 0.55 ± 0.41 g at 1 week, 5.16 ± 0.49 g and 0.64 ± 0.16 g at 2 weeks, and 7.93 ± 0.77 g and 0.97 ± 0.44 g at 4 weeks after surgery.
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Figure 3: Von Frey measurements in SNI and sham rats. The spared nerve injury (SNI) model of neuropathic pain was used. The left or ipsilateral nerve was injured while the right or contralateral nerve was intact. Tactile sensitivity of the spared region of the operated paws was measured from the withdrawal responses to mechanical stimulation with von Frey filaments. 50% response thresholds were calculated according to Chaplan et al. (1994). Data are shown by dividing the contralateral/right paw over the ipsilateral/left paw, at 1, 2, and 4 weeks post SNI or sham surgery. All animals that underwent SNI surgery developed significant tactile allodynia in the ipsilateral/left paw that persisted until they were sacrificed, while sham rats showed no changes, *p < 0.01. n = 8 SNI and sham for 1 week, 4 for 2 weeks, and 6 for 4 weeks. The left foot von Frey thresholds for sham-operated and SNI rats, respectively, were 3.93 ± 0.58 g and 0.55 ± 0.41 g at 1 week, 5.16 ± 0.49 g and 0.64 ± 0.16 g at 2 weeks, and 7.93 ± 0.77 g and 0.97 ± 0.44 g at 4 weeks after surgery.

Mentions: Having found that all DNMTs are expressed in the adult DRG, although in a cell-type dependent pattern, we wondered whether their expression levels are affected following neuropathic injury. Widespread changes in gene expression have been reported in animal models of neuropathic pain, and it is possible epigenetic mechanisms may underlie at least some of these changes. To test this hypothesis we took advantage of the SNI model, a robust and well-established model of neuropathic pain, which involves severing the tibial and peroneal portions of the sciatic nerve (Decosterd and Woolf, 2000). Tactile sensitivity was measured to verify the efficacy of the surgery. von-Frey filament testing was performed on all SNI and sham operated rats prior to experimental use and confirmed the presence of allodynia in all SNI rats but not sham controls. The left (operated) paw von Frey thresholds were 3.93 ± 0.58 g and 0.55 ± 0.41 g at 1 week, 5.16 ± 0.49 g and 0.64 ± 0.16 g at 2 weeks, and 7.93 ± 0.77 g and 0.97 ± 0.44 g at 4 weeks after surgery for sham-operated and SNI rats, respectively (Figure 3). qRT-PCR experiments were done 1, 2, and 4 weeks post surgery using L4 and L5 DRG tissue of control (sham operated) and SNI rats; genes of interest were normalized to the GAPDH reference gene. All data are shown normalized to age matched sham controls. First, the amount of beta actin transcript was quantified relative to GAPDH for all SNI and sham groups to test the stability of GAPDH and verify it’s suitability for use as a reference gene. No significant changes were detected in beta actin transcript levels between SNI and sham at 1, 2, or 4 weeks post surgery, thus confirming its suitability as a reference gene (data not shown).


Expression of DNA methyltransferases in adult dorsal root ganglia is cell-type specific and up regulated in a rodent model of neuropathic pain.

Pollema-Mays SL, Centeno MV, Apkarian AV, Martina M - Front Cell Neurosci (2014)

Von Frey measurements in SNI and sham rats. The spared nerve injury (SNI) model of neuropathic pain was used. The left or ipsilateral nerve was injured while the right or contralateral nerve was intact. Tactile sensitivity of the spared region of the operated paws was measured from the withdrawal responses to mechanical stimulation with von Frey filaments. 50% response thresholds were calculated according to Chaplan et al. (1994). Data are shown by dividing the contralateral/right paw over the ipsilateral/left paw, at 1, 2, and 4 weeks post SNI or sham surgery. All animals that underwent SNI surgery developed significant tactile allodynia in the ipsilateral/left paw that persisted until they were sacrificed, while sham rats showed no changes, *p < 0.01. n = 8 SNI and sham for 1 week, 4 for 2 weeks, and 6 for 4 weeks. The left foot von Frey thresholds for sham-operated and SNI rats, respectively, were 3.93 ± 0.58 g and 0.55 ± 0.41 g at 1 week, 5.16 ± 0.49 g and 0.64 ± 0.16 g at 2 weeks, and 7.93 ± 0.77 g and 0.97 ± 0.44 g at 4 weeks after surgery.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4126486&req=5

Figure 3: Von Frey measurements in SNI and sham rats. The spared nerve injury (SNI) model of neuropathic pain was used. The left or ipsilateral nerve was injured while the right or contralateral nerve was intact. Tactile sensitivity of the spared region of the operated paws was measured from the withdrawal responses to mechanical stimulation with von Frey filaments. 50% response thresholds were calculated according to Chaplan et al. (1994). Data are shown by dividing the contralateral/right paw over the ipsilateral/left paw, at 1, 2, and 4 weeks post SNI or sham surgery. All animals that underwent SNI surgery developed significant tactile allodynia in the ipsilateral/left paw that persisted until they were sacrificed, while sham rats showed no changes, *p < 0.01. n = 8 SNI and sham for 1 week, 4 for 2 weeks, and 6 for 4 weeks. The left foot von Frey thresholds for sham-operated and SNI rats, respectively, were 3.93 ± 0.58 g and 0.55 ± 0.41 g at 1 week, 5.16 ± 0.49 g and 0.64 ± 0.16 g at 2 weeks, and 7.93 ± 0.77 g and 0.97 ± 0.44 g at 4 weeks after surgery.
Mentions: Having found that all DNMTs are expressed in the adult DRG, although in a cell-type dependent pattern, we wondered whether their expression levels are affected following neuropathic injury. Widespread changes in gene expression have been reported in animal models of neuropathic pain, and it is possible epigenetic mechanisms may underlie at least some of these changes. To test this hypothesis we took advantage of the SNI model, a robust and well-established model of neuropathic pain, which involves severing the tibial and peroneal portions of the sciatic nerve (Decosterd and Woolf, 2000). Tactile sensitivity was measured to verify the efficacy of the surgery. von-Frey filament testing was performed on all SNI and sham operated rats prior to experimental use and confirmed the presence of allodynia in all SNI rats but not sham controls. The left (operated) paw von Frey thresholds were 3.93 ± 0.58 g and 0.55 ± 0.41 g at 1 week, 5.16 ± 0.49 g and 0.64 ± 0.16 g at 2 weeks, and 7.93 ± 0.77 g and 0.97 ± 0.44 g at 4 weeks after surgery for sham-operated and SNI rats, respectively (Figure 3). qRT-PCR experiments were done 1, 2, and 4 weeks post surgery using L4 and L5 DRG tissue of control (sham operated) and SNI rats; genes of interest were normalized to the GAPDH reference gene. All data are shown normalized to age matched sham controls. First, the amount of beta actin transcript was quantified relative to GAPDH for all SNI and sham groups to test the stability of GAPDH and verify it’s suitability for use as a reference gene. No significant changes were detected in beta actin transcript levels between SNI and sham at 1, 2, or 4 weeks post surgery, thus confirming its suitability as a reference gene (data not shown).

Bottom Line: Real-time RT PCR analysis revealed robust and time-dependent changes in DNMT transcript expression in ipsilateral DRGs from spared nerve injury (SNI) but not sham rats.Interestingly, DNMT3b transcript showed a robust upregulation that appeared already 1 week after surgery and persisted at 4 weeks (our endpoint); in contrast, DNMT1 and DNMT3a transcripts showed only moderate upregulation that was transient and did not appear until the second week.We suggest that DNMT regulation in adult DRGs may be a contributor to the pain phenotype and merits further study.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology, Northwestern University Feinberg School of Medicine Chicago, IL, USA.

ABSTRACT
Neuropathic pain is associated with hyperexcitability and intrinsic firing of dorsal root ganglia (DRG) neurons. These phenotypical changes can be long lasting, potentially spanning the entire life of animal models, and depend on altered expression of numerous proteins, including many ion channels. Yet, how DRGs maintain long-term changes in protein expression in neuropathic conditions remains unclear. DNA methylation is a well-known mechanism of epigenetic control of gene expression and is achieved by the action of three enzymes: DNA methyltransferase (DNMT) 1, 3a, and 3b, which have been studied primarily during development. We first performed immunohistochemical analysis to assess whether these enzymes are expressed in adult rat DRGs (L4-5) and found that DNMT1 is expressed in both glia and neurons, DNMT3a is preferentially expressed in glia and DNMT3b is preferentially expressed in neurons. A rat model of neuropathic pain was then used to determine whether nerve injury may induce epigenetic changes in DRGs at multiple time points after pain onset. Real-time RT PCR analysis revealed robust and time-dependent changes in DNMT transcript expression in ipsilateral DRGs from spared nerve injury (SNI) but not sham rats. Interestingly, DNMT3b transcript showed a robust upregulation that appeared already 1 week after surgery and persisted at 4 weeks (our endpoint); in contrast, DNMT1 and DNMT3a transcripts showed only moderate upregulation that was transient and did not appear until the second week. We suggest that DNMT regulation in adult DRGs may be a contributor to the pain phenotype and merits further study.

No MeSH data available.


Related in: MedlinePlus