Tuesday, April 24, 2012
Tuesday, February 28, 2012
pn-Fect and Transfection of DRG cultures
In Vitro Gene expression analysis assays are essential for understanding how up or down regulation of related target proteins could result in pathologies. Dorsal Root Ganglion (DRG), Neuronal and Glial Cultures have proven hard to transfect as as many transfection reagents are toxic to these cells. It is important for the study of neuro-diseases that researchers have tools and methods that enable success.
In this study, researchers successfully transfect DRG cultures with IKAP-shRNA using our pn-Fect kit. The own regulation of IKAP in these cultures support findings that helped explain the potential pathology of Familial Dysautonomia (FD; Hereditary Sensory Autonomic Neuropathy; HSAN III): Hunnicutt BJ , Chaverra M , George L , Lefcort F , 2012 IKAP/Elp1 Is Required In Vivo for Neurogenesis and Neuronal Survival, but Not for Neural Crest Migration. PLoS ONE 7(2): e32050. doi:10.1371/journal.pone.0032050.
Cell culture: Dorsal root ganglia were dissected from Embryonic day 5–9.5 chick embryos (E5–9.5) and dissociated by incubation in 0.25% trypsin-EDTA (Gibco) for 7 min at 37 C followed by trituration through fire–pulled glass pipettes. The culture media consisted of Neurobasal medium (Invitrogen) supplemented with B27 (1X,Invitrogen), Glutamax (1X,Invitrogen), Hybrimax Antibiotic\Antimicotic (1:100, Sigma), NGF (10 ng/ml, gift from Dr. Thomas Large). Cells were plated on 8-well Nunc glass chamber slides that were coated with poly-D-lysine (1:100, Sigma) and laminin 20 ug/ml (Gibco). Approximately equal numbers of cells (52,500) were plated per well. Immediately after plating, cells were transfected with IKBKAP-7.4 shRNA or control shRNA via pn-Fect (Neuromics, PN3375). Several ratios of pnfect:DNA were tested with the optimum obtained 1.84:1. The cells were then cultured for approximately 29 h at 37 C, 5.5% C02. After incubation culture cells were fixed and inmunostained as previously described [24]. To determine whether IKBKAP shRNAs altered cell proliferation and/or neuronal differentiation in dissociated DRG cultures, Brdu was added to the cultures and the cells were fixed 24 hrs later (as described in [24]). The number of GFP+/BrdU+ or GFP+/Tuj-1+ cells were quantified for each experiment, and a ratio comparing control vs. IKBKAP shRNAs for each experiment determined. For the BrdU+ experiment, a total of 556 GFP+/control shRNA transfected cells were counted and 357 GFP+/IKBKAP shRNA transfected cells in 3 separate experiment. For determining neuronal differentiation, a total of 2866 GFP+/control shRNA transfected cells and 2913 IKBKAP shRNA transfected cells were counted, over 3 separate experiments.
Images: IKAP regulates neuronal differentiation in the DRG. Reduction in IKAP leads to increased numbers of neurons in the immature DRG (A–C). Embryos at St. 12 were transfected with either control shRNAs or IKBKAP shRNAs and analyzed at St 24/25. Embryos were sectioned, and immunolabled with the neuronal markers Tuj-1 or Ben and the percentage of GFP+ neurons determined. Significantly more IKBKAP shRNA transfected DRG precursor cells differentiated into neurons (arrows in B; IKBKAP shRNA 7.4; n = 3 embryos; p = 0.002; IKBKAP shRNA 1.6 & 4.5, n = 3 embryos; p = 0.001) than Control shRNA transfected DRG precursor cells (n = 5 embryos). (D–H) IKBKAP shRNA-transfected DRG precursor cells (n = 3 embryos; 252 cleaved-Caspase 3+ cells counted) were also more likely to die by apoptosis (compare D & E to F & G) than control shRNA-electroporated cells (n = 3 embryos; 117 cleaved caspase-3+ cells counted). (H) The number of cleaved Caspase 3+ cells was quantified in DRG on both the transfected side of the embryo and the non-transfected side of the embryo and a ratio determined. Significantly more cleaved-Caspase 3+ cells were present in the transfected DRG of IKBKAP shRNA transfected embryos than in the transfected DRG in embryos transfected with control shRNAs; p = 0.006. (I–N). Over-expression of the c-terminus of IKAP prevents neural crest cells from coalescing with the DRG (I, K, L; p = 0.004) but the few that do join, tend not to differentiate into neurons (p = 0.007; J, M, N). Embryos were transfected with either a construct driving expression of the c-terminus of chicken IKAP with a His tag (CT-IKAP-His; L,N) or a control, His-tagged construct (K, M) and analyzed at St. 21. The location and neuronal identity of transfected cells was determined in 3 embryos for each treatment; Control His-plasmid: n = 343 transfected cells counted; CT-IKAP-His: n = 278 transfected cells counted. Statistical analysis by Student t-test. doi:10.1371/journal.pone.0032050.g006.
Check out our transfection reagents and capabilities. We will continue to post publications and data that support and add to our capabilities.
In this study, researchers successfully transfect DRG cultures with IKAP-shRNA using our pn-Fect kit. The own regulation of IKAP in these cultures support findings that helped explain the potential pathology of Familial Dysautonomia (FD; Hereditary Sensory Autonomic Neuropathy; HSAN III): Hunnicutt BJ , Chaverra M , George L , Lefcort F , 2012 IKAP/Elp1 Is Required In Vivo for Neurogenesis and Neuronal Survival, but Not for Neural Crest Migration. PLoS ONE 7(2): e32050. doi:10.1371/journal.pone.0032050.
Cell culture: Dorsal root ganglia were dissected from Embryonic day 5–9.5 chick embryos (E5–9.5) and dissociated by incubation in 0.25% trypsin-EDTA (Gibco) for 7 min at 37 C followed by trituration through fire–pulled glass pipettes. The culture media consisted of Neurobasal medium (Invitrogen) supplemented with B27 (1X,Invitrogen), Glutamax (1X,Invitrogen), Hybrimax Antibiotic\Antimicotic (1:100, Sigma), NGF (10 ng/ml, gift from Dr. Thomas Large). Cells were plated on 8-well Nunc glass chamber slides that were coated with poly-D-lysine (1:100, Sigma) and laminin 20 ug/ml (Gibco). Approximately equal numbers of cells (52,500) were plated per well. Immediately after plating, cells were transfected with IKBKAP-7.4 shRNA or control shRNA via pn-Fect (Neuromics, PN3375). Several ratios of pnfect:DNA were tested with the optimum obtained 1.84:1. The cells were then cultured for approximately 29 h at 37 C, 5.5% C02. After incubation culture cells were fixed and inmunostained as previously described [24]. To determine whether IKBKAP shRNAs altered cell proliferation and/or neuronal differentiation in dissociated DRG cultures, Brdu was added to the cultures and the cells were fixed 24 hrs later (as described in [24]). The number of GFP+/BrdU+ or GFP+/Tuj-1+ cells were quantified for each experiment, and a ratio comparing control vs. IKBKAP shRNAs for each experiment determined. For the BrdU+ experiment, a total of 556 GFP+/control shRNA transfected cells were counted and 357 GFP+/IKBKAP shRNA transfected cells in 3 separate experiment. For determining neuronal differentiation, a total of 2866 GFP+/control shRNA transfected cells and 2913 IKBKAP shRNA transfected cells were counted, over 3 separate experiments.
Images: IKAP regulates neuronal differentiation in the DRG. Reduction in IKAP leads to increased numbers of neurons in the immature DRG (A–C). Embryos at St. 12 were transfected with either control shRNAs or IKBKAP shRNAs and analyzed at St 24/25. Embryos were sectioned, and immunolabled with the neuronal markers Tuj-1 or Ben and the percentage of GFP+ neurons determined. Significantly more IKBKAP shRNA transfected DRG precursor cells differentiated into neurons (arrows in B; IKBKAP shRNA 7.4; n = 3 embryos; p = 0.002; IKBKAP shRNA 1.6 & 4.5, n = 3 embryos; p = 0.001) than Control shRNA transfected DRG precursor cells (n = 5 embryos). (D–H) IKBKAP shRNA-transfected DRG precursor cells (n = 3 embryos; 252 cleaved-Caspase 3+ cells counted) were also more likely to die by apoptosis (compare D & E to F & G) than control shRNA-electroporated cells (n = 3 embryos; 117 cleaved caspase-3+ cells counted). (H) The number of cleaved Caspase 3+ cells was quantified in DRG on both the transfected side of the embryo and the non-transfected side of the embryo and a ratio determined. Significantly more cleaved-Caspase 3+ cells were present in the transfected DRG of IKBKAP shRNA transfected embryos than in the transfected DRG in embryos transfected with control shRNAs; p = 0.006. (I–N). Over-expression of the c-terminus of IKAP prevents neural crest cells from coalescing with the DRG (I, K, L; p = 0.004) but the few that do join, tend not to differentiate into neurons (p = 0.007; J, M, N). Embryos were transfected with either a construct driving expression of the c-terminus of chicken IKAP with a His tag (CT-IKAP-His; L,N) or a control, His-tagged construct (K, M) and analyzed at St. 21. The location and neuronal identity of transfected cells was determined in 3 embryos for each treatment; Control His-plasmid: n = 343 transfected cells counted; CT-IKAP-His: n = 278 transfected cells counted. Statistical analysis by Student t-test. doi:10.1371/journal.pone.0032050.g006.
Check out our transfection reagents and capabilities. We will continue to post publications and data that support and add to our capabilities.
Sunday, February 26, 2012
Silencing NOV in vivo using i-Fect
Implications for Treating Neuropathic or Inflammatory Pain
It has been a awhile since I posted results here for researchers using Neuromics' i-Fect ™ siRNA Transfection Kits. Over the past months, we have enjoyed more growth in use of these kits so I anticipate more positive results to come.
Inflammation plays and evil role in Neuropathic Pain. Sustained neuroinflammation cased by release of pro-inflammatory cytokines and chemokines (including TNF-α, IL-1β, IL-6 and CCL2). Emerging studies have established that the extracellular matrix (ECM) components, particularly matrix metalloproteinases (MMPs) actively participate in the generation and maintenance of pain.
In this study, investigators show how modulating expression of nephroblastoma overexpressed gene (NOV) can mitigate expression of the MMPs and thus regulate Pain: Lara Kular, Cyril Rivat, Brigitte Lelongt, Claire Calmel, Maryvonne Laurent, Michel Pohl, Patrick Kitabgi, Stephane Melik-Parsadaniantz and Cecile Martinerie. NOV/CCN3 attenuates inflammatory pain through regulation of matrix metalloproteinases-2 and -9. Journal of Neuroinflammation 2012, 9:36 doi:10.1186/1742-2094-9-36.
Results: NOV was expressed in neurons of both dorsal root ganglia (DRG) and dorsal horn of the spinal cord (DHSC). After intraplantar CFA injection, NOV levels were transiently and persistently down-regulated in the DRG and DHSC, respectively, occurring at the maintenance phase of pain (15 days). NOV-reduced expression was restored after treatment of CFA rats with dexamethasone. In vitro, results based on cultured DRG neurons showed that siRNA-mediated inhibition of NOV enhanced IL-1beta- and TNF-alpha-induced MMP-2, MMP-9 and CCL2 expression whereas NOV addition inhibited TNF-alpha-induced MMP-9 expression through beta1 integrin engagement. In vivo, the intrathecal delivery of MMP-9 inhibitor attenuated mechanical allodynia of CFA rats. Importantly, intrathecal administration of NOV siRNA specifically led to an up-regulation of MMP-9 in the DRG and MMP-2 in the DHSC concomitant with increased mechanical allodynia. Finally, NOV intrathecal treatment specifically abolished the induction of MMP-9 in the DRG and, MMP-9 and MMP-2 in the DHSC of CFA rats. This inhibitory effect on MMP is associated with reduced mechanical allodynia.
Conclusions: This study identifies NOV as a new actor against inflammatory pain through regulation of MMPs thus uncovering NOV as an attractive candidate for therapeutic improvement in pain relief.
Figure 9. Effect of in vivo endogenous NOV inhibition on MMP-2/-9 expression and mechanical allodynia. In CFA rats, NOV-specific siRNA (2 μg) or control non-silencing siRNA (Ctr) were delivered intrathecally (i.t) daily for 3 consecutive days. (A) NOV protein levels in DHSC. Representative western blot (left panel) and quantification of protein levels normalized to GAPDH (right panel) (**P <0.01, siNOV versus Ctr, n = 6) (B, C) Levels of MMP-9 and MMP-2 mRNA in DRG (B) and DHSC. (C) Transcript levels were quantified by RT-qPCR and values were normalized to rat S26 mRNA level. Data represent the mean value ± SEM of two independent experiments realized with three rats per condition (*P <0.05 siNOV versus Ctr). (D) Representative gelatin zymograph showing MMP-9 and MMP-2 activities in DRG (left panel) and quantification of MMP-2 and MMP-9 gelatinolytic bands (right panel). Data represent the mean ± SEM of six rats per group (**P <0.01 siNOV versus Ctr). (E) Paw withdrawal threshold (g) of CFA rats intrathecally injected with NOV-specific siRNA or control siRNA evaluated using the von Frey test. Data represent the mean ± SEM of eight rats per group (*P <0.05 siNOV- versus Ctr-treated rats), BL: baseline In order to test whether endogenously produced NOV could modulate inflammatory pain, we evaluated the mechanical allodynia of CFA rats treated with NOV. As shown in Figure 9E, intrathecal delivery of siNOV resulted in a significant increase of mechanical allodynia compared to rats injected with control siRNA (*P <0.05, n = 8). These data strongly suggest that endogenously produced NOV influences pain intensity and further support the hypothesis that NOV downregulation could participate in pain processes through
upregulation of MMP-2 and MMP-9.
Learn more about Neuromics' Transfection Reagents!
It has been a awhile since I posted results here for researchers using Neuromics' i-Fect ™ siRNA Transfection Kits. Over the past months, we have enjoyed more growth in use of these kits so I anticipate more positive results to come.
Inflammation plays and evil role in Neuropathic Pain. Sustained neuroinflammation cased by release of pro-inflammatory cytokines and chemokines (including TNF-α, IL-1β, IL-6 and CCL2). Emerging studies have established that the extracellular matrix (ECM) components, particularly matrix metalloproteinases (MMPs) actively participate in the generation and maintenance of pain.
In this study, investigators show how modulating expression of nephroblastoma overexpressed gene (NOV) can mitigate expression of the MMPs and thus regulate Pain: Lara Kular, Cyril Rivat, Brigitte Lelongt, Claire Calmel, Maryvonne Laurent, Michel Pohl, Patrick Kitabgi, Stephane Melik-Parsadaniantz and Cecile Martinerie. NOV/CCN3 attenuates inflammatory pain through regulation of matrix metalloproteinases-2 and -9. Journal of Neuroinflammation 2012, 9:36 doi:10.1186/1742-2094-9-36.
Results: NOV was expressed in neurons of both dorsal root ganglia (DRG) and dorsal horn of the spinal cord (DHSC). After intraplantar CFA injection, NOV levels were transiently and persistently down-regulated in the DRG and DHSC, respectively, occurring at the maintenance phase of pain (15 days). NOV-reduced expression was restored after treatment of CFA rats with dexamethasone. In vitro, results based on cultured DRG neurons showed that siRNA-mediated inhibition of NOV enhanced IL-1beta- and TNF-alpha-induced MMP-2, MMP-9 and CCL2 expression whereas NOV addition inhibited TNF-alpha-induced MMP-9 expression through beta1 integrin engagement. In vivo, the intrathecal delivery of MMP-9 inhibitor attenuated mechanical allodynia of CFA rats. Importantly, intrathecal administration of NOV siRNA specifically led to an up-regulation of MMP-9 in the DRG and MMP-2 in the DHSC concomitant with increased mechanical allodynia. Finally, NOV intrathecal treatment specifically abolished the induction of MMP-9 in the DRG and, MMP-9 and MMP-2 in the DHSC of CFA rats. This inhibitory effect on MMP is associated with reduced mechanical allodynia.
Conclusions: This study identifies NOV as a new actor against inflammatory pain through regulation of MMPs thus uncovering NOV as an attractive candidate for therapeutic improvement in pain relief.
Figure 9. Effect of in vivo endogenous NOV inhibition on MMP-2/-9 expression and mechanical allodynia. In CFA rats, NOV-specific siRNA (2 μg) or control non-silencing siRNA (Ctr) were delivered intrathecally (i.t) daily for 3 consecutive days. (A) NOV protein levels in DHSC. Representative western blot (left panel) and quantification of protein levels normalized to GAPDH (right panel) (**P <0.01, siNOV versus Ctr, n = 6) (B, C) Levels of MMP-9 and MMP-2 mRNA in DRG (B) and DHSC. (C) Transcript levels were quantified by RT-qPCR and values were normalized to rat S26 mRNA level. Data represent the mean value ± SEM of two independent experiments realized with three rats per condition (*P <0.05 siNOV versus Ctr). (D) Representative gelatin zymograph showing MMP-9 and MMP-2 activities in DRG (left panel) and quantification of MMP-2 and MMP-9 gelatinolytic bands (right panel). Data represent the mean ± SEM of six rats per group (**P <0.01 siNOV versus Ctr). (E) Paw withdrawal threshold (g) of CFA rats intrathecally injected with NOV-specific siRNA or control siRNA evaluated using the von Frey test. Data represent the mean ± SEM of eight rats per group (*P <0.05 siNOV- versus Ctr-treated rats), BL: baseline In order to test whether endogenously produced NOV could modulate inflammatory pain, we evaluated the mechanical allodynia of CFA rats treated with NOV. As shown in Figure 9E, intrathecal delivery of siNOV resulted in a significant increase of mechanical allodynia compared to rats injected with control siRNA (*P <0.05, n = 8). These data strongly suggest that endogenously produced NOV influences pain intensity and further support the hypothesis that NOV downregulation could participate in pain processes through
upregulation of MMP-2 and MMP-9.
Learn more about Neuromics' Transfection Reagents!
Monday, February 13, 2012
In Vivo application of RNAi to study pain
This overview is from 2010. I am posting a link because it undercores the need to have transfection reagents that have the ability to deliver small doses of siRNA in vivo.
"One of the biggest challenges in using RNAi in pain research is delivery of siRNA to the CNS in sufficient concentrations. This obstacle exists because siRNA by itself does not cross the blood brain barrier (BBB) and is degraded in the blood by endonucleases. Intravenous or oral administration is, therefore, inadequate to achieve desired protein knockdown. The use of transfection agents and intrathecal delivery has enhanced siRNA uptake by target tissues in recent studies." Zachary J Clark, Gurwattan S. Miranpuri, Daniel K Resnick. In Vivo application of RNAi to study pain. Annals of Neurosciences, Volume 17, Number 3, July 2010.
Please click through the link and you will learn of techniques currently used to delivery siRNA in vivo for pain research.
"One of the biggest challenges in using RNAi in pain research is delivery of siRNA to the CNS in sufficient concentrations. This obstacle exists because siRNA by itself does not cross the blood brain barrier (BBB) and is degraded in the blood by endonucleases. Intravenous or oral administration is, therefore, inadequate to achieve desired protein knockdown. The use of transfection agents and intrathecal delivery has enhanced siRNA uptake by target tissues in recent studies." Zachary J Clark, Gurwattan S. Miranpuri, Daniel K Resnick. In Vivo application of RNAi to study pain. Annals of Neurosciences, Volume 17, Number 3, July 2010.
Please click through the link and you will learn of techniques currently used to delivery siRNA in vivo for pain research.
Saturday, February 4, 2012
siRNA Delivery Group on Linkedin
I wanted to make readers aware of an excellent discussion group on Linkedin named "siRNA Delivery". Included are tip, updates on commercialization and key publications.
Here're some examples:
Here're some examples:
- Alnylam Announces Publication of Pre-clinical Results with ALN-HTT, an RNAi Therapeutic for the Treatment of Huntington’s Disease, in Experimental Neurology
- Delivering siRNA to Neurons
- Life Technologies develops new drug delivery technology
- Recent experiments demonstrate a novel method of liposomal encapsulation of siRNA/RNA/DNA at 4C, could be a promising method to minimize degradation.
Thursday, December 15, 2011
Delivering DNA to Cell Lines and Primary Cells
Neuromics is pleased to introduce DNA-FectTM and DNA-FectTM293 in vivo transfection reagents.
These reagents deliver genes to various established cell lines as well as primary cells, which include HEK293, 293T, 293E, CHO, COS1, HeLa, NIH 3T3, insect cell lines (Sf9 and Sf21) and a variety of other eucaryotic cell lines with low cytotoxicity. GeneExpresso™ MAX reagent, 1.0 ml, is sufficient for 300 to 600 transfections in 24 well plates, or 150 to 300 transfections in 6 well plates.
Capabilities include:
• Proven to deliver DNA to difficult-to-transfect cells
•Stable and easy to use
•Suitable for high-thoroughput (HTS) applications
Image: Comparing DNA-Fect vs Lipofectamine 2000 by FACS Analysis
DNA Delivery Protocol:
These reagents deliver genes to various established cell lines as well as primary cells, which include HEK293, 293T, 293E, CHO, COS1, HeLa, NIH 3T3, insect cell lines (Sf9 and Sf21) and a variety of other eucaryotic cell lines with low cytotoxicity. GeneExpresso™ MAX reagent, 1.0 ml, is sufficient for 300 to 600 transfections in 24 well plates, or 150 to 300 transfections in 6 well plates.
Capabilities include:
• Proven to deliver DNA to difficult-to-transfect cells
•Stable and easy to use
•Suitable for high-thoroughput (HTS) applications
DNA Delivery Protocol:
Thursday, September 29, 2011
β-arrestin siRNA Delivery in vivo and Increased Analgesia
I have reported use of our i-FectTM siRNA delivery kit for gene expression analysis studies of Cav1.2, DOR, hTERT, The β3 subunit of the Na+,K+-ATPase, rSNSR1, NTS1. NAV1.8, , TRPV1, Survivin, Flaviviruses and more.
I would like to congratulate Dr. C.R. Lin and his team at National Taiwan University College of Medicine for silencing β-arrestin expression in vivo and the impact on opioid based analgesia. The results could be good news for improving opioid based pain therapies: C.-H.Yang, H.-W. Huang, K.-H.Chen, Y.-S.Chen, S.-M.Sheen-Chen and C.-R.Lin. Antinociceptive potentiation and attenuation of tolerance by intrathecal β-arrestin 2 small interfering RNA in rats. Br. J. Anaesth. (2011) doi: 10.1093/bja/aer291.
Background: Tolerance to the analgesic effect of opioids complicates the management of persistent pain states. We tested whether the intrathecal infusion of small interfering RNA (siRNA) against β-arrestin 2 would reduce tolerance to chronic morphine use and the severity of precipitated morphine withdrawal.
Methods: Intrathecal β-arrestin 2 (2 μg siRNA per 10 μl per rat) was injected once daily for 3 days. Rats then received a continuous intrathecal infusion of morphine (2 nmol h−1) or saline for 7 days. Daily tail-flick (TF) and intrathecal morphine challenge tests were performed to assess the effect of intrathecal β-arrestin 2 siRNA on antinociception and tolerance to morphine. Naloxone withdrawal (2 mg kg−1) was performed to assess morphine dependence.
Results: In the daily TF test, the antinociception of intrathecal morphine was increased and maintained in rats receiving β-arrestin 2 siRNA compared with the control group (morphine alone). In the probe response test, rats receiving morphine infusion with β-arrestin 2 siRNA treatment showed a significant left shift in their dose–response curve, as measured by per cent maximal possible effect (MPE), such that the AD50 was significantly decreased by a factor of 5.6 when compared with that of morphine-infused rats. In the naloxone-induced withdrawal tests, rats receiving β-arrestin 2 siRNA injection with morphine infusion showed a significant reduction in four of the six signs of withdrawal.
Conclusions: We show here that intrathecal β-arrestin 2 siRNA in rats enhances analgesia and attenuates naloxone-induced withdrawal symptoms. This may warrant further investigation in the context of long-term use of intrathecal opioids for controlling chronic pain.
I would like to congratulate Dr. C.R. Lin and his team at National Taiwan University College of Medicine for silencing β-arrestin expression in vivo and the impact on opioid based analgesia. The results could be good news for improving opioid based pain therapies: C.-H.Yang, H.-W. Huang, K.-H.Chen, Y.-S.Chen, S.-M.Sheen-Chen and C.-R.Lin. Antinociceptive potentiation and attenuation of tolerance by intrathecal β-arrestin 2 small interfering RNA in rats. Br. J. Anaesth. (2011) doi: 10.1093/bja/aer291.
Background: Tolerance to the analgesic effect of opioids complicates the management of persistent pain states. We tested whether the intrathecal infusion of small interfering RNA (siRNA) against β-arrestin 2 would reduce tolerance to chronic morphine use and the severity of precipitated morphine withdrawal.
Methods: Intrathecal β-arrestin 2 (2 μg siRNA per 10 μl per rat) was injected once daily for 3 days. Rats then received a continuous intrathecal infusion of morphine (2 nmol h−1) or saline for 7 days. Daily tail-flick (TF) and intrathecal morphine challenge tests were performed to assess the effect of intrathecal β-arrestin 2 siRNA on antinociception and tolerance to morphine. Naloxone withdrawal (2 mg kg−1) was performed to assess morphine dependence.
Results: In the daily TF test, the antinociception of intrathecal morphine was increased and maintained in rats receiving β-arrestin 2 siRNA compared with the control group (morphine alone). In the probe response test, rats receiving morphine infusion with β-arrestin 2 siRNA treatment showed a significant left shift in their dose–response curve, as measured by per cent maximal possible effect (MPE), such that the AD50 was significantly decreased by a factor of 5.6 when compared with that of morphine-infused rats. In the naloxone-induced withdrawal tests, rats receiving β-arrestin 2 siRNA injection with morphine infusion showed a significant reduction in four of the six signs of withdrawal.
Conclusions: We show here that intrathecal β-arrestin 2 siRNA in rats enhances analgesia and attenuates naloxone-induced withdrawal symptoms. This may warrant further investigation in the context of long-term use of intrathecal opioids for controlling chronic pain.
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