High Fat and Diet Induced Obesity

i-Fect^TM Delivers Again!

Research shows that rats and humans on a high-fat diet (HFD) are less sensitive to satiety signals known to act via vagal afferent pathways. Impaired vagal afferent responsiveness to both gastric satiety hormones (CCK and leptin) and mechanical stimulation raises the possibility that changes in electrophysiological properties may be the underlying mechanism responsible for impaired vagal responsiveness to a wide variety of satiety signals.

Potassium channels play a central role. To demonstrate this researchers used our i-Fect siRNA Transfection Kit to silence TRESK and TASK1 to understand their impact on HFD and vagal responsiveness. Gintautas Grabauskas, Xiaoyin Wu, ShiYi Zhou, JiYao Li, Jun Gao, and Chung Owyang. (2019). High-fat diet–induced vagal afferent dysfunction via upregulation of 2-pore domain potassium TRESK channel. JCI Insight. https://doi.org/10.1172/jci.insight.130402.

Images: (A) Representative recordings of NG neuron responses to intra–superior pancreaticoduodenal artery infusions of CCK-8 (60 pmol/kg) and leptin (60 pmol/kg) in LFD-fed or HFD-fed rats and transfected with control siRNA or TRESK siRNA. Note that CCK-8 generated significantly fewer action potentials in HFD-fed rats compared with those fed an LFD. (B) Summary histograms showing single-unit discharges in response to CCK-8 in rats given an LFD and transfected with control siRNA (n = 11) or TRESK siRNA (n = 6), HFD + control siRNA (n = 12), and HFD treated with TRESK siRNA (n = 10). Data are represented as mean ± SEM. One-way ANOVA with Bonferroni’s test, *P < 0.05 vs. LFD + control siRNA; #P < 0.05 vs. HFD + control siRNA. (C) Summary histogram showing single-unit discharges in response to leptin in rats given an LFD and transfected with control siRNA (n = 11) and TRESK siRNA (n = 5), HFD (n = 12), and HFD treated with TRESK siRNA (n = 10). Data are represented as mean ± SEM. One-way ANOVA with Bonferroni’s test, *P less than 0.05 vs. LFD + control siRNA; #P less than 0.05 vs. HFD + control siRNA. (D) Summary histogram showing CCK-AR and ObR expression in vagal sensory ganglia from LFD- and HFD-fed rats were not significantly different. HPRT was used as a loading control. Data are represented as mean ± SEM. CCK-8, cholecystokinin-8.

Following 2 weeks of high-fat feeding, there was a significant upregulation of TRESK and a modest increase in TASK1 channels in the NG. Silencing studies indicate that the upregulation by TRESK channels is mainly responsible for a global decrease in excitability of vagal sensory neurons, which in turn dampens the response to satiety signals, such as CCK and leptin. 

This make TRESK a potential therapeutic target for treating Obesity.

i-Fect used to Study Angiogenesis in Brain Injury

Silencing Lactate Dehydrogenase A in vivo

Pathologic CNS is characterized by neuronal damage that leads to the release of intracellular components. However, the effect of damaged cells on angiogenesis has not been clarified. This study revealed that LDHA, which is a known damage marker, promotes CNS-specific angiogenesis. LDHA-mediated angiogenesis depends on vimentin on the surface of vascular endothelial cells. The work described here proposes a novel mechanism by which neurodegeneration drives angiogenesis in the CNS.

A mixture of our i-Fect^TM and LDHA siRNA, in this study, were directly injected into mice cortexes: Hsiaoyun Lin, Rieko Muramatsu, Noriko Maedera, Hiroto Tsunematsu, Machika Hamaguchi, Yoshihisa Koyama, Mariko Kuroda, Kenji Ono, Makoto Sawada, Toshihide Yamashita. Extracellular Lactate Dehydrogenase A Release From Damaged Neurons Drives Central Nervous System Angiogenesis. doi.org/10.1016/j.ebiom.2017.10.033.

Images: LDHA is sufficient to evoke CNS angiogenesis. (a) Representative images of CD105-labeled spinal cord sections obtained 7 days after LDHA administration. (b) Length of CD105+ neovessels around the LDHA administration site as indicated in a, n = 5 each. (c) Representative image of a Nissl-stained brain section after controlled cortical impact (CCI). (d) Representative image of the CD105-immunolabelled cerebral cortex obtained 7 days after CCI. (e) Length of CD105+ neovessels around CCI lesions as indicated in d; n = 5 each, all error bars represent the s.e.m. **P < 0.01, Student's t-tests. Scale bars, 200 μm.

The findings reveal unexpected neurovascular interactions in the injured adult CNS that may be relevant to our understanding of neuronal damage, which is a hallmark of many CNS disorders.

i-Fect Deliver Plasmids to the CNS

Important for Gene Expression Studies.
I have posted many examples of how our customers use i-Fect^TM  and other Transfection Solutions for Gene Manipulation Studies. There are also many publications.

Here we feature how i-Fect was used to delivery plasmids to the CNS: Sara Elramah, María José López-González, Matthieu Bastide, Florence Dixmérias, Olivier Roca-Lapirot, Anne-Cécile Wielanek-Bachelet, Anne Vital, Thierry Leste-Lasserre, Alexandre Brochard, Marc Landry & Alexandre Favereaux. Spinal miRNA-124 regulates synaptopodin and nociception in an animal model of bone cancer pain. Scientific Reports 7, Article number: 10949 (2017) doi:10.1038/s41598-017-10224-1...Intrathecal administration of miRNAs and ShRNA To over-express miR-124, we cloned the pre-miRNA sequence of miR-124 into a plasmid. To determine cells expressing this miR-124 encoding plasmid, we added a GFP-coding sequence to the construct under the control of an IRES. Thus, miR-124 over-expressing cells also express GFP. To inhibit synaptopodin expression, we cloned a ShRNA sequence directed against synaptopodin into a plasmid. To determine cells expressing this ShRNA, we added a GFP-coding sequence to the construct under the control of an IRES. Thus, ShRNA expressing cells also expressed GFP. Two micrograms of these plasmids or the corresponding controls, were solubilized in 10 µl of i-Fect reagent (Neuromics, Edina, USA), and injected intrathecally between the L5 and L6 lumbar vertebrae every two days for a total of 3 injections, according to the manufacturer’s instructions and previously published experiments...

Figures: (C and D) Immunostaining of synpo in spinal cord after miR-124 intrathecal injections: only the dorsal horn which receive nociceptive information was quantified (white dash area). Measurement of synaptopodin stained area reveals ability of miR-124 to inhibit endogenous Synpo expression (20/3 and 17/3 denotes number of sections/animals for control and miR-124-injected mice, respectively.

I am confident there will be many more positive reports regarding our Transfection Reagents.

More iFect in-vivo

The parade of publications continues to grow.

Here researchers use our i-Fect^TM Transfection Kit for delivering sh-IRF3 in vivo: Rui Li, Li-guo Wang, Qi Wang, Zhi-hua Li, Ya-li Ma, Qing-Duo Guo. Silencing of IRF3 alleviates chronic neuropathic pain following chronic constriction injury. doi.org/10.1016/j.biopha.2017.01.085... The oligonucleotides for sh-IRF3 were: 5′-CACCGCGTCTAGGCTGGTGGTTATTCGAAAATAACCACCAGCCTAGACGC-3′ −3′. Then, 10 μg sh-IRF3 dissolved in 30 μl i-Fect transfection reagent (Neuromics, Edina, MN, USA) was administered intrathecally once daily for 7...

Fig. Down-regulation of IRF3 attenuated mechanical allodynia and thermal hyperalgesia in CCI rats. (A) The mRNA expression level of IRF3 in the DRG at postoperative day 7. (B) The protein expression level of IRF3 in the DRG at postoperative day 7. (C and D) PWT and PWL were measured 1 day before CCI and 1, 3, 7 and 14 days after intrathecal injection of sh-IRF3 or scramble.

Down-regulation of IRF3 inhibited the production of pro-inflammatory cytokines in the DRG of CCI rats.

These results indicated that IRF3 was involved in the development of neuropathic pain. Down-regulation of IRF3 attenuated neuropathic pain in CCI rats by inhibiting the activation of NF-κB signaling pathway, suggesting that IRF3 may be a novel and potential target for the treatment of neuropathic pain.

Delivering miRNA in vivo

More Transfection Success! Great Research Tools!

Our i-Fect^TM  Transfection Kit is used to study Epigenetics and pain. Here's yet another example: M. Leinders, b, N. Üçeyler, R.A. Pritchard, C. Sommer, L.S. Sorkin. Increased miR-132-3p expression is associated with chronic neuropathic pain. Experimental Neurology. Volume 283, Part A, September 2016, Pages 276–286...The inhibitor and mimetic were administered to awake rats via the it catheters. Prior to injection, active or mismatch inhibitors were mixed with (1:5 w/v) i-Fect™ in vivo transfection reagent(Neuromics, Edina, USA) to final doses of 5, 2 and 1 μg in 10 μl...

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Spinal administration of miR-132-3p antagonists via intrathecal (i.t.) catheters dose dependently reversed mechanical allodyina  and eliminated pain behavior in the place escape avoidance paradigm (p < 0.001). Intrathecal administration of miR-132-3p mimetic dose-dependently induced pain behavior in naïve rats (p < 0.001). Taken together these results indicate a pro-nociceptive effect of miR-132-3p in chronic neuropathic pain.

Finding like these could pave the way for an miRNA like therapy for pain.

Epigenetics and Pain Research

i-Fect Used to Study Impacts

Our i-Fect siRNA, miRNA and shRNA Trasfection Kit was recently used to study the impact of G9a-specific siRNA (AGUAACGGGCAUCAAUGC) on Mu Opioid Receptors: Yuhao Zhang, Shao-Rui Chen, Geoffroy Laumet, Hong Chen and Hui-Lin Pan. Nerve Injury Diminishes Opioid Analgesia through Lysine Methyltransferase-Mediated Transcriptional Repression of µ-Opioid Receptors in Primary Sensory Neurons. First Published on February 25, 2016, doi: 10.1074/jbc.M115.711812... In some SNL rats, G9a-specific siRNA (4 µg) or the negative control siRNA was administered intrathecally. G9a-specific siRNA(AGUAACGGGCAUCAAUGC) or universal negative control siRNA (#SIC001, Sigma-Aldrich) was mixed with i-Fect (Neuromics, Edina, MN) to a final concentration of 400 mg/L for the intrathecal injections...

Figures: G9a knockdown with siRNA reverses the MOR expression in the DRG and the morphine analgesic effect diminished by nerve injury. (A,B) Quantitative PCR (A) and Western blotting (B) analyses show the mRNA and protein levels of MORs in the DRGs of sham and SNL rats treated with control or G9a-specific siRNA (n = 10 rats in each group). The ipsilateral L5 and L6 DRG tissues were removed 24 h after the last siRNA injection. The amount of MOR mRNA and protein was normalized to GAPDH in the same samples, and the mean value of MOR levels in sham control rats was considered to be 1. (C) Time course of the intrathecal morphine effects on the tactile and pressure withdrawal thresholds in sham and SNL rats treated with G9a-specific siRNA or negative control siRNA (n = 9 rats in each group). The withdrawal thresholds after the last siRNA injection were plotted as the baseline control (BL).


Summary: The findings provide new insight into the epigenetic mechanism regulating MOR expression in primary sensory neurons in neuropathic pain. This multidisciplinary approach provides conclusive evidence for G9a as a key chromatin regulator responsible for MOR downregulation in the DRG and the analgesic efficacy of opioids reduced by nerve injury. A better understanding of the epigenetic mechanisms underlying nerve injury-induced downregulation of MORs in primary sensory neurons could help improve the analgesic efficacy of opioids for treating chronic neuropathic pain. G9a inhibitors could be used to enhance the opioid analgesic effect and reduce opioid consumption in patients with chronic neuropathic pain.

EPO Protects Against Cerebral Ischemia

Neuromics' i-Fect ^TM used to Modulate phospho-Connexin 43

In this study data suggest the protective effects of EPO on NUV injuries are highly associated with the increase of p-Cx43, which improves GJIC to reduce neurotoxic substances: Ziyi Zhoua, Xiaobai Weib, Jun Xiang, Junpeng Gao, Lixin Wang, Jinsong You, Yefeng Cai , Dingfang Caid. Protection of erythropoietin against ischemic neurovascular unit injuries through the effects of connexin43. Biochemical and Biophysical Research Communications. doi:10.1016/j.bbrc.2015.02...The strands were incubated at 90°C for 5 min and then at 37°C for 1 h. SiRNA was prepared immediately before administration by mixing the RNA solution (1 μg/μl in annealing buffer) with the transfection reagent i-Fect (v/v: 1/3; Neuromics, Edina, MN, USA) to a final siRNA ...

Highlights
•EPO has protective effects on ischemic NVU injuries.
•EPO up-regulates phosphorylation of Cx43, not total Cx43.
•EPO's protective effects on NUV injuries are p-Cx43-GJIC dependent.

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.

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:

• 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.

Happy reading.

i-Fect and siRNA Delvery to Toll-like receptor 4

I have reported use of our i-Fect^TM siRNA delivery kit for gene expression analysis studies of DOR, hTERT, The β3 subunit of the Na+,K+-ATPase, rSNSR1, NTS1. NAV1.8, Survivin,  Flaviviruses and more.

These represent potential targets for Pain, Cancer and Infectious Disease Therapies.

The latest study involves successful knockdown of the Toll-like receptor 4 (TLR-4):

Wu Fx, Bian Jj, Miao Xr, Huang Sd, Xu Xw, Gong Dj, Sun Ym, Lu Zj, Yu Wf. Intrathecal siRNA against Toll-like receptor 4 reduces nociception in a rat model of neuropathic pain. Int J Med Sci 2010; 7:251-259.

Background: Neuropathic pain is characterized by hyperalgesia, allodynia and spontaneous pain. It often occurs as a result of injury to peripheral nerves, dorsal root ganglions (DRG), spinal cord, or brain. Recent studies have suggested that Toll-like receptor 4 (TLR4) might play a role in neuropathic pain. Methodology/Principal Findings: In this study, we investigated the role of TLR4 in a rat chronic constriction injury (CCI) model and explored the feasibility of treating neuropathic pain by inhibiting TLR4. Our results demonstrated that intrathecal siRNA-mediated suppression of TLR4 attenuated CCI-induced mechanical allodynia and thermal hyperalgesia through inhibiting the activation of NF-κB p65 and production of proinflammatory cytokines (e.g., TNF-α and IL-1β). Conclusions/Significance: These findings suggest that suppression of TLR4 mediated by intrathecally administered siRNA may be a new strategy for the treatment of neuropathic pain.

Images: Screening siRNA for an efficient suppression of TLR4 expression in vitro. HEK-293 cells were co-transfected with both pEGFRC1-TLR4 and either one of three independent siRNA oligonucleotides targeting TLR4 (TLR4-siRNA1-3) or a control siRNA (MM-siRNA). Two days after transfection, EGFP fluorescence was observed under microscope (A) or quantified by flow cytometry (B). (A) EGFP fluorescence under an inverted fluorescence microscope (×100) or cell density under an optical microscope (×100). A, control; B, siRNA1; C, siRNA2; D, siRNA3. (B) The quantification of TLR4-EGFP fluorescence intensity upon siRNA knockdown was evaluated by flow cytometry analysis. Immunofluorescence and flow cytometry results revealed that all 3 siRNAs had efficient inhibition on GFP fluorescence, and TLR4-siRNA2 was the most potent.

siRNA and i-Fect for the Study of Retinal Disease

We continue to add new references to the many ways researchers are using i-Fect^TM to increase the potency of siRNA delivery.

This is a new reference from the book entitled Retinal Degenerative Diseases: Laboratory and Therapeutic Investigations By Robert E. Anderson, Joe G. Hollyfield, Matthew M. Lavail.

In this study, researchers used i-Fect to transfect and immortalized cell line from Mouse cones (661W) expressing ELOVL4. Using siRNA designed to silence the ELOVL4 gene, they used i-Fect + siRNA to transfect cells cultured at a density of 2X10⁵. Knockdown was achieved as confirmed by western blot analysis.

ACIC3 Receptors Knockdown in vivo

Researchers using siRNA complexed with our i-Fect ™ transfection regent have successfully knocked down ASIC3 Receptors in vivo. This publication joins the growing parade (starting with Luo et al, 2005) that refererence successuful modulation of receptors involved in pain using siRNA complexes.

These studies all share animal behavior studies showing a marked change in response to pain stimuli after treatment.

In this study, Dr. Eric Lingueglia and his team found Peripheral ASIC3 channels are thus essential sensors of acidic pain and integrators of molecular signals produced during inflammation where they contribute to primary hyperalgesia.

Emmanuel Deval, Jacques Noël, Nadège Lay, Abdelkrim Alloui, Sylvie Diochot, Valérie Friend, Martine Jodar, Michel Lazdunski and Eric Lingueglia. ASIC3, a sensor of acidic and primary inflammatory pain. The EMBO Journal advance online publication 16 October 2008; doi: 10.1038/emboj.2008.213

Cy3-labelled siRNA no. 1121 and its corresponding scramble (no. 1121S; GCTCACACTACGCAGAGAT) synthesized by MWG Biotech (Germany) were injected in rats by intrathecal bolus to the lumbar region of the spinal cord once a day for 3 days before the induction of inflammation with CFA. Each 10-ml injection corresponded to 2 mg of siRNA complexed with i-Fect siRNA transfection reagent (Neuromics) at a ratio of 1:4 (w:v) (Luo et al, 2005), following the supplier’s suggested protocol. siRNA uptake in lumbar DRGswas monitored by fluorescence microscopy on cryostat sections 24 h after a single intrathecal injection.

Here’s a synopsis of results:
Inflammation was produced by CFA injection, which led to primary heat hyperalgesia, and this hyperalgesia was drastically reduced by the ASIC3 blocker APETx2 injected subcutaneously, which only access cutaneous nociceptors. It was also drastically reduced when, before triggering the inflammation state, intrathecalinjections of an siRNA against ASIC3 had induced a knockdown of ASIC3 expression in lumbar DRGs.

Central Delivery of DsiRNA

Louis Doré-Savard, Geneviève Roussy, Marc-André Dansereau, Michael A Collingwood, Kim A Lennox, Scott D Rose, Nicolas Beaudet, Mark A Behlke and Philippe Sarret. Central Delivery of Dicer-substrate siRNA: A Direct Application for Pain Research. Molecular Therapy (2008); doi:10.1038/mt.2008.98.

Images: Cellular uptake of Texas Red–tagged Dicer-substrate small-interfering RNA (DsiRNA) by spinal nociceptive structures. (a,b) Distribution of fluorescence in lumbar dorsal root ganglia at 24 hours after intrathecal injection of a control siRNA conjugated with Texas Red (1 μg administered twice with a 24-hour interval; n = 3). As seen by confocal microscopy, the staining is not uniformly distributed among the cells. Higher-magnification images also show that the fluorescent signal is detected in the form of small intracytoplasmic hot spots, sparing the nucleus. (c,d) Expression of Texas Red–tagged DsiRNA in a dorsal spinal cord section taken from an L5 segment. Fluorescence clusters are present in the cytoplasm of the cells. Note that the labeling is also detected in neuronal processes. Scale bar: 60 μm in a, 30 μm in b,25 μm in c and 15 μm in d. Courtesy of Dr. Nicolas Beudeat. Published in Molecular Therapy (2008); doi:10.1038/mt.2008.98

Use of Dicer Substrate siRNAs

Dicer-substrate siRNAs (DsiRNAs) have recently been employed for in vivo studies using intraperitoneal and intrathecal routes of administration. “IDT got into RNAi research in collaboration with John Rossi at The City of Hope and the Beckman Research Institute five years ago,” explained Dr. Behlke. In vivo, long dsRNAs are cleaved by the RNase III class endoribonuclease dicer into 21–23 base duplexes having 2-base 3´-overhangs. These species, called small interfering RNAs (siRNAs), enter the RISC and serve as a sequence-specific guide to target degradation of complementary mRNA species.
Typically, siRNAs are chemically synthesized as 21 mers with a central 19 bp duplex region and symmetric 2-base 3´-overhangs on the termini, reported Dr. Behlke. These duplexes are transfected into cells lines, directly mimicking the products made by dicer in vivo. Most siRNA sequences can be administered to cultured cells or to animals without eliciting an interferon response.

“We observed,” added Dr. Behlke, “that the use of slightly longer sequences that were substrates for dicer showed improved potency, which we theorize relates to participation of dicer in RISC loading. We are now focusing on the use of these compounds in vivo.”

IDT recently completed a collaborative study with the laboratory of professor Phillipe Sarret at the Université de Sherbrooke in Quebec. The collaboration studied the use of DsiRNA to knockdown the GPCR NTS2 (neurotensin type 2 receptor) in rat spinal cord and dorsal root ganglia. The RNA duplexes were administered by intrathecal injection in a cationic lipid slurry. Stimulation of NTS2 with a chemical agonist resulted in analgesia. Pain responses were monitored in treated animals by dipping their tails in hot water with and without the chemical agonist.

“The anti-NTS2 DsiRNA treated animals showed a marked difference of response to the test stimulus,” said Dr. Behlke. “We recorded differences of up to five seconds, which is quite a long time for a rat to sit with its tail in hot water. While interesting, this work mainly represents a pilot study to demonstrate the feasibility of using DsiRNA to study pain pathways in rats. We were amazed at the low dose it takes to get knockdown—we used 1 mcg/200 g rat, which is only a 0.005 mg/kg dose.” Modulating CNS disease and affecting brain processes is clearly possible, but better methods of delivery are going to be needed to move this approach from a research tool into the clinic, noted Dr. Behlke.

More on Delivering siRNA in vivo

We are excited about this upcoming presentation at Society for Neuroscience comference presesentation. Neuromics' i-Fect ™ reagent was used to deliver siRNA in vivo to silence the NTS2 gene.

Program#/Poster#:509.6/PP9
Title:Small interfering RNA-mediated selective knockdown of NTS2 receptors reverses neurotensin-induced analgesia in rats
Location:San Diego Convention Center: Halls B-H
Presentation Start/End Time:Monday, Nov 05, 2007, 2:00 PM - 3:00 PM
Authors:*L. DORE-SAVARD1,2, G. ROUSSY1, M.-A. DANSEREAU1, K. BELLEVILLE1, N. BEAUDET1, M. BEHLKE2, P. SARRET1;
1Physiology and Biophysics, Univ. Sherbrooke, Sherbrooke, PQ, Canada; 2Integrated DNA Technologies Inc., Coralville, IA2007

Copyright by the Society for Neuroscience all rights reserved. Permission to republish any abstract or part of any abstract in any form must be obtained in writing by SfN office prior to publication