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.

Neuromics' Transfection Kits-Genes Studied

Delivering siRNA, miRNA, Plasmids and Viral Vectors for Gene Expression Analysis.

I have shared the many genes researchers have studied using our Transfection Kits. These include: β-arrestin, ABCA, ASIC, β-arrestin, CAV1.2, CX3CR1, DOR, EHDAC2, LOVL4, IKBKAP, K+-ATPase, KV1.1, KV9.1 , neuroligin 2, The β3 subunit of the Na+,K+-ATPase, NTS1, NAV1.8, NTS1, NOV, Raf-1, RANK, SNSR1, hTert, NOV, Survivin, TLR4, Troy and TRPV1 and More!

We can now add GPNMB to this list: Lili Hou, Yanfeng Zhang, Yong Yang, Kai Xiang, Qindong Tan, Qulian Guo. Intrathecal siRNA Against GPNMB Attenuates Nociception in a Rat Model of Neuropathic Pain. Journal of Molecular Neuroscience. July 2014...Ten micrograms of siRNA1- GPNMB dissolved in 30 μl i-Fect transfection reagent (Neuromics, Edina, MN, USA) was administered intrathecally once daily for 7 days, starting from 1 day before CCI surgery...

Abstract: Neuropathic pain is characterized by hyperalgesia, allodynia, and spontaneous pain. Recent studies have shown that glycoprotein nonmetastatic melanoma B (GPNMB) plays a pivotal role in neuronal survival and neuroprotection. However, the role of GPNMB in neuropathic pain remains unknown. The aim of the present study was to assess the role of GPNMB in neuropathic pain. In cultured spinal cord neurons, we used two small interfering RNAs (siRNAs) targeting the complementary DNA (cDNA) sequence of rat GPNMB that had potent inhibitory effects on GPNMB, and siRNA1-GPNMB was selected for further in vivo study as it had the higher inhibitory effect. After sciatic nerve injury in rats, the endogenous level of GPNMB was increased in a time-dependent manner in the spinal cord. Furthermore, the intrathecal injection of siRNA1-GPNMB inhibited the expression of GPNMB and pro-inflammatory factors (TNF-α, IL-1β, and IL-6) and alleviated mechanical allodynia and thermal hyperalgesia in the chronic constriction injury (CCI) model of rats. Taken together, our findings suggest that siRNA against GPNMB can alleviate the chronic neuropathic pain caused by CCI, and this effect may be mediated by attenuated expression of TNF-α, IL-1β, and IL-6 in the spinal cord of CCI rats. Therefore, inhibition of GPNMB may provide a novel strategy for the treatment of neuropathic pain.

If you would like to learn how you can optimize your gene expression analysis studies, do not hesitate to e-mail: pshuster@neuromics.com or direct line: 612-801-1007.

i-Fect siRNA Transfection Successes

Meeting your Gene Expression Analysis Requirements.

I have posted many success stories resulting from use of  Neuromics'  Neuromics' i-Fect^TM siRNA Transfection Kit. It has been 3 months since my last posting so here I would like to post some recent highlights.

Here I feature customer publication demonstrating:

1. Down regulation of Neuroligin 2-data showed unexpected upregulation and pronociceptive effects of the “inhibitory” NL2 in neuropathic pain, suggesting a functional shift of NL2 from inhibition to excitation that changed the synaptic ratio toward higher excitation.
2. Role of IGFPB5 for survival of neurons in post-natal development.
3. HDAC role in Anxiety and Alcoholsim

Here're the publications:
Tiphaine Dolique, Alexandre Favereaux, Olivier Roca-Lapirot, Virginie Roques, Claire Léger, Marc Landy, Frédéric Nagy, Unexpected association of the “inhibitory” Neuroligin 2 with excitatory PSD95 in neuropathic pain, PAIN, Available online 25 July 2013, ISSN 0304-3959, http://dx.doi.org/10.1016/j.pain.2013.07.035.
(http://www.sciencedirect.com/science/article/pii/S030439591300403X)...The siRNAs were solubilized in 10 µl of i-Fect reagent (Neuromics, Edina, Minnesota, USA) following Neuromics instructions and published protocol, and applied intrathecally using a Hamilton syringe...

Masaki Ueno,Yuki Fujita, Tatsuhide Tanaka, Yuka Nakamura, Junichi Kikuta, Masaru Ishii & Toshihide Yamashita. Layer V cortical neurons require microglial support for survival during postnatal development. Nature Neuroscience 16, 543–551 (2013) doi:10.1038/nn.3358.
...vehicle (PBS) was delivered intraventricularly through the cisterna magna with a glass pipette while P3 mice were cold anesthetized. Igf1 siRNA (stealth siRNA, Invitrogen) or Igfbp5 siRNA with i-Fect reagent (Neuromics) was delivered intraventricularly through the cisterna magna at P3 twice with a 12-h interval...

Sachin Moonat, Amul J. Sakharkar, Huaibo Zhang, Lei Tanga, Subhash C. Pandey. Aberrant Histone Deacetylase2–Mediated Histone Modifications and Synaptic Plasticity in the Amygdala Predisposes to Anxiety and Alcoholism. Biological Psychiatry. doi.org/10.1016/j.biopsych.2013.01.012
...The siRNAs were dissolved in iFect solution (Neuromics, Edina)...

I will keep updating you as more pubs and data becomes available.

i-Fect Delivers IGF1 siRNA to the Mouse Brain

Intraventricular Injections Used to Study Neuronal Survival During Post Natal Development

Neuromics' i-Fect^TM Transfection Kit continue to be successfully used to deliver siRNA, shRNA and miRNA to cell cultures and the CNS in vivo (via intrathecal, epidural and intraventricular injection). Genes studied include: ABCA, ASIC, β-arrestin, CAV_1.2, IGF1, CX3CR1, DOR, ELOVL4, IKBKAP, K+-ATPase, K_V1.1, K_V9.1 ,The β3 subunit of the Na+,K+-ATPase, NTS1, NAV_1.8, NTS1, NOV, Raf-1, RANK,SNSR1, hTertTRPV1 NOV, Survivin, TLR4, Troy and TRPV1. Related Publications.

It is always an honor for one our products to be referenced in one of the Nature publications. Here researchers use i-Fect to study the impact of microglial derived IGF1 silencing on Neuronal Survival: Masaki Ueno,Yuki Fujita, Tatsuhide Tanaka, Yuka Nakamura, Junichi Kikuta, Masaru Ishii, Toshihide Yamashita. Layer V cortical neurons require microglial support for survival during postnatal development. Nature Neuroscience 16, 543–551 (2013) doi:10.1038/nn.3358. ...vehicle (PBS) was delivered intraventricularly through the cisterna magna with a glass pipette while P3 mice were cold anesthetized. Igf1 siRNA (stealth siRNA, Invitrogen) or Igfbp5 siRNA with i-Fect reagent (Neuromics) was delivered intraventricularly through the cisterna magna at P3 twice with a 12-h interval...

Images: (a) Igf1 expression (blue) and Iba1-positive microglia (brown) in P5 brain. Scale bar represents 400 μm. (b) Magnified view of dotted square in a. Scale bar represents 50 μm. (c,d) IGF1Ra expression (red) in CTIP2-positive (c) and SATB2-positive (d) layer V neurons at P5. Scale bar represents 50 μm. (e) IGF1 protein levels in medium from cultured cortical neurons, microglia and neurons with microglia in transwell systems detected by enzyme-linked immunosorbent assay (ELISA). **P < 0.01 (neuron, microglia + neuron, n = 5; microglia, n = 6 experiments; one-way ANOVA followed by Tukey-Kramer test). (f) The number of cleaved caspase-3–positive neurons in transwell systems treated with LY294002 or H-1356 or transfected to microglia with Igf1 siRNA. *P < 0.05, **P less than  0.01 (n = 3 experiments, one-way ANOVA followed by Tukey-Kramer test). (g) Neuronal phospho-AKT expression in cultured cortical neurons and those with microglia in transwell system. (h) TUNEL-positive cells in H-1356–treated cortex (36 h after treatment). Scale bar represents 100 μm. (i) The number of TUNEL-positive apoptotic cells in each layer in vehicle- (phosphate-buffered saline, PBS) or H-1356–treated mice (36 h after injection). *P < 0.05 (n = 4 brains, one-way ANOVA followed by Tukey-Kramer test). (j) Cleaved caspase-3–labeled cells (red) expressing CTIP2 (green, arrowheads). Scale bar represents 100 μm. (k,l) TUNEL-positive cells in the cortex treated with control or Igf1 siRNA (48 h after treatment). Scale bar represents 100 μm. (m) The number of TUNEL-positive apoptotic cells in each layer in control siRNA– and Igf1 siRNA–treated mice. **P less than 0.01 (n = 5 brains, one-way ANOVA followed by Tukey-Kramer test). Error bars represent s.e.m.

I will continue to post new developments and successes.

i-Fect™ Delivers you siRNA Payload

Delivering siRNA to Dorsal Root Ganglia to Silence K_V Receptors.

Our i-Fect transfection kits continue to be used to optimize delivery in vivo and into hard to transfect cells like primary neurons. In these 2 latest expamples, researchers use i-Fect to deliver siRNA to K_V Receptors in Rat DRGs. Knocking down these receptors enable the study of their role in pain modulation: John H. Winston, Sushil K. Sarna. Developmental Origins of Functional Dyspepsia-Like Gastric Hypersensitivity in Rats. Gastroenterology. Volume 144, Issue 3, March 2013, Pages 570–579.e3. dx.doi.org/10.1053/j.gastro.2012.11.001....intrathecal treatment, 2 μg of the appropriate siRNA was mixed (1:5 vol/vol) with i-Fect transfection reagent (Neuromics, Edina, MN); rats received 2 ug siRNA/10 uL/rat/injection...

Figures. siRNA-mediated knockdown of Kv1.1 expression in thoracic DRG significantly increased gastric sensitivity in naive adult rats. (A) Western blots showed a significant decrease in Kv1.1 protein in thoracic DRG (T8–T12) after intrathecal treatment with Kv1.1 siRNA but not with control siRNA. siRNA treatment did not alter TrpV1 expression (n = 5 rats each; *P < .01 vs control siRNA). (B) Naive rats treated with Kv1.1 siRNA showed a significant increase in VMR to gastric distention (n = 5 rats each, compared with pretreatment baseline; *P < .05). (C) Treatment with control siRNA had no significant effect on gastric hypersensitivity. (D) Patch clamp recordings from freshly dissociated gastric DRG neurons from FD-like and PND 10 saline-treated littermate controls showed a significant decrease in rheobase in FD-like rats (*P < .05), and (E) a significant increase in the number of action potentials elicited by current injection at 3× the rheobase in gastric DRG neurons from FD-like rats (*P < .05). (F) Sample voltage vs time traces showing action potentials evoked at ×1, ×2, and ×3 rheobase. The patch clamp data were obtained from 16 cells from 5 PND 10 saline control rats and 19 cells from 5 FD-like rats.
Tsantoulas C, Zhu L, Shaifta Y, Grist J, Ward JP, Raouf R, Michael GJ, McMahon SB. Sensory neuron downregulation of the Kv9.1 potassium channel subunit mediates neuropathic pain following nerve injury. J Neurosci. 2012 Nov 28;32(48):17502-13. doi: 10.1523/JNEUROSCI.3561-12.2012..."I'd just like to notify you about a recent paper from my team which utilised iFect for in vivo transfection of dorsal root ganglia." Dr. Christoforos Tsantoulas, University of Cambridge...i-Fect-siRNA-mediated knock-down of Kv9.1 in naive rats led to neuropathic pain behaviors...

These add to the many publications referencing use of i-Fect to deliver siRNA. Genes studied include:  DOR, hTERT, The β3 subunit of the Na+,K+-ATPase, rSNSR1, NTS1. NAV1.8, , RANK, Toll-Like Receptors, Kv Recptors, BDNF, Ret, TRPV1, Survivin, Flaviviruses, NOV, Troy β-arrestin, TRPV1 CAV1.2 TLR4 and ASIC.

BDNF and Recovery of Motor Function After Brian Injury

Researchers use i-Fect^TM to Study BDNF Silencing in Mice

Brain injury that results in an initial behavioural deficit is frequently followed by spontaneous recovery. The intrinsic mechanism of this functional recovery has never been fully understood. Here, we show that reorganization of the corticospinal tract induced by target-derived brain-derived neurotrophic factor is crucial for spontaneous recovery of motor function following brain injury:  Masaki Ueno, Yasufumi Hayano1, Hiroshi Nakagawa and Toshihide Yamashita. Intraspinal rewiring of the corticospinal tract requires target-derived brain-derived neurotrophic factor and compensates lost function after brain injury. Brain (2012) doi: 10.1093/brain/aws053. ... motor cortex at 14 days after the injury, using i-Fect™ transfection reagents (Neuromics) according to the manufacture's instruction .

Findings Overview: After destruction of unilateral sensorimotor cortex, intact-side corticospinal tract formed sprouting fibres into the specific lamina of the denervated side of the cervical spinal cord, and made new contact with two types of spinal interneurons—segmental and propriospinal neurons. Anatomical and electrophysiological analyses revealed that this rewired corticospinal tract functionally linked to motor neurons and forelimb muscles. This newly formed corticospinal circuit was necessary for motor recovery, because transection of the circuit led to impairment of recovering forelimb function. Knockdown of brain-derived neurotrophic factor in the spinal neurons or its receptor in the intact corticospinal neurons diminished fibre sprouting of the corticospinal tract. Our findings establish the anatomical, functional and molecular basis for the intrinsic capacity of neurons to form compensatory neural network following injury.

We will continue to post new references to our Transfection Kits.

Using i-Fect to Silence Kv9.1 in vivo

There have been multiple publications referenced here on using Neuromics' i-Fect siRNA Delivery Kit to study the effect of silencing genes known to play a role in Pain Signaling. These include: DOR,The β3 subunit of the Na+,K+-ATPase, NTS1, NAV1.8, TRPV1 NOV, β-arrestin, TRPV1, CAV1.2 and ASIC.

I would like to highlight an exciting new study referencing how knocking down Kv9.1 Potassium Channel Subunit in vivo mediates neuropathic pain: Christoforos Tsantoulas, Lan Zhu, Yasin Shaifta, John Grist, Jeremy P. T. Ward, Ramin Raouf, Gregory J. Michael, and Stephen B. McMahon. Sensory Neuron Downregulation of the Kv9.1 Potassium Channel Subunit Mediates Neuropathic Pain following Nerve Injury. The Journal of Neuroscience, 28 November 2012, 32(48): 17502-17513; doi: 10.1523/​JNEUROSCI.3561-12.2012.

Highlights: Here, we report that the potassium channel subunit Kv9.1 is expressed in myelinated sensory neurons, but is absent from small unmyelinated neurons. Kv9.1 expression was strongly and rapidly downregulated following axotomy, with a time course that matches the development of spontaneous activity and pain hypersensitivity in animal models. Interestingly, siRNA-mediated knock-down of Kv9.1 in naive rats led to neuropathic pain behaviors. Diminished Kv9.1 function also augmented myelinated sensory neuron excitability, manifested as spontaneous firing, hyper-responsiveness to stimulation, and persistent after-discharge. Intracellular recordings from ex vivo dorsal root ganglion preparations revealed that Kv9.1 knock-down was linked to lowered firing thresholds and increased firing rates under physiologically relevant conditions of extracellular potassium accumulation during prolonged activity. Similar neurophysiological changes were detected in animals subjected to traumatic nerve injury and provide an explanation for neuropathic pain symptoms, including poorly understood conditions such as hyperpathia and paresthesias. In summary, our results demonstrate that Kv9.1 dysfunction leads to spontaneous and evoked neuronal hyperexcitability in myelinated fibers, coupled with development of neuropathic pain behaviors.

n vivo RNA interference: Anesthetized rats were subjected to a thoracic laminectomy and a silastic tube was inserted subdurally to lie just rostral to L3 DRG and externalized to deliver bolus injections (one injection per day for 4 consecutive days). Animals were allowed to recover for 5 d before treatment commenced. On the day of injection, siRNA was mixed with i-Fect (Neuromics) to a final concentration of 0.2 μg μl−1, according to published protocols (Luo et al., 2005). For each treatment, 10–20 μl of Kv9.1 siRNA or scrambled control mixture was injected, followed by a 10 μl saline flush. Twenty-four hours after the fourth injection animals were killed and L5 DRGs fresh dissected for qRT-PCR analysis. A separate set of animals were PFA perfused and DRGs retrieved for IHC. Passenger strand sequences for Kv9.1 and scrambled control siRNAs were cuuggaaucuguaggauca and gaggcctaatcgatatgtt, respectively (Dharmacon; “in vivo processing” option).

Intrathecal Kv9.1 siRNA treatment induces pain behaviors in naive rats. A, qRT-PCR quantification of Kv9.1 mRNA in rat PASMC cultures transfected with one of three Kv9.1 siRNA sequences or control siRNA (control, n = 6; siRNA, n = 3 per group; *p < 0.05 vs control, one-way ANOVA with Tukey's). B, qRT-PCR showing Kv9.1 in vivo knock-down in L5 DRG, 4 d after intrathecal delivery of siRNA #1 compared with vehicle or matched scrambled control (vehicle, n = 4; scrambled, n = 5; Kv9.1, n = 7; *p < 0.05, t test). C, IHC for Kv9.1 in scrambled- and siRNA-treated DRG to determine protein knockdown. Graphs illustrate quantification of number of positive myelinated neurons and mean Kv9.1 signal intensity (scrambled, n = 4; siRNA, n = 6; **p < 0.01, ***p < 0.001, t test). D, Kv9.1 siRNA infusion inflicts a reduction in mechanical pain withdrawal thresholds (Kv9.1, n = 7; control, n = 6; *p < 0.05, **p < 0.01, ***p < 0.001 vs scrambled control or baseline, two-way repeated measurements ANOVA with Tukey's). E, There was no change in heat pain thresholds after siRNA treatment. Vertical arrows on x-axis denote siRNA injections. All data represent mean ± SEM.

Kv9.1 knock-down triggers ectopic activity and a form of peripheral wind-up in response to stimulation. A, Schematic illustrating the positions of stimulating and recording electrodes. B, Example recordings from centrally disconnected L4/L5 strands demonstrating SA in Kv9.1 siRNA-treated or nerve-injured rats, but not in control (scrambled siRNA) animals. C, Frequency-dependent SEA (denoted by double arrowheads) in Kv9.1 siRNA-treated (middle) and injured (right), but not control (left) animals. This activity is not locked in time and can be seen in between stimulation events (vertical arrows on top of 5 Hz stimulation traces, only first 5 shown). Also note the prolonged after-discharge (AD) observed in siRNA-treated and injured animals. D, Percentage of units showing SA and SEA in control (n = 269), Kv9.1 siRNA-treated (n = 369) and injured (n = 176) animals (*p < 0.05, **p < 0.01, ***p < 0.001 vs control, χ2 test). E, Firing rate of SEA units at different stimulation frequencies (mean ± SEM; control, n = 4; siRNA, n = 22; injured, n = 17; *p < 0.05 vs control, two-way ANOVA with Tukey's). F, Quantification of AD rate per SEA unit (mean ± SEM; *p < 0.05 vs control, Mann–Whitney test).

Results propose that Kv9.1 downregulation after nerve injury may be the molecular switch controlling myelinated sensory neuron hyperexcitability. Intriguingly, a recent wide-genome association screen in humans identified a Kv9.1 polymorphism associated with susceptibility to develop chronic neuropathic pain after back surgery or leg amputation (Costigan et al., 2010), suggesting that the mechanisms described in our studies will be of direct clinical relevance to human pain. Future efforts to elucidate the precise pathways involved, combined with approaches aiming to compensate loss of Kv9.1 function, may create novel therapeutic opportunities for neuropathic pain management.

RNA Interference Video

This is worth a look!

intra-i-Fect and intravenous delivery of siRNA

Deliver siRNA in-vivo with stunning results! Introductory Special-200 to 600 USD (valid through 9/30/2010)
These intra-i-Fect kits are designed to deliver siRNA in vivo via intravenous injections with high efficiency to specific tissue in rats and mice. The protocol involves these simple steps: prep, mix, dry, hydrate and inject.

Figure: siRNAs knock down profiles of the gene related to cancer, diabetes, obesity, steatosis hepatitis, cirrhosis and a gene specifically expressed in endothelial cells in liver

They are developed using a proprietary platform that uses nano-particles as the delivery vehicle. This platform enables:
•Effective delivery (60%+ knockdown) with no toxicity.
•Scalable to high throughput siRNA based gene screening.
•Consistent and reproducible results.