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.

ABCA siRNA Transfection of Neurons & Astrocytes

Neuromics' i-Fect^TM Transfection Kit successfully meets the challenge of transfecting neurons and astrocytes in culture. The kit has been used for gene expression analysis studies for: DOR, hTERT, The β3 subunit of the Na+,K+-ATPase, rSNSR1, NTS1. NAV1.8, , RANK, TRPV1, Survivin, Flaviviruses, NOV, Troy β-arrestin, TRPV1 CAV1.2 TLR4 and ASIC. Related publication reference use of the kit for both in vitro and in vivo studies

In this study, Dr. Marina Guizzetti and his team use our i-Fect Kit and several other commercially available kits to knockdown ABCG1 and ABCG4: Jing Chen, Xiaolu Zhang, Handojo Kusumo, Lucio G. Costa, c, Marina Guizzettia. Cholesterol efflux is differentially regulated in neurons and astrocytes: Implications for brain cholesterol homeostasis. Biochimica et Biophysica Acta (BBA) - Molecular and Cell Biology of Lipids, 1831 (2), p.263-275, Feb 2013. doi:10.1016/j.bbalip.2012.09.007.
Abstract: Disruption of cholesterol homeostasis in the central nervous system (CNS) has been associated with neurological, neurodegenerative, and neurodevelopmental disorders. The CNS is a closed system with regard to cholesterol homeostasis, as cholesterol-delivering lipoproteins from the periphery cannot pass the blood–brain-barrier and enter the brain. Different cell types in the brain have different functions in the regulation of cholesterol homeostasis, with astrocytes producing and releasing apolipoprotein E and lipoproteins, and neurons metabolizing cholesterol to 24(S)-hydroxycholesterol. We present evidence that astrocytes and neurons adopt different mechanisms also in regulating cholesterol efflux. We found that in astrocytes cholesterol efflux is induced by both lipid-free apolipoproteins and lipoproteins, while cholesterol removal from neurons is triggered only by lipoproteins. The main pathway by which apolipoproteins induce cholesterol efflux is through ABCA1. By upregulating ABCA1 levels and by inhibiting its activity and silencing its expression, we show that ABCA1 is involved in cholesterol efflux from astrocytes but not from neurons. Furthermore, our results suggest that ABCG1 is involved in cholesterol efflux to apolipoproteins and lipoproteins from astrocytes but not from neurons, while ABCG4, whose expression is much higher in neurons than astrocytes, is involved in cholesterol efflux from neurons but not astrocytes. These results indicate that different mechanisms regulate cholesterol efflux from neurons and astrocytes, reflecting the different roles that these cell types play in brain cholesterol homeostasis. These results are important in understanding cellular targets of therapeutic drugs under development for the treatments of conditions associated with altered cholesterol homeostasis in the CNS.

siRNA transfection: ABCA1 SiRNA transfections: Neurons and astrocytes were transfected using the Nucleofector™ technology (Lonza/Amaxa; Walkersville, MD) as per the manufacturer's optimized protocol. In brief, primary neurons immediately after isolation, or astrocytes harvested after 7–10 days in vitro (DIV), were resuspended in Nucleofector solution. Aliquots of neurons or astrocytes were mixed with 200 pmol ABCA1 siRNA or non-targeting siRNA and were transfected using the Nucleofector programs O-007 and T-20 respectively. Exogenous cholesterol efflux was measured 96 h post transfection. ABCA1 down-regulation in ABCA1 siRNA-transfected cells was verified by Western blot. ABCG1 and ABCG4 Stealth RNAi^TM siRNA transfections: on the day of transfection primary astrocytes were switched to a medium (DMEM with 10%FBS) without antibiotics and supplemented with 50 nM ABCG1 or ABCG4 siRNA, lipofectamine RNAiMAX Transfection Reagent, and Opti-MEM I according to the manufacturer's instruction for 24 h followed by the removal of the medium containing transfection reagents. Six days after preparation, primary cortical neurons were shifted to a medium (Neurobasal/B27) without antibiotic; transfection was carried out by adding to the cultures a solution containing 12 nM ABCG1 or ABCG4 SiRNA, i-Fect siRNA Transfection Reagent, and Opti-MEM I for 24 h. Exogenous cholesterol efflux was measured 48 h after the removal of the transfection reagents. The specific silencing of ABCG1 in astrocytes and neurons was confirmed by Western blot and by qRT-PCR; silencing of ABCG4 was confirmed only by qRT-PCR because no specific antibody to ABCG4 is available.

Figures: Effect of ABCG1 and ABCG4 silencing on cholesterol efflux from neurons and astrocytes. A: Primary rat cortical neurons were transfected with a non-target (NT siRNA), an ABCG1 specific siRNA (ABCG1 siRNA) or an ABCG4 siRNA using the i-Fect™ siRNA transfection reagent. B: Primary rat cortical astrocytes were transfected with an ABCG1 siRNA or an ABCG4 siRNA using lipofectamine RNAiMAX. Twenty-four hours after transfection, cells were labeled with 1 μCi/ml [3H]cholesterol for 24 h followed by a 6 h incubation with cholesterol acceptors. [3H]Cholesterol was quantified in the medium and in the cellular lipids (n=11–12). **pb0.01; ***pb0.001 compared to acceptor-matched controls by Student's t test. C: ABCG1 (left) and ABCG4 (right) mRNA levels were quantified by qPCR in neurons transfected with NT siRNA, ABCG1 siRNA or ABCG4 siRNA (n=4). D: the levels of ABCG1, ABCG4 and ABCA1 mRNA were determined by qPCR in ABCG1 siRNA-transfected (left) and ABCG4 siRNA transfected astrocytes (n=4). Representative immunoblots of ABCG1 levels (upper blots) and β-actin levels (lower blots) in neurons transfected with NT siRNA and ABCG1 siRNA (E) and astrocytes transfected with ABCG1 siRNA (F) and the densitometric quantification of ABCG1 levels normalized to β-actin from 4 independent determination (G) are shown.

The efficiency of transfection is higher in astrocytes than in neurons, it is possible that the lack of an effect of ABCG1 siRNA on cholesterol efflux from neurons may be due to the fact that ABCG1 is not sufficiently down-regulated in these cells. Further study on this phenomena is needed and are important for the discovery of druggable targets that could positively modulate cholesterol homeostasis.

i-Fect and the Study of Glomerulosclerosis (Kidney Disease)

in vitro RANK knockdown system to determine whether RANK was necessary for podocyte survival.

Our i-Fect^TM Transfection Kit is widely used and frequently published as a potent tool for gene expression analysis. In this study i-Fect was used to consistently achieved close to 71.6% siRNA transfection efficiency in podocytes: Shuangxin Liu, Wei Shi, Houqin Xiao, Xinling Liang, Chunyu Deng, Zhiming Ye, Ping Mei, Suxia Wang, Xiaoying Liu, Zhixin Shan, Yongzheng Liang, Bin Zhang, Wenjian Wang, Yanhui Liu, Lixia Xu, Yunfeng Xia, Jianchao Ma, Zhilian Li. Receptor Activator of NF-kappaB and Podocytes: Towards a Function of a Novel Receptor-Ligand Pair in the Survival Response of Podocyte Injury. PLoS ONE: Research Article, published 25 Jul 2012 10.1371/journal.pone.0041331......RANK small interference RNA (siRNA) knockdown was performed by using transient transfection of pooled, functionally validated Cy3–labeled RANK siRNA (Invitrogen) [16]. Podocytes that were differentiated for 10 to 12 d were maintained at 10% FBS/RPMI as described above, and transfected using the RANK siRNA transfection reagent (Neuromics). For determination of the transfection efficiency, a Cy3–labeled RANK siRNA was analyzed by flow cytometry. Western blot analysis for RANK was performed with samples from cells 24 to 96 h after the transfection. Several concentrations of RANK siRNA (20, 40, and 60 nM) were tested to determine optimal knockdown conditions...
Data Highlights: To better understand RANK function in immortalized mouse podocytes, we used an in vitro RANK knockdown system to determine whether RANK was necessary for podocyte survival [33]. We consistently achieved close to 71.6% siRNA transfection efficiency in podocytes, as visualized by transfecting a fluorescently tagged Cy3-RANK siRNA. Flow cytometry fluorescence of podocytes not transfected with siRNA-Cy3 was measured as negative control; the value in podocytes was 4.1% (Figure 5A,5B and 5C). Knockdown of RANK was determined to be maximal between days 3 and 4 after transfection (Figure 5D). We did not observe any morphologic changes between cells with or without RANK siRNA knockdown during the 4 d after transfection. To test whether RANK was involved in the apoptosis of podocytes in vitro, we studied podocytes apoptosis before and after stable knockdown of RANK with siRNA. The knockdown of RANK alone did not induce podocytes apoptosis, but increased mildly the apoptosis of podocytes exposed to PA. However, RANKL reduced apoptosis of podocytes transfected with RANK siRNA exposed to PA compared with control siRNA (RANK siRNA 16.5±1.5% versus control siRNA 24.0±1.8%, p<0.01, Figure 5E).

Images: RANKL and RANK Protects Mouse Podocytes from Apoptosis.(A) Mouse podocytes were transfected with RANK siRNA at a concentration of 100 nM. (B) Flow cytometry fluorescence of podocytes not transfected with siRNA-Cy3 was measured as negative control; the value in podocytes was 4.1%. (C) Transfection efficiency was measured by counting fluorescence-positive cells by flow cytometry; the value in podocytes was 71.6%. (D) RANK immunoblotting of podocytes revealed that RANK was downregulated with RANK siRNA. RANK protein was low abundance in the podocytes 3 d after transfection (lane 1 and lane 2). (E) The percentage of apoptotic cells was measured by flow cytometry. In cells transfected with RANK siRNA, RANK knockdown was associated with mild increase in apoptosis compared with control siRNA (24.0±1.8% versus 22.8±1.1%; p>0.05) after podocytes were exposed to PA. However, RANKL (40 ng/ml) decreased PA-induced apoptosis of podocytes with RANK siRNA (RANKL 16.5±1.5% versus control 22.8±1.1%). (F) Quantification of apoptosis of podocytes with PA and RANKL. Apoptosis was measured by flow cytometry in control podocytes without PA induction (G) (8.7±0.97%) and podocytes that were exposed to RANKL (40 ng/ml) without PA after 48 h (H) (5.7±0.81%). (I) PA increased the apoptosis of podocytes. Apoptosis was measured by flow cytometry of podocytes exposed to PA (25 µg/ml) for 48 h (26.3±3.6%). (J) Exogenous RANKL protected podocytes from PA induction apoptosis. RANKL (40 ng/ml) decreased PA-induced apoptosis (15.5±2.2%). All the experiments were conducted in three times. **Compared with control, p<0.01; *Compared with control, p<0.05.

The upregulation of RANKL and RANK, in combination with the significant protective effects of RANKL, indicates that RANK is part of an adaptive, recovery response to podocyte injury. This is the first observation that RANKL, acting through RANK, functions in an injury paradigm in the kidney. These data raise the exciting therapeutic possibility of giving exogenous RANKL to patients with glomerular disease that is characterized by a loss of podocytes, such as membranous nephropathy and focal segmental glomerulosclerosis.

Using MATRa for siRNA Transfection of Carcinoma Cell Lines

MATRa^TM -Magnet Assisted Transfection is an easy-to-handle, very fast and highly efficient technology to transfect cells in culture with siRNA. Multiple successes with the system includes Carcinoma Cell Lines.

Efficient transient transfection of siRNA in head and neck cancer cells. The cell line ANT-1 was transiently transfected with MATra-A (1 µl/1 µg DNA) in a 6 well format (5 x 105 cells/cavity) with siRNA against protein 1 (100 nM). After 24 hours total RNA was isolated and expression of protein 1-specific mRNA determined by RT-PCR (upper lane). SiRNA 13 are three different oligonucleotide sequences. Control for consistent loading and cDNA quality: expression of ubiquitary GAPDH mRNA (lower lane).
Protein 2 expression in head and neck cancer cells GHD-1. GHD-1 cells (5 x 105 cells/cavity of a 6 well plate) were transiently transfected with two different siRNAs against protein 2. Expression of protein 2 was detected with specific antibodies in an immunoblot 72 hours after transfection with MATra-A (1 µl / 1 µg DNA). As control ubiquitary β-actin was detected as well.
Treating the carcinoma cells with specific siRNA caused a clear inhibition of protein 1/protein 2 expression which indicates high transfection efficiencies.
(Data kindly provided by Rauch, Schaffrik, Ahlemann and Gires, LMU Munich and GSF, Munich, Germany).

"After having tested MATra in a variety of experimental set ups we can summarize the following advantages: High transfection efficiency   Easier to handle   High reproducibility Serum compatibility Low sensibility against cell confluence" Dr. Oliver Gires, LMU Munich, Germany

Delivering siRNA in Mice for Studying Opioid-Induced Hyperalgesia

Researchers have successfully delivered siRNA in-vitro and in-vivo using Neuromics' i-Fect ™ siRNA Transfection Reagent. Gene expression studies include: DOR, hTERT, The β3 subunit of the Na+,K+-ATPase, rSNSR1, NTS1. NAV1.8 and more.

Here's a link to all transfection publications: Transfection Kit Pubs

We are pleased to present yet another study and related publication. This includes one of the first successful delivery of siRNA in mice using i-Fect ™ :

Yan Chen, Cheng Yang, and Zaijie Jim Wang. Ca2+/Calmodulin-Dependent Protein Kinase II Is Required for the Initiation and Maintenance of Opioid-Induced Hyperalgesia. The Journal of Neuroscience, January 6, 2010, 30(1):38-46; doi:10.1523/JNEUROSCI.4346-09.2010.

...KN93 and KN92 were administered intrathecally by percutaneous puncture through the L5-L6 intervertebral space, as described previously (Hylden and Wilcox, 1980; Chen et al., 2009). A lateral tail flick was considered as success of the intrathecal injection. To inhibit CaMKII, CaMKII was targeted by small interfering RNA (siRNA). Four days after morphine pellet implantation, mice were treated with CaMKII siRNA (5'-CACCACCAUUGAGGACGAAdTdT-3', 3'-dTdTGUGGUGGUAACUCCUGCUU-5') (Zayzafoon et al., 2005) or Stealth RNAi negative control (Invitrogen) (2 µg, i.t., twice per day for 3 consecutive days). These oligos were mixed with the transfection reagent i-Fect (Neuromics), in a ratio of 1:5 (w/v) (Luo et al., 2005). Mechanical and thermal sensitivity tests were performed daily...

Using i-Fect for treatment of Glioblastomas

Dr. Swapan K. Ray, Department of Pathology, Microbiology, and Immunology, University of South Carolina School of Medicine and his team should positive results in reducing growth of Glioblastomas by knocking down hTERT expression using Neuromics' i-Fect ™ siRNA Transfection Kit. Here's the related pub:

Joseph George, Naren L. Banik, Swapan K. Ray. Combination of hTERT Knockdown and IFN-γ Treatment Inhibited Angiogenesis and Tumor Progression in Glioblastoma. Clin Cancer Res 2009;15(23):7186–95

...with i-Fect transfection reagent (Neuromics) to obtain 5 μg DNA/10 μL of injection volume...

Results: In vitro and in vivo angiogenesis assays showed inhibition of capillary-like network formation of microvascular endothelial cells and neovascularization under dorsal skin of nude mice, respectively. We observed inhibition of intracerebral tumorigenesis and s.c. solid tumor formation in nude mice after treatment with combination of hTERT siRNA and IFN-γ. Western blotting of solid tumor samples showed significant downregulation of the molecules that regulate cell invasion, angiogenesis, and tumor progression.

Conclusions: Our study showed that the combination of hTERT siRNA and IFN-γ effectively inhibited angiogenesis and tumor progression through the downregulation of molecules involved in these processes. Therefore, the combination of hTERT siRNA and IFN-γ is a promising therapeutic strategy for controlling the growth of human glioblastoma.