Tissue Acidosis and Inflammation Related Nociceptive Pain

Important Implications for Inflammatory Pain States

Inflammatory pain is often accompanied by a drop in pH (Acidosis). Here investigators hypothesized that modest drops in extracellular pH leading to calcium fluxes acts like a dynamic switch to rapidly mobilize trkA to the cell membrane surface of adult sensory neurons, which in turn serves to increase the sensitivity of these neurons to NGF. The findings reveal a cellular mechanism whereby even small changes in pH can rapidly shift sensitivity to a critical driver of the inflammatory pain state—NGF. They way they demonstrate this shift is quite ingenious: Geoffrey E. Bray, Zhengxin Ying, Landon D. Baillie, Ruiling Zhai, Sean J. Mulligan, and Valerie M.K. Verge. Extracellular pH and Neuronal Depolarization Serve as Dynamic Switches to Rapidly Mobilize trkA to the Membrane of Adult Sensory Neurons. The Journal of Neuroscience, 8 May 2013, 33(19):8202-8215; doi:10.1523/JNEUROSCI.4408-12.2013.

Images: NGF-responsive adult sensory neurons have a large cytoplasmic pool of high-affinity NGF receptors. Six micrometer cryostat sections of L5 DRG processed for high-affinity NGF binding (left) or trkA immunohistochemistry (right) reveal a large cytoplasmic pool of proteins that are able to bind 20 pm radio-iodinated NGF with high affinity or that are immunoreactive to trkA-selective antibodies. Scale bar, 20 μm.

Here're images showing the pH Related switch and rapid migration of TrkA to the membrane

Images: Acidic pH challenge rapidly mobilizes trkA to the membrane from internal stores. A, Bar graphs summarize relative changes in neuronal cell-surface trkA expression from three separate experiments (detected with immunofluorescence) over neurons exposed to control (pH 7.4) or acidic (pH 6.5) media for 30 min with or without exposure to Golgi collapsing compound BFA and as normalized to the mean signal intensity from the control pH group. Note: A significant increase is observed in the mean levels of trkA mobilized to the neuronal membranes of sensory neurons in response to acidosis when compared with the control pH, a response that is blocked with BFA treatment. B, Immunofluorescence photomicrographs and summary histograms (C) depict degree of FM 1–43FX-stained neuronal membrane internalization in response to conditions as indicated. Note: Significant membrane internalization was only observed in the NGF challenge control group and not in response to acidic pH challenge with or without BFA treatment. (Data normalized to the control pH of each experimental condition and pooled from three separate experiments; A, C, one-way ANOVA with post hoc Tukey's, **< 0.01; ***< 0.001). Scale bar, 20 μm.

The heightened level of trkA activation likely has ramifications on both short- and long-term sensitization processes as it regulates the activity and expression of a wide variety of receptors, ion channels, and signaling molecules (Mantyh et al., 2011). In the short term, it modulates the activity of receptors and ion channels. For example, NGF decreases the threshold of activation of the nociceptive transient receptor potential vanilloid 1 (TRPV1) receptors (Chuang et al., 2001), mediates TRPV1 trafficking to the membrane (Stein et al., 2006), increases purinergic receptor P2X3-mediated currents and Ca²⁺ transients (D'Arco et al., 2007) and for sympathetic neurons, rapidly modulates the activity of at least four voltage-gated currents (Luther and Birren, 2009). In the long term, increased NGF can lead to increased transcription of many nociception-associated genes such as its receptors trkA and p75 (Verge et al., 1989, 1992); the neuropeptides Substance P, calcitonin gene-related peptide (Lindsay and Harmar, 1989; Verge et al., 1995), and PACAP; Jongsma Wallin et al., 2001, 2003); sodium channels (Dib-Hajj et al., 1998; Fjell et al., 1999; Kerr et al., 2001); and P2X3 (Ramer et al., 2001; Simonetti et al., 2006). It is also interesting to note that activation of trkA was linked in our study to a parallel activation of p38MAPK, which has been shown to be linked to NGF-mediated increases in ASIC3 expression during inflammation (Mamet et al., 2003).

Related Reagents:
All Trk Antibodies
Neurotrophin Antibodies
Neurotrophin Proteins
Primary Neurons and Astrocytes-Primary human, rat and mouse neurons and astrocytes by Category

More on the Neurobiology of Itch

I have an earlier posting on neuro-transmission of itch or pruritis. This focused on the role of Toll-like Receptor 3 (TLR 3). This posting looks at the role of Gastrin-Releasing Protein (GRP) and the Gastrin-Releasing Protein Receptor (CRPR) in chronic itch. The findings are important because this condition affects millions worldwide and results in a costly erosion of quality of life.

Here chronic itch was studied in Macque Monkeys over a period of 4 years. The expression patterns of GRP, GRPR and PGP9.5 were accessed by immunohistochemistry: Leigh A Nattkemper, Zhong-Qiu Zhao, Anna J Nichols, Alexandru D P Papoiu, Carol A Shively, Zhou-Feng Chen and Gil Yosipovitch. Over-Expression of the Gastrin-Releasing Peptide in Cutaneous Nerve Fibers and its Receptor in Spinal Cord in Primates with Chronic Itch. Journal of Investigative Dermatology accepted article preview 4 April 2013; doi: 10.1038/jid.2013.166.

Images: Double labeling of PGP9.5 and GRP in skin of primates representing mild, moderate and severe itch. Primates with higher scratching severity showed in increase in the co-localization of PGP9.5 and CRP at the dermal-epidermal junctions (arrows).

In addition to increased PGP9.5/GRP expression in the skin, similar results were shown for expression of GRP/GRPR in DRGs. This makes GRP and its receptor candidate drug targets for chronic itch or pruritis in humans.

Neuron-Glial Cultures-Setting a Higher Bar!

Improved Methods for Long Term, High Denisty Cultures

 

Dr. Randen Patterson and his team at UC Davis have developed new culturing techniques using our e18 Rat Primary Hippocampal Neurons. They have developed a protocol that allows for culturing of E18 hippocampal neurons at high densities for more than 120 days. These cultured hippocampal neurons are (i) well differentiated with high numbers of synapses, (ii) anchored securely to their substrate, (iii) have high levels of functional connectivity, and (iv) form dense multi-layered cellular networks. We propose that our culture methodology is likely to be effective for multiple neuronal subtypes–particularly those that can be grown in Neurobasal/B27 media. This methodology presents new avenues for long-term functional studies in neurons. This is good news indeed: Todd GK, Boosalis CA, Burzycki AA, Steinman MQ, Hester LD, et al. (2013) Towards Neuronal Organoids: A Method for Long-Term Culturing of High-Density Hippocampal Neurons. PLoS ONE 8(4): e58996. doi:10.1371/journal.pone.0058996.

 

Protocol Highlights: 
Substrate Preparation
1. On the day of plating, prepare 25 mm coverslips by removing them from 70% ethanol storage solution and propping them up at an angle in each well of a 6-well culture plate to allow drying. [No more than 5 plates (30 coverslips) should be dried simultaneously for 15–25 minutes in culture hood to avoid over-drying.]
2. Once dry, shake slips down flat into their respective wells and coat with 1 mL 0.1% poly-D-lysine, taking care to form a liquid meniscus on each slip. Carefully transfer coverslips into incubator, taking care to preserve meniscus.
3. Incubate for 1 hr at 37°C. [Keeping poly-D-lysine meniscus on top of coverslip is important; this serves to avoid poly-D-lysine coating under coverslip surface that may lead to problematic flotation of coverslip.] After incubation, remove poly-D-lysine and rinse each coverslip three times with 2 mL sterile deionized water. Take care to ensure coverslips do not completely dry at any point during the rinse. After the third and final rinse, leave coverslips in 2 mL sterile deionized water for at least 1 hr. Remove water just before plating, again, make sure to avoid over-drying. [This critical step requires attention. Take care to aspirate off all sterile water remaining from the final rinse, but also use caution as not to over-dry the coverslips. Ultimately, the coverslip must be mostly dry as to allow for the meniscus formation during plating (Fig. 1, Step 12), whereas over-drying can result in the neurons peeling off the glass coverslips days to weeks after plating.]

Fig 1: Neuron-Glial Culturing Steps
Preparation of Isolated Neurons (Numbers in Fig. 1 correspond to numbers below).
1. Store tissue at 4°C until ready to use. If dissecting your own cultures, upon isolation of the tissue, store in an appropriate storage media.
2. When ready to plate, make 2 mL of enzymatic solution without B27. In our case, we used Hibernate E-Ca, containing 4 mg (2 mg/mL) of papain. If making your own solution, use a commercially available papain dissociation kit. Make sure to sterile filter solution with 0.2 micron filter after adding papain if source of enzyme is not sterile.
3. Remove the storage media from the dissected tissue and transfer into sterile 15 mL screw-cap tube; be careful not to disturb or remove tissue from original tube. Save the storage media, do not discard.
4. Add 2 mL of media made in Step 2 to tissue (in our case, Hibernate E-Ca containing 2 mg/mL of papain). Incubate for 35 min at 37°C. [Be sure to add Hibernate E-Ca containing papain slowly as to avoid disturbing tissue.]
5. Remove enzymatic solution from tissue, again, take care not to disturb or remove tissue. Add back 1 mL of storage media saved in 15 mL tube.
6. Using a 1 mL pipettor with a sterile plastic pipette tip (tissue can adhere to glass pipettes), aspirate the tissue with the medium into the pipette and immediately dispense contents back into same container. Take care not to create bubbles. [This is another critical step that requires attention. Take care to make sure pipette tip remains in a stable position (as shown in Fig. 1, Step 6). Maintain slow, steady speed when both drawing in and re-dispensing media containing tissue.]
7. Repeat this trituration step 10–12 times or until most all the tissue is dissociated and the cells are dispersed. [Under close examination cell dispersion is highly visible. Stop pipetting immediately upon cell dispersion.]
8. Slowly transfer contents of the tissue tube into a new sterile 15 mL screw-cap tube.
9. Use the remainder of storage medium saved in Step 3 and rinse the interior of the tissue tube before adding it to the sterile 15 mL screw cap tube containing dispersed cells from step 7. [This step helps ensure minimal wastage, as any remaining cells should be saved with this extra rinse.]
10. Spin dispersed cells at 1,100 rpm (200Xg) for 1 min.
11. Discard the supernatant while being careful not to remove any of the cells from cell pellet.
12. Flick tube a few times to loosen the cell pellet. Re-suspend pellet in 2.4 mL of pre-warmed B27/Neurobasal/0.5 mM glutamine medium. Re-suspend by gently pipetting up and down. For E18 Hippocampus, medium includes 25 µM glutamate.
13. Plate cells within a meniscus (approx. 10 mm diameter) at a minimum of 40 µL per 25 mm coverslip. Take care not to disturb meniscus. [Periodically pipette up and down throughout plating process (no more than once every plate per 6 coverslips) to help maintain equal cell density. Again, plating with meniscus formation is critical.]
14. Incubate plated cells at 37°C with 5% CO2 and/or 9% or 20% oxygen for 1 hr.
15. Add 1.5 mL per well of pre-warmed 1:1 ACM/NbActiv4. [Slow and steady media addition rate and proper pipette position are necessary for successful plating density consistency. Position pipette tip at 45° angle along middle of 6-well interior sidewalls, dispense 1.5 mL as slowly and steadily as possible (see Steps 1–14).]
16. Incubate cells at 37°C with 5% CO2 and/or 9% or 20% oxygen.
17. Add Cytosine β-D-arabinofuranoside (Ara-C) to a final concentration of 5 µM, 5–6 days after plating to curb glial proliferation. [Remove 1/3 of media from each well and replace with equal volume containing final concentration of Ara-C]
18. After 4 days or longer, neurons are well differentiated. If further culture is desired, change 1/3 of medium with fresh, pre-warmed 1:1 ACM/NbActiv4 every 7–8 days.

Images: 40X Confocal Images of 30 DIV Hippocampal Cultures. Immunofluorescence detection of MAP-2 (green) and GFAP (red) in 30 DIV (A–C) cultured E18 hippocampal cells using a 40X objective. These images (A and B) clearly depict the intimate physical contact between glia processes and dendritic arbors. Under closer examination (CI and CII), it is clear that the dendrites have grown both bellow (blue arrows) and above (white arrows) glial processes, forming a highly interconnected three-dimensional network by 30 DIV. doi:10.1371/journal.pone.0058996.g004.
All Primary Neuron Assay Customer Publications

 

Related Content:

• Primary Neurons and Astrocytes-Primary human, rat and mouse neurons and glia/astrocytes.
• Neuronal-Glial Markers-Astrocytes, Glia, Microglia, Olidogodendrocytes, Progenitors and Schwann Cell Markers
• Cells, Cell Culturing Systems and Cell Based Assay Reagents-Cells, Petaka Cell Culturing Systems, Biolumonics Kits, Media, 3-D Scaffolds and Related Reagents Designed to Optimize Cell Based Assays

If you have any questions on optimizing your cell cultures, do not hesitate to contact me @ pshuster@neuromics.com or 612-801-1007

Small Molecules-Peptides for Neuroscience Research

Agonists, Antagonists, Inhibitors and Ligands for Studying Neuromodulation

Our friends at R and D Systems/Tocris Bioscience have made available to us select Small Molecules/Peptides. Our focus will be on providing agonists, antagonists, inhibitors and ligands that complement our Neuroscience and Pain Research products and expertise.

2-APB=TRP Channel Modulator

We will be adding about 10 new molecules/peptides per month. Here's a sampling our our most recent additions:

Name	Type	Bioactivity
(±)-trans-ACPD	Agonist	Potent NMDA agonist. Also group II mGluR agonist
(S)-(-)-5-Fluorowillardiine	Agonist	Very potent AMPA agonist
(S)-4-Carboxyphenylglycine	Antagonist	Competitive group I mGluR antagonist/weak group II agonist
2-APB	Modulator	TRP channel modulator. Also IP3 receptor antagonist
2-Methylthioadenosine triphosphate tetrasodium salt	Agonist	P2 purinergic agonist
AM 404	Modulator	Vanilloid receptor agonist. Also anandamide transport inhibitor
BRL 52537 hydrochloride	Ligand	Potent and selective κ opioid receptor agonist
CNQX	Antagonist	Potent AMPA/kainate antagonist
Clocinnamox mesylate	Antagonist	Irreversible μ-opioid receptor antagonist
Endomorphin-1	Agonist	Potent and selective μ opioid receptor agonist
Endomorphin-2	Agonist	Potent and selective μ opioid receptor agonist
FIT	Agonist	Irreversible δ opioid receptor agonist
GBR 13069 dihydrochloride	Agonist	Potent dopamine uptake inhibitor
L-NIO dihydrochloride	Inhibitor	Potent eNOS inhibitor
L-Quisqualic acid	Agonist	Very potent group I mGluR agonist
N-Benzylnaltrindole hydrochloride	Agonist	Opioid receptor selective non-peptide antagonist
Nociceptin	Inhibitor	Endogenous NOP agonist
O-Phospho-L-serine	Antagonist	Group III mGluR agonist; enhances neuronal differentiation
Ro 51	Antagonist	Potent P2X3, P2X2/3 antagonist
cis-ACPD	Agonist	Potent NMDA agonist. Also group II mGluR agonist

We will be posting new additions and related data and publications

P2X3 Receptors and Migraine

P2X3 Receptors of Trigeminal Sensory Neurons and Familial Hemiplegic Migraine Type 1 (FHM-1).

Our P2X Receptor Markers continue to be used in interesting and novel ways. Here researchers use our P2X3 Receptor Antibody to study expression using primary rat ganlia cultures: Swathi K. Hullugundi,Michel D. Ferrari, Arn M. J. M. van den Maagdenberg, Andrea Nistri. Andrea Nistri. The Mechanism of Functional Up-Regulation of P2X3 Receptors of Trigeminal Sensory Neurons in a Genetic Mouse Model of Familial Hemiplegic Migraine Type 1 (FHM-1). PLoS ONE 8(4): e60677. doi:10.1371/journal.pone.0060677

Abstract: A knock-in (KI) mouse model of FHM-1 expressing the R192Q missense mutation of the Cacna1a gene coding for the α1 subunit of CaV2.1 channels shows, at the level of the trigeminal ganglion, selective functional up-regulation of ATP -gated P2X3 receptors of sensory neurons that convey nociceptive signals to the brainstem. Why P2X3 receptors are constitutively more responsive, however, remains unclear as their membrane expression and TRPV1 nociceptor activity are the same as in wildtype (WT) neurons. Using primary cultures of WT or KI trigeminal ganglia, we investigated whether soluble compounds that may contribute to initiating (or maintaining) migraine attacks, such as TNFα, CGRP, and BDNF, might be responsible for increasing P2X3 receptor responses. Exogenous application of TNFα potentiated P2X3 receptor-mediated currents of WT but not of KI neurons, most of which expressed both the P2X3 receptor and the TNFα receptor TNFR2. However, sustained TNFα neutralization failed to change WT or KI P2X3 receptor currents. This suggests that endogenous TNFα does not regulate P2X3 receptor responses. Nonetheless, on cultures made from both genotypes, exogenous TNFα enhanced TRPV1 receptor-mediated currents expressed by a few neurons, suggesting transient amplification of TRPV1 nociceptor responses. CGRP increased P2X3 receptor currents only in WT cultures, although prolonged CGRP receptor antagonism or BDNF neutralization reduced KI currents to WT levels. Our data suggest that, in KI trigeminal ganglion cultures, constitutive up-regulation of P2X3 receptors probably is already maximal and is apparently contributed by basal CGRP and BDNF levels, thereby rendering these neurons more responsive to extracellular ATP.

Images: Examples of TNFR2 and P2X3 co-exexpression in (wildtype) WT and R192Q (knockin) KI neurons. Left panel shows P2X3 expression (green), and right panel shows TNFR2 staining (red). B, Histograms quantifying % of cells co-expressing TNFR2 and P2X3: both WT and KI cultures show similar TNFR2 and P2X3 co-expression. N = 3 independent experiments (6 mice). C, Representative traces of currents induced by application of α,β-meATP (10 µM, 2 s) to WT or R192Q KI neurons in control conditions or after 4 h TNFα application. D, Histograms show average peak amplitudes of P2X3 receptor-mediated currents: WT control (open bar), n = 30; WT TNFα (stippled bar), n = 38; KI control (grey bar), n = 34; KI TNFα (stippled gray bar), n = 34; ** = p<0 .006="" i="" nbsp="" p="">doi:10.1371/journal.pone.0060677.g001.

Understanding the interplay between TNFR2 and P2X3 could lead to a better understanding of the root causes of migraines. This could open up yet more potential drug targets for this insidious condition.

Check out these related reagent categories:
All Purinergic Receptor Antibodies
Pain and Inflammation Research Antibodies 
Neurotransmission Research Antibodies
Primary Neurons and Astrocytes-Primary human, rat and mouse neurons and astrocytes

Science Behind Elecroacupuncture for Treating Shingles

Postherpetic neuralgia (PHN) or shingles, caused by caused by herpes zoster, causes nerve damage in the skin and results in abnormal electrical signals to the brain,  and may persist or recur for months, years or for life.

Electroacupuncture (EA) is effective in relieving pain in patients with PHN. Researchers in this study determined the beneficial effect of EA and the potential mechanisms in a rat model of PHN. They use our TRPV1 antibody to track expression in the Dorsal Root Ganglia (DRG) and Dorsal Horn (DH): Cai-hua Wu, Zheng-tao Lv, Yin Zhao, Yan Gao, Jia-qing Li, Fang Gao, Xian-fang Meng, Bo Tian, Jing Shi, Hui-lin Pan and Man Li. Electroacupuncture improves thermal and mechanical sensitivities in a rat model of postherpetic neuralgia. Molecular Pain 2013, 9:18 doi:10.1186/1744-8069-9-18


Conclusions: EA treatment improves thermal perception by recovering TRPV1-positive sensory neurons
and nerve terminals damaged by RTX. EA Also reduces RTX-induced tac tile allodynia by attenuating the damage of myelinated afferent nerves and their abnormal sprouting into the spinal lamina II. Our study provides new information about the mechanisms of the therapeutic actions of EA in the treatment of PHN.

Figure 2:  Effect of EA on RTX-induced deletion of TRPV1-immunoreactive neuron s in the DRG. A, Representative images showing TRPV1-immunoreactive neurons in the lumbar DRG of vehicle ( a ) , RTX ( b ), RTX plus 2 Hz EA ( c ), RTX plus 15 Hz EA ( d ), RTX plus 100 Hz EA ( e ), and RTX plus sham EA ( f ) groups. Scale bar, 50 μ m. B, Summary data show the number of TRPV1 immunoreactive neurons in different groups. Data are expresse d as means ± SEM (n = 6 rats in each group). *P < 0.05, compared with the vehicle group; # P< 0.05, compared with the sham EA group.

Figure 3: Effect of EA on RTX-induced deletion of TRPV1 immunoreactive central terminals in the spinal dorsal horn. A, Representative images showing TRPV1 immunoreactive central terminals of afferent fibers in the spinal dorsal horn of vehicle (a) ,RTX (b), RTX plus 2 Hz EA (c), RTX plus 15 Hz EA (d), RTX plus 100 Hz EA (e), and RTX plus sham EA (f) groups. Scale bar, 50 μm. B, Summary data show the area of TRPV1 immunoreactive central terminals in different groups. Data are expressed as means ± SEM (n= 6 rats in each group). *P < 0.05, compared with the vehicle group; # P < 0.05, compared with the sham EA group. 

I am always interested in how our Pain and Inflammation Research Markers are used to help understand the science behind pain therapies and also discovery of new therapies. There are multiple postings on these subjects with many more to come.

hN2 Human Neurons for Toxicity Screening

Our Human hN2 Neurons proving excellent platforms for Neurotoxicology Studies. I would like to share recent publication and that confirms the potential of these solutions for use in your toxicology assays.

Xiugong Gao, Hsiuling Lin, Radharaman Ray, Prabhati Ray. Toxicogenomic Studies of Human Neural Cells Following Exposure to Organophosphorus Chemical Warfare Nerve Agent VX. Neurochemical Research. February 2013.

Abstract: Organophosphorus (OP) compounds represent an important group of chemical warfare nerve agents that remains a significant and constant military and civilian threat. OP compounds are considered acting primarily via cholinergic pathways by binding irreversibly to acetylcholinesterase, an important regulator of the neurotransmitter acetylcholine. Many studies over the past years have suggested that other mechanisms of OP toxicity exist, which need to be unraveled by a comprehensive and systematic approach such as genome-wide gene expression analysis. Here we performed a microarray study in which cultured human neural cells were exposed to 0.1 or 10 μM of VX for 1 h. Global gene expression changes were analyzed 6, 24, and 72 h post exposure. Functional annotation and pathway analysis of the differentially expressed genes has revealed many genes, networks and canonical pathways that are related to nervous system development and function, or to neurodegenerative diseases such as Alzheimer’s disease, Huntington’s disease, and Parkinson’s disease. In particular, the neuregulin pathway impacted by VX exposure has important implications in many nervous system diseases including schizophrenia. These results provide useful information valuable in developing suitable antidotes for more effective prevention and treatment of, as well as in developing biomarkers for, VX-induced chronic neurotoxicity.

Images: hN2 Neurons at 18, hrs, 72 hrs and 6 Days.

Image: hN2 cells grown in culture for 4 days and stained with our chicken polyclonal to Neurofilament light or low molecular weight chain NF-L, a marker of neurons. Many of the differentiating cells show strong cytoplasmic and clearly fibrillar staining for NF-L. Blue stain is DAPI and reveals cell nuclei of some non neuronal cells in this culture

I will continue to post updates on new applications of these potent, pure and easy to grow human neurons.

hNP1 Neural Progenitors to Sensory Neurons

A Source for SN-Related Neural Circuits and for Designing Therapeutic Models for Related Diseases.

Researchers have successfully differentiated our hNP1^TM Neural Progenitors into Sensory Neurons. These cells can be passaged 10X+ prior to differentiation. This means basic and drug discovery researchers now have a source to generate large quantities of SNs: Xiufang Guo, Severo Spradling, Maria Stancescu, Stephen Lambert, James J. Hickman. Derivation of sensory neurons and neural crest stem cells from human neural progenitor hNP1. Biomaterials, In Press, Corrected Proof, Mar 2013.doi:10.1016/j.biomaterials.2013.02.061.

Abstract: Although sensory neurons constitute a critical component for the proper function of the nervous system, the in vitro differentiation of functional sensory neurons from human stem cells has not yet been reported. This study presents the differentiation of sensory neurons (SNs) from a human neural progenitor cell line, hNP1, and their functional maturation in a defined, in vitro culture system without murine cell feeder layers. The SNs were characterized by immunocytochemistry and their functional maturation was evaluated by electrophysiology. Neural crest (NC) precursors, as one of the cellular derivatives in the differentiation culture, were isolated, propagated, and tested for their ability to generate sensory neurons. The hSC-derived SNs, as well as the NC precursors provide valuable tools for developing in vitro functional systems that model sensory neuron-related neural circuits and for designing therapeutic models for related diseases.

Images: Phase contrast images of the cultures before and after the sensory neuron induction. A) hNP1 culture before sensory induction. B) hNP1 culture 10 days after sensory induction. C) hNP1 culture 30 days after sensory induction. Neuronal clusters and axonal bundles, which resemble rat DRG cell cultures, were typically observed. D) For comparison, an image of a rat embryonic DRG cell culture at 7 DIV is provided. doi.org/10.1016/j.biomaterials.2013.02.061

Images: Generation of Schwann cells from the differentiated culture. Immunostaining of a day 38 culture with the Schwann cell marker S100 demonstrating a significant number of Schwann cells in the culture. Schwann cells were located either within the neuronal clusters (A) or along the axonal bundles (B). The neuronal clusters and axonal bundles were marked by Peripherin immunostaining. doi.org/10.1016/j.biomaterials.2013.02.061

We are developing human cell based assays for High Content and Throughput Screening and will continue to post updates.

i-Fect™ Delivers Your 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.

MSCGro Media-locity

We can say our MSCGro^TM Media Mesenchymal stem Cell Media stacks up well vs our competition. The proof, though, is in the data and what our customers say. Here's a testimonial: ""Your MSC-Gro^TMmedia worked great. We carried out a simple pilot study where we plated an equal number of cells from a few CAF samples in both our traditional media and yours. We took a look at the cells on a daily basis, and by 3-4 days we saw a clear increase in cell numbers using your media." Obdulio Piloto, Ph.D., CSO of Conversantbio Inc.

And here is data:

Check out our MSCGro Media for yourself and if you re not delighted with the results, we will refund 100% of your purchase.

Increasing Returns in Drug Discovery

"Harnessing the Power of Cells"
 

Cell based assays presentation v1_03_2012 from Pete Shuster

This presentation provides an executive overview of how our Solutions can improve Drug Discovery processes.

Why Consider our Solutions?

Research proven solutions used by large Pharmas, Biotechs, Academic and Government Labs. 2500+ customers-Publications/Testimonials
Lower per well costs-we can aggressively discount cells and media
Customer success focused                                  
Expert technical support
Detailed protocols and methods
Quick replacements and reorders
Large expert resource network
Published expert input
Non confidential; customer feedback and data sharing through social media outlets and blogs

Osteoblasts Off

I am pleased to announce we now have unlabeled and FITC-labeled Osteoblasts. These are differentiated from our Umbilical Cord Blood derived Human Mesenchymal Stem Cells.

They are designed for:

• Osteogenesis/Bone Formation Studies
• Compound/Small Molecule Testing
• Gene Expression Analysis

Images: (A) Human cord-blood MSCs were expanded in low-serum MSC-Gro^TM to confluence as shown here. (B) were differentiated in osteogenic MSC-Gro^TM. Early stage osteoblasts are shown here; the arrow shows early formation of mineralized matrix. (C)&(D) Mature osteoblasts stained positive for Alizarin red. Phase contrast image at 200 x, scale bar is 50 mmeters.

I will continue to update you on new Cell Based Assay Solutions.

New Near Infrared Apoptosis Detection!

Detecting apoptosis in cells, tissue and living animals

In vitro and in vivo apoptosis detection assays are widely used in basic research and drug discovery. Neuromics has a wide range of kits providing capablilties to meet you unique requirements. We are pleased to offer even more options with our new Far Infrared Kits:
660 Polycaspase in vitro Apoptosis Detection Kit 25-50 Tests $199
660 Caspase-1  in vitro Apoptosis Detection Kit 25-50 Tests $199
NIR FLIVO™ 690 in vivo Apoptosis Tracer Kit 20 Tests-$399
NIR FLIVO™ 747 in vivo Apoptosis Tracer Kit 20 Tests-$399

Seeing is Believing

Images: To demonstrate the capabilities of the NIR-FLIVO® 747 apoptosis tracer, adult wild-type Balb/c mice were either inoculated with HSV-1 virus, which is known to induce apoptosis in the brain, or given a sham treatment. Seven days after viral inoculation, the mice were injected intravenously with either the NIR-FLIVO® 747apoptosis tracer (cat. KF17368), the NIR-FLIVO® 747 free dye (cat. KF17370, DyLight®747), or no reagent. Seven hours after reagent injection, the animals were imaged with a Carestream In-Vivo FX Pro imaging system. Strong caspase activity was located in the brain of the animal treated with HSV-1 and injected with NIR-FLIVO® 747 tracer (cat. KF17367). Minimal signal was detected in the liver region of the HSV-1-treated animal injected with the free dye control (HSV-Infected; NIR747FreeDye+) and of the uninfected mouse injected with NIR-FLIVO®747 tracer; the liver is the route of clearance for FLIVO® tracers. All reagent-injected mice show fluorescence signal in the tail where reagent is likely to pool after IV injection.

Images: Brain abscesses were induced in mice following the intracerebral inoculation of live S. aureus. Animals received intravenous injections of the pan-caspase tracer NIR-FLIVO™ 690 as an apoptosis probe or DyLight®690 (carboxylic acid form) as a control at 17 h post-infection, whereupon signals were acquired 1 h later from brain tissues immediately ex vivo using an IVIS Spectrum (Caliper Life Sciences). The same instrument settings were used to acquire both images. Strong caspase activity, detected with NIR-FLIVO™ 690 (arrows, image on right), was associated with brain abscesses, whereas minimal signal was detected in animals injected with the carboxylic acid control (image on left).

Check them all out
Apoptosis Research Reagents-Detection kits, antibodies and proteins

Everything You Wanted to Know About Stem Cells

Unforgettable way to learn about Stem Cells.

Rap on Dr. Jonathan Garlick Rap on.

If you are doing stem cell related research, check out our:

hNP1™ Human Neural Progenitor & hN2™ Neuron Discovery Kits Derived from H9 (WA09) ECSs-Consistent, Easy to Use & Cost Effective	Neural Stem Cells Media
hMPro™ Human Mesenchymal Progenitors (hMPCs) hESC Derived	Human Mesenchymal Stem Cells (hMSCs) hMSCs Derived from Umbilical Cord Blood
MSCGro™ Mesenchymal Stem Cell Media Proven and Potent Culture Growth and Differentiation Media for Mesenchymal Stem Cells (MSCs)	Cell Cryopreservation Media Maximizes cell recovery, attachment and growth
Expansion/Differentiation Kits SC to Oligodendrocytes and Dopaminergic Neurons Kits	Bioluminomics™ In Vitro Assays Brought to you by HemoGenix®
3-D Cell Based Assay Solutions Nanofibers, Hydrogels and Extracellular Matrix (ECM) Proteins	Stem Cell Research Antibodies
Stem Cell Research Proteins	Rat Neurosphere Neuroprogenitor Tissue Tissue provided live, unseparated, fresh from E18 rat cortex/hippocampus including subventricular zone.

Schwann Cells Markers

Important tool for studying diseases that cause PNS abnormalities or degeneration.

It is a goal of mine to have the best and brightest collection of Neuron-Glial Markers. Here I feature use of one of our Schwann Cell Markers. In this study our p75/NGF antibody is used as a control to determine differences in myelin sheath structure in new born pigs having the Cystic Fibrosis Mutant vs Normal Gene Expression (controls). Leah R. Reznikov, Qian Dong, Jeng-Haur Chena, Thomas O. Moninger, Jung Min Park, Yuzhou Zhang, Jianyang Du, Michael S. Hildebrand, Richard J. H. Smith, Christoph O. Randak, David A. Stoltz, and Michael J. Welsh. CFTR-deficient pigs display peripheral nervous system defects at birth. www.pnas.org/cgi/doi/10.1073/pnas.1222729110.

Images: CFTR is functionally active in Schwann cells. (A) Primary cultures of porcine Schwann cells were used 4 wk after seeding when they had developed the specific bipolar morphology and a phase-bright cell body under differential interference contrast microscopy. Schwann cells were positive for the phenotypic markers S100 and p75. (B) Whole-cell current recorded in the presence of PKA and ATP in the pipette solution and 1 min after adding 100 μM of CFTR inhibitor GlyH-101 to the bath solution. (Left) Example of currents from one cell; Inset shows voltage-pulse protocol. (Upper Right) Example of current-voltage relationship. (Lower Right) Data from five CFTR+/+ Schwann cells and seven CFTR−/− Schwann cells. *P = 0.003 (Mann–Whitney rank sum test).

Images: Fig. 1. CFTR is expressed in trigeminal nerve Schwann cells. Data are confocal microscopic images of trigeminal nerve immunostained for CFTR (green) and marker indicated above middle panels (red). Nuclei were stained with DAPI (blue). (A) Transverse cross-section with axons stained with β-tubulin III antibody. (B) CFTR−/− trigeminal nerves immunostained for CFTR and β-tubulin III.  (C) Sagittal crosssection immunostained with fluoromyelin. (D) Section stained with S100 antibody, a marker of Schwann cells. (E) Section stained with p75 antibody, also a marker of Schwann cells. (Scale bar, 20 μm.).

I will continue to post new customer pubs and other shared customer data featuring use of our Neuron-Glial Markers.

Epidermal Nerve Fibers in Neuropathic Pain Model

Re-innervation in spared nerve injury (SNI) vs normal rats
Neuromics' Pain and Inflammation Research Antibodies are frequently used to help researchers find root causes of neuropathic pain. This is a fascinating study of re-innervation patterns of epidermal and dermal nerve fibers in a rat neuropathic pain model (Note: our Guinea Pig Polyclonal P2X3 Antibody was used in this study): Liron S. Durakua, Mehdi Hossaini, Barthold N. Schüttenhelm, b, Joan C. Holstege, Martijn Baas, Tom J.H. Ruigrok, Erik T. Walbeehm. Re-innervation patterns by peptidergic Substance-P, non-peptidergic P2X3, and myelinated NF-200 nerve fibers in epidermis and dermis of rats with neuropathic pain. Experimental Neurology Volume 241, March 2013, Pages 13–24. doi.org/10.1016/j.expneurol.2012.11.029.

Non-footpad area vs. footpad.The distribution pattern between the center non-footpad area and footpad is compared for the different subgroups of sensory skin fibers. For epidermal Sub P-IR and upper dermal NF-200-IR fibers there is an equal density of fibers in the center and footpad area suggesting a homogenous distribution over the foot sole of a rat. Whereas for epidermal P2X3-IR fibers there is a significant lower number of P2X3-IR fibers in the footpads as compared to the center non-footpad area, suggesting a heterogeneous distribution in the foot sole for this type of sensory fibers. N = 5 per group. Unpaired t-test. ***: p < 0,001. Scale bar: 250 μm. doi.org/10.1016/j.expneurol.2012.11.029

Illustration of the skin innervation in normal and SNI situation.Illustration showing the innervation pattern of subgroups of sensory skin fibers in the normal situation and in the SNI model 10 weeks PO. In the naïve animals the peptidergic CGRP and non-peptidergic P2X3 fibers have a lower density in the footpads as compared to the non-footpad areas, while the Substance P and NF-200 fibers have an equal distribution over the complete foot sole. In the SNI model there is a significant increase of CGRP epidermal fibers in the medial and lateral area of the foot sole and there is a complete re-innervation of the center area. In addition the footpads in the SNI model are hyper-innervated with CGRP fibers. Substance P and P2X3 and NF-200 fibers show no increased density in the uninjured medial and lateral area after 10 weeks PO. In addition, Substance P and P2X3 fibers hardly re-innervate the center area; however, the NF-200 fibers have the same density of fibers in the denervated area as in the normal situation. In the SNI model the medial, center and lateral area have all an increase in LC's, with the center area being the most prominent one. In addition the epidermis thickness of the SNI model has decreased significantly in all the three areas after 10 weeks PO.
Related Postings: http://neuromics.blogspot.com/search/label/Neuropathic%20Pain.

QCing our hMSC Derived Chondrocytes

We routinely internally test our Human Mesenchymal Stem Cells (hMSCs) and terminally differentiated cells. I would like to share the latest on our hMCS Derived Human Chondrocytes.

Msc derived human chondrocytes in culture-01-2013 from Pete Shuster
We will soon be adding QC and related images for our Osteoblasts and Endothelial Cells.

Stem Cells, Duchenne Muscular Dystrophy (DCM) & Cardiomyopathies

Transplanting Aorta-derived mesoangioblasts (ADMs) to Prevent ADM Related Cardiomyopathy-ADMs can be induced to express cardiac markers, including Nkx2.5, cardiac tropomyosin, cardiac troponin I, and -actinin, and adopt cardiomyocyte morphology. Transplantation of ADMs into the heart of mdx/utrn−/− mice prior to development of DCM prevented onset of cardiomyopathy, as measured by echocardiography, and resulted in significantly higher CD31 expression, consistent with new vessel formation. Dystrophin-positive cardiomyocytes and increased proliferation of endogenous Nestin cardiac stem cells (Neuromics' Chicken Polyclonal Nestin antibody was used as a marker to determine presence of these cells) were detected in ADM-injected heart: JU LAN CHUN, ROBERT O’BRIEN, MIN HO SONG, BLAKE F.WONDRASCH, SUZANNE E. BERRY.Injection of Vessel-Derived Stem Cells Prevents Dilated Cardiomyopathy and Promotes Angiogenesis and Endogenous Cardiac Stem Cell Proliferation in mdx/utrn−/− but Not Aged mdx Mouse Models for Duchenne Muscular Dystrophy. STEM CELLS TRANSLATIONAL MEDICINE 2013;2:000–000. http://dx.doi.org/10.5966/sctm.2012-0107.

Figure 6. Nestin and cardiac myocytes in ADM-transplanted mdx/utrn / heart. Fluorescent microscopy was used to visualize nestin interstitial and striated cells in the heart. (A): There were significantly fewer nestin interstitial stem cells in ADM-injected (dko/ADMs, n 5) mdx/utrn / heart in comparison with age-matched wild-type heart injected with saline (WT/HBSS, n 4). p values were obtained using Student’s t test. No difference was observed between WT/HBSS versus dKO/HBSS (p .0538) or dKO/HBSS versus dKO/ADMs (p .3843). (B–F): Nestin striated cells (green, indicated by white arrows) were observed in four of five mdx/utrn / hearts transplanted with ADMs. (C): A cluster of nestin striated cells (green, indicated by white arrows) and surrounding tissue containing nestin interstitial stem cells (also green, indicated by yellow arrowheads). (D–F): The same cluster of nestin cells (green) shown in (C), at higher magnification, expressed cardiac troponin I (red [E, F]). (G): Some nestin striated cells were also observed in one of four mdx/utrn / hearts injected with saline. (H): Nestin striated cells were not present in saline-injected wild-type heart. Abbreviations: ADM, aorta-derived mesoangioblast; cTpI, cardiac troponin I; DAPI, 4 ,6-diamidino-2-phenylindole; dKO, double knockout mdx/utrn / ; HBSS, Hanks’ balanced saline solution; WT, wild-type.

Conclusion: ADMs delay or prevent development of DCM in dystrophin-deficient heart, but timing of stem cell transplantation may be critical for achieving benefit with cell therapy in DMDcardiac muscle.
Stem Cell Research Reagents.

Shiny, New GF nanoparticle labeled hMSCs

Human Mesenchymal Stem Cells-Green Fluorescent Nanoparticle-labeled-These cells are generated by chemical transfection of CdSe/ZnS nanoparticles, 100 nm in diameter and show a strong fluorescent signal (Excitation maximum= 515 nm; Emission maximum= 520-550 nm) detectable by standard FITC filters.

Image: FITC analysis of cells during fourth passage following transfection. Image obtaining using FITC filter on Olympus CKX41 microscope at 200 X equipped with a Retiga 2000 digital camera and Q-Capture software program. Scale bar is 25 micrometers. Fluorescent nanoparticles are less prevalent within the cytoplasm than earlier passages. Technical Information & Data.

They retain GFNP labeling through 3+ passages and can be differentiation into chondrogenic, adipogenic or osteogenic lineages. Excellent for regeneration, toxicity and related studies. Technical Information & Data.

Image: Osteogenic differentiation of GF nanoparticle-labeled MSCs. Image obtaining using phase contrast Olympus CKX41 microscope at 100 X equipped with a Retiga 2000 digital camera and Q-Capture Pro software program. Scale bar is 25 micrometers. Note the presence of mineral deposits indicating differentiation into osteoblasts. Chondrogenic and adipogenic differentiation was also apparent.

• All Stem Cell Research Reagents.

Pain and the Interplay Between P2Y Receptors with P2X3

P2Y2-P2X3 crosstalk in DRG neurons

Alfredo Ribeiro-da-Silva and his team use our Purinergic Receptor Antibodies to the study role of their expression in Nociceptive and Neuropathic Pain. They referenced their use in 8 publications.

Based on their research, they suspect changes in P2X3 function under pathological conditions are more complex than simple up- or down-regulation of expression at the protein level. This would have profound implications for the dicovery of drugs that target P2X3 expression levels: Gary Mo, Jennifer C. Peleshok, Chang-Qing Cao, Alfredo Ribeiro-da-Silva and Philippe Séguéla. Control of P2X3 channel function by metabotropic P2Y2 UTP receptors in primary sensory neurons. Molecular Pharmacology Fast Forward. Published on December 18, 2012 as doi:10.1124/mol.112.082099.

Images: P2X3 and P2Y2 receptors are co-expressed in rat DRG sensory neurons.(A) DRG sections labeled for P2Y2 (green) and P2X3 (red) and merged (yellow). P2Y2 immunoreactivity is broadly distributed in small-diameter neuronal somata and co-localized with P2X3 (arrows). P2Y2 immunoreactivity can be detected in cells negative for P2X3 (arrow heads). (B) P2Y2 (green) and P2X3 (red) are also colocalized (yellow) in peripheral nerve fibers.

The two receptors were found to be colocalized both in cell bodies and nerve fibers, indicating co-trafficking to peripheral and central terminals. The immunolocalization evidence presented here provides valuable information on the physiological relevance of crosstalks between ionotropic and metabotropic ATP receptors in sensory pathways, yet much work is still needed to fully comprehend the role of nucleotide signaling in pain, especially in pathological conditions. Changes in P2Y2 receptor expression in response to inflammation have been documented (Malin et al., 2008), therefore it will be worth investigating the functional impact of P2Y2-P2X3 interactions on ATP signaling under chronic pain conditions.

I will keep you posted on any new developments.