Focus on Vision Systems Research

New Reagents, Publications and Data

We are intensifying our focus on Vision System Research with the addition of new antibodies. We are offering 50 USD off. They include: PSD-95/SAP90, Rhodopsin (A531), Rhodopsin (B630) and select Calcium (Ca2+) Signaling-Binding antibodies.

Image: Rhodopsin staining of pig retinal sections (green) and counter-stained with NF-M (red) and DNA (blue). Rhodopsin is most abundant in the outer segments of retina (OS), NF-M is abundant in the optic nerve fiber layer (ONFL), but seen in processes and neurons in other regions also. Other layers are pigmented epithelium (PE), outer and inner nuclear layers (ONL, INL), outer and inner plexiform layers (OPL, IPL) and ganglion cell layer (GCL). Inset: Bovine retinal extracts blotted with rhodopsin. Protocols on data-sheet.
Our ability to effectively serve researchers is confirmed by the growing parade of publications. I would like to highlight a recent publication by Dr. Sal Salvatore and his colleagues. It features use of our Shank 1a antibody. They are the first to show Shank 1 expression in the mammalian retina revealing Shank 1 immunoreactivity within both synaptic layers of the retina: Salvatore L. Stella Jr, Alejandro Vila, Albert Y. Hung, Michael E. Rome, Uyenchi Huynh, Morgan Sheng, Hans-Juergen Kreienkamp, Nicholas C. Brecha. Association of Shank 1A Scaffolding Protein with Cone Photoreceptor Terminals in the Mammalian Retina. PLoS ONE 7(9): e43463. doi:10.1371/journal.pone.0043463.

Images: Shank 1A immunoreactivity is in both the inner plexiform layer (IPL) and outer plexiform layer (OPL) of the mouse YFP-16 line retina. A–C: A. Image of a retinal section immunostained for Shank 1A. B. Mouse YFP-16 line vertical retinal section. C. Shank 1A (red) immunolabeling and YFP (yellow). Shank1A expression is restricted to the OPL and IPL. A regular pattern of Shank 1A immunolabeling appears in the OPL, which is indicative of cone photoreceptor terminals. D–E: High magnification zoom of the OPL demonstrates that Shank 1A puncta (red) are distal to the dendrite tips (yellow) of YFP labeled cone bipolar cells, suggesting that Shank 1A is expressed presynaptic to the YFP cone bipolar cell dendrite. G–L: High magnification zoom of the IPL demonstrates that Shank 1A puncta are likely expressed postsynaptically to bipolar cell terminals. G. Shank 1A immunoreactive puncta. H. YFP labeled neurons and processes within the IPL region. I. PKCα labeled rod bipolar cell axons and terminals. J. Combined Shank 1A (red) and PKCα (blue) immunolabeling illustrate that shank 1A puncta are postsynaptic to rod bipolar cell terminals in the IPL. K. Combined Shank 1A (red) immunolabeling and YFP (yellow) in the IPL demonstrate that Shank 1A puncta are postsynaptic to cone bipolar cell terminals in the IPL. L. Combined triple fluorescent image of Shank 1A (red), PKCα (blue), and YFP (yellow) in the IPL. OPL = outer plexiform layer, INL = inner nuclear layer, IPL = inner plexiform layer, and GCL = ganglion cell layer. Scale bars = 10 µm. doi:10.1371/journal.pone.0043463.g001

More Pubs
Diego C. Fernandez, Laura A. Pasquini, Damián Dorfman, Hernán J. Aldana Marcos, Ruth E. Rosenstein. Early Distal Axonopathy of the Visual Pathway in Experimental Diabetes. doi:10.1016/j.ajpath.2011.09.018

...a goat polyclonal anti–platelet-derived growth factor receptor-α (PDGFR-α) antibody (1:100; Neuromics, Edina, MN)
Raoul Torero Ibad, Jinguen Rheey, Sarah Mrejen, Valérie Forster, Serge Picaud, Alain Prochiantz, and Kenneth L. Moya. Otx2 Promotes the Survival of Damaged Adult Retinal Ganglion Cells and Protects against Excitotoxic Loss of Visual Acuity In Vivo. The Journal of Neuroscience, 6 April 2011, 31(14): 5495-5503; doi: 10.1523/​JNEUROSCI.0187-11.2011...For antibody neutralization experiments, anti-Otx2 antibody (Neuromics) was dialyzed against PBS, and then Otx2 (25 ng in 500 mul) was incubated with anti-Otx2 (0.5 mug) in culture medium at 37C...

Hoon Shim, Chih-Ting Wang, Yen-Lin Chen, Viet Q. Chau, Kevin G. Fu, Jianqi Yang, A. Rory McQuiston, Rory A. Fisher, and Ching-Kang Chen. Defective Retinal Depolarizing Bipolar Cells (DBCs) in Regulators of G-protein Signaling (RGS) 7 and 11 Double Null Mice. JBC Papers in Press. Published on February 27, 2012 as Manuscript M112.345751. The latest version is at http://www.jbc.org/cgi/doi/10.1074/jbc.M112.345751...Animals were sacrificed by CO2 inhalation and the eyeballs were immediately enucleated. After removal of cornea and lens the resulting eyecups were immersionfixed in 4% paraformaldehyde in 1X PBS at room temperature for 15 minutes. This short fixation time ensured good mGluR6 and Gb5 signals at the OPL. After cryoprotection in 30% sucrose in 1X PBS, the eyecups were embedded in TBS (Richard Allan Scientific, Kalamazoo, MI), sectioned at 20μm thickness, and stained...

I will keep you posted on progress.

ASIC3 and Osteoarthritis

ASIC3 modulates pain and disease progression

Neuromics' foundation is built on providing reagents for pain researchers. I have posted the twists and turns via key publications and related data. Here's yet another success story with one of our Pain and Inflammation Research Antibodies.

Acid sensing ion channels (ASICs) are sodium-selective ion channels activated by low extracellular pH, and belong to the degenerin/epithelial Na+ channel superfamily. ASIC3  is the most sensitive to such a pH change [2,3], abundantly expressed in dorsal root ganglia (DRG) [4], and strongly correlated with pain. Here researchers show the role of ASIC3 in osteoarthritis: Masashi Izumi, Masahiko Ikeuchi, Qinghui Ji, Toshikazu Tani. Local ASIC3 modulates pain and disease progression in a rat model of osteoarthritis. Journal of Biomedical Science 2012, 19:77 doi:10.1186/1423-0127-19-77.
Highlights: OA was induced via intra-articular mono-iodoacetate (MIA) injection, and pain related behaviors were evaluated including weight bearing measured with an incapacitance tester and paw withdrawal threshold in a von Frey hair test. OA rats showed not only weight-bearing pain but also mechanical hyperalgesia outside the knee joint (secondary hyperalgesia). ASIC3 expression in knee joint afferents was significantly upregulated approximately twofold at Day 14. Continuous intra-articular injections of APETx2 inhibited weight distribution asymmetry and secondary hyperalgesia by attenuating ASIC3 upregulation in knee joint afferents. Histology of ipsilateral knee joint showed APETx2 worked chondroprotectively if administered in the early, but not late phase.

Images: Fast Blue labeling and immunohistochemistry staining for ASIC3 : (a-b) Naïve- model, (c-d) OA-model, (e-f) APETx2 administration to OA-model in early phase. Photos in each row are the same DRG. In (b),(d),(f), large arrows indicate Fast Blue labeled, ASIC3 immunoreactive (ASIC3-ir) DRG cells, while ASIC3-ir cells that were not labeled by Fast Blue are indicated by small arrowheads. More than 100 FB-labeled neurons were analyzed from 4 rats in each group. The percentage of ASIC3-ir knee joint afferents was 18 ± 3% (mean ± SD) in naïve models, 46 ± 4% in OA-models (p = 0.003), and 20 ± 5% in the early-phase APETx2 group (p = 0.006), respectively. Scale bar: 50 μm

Protocol: The [DRG] sections were blocked in 3% normal goat serum for 1 h, then incubated in primary antibody of ASIC3 (Neuromics; Edina, MN, GP 14015, 1:500) overnight in a humid chamber. The next day, the sections were incubated in the secondary antibody (Vector; Burlingame, CA, FI-7000, 1:500, FITC tagged) for 2 h. All antisera used were diluted in PBS containing 1% normal goat serum and 0.05% Triton X-100. Before, between, and after each incubation step, the sections were washed 3 times for 5 min in PBS. Finally, all sections were mounted with Vectashield (Vector, Burlingame, CA).
1. Waldmann R, Champigny G, Bassilana F, Heurteaux C, Lazdunski M: A proton-gated cation channel involved in acid-sensing. Nature 1997, 386:173–177. 2. Lingueglia E: Acid-sensing ion channels in sensory perception. J Biol Chem 2007, 282:17325–17329. note: see http://neuromics.net/weblog/post/tag/dr-eric-lingueglia/ for research using our siRNA transfectio reagent for ASIC3 gene expression analysis. 
3. Wemmie JA, Price MP, Welsh MJ: Acid-sensing ion channels: advances, questions and therapeutic opportunities. Trends Neurosci 2006, 29:578–586.
4. Voilley N, de Weille J, Mamet J, Lazdunski M: Nonsteroid anti-inflammatory drugs inhibit both the activity and the inflammation-induced expression of acid-sensing ion channels in nociceptors. J Neurosci 2001, 21:8026–8033.

hMSCs+Petaka=Excellent SC Cultures

I have been promoting the capabilities of our new Petaka^TM Cell Culturing Systems. My confidence that you will be delighted with these capabilities is based, in part, on the success Dr. Jim Musick, CEO, Vitro Biopharma had culturing our UCB derived Human Mesenchymal Stem Cells.

These cells work "hand in glove" with Petaka. The system enabled Jim to:

• Culture the cells with out the addition of CO₂ or humidity (no environmental dehydration)-no incubator required.
• Use less growth media
• Drive the cells into dormancy enabling them to be maintaned, shipped and stored at Room Temperature.

Images: hMSCs in Petaka and cell dormancy and recovery.

Additional Resources:

Petaka Presentations-Petaka features and capabilities. Includes: culturing stem cells, other primary cells and cell lines; customizing phsiological conditions to match cells' in vivo environment and cell culture pausing for room temperature handling and shipping.

Petaka Cell Culturing Video Protocols
Petaka Cell Harvesting Video Protocols
Petaka Cell Maintenance, Shipping and Recovery Video Protocols

Petaka means better cultures at a lower cost. I will continue to post new data confirming this here.

TRPV1, SCI and Autonomic Dysreflexia

Acute autonomic dysreflexia (AD) is a reaction of the autonomic (involuntary) nervous system to overstimulation. It is characterised by severe paroxysmal hypertension (episodic high blood pressure) associated with throbbing headaches, profuse sweating, nasal stuffiness, flushing of the skin above the level of the lesion, bradycardia, apprehension and anxiety, which is sometimes accompanied by cognitive impairment.  

My friend Matt Ramer and his team at University of British Columbia use our antibodies as markers for sensory neurons. In this important study they use our TRPV1 (Neuromics, Edina, MN, USA; 1:2,000)...Substance P (Neuromics, 1:1,000) and   β-III-tubulin (Neuromics; 1:500) to label neuronal profiles in the DRG: Leanne M. Ramer, A. Peter van Stolk, Jessica A. Inskip, Matt S. Ramer and Andrei V. Krassioukov. Plasticity of TRPV1-expressing sensory neurons mediating autonomic dysreflexia following spinal cord injury. DOI=10.3389/fphys.2012.00257.
Highlights: Spinal cord injury (SCI) triggers profound changes in visceral and somatic targets of sensory neurons below the level of injury. Despite this, little is known about the influence of injury to the spinal cord on sensory ganglia. One of the defining characteristics of sensory neurons is the size of their cell body: for example, nociceptors are smaller in size than mechanoreceptors or proprioceptors. In these experiments, we first used a comprehensive immunohistochemical approach to characterize the size distribution of sensory neurons after high- and low-thoracic SCI. Male Wistar rats (300 g) received a spinal cord transection (T3 or T10) or sham-injury. At 30 days post-injury, dorsal root ganglia (DRGs) and spinal cords were harvested and analyzed immunohistochemically. In a wide survey of primary afferents, only those expressing the capsaicin receptor (TRPV1) exhibited somal hypertrophy after T3 SCI. Hypertrophy only occurred caudal to SCI and was pronounced in ganglia far distal to SCI (i.e., in L4-S1 DRGs). Injury-induced hypertrophy was accompanied by a small expansion of central territory in the lumbar spinal dorsal horn and by evidence of TRPV1 upregulation. Importantly, hypertrophy of TRPV1-positive neurons was modest after T10 SCI. Given the specific effects of T3 SCI on TRPV1-positive afferents, we hypothesized that these afferents contribute to autonomic dysreflexia (AD). Rats with T3 SCI received vehicle or capsaicin via intrathecal injection at 2 or 28 days post-SCI; at 30 days, AD was assessed by recording intra-arterial blood pressure during colo-rectal distension (CRD). In both groups of capsaicin-treated animals, the severity of AD was dramatically reduced.

Images: High-thoracic (T3) spinal cord injury had no effect on medium-to-large sized neurons in the L4/L5 DRG expressing heavy neurofilament (NF200). (A) NF200-positive neurons did not undergo SCI-induced hypertrophy, nor did the proportion of neurons expressing NF200 change. (B) Hypertrophy of TRPV1-expressing DRG neurons was not accompanied by increased co-localization of TRPV1 and NF200. Ganglia were harvested 3 months after sham-injury (gray) or complete T3 SCI (black). Arrow: DRG neuron immunopositive for both TRPV1 and NF200. Scale bar = 70 μm.

Conclusion: Previous work has identified numerous mechanisms that might contribute to induction and progression of AD, and the list of putative mechanisms includes injury-induced changes in the vasculature and multiple components of the spinal sensory-sympathetic circuitry caudal to SCI (Krenz and Weaver, 1998; Krassioukov et al., 1999; Krenz et al., 1999; Brock et al., 2006; McLachlan and Brock, 2006). In terms of sensory plasticity, prior findings demonstrate that severity of AD is closely correlated to the extent of intraspinal nociceptor sprouting (Cameron et al., 2006). However, this is the first study to demonstrate AD mediated by a specific subset of afferents that exhibit pronounced somatic, but only slight central, injury-induced plasticity. Given the array of pronounced changes in peripheral targets of sensory neurons after SCI, it is not surprising that they respond to injury. Plasticity occurring outside the CNS may represent a new and more accessible target for limiting sensory-autonomic dysfunction following SCI.

I will keep you posted as more progress is made towards finding therapeutic targets AD.

Nucleostemin and Liver Regeneration

Our Nucleostemin Stem Cell/Cell Regeneration Marker is widely used and published. I have multiple posting highlighting results of the marker in action. This includes use for staining Tendon Progenitors/Stem Cells (TSCs).

Here researchers use the antibody as a marker for injury induced liver regeneration: Haruhiko Shugo, Takako Ooshio, Masako Naito, Kazuhito Naka, Takayuki Hoshii, Yuko Tadokoro, Teruyuki Muraguchi, Akira Tamase, Noriyuki Uema, Taro Yamashita, Yasunari Nakamoto, Toshio Suda, Shuichi Kaneko, and Atsushi Hirao.Nucleostemin in Injury-Induced Liver Regeneration. Stem Cells and Development. -Not available-, ahead of print. doi:10.1089/scd.2011.0725...Membranes were then incubated with a goat anti-NS antibody (1:1,000; Neuromics, Edina, MN)...

Abstract: The high regenerative capacity of liver contributes to the maintenance of its size and function when injury occurs. Partial hepatectomy induces division of mature hepatocytes to maintain liver function, whereas severe injury stimulates expansion of undifferentiated hepatic precursor cells, which supply mature cells. Although several factors reportedly function in liver regeneration, the precise mechanisms underlying regeneration remain unclear. In this study, we analyzed expression of nucleostemin (NS) during development and in injured liver by using transgenic green fluorescent protein reporter (NS-GFP Tg) mice. In neonatal liver, the hepatic precursor cells that give rise to mature hepatocytes were enriched in a cell population expressing high levels of NS. In adult liver, NS was abundantly expressed in mature hepatocytes and rapidly upregulated by partial hepatectomy. Severe liver injury promoted by a diet containing 3,5-diethoxycarbonyl-1,4-dihydrocollidine induced the emergence of NS-expressing ductal epithelial cells as hepatic precursor cells. NS knockdown inhibited both hepatic colony formation in vitro and proliferation of hepatocytes in vivo. These data strongly suggest that NS plays a critical role in regeneration of both hepatic precursor cells and hepatocytes in response to liver injury.

Check out Neuromics' Stem Cell Research Reagents.

Vagal Nerve Stimulation and Mechanisms for Treating Depression

How does VNS work?

Vagal Nerve Stimulation (VNS) has proven effective in treating severe, intractable depression. It is a procedure that sends electrical impulses into your brain in an effort to improve depression symptoms. Side effects with VNS Therapy include temporary hoarseness or a slight change in voice tone, increased coughing, shortness of breath upon physical exertion, and a tickling in the throat (Rush AJ, Sackeim HA, Marangell LB, et al.).

I wanted to post highlights of a recent study that for the first time shows that VNS increases brain-derived neurotrophic factor (BDNF) in brain or activate, via phosphorylation, its receptor, TrkB: Havan Furmaga, Flavia Regina Carreno, and Alan Frazer. Vagal Nerve Stimulation Rapidly Activates Brain-Derived Neurotrophic Factor Receptor TrkB in Rat Brain. PLoS One. 2012; 7(5): e34844. Published online 2012 May 1. doi: 10.1371/journal.pone.0034844.

Images: Vagal nerve stimulation (VNS) rapidly induces TrkB activation in rat hippocampus. Effects of acute (2 hr) VNS, desipramine (DMI, 10 mg/kg, i.p) or fluoxetine (15 mg/kg, i.p) on TrkB phosphorylation at different tyrosine residues in rat hippocampus.Phospho-TrkB values are normalized against total-TrkB values. One-way MANOVA, Student's Newman-Keuls post-hoc test, *P<0 .05=".05" br="br" group.="group." n="4−8" per="per">

It is this phosphorylation of TrkB that could make VNS a distinct and effective treatment. This VNS induced mechanism is distinct from the effect of standard antidepressant drugs.

Further understanding could result in therapies that are less intrusive, less costly and more effective for treating severe depressive disorders. I will continue to post new findings here.

Mu Opioid Receptor, Aging and Immune Response

Mu Opioid Receptors (MOR) play an integral role in modulating perception of pain. Our Opioid Receptor and Opioid Neuropeptide Antibodies have been widely used and extensively published by Pain Researchers.

Here researchers determine a pivotal role of synaptic IGF-1R/Fyn signaling controlled by MOR downstream signaling cascades were crucial for the age-dependent neuroimmune modulation following traumatic stress: Hui Zhao, Xiaocong Zhao, Xiaoding Cao and Gencheng Wu. Age-Dependent Neuroimmune Modulation of IGF-1R in the Traumatic Mice. Immunity & Ageing 2012, 9:12 doi:10.1186/1742-4933-9-12.

Highlights: Fyn activity, as well as its molecular connection with IGF-1R was dependent on MOR, on account of that their coupling was obviously not able to be observed when lack of MOR, accordingly, there was not improvement from the immuno-suppression mediated by traumatic stress in MOR (−/−) mice. Likewise, there was not remarkable change in MOR expression, as well as the association of MOR with IGF-1R or Fyn in the synaptic zone. Then, it is plausible that a characteristic feature of IGF-1R was probably due to age-dependent Fyn activation that was triggered by MOR signaling cascades, this specialized process was mainly concentrated within synaptic zone and might contribute to the recovery from traumatic stress mediated immuno-suppression.

Images: MOR expression during traumatic stress. 2-month and 1-year mice were killed 1 and 3 days after traumatic stress (n = 5 for each group), synaptoneurosome from frontal cortex was prepared, Western blot analysis was used to detect MOR expression (A). Immunoprecipitation was used to analyze alterations of MOR and IGF-1R/Fyn interaction. The immunoprecipitation antibody was anti-Fyn (B) or anti-IGF-1R (D) and the immunoblotting antibody was anti-MOR. Panel C and E depict quantitative analysis of B and D respectively. Data are presented as percentage of control, values represent mean ± SD for 3 independent experiments. Con: control; T3: 3 days after trauma.

I will continue to post new applications for our MOR antibodies.

Neuron-Glial Antibodies Hit the Mark!

Neuromics' Neuron-Glial Markers offerings include:

Glial-Astrocyte Markers	Neural Progenitor Markers
Neuron/Synapse Markers	Oligodendrocyte, Oligodendroglial Oligodendrocyte Lineage Markers
Schwann Cell or PNS Neuronal Markers -Peripheral Nervous System (PNS) Related

They are widely used and frequently published. Customers have also shared data which is included in product descriptions. Here is a listing of the most recent pubs:
Wiebke Kallenborn-Gerhardt, Katrin Schröder, Domenico Del Turco, Ruirui Lu, Katharina Kynast, Judith Kosowski, Ellen Niederberger, Ajay M. Shah, Ralf P. Brandes, Gerd Geisslinger, and Achim Schmidtko. NADPH Oxidase-4 Maintains Neuropathic Pain after Peripheral Nerve Injury. The Journal of Neuroscience, 25 July 2012, 32(30): 10136-10145; doi: 10.1523/​JNEUROSCI.6227-11.2012.

Images: MPZ and PMP22 expression in the sciatic nerve of WT and Nox4−/− mice after SNI. A, Western blot analysis of the myelin-specific proteins MPZ and PMP22 in the day 14 SNI sciatic nerve (proximal nerve stump) and the uninjured control sciatic nerve. GAPDH was used as loading control. Note that MPZ and PMP22 protein expression is significantly decreased after SNI in WT mice but not in Nox4−/− mice. n = 3 mice per group. Data are presented as mean ± SEM (*p < 0.05). B, Immunostaining of the day 14 SNI sciatic nerve shows increased MPZ immunoreactivity in Nox4−/− mice compared with WT mice, whereas immunoreactivity of the neuronal marker NF200 is similar in both genotypes. Scale bar, 10 μm.

Maria Maddalena Valente, Valeria Bortolotto, Bruna Cuccurazzu, Federica Ubezio, Vasco Meneghini, Maria Teresa Francese, Pier Luigi Canonico, Mariagrazia Grilli.Alpha2delta ligands act as positive modulators of adult hippocampal neurogenesis andprevent depressive-like behavior induced by chronic restraint stress. Molecular Pharmacology Fast Forward Published on May 9, 2012 as doi:10.1124/mol.112.077636...chicken anti-nestin polyclonal (1:4,000, Neuromics, Edina, Minnesota)...

Frances Y. Cheng, Xi Huang, Anuraag Sarangi, Tatiana Ketova, Michael K. Cooper, Ying Litingtung, Chin Chiang. Widespread Contribution of Gdf7 Lineage to Cerebellar Cell Types and Implications for Hedgehog-Driven Medulloblastoma Formation. PLoS ONE 7(4): e35541. doi:10.1371/journal.pone.0035541...rabbit anti-GFAP (Neuromics, 1:500), mouse anti-GFAP (Neuromics, 1:200)...

Juan M Jimenez-Andrade and Patrick W Mantyh. Sensory and sympathetic nerve fibers undergo sprouting and neuroma formation in the painful arthritic joint of geriatric mice. Arthritis Research & Therapy 2012, 14:R101...to label primary afferent sensory nerve fibers, an antibody against neurofilament 200 kDa (NF200, chicken anti neurofilament 200 kDa; NF200, 1:5000; Neuromics; catalog #CH22104)...

I will be posting new developments.

Converting Human Pluripotent Stem Cells into Nociceptors

Methods for differentiating induced pluripotent stem cells (iPSCs) into specific cell types are a requirement for converting the "promise of iPSCs" into reality. The knowledge derived from this research can be leveraged for high throughput  Drug Discovery and ultimately the development of therapies. I am excited to highlight results recently published by Dr. Lorenz Studer and his team at Memorial Sloan-Ketterling: Stuart M Chambers, Yuchen Qi, Yvonne Mica, Gabsang Lee, Xin-Jun Zhang, Lei Niu, James Bilsland, Lishuang Cao, Edward Stevens, Paul Whiting, Song-Hai Shi, Lorenz Studer. Combined small-molecule inhibition accelerates developmental timing and converts human pluripotent stem cells into nociceptors. Nature Biotechnology 30, 715–720 (2012) doi:10.1038/nbt.2249.

Abstract: Considerable progress has been made in identifying signaling pathways that direct the differentiation of human pluripotent stem cells (hPSCs) into specialized cell types, including neurons. However, differentiation of hPSCs with extrinsic factors is a slow, step-wise process, mimicking the protracted timing of human development. Using a small-molecule screen, we identified a combination of five small-molecule pathway inhibitors that yield hPSC-derived neurons at >75% efficiency within 10 d of differentiation. The resulting neurons express canonical markers and functional properties of human nociceptors, including tetrodotoxin (TTX)-resistant, SCN10A-dependent sodium currents and response to nociceptive stimuli such as ATP and capsaicin. Neuronal fate acquisition occurs about threefold faster than during in vivo development(1), suggesting that use of small-molecule pathway inhibitors could become a general strategy for accelerating developmental timing in vitro. The quick and high-efficiency derivation of nociceptors offers unprecedented access to this medically relevant cell type for studies of human pain

Figure – LSB3i Differentiation model. Early LSB inhibits trophectoderm, mesendoderm, and non-neural ectoderm cell fates yielding neuroectoderm. CHIR99021, SU5402 and DAPT induce and accelerate neural crest stem cell identity by day 8 and promote rapid differentiation of the neural crest stem cells to nociceptors expressing peptidergic markers by day 10.

Note: Neuromics' TRPV1 Antibody was used as a marker for mature nociceptors.

Check out Supplementary Data for more. I will continue to post links to methods here and @ Neuromics' Stem Cell Research Reagents.

mGluR Antibodies in Action

Metabotropic glutamate receptors (mGluRs) are involved in normal brain function and can be perturbed in many neuropathologic conditions. This makes mGluR Markers important tools for Neuroscience Research.

Neuromics has a comphrensive catalog of these markers. They are widely used and frequently reference in pubs-Neuromics' mGluR customer publications. I get excited when a mGluR we recently manufactured is referenced in a publication: Hoon Shim, Chih-Ting Wang, Yen-Lin Chen, Viet Q. Chau, Kevin G. Fu, Jianqi Yang, A. Rory McQuiston, Rory A. Fisher, and Ching-Kang Chen. Defective Retinal Depolarizing Bipolar Cells (DBCs) in Regulators of G-protein Signaling (RGS) 7 and 11 Double Null Mice. JBC Papers in Press. Published on February 27, 2012 as Manuscript M112.345751. The latest version is at http://www.jbc.org/cgi/doi/10.1074/jbc.M112.345751...Animals were sacrificed by CO2 inhalation and the eyeballs were immediately enucleated. After removal of cornea and lens the resulting eyecups were immersionfixed in 4% paraformaldehyde in 1X PBS at room temperature for 15 minutes. This short fixation time ensured good mGluR6 and Gb5 signals at the OPL. After cryoprotection in 30% sucrose in 1X PBS, the eyecups were embedded in TBS (Richard Allan Scientific, Kalamazoo, MI), sectioned at 20μm thickness, and stained...

Images: Absence of Gb5 immunoreactivity in mGluR6-containing puncta in retinas of RGS7 and RGS11 double knockout (711dKO) mice. Retinal sections from wild-type control (WT) and 711dKO (DKO) mice stained for metabotropic glutamate receptor 6 (mGluR6, 1:200) and Gb5 (CT215, 1:250) demonstrates superimposed signals in the WT but absence of Gb5 signal in the DKO. Scale bar equals 10μm.

I will continue to post new developments reagrding this important category of Neurotransmission Research Antibodies.

FLICA™ in vitro Caspase Kits for Apoptosis Assays

Great solution for measuring neurotoxicity

Here's a recent pub confirming the capabilities of our FLICA™ in vitro Caspase Apoptosis Detection Kits: Minghai Zhou, Gregory Ottenberg, Gian Franco Sferrazza, and Corinne Ida Lasmézas Highly neurotoxic monomeric α-helical prion protein. PNAS 2012 109 (8) 3113-3118; published ahead of print February 7, 2012, doi:10.1073/pnas.1118090109. ...PFA and stained for caspases using FLICA polycaspase assay (Neuromics) according to the manufacturer's instructions. Caspases...assayed on live cells using the caspase- specific FLICA assay (Neuromics). Cells were photographed using a Nikon inverted epifluorescence...

Images: FLICA was used to assess cell death in primay rat hippocampal neurons.Cells were plated on 25-mm poly-l-lysine-coated coverslips at 300,000 cells per coverslip. Cells were used at 4 or 8 days in vitro. Composite imagae (A) 3 out of 4 cells are apoptotic (green). No cells were necrotic as both of the PI-positive cells were FLICA-positive; they had compromised membranes and were probably in the late stages of apoptosis rather than necrosis. (B) 3 Caspase-positive cells fluoresce green.

I would like to underscore that we have solutions to cover most your needs for Apoptosis and Cytoxicity Assays. I will continue to post updates on customer data and publications.

eSC Derived human Mesenchymal Progenitor Pubs

We have added hMPro^TM Mesenchymal Progenitors to our Stem Cell Research Reagents category. These progenitors coupled with our UBC derived Human Mesenchymal Stem Cells give more options to basic and drug discovery researchers. Since they serve as potent research tools, it is a strategy of ours to continue to broaden our offerings in this products category.

Publications matter when assessing the capabilities of bio-reagents. I would like to share several that prove the utility of these cells:  David L. Simpson, Nolan L. Boyd, Sunjay Kaushal, Steve L. Stice, Samuel C. Dudley Jr. Use of human embryonic stem cell derived-mesenchymal cells for cardiac repair. Biotechnology and Bioengineering Volume 109, Issue 1, pages 274–283, January 2012. DOI: 10.1002/bit.23301

Nolan L. Boyd, Ph.D., Kelly R. Robbins, Ph.D., Sujoy K. Dhara, D.V.M., Ph.D., Franklin D. West, Ph.D., and Steven L. Stice, Ph.D. Human Embryonic Stem Cell–Derived Mesoderm-like Epithelium Transitions to Mesenchymal Progenitor Cells. Tissue Eng Part A. 2009 August; 15(8): 1897–1907. Published online 2009 January 15. doi: 10.1089/ten.tea.2008.0351.

Images: (A) Phase contrast image of hMPro™ mesenchymal progenitor cells in culture. (B) Confocal image of hMPro™ cells stained for the early smooth muscle marker (αSMA;green), F-actin (red) and nuclei (blue). Exposure to 10ng/mL of transforming growth factor beta 1 (TGF-β1) for 12 days induces expression of alpha smooth muscle actin (αSMA) in WA09-derived mesenchymal progenitor cells, suggesting their ability to differentiate along the smooth muscle lineage.

These cells have the ability to:

•Form adherent monolayers – ideal for 96- and 384-well formats for high throughput and high content cell-based assays.
•Differentiate into a wide variety of mesenchymal subtypes, including osteogenic and chondrogenic lineages (but not adipogenic)– ideal for drug discovery for a wide variety of biological targets and basic research in bone, cartilage, metabolic and immunological diseases.
•Passage up to 10X.

I will keep you posted on new applications.

TLR3s and the Synaptic Transmission of Itch

Synaptic Transmission Markers Trifecta

Itch, also known as pruritus, is a common, intractable symptom of several skin diseases, such as atopic dermatitis and xerosis. This chronic condition erodes quality of life.

The authors of this publication have made an important discovery that could prove a target for treating chronic itch. They also used 3 of our markers to confirm this discovery-guinea pig anti-TRPV1, guinea pig anti-SP antibody and rabbit anti-CGRP antibodies: Tong Liu, Temugin Berta, Zhen-Zhong Xu,Chul-Kyu and Ru-Rong Ji. TLR3 deficiency impairs spinal cord synaptic transmission, central sensitization, and pruritus in mice. J Clin Invest. 2012 June 1; 122(6): 2195–2207. Published online 2012 May 8. doi:10.1172/JCI45414.

Highlights: Scratching behaviors induced by histamine-dependent and -independent pruritogens are markedly reduced in mice lacking the Tlr3 gene. TLR3 is expressed mainly by small-sized primary sensory neurons in dorsal root ganglions (DRGs) that coexpress the itch signaling pathway components transient receptor potential subtype V1 and gastrin-releasing peptide. Ttreatment with a TLR3 agonist induces inward currents and action potentials in DRG neurons and elicited scratching in WT mice but not Tlr3–/– mice. Furthermore, excitatory synaptic transmission in spinal cord slices and long-term potentiation in the intact spinal cord were impaired in Tlr3–/– mice but not Tlr7–/– mice. Consequently, central sensitization–driven pain hypersensitivity, but not acute pain, was impaired in Tlr3–/– mice. In addition, TLR3 knockdown in DRGs also attenuated pruritus in WT mice. Finally, chronic itch in a dry skin condition was substantially reduced in Tlr3–/– mice. This demonstrates a critical role of TLR3 in regulating sensory neuronal excitability, spinal cord synaptic transmission, and central sensitization.

Images: Impaired scratching behaviors and reduced c-Fos expression in the spinal cords in Tlr3–/– mice. (A and B) Scratches in every 5 minutes (left) and 0–30 minutes (right) induced by intradermal injection of 50 μl compound 48/80 (100 μg) and CQ (200 μg). Note a reduction of both histaminergic (compound 48/80) and nonhistaminergic (CQ) itch in Tlr3–/– mice. *P < 0.05, Student’s t test; n = 11–13 mice for each group. Mean ± SEM. Two-way repeated-measures ANOVA analysis also shows a significant difference in the time course of compound 48/80– and CQ-induced scratching between the 2 groups (P < 0.05). (C) c-Fos–like immunoreactivity in the dorsal horn of the cervical spinal cord in WT and Tlr3–/– mice 2 hours after intradermal injection of compound 48/80 (48/80) or CQ. Right panels show the number of c-Fos–positive neurons in the dorsal horn. Scale bars, 100 μm. *P < 0.05, Student’s t test; n = 4–6 mice. All the data are mean ± SEM.

Images: Expression of TLR3 in a subset of small-sized DRG neurons. (A) Single-cell RT-PCR analysis from dissociated small-sized DRG neurons showing the distinct and overlapped distribution patterns of TLR3 and TLR7 in DRG neurons. The lanes were run on the same gel but were noncontiguous. M, marker; NC, negative control. (B) Single-cell RT-PCR analysis from dissociated small-sized DRG neurons showing colocalization of TLR3 with TPRV1 and GRP. Similar results were obtained from 3 independent experiments in 30 cells collected from different animals. (C) Double immunostaining in DRGs showing co-colocalization of TLR3 and GRP. Red and yellow arrows indicate GRP+ only and double-labeled neurons, respectively. Scale bars: 50 μm. (D) Cell size distribution frequency of TLR3+ and GRP+ neurons. (E) Double immunostaining in cultured DRG neurons showing co-colocalization of TLR3 with TRPV1 but not with NF200. Green arrows indicate NF200+ or TRPV1+ neurons, red arrows indicate TLR3+ neurons, and yellow allows indicate double-labeled neurons. Scale bars: 50 μm. (F) A Venn diagram showing the relationship of TLR3+, GRP+, and TRPV1+ populations in a DRG. Note that all TLR3+ cells also express GRP and TRPV1.

Nociceptive DRG neurons are involved in itch. TRPV1 and CGRP are indispensible for itch sensation. Given that all TRL3+ cells express these proteins, TRL3 could be a viable target for treating itch. 

Laryngotracheal Transplantations Using Nanofiber Solutions Scaffolds

Nanofibers Solutions work in transplants-imagine how well they will work in your 3-D based cell based assays.

3-D Cell Based Assays for Drug Discovery are the future. Like any new model, adoption rates are a function of how well the new solutions works. "The proof is in the pudding".

Here're highlights of a historic event based on transplants using nanofiber engineered laryngotrachea : Collaboration between Nanofiber Solutions and the Karolinska Institutet produces first synthetic laryngotracheal implants seeded with the patient’s stem cells to be successfully transplanted into human patients in Russia.
COLUMBUS, Ohio, June 26, 2012 – Nanofiber Solutions, LLC, an Ohio-based developer, manufacturer and marketer of 3-D synthetic scaffolds to advance basic research, tissue engineering and regenerative medicine announced today the first and second successful transplants of its tissue engineered laryngotracheal implants seeded with cells from the patients’ bone marrow.

The surgeries were performed June 19th and 21st at the Krasnodar Regional Hospital (Russia) by Dr. Paolo Macchiarini, Professor of Regenerative Surgery at the Karolinska Institutet (Stockholm, Sweden), and colleagues. Dr. Macchiarini led an international team that included Dr. Vladimir Porhanov, head of Oncological and Thoracic Surgery at Kuban State Medical University (Russia), Dr. Jed Johnson, Nanofiber Solution’s Chief Technology Officer who created the synthetic organs, Harvard Bioscience (Boston, USA) who produced the bioreactor, and Dr. Alessandra Bianco at University of Rome, Tor Vergata, who performed mechanical testing during scaffold development.

Both patients, a 33 year-old mother from St. Petersburg and a 28 year-old man from Rostov-on-Don, were in au to accidents and suffered from a narrowing of the laryngotracheal junction for which they already had failed previous surgeries. Transplantation was the last option for the patients to have normal quality of life. Immediately following transplantation, both patients were able to speak and breathe normally.

 Nanofiber Solutions, lead by Dr. Johnson, designed and built the nanofiber laryngotracheal scaffolds specifically to match the dimensions of each patient’s natural larynx and trachea, while Harvard Bioscience provided a bioreactor used to seed the scaffold with the patients’ own stem cells.  Although this procedure represents the world’s first and second successful use of synthetic synthetic laryngotracheal implants, it is Nanofiber Solution’s second and third successful organ implants using their synthetic scaffolds within the last year.

Nanofiber Solutions’ scaffolds mimic the body’s physical structure and allow for a more successful seeding, growth and differentiation of stem cells. Because the cells used to regenerate the larynx and trachea were the patients’ own, doctors report there has been no rejection of the transplants and the patients are not taking immunosuppressive drugs. (more).

Capabilities of 3-D nanofiber scaffolds for cell based assays:

Human brain tumor biopsy showing migrating tumor cells along the alligned nanofiber.

Nanofibers are optically transparent to allow for live-cell imaging and real time quantification of cell mobility using an inverted microscope

Nanofibers mimic the 3D topography found in vivo which produces a more realistic cellular response to therapeutics.

More realistic cellular behavior means you can use fewer animals and decrease time-to-market for drug discovery and development.

Nanofibers can easily be coated with ECM proteins using existing protocols for standard lab ware.

Cells can be easily removed for protein or gene analysis using trypsin, EDTA, etc.

We will continue posting relevant press releases, pubs and data that prove the capabilities of these important solutions.

100% Animal Free FGF Basic

New ISO-kine^TM FGF basic-only 39 USD-10 ug.

Fibroblast Growth Factor (FGF) Recombinant Proteins are widely used as tools in Stem Cell Research. They have the capabilities to catalyze growth and differentation as well as maintain cell stasis. We continue to offer new FGFs to make sure we meet the unique demands of our cutsomers and collaborators  .

Our E. Coli derived Human FGF (146 aa) recombinant protein is our #1 seller. As Stem Cell research moves from the bench top to the bedside, there will be a growing need for 100% animal free proteins. We are please to announce we have added a 100% animal free FGF (146 aa) basic that has bio-activity comparable to our top selling options

Image: ISOKine^TM bFGF vs our potent and proven e-coli derived bFGF.

I anticipate offering more ISOKine^TM Bio-risk free Stem Cell Research growth factors to meet growing demand. This growth will be driven by their unique advantages. These include low protease activity and secondary metabolite content and simple protein content all of which aid in downstream processing. These products also have the G.R.A.S. (Generally Recognised As Safe) status from FDA.

TRPA1 and Tooth Pain

Our TRPV (Vanilloid); TRPM; TRPA and TRPCs have proven excellent for studying

Transient receptor potential ankyrin 1 (TRPA1) is activated by noxious cold (<17°C) and contributes to cold and mechanical hypersensitivity after inflammation and nerve injury: Yun Sook Kim, PhD, Hoon Kap Jung, DDS, Tae Kyung Kwon, DDS, Chin Soo Kim, DDS, PhD, Jin Hyun Cho, DDS, PhD, Dong Kuk Ahn, DDS, PhD, Yong Chul Bae, DDS, PhD.Expression of Transient Receptor Potential Ankyrin in Human Dental Pulp. Journal of Endodontics. Available online 8 June 2012. doi.org/10.1016/j.joen.2012.04.024.


Highlights: TRPA1 was expressed in a large number of axons branching extensively in the peripheral pulp and in a few axons within the nerve bundles in the core of the coronal pulp and in the radicular pulp. Under electron microscopy, TRPA1 immunoreactivity was typically localized near the plasma membrane of unmyelinated axons in the peripheral pulp, suggesting that in these axons it may act as a functional receptor. The proportion of axons expressing TRPA1 in neurofilament 200–positive axons significantly increased in the painful pulp compared with the normal pulp. TRPA1 was also densely expressed in the processes and the cell body of odontoblasts. A large number of axons coexpressed TRPA1 and Nav1.8.

Images: Immunofluorescent staining for (A) TRPA1 in the human normal dental pulp is completely abolished by (B) preadsorption with a control peptide, proving the specificity of the TRPA1 antiserum (×200, scale bars = 50 µm). doi.org/10.1016/j.joen.2012.04.024.

Related Reagents:

All TRP Antibodies                       
Pain and Inflammation Research Antibodies                     
Neurotransmission -Neurotransmission Research Antibody Categories

Primary Neurons and Astrocytes-Primary human, rat and mouse neurons and astrocytes

Stem Cell Differentiation Flow Charts

Installment #1: Mesoderm Derived Stem Cells

We would like to thank our partners and customers for their input on how to best add value to our Stem Cell Research Reagents. Based on this input, we are in the process of building a new website focused on providing stem cell technical content. Better content should help catalyze better research and drug discovery related decisions.

Here're several stem cell differentiation flow charts that map the genesis of pluripotent and multipotent cells and identify the cell types that result from their differentiation.

This chart was generated using input and publications generously provided by Dr. Henry Young, Tenured Full Professor at Mercer University School of Medicine.

I anticipate the launch date for our Stem Cell Related website to occur in early Q3 2012.

In the meantime, we will posting relevant data, publications and other information here. This will include protocols and related growth factors for generating specific cell types like dopaminergic neurons from neural progenitors. Stay tuned.

Stress, Depression and Alpha2delta Ligands

Implications for treating Stress Related Depression.

Stress related disorders like Post Traumatic Stress Syndrome (PTSD), Major Depressive Disorder (MDD) and Generalized Anxiety Disorders (GAD) dysregulate neurogenesis. This disregulation can lead to disorders like chronic depression. Indeed, stress hurts.

In this study (which includes use of our Neural Progenitor Marker-Nestin), researchers show for the first time that the alpha2delta (α2δ) ligands gabapentin [1- (aminomethyl)cyclohexaneacetic acid; GBP] and pregabalin [S-[+]-3-isobutylGABA or (S)-3- (aminomethyl)-5-methylhexanoic acid; PGB] can produce a concentration-dependent increase in the number of newborn mature and immature neurons generated in vitro from adult hippocampal neural progenitor cells (NPC), and, in parallel, a decrease in the number of undifferentiated precursor cells. These effects were confirmed in vivo, since a significantly increased number of adult generated neurons was observed in the hippocampal region of mice chronically treated with PGB [10 mg/kg, i.p., 21 days] compared to vehicle-treated mice. Moreover, we demonstrated that PGB administration prevented the appearance of depression-like behaviours induced by chronic restraint stress and, in parallel, promoted hippocampal neurogenesis in adult stressed mice. Finally, we provided data suggesting the potential involvement of the α2δ1 subunit and NF-κB signaling pathway in the drug-mediated proneurogenic effects. The new pharmacological activities of α2δ ligands may help explaining their therapeutic activity as add-on therapy in major depression and on depressive symptoms in posttraumatic stress disorder and generalized anxiety disorders. Furthermore these data contribute to the identification of novel molecular pathways which may represent potential targets for pharmacological modulation in depression: Maria Maddalena Valente, Valeria Bortolotto, Bruna Cuccurazzu, Federica Ubezio, Vasco Meneghini, Maria Teresa Francese, Pier Luigi Canonico, Mariagrazia Grilli.Alpha2delta ligands act as positive modulators of adult hippocampal neurogenesis andprevent depressive-like behavior induced by chronic restraint stress. Molecular Pharmacology Fast Forward Published on May 9, 2012 as doi:10.1124/mol.112.077636.

Images: Effect of α2δ ligands on neuronal differentiation and proliferation of hippocampus-derived neural progenitor cells. (A) Representative fluorescence microscopy image of a hippocampal neurosphere immunolabelled for nestin (green) and SRY-related HMG-box gene 2 (Sox-2) (red), markers of undifferentiated NPC. Magnification = X600. Scale bar = 56 μm. (B) After 24 h in absence of growth factors, hippocampal Neural Progenitor Cells (NPC) differentiated giving rise to four different cell populations identified by double Microtubule Associated Protein-2 (MAP-2) and nestin immunolabelling: MAP-2+/nestin- mature neurons, MAP-2+/nestin+, MAP-2-/nestin+ and MAP-2-/nestin- cells. Data are expressed as mean ± S.D. of n=9 experiments, run in triplicates. Gabapentin (GBP) and pregabalin (PGB) promote neuronal differentiation of adult hippocampal NPC. GBP (C-F) and PGB (G-J) significantly increased, in a concentrationdependent manner, the percentage of MAP-2+/nestin- (C, G) and MAP-2+/nestin+ (D, H) cells and decreased the percentage of MAP-2-/nestin- cells (F, J), with no effect on MAP-2-/nestin+ cells (E, I). Data are expressed as mean ± S.D. of n = 3 experiments, run in triplicates. *, p < 0.05; **, p < 0.01; ***, p < 0.001 vs vehicle (Student’s t-test). (K-M) Representative fluorescence microscopy images of MAP-2 immunolabelling (green) in cells derived from hippocampal NPC after 24 h treatment with vehicle (K), 1 nM GBP (L) and 1 nM PGB (M). Nuclei are stained with Draq5(blue). Magnification = X400. Scale bar = 75 μm. (N) Adult hippocampal NPC were treated with vehicle or 1 nM PGB for 6, 24, 48, 72, 96 h and proliferation rate was assessed. PGB had no effect on NPC proliferation, when compared to vehicle. Data, expressed as counts per second (CPS), represent the mean ± S.D. of experiments run in triplicates.

This study is good news. Results demonstrate the new pharmacological activity of α2δ ligands may

potentially explain their efficacy as add-on therapy in MDD, as well as on depressive symptoms
in PTSD and GAD. This knowledge will help the discovery of refined therapies for these debilating disorders.

Serotonin and Pain

Brainstem facilitations and descending serotonergic controls contribute to visceral nociception but not pregabalin analgesia in rats.

Pregabalin is used to treat Neuropathic Pain. Here the authors show state-dependent pregabalin analgesia in neuropathy does not apply to visceral pain. Our rabbit polyclonal Mu Opioid Receptor Antibody is used to demonstrate intra-RVM Derm-SAP locally ablates a substantial proportion of MOR and serotonergic cells: Shafaq Sikandar, Kirsty Bannister, Anthony H. Dickenson. Brainstem facilitations and descending serotonergic controls contribute to visceral nociception but not pregabalin analgesia in rats. Neuroscience Letters. Volume 519, Issue 1, 21 June 2012, Pages 31–36.... For MOR staining, a rabbit polycolonal μ-opioid receptor primary antibody (1:10,000 TTBS; Neuromics, MN, USA, RA10104)...

These findings are important for understanding the limitations of Preglabin as an analgesic for viceral pain. Check out all our Opioid Publications.

Ready for Prime Time Stem Cell Markers

Researchers frequently reference use of Neuromics' Stem Cell Markers in publications. This is an important affirmation for us as these tools are critical for determining the differentiation state of Stem Cells. In this publication, the authors use our Mouse Monoclonal Nestin Antibody to understand the mechanisms underlying neural progenitor differentiation and neuronal fate. This understanding is an important precursor for using these cells in Regenerative Medicine: Serafí Cambray, Charles Arber, Graham Little, Antonios G. Dougalis, Vincenzo de Paola, Mark A. Ungless, Meng Li and Tristan A. Rodríguez. Activin induces cortical interneuron identity and differentiation in embryonic stem cell-derived telencephalic neural precursors. Nature Communications 3, Article number: 841 doi:10.1038/ncomms1817. Received 10 January 2011 Accepted 29 March 2012 Published 15 May 2012.

In this study, the authors show that Activin provides telencephalic neural precursors with positional cues that specifically promote the acquisition of a calretinin interneuron fate by controlling the expression of genes that regulate cortical interneuron identity. This work demonstrates a novel means for regulating neuronal differentiation and specification of subtype identity.

Images: (a)immunostaining (left panels) and quantifications (right panel) indicating that Shh promotes and cyclopamine inhibits proliferation in neural precursors (Nestin+/β-III-tubulin+ cells in cyclopamine 49±4.3/35.1±1.8%, Shh 80.3±3.2/14.8±0.7% and control cultures 68.4±7.2/20.1±2.8%; n=3, mean±s.e.m.). (b) Relative expression levels of Gli1 and Ptch1 during the first 5 days of Activin or control treatment. (c) Immunoblot analysis of Gli1 and Cyclin D1 levels during the first 4 days of Activin or control treatment illustrating how Activin represses the expression of these proteins. (d) Normalized mRNA levels of Gli1 and Ptch1 after 24 h exposure to the indicated conditions illustrating how Shh induces the expression of these genes and Activin inhibits their expression (n=3, mean±s.e.m. Student's t-test. *P<0.005 and **P<0.05). ESCs were differentiated for 5 days as a monolayer, then replated into poly-D-lysine/laminin-coated dishes and cultured in NBB27 media (controls), NBB27+10 ng ml−1 Activin, NBB27+100 ng ml−1 Shh, NBB27+10 μm cyclopamine or NBB27 + 10 ng ml−1 Activin + 100 ng ml−1 Shh. Scale bar=50 μm.

Image: 

Image: Model for how Activin induces the differentiation and CGE fate in telencephalic neuronal precursors.


The protocol described in this manuscript represents a method to obtain an enriched source of calretinin interneurons from both mouse and human ESCs. Therefore, our work significantly contributes to the aim of generating the diverse neuronal subtypes required for the safe and successful use of ESCs in regenerative medicine.

The capabilities of stem cells markers matter in developing protocols for use in Regenerative Medicine.

Cannabinoid Type-1 Receptors and Chemotherapy Related Pain

Chemotherapy can induce painful peripheral neuropathy and also have a toxic effect on peripheral nerves. The authors here show that that cisplatin produces hyperalgesia and toxicity to sensory neurons as indicated by neurochemical, morphological, and functional measures. Increasing AEA signaling at CB1 receptors not only reduced the hyperalgesia but reduced the neurotoxicity of cisplatin as well:  Iryna A. Khasabova,Sergey Khasabov, Justin Paz, Catherine Harding-Rose, Donald A. Simone, and Virginia S. Seybold. Cannabinoid Type-1 Receptor Reduces Pain and Neurotoxicity Produced by Chemotherapy. The Journal of Neuroscience, 16 May 2012, 32(20): 7091-7101; doi: 10.1523/​JNEUROSCI.0403-12.2012.

The authors use our guinea pig TRPV1 antibody to measure cisplatin hyperalgesia vs treated and control mice.

Images: URB597 attenuated effects of cisplatin on protein expression in DRGs. A, TRPV1- and ATF-3-ir were detected by immunofluorescence in L3–L5 DRGs from mice treated with vehicle, cisplatin, or cisplatin plus URB597. Cisplatin (1 mg/kg of body weight, daily for 7 d, i.p.) increased the occurrence of TRPV1- and ATF3-ir in neurons. Co-injection of URB597 (0.3 mg/kg daily, i.p.) with cisplatin attenuated the effect cisplatin on protein-ir. Scale bars: 10 μm (for images within each antigen). B, Quantitative summary of the effect of treatments on TRPV1-ir in neurons. Data are expressed as the mean ± SEM. aSignificantly different from each other group (p < 0.05, one-way ANOVA with Student–Newman–Keuls test; n = 4 mice/treatment). C, Quantitative summary of the effect of treatments on ATF-ir in neurons. Data are expressed as the median and 25th and 75th percentile range. *Significantly different from vehicle control and cisplatin plus URB597 groups (n = 6 mice/treatment; p < 0.001, Kruskal–Wallis ANOVA on ranks test).

Related Reagents:

VR1 N-Terminus (TRPV1)
VR1 (TRPV1)-Goat
VR1 C-Terminus (TRPV1) - mouse specific
All TRP Antibodies
Pain and Inflammation Research Antibodies
Neurotransmission -Neurotransmission Research Antibody Categories

Pinpointing Neuropathic Pain

I am pleased to feature our Guinea Pig P2X3 Antibody as a tool for studying the root causes of Neuropathic Pain.

In this study, the authors showed ablation of nonpeptidergic fibers in a chronic constriction injury model caused significant sympathetic and parasympathetic P2X3 fiber sprouting, and led to an exacerbated pain response. This was an unexpected finding, as it has been suggested that nonpeptidergic fibers play a major role in mechanical pain, and suggests that these fibers play a complex role in the development of neuropathic pain: Anna M.W. Taylora, Maria Osikowicza, Alfredo Ribeiro-da-Silva. Consequences of the ablation of nonpeptidergic afferents in an animal model of trigeminal neuropathic pain. PAIN. Volume 153, Issue 6, June 2012, Pages 1311–1319. doi.org/10.1016/j.pain.2012.03.023....Sections were then incubated for 48 hours at 4°C with a guinea pig polyclonal anti-P2X 3 antibody (1:25,000; Neuromics, Edina, MN, USA), diluted in PBS-T. Following primary antibody incubation, sections were treated with a biotin-conjugated...

Image: Example of  Non-petidergic fibers expressing P2X3 receptor for ATP are present in lamina II of the contralateral dorsal horn (arrows). The P2X3-positive fibers are eliminated from the rat dorsal horn ipsilateral to the rhizotomy (arrow heads). Spinal cord segment C6. Scale bar 400 um. P2X3 antibody dilution 1:25,000. Biomédica vol.24 no.2.

Check out our related reagents categories:

• All Purinergic Receptors
• Pain and Inflammation Research Antibodies
• Neurotransmission Research Antibodies
• Primary Neurons and Astrocytes-Primary human, rat and mouse neurons and astrocytes

 I will continue to post important findings related to the root causes of Neuropathic Pain.

Mesechymal Stem Cell DifferentiationPathways

As a provider of Human Mesenchymal Stem Cells (hMSCs), MSCGro™ Mesenchymal Stem Cell Media and Stem Cell Markers, I receive questions related to differentiation. Specifically, researchers desire to drive these cells to specific progenitor and cell phenotypes like Osteocytes, Adipocytes and Chondroytes.

I would like to share a pathway map that gives a snapshot of these pathways:

Regenerative Biology of the Spine and Spinal Cord. Edited by: Rahul Jandial, Mike Y. Chen, Bihong T. Chen and Joseph Ciacci. ISBN: 978-1-4614-4089-5. Publication date: May 25, 2012. Series: Special Books

I will continue to post information that will enable the researchers to harness the power of Mesenchymal Stem Cells.

Arthritic Pain, Nerve Sprouting and Neuroma Formation

Our friends, Drs Mantyh and Jimenez Andrade, have published important findings on the potential root cuases of age related Arthritic Pain: Juan M Jimenez-Andrade and Patrick W Mantyh. Sensory and sympathetic nerve fibers undergo sprouting and neuroma formation in the painful arthritic joint of geriatric mice. Note: our Chicken Polyclonal NF200 Antibody was used to label primary afferent sensory nerve fibers.

Overview: Introduction: Although the prevalence of arthritis dramatically increases with age, the great majority of preclinical studies concerning the mechanisms that drive arthritic joint pain have been performed in young animals. One mechanism hypothesized to contribute to arthritic pain is ectopic nerve sprouting; however, neuroplasticity is generally thought to be greater in young versus old nerves. Here we explore whether sensory and sympathetic nerve fibers can undergo a significant ectopic nerve remodeling in the painful arthritic knee joint of geriatric mice.

In other words, there is a clear root reason that the pain arthritis sufferers experience increases overtime. Here's some related data. Notice the increase in labeled nerves:

Images. Sensory nerve fiber sprouting and formation of neuroma-like structures in the painful geriatric arthritic knee joint. Schematic of a frontal view of a cross-sectioned mouse knee joint (A). The red square illustrates the synovial region from which the confocal images were obtained. Representative confocal images of calcitonin gene-related peptide (CGRP+), neurofilament 200 kDa (NF200+) sensory nerve fibers (yellow/orange) and growth associated protein (GAP43; marker of fibers undergo regeneration, yellow/orange) and DAPI labeled nuclei (blue) in knee joint sections (20 μm thick) of vehicle-injected (B,D,F) and CFA-injected (C,E,G ) mice. In vehicle-injected mice, a low level, regular pattern of innervation by CGRP+ and NF200+ fibers is observed in the synovial space of the knee joint. Twenty-eight days following the initial CFA injection, a significant number of CGRP+ and NF200+ nerve fibers have sprouted and have a disorganized appearance as compared to vehicle-injected mice. Note that, CGRP+ and NF200+ sprouted nerve fibers are localized in the synovium and are not observed in the meniscus of the joint. Furthermore, there was formation of neuroma-like structures in the synovium of the geriatric mice injected with CFA (G).

Related Reagents: Neuronal-Glial Markers-Astrocytes, Glia, Microglia, Olidogodendrocytes, Progenitors and Schwann Cell Markers.

Primary Hippocampal Neurons Performing!

I advertise our Primary Neurons and Astrocytes as being easy to culture, grow and maintain. We confirm this via the data/images our customers generously share and the many publications referencing use of the cells.

I would like to thank George Kenneth Todd (Patterson Lab at UC Davis) for these wonderful images of our e18 Primary Rat Hippocampal Neurons. These were taken at day 67!

Prior to staining, the cells were treated with Wnt5 for 2 min, then Wnt5 + Wnt3 for an additional 2 min during Calcium Imaging experiments. The cells were then fixed and stained for IP3R (green), Frizzled2 (blue), and B-Catenin (red), and the confocal images were captured at 10x. Notice the parallel neurites formation in image 3.


For staining culture options, check out our neuron-glial-astrocyte markers.