Pain Receptors and Diabetic Neuropathy

We Got the Markers
Our Markers for Pain Research are widely used and frequently published. They are often referenced in publications on Diabetic nerve pain.

Here are some of my past blog postings:

• Cytokines and Neuropathic Pain
• Epidermal Nerve Fibers in Neuropathic Pain Model
• Pain Research and Gene Expression Analysis

Here's a model of the actual nerve receptors behind both intractable pain and loss of sensation.

A: Simplified model of nociception under normal conditions. Free nerve endings transduce a painful stimulus into a neural signal, which propagates to DRG centrally and eventually synapses on a nociceptive neuron within the DH of the spinal cord.B: Proposed model of nociception under conditions of diabetic hyperalgesia and allodynia. A pain signal augmented by upregulated pronociceptive ion channels in sensory neurons is carried toward the DH, where it is further augmented by a hypoactive GABAergic system and subsequently diminished inhibition from an inhibitory interneuron. NMDAR, N-Methyl-D-aspartate receptors. https://doi.org/10.2337/dbi15-0006

We will continue to post findings on the root causes and potential treatment for Neuropathy.

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

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

TRPV1 & P2X3-Daily Double

Low pH and Chronic Muscle Pain

Our Pain and Inflammation Antibodies are routinely used for chronic pain. I would like to highlight a recent publication referencing use of our  Guinea Pig TRPV1 and Pig 2X3 Antibodies and Blocking Peptides:

James P. Lund,Somayeh Sadeghi,Tuija Athanassiadis, Nadia Caram Salas, François Auclair, Benoît Thivierge, Isabel Arsenault, Pierre Rompré, Karl-Gunnar Westberg, and Arlette Kolta.Assessment of the Potential Role of Muscle Spindle Mechanoreceptor Afferents in Chronic Muscle Pain in the Rat Masseter Muscle. PLoS One. 2010; 5(6): e11131. Published online 2010 June 15 doi:10.1371/journal.pone.0011131.

Conclusions/Significance

Low pH leads to changes in several electrical properties of MSA, including initiation of ectopic action potentials which could propagate centrally but could also invade the peripheral endings causing glutamate release and activation of nearby nociceptors within the spindle capsule. This peripheral drive could contribute both to the transition to, and maintenance of, persistent muscle pain as seen in some “functional” pain syndromes.

Images: Photomicrographs of trigeminal ganglion neurons stained with TRPV1 and P2X3.

Related Reagents:

Pain and Inflammation Research Antibodies

Neurotransmission

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

Purinergic Receptor Pubs

Our Purinergic Receptor Antibodies have recently been referenced in several publications.

In the first study 5 of our antibodies are used. It investigates the expression of purinergic P2 receptors in oxygen-induced retinal neovascularization.

Sylvia Sarman; Jorge Mancini; Ingeborg van der Ploeg; J. Oscar Croxatto; Anders Kvanta; Juan E. Gallo. Involvement of Purinergic P2 Receptors in Experimental Retinal Neovascularization. Current Eye Research. 10.1080/02713680701885470 .

...Five eyes (5 animals) from either hyperoxa- or non-hyperoxia-treated mice were enucleated and fixed for 48 hr at 4°C in paraformaldehyde (Sigma-Aldrich, St Louis, MO, USA). Afterward, eyes were immersed for cryoprotection in graded sucrose solution (5% overnight, 10%, 15%, and 20%) and interlocked with resin. Fourteen micron sections were obtained (Shandon AS325 Retraction Microtome, Thermo Scientific, Waltman, MA, USA) and fixed on polylysine-treated glass slides. After overnight incubation in primary antibody (P2X1 1:1000, P2X2 1:1000,P2X3 1:1000, and P2Y2 1:800) (Neuromics, Minneapolis, MN, USA) at 4°C, sections were treated with biotinylated secondary antibodies followed by an avidin peroxidase complex step (Vectastin Elite ABC, Vector Laboratories, Burlingame, CA, USA). Finally, a color reaction was obtained using 3,3'-diaminobenzidine (DAB)/nickel-enhanced solution for staining (Sigma-Aldrich, St Louis, MO, USA). At least 6 sections per retina were analyzed. For immunofluorescence, the sections were labeled with lissamine rhodamine-conjugated goat anti-rabbit Ig-G or with fluorescein-5-isothiocyanate-conjugated goat anti-guinea pig Ig-G (Jackson Immunoresearch Laboratories, West Grove, PA, USA). At least 6 sections per retina were analyzed. Visualization was done using a Nikon Fluorescence Eclipse Microscope (Tokyo, Japan), and photographs were taken with a Nikon DN 100 Digital Camera (Tokyo, Japan)...

Images: Expression of P2X and P2Y receptors in normal mice and in a model of ischemia-induced pathological retinal angiogenesis as assessed by immunohistochemistry. (A) P2X2 receptor expression was found in the outer plexiform layer of control mice. (B) In mice
treated with oxygen, the expression was similarly found in the outer plexiform layer. In addition, a strong signal was also found in the inner plexiform layer. This induction was noted in all slides analyzed. (C) Retina without primary antibody for P2X2. (D) In control mice,
weak P2Y2 receptor expression was seen in the ganglion cell and in the nerve fiber layer. (E) In mice treated with oxygen, P2Y2 expression
showed a similar overall distribution as control mice. The signal was, however, strongly up-regulated. This increase was noted in all slides analyzed. (F) Retina without primary antibody for P2Y2. Black bar = 20 μm.

The second studies P2Xs' role in neurotransmision.

Elsa Fabbretti*, Elena Sokolova*, Lara Masten, Marianna D’Arco, Alessandra Fabbro, Andrea Nistri and Rashid Giniatullin. Identification of negative residues in the P2X3 ATP receptor ectodomain as structural determinants for desensitization and the Ca2+ sensing modulatory sites. JBC Papers in Press. Published on October 8, 2004 as Manuscript M409772200.

...Western immunoblots of transfected or untransfected HEK cells were performed as recently reported (19). In brief, these were lysed using a buffer containing 100 mM Tris HCl (pH 6.8), 200 mM dithiothreitol, 4 % SDS, 20 % glycerol and a cocktail of protease inhibitors (Sigma), and separated on 10 % polyacrylamide gel. After blocking with Tris saline buffer (TBS) containing milk, Tween 20 and preimmune serum, proteins were incubated overnight at 4°C with an anti-P2X3 antibody (Neuromics; 1:2000). Immunocomplexes were incubated for 1 h with a peroxidase-conjugated secondary antibody (Sigma; 1:4000) and detected with a chemiluminescence ECL kit (Amersham). Negative controls were obtained by mock transfection of HEK cells with the pEGFP-N1 plasmid (CLONTECH). Controls for efficient loading of different lysate materials were carried out by using an anti-β actin antibody (mouse monoclonal, 1:2000, Sigma)...

Related Reagents:

P2X1-Rabbit Antibody
P2X2-Guinea Pig Antibody
P2X2-Rabbit Antibody
P2X3-Rabbit Antibody
P2X3-Guinea Pig Antibody
Related Reagents:
All Purinergic Receptor Antibodies
All Pain and Inflammation Antibodies
Vision and Retina Research Antibodies