Allodynia & Cold and Hot Plate test- Transpharmation's resea


Bioseb is happy and proud to present the latest research from Transpharmation and The Royal Veterinary College, London, UK, by Ran Magnusdottir, Amy Fisher and Neil Upton, who used Bioseb's Cold/Hot Plate test for their research:

Reversal of cold and mechanical allodynia with pregabalin
in a rodent model of chemotherapy induced peripheral neuropathy

Background: Chemotherapy- induced peripheral neuropathy (CIPN) is a dose limiting side effect in the use of the platinum-based antineoplastic drug oxaliplatin. Currently there is no treatment available to reverse the neurotoxicity which presents as pain, sensory loss and cold allodynia in up to 80% of patients.

Bioseb's Cold Hot Plate Test - Transpharmation's Study - Alloynia curves
Click to see
Transpharmation's curves
Results: CIPN was induced in 10 male C57BL/6 mice (6 weeks) with a single intraperitoneal injection of oxaliplatin (15mg/kg i.p.). Signs of cold and mechanical allodynia were assessed by Cold/Hot plate (Bioseb, France) at 20°C and hand-held von Frey (vF) hairs from baseline to 20 days after injection. Mechanical and cold allodynia were established 3 days post Oxaliplatin injection and remained stable for 14 days. At day 15, pregabalin (3mg/kg p.o.) reversed mechanical allodynia to baseline scores at 2 hours (H) post dosing, and cold allodynia at 1H and 2H post dosing. Following a 2 day wash out where scores returned to neuropathic baseline, pregabalin (10 mg/kg p.o.) returned scores for mechanical and cold allodynia to baseline scores at both 1H and 2H. Cold testing was performed either immediately after vF or alone with the same results, showing no iatrogenic effects of vF on cold sensitivity. Correlation analysis of the responses to cold and mechanical stimuli showed no clear trend, indicating that oxaliplatin induced peripheral neuropathy affects the two modalities in different ways.

Conclusions: Oxaliplatin induced peripheral neuropathy as measured by cold and mechanical allodynia is reversible by a single dose of pregabalin.

For more information, please contact:
Amy S. Fisher, PhD - Principal Scientist, Transpharmation Ltd
amy.fisher@transpharmation.co.uk - Tel: +44 (0)17 0764 2162
Web: www.transpharmation.co.uk

Bioseb's Hot/Cold plate is becoming a must when studying rodent's Cold hyperalgesia/allodynia. Focussing on Oxaliplatin-induced Thermal Hypersensitivity, it has been proven to be a valid Behavioural test in numerous papers, such as :

• Oxaliplatin-induced cold hypersensitivity is due to remodelling of ion channel expression in nociceptors. (2011)
Oxaliplatin-induced cold hypersensitivity is due to remodelling of ion channel expression in nociceptors.
J. Descoeur, V. Pereira, A. Pizzoccaro, A. Francois, B. Ling et al. (Team of Dr Bourinet)
Institut de Génomique Fonctionnelle, CNRS, UMR-5203, Département de Physiologie, Montpellier, France.
Published in "EMBO Molecular Medicine" (2011-05-24)

Cold hypersensitivity is the hallmark of oxaliplatin-induced neuropathy, which develops in nearly all patients under this chemotherapy. To date, pain management strategies have failed to alleviate these symptoms, hence development of adapted analgesics is needed. Here, we report that oxaliplatin exaggerates cold perception in mice as well as in patients. These symptoms are mediated by primary afferent sensory neurons expressing the thermoreceptor TRPM8. Mechanistically, oxaliplatin promotes over-excitability by drastically lowering the expression of distinct potassium channels (TREK1, TRAAK) and by increasing the expression of pro-excitatory channels such as the hyperpolarization-activated channels (HCNs). These findings are corroborated by the analysis of TREK1-TRAAK null mice and use of the specific HCN inhibitor ivabradine, which abolishes the oxaliplatin-induced cold hypersensibility. These results suggest that oxaliplatin exacerbates cold perception by modulating the transcription of distinct ionic conductances that together shape sensory neuron responses to cold. The translational and clinical implication of these findings would be that ivabradine may represent a tailored treatment for oxaliplatin-induced neuropathy.

• Anticancer Activity of Methyl-substituted Oxaliplatin Analog. (2012)
Anticancer Activity of Methyl-substituted Oxaliplatin Analog.
U. Jungwirth, D. Xanthos, J. Gojo, A. Bytzek, W. Korner et al.
Medical University of Vienna, Institute of Cancer Research, Vienna, Austria.
Published in "Molecular pharmacology" (2012-05-30)

Oxaliplatin is successfully used in systemic cancer therapy. However, resistance development and severe adverse effects are limiting factors for curative cancer treatment with oxaliplatin. The purpose of this study was to comparatively investigate in vitro and in vivo anticancer properties as well as the adverse effects of two methyl-substituted enantiomerically pure oxaliplatin analogs [[(1R,2R,4R)-4-methyl-1,2-cyclohexanediamine] oxalatoplatinum(II) (KP1537), and [(1R,2R,4S)-4-methyl-1,2-cyclohexanediamine]oxalatoplatinum(II) (KP1691)] and to evaluate the impact of stereoisomerism. Although the novel oxaliplatin analogs demonstrated in multiple aspects activities comparable with those of the parental compound, several key differences were discovered. The analogs were characterized by reduced vulnerability to resistance mechanisms such as p53 mutations, reduced dependence on immunogenic cell death induction, and distinctly attenuated adverse effects including weight loss and cold hyperalgesia. Stereoisomerism of the substituted methyl group had a complex and in some aspects even contradictory impact on drug accumulation and anticancer activity both in vitro and in vivo. To summarize, methyl-substituted oxaliplatin analogs harbor improved therapeutic characteristics including significantly reduced adverse effects. Hence, they might be promising metal-based anticancer drug candidates for further (pre)clinical evaluation.

• P2X7 Cell Death Receptor Activation and Mitochondrial Impairment in Oxaliplatin-Induced Apoptosis and Neuronal Injury: Cellular Mechanisms and In Vivo Approach (2013)
P2X7 Cell Death Receptor Activation and Mitochondrial Impairment in Oxaliplatin-Induced Apoptosis and Neuronal Injury: Cellular Mechanisms and In Vivo Approach
F. Massicot, G. Hache, L. David, D. Chen, C. Leuxe et al.
Faculté des Sciences Pharmaceutiques et Biologiques, Université Paris Descartes, Paris, France
Published in "PLOS One" (2013-06-27)

Limited information is available regarding the cellular mechanisms of oxaliplatin-induced painful neuropathy during exposure of patients to this drug. We therefore determined oxidative stress in cultured cells and evaluated its occurrence in C57BL/6 mice. Using both cultured neuroblastoma (SH-SY5Y) and macrophage (RAW 264.7) cell lines and also brain tissues of oxaliplatin-treated mice, we investigated whether oxaliplatin (OXA) induces oxidative stress and apoptosis. Cultured cells were treated with 2–200 µM OXA for 24 h. The effects of pharmacological inhibitors of oxidative stress or inflammation (N-acetyl cysteine, ibuprofen, acetaminophen) were also tested. Inhibitors were added 30 min before OXA treatment and then in combination with OXA for 24 h. In SH-SY5Y cells, OXA caused a significant dose-dependent decrease in viability, a large increase in ROS and NO production, lipid peroxidation and mitochondrial impairment as assessed by a drop in mitochondrial membrane potential, which are deleterious for the cell. An increase in levels of negatively charged phospholipids such as cardiolipin but also phosphatidylserine and phosphatidylinositol, was also observed. Additionally, OXA caused concentration-dependent P2X7 receptor activation, increased chromatin condensation and caspase-3 activation associated with TNF-α and IL-6 release. The majority of these toxic effects were equally observed in Raw 264.7 which also presented high levels of PGE2. Pretreatment of SH-SY5Y cells with pharmacological inhibitors significantly reduced or blocked all the neurotoxic OXA effects. In OXA-treated mice (28 mg/kg cumulated dose) significant cold hyperalgesia and oxidative stress in the tested brain areas were shown. Our study suggests that targeting P2X7 receptor activation and mitochondrial impairment might be a potential therapeutic strategy against OXA-induced neuropathic pain.

• The Nav1. 9 Channel Is a Key Determinant of Cold Pain Sensation and Cold Allodynia (2015)
The Nav1. 9 Channel Is a Key Determinant of Cold Pain Sensation and Cold Allodynia
Lolignier S, Bonnet C, Gaudioso C, Noël J, Ruel J et al.
Pharmacologie Fondamentale et Clinique de la Douleur, Clermont Université, Université d'Auvergne, Clermont-Ferrand, France;
Published in "Cell Rep. " (2015-05-19)

Cold-triggered pain is essential to avoid prolonged exposure to harmfully low temperatures. However, the molecular basis of noxious cold sensing in mammals is still not completely understood. Here, we show that the voltage-gated Nav1.9 sodium channel is important for the perception of pain in response to noxious cold. Nav1.9 activity is upregulated in a subpopulation of damage-sensing sensory neurons responding to cooling, which allows the channel to amplify subthreshold depolarizations generated by the activation of cold transducers. Consequently, cold-triggered firing is impaired in Nav1.9(-/-) neurons, and Nav1.9 null mice and knockdown rats show increased cold pain thresholds. Disrupting Nav1.9 expression in rodents also alleviates cold pain hypersensitivity induced by the antineoplastic agent oxaliplatin. We conclude that Nav1.9 acts as a subthreshold amplifier in cold-sensitive nociceptive neurons and is required for the perception of cold pain under normal and pathological conditions.

• A Polyamine-Deficient Diet Prevents Oxaliplatin-Induced Acute Cold and Mechanical Hypersensitivity in Rats (2013)
A Polyamine-Deficient Diet Prevents Oxaliplatin-Induced Acute Cold and Mechanical Hypersensitivity in Rats
J.Ferrier, M.Bayet-Robert, B.Pereira, L.Daulhac
FACULTE DE MEDECINE, CLERMONT FERRAND, FRANCE
Published in "PLOS ONE" (2013-11-30)

Background
Oxaliplatin is an anticancer drug used for the treatment of advanced colorectal cancer, but it can also cause painful peripheral neuropathies. The pathophysiology of these neuropathies has not been yet fully elucidated, but may involve spinal N-methyl-D-aspartate (NMDA) receptors, particularly the NR2B subunit. As polyamines are positive modulators of NMDA-NR2B receptors and mainly originate from dietary intake, the modulation of polyamines intake could represent an interesting way to prevent/modulate neuropathic pain symptoms by opposing glutamate neurotransmission.
Methods
The effect of a polyamine deficient diet was investigated in an animal model of oxaliplatin-induced acute pain hypersensitivity using behavioral tests (mechanical and cold hypersensitivity). The involvement of spinal glutamate neurotransmission was monitored by using a proton nuclear magnetic resonance spectroscopy based metabolomic approach and by assessing the expression and phosphorylation of the NR2B subunit of the NMDA receptor.
Results
A 7-day polyamine deficient diet totally prevented oxaliplatin-induced acute cold hypersensitivity and mechanical allodynia. Oxaliplatin-induced pain hypersensitivity was not associated with an increase in NR2B subunit expression or phosphorylation, but with an increase of glutamate level in the spinal dorsal horn which was completely prevented by a polyamine deficient diet. As a validation that the oxaliplatin-induced hypersensitivity could be due to an increased activity of the spinal glutamate system, an intrathecal administration of the specific NR2B antagonist, ifenprodil, totally reversed oxaliplatin-induced mechanical and cold hypersensitivity.
Conclusion
A polyamine deficient diet could represent a promising and valuable nutritional therapy to prevent oxaliplatin-induced acute pain hypersensitivity.

• Assessment of thermal sensitivity in rats using the thermal place preference test: description and application in the study of oxaliplatin-induced acute thermal hypersensitivity and inflammatory pain models. (2014)
Assessment of thermal sensitivity in rats using the thermal place preference test: description and application in the study of oxaliplatin-induced acute thermal hypersensitivity and inflammatory pain models.
Balayssac D, Ling B, Ferrier J, Pereira B, Eschalier A, Authier N.
Faculties of Medicine and Pharmacy, Clermont Université, France
Published in "Behav Pharmacol." (2014-04-25)

Thermal sensitivity is an essential characteristic of some painful states, including oxaliplatin-induced neuropathy. The thermal place preference test (TPPT) was designed to finely assess thermal sensitivity in rodents. The TPPT monitors the time spent by unrestrained rodents on a test plate at fixed temperatures (5-50°C) compared with an adjacent reference plate at a neutral temperature (25°C). Here, we report the results of a study designed (i) to validate the optimal methodological parameters for measuring thermal sensitivity in rats, (ii) to assess the thermal sensitivity of healthy rats and animal models of pain and (iii) to explore the pharmacological effects of analgesic drugs. The most reproducible conditions occurred when the TPPT was performed in the morning and in the dark for 3 min with the reference plate set to 25°C. The temperature preferences of healthy rats were more than 17°C and less than 40°C. When compared with control animals, oxaliplatin-treated rats showed thermal hypersensitivity at 12, 20 and 35°C, and carrageenan-treated rats showed thermal hypersensitivity at 15 and 45°C. Duloxetine (2.5 mg/kg, intraperitoneal) reversed oxaliplatin-induced cold hypersensitivity (20°C) and morphine (1 mg/kg, intravenous) reversed carrageenan-induced heat hypersensitivity (45°C). We conclude that the TPPT enables a fine-grained assessment of thermal sensitivity that is relevant to the pathophysiological exploration of animal pain models and to the pharmacological assessment of analgesic drugs.



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