Central and peripheral mechanisms of pain: behavioral and immunoihistochemical investigations for the study of innovative therapeutic approaches in animal models

Pain represents a complex and diversified phenomenon which starting from a foundamental function of “alert” can be transformed in a chronic condition, a pathology itself, strongly invalidating life. Chronic pain is one of the more difficult health problem to be solved with a very huge social component.
Animal models represent a great advantage for the comprehension of physiopathological mechanisms underlying acute and chronic pain in humans. Pain is experimentally induced through nociceptive stimuli of different origin. Although many results on mechanisms underlying chronic pain have recently been acquired as well as a higher aknowledge of the great number of molecules involved, it is not established how these molecules interact at the different levels of the nervous system and how they are modulated by other systems.
The lack of complete knowledge of the neurobiology of pain, specifically the passage from acute to chronic pain, is related to the lack of pharmacological treatments. Actually, drugs represent only palliative therapies and are often associated to tolerance and side effects.
The aim of our laboratory is to investigate, in animal models, neural processes undelying different types of pain, specifically inflammatory and neuropathic. Behavioral responses to thermal, chemical and mechanical nociceptive stimuli are investigated and the effects of pharmacological treatments acting on neuropeptides and/or neurotransmitters involved in pain modulation are evaluated.
The study of the behavioral responses to different stimuli allows us to take into account a number of factors influencing pain perception, such as age, sex, environmental factors etc. Behavioral modulation by drugs administered centrally (intracerebroventricularly or intrathecally), systemically or peripherally, may help to specify the neural component involved (cerebral, spinal etc.), and to improve the comprehension of pain mechanisms.
Our approach is fundamentally a pharmacological-behavioral approach. In addition the purpose is to correlate behavioral observations with structural and functional changes through other investigative approaches, e.g. immunohistochemical and immunofluorescence, carried out in our and other laboratories through scientific cooperations.
A very new pharmacological approach recently carried out in our laboratory with encouraging results (ref.2,4,6) is the use of very low, not toxic doses of bacterial toxins to counteract inflammatory and neuropathic pain.
In particolar, the working projects focuse on the investigation of the effects of central and/or peripheral administration of :
a) different serotypes of botulinum neurotoxins (BoNTs) from Clostridium botulinum interacting with synaptic neurotransmission (see Siro Luvisetto).
b) the Cytotoxic Necrotizing Factor 1 (CNF1), a protein toxin from Escherichia coli.

a) Botulinum Neurotoxins
We have demonstrated the ability of BoNT/A to reduce formalin-induced pain as well as neuropaty induced by sciatic nerve ligation. Work is now in progress to determine, through immunofluorescence techniques, possible differences in the expression of neuronal and glial markers involved in degenerative and regenerative processes following the neuropathy induction. When neuropathy is induced in animals, a complex process occurs that, together with axonal degeneration, is associated to infiltration of cells of the immune system such as macrophages and astroglia. The phenomena related to degeneration of peripheral nerves and regeneration processes play a great role also in a therapeutic perspective.

Fig.1: BoNT/A (3.75, 7.5, 15 picogr/mouse, red lines) exerts analgesic effects on both phasic and tonic phases of formalin-induced inflammatory pain. Left: time course of licking behaviour induced by formalin. Right: total licking time during the first (0-10) and second (10-40) phases characterizing the formalin test.
The study of the intracellular signaling, e.g. p38 mitogen-activated protein kinase (p38 MAPK) pathway, which is critycally involved in the previous mentioned processes, as well as macrophages' localization with mono and policlonal antibodies, are carried out toghether with behavioural and functional investigations.
Fig.2: Analgesic effect of BoNT/A (3.75, 7.5, 15 picogr/mouse) on neuropathic pain in mice.
The arrow indicates the injection of BoNT/A, subcutaneously administered into the paw.
b) CNF1
The peculiarity of the use of CNF1 in treating pain is its mecchanism of action. CNF1 is crucially linked to the activation of the Rac GTPase, a key regulatory protein that is switched on by the toxin, leading to remodeling of the actin cytoskeleton in intact cells. Results obtained provide new insights into the comprehension of inflammatory pain through the investigation of the intracellular mechanisms involved and represent an innovative and encouraging approach to set up a strategic treatment. Further experiments are planned to investigate the interaction with opioid system and the effects on neuropathic pain.

Fig.3: Mechanism of action of CNF1

With the aim to identify new drugs able to selectively modulate pain transmission, our group, in collaboration with EBRI (European Brain Research Institute) and LLG (Lay Line Genomics), has focused the attention on the NGF/TrkA complex as possible target for inhibiting pain (ref.1). The common project is still in progress.

Principal Investigator
Flaminia Pavone, CNR senior researcher
flaminia.pavone@cnr.it - Ph. +39 06 501703271

Research Group
Siro Luvisetto, CNR Researcher
Sara Marinelli, CNR Researcher, temporary contract
Valentina Vacca, Post-Doc
Alba Finocchiaro, PhD student
Elena Fiori, PhD student
Silvia Squillace, student
Jessica Tofanicchio, student

• Istituto di Neurobiologia e Medicina Molecolare, CNR
• Lay Line Genomics, Rome, Italy
• Dipartimento di Neuroscienze, Università di Tor Vergata, Rome, Italy
• Dipartimento di Genetica e Biologia Molecolare, Università "La Sapienza", Rome, Italy
• Dipartimento di Fisiologia, Sezione di Neuroscienze e Fisiologia Applicata, University of Siena, Italy
• Dipartimento di Scienze Biomediche Sperimentali, University of Padua, Italy
• European Brain Research Institute, Rome, Italy
• Drug Dept., Division of Pharmacology of Degenerative and Aging Processes, Health High Institute, Rome, Italy
• Institute of Pharmacology, Polish Academy of Sciences, Cracow, Poland

Selected references
- Vacca V., Marinelli S., Pieroni L., Urbani A., Luvisetto S., Pavone F. 17beta-estradiol counteracts neuropathic pain: a behavioural, immunohistochemical and proteomic investigation on sex-related differences in mice. Sci Rep. 2016 Jan 8;6:18980. doi: 10.1038/srep18980.
- Boccella S., Vacca V., Errico F., Marinelli S., Squillace M., Guida F., Di Maio A., Vitucci D., Palazzo E., De Novellis V., Maione S., Pavone F., Usiello A. D-aspartate modulates nociceptive-specific neuron activity and pain threshold in inflammatory and neuropathic pain condition in mice. Biomed Res Int. 2015;2015:905906. doi:10.1155/2015/905906.

- Luvisetto S., Gazerani P., Cianchetti C., Pavone F (2015). Botulinum Toxin Type a as a Therapeutic Agent against Headache and Related Disorders. Toxins (Basel) 23, 3818-3844. Review.
- Marinelli S., Eleuteri C., Vacca V., Strimpakos G., Mattei E., Severini C., Pavone F., Luvisetto S. (2015). Effects of age-related loss of P/Q-type calcium channels in a mice model of peripheral nerve injury. Neurobiology of Aging 36, 352-364.
- De Angelis F., Marinelli S., Fioretti B., Catacuzzeno L., Franciolini F., Pavone F., Tata AM. M2 receptors exert analgesic action on DRG sensory neurons by negatively modulating VR1 activity. J Cell Physiol. 2014 Jun;229(6):783-90.
- Marinelli S., Nazio F., Tinari A., Ciarlo L., D'Amelio M., Pieroni L., Vacca V., Urbani A., Cecconi F., Malorni W., Pavone F. Schwann cell autophagy counteracts the onset and chronification of neuropathic pain. Pain. 2014 Jan;155(1):93-107.
- Pavone F., Ueda H. Is BoNT/B useful for pain treatment? Pain. 2014 Apr;155(4):649-50.
- Vacca V., Marinelli S., Pieroni L., Urbani A., Luvisetto S., Pavone F. Higher pain perception and lack of recovery from neuropathic pain in females: a behavioural, immunohistochemical, and proteomic investigation on sex-related differences in mice. Pain. 2014 Feb;155(2):388-402.
- Tiveron C., Fasulo L., Capsoni S., Malerba F., Marinelli S., Paoletti F., Piccinin S., Scardigli R., Amato G., Brandi R., Capelli P., D'Aguanno S., Florenzano F., La Regina F., Lecci A., Manca A., Meli G., Pistillo L., Berretta N., Nisticò R., Pavone F, Cattaneo A. ProNGF\NGF imbalance triggers learning and memory deficits, neurodegeneration and spontaneous epileptic-like discharges in transgenic mice. Cell Death Differ. 2013 Aug;20(8):1017-30.
- Vacca V., Marinelli S., Luvisetto S., Pavone F. Botulinum toxin A increases analgesic effects of morphine, counters development of morphine tolerance and modulates glia activation and µ opioid receptor expression in neuropathic mice. Brain Behav Immun. Brain Behav Immun. 2013 Aug;32:40-50.
- Vacca V., Marinelli S., Pieroni L., Urbani A., Luvisetto S., Pavone F. Higher pain perception and lack of recovery from neuropathic pain in females: a behavioural, immunohistochemical, and proteomic investigation on sex-related differences in mice. Pain. 2014 Feb;155(2):388-402. doi: 10.1016/j.pain.2013.10.027. Epub 2013 Nov 11.
- Caprioli A., Coccurello R., Rapino C., Di Serio S., Di Tommaso M., Vertechy M., Vacca V., Battista N., Pavone F., Maccarrone M., Borsini F., The novel reversible fatty acid amide hydrolase inhibitor ST4070 increases endocannabinoid brain levels and counteracts neuropathic pain in different animal models. J Pharmacol Exp Ther J Pharmacol Exp Ther. 2012 Jul; 342(1):188-95.

- Capsoni S., Marinelli S., Ceci M., Vignone D., Amato G., Malerba F., Paoletti F., Meli G., Viegi A., Pavone F., Cattaneo A. Intranasal "painless" human Nerve Growth Factors slows amyloid neurodegeneration and prevents memory deficits in App X PS1 mice. PLoS One. 2012;7(5):e37555.

- Covaceuszach S., Marinelli S., Krastanova I., Ugolini G. Pavone F., Lamba D., Cattaneo A. Single cycle structure-based humanization of an anli-nerve growth factor therapeutic antibody. PLoS One. 2012;7(3):e32212.
- D'Amato F.R., Pavone F. Modulation of nociception by social factors in rodents: contribution of the opioid system. Psychopharmacology (Berl). 2012 Nov;224(1):189-200.
- Razzoli M., Bo E., Pascucci T., Pavone .F, D'Amato F.R., Cero C., Sanghez V., Dadomo H., Palanza P., Parmigiani S, Ceresini G., Puglisi-Allegra S., Porta M., Panzica G.C., Moles A., Possenti R., Bartolomucci A. Implication of the VGF-derived peptide TLQP-21 in mouse acute and chronic stress responses. Behav Brain Res. 2012 Apr 15;229(2):333-9.
- Marinelli S., Vacca V., Ricordy R., Uggenti C., Tata A.M., Luvisetto S., Pavone F. The Analgesic Effect on Neuropathic Pain of Retrogradely Transported botulinum Neurotoxin A Involves Schwann Cells and Astrocytes. PLoS One. 2012;7(10):e47977.
- Vacca V., Marinelli S., Eleuteri C., Luvisetto S., Pavone F. Botulinum neurotoxin A enhances the analgesie effects on inflammatorv pain and antagonizes tolerance induced by morphine in mice. Brain Behav Immun. 2012 Mar;26(3):489-99.
- De Leonibus E., Costantini V.J., Massaro A., Mandolesi G., Vanni V., Luvisetto S., Pavone F., Oliverio A., Mele A. Cognitive and neural determinants of response strategy in the dual-solution plus-maze task. Learn Mem. 2011;18(4):241-4.
- Capsoni S., Covaceuszach S., Marinelli S., Ceci M., Bernardo A., Minghetti L., Ugolini G., Pavone F., Cattaneo A. Taking pain out of NGF: a "painless" NGF mutant, linked to hereditary sensory autonomic neuropathy type V, with full neurotrophic activity. PLoS One. 2011;6(2):e17321.
- Mika J., Rojewska E., Makuch W., Korostynski M., Luvisetto S., Marinelli S., Pavone F., Przewlocka B. The effect of botulinum neurotoxin A on sciatic nerve injury-induced neuroimmunological changes in rat dorsal root ganglia and spinal cord. Neuroscience. 2011;175:358-66.
- Pavone F.,Luvisetto S.. Botulinum Neurotoxin for Pain Management: Insights from Animal Models. Toxins 2010; 2(12), 2890-2913.
- Cobianchi S., Marinelli S., Florenzano F., Pavone F., Luvisetto S. Short- but not long-lasting treadmill running reduces allodynia and improves functional recovery after peripheral nerve injury. Neuroscience. 2010;168(1):273-87.
- Covaceuszach S., Capsoni S., Marinelli S., Pavone F., Ceci M., Ugolini G., Vignone D., Amato G., Paoletti F., Lamba D., Cattaneo A. In vitro receptor binding propertiesof a "painless" NGF mutein, linked to hereditary sensory autonomic neuropathy type V. Biochem Biophys Res Commun. 2010;391(1):824-9.- Marinelli S., Luvisetto S., Cobianchi S., Makuch W., Obara I., Mezzaroma E., Caruso M., Straface E., Przewlocka B., Pavone F. Botulinum neurotoxin type A counteracts neuropathic pain and facilitates functional recovery after peripheral nerve injury in animal models. Neuroscience. 2010; 171(1):316-28.
- Nalepa I., Vetulani J., Borghi V., Kowalska M., Przewlocka B., Roman A., Pavone F. Changes induced by formalin pain in central alpha1-adrenoceptor density are modulated by adenosine receptor agonists. J Neural Transm. 2010;117(5):549-58.
- Ugolini G., Marinelli S., Covaceuszach S., Cattaneo A., Pavone F. The function neutralizing anti-TrkA antibody MNAC13 reduces inflammatory and neurophatic pain. Proc. Natl. Acad. Sci., USA, 104(8), 2985-90, 2007.
- Luvisetto S, Marinelli S, Cobianchi S, Pavone F.: Anti-allodynic efficacy of botulinum neurotoxin A in a model of neuropathic pain. Neuroscience. Mar 2;145(1)1-4, 2007.
- Luvisetto S, Marinelli S, Panasiti MS, D'Amato FR, Fletcher CF, Pavone F, Pietrobon D. Pain sensitivity in mice lacking the Ca(v)2.1alpha1 subunit of P/Q-type Ca2+ channels. Neuroscience. Oct 27;142(3), 823-32, 2006.
- Luvisetto S, Marinelli S, Lucchetti F, Marchi F, Cobianchi S, Rossetto O, Montecucco C, Pavone F. Botulinum neurotoxins and formalin-induced pain central vs. peripheral effects in mice. Brain Res., 1082(1), 124-31, 2006
- Nalepa I., Vetulani J., Borghi V., Kowalska M., Przewlocka B., Pavone F. Formalin hindpaw injection induces changes in the [(3)H]prazosin binding to alpha(1)-adrenoceptors in specific regions of the mouse brain and spinal cord. J. Neural Transm., 112(10), 1309-13319, 2005.
- Luvisetto S, Marinelli S, Rossetto O, Montecucco C, Pavone F. Central injection of botulinum neurotoxins behavioural effects in mice. Behav Pharmacol. 2004 May;15(3), 233-40.
- Borghi V., Przewlocka B., Labuz D., Maj M., Obara I., Pavone F. Formalin induced pain and mu-opioid receptor density in brain and spinal cord are modulated by A1 and A2a adenosine agonist in mice. Brain Research, 956 339-348, 2002.
- Capone F., Aloisi A.M., Carli G., Sacerdote P. and Pavone F. Oxotremorine-induced modifications of the behavioral and neuroendocrine responses to formalin pain in male rats. Brain Research, 830, 292-300, 1999.
- D'Amato F.R., Mazzacane E., Capone F., Pavone F. Effects of postnatal manipulation on nociception and morphine sensitivity in adult mice. Brain Res. Dev. Res., 117(1), 15-20, 1999.
- Przewlocka B., Mika J., Capone F., Machelska H. and Pavone F. Intrathecal oxotremorine affects formalin-induced behavior and spinal nitric oxide synthase immunoreactivity in rats. Pharmacol. Biochem and Behav. 62(3), 1999, 531-536.

- "Physostigmine derivatives with acetylcholinesterase inhibition properties, and the relative production process"(C.N.R. Bollettino Ufficiale - Norme tecniche e Brevetti - A.XVIII-N.98,1984) (European Patent n°.89116950.0-1990).
Inventors: Mario Brufani, Claudio Castellano, Maurizio Marta, Alberto Oliverio, Flaminia Pavone, Massimo Pomponi.
- "Molecules that are able to inhibit the binding between NGF and TrkA receptor as analgesics with prolonged effect" Italian patent: RM2005A000290, European Patent: WO 2006/131951 A2.
Inventors: Flaminia Pavone, Sara Marinelli, Antonino Cattaneo, Gabriele Ugolini.
- "Method for the potentiation of opioid analgesics effects on pain". Italian Patent:RM2005A000332. European Patent: WO 2006/137106 A2.
Inventors: Flaminia Pavone, Sara Marinelli, Antonino Cattaneo, Gabriele Ugolini.
- "Fattori proteici dermonecrotizzanti di origine batterica e relativi usi in campo medico". Italian Patent: RM2005A000422.
Inventors: Alessia Fabbri, Loredana Falzano, Carla Fiorentini, Siro Luvisetto, walter Malorni, Sara Marinelli, Flaminia Pavone, Elisabetta Straface