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      Spinal autophagy is differently modulated in distinct mouse models of neuropathic pain

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          Abstract

          Background

          Autophagy is a homeostatic degradative process essential for basal turnover of long-lived proteins and organelles as well as for removal of dysfunctional cellular components. Dysregulation of the autophagic machinery has been recently associated to several conditions including neurodegenerative diseases and cancer, but only very few studies have investigated its role in pain processing.

          Results

          We previously described autophagy impairment at the spinal cord in the experimental model of neuropathic pain induced by spinal nerve ligation (SNL). In this study, we characterized the main autophagic markers in two other common experimental models of neuropathic pain, the chronic constriction injury (CCI) and the spared nerve injury (SNI). The different modulation of LC3-I, Beclin 1 and p62 suggested that autophagy is differentially affected in the spinal dorsal horn depending on the type of peripheral injury. Confocal analysis of p62 distribution in the spinal dorsal horn indicated its presence mainly in NeuN-positive cell bodies and occasionally in glial processes, thus suggesting a predominant expression in the neuronal compartment. Finally, we investigated the consequences of autophagy impairment on pain behaviour by using the autophagy blocker cloroquine. Intrathecal chloroquine injection in naïve mice induced spinal accumulation of LC3 and p62 paralleled by significant mechanical hypersensitivity thus confirming the block in autophagosome clearance and suggesting the participation of the autophagic process in spinal mechanisms of pain processing. Altogether, our data indicate that spinal autophagy is differentially altered in different experimental pain models of neuropathic pain and that this process may be relevant for pain control.

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          Most cited references42

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          Guidelines for the use and interpretation of assays for monitoring autophagy.

          In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. A key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process vs. those that measure flux through the autophagy pathway (i.e., the complete process); thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from stimuli that result in increased autophagic activity, defined as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (in most higher eukaryotes and some protists such as Dictyostelium) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the field understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field.
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            Spared nerve injury: an animal model of persistent peripheral neuropathic pain.

            Peripheral neuropathic pain is produced by multiple etiological factors that initiate a number of diverse mechanisms operating at different sites and at different times and expressed both within, and across different disease states. Unraveling the mechanisms involved requires laboratory animal models that replicate as far as possible, the different pathophysiological changes present in patients. It is unlikely that a single animal model will include the full range of neuropathic pain mechanisms. A feature of several animal models of peripheral neuropathic pain is partial denervation. In the most frequently used models a mixture of intact and injured fibers is created by loose ligation of either the whole (Bennett GJ, Xie YK. A peripheral mononeuropathy in rat that produces disorders of pain sensation like those seen in man. Pain 1988;33:87-107) or a tight ligation of a part (Seltzer Z, Dubner R, Shir Y. A novel behavioral model of neuropathic pain disorders produced in rats by partial sciatic nerve injury. Pain 1990;43:205-218) of a large peripheral nerve, or a tight ligation of an entire spinal segmental nerve (Kim SH, Chung JM. An experimental model for peripheral neuropathy produced by segmental spinal nerve ligation in the rat. Pain 1992;50:355-363). We have developed a variant of partial denervation, the spared nerve injury model. This involves a lesion of two of the three terminal branches of the sciatic nerve (tibial and common peroneal nerves) leaving the remaining sural nerve intact. The spared nerve injury model differs from the Chung spinal segmental nerve, the Bennett chronic constriction injury and the Seltzer partial sciatic nerve injury models in that the co-mingling of distal intact axons with degenerating axons is restricted, and it permits behavioral testing of the non-injured skin territories adjacent to the denervated areas. The spared nerve injury model results in early ( 6 months), robust (all animals are responders) behavioral modifications. The mechanical (von Frey and pinprick) sensitivity and thermal (hot and cold) responsiveness is increased in the ipsilateral sural and to a lesser extent saphenous territories, without any change in heat thermal thresholds. Crush injury of the tibial and common peroneal nerves produce similar early changes, which return, however to baseline at 7-9 weeks. The spared nerve injury model may provide, therefore, an additional resource for unraveling the mechanisms responsible for the production of neuropathic pain.
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              A peripheral mononeuropathy in rat that produces disorders of pain sensation like those seen in man.

              A peripheral mononeuropathy was produced in adult rats by placing loosely constrictive ligatures around the common sciatic nerve. The postoperative behavior of these rats indicated that hyperalgesia, allodynia and, possibly, spontaneous pain (or dysesthesia) were produced. Hyperalgesic responses to noxious radiant heat were evident on the second postoperative day and lasted for over 2 months. Hyperalgesic responses to chemogenic pain were also present. The presence of allodynia was inferred from the nocifensive responses evoked by standing on an innocuous, chilled metal floor or by innocuous mechanical stimulation, and by the rats' persistence in holding the hind paw in a guarded position. The presence of spontaneous pain was suggested by a suppression of appetite and by the frequent occurrence of apparently spontaneous nocifensive responses. The affected hind paw was abnormally warm or cool in about one-third of the rats. About one-half of the rats developed grossly overgrown claws on the affected side. Experiments with this animal model may advance our understanding of the neural mechanisms of neuropathic pain disorders in humans.
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                Author and article information

                Contributors
                berliocchi@unicz.it
                mariamaiaru@hotmail.it
                giuseppe.varano@hotmail.it
                rossella.russo@unical.it
                mtcorasa@unicz.it
                g.bagetta@unical.it
                cristina.tassorelli@mondino.it
                Journal
                Mol Pain
                Mol Pain
                Molecular Pain
                BioMed Central (London )
                1744-8069
                2 February 2015
                2 February 2015
                2015
                : 11
                : 3
                Affiliations
                [ ]Department of Health Sciences, University “Magna Græcia” of Catanzaro, 88100 Catanzaro, Italy
                [ ]Centre of Neuropharmacology of Normal and Pathological Synaptic Plasticity, University Consortium for Adaptive Disorders and Head Pain, 87036 Rende, Cosenza, Italy
                [ ]Department of Pharmacy, Health and Nutritional Sciences, Section of Preclinical and Translational Pharmacology, University of Calabria, 87036 Rende, Cosenza, Italy
                [ ]C. Mondino National Neurological Institute, Pavia, Italy
                [ ]Department of Brain and Behavioral Sciences, University of Pavia, Pavia, Italy
                Article
                722
                10.1186/1744-8069-11-3
                4417307
                25645145
                e8daa0b3-8fe7-4bcd-bde4-0d656587a930
                © Berliocchi et al.; licensee BioMed Central. 2015

                This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License ( http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver ( http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

                History
                : 14 September 2014
                : 6 January 2015
                Categories
                Research
                Custom metadata
                © The Author(s) 2015

                Molecular medicine
                spinal nerve ligation,chronic constriction injury,spared nerve injury,neuropathic pain,autophagy,lc3,sqstm1/p62,beclin 1,chloroquine

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