Neuropharmacology and AnalgesiaA single subcutaneous injection of ozone prevents allodynia and decreases the over-expression of pro-inflammatory caspases in the orbito-frontal cortex of neuropathic mice
Introduction
Neuropathic pain is caused by a primary lesion or dysfunction in both the peripheral and central nervous systems, often leading to a long-lasting increase in the excitability of spinal dorsal horn neurons (central sensitization) (Bonica, 1970, Zimmermann, 2001). Examples of neuropathic pain include carpal tunnel syndrome, trigeminal neuralgia, post-herpetic neuralgia, disc herniation, phantom limb pain, complex regional pain syndromes and the various peripheral neuropathies. The hallmarks of neuropathic pain are chronic allodynia and hyperalgesia. Allodynia is defined as pain resulting from a stimulus that ordinarily does not elicit a painful response. Hyperalgesia is defined as an increased sensitivity to normally painful stimuli (Bonica, 1970).
Recent studies indicate that apart from the better-explored spinal cord neuroplastic changes in neuropathic pain conditions (Ossipov et al., 2007, Kozai et al., 2007), supraspinal brain regions are also affected in spared nerve injury and spinal nerve transection models in rodents (Apkarian et al., 2006, Xie et al., 2006). Human brain imaging studies have revealed new roles of cortical neuronal networks in chronic pain, including the activation of the synaptic excitatory and inhibitory neurotransmission, gene activation of neurotrophic factors and protein involved in receptor glutamate formation, new cortical connections and possible GABAergic neuron apoptosis (Zhuo, 2008). Moreover, patients with chronic back pain showed 5–10% less neocortical gray matter volume than control subjects (Apkarian et al., 2004) and neuronal or glial apoptosis (possibly caused by excitatory and inflammatory pathways) has also been considered in such a morpho-functional frontal atrophy. Many different drugs have been used in the treatment of neuropathic pain. These include a wide range of drugs with peripheral and central neuronal or non-neuronal targets, such as antiepileptic drugs, opioids and antidepressant agents. Furthermore, utilization of these molecules may be limited by pharmacokinetic factors and pose a high risk for central side effects (Gidal, 2006, Frank et al., 2008). The ameliorative effect of ozone has been shown in patients affected by low back pain in lumbar and cervical disk herniation, without any significant complications when topically applied (Gallucci et al., 2007, Bonetti et al., 2005, Muto et al., 2004, Barber et al., 1999, Hernandez et al., 1995, Ginanneschi et al., 2006, Alexandre et al., 2005).
Ozone is an unstable and irritant gas with a pungent odour which has a strong oxidizing power and good antiseptic, disinfectant and antiviral properties (Wells et al., 1991). Ozone has also been proposed as an immunomodulator and activator of cellular metabolism which shows long-term anti-inflammatory effects and reduces inflammation with an apparent low toxicity (Chang et al., 2007). Ozone therapy increases the endogenous antioxidant system activities in endotoxic and septic shock models (Zamora et al., 2005). However, there is no study aimed at identifying the possible molecular mechanisms of the anti-allodynic/hyperalgesic effect of ozone and the possible involvement of some inflammatory and apoptotic pathways at supraspinal limbic level (i.e. the orbito-frontal cortex).
The caspase family comprises at least 13 different cysteine proteases that are mainly involved in the apoptotic pathways (Cohen, 1997). Nevertheless, to a greater extent than apoptosis, caspase-1 plays a pivotal role in regulating cellular export of pro-inflammatory cytokines (i.e. IL-1β and IL-18) and is present in several central nervous system disease inflammatory processing, such as autoimmune demyelination (Furlan et al., 1999), spinal cord trauma or inflammation (Clark et al., 2006) and neuropathic and chronic inflammatory pain (Joseph and Levine, 2004).
In this study we have addressed the issue of whether peripheral neuropathy induced by the spared nerve injury of the sciatic nerve could modify the expression of pro-inflammatory and pro-apoptotic caspases (caspase-1, caspase-3, caspase-12 and caspase-8) in the orbito-frontal cortex, together with allodynia and hyperalgesia development. Furthermore, we have studied whether a single subcutaneous administration of ozone has anti-allodynic and anti-hyperalgesic effects, and whether or not it is capable of modifying caspase expression and IL-1β immunoreactivity in the orbito-frontal cortex of SNI mice.
Section snippets
Animals
Male C57BL/6N mice (35–40 g) were housed 3 per cage under controlled illumination (12-h light/12-h dark cycle; light on 06:00 h) and environmental conditions (room temperature 20–22 °C, humidity 55–60%) for at least 1 week before the commencement of experiments. Mouse chow and tap water were available ad libitum. The experimental procedures were approved by the Animal Ethics Committee of the Second University of Naples. Animal care was in compliance with the IASP and European Community (E.C.
Ozone, but not oxygen, prevents the development of mechanical allodynia
The nociceptive response to mechanical stimulation (mechanical paw withdrawal threshold, PWT) was measured every 30 min for 3 h before surgery and 14 days after spared nerve injury of the sciatic nerve. The mean PWT was calculated from six consecutive trials and averaged from each group of mice. In naive mice, the behavioural change of mechanical allodynia did not undergo significant modification during analgesimeter registration (mean ± S.E.M.: 9.75 ± 0.53 g). Spared nerve injury of the sciatic
Discussion
This study offers first evidence that caspase-1, caspase-8 and caspase-12 gene expression and IL-1β protein were significantly increased in the orbito-frontal cortex of mouse 14 days after spared nerve injury of the sciatic nerve. Unlike the above mentioned caspases, the expression of caspase-3; the “canonical” protease effector of cell death, decreased in the orbito-frontal cortex in spared nerve injury mice. The caspase family comprises thus far 13 different cysteine proteases that are mainly
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