The development of metabolic stable non

The development of metabolic stable, non-peptidergic galanin agonists could be the basis to clinical diagnosis and treatment of tumors with high levels of galanin receptor expression like gliomas and pituitary tumors. As for gliomas, penetration of the blood brain barrier by the compounds might be possible since the barrier is frequently disturbed in glial tumors. In addition, local injection of radiolabeled peptide analogues has already been tested for therapy of CNS tumors (Hofer et al., 2001, Merlo et al., 1999).
Acknowledgments
Introduction Galanin is a neuropeptide containing 29 (30 in human) amino Galanthamine residues [8]. The neuropeptide is widely distributed in the central nervous system and peripheral tissues [8], [10]. There are three types of galanin receptors, galanin receptor 1 (GalR1), galanin receptor 2 (GalR2), galanin receptor 3 (GalR3), and all the galanin receptors are G protein coupled receptors [1], [10]. It is well known that galanin and its receptors play a lot of physiological functions, such as endocrine modulation, learning and memory, and nociceptive modulation [2], [10], [18]. Periaqueductal grey (PAG) is one of the most important brain structures involved in the endogenous anti-nociceptive system [12]. It has been demonstrated that there are galanin existed in PAG, as well as GalR2 [6], [10], [14]. Ca(2+)/calmodulin-dependent kinase II (CaMKII) has been demonstrated to be involved in nociceptive modulation [3], [9]. Activation of GalR2 induces the increase in the content of Ca2+, the increased content of Ca2+ may then activate CaMKII and induce nociceptive modulation [10]. Previous studies in our laboratory have demonstrated that galanin and its receptors play important roles in nociceptive modulation in PAG [15], [16]. The present study was performed to explore the effect of the GalR2 antagonist M871 on the galanin-induced antinociception in PAG, and whether CaMKII is involved in the galanin-induced antinociception.
Methods All experiments were performed on freely moving male Sprague-Dawley rats weighting 200–250g (Experimental Animal Center of LUYE Pharma Company, Yantai, China). The rats were housed in cages with free access to food and water, and maintained in a climate-controlled room on a normal day/night cycle. Behavioral tests were performed during the day cycle. All experiments were conducted according to the guidelines of the International Association for the Study of Pain [19] and every effort was made to minimize both the animal suffering and the number of animals used. All rats were accustomed to the testing conditions for five to six days before the starting of the experiment to minimize the stress induced by handling. The hindpaw withdrawal latencies (HWLs) during thermal and mechanical stimulation were measured as described previously [5], [7]. The hot-plate test was used to assess the HWL to thermal stimulation. The entire ventral surface of the rat’s hindpaw was placed manually on a hot-plate which was maintained at a temperature of 52°C (51.8–52.4°C). The time to hindpaw withdrawal was measured in seconds and referred to as the HWL to thermal stimulation. The Randall Selitto test (Ugo Basile, Type 7200, Italy) was used to assess the HWL to mechanical stimulation. A wedge-shaped pusher at a loading rate of 30g/s was applied to the dorsal surface of a hindpaw. The latency required to initiate the withdrawal response was assessed and expressed in seconds. The HWL was tested 3 times and the average values obtained were regarded as the basal HWL. Then intra-PAG injection was performed over one minute. The HWLs recorded during subsequent experiments were expressed as percentage changes from the basal level (% change of HWL) for each rat. Each rat was tested by both types of stimulation. A cut-off limit of 15s was set up to avoid tissue damage. Animals were anesthetized by intraperitoneal injection of sodium pentobarbital (50mg/kg) and were placed on a stereotaxic instrument. Two stainless steel guide cannulas of 0.8mm outer diameter were directed to the both sides of PAG (B, −4.8mm; L or R, 0.7mm; V: 4.5mm. B, anterior (+) or posterior (−) to Bregma; L or R, left or right to midline; V, ventral to the surface of skull) according to the Paxinos and Watson [13] and was fixed to the skull by dental acrylic. There were more than 3 days for rats to recover from the operation. On the day of experiment, a stainless steel needle with 0.4mm outer diameter was directly inserted into the guide cannula with 1.5mm beyond the tip of the latter. One micro-liter of solution was infused into PAG over 1min and the injection needle was left in the site for 1min after each injection.