The Effect Of Formalin-induced Pain On CCK Immunoreactivity In Rat Sensory Neurones

CCK is a kind of brain-gut peptide that is abundantly distributed in both gastrointestinal tract and the central and peripheral nervous systems. In the central nervous system, the sulfated octapeptide fragment of cholacystokinin (CCK-8s) is engaged in numerous physiological and pathological processes including behavior, anxiety, learning/memory processes and pain. CCK-8s is one of the strongest endogenous anti-opioid substances and suppresses morphine tolerance which results from long term use of morphine. Here we observe the effect of formalin-induced pain on CCK immunoreactivity in rat sensory neurons through immunohistochemistry technique, and test the effect of CCK-8s on membrane potential of DRG neurons through intracellular recording technique. Results are as follows:1. The Effect of Formalin-induced Pain on positive rate of CCK Immunoreactivity in Rat DRG Neurons.After 1h of subcutaneous injection of saline in one paw of rats, we observe that CCK immunoreactivity in few small, moderate and big neurons on DRG are weak positive both in injection and non-injection side. Positve rate of CCK immunoreactivity of small neurons on DRG are 24.66%±4.81% and 26.21%±4.23% in injection and non-injection respectively(t=0.22,P>0.05); and positve rate of CCK immunoreactivity of moderate and big neurons on DRG are 3.99%±1.75% and 4.44%±1.54% respectively(t=0.16, P>0.05), no remarkable difference reveals.After 1h of subcutaneous injection of formalin in one paw of rats, positive rate of CCK immunoreactivity of small neurons on DRG is 60.37%±6.92% in injection side which is greater than that of saline group(t=4.21,P<0.01). and positive rate of CCK immunoreactivity of moderate and big neurons on DRG is 26.96%±4.01%, higher than that of saline group (t=5.24, P<0.01). In non-injection side, positive rate of CCK immunoreactivity of small neurons on DRG is 25.22%±5.08%, no differences reveal compared with saline group(t=0.08,P>0.05); positive rate of CCK immunoreactivity of moderate and big neurons on DRG is 11.73%±2.73% which is a remarkable difference compared with saline group(3.99%±1.75%) (t=2.38, P<0.05). Compare positive rates of CCK immunoreactivity of small neurons on DRG of injection side and non-injection side, the difference is significant (t=8.94,P<0.01), so do the moderate and big neurons on DRG(t=2.81,P<0.05).After 3h of subcutaneous injection of formalin in one paw of rats, positive rate of CCK immunoreactivity of small neurons on DRG is 49.78%±3.68% in injection side which is slightly lower than that of injection of formalin after 1h (t=1.35,P>0.05). and positive rate of CCK immunoreactivity of moderate and big neurons on DRG is 30.17%±2.87% which is slightly higher than that of injection of formalin after 1h (26.96%±4.01%) (t=0.65 P>0.05). Compare positive rates of CCK immunoreactivity of small neurons on DRG of injection side and non-injection side, the difference is apparent(t=4.36,P<0.01), so do the moderate and big nerons on DRG(t=4.44,P<0.01).2. The Effect of Formalin-induced Pain on CCK Immunoreactivity in Rat DRG Neurons.After 1h of subcutaneous injection of saline in one paw of rats, the semi-quantitative optical density average values of CCK immunoreactivity neurons on DRG are 0.163±0.002 and 0.168±0.003 in injection side and non-injection side respectively(t=1.49,P>0.05), no remarkable difference reveals.After 1h of subcutaneous injection of formalin in one paw of rats, the semi-quantitative optical density average value of CCK immunoreactivity neurons on DRG is 0.236±0.012 in injection side which is higher than that of saline group after 1h (0.163±0.002) (t=5.49,P<0.01). Compare non-injection side of formalin with non-injection side of saline, the semi-quantitative optical density average values of CCK immunoreactivity neurons on DRG, the results are similar (t=0.92,P>0.05). the value in formalin injection side is greater than that of non-injection side(t=6.65,P<0.01).After 3h of subcutaneous injection of formalin in one paw of rats, the semi-quantitative optical density average value of CCK immunoreactivity neurons on DRG is 0.209±0.006 while it is 0.236±0.012 after 1h in injection side(t=2.01,P>0.05). In non-injection side, this value of CCK immunoreactivity neurons on DRG is 0.168±0.003 while it is 0.173±0.005 after 1h (t=0.92,P>0.05). the value in injection side of formalin is (0.209±0.006) greater than that of non-injection side(0.168±0.003) (t=7.33,P<0.01).3. The Effect of Formalin-induced Pain on CCK Immunoreactivity in Spinal Cord Neurons.After subcutaneous injection of saline in one paw of rats, we observe that CCK immunoreactivity in few neurons in spinal cord is positive. The semi-quantitative optical density average values of CCK immunoreactivity neurons are 0.261±0.007 and 0.263±0.007 in injection side and non-injection side respectively(t=0.44,P>0.05).After 1h of subcutaneous injection of formalin in one paw of rats, the number of positive neurons of CCK immunoreactivity in spinal cord neurons is obviously increasing and greater than that of non-injection side. The semi-quantitative optical density average values of CCK immunoreactivity neurons are 0.397±0.014 and 0.295±0.007 in injection side and non-injection side respectively(t=8.19,P<0.01), the difference is obvious . The value(0.397±0.014) in injection side of formalin is greater than that of (0.261±0.007) injection side of saline (t=8.85,P<0.01), and the value (0.295±0.007) in non-injection side of formalin is greater than that of (0.263±0.007) non-injection side of saline (t=3.31,P<0.01).After 3h of subcutaneous injection of formalin in one paw of rats, the number of positive neurons of CCK immunoreactivity in spinal cord becomes fewer than that of after 1h. The semi-quantitative optical density average values of CCK immunoreactivity neurons are 0.366±0.009 and 0.303±0.005 in injection side and noninjection side respectively(t=8.19,P<0.01), the difference is of significance; the value(0.366±0.009) in injection side of formalin is lower than it (0.397±0.014)in injection side of formalin after 1h (t=1.86,P>0.05); and the value (0.303±0.005) in non-injection side of formalin is greater than that of(0.295±0.007)in non-injection side of formalin after 1h (t=0.96,P>0.05), but the difference is not significant.4.CCK-8s evoked membrane depolarization in rat DRG neuronsAbout 45.6% (26/57) CCK-8s (10-8~10-6 mol/L) evoked a depolarizing response. The membrane depolarization induced by CCK-8s was concentration dependent and abolished by prolumide (10-4 mol/L), a selective antagonist of CCK-A receptor (n=8). LY225910 (10-4 mol/L), a selective antagonist of CCK-B receptor (n=9), had no effect on CCK-8s evoked membrane depolarization.Results indicate:1) The positive rate and semi-quantitative optical density average value of CCK immunoreactivity neurons on DRG and in spinal dorsal horn neurons increase with formalin induced pain, this reveals that CCK participates in modulation of pain sensory conduction;2) There are possibly CCKA receptor and CCKB receptor on DRG neurons. Stimulating CCKA receptor can lead to depolarization reaction in DRG neurons, but CCKB cannot, which indicates that CCKA and CCKB play different physiological roles;3) The CCK neuropeptidein exists in the plasma of DRG neurons, and CCKA and CCKB on the membrane of DRG neurons, so we can conclude that presynaptic CCKA and CCKB receptors exist in the presynaptic membrane of primary sensory afferent endings.