Formalin-induced Pain In Rats Neurons Dopamine-¦Â-hydroxylase Expression Changes And Its Regulatory Mechanism

PART ONE THE CHANGES OF DBH AND AP-2 ALPHA EXPRESSION IN THE SPINAL CORD AND BRAIN OF FORMALIN-INDUCION RAT PAIN MODELObjective The aim of the present study was to investigate the changes of dopamine-β-hydroxylase (DBH) and activator protein 2-α(AP-2α) expression in spinal cord, locus coeruleus (LC), cerebellar Purkinje cells (PCs), and the behavioral changes in formalin-induced pain rat, so as to explore the mechanism of modulation of pain stress resulting from formalin injection.Methods①One hundreds and ten Wistar rats were randomly divided into control and experiment group. The animals of experiment groups were injected subcutaneously with formalin into the plantar surface of the right hidpaw. The animals of control group were injected with saline into the same site. The tissue was properly taken at an interval of 1, 6, 24 h, 3 d, 7 d after formalin injection.②To observe body surface changes of the injected region and the behavioral changes in the experiment animals. ③After formalin injection, HE staining, Nissl staining, transmission electron microscopy were used to detect the morphological changes of the spinal cord, LC and cerebellar PCs;④Immunohistochemical staining (IHC), double immunofluorescence staining were used to detect the changes of DBH/ AP-2αand its mRNA expression in spinal cord, LC, cerebellar PCs of formalin-induced pain rat.⑤Western blotting and RT-PCR were used to detect the changes of DBH/ AP-2αand its mRNA expression in spinal cord, LC and cerebellum of formalin-induced pain rat.⑥Computing-image analysis system were used to detect the changes of DBH/ AP-2αstaining intensity and the number of DBH- positive neurons in the experimental groups.⑦The data were statistically analyzed with SPSS software.Results①After formalin injection, the nociceptive behavior of rats to the stress of pain showed biphasic pain response and imflammation appeared in the injected region of rats. However, there was no obviously morphological change observed in the expertment groups by HE staining. The nissl staining showed that the number of nissl body increased and the staining enhanced after formalin injection. Only were the increased endocytoplasmic reticulum and Golgi's complex seen in the neurons at ultrastructural level after formalin injection. ②The PCs in both control group and experiment groups showed positive staining of DBH/ AP-2α. However, the staining density of the PCs in experimental groups were signifcantly increased compared with that in control group(P<0.05). The grey value of DBH/ AP-2α-positive staining reached its peak on day 3 after formalin-injection and began to decrease on day 7, but still higher than that in the control group (P<0.05). The rules of AP-2αexpression changes were similar to those of DBH expression changes in cerebellar PCs of painful rats. The results of Western blotting and RT-PCR confirmed that the rules of DBH/ AP-2αand its mRNA expressin changes were consistant with those of DBH/ AP-2αexpression changes in IHC. Co-existence of DBH and AP-2αin the PCs was observed by means of double immunofluorescence staining.③A small number of DBH-positive neurons sparsely distributed in the LC of normal spinal cord. In the formalin-treated group, however, much more darkly-stained DBH-positive neurons appeared in the LC. The grey value and the number of DBH-positive neurons reached its peak on day 3 after formalin-injection (P<0.05). On the day 7 after formalin injection, it began to decrease, but still higher than that in the control group (P<0.05). The grey value change of AP-2αexpression was similar to that of DBH expression in the LC of pain model rat.④A small number of DBH-positive neurons sparsely distributed in the anterior horn of the normal spinal cord. However, in the formalin-treated group, much more darkly-stained DBH-positive neurons increased in the anterior horn, and appeared in the intermediate zone and the dorsal horn, which reached its peak on day 3 after formalin-injection. The grey value and number of DBH-positive neurons on day 7 post-injection began to decrease, but still higher than that in the control group. Compared with control group, the number of noradrenergic neurons in spinal cord of formalin-treated rat significantly increased (P<0.05). The increasing of DBH expression was also confirmed by western blotting and RT-PCR. Double immunofluorescence staining showed that DBH and AP-2αco-existed in the neurons of the spinal cord. The change of AP-2αexpression was similar to that of DBH in the spinal cord of pain model rat.Conclusions①The PCs showed positive staining of DBH. However, the staining density of the PCs in experimental groups was signifcantly increased compared with control group (P<0.05). The results indicated that NA in PCs may be involved in the formalin-induced pain and/or behavior regulation.②The grey value and number of DBH-positive neurons were increased after formalin-injection in LC (P<0.05). The results indicated that noradrenaline in LC cells may be involved in the pain and behavior regulation.③After formalin injection, the grey value of DBH-positive neruons were increased, and much more DBH-positive neurons increased in anterior horn and appeared in intermediate zone, and the dorsal horn. The results indicated that some non-noadrenergic neurons with different chemical properties might convert into noadrenergic neurons under pain stimulation. The neurons have the property of chemical plasticity.④The changes of AP-2αexpression were similarly to that of DBH in the PCs, LC cells and spinal cord neurons of pain model rat. Double immunofluorescence staining showed that DBH and AP-2αco-existed in the cells of the spinal cord and PCs. The results indicated that AP-2αmay enhance the DBH synthesis. PART TWO THE CHANGES OF BEHAVIOR AND NA-ENERGIC NEURONS AFTER INJECTION WITH NA/DBH INHIBITOR IN THE FORMALIN-INDUCED PAIN RATSObjective To investigated the changes of behavior and the expression of DBH in NA-energic neurons of the formalin-induced pain rats, after intrathecal injection with NA or DBH inhibitor (U-14,624),.Methods①Sixty six Wistar rats were randomly divided into six groups (U-14624,NA,NS,PFA+NS,PFA+U-14624,PFA+NA). The tissue was properly taken at an interval of 1 h, 6 h, 24 h, 3 d, 7 d after injection at right footplate.②The animals were intrathecal injected with diverse reagents by intrathecal placed tubes (NS and PFA+NS groups with NA, PFA+NA and NA groups with NA, U-14624 and PFA+U-14624 groups with U-14624).③At 10 min after intrathecal injection, the animals of PFA+NS, PFA+U-14624 and PFA+NA groups were subcutaneous injected with 0.1 ml 3% formalin at the right footplate. The animanls of control group (NA, U-14624 and NS groups) were subcutaneous injected with equivalence saline at the same site.④The changes of injected region body surface and the changes of behavior. Were observed in rats The scores of behavior and the foundation pain threshold were recorded and statistical analyzed.⑤At 24 h after formalin injection, HE staining, Nissl staining, transmission electron microscope were used to detect the change of morphology in the spinal cord.⑥Immunohistochemical staining, Western blotting and RT-PCR were used to detect the changes of DBH and its mRNA expression in spinal cord.⑦Computing-image analysis system and statistical analysis were used to analyse the experiment results.Results:①After intrathecal injection, the animals had no respiratory depression, death and spinal injury reduced hind limb paralysis.②The animals showed typical two biphasic pain response after formalin injection.③There were no significant behavior changes after intrathecal injection with NA (the NA group, Vs the NS groups, P>0.05). The scores of behavior in PFA+NA groups showed that there was significantly analgesia effect in the first and second stages nociceptive pain reaction after formalin injection (P<0.05). The pain sensibility of animals was decreased after intrathecal injected with NA on the formalin-induced pain model rats (P<0.05).④There were no significant behavior changes after intrathecal injection with U-14624 (the U-14624 group, Vs the NS groups, P>0.05). The scores of behavior in PFA+ U-14624 groups showed that there was no significant effect in the first stage nociceptive pain reaction after formalin injection (P>0.05), but there was significantly enhancement on the pain sensibility of animals in the second nociceptive pain reaction(Vs the PFA group, P<0.05). The pain sensibility of animals were decreased after intrathecal injection with NA on the formalin-induced pain model rats (PFA+NA) (P<0.05).⑤At 24 h after formalin injection in the PFA+NA groups, the IHC showed that the number and stainning intensity of DBH-positive neurons were increased and enhanced compared with the control group (NS, NA groups) (P<0.05), while decreased and lower than the formalin-induced pain model rats (PFA group) (P<0.05). There changes were also confirmed by western blotting and RT-PCR.⑥At 24 h after formalin injection in the PFA+U-14624 groups, the IHC showed that the number and stainning intensity of DBH-positive neurons increased and enhanced obviously compared with the control groups (NS, NA groups) (P<0.05), and its be no significantly decreased and lower than the formalin-induced pain model rats (PFA+NS groups) (P>0.05). There changes were also confirmed by western blotting and RT-PCR.Conclusion After intrathecal injection with NA, there was significant analgesia in the formalin-induced pain model rats, and significant feedback suppression to the activity of DBH. After intrathecal injected with DBH inhibitor, the behavior detection showed that there was significant effect in the first stage of pain model rats, and significant change in the second stage of the pain model rats. The results showed that U-14624 had no influence on the algesia by direct effect, U-14624 maybe inhibit the activity of DBH, then decrease the synthesis of NA, result in the behavior changes of the pain model rat. The IHC result showed that the DBH inhibitor (U-14624) had no effect on the quatitative of DBH. The results showed NA in the spinal cord maybe involved in the pain regulation and analgesia, and decrease the pain sensitive of the pain model rats. PART THREE EFFECTS OF NA AND DBH INHIBITOR ON THE ELECTROPHYSIOLOGICAL OF RAT PURKINJE CELLS AND ANTERIOR HORN NEURONSABSTRACTObjective The aim of the present study was to investigate the resting membrane potential (RMP) and spontaneous action potential (SAP) of rat cerebellar PCs and anterior horn neurons after perfusion with NA and DBH inhibitor (U-14624).Methods Patch clamp technique of cerebellar slice and/or spinal cord slice were used to observe the RMP and SAP changes of rat cerebellar PCs and anterior horn neurons perfusion with ACSF containing NA and/or DBH inhibitor.Results①The suitable neurons for patch clamp technique were easily obtained by cerebellar slice and/or spinal cord slice compared with single cell isolation.②In control spinal cord anterior horn neurons, the amplitude of RMP was -43.65±5.21 mV (n=48) and the frequency of SAP was 1.62±0.26 Hz.③In cerebular PCs, the amplitude of RMP was -50.71±2.21 mV (n=39) and the SAP has two types (simple spike and complex spike). The frequency of complex spike was 2.86±0.31 Hz. The frequency of simple spike was 35.37±5.63 Hz.④After perfusion with NA to the spinal cord anterior horn neurons, the amplitude of RMP was -48.39±7.87 mV (n=24) and the frequency was 3.04±0.52 Hz;⑤After perfusion with NA, the electrical activity of the cerebellar PCs became immediately. The amplitude of RMP was -54.29±2.48 mV (n=22), Most SAP of the PCs were depression (68.18%), some were excition (22.73%) and few was biphase (9.09%).⑥After a few minitues perfusion with DBH inhibitor (U-14624) , the amplitude of RMP was -38.39±4.19 mV and the frequency of SAP was 0.89±0.20 Hz.⑦After perfusion with DBH inhibitor (U-14624) to the cerebellar PCs, there was no obviously change in the RMP. The high-intensity discharge of SAP come out immediately, and the frequency of SAP was 30.74±13.20 Hz. At 5-20 min after perfusion, most of the SAP was excition (60%), some were depression (33.33%) and a few were diphase (6.67%).Conclusions①By cerebellum slice and/or spinal cord slice, the neurons could maintain the structure and function, and which electrical actibity could be recorded easily. ②The changes of RMP and SAP were inhibited in anterior horn neurons by NA. The changes of RMP and SAP were complicted in cerebellar PCs by NA, the inhibition was the main effect. Through the neural network or directly combination to the receptors of the neurons, the RMP and SAP of spina cord anterior horn neurons were influenced by NA.③The SAP of rat cerebellar PC was potentiated in few minutes after perfusion with DBH inhibitor. This change may be correlated to direct effect. The changes of SAP were mainly excition on rat cerebellar PCs by DBH inhibitor, however, it was inhibition in anterior horn neurons by DBH inhibitor. These may be correlated to the inhibitor of DBH activity and the decreasing of NA synthesis.The mechanism of electrophysiological changes in rat cerebellar PCs by NA and/or DBH inhibitor were analysised. It was provided the way foundation for studying the physiological functions of two cells.