GONG Qing-juan，CHEN Jin-sheng，HUANG Qiao-dong，LU Zhen-he

(Department of Pain Medicine，The Second Affiliated Hospital，Guangzhou Medical College，Guangzhou 510260，China.E-mail:luzhh@126.com)

Src family kinases(SFKs)belong to the family of non-receptor protein tyrosine kinases and are expressed in the central nerve system(CNS)[1].In the developed CNS，some data indicate that SFKs act as a point of convergence for various signaling pathways and might be involved in the processes underlying physiological and pathological plasticity，such as learning and memory，epilepsy and neurodegeneration[1].Recently，it has been reported that SFKs are selectively activated in spinal microglia after peripheral nerve injury，and intrathecal administration of PP2，an inhibitor of all SFK members，suppresses mechanical hypersensitivity in nerve-injured rats[2]，suggesting that microglial SFKs are crucial for neuropathic pain.Long-term potentiation(LTP)，a phenomenon of persistent increase in synaptic strength produced by repetitive presynaptic stimulation，was first described in hippocampus and is considered as a fundamental mechanism of memory storage[3].Besides the hippocampus，LTP has also been found in the spinal dorsal horn[4].Spinal LTP of C-fiber evoked field potentials can be induced by tetanic stimulation of sciatic nerve，natural noxious stimulation on peripheral tissues or nerve injury.Accordingly，it is believed to underlie pathological pain[5]，manifested as allodynia，hyperalgesia and spontaneous pain.Our previous works have shown that spinal application of ATP induced LTP[6].In the present study，the effects of SFKs on ATP-induced LTP in spinal dorsal horn were investigated by the techniques of Western blotting，immunohistochemistry and electrophysiology.

MATERIALS AND METHODS

1 Animal preparation

Experiments were performed on male Sprague-Dawley rats(250-280 g).Urethane(1.5 g/kg，ip)was used to induce and maintain anesthesia.The trachea was cannulated，and the animals breathed spontaneously.One carotid artery was cannulated to continuously monitor the mean arterial blood pressure，which was maintained from 80 to 120 mmHg.A laminectomy was performed to expose the lumbar enlargement of spinal cord.The left sciatic nerve was dissected free for bipolar electrical stimulation with platinum hook electrodes.All exposed nervous tissues were covered with warm paraffin oil except the spinal lumbar enlargement，onto which the drugs will be applied.Colorectal temperature was kept constant(37-38℃)by means of a feedback-controlled heating blanket.At the end of the experiments，the animals were killed with an overdose of urethane.

2 Drug preparation

ATP(Sigma)was dissolved in double-distilled water and diluted with artificial cerebrospinal fluid(ACSF)to make the final concentration of 3×10-4mol/L just prior to use.4-Amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3，4-d]pyrimidine(PP2，Calbiochem)，2-oxo-3-(4，5，6，7-tetrahydro-1H-indol-2-ylmethylene)-2，3-dihydro-1H-indole-5-sulfonic acid dimenthylamide(SU6656，Calbiochem)and 4-amino-7-phenylpyrazolo[3，4-d]pyrimidine(PP3，Calbiochem)were first dissolved in dimethyl sulphoxide(DMSO)to make a stock concentration of 50 mmol/L，aliquoted in small volumes and stored at-80℃.The stock solution was subsequently diluted with 0.9%saline to make final concentrations immediately before administration.The final concentration of DMSO was＜0.5%.Previous works have shown that spinal application of 0.5%DMSO does not affect C-fiber evoked field potentials[7].

3 Methods

3.1 Western blotting The dorsal quadrants of L4-L5 spinal cord on the stimulated side were isolated and put into liquid nitrogen immediately，followed by homogenization in 1.5 ×10-2mol/L Tris buffer，pH 7.6.The tissues were sonicated on ice，and then centrifuged at 13 000×g for 15 min at 4℃to prepare the supernatant containing protein samples.Proteins were separated by gel electrophoresis(SDS-PAGE)and transferred onto a PVDF membrane(Bio-Rad).The blots were blocked with 5%W/V non-fat dry milk in TBST for 1 h at room temperature and then incubated with primary antibodies，including rabbit polyclonal anti-rat p-SFKs(Tyr416)antibody(1∶1 000，Cell Signaling Technology)and rabbit polyclonal anti-rat β-actin antibody(1∶1 000，Cell Signaling Technology)，overnight at 4℃ with gentle shaking.The blots were washed 3 times for 15 min each with washing buffer(TBST)and then incubated with secondary antibody horseradish peroxidase(HRP)-conjugated goat antirabbit IgG(1∶1 000，Santa Cruz)for 2 h at room temperature.After incubated with the secondary antibody，the membrane was washed，again，as above.The immune complex was detected by ECL(Amersham)and exposed to X-ray film(Kodak).The band intensities on the film were analyzed by densitometry with a computer-assisted imaging analysis system(Kontron IBAS 2.0).To calculate the phosphorylated versus nonphosphorylated form of protein，the same membrane was stripped with stripping buffer(6.75 ×10-2mol/L Tris，pH 6.8，2%SDS，and 0.7% β-mercaptoethanol)for 30 min at 50℃and reprobed with rabbit polyclonal anti-rat SFKs antibody(1∶1 000，Cell Signaling Technology)，and detected as above.

3.2 Electrophysiological recording Recording of C-fiber-evoked field potentials in spinal dorsal horn was performed according to our previous described method[4，6].Briefly，following electrical stimulation of the sciatic nerve，field potentials were recorded in the spinal dorsal horn(L4 and L5 segments)at the depth of 50-500 μm from the dorsal surface with a tungsten microelectrode(World Precision Instruments;impedance 1-2 MΩ，exposed tip diameter∶1-2 μm).An A/D converter card(DT2821-F-16SE，Data Translation)was used to digitize and store data in a computer at a sampling rate of 10 kHz.Single square pulses(0.5 ms duration，in 1 min intervals)delivered to the sciatic nerve were used as test stimuli.The strength of stimulation was adjusted to 1.5-2 times the threshold for C-fiber response.Tetanic stimulation(0.5 ms duration，40 V，100 Hz，given in 4 trains of 1 s duration at 10 s intervals)was used to induce LTP[4，8，9].The distance from the stimulation site at the sciatic nerve to the recording site in lumbar spinal dorsal horn was around 11 cm.

3.3 Immunohistochemistry Following electrophysiological recordings，the rats were perfused with 300 mL saline followed by 350 mL of cold 0.1 mol/L phosphate-buffer saline(PBS)containing 4%paraformaldehyde.The L4-L5 of spinal cord were removed，postfixed for 3 h，and then replaced with 30%sucrose for 2 d at 4 ℃.Transverse spinal sections(free-floating，25 μm)were cut in a cryostat(Leica CM3050S)and processed for immunofluorescence，according to the methods as described previously[6].Briefly，the sections were blocked with 3%donkey serum in 0.3%Triton for 1 h at room temperature and incubated over night at 4 ℃ with anti-p-SFKs antibody(1∶500).The sections were then incubated for 1 h at room temperature with Cy3-conjugated secondary antibody(1∶300，Jackson immunolab).For double immunofluorescence，spinal sections were incubated with a mixture of anti-p-SFKs receptors antibody and monoclonal neuronal-specific nuclear protein(NeuN，neuronal marker，1∶500，Chemicon)，glial fibrillary acidic protein(GFAP，astrocyte marker，1∶500，Chemicon)，Iba-1(microglia marker，1∶500，Serotec)over night at 4℃，followed by a mixture of FITC-(1∶200;Jackson ImmunoResearch)and CY3-conjugated secondary antibodies for 1 h at room temperature.The stained sections were examined with an Olympus IX71 fluorescence microscope(Olympus Optical)and the images were captured with a CCD spot camera.The area of p-SFKs-immunoreactive(IR)per section was measured in the spinal dorsal horn using a computerized image analysis system(NIH ImageJ).A density threshold was set above background level firstly to identify positively stained structure.In each rat，4 to 6 sections of the spinal cord at each time point were selected randomly.An average percentage of area of p-SFKs-IR，relative to the total area of the spinal dorsal horn of the sections，was obtained for each animal across the different tissue sections.Six rats were included in each group for quantification of the immunohistochemical results[10，11].The measurement of the area of p-SFKs-IR was performed by a person who did not know the experiment design.

4 Statistical analysis

For the data of electrophysiology，the amplitudes of C-fiber evoked field potentials were determined offline by parameter extraction.In each experiment，amplitudes of 5 consecutive field potentials recorded were averaged.The mean amplitudes of the averaged responses prior to drug application or tetanic stimulation served as baseline control.The summary data from different animals were expressed as mean±sE(±sE).Statistical tests were performed with SPSS 10.0.The relative densities of Western blotting at different time points after ATP application were compared to those in control animals，using ANOVA with the least significant difference test(LSD-t).Other data were compared with nonparametric test(Friedman ANOVA for repeated measurements).P＜0.05 was considered significant.

RESULTS

1 Spinal application of ATP activated SFKs in microglia

Evidence has shown that activation of SFKs in spinal dorsal horn plays an important role in the development of neuropathic pain[2].We therefore examined whether SFKs are activated after LTP induction by ATP.The results showed that the levels of p-SFKs in spinal dorsal horn were significantly increased 30 min and 60 min after ATP application，compared with the ACSF group(Figure 1，F(xiàn)igure 2A-D).To determine the cell types that expressed the activated SFKs，we performed double immunofluorescence staining 30 min after spinal application of ATP and found that p-SFKs was co-localized exclusively with Iba-1，but not with GFAP or NeuN(Figure 3A-C)，indicating that ATP may activate SFKs only in microglia but not in astrocyte or neuron.

Figure 1.The level of p－SFKs was significantly increased after ATP application in spinal dorsal horn.A:the bands showed the phosphorylated and total SFKs and β－actin from protein samples of ipsilateral spinal dorsal horn(L4－L5)30 min and 60 min after ATP application;B:the bar chart showed the p－SFKs levels were normalized by total SFKs levels and expressed as percentages of their controls. ± sE.n=3.＊＊ P ＜ 0.01 vs naive or control.

Figure 2.The level of p－SFKs in spinal dorsal horn was increased after LTP induction by ATP.A－C:representative images showed the immunofluorescence staining of p－SFKs from an ACSF control rat(A)and from the rats in which LTP was induced by ATP for 30 min(B)and 60 min(C).Scale bar:200 μm.D:quantification of p －SFKs－immunoreactive positive area in spinal dorsal horn in ACSF control rats，and in rats that LTP was induced 30 min or 60 min after the application of ATP.±sE.n=6.＊＊P＜0.01 vs control.

Figure 3.p－SFKs expressed in microglia of spinal dorsal horn after LTP induction by ATP.The results of double immunofluorescence staining of p－SFKs(red，A，B，C)with Iba－1，a microglia marker(green，A)，NeuN，a neuronal marker(green，B)，and GFAP，an astrocyte marker(green，C)showed that p－SFKs exclusively co－localized with Iba －1(yellow in A).Scale bar:200 μm.

2 Inhibition of SFKs prevented ATP－induced spinal LTP

To evaluate the role of activation of SFKs in spinal LTP induction，PP2，a specific inhibitor of SFKs was applied onto spinal dorsal surface at recording segments 30 min before ATP.We found that ATP(3×10-4mol/L)induced spinal LTP of C-fiber-evoked field potentials(Figure 4C)and PP2(1×10-4mol/L)completely blocked LTP induced by ATP(Figure 4A)，whereas PP3(1×1 0-4mol/L)，an inactive form of PP2，had no influence on LTP induction(Figure 4B).To confirm the role of SFKs in ATP-induced spinal LTP，another SFKs inhibitor SU6656 was tested with the same protocol.We found that in the rats pretreated with SU6656(2×10-4mol/L)，ATP did not induce spinal LTP(Figure 4A).PP2 and SU6656 did not affect the baseline of C-fiber response[12].The results indicate that the activation of SFKs contributes to ATP-induced spinal LTP.

Figure 4.The effect of SFKs in ATP－induced spinal LTP.A:spinal application of ATP did not induce LTP of C－fiber－evoked field potentials in the rats pretreated with SFKs inhibitor (PP2 or SU6656).B:spinal application of PP3 had no effect on ATP－induced LTP.C:spinal application of ATP induced LTP.

DISCUSSION

In this study，we found that spinal application of ATP significantly increased the level of p-SFKs，and p-SFKs were only localized in spinal microglia.Spinal application of SFKs inhibitors(PP2 and SU6656)prevented ATP-induced LTP.These results indicate that SFKs may play a key role in ATP-induced LTP of C-fiber evoked field potentials in the spinal dorsal horn.

Src，Lyn，F(xiàn)yn，Lck and Yes are 5 members of the SFK family and express in the CNS.As non-receptor protein tyrosine kinases，SFKs are involved in neuronal development and synaptic plasticity[1]，and play a key role in regulating the functions of glial cells，including microglia，a type of immune cell that resides in the CNS.Accumulating evidence has shown that the activation of glial cells is crucial for the pathogenesis of pathological pain produced by nerve injury[13，14].The activated microglia may contribute to pain hypersensitivity by releasing the pro-inflammatory cytokines such as TNF-α and IL-1β.Furthermore，the activation of glial cells is necessary for the spinal LTP induction in vivo[15]and in vitro[16].Recent data indicate that the level of p-SFKs in spinal microglia is increased after LTP induced by high frequent stimulation[12].Similar results were also found in our study.It has been demonstrated that SFKs are activated in spinal microglia[2]and can induce upregulation of P2X4receptors after peripheral nerve injury[17].Our previous study has shown that ATP bound to intrinsic P2X4receptors and enhanced the expression of P2X4receptors，contributing to LTP in the spinal dorsal horn[6].Therefore，it is possible that SFKs may play an important role in ATP-induced spinal LTP by upregulating the expression of P2X4receptors，and further studies are needed to confirm how the activation of microglial SFKs regulates synaptic plasticity in the spinal dorsal horn.

ACKNOWLEDGEMENTS

The authors are very grateful to Professor LIU Xian-guo(Department of Physiology，Zhongshan School of Medicine，Sun Yat-sen University)for his support and help in our study.

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