The Contributions Of INOS In The Spinal Dorsal Horn In A Rat Model Of Postherpetic Neuralgia

Background and objective Pain is one of the most unbearable and miserable experience for people with disease,especially like postherpetic neuralgia(PHN).The main clinical manifestation of PHN is allodynia(pain occurning normally non-painful stimulation) and hyperalgesia(increased sensitivity to normally non-painful stimulation).Pain is often prolonged and persists for more than 3 months. The group attacked by PHN is mainly composed of the old people. However, China is becoming an aging society. PHN will severely threaten the health of older Chinese,as well as the quality of their life. PHN responds poorly to using antiviral drugs,anticonvulsants,electric acupuncture and moxibustion and Chinese medicine clinically;and so far,there is not any effective method to prevent or treat PHN.In fact,PHN is a special kind of neuropathic pain,but the neural mechanism underlying PHN remains unclear.According to classic views, the pain pathway and mechanism of PHN have been simply described as inflammation and denaturation of peripheral nerves[3].However,more and more recent studies show that central nervous system is also identified to be strongly involved in the creation and maintenance of PHN.The spinal dorsal horn,where there are abundant second order neurons(the major is specific algesia neurons), is the key of pain pathophysiology regulating. In our experiment,we shall view the spinal dorsal horn as our entry point, and intend to explore and research the neuropathic and molecular mechanism underlying PHN.In our pre-experiment,we have discovered that expression of iNOS considerably upregulated during the processing of allodynia and hyperalgesia in a rat model of PHN.Thus,we guess whether iNOS expession has related to the neuropathic and molecular mechanism of pain?How does it impact on the processing of performing allodynia and hyperalgesia?Whether it can exert significant analgesia effect in VZV infected rats by using of intrathecal treatment with selective or competitive iNOS inhibitors? How does it guide to clinical treatment?In order to solve these question above,we designed our experiment.Methods ⑴Making animal models of postherpetic neuralgia: Rats were divided into VZV group, Mock infected group and Naive group in random(n=10/group).VZV was propagated on African green monkey kidney fibroblast cells(CV-1) cells and harvested in phosphate-buttered saline(PBS,pH7.3) when the cells exhibited 80% cytopathic effect.The plantar surface of rats hindpaws were injected with 50μl inoculum containing VZV-infected cells(as VZV group). Control rats were injected with uninfected cells(as Mock infected group),or 0.9% of sodium chloride injection(as Naive group)[2].A few days afterwards,when VZV group come into appearing allodynia and hyperalgesia,we treat them with injecting anticonvulsants and observe whether the hindpaws withdrawal threshold will change or even recovery?⑵Immunofluorescence histochemical staining and double-labeling immunofluorescence: ①Tissue preparation :after rats were anesthetized and perfused transcardially with paraformaldehyde solution, the lumbosacral enlargement of spinal cord was eviscerated and transferred into 20% sucrose in PBS for cryoprotection,and the lumbosacral enlargement was cut into longitudinal 15μm thick sections in frozen section machine and mounted onto glass-slides.Ten tissue selections from each rat were selected(10/group).And then being blocked with ten percent normal oat serum,these selections were incubated with corresponding antibodies.②After washed with PBS and0.3%Triton-100,the lumbosacral enlargement sections were incubated with mouse anti-GFAP IgG(1:500) and mouse anti-OX-42 IgG(1:200)for 2 days at 4 ℃.③Rabbit anti-iNOS IgG(1:400) and nNOS IgG(1:400) were respectively double labelled with mouse anti-GFAP IgG(1:500) and mouse anti-OX-42 IgG(1:200) in the lumbosacral enlargement sections.④After staining,observed the sections with laser scanning microscope[2, 4].⑤Finally,watched whether the staining density of the spinal dorsal horn in the sections will change,composed with control group?⑶Western blot analysis:Rats were anesthetized and the lumbosacral enlarement of the spinal cord was rapidly eviscerated on the surface of ice fragments. The tissue was mechanically homogenized and centrifuged.The object protein concentrations of the supernatant were determined using BCA Protein Assay Kit,then separated by SDS-PAGE electrophoresis(25μg/well) and transferred onto nitrocellulose membranes.The membranes were placed in a blocking solution,and incubated overmight with rabbit anti- iNOS IgG(1:200) 、 rabbit anti-nNOS IgG(1:200) 、 and rabbit anti-eNOS IgG IgG(1:200)respectively.After washing,the membranes were incubated in peroxidase-conjugated secondary antibody(1:800) for 2 hours,and then the membranes were detected by the enhanced chemiluminescence detection method.The densities of proteins blots were analyzed by using the labworks software and normalized to β-actin levels[3, 4].⑷Intrathecal catheter insertion and drug administration:VZV group rats were anesthetized.PE-10 catheter was inserted into lumbosacral enlargement of the spinal subarachnoid space in rat by using a catheter-through-technique,then tunnelled under the skin between two ears[5, 6].When the hind paws withdrawal threshold of VZV group significantly decreased and reached the lowest value at 14 d,the treatment group received intrathecal injection of PTIO(NO scavenger,30μg),L-NAME(i NOS broad-spectrum inhibitor 50μg),L-NIL(iNOS specific inhibitor 100μg),7-NINA(nNOS inhibitor,45μg)or L-NIO(eNOS inhibitor,25μg),while the same volume of 0.9% of sodium chloride injection was injected in control group,then observed the effect of analgesia.Finally,detected paws withdraw threshold of rats namely,10 min、20min、30min、40min、50min and 60 min after injected, and collected data.Results :⑴Compared to Naive group and Mock infected group, allodynia of VZV group was observed approximately 5 days post-infection(11.4±1.6g), reached the lowest value at 14 d(5.8±1.4g),and continued undiminished to 8 weeks post-infection.(n =10/group; P < 0.05).This progress of the paw withdraw threshold persisted until 8th week,and approached normal level until 12 th week.There was any rash in the injury skin and any effect of analgesia after using antiviral therapy,the same as the manifestation of patients suffered PHN.⑵While the allodynia and hyperalgesia of rats developed,the staining density of iNOS in spinal dorsal horn of rats has increased significantly in VZV group,compared to Naive group and Mock infected group. And one more thing,iNOS-immunopositive structure was concentrated in lamina I and II of spinal dorsal horn;and i NOS-LI was localized in GFAP-immunopositive cells but not in OX42-immunopositive cells or NeuN-immunopositive cells in spinal cord of VZV infected rats[1]. Bar = 25 μm.⑶The expression of iNOS but not nNOS or eNOS was increased in the spinal cord of VZV infected rats, which was related to mechanical allodynia. Compared to Naive rats(0.06 ± 0.02)and Mock infected rats(0. 05 ± 0.01),Western blot analysis showed that spinal i NOS expression was significantly increased in VZV infected rats(0.68 ± 0.13). aP < 0.05, bP < 0.01 vs Naive rats and Mock infected rats.The expression level of iNOS was found to be significantly correlated to the paw withdrawal threshold in VZV infected rats(P < 0.001, r = – 0.89). With regard to nNOS or eNOS expression in spinal cord, there was no difference among Naive rats, Mock infected rats and VZV infected rats. In VZV infected rats, nNOS or eNOS expression was unchanged through the period tested.[1](n = 10/group/day)⑷When the hindpaws withdrawal threshold of VZV group significantly decreased and reached the lowest value at 14 th day[1],the treatment group received intrathecal injection of PTIO( NO scavenger,30μg),L-NAME(iNOS broad-spectrum inhibitor 50μg),L-NIL(iNOS specific inhibitor 100μg),all of rats in this group achieved analgesia.However,there was no change of hindpaws withdrawal threshold of VZV group received intrathecal injection of7-NINA(nNOS inhibitor,45μg)or L-NIO(eNOS inhibitor,25μg).(n=10/group)Conclusions : ⑴PHN is not caused by acute replication of herpes virus simply.PHN is a specific kind of neuropathic and chronic pain,caused by the infection of herpes virus,while the mechanism underlying PHN is not simpliy thereplication of the virus.In our experiment,there was not any rash on the skin of rats.After the use of antiviral drugs,there was no effect of analgesia.⑵The place of NOS regulating chronic pain mainly focus on the laminaI and laminaII in the spinal dorsal horn.The spinal dorsal horn is located in the second order neurons on the way of pain conductivity. The outermost layer of the spinal dorsal horn is called lamina I or the marginal layer,where most of the neurons are specific pain-sensing neurons,receiving pain signals transmitted from Aδ fibers of skin or internal organs.The lamina II,also called gelatinous layer which receives peripheral chronic pain signals transmitted from C fibers,is adjoining the marginal layer.Function of NOS for regulating pain sensory transmission is performed here.⑶There is a significant negative correlation between the expression of NOS and pain threshold value. After the binding site in the promoter of iNOS combined with NF-κ B,NOS is stimulated.The increased activity of NOS can produce excess NO,which indirectly activates c-fos,and a series of extensive biological effect can also be caused. In this way, altered synaptic plasticity and central sensitization produce hyperalgesia,thus the threshold value of rats with PHN decreased.⑷It is iNOS rather than nNOS or eNOS that acts as the role of regulator in mechanism underlying PHN. In drug management test,the paw withdraw threshold recovered to its normal level gradually after the use of i NOS scavenger or inhibitor in rat model,while there was not any change after the use of nNOS or eNOS inhibitors.This illustrates that there may be a relationship between expressing activity of iNOS in spinal dorsal horn and the change of mechanical algesthesia threshold.Our experiment found that the expression of iNOS in spinal cord is likely to participate and play an important role in performing allodynia and hyperalgesia in a rat model of postherpetic neuralgia.These findings have enriched our knowledge of thepathophysiology and mechanism underlying PHN, and supplied a more novel and reasonable theoretical basis for the prevention and treatment of PHN.What’s more,our study will be beneficial for developing A new generation of analgesic drugs and will bring prospect to the people with PHN clinically.