The Elevated Spinal Endocannabinoids Activated Astrocytes And Contributed To The Development Of Neuropathic Allodynia

Under physiological conditions,pain can be produced in response to potential tissue damage,which is essential to maintain the integrity of the body and health.However,the occurrence of spontaneous or persistent pain(chronic pain)with inappropriate stimulation can seriously affect the quality of life.According to the Reports of American Academy of Medicine,more Americans suffer from chronic pain than heart disease,diabetes and cancer combined.As the most common chronic pain,neuropathic pain has attracted close attention because of its high incidence and high disability rate.Therefore,neuropathic pain is an important problem to be solved in the medical field.Neuropathic pain is characterized by spontaneous pain,hyperalgesia and allodynia.The endocannabinoid system(e CBs)is mainly composed of e CBs(such as AEA,2-AG,OEA etc),cannabinoid hydrolase(FAAH,MAGL etc)and receptors(CB1,CB2,TRPV1,PPAR-γ etc).Previous studies reported the analgesic effect of e CBs in pain modulation,but some recent research suggested that it may inhibit the function of inhibitory interneurons to induce pain.Starowicz and Przewlocka speculated that the increased content of e CBs in the spinal dorsal horn after nerve injury may promote the occurrence of neuropathic pain in some circumstances.Spinal cord astrocytes play an important role in the occurrence and development of chronic pain,including inflammatory pain,neuropathic pain and cancer pain.It has been confirmed that there are CB1 receptors on astrocytes,which can be activated by e CBs released by neurons.The glutamate released from astrocytes can activate the neuronal extracellular NMDA receptors.This study intends to use pain behavior testing and immunofluorescence staining techniques to clarify that upregulation of endocannabinoids in spinal dorsal horn may activate spinal astrocytes and contribute to the development of neuropathic allodynia.Part 1: The co-localization of cannabinoid receptor CB1 and astrocytes in the spinal dorsal hornObjective To observe the expression of cannabinoid receptor CB1 in astrocytes of the spinal dorsal horn.Methods Four male C57BL/6 mice were anesthetized with chloral hydrate and perfused through the heart by PBS,and fixed by paraformaldehyde.The lumbar enlargement of spinal cord was cut and fixed in 4% phosphate-buffered paraformaldehyde,then dehydrated by sucrose at 4 °C.Twenty mm thick transverse sections were made using frozen microtome.Three slices of each mouse`s spinal cord were randomly picked for GFAP/CB1 double immunofluorescence.The expression of CB1 and GFAP positive astrocytes in the spinal dorsal horn were observed by confocal microscope.Results The cannabinoid receptor CB1 immunostainings were co-localized with GFAP positive astrocytes in the spinal dorsal horn.Part 2: The effects of elevated intrathecal cannabinoid on the mechanical paw withdrawal threshold of ratsObjective To observe the pain behavior changes of rats after intrathecal injection of cannabinoid.Methods Thirty six male C57BL/6 mice were randomly divided into 2-AG group(n=6),acetonitrile control group(n=6),CP55940 group(n=6),DMSO solvent control group(n=6),JZL195 group(n=6)and DMSO solvent control group(n=6).All rats were implanted with intrathecal catheters.After the lidocaine test,the successful animal model was given intrathecal injection through the catheters.After 1,3,5,7,14 and 21 days,respectively,the mechanical paw withdrawal threshold were measured.Results Compared with the solvent control group,intrathecal injection of exogenous cannabinoid 2-AG,cannabinoid receptor agonist CP55940 or cannabinoid hydrolase inhibitor JZL195 produced significant mechanical allodynia in the first day(P < 0.01).The mechanical allodynia lasted for at least 21 days after administration(P < 0.001).Part 3: The effects of elevated intrathecal cannabinoid on the activation of astrocytes in spinal dorsal hornObjective To observe the activation of astrocytes in spinal dorsal horn after intrathecal injection of cannabinoid.Methods Seventy five rats were randomly divided into 2-AG group(n=21),CP55940 group(n=21)and JZL195 group(n=21).Three rats in each group were taken as vertical control group.All rats were implanted with intrathecal catheters.After the lidocaine test,the successful animal models were given intrathecal injection.After 1,3,5,7,14 and 21 days,the lumbar enlargement of spinal cord was cut and fixed in 4% phosphateusing frozen microtome.Three slices of each mouse`s spinal cord were randomly picked for GFAP/CB1 double immunofluorescence.The expression of CB1 and GFAP positive astrocytes in the spinal dorsal horn were observed by confocal microscope.Results Compared with the vertical control group,intrathecal injection of exogenous cannabinoid 2-AG,cannabinoid receptor agonist CP55940 or cannabinoid hydrolase inhibitor JZL195 induced the activation of astrocytes since the third day(P < 0.05).The activation of astrocytes lasted for at least t21 days after administration(P < 0.01).Part 4: The effects of knocking out CB1 receptors to the activation of astrocytes in the dorsal horn and mechanical allodynia in response to the nerve injuryObjective To observe the effects of knocking out CB1 receptors to the activation of astrocytes in the dorsal horn and mechanical allodynia in response to the nerve injury.Methods Nine male GFAP-CB1-KO mice and 9 male GFAP-CB1-WT mice were used.Three KO mice and 3 WT mice were randomly selected for control immunofluorescence labeling.Sciatic nerve ligation(CCI model)was performed in the remaining 12 rats.Seven days after CCI,3 KO mice and 3 WT mice were randomly selected for immunofluorescence labeling after behavioral test.The rest 3 KO mice and 3 WT mice were used for the behavioral test and immunofluorescence labeling at 14 days after CCI.Results Compared with WT+CCI mice,the KO+CCI mice did not develop mechanical allodynia,if any,after nerve injury(P < 0.001).The astrocytes in the KO+CCI mice did not show obvious activation in the spinal dorsal horn after nerve injury(P < 0.01).Summary 1.The cannabinoid receptor CB1 immunostainings were co-localized with GFAP positive astrocytes in the spinal dorsal horn.2.Intrathecal injection of 2-AG,CB1 agonist CP55940 or cannabinoid hydrolase inhibitor JZL195 in normal rats produced long lasting mechanical allodynia.3.Intrathecal injection of 2-AG,CB1 agonist CP55940 or cannabinoid hydrolase inhibitor JZL195 in normal rats induced long lasting activation of astrocytes in the spinal dorsal horn.4.Knocking out CB1 receptors from astrocytes prevented the activation of astrocytes in the dorsal horn and the development of mechanical allodynia in response to the nerve injury.Conclusions The elevated endocannabinoid in the spinal dorsal horn after peripheral nerve injury may activate the astrocytes through CB1 receptors and contribute to the development of neuropathic allodynia.