The Role Of Increased Blood-spinal Cord Barrier Permeability In The Pathogenesis Of Bone Cancer Pain

Background Cancer pain,a common clinical manifestation among terminal-stage cancer patients,is a misery torturing the life quality among those patients.Unfortunately,a number of cancer pain patients do not receive satisfactory relief,which partly ascribes to the CNS adverse effect and drug tolerance of opiods.BBB/BSCB is a dynamic barrier located in the interface of peripheral circulation and CNS,and a bridge to implement the selective transportation of substance between periphery and CNS.Many CNS disorders have been found to be associated with BBB/BSCB impairment.Recent studies have suggested that some pathological pains,e.g.inflammatory pain and neuropathic pain could induce BBB/BSCB leakage,yet the relationship between BBB/BSCB and bone cancer pain still remains elusive.The current study is the first one aiming to investigate the role of increased BSCB permeability in the pathogenesis of bone cancer pain and further carry out interventions targeted on detrimental BSCB,based on the establishment of C3H/HeN bone cancer pain mice model.This study would provide new insights for clinical medications for bone cancer pain.Methods 1.C3H/HeN mice bone cancer pain model was adopted in our study.To determine the success of model establishment,NSF and PWMT were used to assess pain behaviors,and HE staining of the ipsilateral femur was used to observe the growth of sarcoma.The BBB/BSCB leakage was tested by Evans blue dye.Expressions of tight junction proteins in the spinal level were measured through Western blotting and immunofluorescence.2.ELISA or Western blotting and/or immunofluorescence were used to detect the peripheral/spinal TNF-α,IL-1β,MMP-9,VEGF-A,VEGFR-1 and VEGFR-2 expression levels.3.After intraperitoneal or intrathecal administration of non-selective VEGFR-1 and VEGFR-2 inhibitor Axitinib and selective VEGFR-2 inhibitor Ki8751,we used PWMT to assess analgesic effect of two drugs,Evans blue dye to measure permeability of BSCB,Western blotting to detect the expression of VEGFR-1,VEGFR-2 and tight junction proteins in the spinal cord,and immunofluorescence to stain the VEGFR-1,VEGFR-2 and IL-1β in the medullary cavity,and activated status of astrocytes as well as microglia in the spinal cord.The transcription level of Shh and related genes were tested by RT-PCR.Results 1.Inoculation of sarcoma cells into the right femur medullar in mice induced stable increased NSF and decreased PWMT 14 days after the establishment of cancer pain model,and HE staining showed abundant tumor cells infiltrate and invade into the medullary cavity,accompanied by increased permeability of BSCB and downregulated ZO-1 and Claudin-5.These alterations lasted to the end-point of our observing time(28 days).2.ELISA data implied no significant changes of peripheral TNF-α,IL-1β,MMP-9 and VEGF-A levels.Western blotting and immunofluorescence data suggested that the expression of VEGFR-1 in spinal cord started to increase 4 days after the inoculation and VEGFR-2 level upregulated since day 14,both of which mainly expressed on the vascular endothelial cells.3.Intraperitoneal or intrathecal administration of high dose Axitinib and Ki8751(Axitinib 40 mg/kg and Ki8751 30 mg/kg for intraperitoneal injection,3nmol of both for intrathecal injection)significantly ameliorated mechanical allodynia of bone cancer pain mice,decreased the BSCB leakage,upregulated the expressions of Tight junction proteins,and reduced activated microglia as well as IL-1β in ipsilateral femur medullar 2 hours after the injection.The analgesic effect of intraperitoneal injection lasted for at most 24 hours while intrathecal administration lasted for 72 hours,which further suppressed the activated astrocytes and Shh signal pathway in spinal cord.Conclusion The permeability of BSCB increased during the pathogenesis of bone cancer pain,which might be mediated via spinal VEGFA-VEGFRs pathway.Inhibition of VEGFR-1 and/or VEGFR-2 in the BSCB endothelial cells alone or together with VEGFR-1 and/or VEGFR-2 of sarcoma cells in the femur medullary cavity could protect the integrity of BSCB,thus resulting in pain relief.