Differential Lumbar Spinal Cord Responses among Wild Type, CD4 Knockout, and CD40 Knockout Mice in Spinal Nerve L5 Transection-Induced Neuropathic Pain

Abstract

BackgroundOur previous studies have indicated that both lumbar spinal cord-infiltrating CD4+ T cells and microglial CD40 contribute to the maintenance of mechanical hypersensitivity in a murine model of neuropathic pain spinal nerve L5 transection (L5Tx). To further delineate the CD4 and CD40-mediated mechanisms involved in the development of L5Tx-induced neuropathic pain behaviors, we examined the lumbar spinal cord mononuclear cells of wild type (WT) BALB/c, BALB/c-CD4 knockout (KO), and BALB/c-CD40 KO mice via flow cytometry.ResultsIn WT mice, L5Tx induced significant but transient (at day 3 and/or day 7) increases of the total numbers of mononuclear cells, microglial cells (CD45loCD11b+), and infiltrating leukocytes (CD45hi) in the ipsilateral side of the spinal cord. In CD4 KO mice, significant elevation of microglia was detected only on day 7 post-L5Tx, while no significant increase in infiltrating leukocytes post-L5Tx was observed. CD40 KO mice did not exhibit any of the changes observed in WT mice. Furthermore, neutralizing CD40 antibody treatment indicated an early involvement of CD40 signaling in the development of L5Tx-induced mechanical hypersensitivity.ConclusionsAltogether, data indicate that both CD4 and CD40 play a role in L5Tx-induced leukocyte infiltration into the lumbar spinal cord but have differential contributions to spinal cord microglial activation following peripheral nerve injury.

Phenotypic Identification Of Spinal Cord-Infiltrating CD4+ T Lymphocytes In A Murine Model Of Neuropathic Pain

“…For instance, it is known that nitric oxide (NO) can induce GFAP expression [36] and that the Th1-related cytokines IFN-γ and TNF-α are known to be involved in NO production in the CNS [37]. Further, our previous observation of a reduction in the L5Tx-induced increase of microglia in the lumbar spinal cord in CD4 KO mice coupled with our GFAP results presented here suggest that CD4 + T lymphocytes may regulate astrocytic responses via microglia-mediated mechanisms [32]. Comprehensive in vitro and in vivo studies could be used to establish the interactions between Th1 CD4 + T lymphocytes and astrocytes in the future.…”

“…The discrepancy in the development of behavioral hypersensitivity between CD40 KO and CD154 KO mice suggest that such an alternative CD40 ligand may be involved in CD40-mediated pro-nociceptive effects. In addition, we previously did observe a reduction in the L5Tx-induced increase of microglia in the lumbar spinal cord in CD4 KO mice [32]. Thus, further studies are necessary to explore other factors involved in the interaction between CD4 + Th1 lymphocytes and microglia.…”

An optimized method to process mouse CNS to simultaneously analyze neural cells and leukocytes by flow cytometry

“…Importantly, we also validated that the collagenase D + DNase I digestion does not affect the detection of numerous immune markers (KLRG1, NKG2D, TCR beta chain, CD44, CD62L, NK1.1, CD127, TCR gamma/delta) by comparing splenocytes from undigested vs. collagenase D + DNase I digested organs (data not shown). As expected and observed in numerous studies of CNS injury models (EAE/neuropathic pain) (Berghmans et al, 2012;Cao et al, 2012), the number of hematopoietic cells was dramatically increased in EAE mice (3.8 millions) compared to naïve animals (310,000 cells) (Fig. 4).…”

“…Some groups did not enzymatically digest CNS tissues (Berghmans et al, 2012;Cao et al, 2012); others digested samples with trypsin (Phares et al, 2009). Moreover, these groups got rid of myelin and cell debris using the Percoll TM gradient step (Berghmans et al, 2012;Cao et al, 2012;Jin et al, 2007;Phares et al, 2009).…”

“…In several studies, experimental animals have been pooled to obtain sufficient numbers of cells to perform flow cytometry analysis (Berghmans et al, 2012;Cao et al, 2012;Phares et al, 2009). Some groups did not enzymatically digest CNS tissues (Berghmans et al, 2012;Cao et al, 2012); others digested samples with trypsin (Phares et al, 2009).…”