Non-Invasive Monitoring Of CNS MHC Ⅰ Molecules And Evaluation Of The Therapeutic Potential Of A TLR4-Suppressing-Peptide In Ischemic Stroke Mice

Although central nervous system (CNS) was thought to be privileged and major histocompatibility complex (MHC) molecules were barely expressed by mature CNS for decades, a growing body of reports has demonstrated that MHC Ⅰ can be expressed by some neuronal populations on the condition of brain infection or injury. Recent studies suggested that neuronal MHC Ⅰ was closely associated with ischemic stroke. It was reported that not only of H-2K~b and H-2D~b, but also of PirB and downstream signaling were elevated after stroke unset 7 days, which exacerbated brain injury after ischemia. As the second leading cause of death plus disablement worldwide, stroke is in great needed for a non-invasive, clinically feasible and economical method for the evaluation of early acute phase brain damage, which is crucial to diagnosis patients choosing treatments. To date, there is no research on brain spatial-temporal expression pattern of MHC Ⅰ molecules during ischemic acute phase, and whether CNS induced MHC Ⅰ molecules will be applicable for stroke injury imaging is still unknown.In this study, the expression patterns of MHC Ⅰ molecules in C57BL/6 mouse were investigated during the acute phase of ischemic stroke. Then a peptide named H2BP with both H-2K~b and H-2D~b strong binding abilities was screened by bioinformatic methods. H2BP peptide served quite well as novel probe in in-vivo near-infrared fluorescence (NIRF) imaging in stroke mice. Our partial research results were as follows:1. After reperfusion 6 or 24 hours in mouse MCAO models, MHC Ⅰ molecules showed a much greater elevation in their ischemic hemisphere than contralateral side. In the photothrombotic ischemic mice cerebral cortex, MHC Ⅰ molecules were reached their expression peaks within 3 hours, during 3 to 24 hour the dramatic distinction between two hemispheres showed slowly shrank. Western blots of each tested brains all showed two specific MHC Ⅰ bands of 45 and 55 kDa, and 55 kDa band revealed more significant difference between hemispheres than 45 kDa one did. Cultured neuron oxygen glucose deprivation (OGD) in vitro ischemic model displayed elevated MHC Ⅰ expression within 24 hours after injury insult.2. A peptide named H2BP was screened by bio informatics’methods. Prediction of H2BP-MHC class I binding using artificial neural networks (ANNs) revealed that H2BP interaction with both H-2K~b and H-2D~b were strong binding type. When docked with CNS potential receptor LY49A, H2BP-MHC Ⅰ complex showed weaker binding ability than wide-type epitope gp33 formed gp33-MHC Ⅰ complex did. That in silico data suggest H2BP-MHC Ⅰ complex may shared the same affinity-changing pattern with TCR of peripheral immune system.3. FITC-H2BP with FACS was used to distinguish spleen cells of C57BL/6 mice from that of Babl/c mice. Results demonstrated that FITC-H2BP recognized H-2K~b and H-2D~b other than H-2Kd or H-2Dd on the cell surface. More intensive FACS study of H-2K~b-/-D~b-/- and C57BL/6 wide-type mice cells was done, whose results displayed well distinguishing ability of FITC-H2BP, in the meantime FITC-control peptide can not tell the differences between groups. A series of experiments utilized in vitro ischemic OGD model demonstrated that FITC-H2BP was specific binding to neurons underwent ischemia. While a FITC-conjugated scrambled control peptide showed no binding. FITC-H2BP preferentially homed to ischemic stroke tissue after intravenous administration into the ischemic mice and Cy5.5-H2BP probe showed low cytotoxicity to neurons, which together making Cy5.5-H2BP a promising imaging probe for in vivo study.4. Strong fluorescence was seen over the ipsilateral side of ischemic mice injected with Cy5.5-H2BP probe on both in vivo Near-infrared fluorescence (NIRF) image and ex vivo NIRF image while slightly higher fluorescence intensities between hemispheres were detected in mice received Cy5.5-control probe. Significantly higher fluorescence signals were found over the ipsilateral hemisphere compared to its counterpart after Cy5.5-H2BP probe administrated 5 to 24 hours. Moreover, an obvious accumulation of Cy5.5 signal was detected in the ischemic brain other than organs such as kidney when mice received Cy5.5-H2BP.5. Our work demonstrated that OGD injury induced neuronal TLR4 were expressed mainly in neuron cell bodies and dendrites, and MD2MP peptide showed specific binding with TLR4 on mouse cell line RAW264.7 surface. In addition, MD2MP was able to suppress TLR4 signaling in LPS or OGD stimulated neurons. MD2MP peptide showed protective effects in OGD injured neurons and ischemic mice.Taken together, these results suggest that MHC Ⅰ is a potential target for molecular imaging in ischemic stroke, besides H2BP peptide is quite suitable as mouse non-invasive in vivo ischemical monitoring probe. Moreover, MD2MP showed a promising therapeutical utilization in the future of stroke therapy.