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The bloodCspinal cord barrier (BSCB) regulates molecular exchange between blood and spinal cord. proteins, accumulation in motor neurons of cyotoxic thrombin and fibrin and motor neuron loss. Barrier disruption in perciyte-deficient mice coincided with further reductions in ZO-1 and occludin. These data suggest that pericytes contribute to proper function of the BSCB at the capillary level. Regional reductions in spinal cord pericytes may provide a cellular basis for heightened spinal cord FTY720 ic50 barrier capillary permeability and motor neuron loss. (Pan mice on a 129S1/SvlmJ background were generated and maintained as described (Tallquist mice. Adult 13-month-old mice and 13-month-old nontransgenic 129S1/SvlmJ littermates were utilized for neuronal analysis. Tissue Preparation FTY720 ic50 Animals were anesthetized with intraperitoneal injection of 100?mg/kg ketamine and 10?mg/kg xylazine and transcardially perfused with phosphate-buffered saline (PBS) containing 5?mM EDTA (Sigma-Aldrich, St Louis, MO, USA). In a separate set of studies, mice were intravenously injected with 0.1?mL of Alexa Fluor 546-conjugated cadaverine diluted 0.5?for 20 minutes in 18% dextran solution (MW: 70,000?Da; Sigma-Aldrich) in PBS containing 2% FBS. Following centrifugation, floating glial and neuronal elements were gently aspirated and the vascular pellet was resuspended in PBS containing 2% FBS. Isolated microvessels were then passed through a 100-(PDGFR(tomato) lectin (Vector Labs) diluted 1:100. Sections treated with lectin were then incubated in Dylight 649-conjugated streptavidin (Vector Labs) to FTY720 ic50 detect endothelial-specific fluorescent signal. Nuclei were detected with Hoechst 33342 (Invitrogen) diluted 1:10,000. In a separate set of studies, 14?(tomato) lectin (Vector Labs) diluted 1:100. FTY720 ic50 Tissue sections and microvessel preparations were mounted with fluorescent mounting media (Dako) and coverslipped. All slides were scanned with a custom built Zeiss 510 meta confocal laser scanning microscope with a Zeiss Apochromat 25/0.8 NA water immersion objective (Car Zeiss Microimaging, Thornwood, NY, USA). A 488-nm argon laser was used to excite Alexa Fluor 488 and the emission was collected through a 500 to 550?nm band pass (bp) filter. A 543-nm HeNe laser was used to excite Cy3, Alexa Fluor 546 and DyLight 549 and the emission was collected through a 560 to 615?nm bp filter. A 633-nm HeNe laser was used to excite DyLight 649 and the emission was collected through a 650 to 700?nm bp filter. A 800-nm tuned Ti:sapphire laser (Specra Physics, Irvine, CA, USA) was used to excite Hoechst 33342 and the emission was collected through a 435 to 485?bp filter. Image Analysis All image analysis was conducted utilizing NIH ImageJ software. All image analyses were performed by a blinded investigator. For pericyte coverage analysis, Rabbit Polyclonal to OR2T10 CD13-, PDGFRmice, three randomly selected fields per section from three nonadjacent sections (100?represents experimental time. Statistical Analysis All data were analyzed using multifactorial analysis of variance (ANOVA) followed by Tukey tests. Correlations were determined using Pearson’s correlation analysis. A value 0.05 was considered statistically significant in all studies. Results BloodCSpinal Cord Barrier Pericyte Coverage and Number Are Reduced in Comparison to Brain Pericytes represent a heterogenous cell population not covered by a single cell marker that is specific or inclusive (Armulik (Bell (Figures 1D and E) in the brain regions was 80% (Armulik (PDGFR(PDGFRmice (Bell mutants and may serve as a valuable model to deduce BSCB pericyte function. Open in a separate window Figure 5 Exacerbation of pericyte deficiency leads to overt bloodCspinal cord barrier disruption and leakage of endogenous plasma proteins. (A) Confocal microscopy analysis of CD13-positive pericytes (green) and collagen IV-positive capillary profiles (red) in 6-month-old mouse cervical, thoracic, and lumbar spinal cord anterior horn. (B) Quantification of regional CD13-positive pericyte coverage of collagen IV-positive anterior horn spinal cord capillaries. Means.e.m., cervical, thoracic, and lumbar spinal cord. (D, E) Quantification of IgG (D) and fibrin (E) extravascular deposits in the spinal cord regions in tissue sections from 2-month-old B6SJL/F1 mice. Means.e.m., lumbar spinal cord. Arrows indicate neuronal accumulation. We next evaluated whether BSCB pericyte deficiency directly exacerbates capillary leakage and motor neuron accumulation of the endogenous plasma proteins IgG and Fibrin (Figure 5C). Quantification of extravascular IgG (MW: 150,000?Da) and fibrin (MW: 53,000 to 73,000?Da) demonstrated significant increases in spinal cord accumulation in cervical, thoracic, and lumbar regions of mice (Figures 5D and 5E). No strain-specific differences in deposition of plasma-derived IgG (Figure 5D) or fibrin (Figure 5E) were detected between nontransgenic mouse lines. Increased spinal barrier disruption led to neuronal accumulation of several potentially neurotoxic plasma proteins including thrombin (MW: 36,000?Da) (Figure 5F) and fibrin (Figure 5G) in lumbar spinal cord of mice. Similar results were obtained in cervical and thoracic spinal cord (data not shown). In contrast, plasma proteins were not.