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Retrobulbar administration of TPCA-1 causes no obvious toxicity to blood cells.

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posted on 2014-01-28, 03:23 authored by Huayi Lu, Qingxian Lu, Subhash Gaddipati, Ramesh Babu Kasetti, Wei Wang, Manolis Pasparakis, Henry J. Kaplan, Qiutang Li

(A–F) Flow cytometric analyses of blood cell population. Single cell suspension was prepared from spleen, thymus and bone marrow of the mice at 7 days after laser-injury followed immediately by a single retrobulbar injection of 56 μg TPCA-1 in 20 μL vehicle solution (PBS/20% DMSO) or control vehicle only in 20 μL. The representative plots are shown and numerical values represent the average (±SD, n>5). Statistics were performed using ProStat 5.5 program, and showing no significant difference in all the groups. P≤0.05 is considered as significant. (A, B) The CD4+ and CD8a+ T cells from the TCRβ-gated populations in spleen (A) and thymus (B) were calculated and plotted. (C, D) Spleen (C) and bone marrow (D) cells were isolated from TPCA-1 treated (+) and untreated (−) mice and stained with anti-B220, anti-IgM, and anti-IgD antibodies. Data in (C) show the percentile of mature (IgDhighIgMlow) and immature (IgDlowIgMhigh) B cells in the B220-gated splenocytes, and data in (D) show the percentages of mature recirculating (R2, IgDhighIgMint), transitional immature (R3, IgMhighIgDint), immature (R4, IgMhighIgD) and precursor (R5, IgMIgD) B cell populations in the B220-gated bone marrow cells. (E) Spleen cells were isolated from TPCA-1 treated (+) and untreated (−) mice and stained with anti-CD11b, anti-Ly6C and anti-Ly6G antibodies. The numerical values are shown as the average of neutrophil (Ly6G+/Ly6Cint), monocyte (Ly6G/Ly6C+) and mature macrophage (Ly6G/Ly6C) from the CD11b gate in the spleens. (F) Spleen cells were stained with 7AAD to detect dead cells. The numerical values are shown as the average of the CD11b+ 7AAD+ populations in splenic lymphoid cells. (G) Hematoxylin and eosin (H&E) histological staining was performed to evaluate toxicity to liver and spleen at 7 days after treatment. The scale bar represents 50 μm in length. (H) Relative cell viability of HEK293T cells (left panel) and ARPE-19 cells (right panel) was shown at 12 hours after being cultured with 50 μM TPCA-1 or 10 ng/mL TNFα or combination of both. The cells were pretreated with 50 μM TPCA-1 for 30 minutes prior to addition of 10 ng/mL TNFα for combination treatment. Cell viability was measured using MTT assay. Data are represented as the mean±SD from three independent experiments. Asterisk indicates Two-tail T-test: *** p<0.005.