Article Figures & Data
Figures
- Fig. 1.
Schematic representation of the experiments described herein. A, Schematic representation and anatomic correlates of intra-arterial injection of hyperosmotic media and chemotherapeutic agents used to treat primary brain lymphomas with blood–brain barrier disruption. Hemispheric opening of the BBB was achieved after this procedure. B, The success of the procedure was quantified by CT scans taken approximately after completing the injection protocol. Note the hemisphere enhancement by iodinated contrast media indicated by arrows.
- Fig. 2.
Detection of putative BBB markers by 2D gel electrophoresis of human blood proteins before and after osmotic opening of the BBB by intra-arterial mannitol. The samples used for loading were taken before mannitol injection and after chemotherapy. The timing of the injection of the osmotic agent and the introduction of the chemotherapic agent (methotrexate) are shown in the timeline. Protein signals that remained unchanged are indicated byarrows. The region in which significant changes were observed is boxed by a dashed line. Note the appearance of a distinct spot after BBB disruption. This spot corresponded to a protein of approximate molecular weight of 14 kDa and a pI of 5.5.
- Fig. 3.
Time course of serum protein changes after BBB disruption. S-100β, TTR, haptoglobin, and NSE were measured at the time indicated by the inset. Note that S-100β and TTR increased significantly after BBB opening but with different kinetics. NSE and haptoglobin-1 (Hapto-1) remained unchanged throughout the procedure. The timeline is color-coded to match the histogram bars. The mean ± SD of three experiments is shown; *p < 0.05. Note that the values for TTR and haptoglobin-1 are expressed as percentage change of spot intensity (see Materials and Methods), whereas NSE and S-100β were measured by immunodetection techniques, and the values are expressed in nanograms per milliliter. The values for S-100β were scaled for clarity (100×). TTR, Transthyretin; NSE, neuron-specific enolase; MTX, methotrexate;NICU, neurointensive care unit.
- Fig. 4.
Immunological analysis of protein changes induced by BBB disruption. Top panel, Denaturated proteins were run in parallel with purified TTR (left lane). Western blot analysis revealed a significant increase of immunosignal for both low molecular weight isoforms. Quantitative analysis was performed on the same samples by RID (bottom panels); note the progressive increase of the immunoprecipitation signal surrounding the sample port (see Materials and Methods for details). The numeric values represent TTR levels extrapolated from these measurements and are expressed as micrograms per milliliter.
- Fig. 5.
Non-SDS separation of pre-BBBD and post-BBBD samples. Low molecular weight proteins obtained after separation with a sucrose gradient (cutoff 50 kDa) were loaded on a non-denaturating gel. Note the appearance of a 15 kDa molecular weight band after BBB disruption. Also note that this band corresponds to the monomeric form of standard TTR loaded on a separate gel.
- Fig. 6.
Different distribution between S-100β and TTR in the brain. In the brain, S-100β is synthesized primarily by the astrocytes surrounding the BBB, whereas TTR is synthesized by the choroid plexuses and is found in the ventricular CSF. This topographic segregation may explain the different roles of these markers. See Discussion.
Tables
Protein fragments obtained from MALDI-mass spectroscopy Transthyretin amino-acid sequence VLDAV R GSPAIN VAV HVFR KAADDT WEPFASGK MASHRLLLLC LAGLVFVSEA GPTGTGESKC AADDT WEPFASGK PLMVKVLDAV RGSPAINVAV HVFRKAADDT TS ESGELHGLTT EEEFVEGIYK WEPFASGKTS ESGELHGLTT EEEFVEGIYK TS ESGELHGLTT EEEFVEGIYK VEIDTK VEIDTKSYWK ALGISPFHEH AEVVFTANDS VEIDTK GPRRYTIAAL LSPYSYSTPA VVTNPKE SYWK ALGISPFHEH AEVVFTANDS GPR
