The experiment for using animals in this study was approved by The Animal Care and Use Committee at Northwestern University. All procedures were performed in accordance with the standards of NIH guidelines for the care and use of laboratory animals.
Recombinant rat SCF and recombinant human G-CSF were provided by Amgen (Thousand Oaks, CA, USA); and I125-labeling was performed by MP Biochemicals, (Irvine, CA, USA). Radiochemical purity of [125I]-SCF (2.47 µCi/µg) and [125I]-GCSF (2.47 MBq/mg) was examined for purity by TCA precipitation, which was greater than 99% before use.
Thirty Sprague-Dawley rats (2 months old) were intraperitoneally anesthetized with ketamine (40 mg/kg) and xylazine (5 mg/kg). Polyethylene catheters (PE-10) were inserted into the femoral artery and vein for collecting blood samples and administering the radioisotopes, respectively. Arterial blood pressure, blood gases, and body temperature were monitored throughout the experiment. Heat lamps were used to maintain body temperature close to 37 °C.
Twenty to thirty µCi of [125
I]-SCF (n=15) or [125
I]-GCSF (n=15) was injected into the femoral vein as an intravenous bolus. Arterial blood samples were collected at 10, 30, 60, and 120 min (n=3–4/time point/cytokine) after drug injection. Plasma was used for [125
I] radioactivity counting by liquid scintillation counting with appropriate quench correction. After collecting blood samples, the rats were sacrificed at 10, 30, 60, and 120 min (n=3–4/time point for each cytokine). The rats were then decapitated, and the brains were removed and stored at − 40°C. The brains were homogenized, and mixed with 3 times their weight in saline after thaw. Aliquots of the brain samples were used for determination of tissue radioactivity by liquid scintillation counting. The plasma and brain tissue radioactivity counts were divided by the sample weights to give tissue radioactivity concentrations in nCi/g. The plasma (Cp
) and brain (Am
) radioactivity concentrations were used to determine the influx constant according to a graphical method of analysis described elsewhere (Patlak et al., 1983). With this method, the values of the integrated plasma activity divided by the final plasma value are plotted against the tissue radioactivity value divided by the final plasma value. An apparent K1
for each animal was also calculated using the equation
presents the tissue plasma space and any rapidly equilibrating space, with units of ml/g. Cp
(T) is the final plasma value. K1
, the blood-to-brain tissue transfer constant, was presented with a value of ml/g/min.
Immunofluorescent staining, which has been described elsewhere (Zhao, et al., 2003
), was used for determining SCF and G-CSF receptor expression in the capillaries of rat brain. Brain sections (cut by a cryostat, 10µm thick) were collected from Sprague-Dawley rats (3 month old). The sections were fixed with 4% formaldehyde in phosphate-buffered saline (PBS). After blocking nonspecific binding with goat normal serum, sections were incubated with primary antibodies: rabbit anti-cKit or rabbit anti-GCSFR (1:50, Santa Cruz Biotechnology, Santa Cruz, CA, USA) over night at 4°C. After 2-hrs incubation with Cy3-conjucted goat anti-rabbit (1:400, Jackson ImmunoResearch Lab, West Grove, PA, USA) in dark, counterstaining was performed with DAPI (1:5000, Sigma, St. Louis, MO, USA). Immunofluorescent staining was photographed with a fluorescent microscope (Nikon, Eclipse TE 2000-S).
For western blots, adult rat brain microvascular endothelial cells (SV-40 cell line, gift from Institute for Biological Sciences, Canada) were grown in M199 media contain 10% FBS. The cells were harvested and lysed with RIPA buffer containing a protease inhibitor cocktail (Pierce, Rockford, IL). Twenty micrograms proteins were loaded to 8% or 12% SDS-PAGE polyacrylamide gel and transferred to a PVDF membrane (Bio-Rad, Hercules, CA). Membranes were blocked with 5% non-fat milk and incubated with rabbit anti-c-Kit or rabbit anti-G-CSFR polyclonal antibodies (1:200, Santa Cruz Biotechnology, Santa Cruz, CA), followed by incubation with a peroxidase conjugated secondary antibody (Jackson Immunoresearch, West Brove, PA). Bands were visualized by chemiluminescent substrate (Pierce, Rockford, IL) according to the manufacturer's instructions. Immunoblotting with a mouse anti-β-actin (Sigma, Saint Louis, MO) was used as internal control.