Apolipoprotein E does not cross the blood-cerebrospinal fluid barrier, as revealed by an improved technique for sampling CSF from mice

Abstract

Apolipoprotein E (apoE) is a 34-kDa glycoprotein that is important in lipoprotein metabolism both peripherally and centrally. Because it is primarily produced in the liver, apoE observed in the brain or cerebrospinal fluid (CSF) could have originated in the periphery; i.e., circulating apoE may cross the blood-brain barrier (BBB) and/or enter CSF and be taken up by brain cells. To determine whether this occurs, a second-generation adenovirus encoding human apoE3 was administered intravenously (iv) to C57BL/6J mice, and the detection of human apoE3 in the CSF was used as a surrogate measure of central availability of this protein utilizing an improved method for sampling CSF from mice. This improved technique collects mouse CSF samples with a 92% success rate and consistently yields relatively large volumes of CSF with a very low rate of blood contamination, as determined by molecular assessment of apolipoprotein B, a plasma-derived protein that is absent in the central nervous system. Through this improved method, we demonstrated that in mice receiving the administered apoE3 adenovirus, human apoE3 was expressed at high levels in the liver, leading to high levels of human apoE3 in mouse plasma. In contrast, human apoE3 levels in the CSF, as assessed by a sensitive ELISA, were essentially undetectable in human apoE3 adenovirus-treated mice, and comparable to levels in LacZ adenovirus-treated control mice. These data indicate that apoE in the CSF cannot be derived from the plasma pool and, therefore, must be synthesized locally in the brain.

Fig. 1.

A: photo of the stereotaxic instrument with the electrode holder bent at 90° at its distal end. B: an amplified view of the tapered and sharpened tip of the capillary tube. C: position of the mouse during the procedure. The incisor bar in the stereotaxic device is positioned so that the mouse's neck is maximally ventroflexed. D and E: Close-up views of the membrane of exposed cisternum magnum before and after penetration of the capillary tube.

Fig. 2.

Determination of apolipoprotein B (apoB) levels by Western blot analysis. Twofold serial dilutions of plasma, corresponding to 2.5–0.078% plasma in water (vol/vol), were prepared as standards using the plasma from one mouse. These standards were run in parallel. A level as low as 0.078% blood contamination (lane 6) was observed as clear bands. Two cerebrospinal fluid (CSF) samples (lanes 7 and 8, 1:4 dilution, 20 μl/lane) from two individual mice were used as examples to determine potential blood contamination. No apoB-immunoreactive bands (apoB100 and apoB48) were observed, indicating no blood contamination in either CSF sample.

Fig. 3.

Intravenous administration of adenovirus encoding human apoE3 protein (AdhapoE3) induced a high level of human apoE3 in mouse liver, when compared with adenovirus encoding LacZ cDNA (AdLacZ). Representative bands from Western blot revealed that human apoE3 was in the liver of AdhapoE3-treated mice (E3–1 and E3–2) but not in that of AdLacZ-treated controls (L-1 and L-2). Values are expressed as means ± SE; n = 4–5. **P < 0.01, compared with AdLacZ-treated mice.

Fig. 4.

Intravenous administration of adenovirus encoding human apoE3 protein induced a high level of human apoE3 in plasma, but not in CSF, when compared with LacZ adenovirus. Values are expressed as means ± SE; n = 9–10, **P < 0.01, compared with LacZ adenovirus-treated mice. The box within the figure depicts the ELISA standard curve.