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Reelin expression in brain endothelial cells: an electron microscopy study.

Perez-Costas E, Fenton EY, Caruncho HJ - BMC Neurosci (2015)

Bottom Line: As expected, at the electron microscope reelin labeling was observed in neurons of the cortex, where most of the labeling was associated with the rough endoplasmic reticulum.Importantly, reelin was also observed in some endothelial cells located in small capillaries, which confirmed the findings obtained at the light microscope.In addition, some scarce labeling was observed in the nuclear membrane.

View Article: PubMed Central - PubMed

Affiliation: Department of Psychology, University of Alabama at Birmingham, College of Arts and Sciences, Campbell Hall 415, 1720 2nd Avenue South, Birmingham, Alabama, 35294, USA. epcostas@uab.edu.

ABSTRACT

Background: Reelin expression and function have been extensively studied in the brain, although its expression has been also reported in other tissues including blood. This raises the possibility that reelin might be able to cross the blood-brain barrier, which could be functionally relevant. Up-to-date no studies have been conducted to assess if reelin is present in the blood-brain barrier, which is mainly constituted by tightly packed endothelial cells. In this report we assessed the expression of reelin in brain capillaries using immunocytochemistry and electron microscopy.

Results: At the light microscope, reelin immunolabeling appeared in specific endothelial cells in brain areas that presented abundant diffuse labeling for this protein (e.g., layer I of the cortex, or the stratum lacunosum moleculare of the hippocampus), while it was mostly absent from capillaries in other brain areas (e.g., deeper cortical layers, or the CA1 layer of the hippocampus). As expected, at the electron microscope reelin labeling was observed in neurons of the cortex, where most of the labeling was associated with the rough endoplasmic reticulum. Importantly, reelin was also observed in some endothelial cells located in small capillaries, which confirmed the findings obtained at the light microscope. In these cells, reelin labeling was located primarily in caveolae (i.e., vesicles of transcytosis), and associated with the plasma membrane of the luminal side of endothelial cells. In addition, some scarce labeling was observed in the nuclear membrane.

Conclusions: The presence of reelin immunolabeling in brain endothelial cells, and particularly in caveolar vesicles within these cells, suggests that reelin and/or reelin peptides may be able to cross the blood-brain barrier, which could have important physiological, pathological, and therapeutic implications.

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Reelin labeling is not present in all endothelial cells. A) Low magnification image of an unlabeled brain capillary in the cortex. The wall of this brain capillary is lined by at least two endothelial cells, as shown by the presence of their nuclei (n). Note also the presence of unlabeled myelinated (m) and non-myelinated (p) neuronal processes. The basement membrane was also clearly present (white arrowheads). B-C) Detail images of areas indicated with dashed-lined boxes in A. In B, a caveolar vesicle is clearly unlabeled (white arrow). In B and C, the luminal side of the endothelial cell membrane is also devoid of labeling (white stars). m: myelinated process; n: nucleus; p: neuronal process. White arrowheads indicate the membrane in the abluminal side Scale bars: 2 microns in A; 0.5 microns in B-C.
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Fig4: Reelin labeling is not present in all endothelial cells. A) Low magnification image of an unlabeled brain capillary in the cortex. The wall of this brain capillary is lined by at least two endothelial cells, as shown by the presence of their nuclei (n). Note also the presence of unlabeled myelinated (m) and non-myelinated (p) neuronal processes. The basement membrane was also clearly present (white arrowheads). B-C) Detail images of areas indicated with dashed-lined boxes in A. In B, a caveolar vesicle is clearly unlabeled (white arrow). In B and C, the luminal side of the endothelial cell membrane is also devoid of labeling (white stars). m: myelinated process; n: nucleus; p: neuronal process. White arrowheads indicate the membrane in the abluminal side Scale bars: 2 microns in A; 0.5 microns in B-C.

Mentions: Our electron microscopy study confirmed and extended our findings. As expected, reelin immunostaining was found in neurons, where labeling was located in discrete regions of the rough endoplasmic reticulum (Figures 2A-C), which is in agreement with previous studies [31-35], and is also consistent with the fact that reelin is an extracellular matrix protein expressed through the secretory pathway. Importantly, we have also confirmed that some endothelial cells associated with small capillaries contain reelin immunostaining (Figure 3), while others appear devoid of labeling (Figure 4). High magnification electron micrographs allowed us the identification of the subcellular distribution of reelin labeling, which was mostly located inside vesicles of transcytosis (Figures 3B-C, E-F). In fact, we were able to observe almost all stages of the transcytosis vesicles, from the formation of one of these vesicles accumulating reelin immunostaining (Figure 3B), to reelin-labeled caveolar vesicles located close to the lumen of the capillary or even showing the opening neck (Figures 3C, E-F). In addition, reelin labeling was also found in the endothelial plasma membrane bordering the capillary lumen (Figures 3E-F), but not in the abluminal side (Figures 3B-C, E-F). Furthermore, we found some punctual labeling in the nuclear envelope, which may suggest a possible synthesis of reelin by endothelial cells; although we did not observe immunolabeling in the Golgi complex of these cells, which is poorly developed. Finally, we also found capillaries completely devoid of reelin labeling (Figure 4) in the same electron microscopy sample of the cortex, confirming our light microscopy observation that reelin appears in some (but not all) capillaries within the same area.Figure 2


Reelin expression in brain endothelial cells: an electron microscopy study.

Perez-Costas E, Fenton EY, Caruncho HJ - BMC Neurosci (2015)

Reelin labeling is not present in all endothelial cells. A) Low magnification image of an unlabeled brain capillary in the cortex. The wall of this brain capillary is lined by at least two endothelial cells, as shown by the presence of their nuclei (n). Note also the presence of unlabeled myelinated (m) and non-myelinated (p) neuronal processes. The basement membrane was also clearly present (white arrowheads). B-C) Detail images of areas indicated with dashed-lined boxes in A. In B, a caveolar vesicle is clearly unlabeled (white arrow). In B and C, the luminal side of the endothelial cell membrane is also devoid of labeling (white stars). m: myelinated process; n: nucleus; p: neuronal process. White arrowheads indicate the membrane in the abluminal side Scale bars: 2 microns in A; 0.5 microns in B-C.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4374371&req=5

Fig4: Reelin labeling is not present in all endothelial cells. A) Low magnification image of an unlabeled brain capillary in the cortex. The wall of this brain capillary is lined by at least two endothelial cells, as shown by the presence of their nuclei (n). Note also the presence of unlabeled myelinated (m) and non-myelinated (p) neuronal processes. The basement membrane was also clearly present (white arrowheads). B-C) Detail images of areas indicated with dashed-lined boxes in A. In B, a caveolar vesicle is clearly unlabeled (white arrow). In B and C, the luminal side of the endothelial cell membrane is also devoid of labeling (white stars). m: myelinated process; n: nucleus; p: neuronal process. White arrowheads indicate the membrane in the abluminal side Scale bars: 2 microns in A; 0.5 microns in B-C.
Mentions: Our electron microscopy study confirmed and extended our findings. As expected, reelin immunostaining was found in neurons, where labeling was located in discrete regions of the rough endoplasmic reticulum (Figures 2A-C), which is in agreement with previous studies [31-35], and is also consistent with the fact that reelin is an extracellular matrix protein expressed through the secretory pathway. Importantly, we have also confirmed that some endothelial cells associated with small capillaries contain reelin immunostaining (Figure 3), while others appear devoid of labeling (Figure 4). High magnification electron micrographs allowed us the identification of the subcellular distribution of reelin labeling, which was mostly located inside vesicles of transcytosis (Figures 3B-C, E-F). In fact, we were able to observe almost all stages of the transcytosis vesicles, from the formation of one of these vesicles accumulating reelin immunostaining (Figure 3B), to reelin-labeled caveolar vesicles located close to the lumen of the capillary or even showing the opening neck (Figures 3C, E-F). In addition, reelin labeling was also found in the endothelial plasma membrane bordering the capillary lumen (Figures 3E-F), but not in the abluminal side (Figures 3B-C, E-F). Furthermore, we found some punctual labeling in the nuclear envelope, which may suggest a possible synthesis of reelin by endothelial cells; although we did not observe immunolabeling in the Golgi complex of these cells, which is poorly developed. Finally, we also found capillaries completely devoid of reelin labeling (Figure 4) in the same electron microscopy sample of the cortex, confirming our light microscopy observation that reelin appears in some (but not all) capillaries within the same area.Figure 2

Bottom Line: As expected, at the electron microscope reelin labeling was observed in neurons of the cortex, where most of the labeling was associated with the rough endoplasmic reticulum.Importantly, reelin was also observed in some endothelial cells located in small capillaries, which confirmed the findings obtained at the light microscope.In addition, some scarce labeling was observed in the nuclear membrane.

View Article: PubMed Central - PubMed

Affiliation: Department of Psychology, University of Alabama at Birmingham, College of Arts and Sciences, Campbell Hall 415, 1720 2nd Avenue South, Birmingham, Alabama, 35294, USA. epcostas@uab.edu.

ABSTRACT

Background: Reelin expression and function have been extensively studied in the brain, although its expression has been also reported in other tissues including blood. This raises the possibility that reelin might be able to cross the blood-brain barrier, which could be functionally relevant. Up-to-date no studies have been conducted to assess if reelin is present in the blood-brain barrier, which is mainly constituted by tightly packed endothelial cells. In this report we assessed the expression of reelin in brain capillaries using immunocytochemistry and electron microscopy.

Results: At the light microscope, reelin immunolabeling appeared in specific endothelial cells in brain areas that presented abundant diffuse labeling for this protein (e.g., layer I of the cortex, or the stratum lacunosum moleculare of the hippocampus), while it was mostly absent from capillaries in other brain areas (e.g., deeper cortical layers, or the CA1 layer of the hippocampus). As expected, at the electron microscope reelin labeling was observed in neurons of the cortex, where most of the labeling was associated with the rough endoplasmic reticulum. Importantly, reelin was also observed in some endothelial cells located in small capillaries, which confirmed the findings obtained at the light microscope. In these cells, reelin labeling was located primarily in caveolae (i.e., vesicles of transcytosis), and associated with the plasma membrane of the luminal side of endothelial cells. In addition, some scarce labeling was observed in the nuclear membrane.

Conclusions: The presence of reelin immunolabeling in brain endothelial cells, and particularly in caveolar vesicles within these cells, suggests that reelin and/or reelin peptides may be able to cross the blood-brain barrier, which could have important physiological, pathological, and therapeutic implications.

Show MeSH
Related in: MedlinePlus