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. 2008 Jan;18(1):71-85.
doi: 10.1111/j.1750-3639.2007.00104.x. Epub 2007 Oct 9.

Vascular endothelial growth factor and nitric oxide production in response to hypoxia in the choroid plexus in neonatal brain

Affiliations

Vascular endothelial growth factor and nitric oxide production in response to hypoxia in the choroid plexus in neonatal brain

Viswanathan Sivakumar et al. Brain Pathol. 2008 Jan.

Abstract

Damage to the choroid plexus in 1-day-old Wistar rats subjected to hypoxia was investigated. The mRNA and protein expression of hypoxia-inducible factor-1alpha (HIF-1alpha), endothelial, neuronal, inducible nitric oxide synthase (eNOS, nNOS, iNOS), and vascular endothelial growth factor (VEGF) along with nitric oxide (NO) production and VEGF concentration was up-regulated significantly in hypoxic rats. Ultrastructurally, the choroid plexus epithelial cells showed massive accumulation of glycogen. A striking feature was the extrusion of cytoplasmic fragments from the apical cell surfaces into the ventricular lumen following the hypoxic insult. Intraventricular macrophages showed increased expression of complement type 3 receptors, major histocompatibility complex class I and II antigens, and ED1 antigens. Following an intravenous injection of horseradish peroxidase (HRP), a large number of intraventricular macrophages were labeled suggesting enhanced leakage of the tracer from the blood vessels in the choroid plexus connective tissue stroma into the ventricular lumen. We suggest that increased production of NO in hypoxia is linked to the structural alteration of the choroid plexus, and along with VEGF, may lead to increased vascular permeability. Melatonin treatment reduced VEGF and NO levels as well as leakage of HRP suggesting its potential value in ameliorating damage in choroid plexus pathologies.

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Figures

Figure 1
Figure 1
Reverse transcription polymerase chain reaction (RT‐PCR) analysis of hypoxia‐inducible factor‐1α (HIF‐1α), vascular endothelial growth factor (VEGF), endothelial, inducible, and neuronal nitric oxide synthase (eNOS, iNOS, and nNOS) gene expression in the choroid plexus of rats subjected to hypoxia at postnatal day 1. Left panel represents 1.5% agarose gel stained with ethidium bromide of RT‐PCR products of the above‐mentioned mRNA in the choroid plexus of rats at 3, 24 h, 3, 7, and 14 days after the hypoxic exposure and their corresponding controls (C). Right panel shows the graphical representation of fold changes quantified by normalization to the β‐actin as an internal control. Each bar represents the mean ± SD. Differences in the mRNA levels are significant (*P < 0.05) after the hypoxic exposure when compared with controls.
Figure 2
Figure 2
Western blotting of hypoxia‐inducible factor‐1α (HIF‐1α), vascular endothelial growth factor (VEGF), endothelial, inducible, and neuronal nitric oxide synthase (eNOS, iNOS, and nNOS) protein expression in the choroid plexus tissue supernatants of rats at 3, 24 h, 3, 7, and 14 days after the hypoxic exposure and their corresponding controls (C). Upper panel shows HIF‐1α (120 kDa), VEGF (25 kDa), eNOS (140 kDa), iNOS (130 kDa), and nNOS (155 kDa) immunoreactive bands. Lower panel represents bar graphs (A, HIF‐1α; B, VEGF; C, eNOS; D, iNOS; E, nNOS) showing significant changes in the optical density after hypoxic exposure.
Figure 3
Figure 3
Vascular endothelial growth factor (VEGF) concentration (A) and nitric oxide (NO) production (B) in the postnatal rat choroid plexus of the control (C) and at 3, 24 h, 3, 7, and 14 days after hypoxia as determined by enzyme immunoassay and NO assay, respectively. Data represent mean ± SD. Significant differences between control and hypoxic rats are indicated by *P < 0.05. C and D show significant (#P < 0.05) reduction in levels of VEGF and NO at 3 and 24 h in the choroid plexus after melatonin administration in hypoxic rats.
Figure 4
Figure 4
Endothelial nitric oxide synthase (eNOS) expression is detected in some blood vessels in the choroid plexus (CP) in a 1‐day‐old control rat (A) but not in the CP epithelial cells. Expression of eNOS is, however, enhanced at 3 h in the blood vessels (arrowheads) and the CP epithelial cells after the hypoxic exposure (B). The CP epithelial cells express eNOS immunoreactivity which is especially intense at their apical surfaces as focal dense spots (arrows, B). Weak neuronal nitric oxide synthase (nNOS) expression is detected in the choroid plexus in a 1‐day‐old control rat (C). It is noticeably enhanced at 24 h after the hypoxic exposure (D). Expression of inducible nitric oxide synthase (iNOS) is barely detected in the choroid plexus of a 1‐day‐old control rat (E). It is markedly induced at 24 h (F) in the epithelial cells of hypoxic rats. Many intraventricular macrophages (arrows) in hypoxic rats also exhibit intense iNOS expression in F. VEGF expression is greatly enhanced at 3 days (H) after the hypoxic exposure when compared with the corresponding controls (G).
Figure 5
Figure 5
Few OX‐42‐positive intraventricular macrophages are associated with the choroid plexus in a 4‐day‐old control rat (A, arrows). At 3 days after exposure to hypoxia, a large number of OX‐42‐positive cells can be seen (B, arrows). Scattered ED1‐positive cells (C, arrows) in a 4‐day‐old control rat as compared with a large number of ED1‐positive cells at 3 days after the hypoxic exposure (D, arrows). OX‐18‐positive intraventricular macrophages in a 4‐day‐old control rat (E, arrows) and at 3 days after hypoxia (F, arrows). The numbers of OX‐18‐positive cells are drastically increased in the hypoxic rats. Many OX‐6‐positive cells can be seen at 3 days after hypoxic exposure (H, arrows) as compared with the corresponding controls in which occasional OX‐6‐positive epiplexus cells are identified (G, arrow).
Figure 6
Figure 6
Periodic acid Schiff‐stained choroid plexus (CP) epithelial cells in a 1‐day‐old control rat (A). Note the very light staining indicating a scarcity of glycogen accumulation in the cells. Intense PAS staining in B indicates a marked increase in glycogen accumulation in the CP epithelial cells (arrows) in hypoxic rats (B).
Figure 7
Figure 7
A. Choroid plexus of a control rat at 1 day of age. The epithelial cells (EC) show mitochondria, cisternae of rough endoplasmic reticulum, and some dense granules. The projecting microvilli are slender and uniform in diameter. B. Choroid plexus in a rat at 3 h after the hypoxic exposure. Note the massive accumulation of glycogen in the EC in B. Some cytoplasmic vacuoles (arrow) may be seen. C shows an enlarged view of glycogen (GL) accumulation in an epithelial cell at 3 h after hypoxic exposure. D. A choroid plexus EC at 24 h after the hypoxic exposure shows a sessile protrusion (asterisk) into the ventricular lumen. E. Many cytoplasmic fragments or profiles (asterisks) appear to be detached from the EC surfaces at 24 h after the hypoxic exposure. F. Intercellular spaces (IS) are widened between the EC at 3 days after hypoxic exposure. The tight junction (arrows) between the epithelial cells remains intact (F). G. An intraventricular macrophage in a 2‐day‐old control rat is in close association with the microvilli of the choroid plexus epithelial cells. H. An intraventricular macrophage at 24 h after the hypoxic exposure. Note the occurrence of large cytoplasmic vacuoles (V). One of them (asterisk) contains flocculent materials.
Figure 8
Figure 8
Choroid plexus (CP) in a control (A), hypoxic (B), and hypoxia + melatonin (C) administered rat showing horseradish peroxidase (HRP) reaction product. In A and C, massive accumulation of HRP reaction product is seen in the choroid plexus tissue in the epithelial cells and intercellular spaces, whereas in B, HRP accumulation is localized only in the intraventricular macrophages (arrows).

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