Induction of inhibitory factor kappaBalpha mRNA in the central nervous system after peripheral lipopolysaccharide administration: an in situ hybridization histochemistry study in the rat

Abstract

In this study we investigate the mRNA expression of inhibitory factor kappaBalpha (IkappaBalpha) in cells of the rat brain induced by an intraperitoneal (i.p.) injection of lipopolysaccharide (LPS). IkappaB controls the activity of nuclear factor kappaB, which regulates the transcription of many immune signal molecules. The detection of IkappaB induction, therefore, would reveal the extent and the cellular location of brain-derived immune molecules in response to peripheral immune challenges. Low levels of IkappaBalpha mRNA were found in the large blood vessels and in circumventricular organs (CVOs) of saline-injected control animals. After an i.p. LPS injection (2.5 mg/kg), dramatic induction of IkappaBalpha mRNA occurred in four spatio-temporal patterns. Induced signals were first detected at 0.5 hr in the lumen of large blood vessels and in blood vessels of the choroid plexus and CVOs. Second, at 1-2 hr, labeling dramatically increased in the CVOs and choroid plexus and spread to small vascular and glial cells throughout the entire brain; these responses peaked at 2 hr and declined thereafter. Third, cells of the meninges became activated at 2 hr and persisted until 12 hr after the LPS injection. Finally, only at 12 hr, induced signals were present in ventricular ependyma. Thus, IkappaBalpha mRNA is induced in brain after peripheral LPS injection, beginning in cells lining the blood side of the blood-brain barrier and progressing to cells inside brain. The spatiotemporal patterns suggest that cells of the blood-brain barrier synthesize immune signal molecules to activate cells inside the central nervous system in response to peripheral LPS. The cerebrospinal fluid appears to be a conduit for these signal molecules.

Figure 1

Representative film autoradiographs at the level of SFO show patterns of IκBα mRNA hybridization in animals killed after saline injection (Sal; A) and at 0.5 hr (B), 2 hr (C), and 12 hr (D) after the LPS injection. The arrows in B and C point to blood vessels. Ch Plx, choroid plexus; 3V, third ventricle; V Ep, ventricular ependyma.

Figure 2

Low-power dark-field photomicrographs at the level of AP show IκBα mRNA labeling in animals sacrificed after saline injection (Sal; A), and at 0.5 hr (B), 1 hr (C), 2 hr (D), 4 hr (E), and 12 hr (F) after LPS injection.

Figure 3

High-magnification bright-field photomicrographs show labeling of IκBα mRNA in blood vessels (BV) at the base of the brain in animals killed after saline injection (Sal; A), and at 0.5 hr (B), 2 hr (C), and 12 hr (D) after the LPS injection. Arrows point to cells of the blood vessels, and arrowheads point to cells in the arachnoid membrane.

Figure 4

High-magnification bright-field photomicrographs show labeling of IκBα mRNA in the choroid plexus in animals killed after saline injection (Sal; A) and at 0.5 hr (B) and 2 hr (C) after the LPS injection. Arrows point to cells of the blood vessels (BV), and arrowheads point to cells in the choroidal ependyma (Ch Ep).

Figure 5

High-magnification bright-field color photomicrographs show hybridization of IκBα mRNA (arrows point to clusters of silver grains where cells are positive) and immunohistochemical labeling (brown reaction product) of platelet and endothelial cell adhesion molecule (PECAM) (A), glial fibrillary acidic protein (GFAP) (B), OX-42 (C), and ED2 (D). In the OX-42 and ED2-labeled sections, the silver grains did not colocalize with the immunostaining. The section in D was counter stained with cresyl violet to show that the silver gains are over an adjacent Nissl-stained cell.