Time-course changes of nerve growth factor, corticotropin-releasing hormone, and nitric oxide synthase isoforms and their possible role in the development of inflammatory response in experimental allergic encephalomyelitis

Abstract

In this paper we report a time-course study of development of experimental allergic encephalomyelitis in Lewis rats, by monitoring neuroendocrine regulation of the hypothalamus-pituitary-adrenal axis through corticotropin-releasing hormone mRNA expression, inflammatory cellular infiltrate, macrophagic and neuronal nitric oxide synthase, nerve growth factor (NGF), and NGF p75 and trkA receptors in the brain and spinal cord. We analyzed animals during 20 days after immunization, a time interval that corresponds to the acute immunological phase. We have described a severe, early fall of corticotropin-releasing hormone mRNA expression, which could account for the decreased response of the hypothalamus-pituitary-adrenal axis to inflammatory stress. During this period, an increase of neuronal nitric oxide synthase was observed in the cerebral cortex and spinal cord, and macrophagic nitric oxide synthase positive cells were found in the inflammatory cellular infiltrate, which was abundant in perivascular and submeningeal areas 20 days after immunization. Concomitantly, we found a dramatic up-regulation of NGF receptors on the wall of blood vessels and adjacent neurons in perivascular areas. NGF content also had increased in some brain areas, such as the thalamus, while it had decreased in others, like the spinal cord and medulla oblongata, at time points in which the most serious cellular infiltrate was found.

Figure 1

Body weight of experimental rats (A) and clinical score of EAE rats (B). Statistical analysis: ANOVA and Dunnett’s test; ∗, P < 0.05.

Figure 2

(A) Toluidine blue stained section from mesencephalon of EAE rat 20 days after immunization. Arrows indicate large neurons in the mesencephalic nucleus of the V nerve. Intense perivascular cellular infiltrates were found in this area. Numerous diaminobenzidine-positive elements, probably neurophils (B and C) and iNOS-like positive cells (D and E) were observed in perivascular and intraparenchymal inflammatory cellular infiltrates. (F and G) Mast cells stained metachromatically violet by toluidine blue in the thalamus. (H–L) p75-immunoreactive neurons in the cerebral cortex of EAE rats 20 days after immunization. Small, stellar, p75-positive cells adherent to the wall (M), and fibers around blood vessels (N) were observed all over the brain and spinal cord. (O–R) trkA-immunoreactive cells in the cerebral cortex of EAE rats 20 days after immunization. Asterisks indicate the lumen of blood vessels. (Bars: A, I, and O, 50 μm; B and C, 25 μm; D–G, L–N, and P–R, 25 μm; H, 10 μm.)

Figure 3

(A, C, and D) NGF content in different brain areas during development of EAE and CFA-induced inflammation. (B) Number of thalamic mast cells in the thalamus during EAE. Mast cells have been counted in 5 matching sections from the thalamus of each animal, and the relative number has been normalized according to the sampled area. Statistical analysis: ANOVA and Dunnett’s test; ∗, P < 0.05, ∗∗, P < 0.01, ∗∗∗, P < 0.001; a, Student’s t test, P < 0.05.

Figure 4

nNOS mRNA expression in single neurons in the spinal cord (A) and cerebral cortex (B) during EAE, CFA-induced arthritis, and in control rats. Statistical analysis: ANOVA and Dunnett’s test, ∗, P < 0.05, ∗∗, P < 0.01; a, Student’s t test, P < 0.05.

Figure 5

(A) CRH and (B) nNOS mRNA expression in the PVN during EAE (•), CFA-induced arthritis (○), and in control rats (stippled band). Statistical analysis: ANOVA and Dunnett’s test, ∗, P < 0.05, ∗∗, P < 0.01; a, Student’s t test, P < 0.05.