Cognitive deficit associated with cholinergic and nerve growth factor down-regulation in experimental allergic encephalomyelitis in rats

Abstract

Clinical symptoms in multiple sclerosis include cognitive dysfunction. Difficulties in learning and remembering new information represent the most common cognitive deficit and are associated with a general and progressive brain pathology. Possible pathogenetic mechanisms for neuronal damage such as neuroprotective strategies are under active investigation also in experimental allergic encephalomyelitis, the most widely used experimental model for multiple sclerosis. In this paper we demonstrate that a selective deficit in learning and memory performance, as investigated by the Morris water maze test, is a consistent feature in rat encephalomyelitis, which correlates with a decline in choline acetyltransferase activity and nerve growth factor mRNA level in cerebral cortex, hippocampus, and basal forebrain. Treatment aimed to restore acetylcholine content through chronic administration of selective acetylcholinesterase inhibitors (rivastigmine and donepezil) restores cognitive performance, choline acetyltransferase activity, and nerve growth factor mRNA expression.

Fig. 1.

Design of the study (A) and clinical score of EAE in Lewis female rats (B) included in experiment 1. Animals were tested for behavioral performance (Morris water maze test) at the indicated time, and all animals were then killed for biochemical, molecular, and morphological analysis. Animals developed severe EAE 10–14 dpi. Only nonrelapsing animals were included in the study.

Fig. 2.

The graphs report performances in the water maze task, as indicated by latency (A) and velocity (B) in the visible and hidden platform trials, and in the spatial orientation trial (C) in control, EAE, and EAE+rivastigmine animals at 90 dpi. In A and B, each point represents the average over the two trials within each block. In C, the percentage of time spent in the target quadrant is reported. Repeated overall ANOVA measures between EAE and control animals indicated a significant group effect (F = 5.66, P = 0.0365) that was also confirmed in the spatial orientation trail. Rivastigmine treatment restores behavioral capability in the spatial orientation test (Student's t test; *, P < 0.05).

Fig. 3.

Morphological study of cholinergic neurons in the basal forebrain (horizontal diagonal band nucleus). (A–D) Immunohistochemical visualization of ChaT-positive neurons to show codistribution with microglial cells (OX42-positive; A and B) and astrocytes (GFAP-positive; C and D) in control (A and D) and EAE (B and D) 90-dpi animals. A slight astrocytosis is observed in EAE animals. (E and F) Colocalization of p75^NTR in ChaT-positive neurons. A wider expression of p75^NTR is observed in dendrites in EAE animals (F; 90 dpi) compared with control animals (E), suggesting an increased expression of the receptor. Representative images (n = 5 per group) obtained by confocal laser microscopy are shown.

Fig. 4.

Decline of ChaT activity in different brain areas (basal forebrain, cerebral cortex, and hippocampus) in EAE rats, expressed as the percentage variation compared with control animals at two different times (30 and 90 days) after immunization. There is a significant decrease in the ChaT activity at both 30 and 90 dpi in all investigated areas. The recovery in rivastigmine-treated EAE rats is also shown. Statistical analysis was by one-way ANOVA and Dunnet's test (P < 0.01).

Fig. 5.

NGF mRNA significantly decreases in the cortex of EAE animals as measured by RT semiquantitative PCR. Rivastigmine and donepezil induce a partial recovery in NGF mRNA level. Linear regressions for NGF and GAPDH housekeeping genes indicate the linear relationship between the cDNA amplified and the PCR product obtained. Data obtained from three to four animals per groups were statistically analyzed by one-way ANOVA and Dunnett's test (*, P < 0.05).