Histological correlates of hippocampal magnetization transfer images in drug-resistant temporal lobe epilepsy patients

Abstract

Objective: Temporal lobe epilepsy patients (TLE) often present with hippocampal atrophy, increased T2 relaxation, and reduced magnetization transfer ratio (MTR) in magnetic resonance images (MRI). The histological correlates of the reduced hippocampal MTR are so far unknown. Since MTR is dependent on the tissue's macromolecules, our aim was to evaluate the correlations between cellular populations, extracellular matrix molecules and the MTR in TLE patients.

Methods: Patients with TLE (n = 26) and voluntaries (=20) were scanned in a 3 Tesla MRI scanner, and MTR images were calculated from 3DT1 sequences with magnetization pulse on resonance. Immunohistochemistry for neurons, reactive astrocytes, activated microglia, and extracellular matrix chondroitin sulfate were performed in formalin fixed, paraffin embedded tissues of TLE and autopsy controls (n = 10). Results were considered significant with adjusted p < 0.05.

Results: Compared to the respective controls, TLE patients had reduced hippocampal MTR, increased reactive astrocytes and activated microglia, increased extracellular chondroitin sulfate, and reduced neuron density, compares to controls. MTR correlated positively with neuron density in CA3 and with chondroitin sulfate in CA3 and CA1. Multiple linear regressions reinforced the correlations between chondroitin sulfate and MTR.

Significance: Our data indicate that extracellular matrix molecules are the most significant histological correlates of magnetization transfer ratio in the hippocampus of TLE patients.

Keywords: Extracellular matrix; Hippocampal sclerosis; Magnetization transfer ratio; Neuron density; Temporal lobe epilepsy.

Fig. 1

Quantitative magnetic resonance evaluation of the hippocampi from controls (radiological controls, blue boxplots) and TLE patients (red boxplots). (A) There was no difference between controls and ipsilateral (dark red boxplot) or contralateral (light red boxplot) hippocampi from TLE regarding volume. (B) Only the ipsilateral hippocampus of TLE patients presented with increased T2 relaxation time and (C) reduced magnetization transfer, when compared to controls. There was no difference between the ipsilateral and contralateral hippocampus of TLE cases. The asterisks indicate statistical difference, the line inside the boxplots indicate median and the dot indicate mean. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 2

Semi-quantitative evaluation of hippocampal sections from controls (histological controls, blue boxplots) and TLE patients (red boxplots) submitted to immunohistochemistry. (A) All hippocampal subfields but the subiculum of TLE cases presented with neuron loss. (B) Only CA1 and the subiculum of TLE had significant astroglial reaction, when compared to controls. (C) Following the neuron density changes, all hippocampal subfields but the subiculum had activated microglia, when compared to controls. (D) Chondroitin sulfate proteoglycan was seen in higher levels in all hippocampal subfields of TLE cases, compared to controls. The asterisks indicate statistical difference, the line inside the boxplots indicate median and the dot indicate mean. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 3

Representative immunohistochemistry micrographs of the pathological changes seen in the CA1 subfield from TLE samples. (A) NeuN staining, showing reduced neuron density in TLE CA1. (B) GFAP staining showing fibrous astrogliosis, as defined by ILAE, in a TLE case. Two reactive astrocytes are indicated by arrows. (C) Increased activation of microglial cells, marked by the expression of MHC class II HLA-DR protein. One activated microglia is pointed by the black arrow. (D) Fibrous aggregates of extracellular chondroitin sulfate proteoglycans, showed by CS-56 antigen immunohistochemistry. The bar in (D) indicates 100 µm.

Fig. 4

Scatterplot and regression between histological and magnetization transfer data of TLE patients. Whole hippocampal MTR from TLE cases correlated positively with neuron density in CA3 (A) and with chondroitin sulfate levels in CA3 (B), CA1 (C), and the subiculum (D).