Satoshi Fujiwara1, Sosuke Yoshinaga1, Shigeto Iwamoto1, Sayaka Shibata2, Aiko Sekita2, Nobuhiro Nitta2, Tsuneo Saga2, Ichio Aoki2, and Hiroaki Terasawa1
1Faculty of Life Sciences, Kumamoto University, Kumamoto, Japan, 2Institute for Quantum Life Science, National Institutes for Quantum and Radiological Science and Technology (QST), Chiba, Japan
Synopsis
Neuroinflammation
is initiated by many types of neural disorders
as a defensive response of the innate immune system in the central nervous system
(CNS). Neuroinflammation is typically accompanied by the disruption of Ca2+
homeostasis. Manganese chloride (MnCl2) is a useful positive MRI
contrast agent that enters activated cells through Ca2+ channels,
and is utilized in Manganese-Enhanced MRI (MEMRI) for functional neuroimaging. We sought to determine whether MEMRI could be
used to assess the cellular/molecular alterations caused by acute
neuroinflammation in vivo, by
focusing on Mn2+ accumulation in the rodent brain.
Introduction
Acute neuroinflammation, initiated by trauma, infection, stroke and toxins, is the rapid and short-lived activation of the innate immune system in the CNS [1]. These inflammatory responses are commonly characterized by highly activated glial cells, which produce reactive oxygen species (ROS) and release proinflammatory cytokines. Proinflammatory cytokines and ROS are known to disrupt Ca2+ homeostasis in neuronal cells [2]. Therefore, it is possible that the disruption of Ca2+ homeostasis could be a good biomarker for the in vivo functional/molecular imaging of neuroinflammation. A technique referred to as MEMRI utilizes MnCl2 as a contrast agent for the in vivo imaging of brain function in animals. Mn2+ is an analogue of Ca2+, and it can enter excitable neurons and reactive glial cells, mainly via voltage-gated calcium channels [3–5]. MEMRI reflects the Ca2+ influx into excitable neurons and activated glial cells, we hypothesized that neuroinflammation could be detectable by MEMRI. In this study, we used a rat model, in which experimental acute neuroinflammation was triggered by an intracerebroventricular injection of lipopolysaccharide (LPS). We sought to determine whether MEMRI could be used to assess the alterations caused by acute neuroinflammation in vivo, by focusing on Mn2+ accumulation in the rodent brain.Materials and Methods
Nine male Sprague-Dawley rats were randomly divided into two groups, consisting of LPS treated (n = 4) and control (n = 5) rats. All MRI scans were performed on a 7T scanner, with a volume resonator for transmission and an 8 channel rat brain phased array coil for reception (Bruker). During the MRI scan, all rats were anesthetized with 2.0% isoflurane. At 0 h, baseline T1-weighted and T2-weighted MRI scans were performed for all of the animals (Fig. 1). For the LPS-treated group, LPS was injected via the left cerebral ventricle, while for the control group, to assess the effects of manipulation, the same operation was performed, but only saline was injected. Sterile saline was also administered to the contralateral side in both groups. At 48 h after the LPS or saline injection, we measured the T1-weighted and T2-weighted images, and performed quantitative T1 and T2 mapping without contrast agents. Thereafter, 50 mM of MnCl2 (100 mg/kg, Sigma Aldrich) was slowly administered through the tail vein for 1 h. MEMRI data were acquired exactly 24 h after MnCl2 administration for all groups. Finally, perfusion fixation was performed, and the brain was carefully extracted for immunohistochemical staining with HE, Iba1, ED1 and GFAP. Results
The volumetric MRI analysis revealed that the
LPS administration caused a significant increase in the volume of the
LPS-injected lateral ventricle. In the third ventricle, no apparent volumetric
changes were observed in any groups (Fig. 2 and Table 1). The T1- and
T2-weighted images acquired before MnCl2 administration showed no
significant differences between the LPS-treated and control groups, in terms of
the signal intensity in the brain parenchyma (e.g., hippocampus and
hypothalamus) (Fig. 2).
MEMRI signal enhancement at 24 h after the
MnCl2 injection was observed throughout the cerebral cortex,
thalamus and hippocampus, for the saline-treated control group (Fig. 3A, right).
In the LPS-treated animals, the signal intensity in the hippocampus was even
more enhanced, in comparison with that of the controls (Fig. 3A, left). To
quantitatively evaluate the accumulation of Mn2+ in brain tissues,
we performed T1 mapping using the saturation recovery RARE sequence, before and
after the MnCl2 administration (Fig. 3B). The ROIs were defined in
the hippocampus (CA2 and CA3) of each animal, based on the T1-weighted MEMRI
and a rat brain atlas [6] (Fig. 3A). The ratio of R1 before and after Mn2+
injection (R1(post) / R1(pre)) in the LPS-treated hemisphere was statistically
larger in the hippocampus (CA2) (1.96 ± 0.15), in comparison with the control
(Fig. 3B and 3C, p < 0.05). This indicates that the increase in R1,
reflecting the high Mn2+ accumulation, is attributable to the
LPS-induced neuroinflammation in the hippocampus.
To investigate whether the LPS injection
affected the brain tissue, we performed HE staining, Iba1 and ED1 immunostaining
to detect activated microglia/macrophages, and GFAP immunostaining to detect
reactive astroglia (Fig. 4). Iba1-positive cells and ED1-positive cells were
observed in throughout the hippocampus and in part of the cerebral cortex surrounding
the lateral ventricle. In contrast, the numbers of Iba1- and/or ED1-positive
cells for the control group were much lower than those for the LPS-treated
group. The GFAP immunostaining, which confers a brown color to the astroglia,
was similar for both groups.Discussion
In the hippocampus, the MEMRI signal of the neuroinflammation model
(induced by intracerebroventricular
administration of LPS) was significantly higher than that of the normal model,
and the enhanced area corresponded to the areas with Iba1- and ED1-positive
microglia. This result indicates that MEMRI signal enhancement, due to the
accumulation of activated microglia as well as enhanced Mn2+ influx
via Ca2+ channels, may reflect LPS-induced acute neuroinflammation.Conclusion
MEMRI is an attractive non-invasive method for the detection of inflammatory CNS disorders.Acknowledgements
The
authors thank Dr. Daisuke Kokuryo and Dr. Jeff Kershaw (QST) for helpful
advice.References
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