Science & Progress: For the first time, we have imaging technology to visualize neuroinflammation in the brain!

*For medical professionals only

With the acceleration of the global aging process, neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease and multiple sclerosis have become major health issues that need to be solved urgently in an aging society [1-3

A growing body of research shows that chronic neuroinflammation caused by the continuous activation of microglia and astrocytes strongly affects neurodegenerative diseases and promotes disease progression

Based on the above vision, how to achieve continuous monitoring of the inflammatory response of glial cells in the brain and provide biomarkers for diagnosing neurodegenerative diseases has become a hot topic

Recently, a team of researchers led by Silvia De Santis and Santiago Canals of the UMH-CSIC Institute of Neuroscience in Spain published a groundbreaking research in

They successfully used magnetic resonance imaging to record the activation of microglia and astrocytes in vivo [7].

Screenshot of the first page of the paper

Given the difficulty of biopsy sampling of neurological disorders, so far, the diagnosis of neurological diseases has mainly relied on imaging techniques such as CT, MRI, and PET

Currently, the "gold standard" for diagnosing neuroinflammation is based on a positron emission scan (PET) test

The diffusion-weighted magnetic resonance imaging (dw-MRI) captures the imaging principle of the random movement of water molecules in the brain parenchyma, making it have a unique non-invasive, high-spatial resolution imaging ability

Based on this characteristic, researchers have conducted a series of studies around MRI sequences, but the current design mainly targets white matter and axons, and it is impossible to distinguish which type of cells
these structures come from.

Since different types of neurodegenerative diseases have different mechanisms, involve specific cell populations, and the specific role played by different types of cell populations in the etiology and progression of the
disease, cell type specificity for imaging is critical.

However, to date, no technique has been able to achieve specific imaging
of microglia and/or astrocytes in vivo.

Existing research has measured the diffusion characteristics of specific tissue gaps and even cell types by combining advanced dw-MRI sequences with mathematical models based on knowledge of brain parenchymal cell morphology [9].

Based on the above ideas, the Santis team used knowledge of microglia and astrocyte morphology to develop a new set of magnetic resonance imaging sequences to record the activation responses of small glial cells and astrocytes in gray matter by constructing microstructured multi-chamber tissue models
.

Let's take a look at how scientists cleverly implemented this idea
.

First, the researchers established a gray matter diffusion model
based on the corresponding sphere size and stick branches based on the histological morphology of microglia and astrocytes.

In simple terms, microglia in the activated state are "amoeba-like", and their models are small spheroids and rod-like branches, small spheres simulate the degree of water diffusion in microglia cell bodies at dw-MRI, and the rod-like structure of radial branches mimics the diffusion parameters of microglia emitting branches; Large spheres mimic astrocytes
in the activated state.

Microglia gray matter diffusion model (top) and astrocyte gray matter diffusion model (bottom)

Finally, by collecting and analyzing the sphere radius representing glial cell size, the stick fraction representing the branch emitted by glial cells, the stick dispersion degree representing the number of branches emitted during the occurrence or resolution of inflammation, and the tissue fraction representing the number of glial cells during the occurrence or resolution of inflammation.
To distinguish between microglia and/or astrocytes in gray matter activated or not
.

After the model parameters were determined, the scientists used tissue immunochemical staining and dw-MRI to compare and verify
the model.

Design of experiments

First, the scientists used Iba-1⁺ immunohistochemical staining and dw-MRI to compare and analyze the activation response of
microglia.

At different time points after the injection of the inflammatory inducer lipopolysaccharide (LPS), histomorphological analysis of tissue Iba-1⁺ cells showed that within 8 hours, microglia responded rapidly and there were significant changes in tissue density, cell size, and process dispersion until 24 h
.

The above glial morphological changes
were not detected when LPS and microglia depletion (PLX5622) were used simultaneously, or after 2 weeks of self-disappearance of the LPS-induced inflammatory response.

Iba-1⁺ immunohistochemical staining

In the figure below, the stick fraction, small sphere radius, and stick dispersion of diffusion-weighted magnetic resonance imaging have time-dependent changes consistent with Iba-1⁺ cells, indicating that the activation response of microglia in vivo can be captured using dw-MRI
.

dw-MRI

In addition, when looking at the differences between individuals, they found that there was a strong correlation between the parameters recorded by the MRI and their corresponding histological changes at all measured time points (8h and 24h), with the correlation coefficient r 2=0.
997 (p=0.
0026) at 8h and the correlation coefficient r²=0.
9381 (p=0.
0314)
at 24h.

Iba-1⁺ immunohistochemical staining and dw-MRI correlation analysis

The above results demonstrate that dw-MRI can record the microglial activation response in vivo and its specific signals
after recovery.

Next, the researchers used GFAP immunohistochemical staining and dw-MRI to compare and analyze the activation response process
of astrocytes.

Unlike microglia, after 8 h of LPS injection, immunohistochemical staining showed that the density and morphology of astrocytes did not change significantly, but their volume increased significantly after 24 h, and the phenomenon disappeared after 2 weeks of inflammation on their own
.

Moreover, after the injection of microglial depletion (PLX5622), the phenomenon of a significant increase in astrocyte volume persists (Figure C
).

The large bulb volume parameters of dw-MRI undergo time-dependent changes consistent with GFAP (Figure D
).

When looking at the differences between individuals, they also found a strong correlation between the parameters recorded by the MRI and their corresponding histological changes in the sample at all measurement time points, with a correlation coefficient r²=0.
926 (p=0.
0375).

GFAP immunohistochemical staining and dw-MRI correlation analysis

In general, the causes of inflammatory diseases of the nervous system can be roughly divided into two categories: one is induced by spontaneous apoptosis and/or necrosis of neuronal cells, which in turn promotes the inherent immunity of the nervous system to accelerate the progression of diseases; The other category is substances other than neuronal cell degeneration, which cause a simple inflammatory response
in brain tissue.

It is not difficult to see that separating the inflammatory response with neurodegeneration from the simple inflammatory response is of great significance for the differential diagnosis of
the cause.

The researchers then tried to see if they could distinguish
the two with the model they developed.

They injected the left cerebral hemisphere of a group of animals with the neurotoxin Amanita ammonia (IBO) and the right cerebral hemisphere with physiological saline, and the left side of the brain developed neurodegeneration, and the right side of the brain served as a control of
itself in the physiological state.

Comparative analysis of MRI imaging and NeuN immunohistochemical staining is performed after 2 weeks
.

Design of experiments

The experimental results show that unlike the simple inflammatory response (microglial activation) caused by LPS, which disappears after 2 weeks, the microglial activation state caused by IBO-mediated neuronal degeneration persists (Figure A), and the resulting MRI parameters are also significantly different
.

For example, the dw-MRI stick fraction and tissue fraction parameters of the IBO treatment group were significantly reduced.
However, the small sphere radius, small sphere fraction, and stick dispersion were significantly higher (Figure C
).

The above results show that the simple inflammatory response and the inflammatory response with neurodegeneration can be distinguished by
the small sphere parameters and tissue density parameters in the dw-MRI results.

To further verify the reliability of the model, the researchers tried to observe the corresponding histological changes and desirable changes in MRI parameters after using the neuroprotective agent minocycline
.

The results were also ideal, with Neun staining demonstrating the protective effect of minocycline on IBO-induced neuronal degeneration, and the MRI parameter tissue density also captures this effect (Figure D
).

The above results suggest that dw-MRI can distinguish between a simple inflammatory response and an inflammatory response
with neurodegeneration.

Neun immunohistochemical staining and dw-MRI correlation analysis

To further explore the reproducibility and effectiveness of the model's transition to clinical applications, the researchers validated
it in humans.

The results show that the coefficient of variation (CoV) values of the model in the same subjects are between 1.
5-8%, and the CoV values between different subjects are between 2.
6-15%, which are within the range of MRI measurements used in clinical routines, indicating that this model has diagnostic value
.

In addition, multiple linear regression models show a significant correlation between histological results and MRI parameters (p=0.
03
).

Multiple linear regression models

Overall, this study shows that dw-MRI has great potential in revealing the diagnosis and differential diagnosis of neurodegenerative diseases that accompany inflammatory responses, providing researchers and patients with new, non-invasive, gliocyte-centric MRI biomarkers that greatly facilitate
research into diseases associated with glial cell activation.

Given that inflammation is a potential cause of many neurological disorders, the technique developed by Santis' team to detect the activation response of glial cells promises to be a powerful marker of early diagnosis and/or prognosis
.

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