A Deeper Dive

Multimodal Neuroimaging

With multimodal neuroimaging, multiple features of the brain can be assessed non-invasively. Specifically, by utilizing various sequences one can measure regional volume, microstructural properties, and estimates of myelin content.

Expand each property below to learn more.

Captured through relaxation contrast, such as T1-weighted images (i.e. MPRAGE)
Using the Pig Brain Atlas, the brain can be segmented into:
- White matter
- Gray matter
- Cerebral spinal fluid
- Individual regions
Utilized to identify discrepancies in absolute & relative volume to assess volumetric growth trajectories

Captured through diffusion weighted imaging (DWI) which models the diffusion of water through the brain
Several diffusion metrics can be derived to estimate fiber direction and integrity
Axial diffusivity (AD) = diffusion rate along the fiber
Radial diffusivity (RD) = diffusion across the fiber
Mean diffusivity (MD) = average diffusion rate
Fractional anisotropy (FA) = directional coherence of water movement

Acquired through myelin water imaging (MWI)
Myelin sheath is a layer of fatty tissue that surrounds an axon to ensure efficient and effective communication
Within the lipid bilayers of myelin, there is water trapped that has different relaxometry properties compared to free floating molecules
A myelin water fraction (MWF) can be derived to map myelin in the brain

The Challenge

However, although multimodal neuroimaging is useful to gain insights on how environmental influences may impact the brain longitudinally, there remains a major challenge.

How do we know the true biological significance of MRI results?

Microscope Using various histological measures, we can assess the microstructure of different regions.

Our lab has particularly been interested in the hippocampus - a mixed gray matter and white matter region important for memory function.

11-weighted coronal anatomical mri and gallyas histological stain of hippocampus — **Figure 1.** On the left is a T1-weighted coronal slice of the pig brain. This is an anatomical MRI image typically used for volumetric assessment of individual regions of interest. Within this image the gray regions represent gray matter, while the white regions signify white matter. **A box is outlining a section of the left hippocampus with a portion of the thalamus.** To the right is a zoomed in section of the hippocampal and thalamic tissue that is stained with the **Gallyas silver impregnation method**. This method stains myelin fibers a dark brown color which is easily distinguishable from the light background, making myelin quantification possible. *Abbreviations: MRI, magnetic resonance imaging.*

Using different stains, we can obtain different microstructural information about the tissue.

Two histological stains that have been done on pig hippocampus tissue.

Individual myelin fibers are darkly stained with this method allowing quantification of myelin density within a tissue.

Individual neuronal cell bodies are stained a blue-purple color allowing quantification of cell density and distinction of various cellular types.

Current Work

Recently, our laboratory was interested in determining if there are any sex-specific neurodevelopmental differences in pigs.

Two pigs, one male and one female with a brain showing the assesments and stained hippocampus section. — **Figure 2.** Our laboratory was interested in identifying if there are any sex differences in neurodevelopment in 4-week pigs. The four icons represent 4 different ways that we can assess neurodevelopment. Starting from the left moving right we can use the following methods: **volumetric assessment** with T1-weighted images, **microstructure assessment** with diffusion tensor imaging, **myelin assessment** with myelin water fraction, and estimates of **myelin quantification** using the Gallyas silver impregnation histological method.

Using multimodal neuroimaging and histology several sex-specific differences were identified in the hippocampus.

A boxplot graph representing results of myelin water fraction assessment in the hippocampus of male and female pigs. — **Figure 3.** Myelin water fraction (MWF) differences between sexes in the hippocampus. (A) Differences between males (n = 11) and females (n =10) were observed in MWF in the whole hippocampus (𝑚𝑎𝑙𝑒𝑠: 0.074; 𝑓𝑒𝑚𝑎𝑙𝑒𝑠: 0.079; 𝑃 = 0.022). Female pigs were observed to have higher values of MWF in the hippocampus compared to male pigs. (B) Additionally, differences between males (n = 11) and females (n =10) were observed in MWF in the left hippocampus (𝑚𝑎𝑙𝑒𝑠: 0.072; 𝑓𝑒𝑚𝑎𝑙𝑒𝑠: 0.078; 𝑃 = 0.038), but not the right hippocampus (𝑚𝑎𝑙𝑒𝑠: 0.076; 𝑓𝑒𝑚𝑎𝑙𝑒𝑠: 0.080; 𝑃 = 0.115). Significance (𝑃 < 0.05) between means is denoted by an asterisk (*). *Abbreviations: Hippo, hippocampus.*

Specifically, female pigs were observed to have higher MWF values in the hippocampus as a whole (A) and more specifically in the left hippocampus (B) compared to male pigs.

Furthermore, through multiple regression analyses, it was determined that AD and MD, measures derived from diffusion tensor imaging, were positively associated with myelin density independent of sex.

Read the full article to learn more

Pig Imaging Group

Beckman Institute for Advanced Science and Technology