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A breakthrough in creating transparent brains

A transparent 3-D view of amyloid beta plaques (green) and blood vessels (red) in a region of cerebral cortex from a 20-month-old AD model mouse (credit: RIKEN)

Researchers at the RIKEN Brain Science Institute in Japan have created a new technique for converting brain tissue into transparent tissue to reveal 3D brain anatomy at very high resolution.

The researchers say they have used the new technique, called ScaleS, to provide new insights into Alzheimer’s disease plaques and for large-scale connectomic mapping and 3D neural circuit reconstruction.

CLARITY_stained

Three-dimensional view of stained hippocampus showing fluorescent-expressing neurons (green), connecting interneurons (red) and supporting glia (blue), created with the Stanford CLARITY technique (credit: Deisseroth lab)

Previous techniques, such as Stanford’s CLARITY, for creating transparent brain samples — a process called “optical clearing — are useful for microscopy, but the transparency process itself can damage the structures under study, the researchers note. The structures are also not firm enough for electron microscopy, which is used to provides images at a finer level.

These limitations are now overcome with ScaleS, according to the researchers. The internal structures also maintain their original shape and are firm enough to permit the micron-thick slicing necessary for more detailed analyses.

Published in Nature Neuroscience, the new technique combines sorbitol in the right proportion with urea. “We could create transparent brains with minimal tissue damage,” said lead scientist Atsushi Miyawaki. The technique can handle “both florescent and immunohistochemical labeling techniques, and is even effective in older animals,” he added.

Miyawaki believes that the quality and preservation of cellular structures viewed by electron microscopy with ScaleS is unparalleled.

New Alzheimer’s disease findings

A 2-D version of a 3-D image of amyloid beta plaques in entire brain hemispheres of an Alzheimer’s disease model mouse created with ScaleS. The inset (bottom right) shows a high-magnification volume rendering of a representative senile plaque (credit: RIKEN)

The researchers were able to use ScaleS to visualize the mysterious “diffuse” plaques seen in the postmortem brains of Alzheimer’s disease patients that are typically undetectable using 2D imaging. They found that contrary to current assumptions, the diffuse plaques proved to be not isolated; they showed extensive associations with microglia — mobile cells that surround and protect neurons.

The researchers also examined 3-D images of active microglial cells and amyloid beta plaques. While some scientists suggest that active microglial cells are located near plaques, a detailed 3D reconstruction and analysis using ScaleS clearing showed that association with active microglial cells occurs early in plaque development, but not in later stages of the disease, after the plaques have accumulated.

“Clearing tissue with ScaleS followed by 3D microscopy has clear advantages over 2D stereology or immunohistochemistry,” states Miyawaki. “Our technique will [also be useful] for examining normal neural circuits and pinpointing structural changes that characterize other brain diseases.”

Abstract of ScaleS: an optical clearing palette for biological imaging

Optical clearing methods facilitate deep biological imaging by mitigating light scattering in situ. Multi-scale high-resolution imaging requires preservation of tissue integrity for accurate signal reconstruction. However, existing clearing reagents contain chemical components that could compromise tissue structure, preventing reproducible anatomical and fluorescence signal stability. We developed ScaleS, a sorbitol-based optical clearing method that provides stable tissue preservation for immunochemical labeling and three-dimensional (3D) signal rendering. ScaleS permitted optical reconstructions of aged and diseased brain in Alzheimer’s disease models, including mapping of 3D networks of amyloid plaques, neurons and microglia, and multi-scale tracking of single plaques by successive fluorescence and electron microscopy. Human clinical samples from Alzheimer’s disease patients analyzed via reversible optical re-sectioning illuminated plaque pathogenesis in the z axis. Comparative benchmarking of contemporary clearing agents showed superior signal and structure preservation by ScaleS. These findings suggest that ScaleS is a simple and reproducible method for accurate visualization of biological tissue.

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Low vitamin D associated with faster decline in cognitive function

(Credit: iStock)

A research team has found that Vitamin D insufficiency was associated with faster decline in cognitive functions among a group of ethnically diverse older adults, according to an open-access paper published in JAMA Neurology.*

According to the researchers — Joshua W. Miller, Ph.D., of Rutgers University, New Brunswick, N.J., and coauthors from the University of California, Davis — Vitamin D may influence all organ systems, not just calcium absorption and bone health.

Both the vitamin D receptor and the enzyme that converts 25-hydroxyvitamin D (25-OHD) to the active form of the vitamin are expressed in all human organs, including the brain. So research has increasingly examined the association between vitamin D status and a variety of health outcomes, including dementia and age-associated cognitive decline.

JAMA |Vitamin D and Cognitive Decline in Multiethnic Cohort of Older Adults

The authors report:

  • The average 25-OHD level among participants was 19.2 ng/mL, with 26.2 percent of participants being vitamin D deficient and 35.1 percent vitamin D insufficient.
  • Average 25-OHD levels were lower for African American and Hispanic participants compared with their white counterparts (17.9, 17.2 and 21.7 ng/mL, respectively).
  • Average 25-OHD levels were lower in the dementia group compared with mild cognitive impairment and cognitively normal groups (16.2, 20.0 and 19.7 ng/mL, respectively.
  • During an average follow-up of 4.8 years, rates of decline in episodic memory and executive function among vitamin D deficient and vitamin D insufficient participants were greater than those with adequate vitamin D status after adjusting for a variety of patient factors.
  • Vitamin D status was not significantly associated with decline in semantic memory or visuospatial ability.

The authors note limitations to their study including that they did not directly measure dairy intake, sun exposure or exercise, each of which can influence vitamin D levels.

“Our data support the common occurrence of VitD [vitamin D] insufficiency among older individuals. In addition, these data show that African American and Hispanic individuals are more likely to have VitD insufficiency or deficiency.

“Independent of race or ethnicity, baseline cognitive ability, and a host of other risk factors, VitD insufficiency was associated with significantly faster declines in both episodic memory and executive function performance, which may correspond to elevated risk for incident AD [Alzheimer disease] dementia.

“Given that VitD insufficiency is medically correctable, well-designed clinical trials that emphasize enrollment of individuals of nonwhite race/ethnicity with hypovitaminosis D could be useful for testing the effect of VitD replacement on dementia prevention,” the study concludes.

* The researchers examined baseline vitamin D status and change in subdomains of cognitive function as measured on assessment scales in an ethnically diverse group of 382 older adults.

Serum (blood) 25-OHD was measured and vitamin D status was defined as follows: deficient was less than 12 ng/mL; insufficient was 12 to less than 20 ng/mL; adequate was 20 to less than 50 ng/mL; and high was 50 ng/mL or higher.

Study participants were an average age of 75.5 years and nearly 62 percent were female, while 41.4 percent of the group was white, 29.6 percent were African American and 25.1 percent were Hispanic. At study enrollment, 17.5 percent of the participants had dementia, 32.7 percent had mild cognitive impairment and 49.5 percent were cognitively normal.

Abstract of Vitamin D Status and Rates of Cognitive Decline in a Multiethnic Cohort of Older Adults

Importance:  Vitamin D (VitD) deficiency is associated with brain structural abnormalities, cognitive decline, and incident dementia.

Objective:  To assess associations between VitD status and trajectories of change in subdomains of cognitive function in a cohort of ethnically diverse older adults.

Design, Setting, and Participants:  Longitudinal multiethnic cohort study of 382 participants in an outpatient clinic enrolled between February 2002 and August 2010 with baseline assessment and yearly follow-up visits. Serum 25-hydroxyvitamin D (25-OHD) was measured, with VitD status defined as the following: deficient, less than 12 ng/mL (to convert to nanomoles per liter, multiply by 2.496); insufficient, 12 to less than 20 ng/mL; adequate, 20 to less than 50 ng/mL; or high, 50 ng/mL or higher. Subdomains of cognitive function were assessed using the Spanish and English Neuropsychological Assessment Scales. Associations were evaluated between 25-OHD levels (as continuous and categorical [deficient, insufficient, or adequate]) and trajectories of cognitive decline.

Main Outcomes and Measures:  Serum 25-OHD levels, cognitive function, and associations between 25-OHD levels and trajectories of cognitive decline.

Results:  Participants (N = 382 at baseline) had a mean (SD) age of 75.5 (7.0) years; 61.8% were women; and 41.4% were white, 29.6% African American, 25.1% Hispanic, and 3.9% other race/ethnicity. Diagnosis at enrollment included 17.5% with dementia, 32.7% with mild cognitive impairment, and 49.5% cognitively normal. The mean (SD) 25-OHD level was 19.2 (11.7) ng/mL, with 26.2% of participants being VitD deficient and 35.1% insufficient. The mean (SD) 25-OHD levels were significantly lower for African American and Hispanic participants compared with white participants (17.9 [15.8] and 17.2 [8.4] vs 21.7 [10.0] ng/mL, respectively; P < .001 for both). The mean (SD) 25-OHD levels were similarly lower in the dementia group compared with the mild cognitive impairment and cognitively normal groups (16.2 [9.4] vs 20.0 [10.3] and 19.7 [13.1] ng/mL, respectively; P = .006). The mean (SD) follow-up was 4.8 (2.5) years. Rates of decline in episodic memory and executive function among VitD-deficient (episodic memory: β = −0.04 [SE = 0.02], P = .049; executive function: β = −0.05 [SE = 0.02], P = .01) and VitD-insufficient (episodic memory: β = −0.06 [SE = 0.02], P < .001; executive function: β = −0.04 [SE = 0.02], P = .008) participants were greater than those with adequate status after controlling for age, sex, education, ethnicity, body mass index, season of blood draw, vascular risk, and apolipoprotein E4 genotype. Vitamin D status was not significantly associated with decline in semantic memory or visuospatial ability. Exclusion of participants with dementia did not substantially affect the associations between VitD status and rates of cognitive decline.

Conclusions and Relevance:  Low VitD status was associated with accelerated decline in cognitive function domains in ethnically diverse older adults, including African American and Hispanic individuals who exhibited a high prevalence of VitD insufficiency or deficiency. It remains to be determined whether VitD supplementation slows cognitive decline.

Vitamin D3 sources and dosage (update Sept. 17, 2015)

The U.S. National Institutes of Health explains that Vitamin D can be obtained from “sun exposure, food, and supplements,” notes food sources, and makes recommended dietary allowances (RDA). Some sources, such as “The Real RDA for Vitamin D Is 10 Times Higher Than Currently Recommended,” recommend higher RDA.