Florida Researchers Study Traumatic Brain Injury With Stereo Investigator

journal.pone.0053376.g003

Figure 3 from “Hippocampal CA3 cell loss and downregulation of cell proliferation.”

If a head gets hit hard enough, the trauma occurs instantly. Neurons die, the brain swells as microglia cells rush to the damaged area, and the protective armor known as the blood brain barrier might even rupture. But it doesn’t end there. Long term effects include cognitive impairment, loss of sensory processing, and susceptibility to neurodegenerative diseases like Alzheimer’s.

Researchers at the University of South Florida say patients suffering from chronic Traumatic Brain Injury (TBI) experience a “cascade of events” marked by long-term neuroinflammation, cell loss, and impaired cell proliferation that may manifest over time.

“While TBI is generally considered an acute injury, a chronic secondary cell death perturbation (i.e., neuroinflammation) and a diminished endogenous repair mechanism (i.e., cell proliferation) accompany the disease pathology over long-term,” the authors say in their paper published this month in PLOS ONE.

The scientists used unbiased stereology to analyze activated microglia cells, cell proliferation, and differentiation into immature neurons in several regions of the brains of rats which had experienced TBI eight weeks prior.

They used Stereo Investigator with the Cavalieri estimator probe and the optical fractionator probe to estimate the quantity and volume of stained cells in the cortex, striatum, thalamus, fornix, cerebral peduncle, and corpus callosum, as well as the subgranular zone and the subventricular zone in both hemispheres of the brain.

Eight weeks after the TBI occurred, the researchers found an increased level of active microglia cells at the direct site of the TBI as well as surrounding regions. They also report a decrease in hippocampal neurons, and low levels of cell proliferation in the neurogenic niches.

“Our overarching theme advances the concept that a massive neuroinflammation after TBI represents a second wave of cell death that impairs the proliferative capacity of cells, and impedes the regenerative capacity of neurogenesis in chronic TBI,” the authors say in their paper.

They go on to suggest a “multi-pronged treatment targeting inflammatory and cell proliferative pathways” may help alleviate the pathological effects of chronic TBI.

Read the full paper “Long-Term Up-regulation of Inflammation and Suppression of Cell Proliferation in the Brain of Adult Rats Exposed to Traumatic Brain Injury Using the Controlled Cortical Impact Model” on PLOS ONE.

{Acosta S.A., Tajiri N., Shinozuka K., Ishikawa H., Grimmig B., et al. (2013). Long-Term Up-regulation of Inflammation and Suppression of Cell Proliferation in the Brain of Adult Rats Exposed to Traumatic Brain Injury Using the Controlled Cortical Impact Model. PLoS ONE 8(1): e53376. doi:10.1371/journal.pone.0053376}

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Study Review: Dr. Daniel Peruzzi Highlights Best Publication Practices by Analyzing a New Stereological Study

Dr. Daniel Peruzzi, staff scientist, shares his thoughts below:

Customers often ask Staff Scientists at MBF Bioscience why it is sometimes difficult to reproduce certain published stereological results. For example, we get the question, “The estimates that I make of cell number in the region I’m researching do not match numbers reported in the literature. Can you help me understand why?”

To solve this dilemma, we encourage our customers to publish relevant information about their stereological methods to help others reproduce their findings. That’s why it was a pleasure for me to read the recent paper “Postnatal development of the rat amygdala: a stereological study in rats,” by Chareyron, Banta Lavenex, and Lavenex, 2012, reporting stereological estimates of the volume and number of cells contained in the nuclei of the developing rat amygdala.

The authors did an excellent job reporting their stereological methods, including where they researched, and how they performed stereological sampling. As a result, their conclusions are convincing and more easily reproduced by others. I was impressed by both their methods and their findings. In this article I will first summarize their research and then go on to discuss how they reported the particulars of their stereology methods. My main purpose is to point out their stereological reporting and how well thought out their stereologic methods were.

Continue reading “Study Review: Dr. Daniel Peruzzi Highlights Best Publication Practices by Analyzing a New Stereological Study” »

John Hopkins University Scientists Quantify Neurons with Stereo Investigator

 

Rats lose brain cells as they get older. But that doesn’t mean they can’t find their way through a water maze as quickly as their younger cohorts can.

Using unbiased stereology to quantify neurons in the prefrontal cortex of young and old rats, scientists at John Hopkins University in Baltimore found the total neuron number in the dorsal prefrontal cortex (dPFC) decreases with age. But despite the lost neurons, not all of the aged rats showed spatial learning impairment.

Led by Dr. Alexis Stranahan, the researcher team used Stereo Investigator with the Optical Fractionator to quantify total neuron number and the number of interneurons positively stained with antibodies to glutamic acid decarboxylase 67 (GAD67) in both the dorsal and ventral prefrontal cortex. They also used Stereo Investigator to outline cytoarchitectural boundaries in these regions of the rat brain.

To measure the efficiency of the rats’ spatial memory, the researchers used the Morris Water Maze. Trained to find a target platform while swimming in a pool of water, the rats were rated on their speed, distance traveled, and the time they spent in each area of the pool.

Their stereological analysis only revealed neuron count changes in the dPFC. No changes were observed in the vPFC; “and age-related neuronal loss was not associated with spatial memory performance,” the authors state in their paper, which was published online last February in the Journal of Comparative Neurology and will appear in the April 15 issue.

“We believe that when these data are taken together with the current observation that both aged-impaired and aged-unimpaired rats exhibit decreased neuron number in the dorsal prefrontal region, to the extent that such neuron loss is detrimental in this behavioral model, some compensatory mechanisms might be recruited to maintain the performance of unimpaired rats,” according to the study.

Read the full paper here.

 

Reference:

Stranahan, A. M., N. T. Jiam, A. M. Spiegel and M. Gallagher (2012).
“Aging reduces total neuron number in the dorsal component of the rodent prefrontal cortex.”
The Journal of Comparative Neurology 520(6): 1318-1326.

Our Webinar on Using Unbiased Stereology is Now Available to View Online

 

Our most recent webinar on ‘Using Unbiased Stereology to Accurately Determine the Number of Cells in a Region of Interest’ is now available to view on our website.

The Optical Fractionator is the most commonly used stereological probe in the life sciences.  In our webinar Drs. Jose Maldonado and Dan Peruzzi go over the theory behind the Optical Fractionator probe.

Learn the correct stereological protocol to use when running the Optical Fractionator probe as Drs. Maldonao and Peruzzi give practical advice for planning an experiment using the Optical Fractionator.  Discover how to count cells in order to get an estimate that is precise enough for your research while avoiding spending unnecessary time counting.  Use the theoretical framework presented in this webinar to support the practical application of the Optical Fractionator in your laboratory’s research.

Click here to view the webinar.

Register for Our Upcoming Stereology Webinar

The Optical Fractionator probe in Stereo Investigator is an extremely effective tool for stereological cell quantification. It is our goal to provide our customers with the most efficient methods and tools for their research needs.

Join our staff scientists Drs. Jose Maldonado and Daniel Peruzzi on Wednesday, February 22nd at 12:00pm EST for a webinar on using on using unbiased stereology to accurately determine the number of cells in a region of interest.  Our knowledgeable staff scientists, who use the Optical Fractionator probe extensively in their own research, will explain how the Optical Fractionator probe can be applied to your research.

 

Please click here to register and read the full abstract.

UCLA Scientists use Stereo Investigator to Quantify Juvenile Neurogenesis in Mice

In the period of juvenile life, between birth and adulthood, a mouse brain adds a significant number of new neurons; nearly doubling their number in some regions. Researchers at the University of California Los Angeles published their findings last week in Frontiers in Behavioral Neuroscience.  Their findings showed that these new neurons may aid in the development of several cognitive skills.

Using a transgenic mouse model that lacked the ability to make new neurons after birth, the way a normal mouse does, the researchers were able to quantify the number of neurons contributed to the brain by postnatal, juvenile, and adult neurogenesis.

At age intervals between 14 days and 24 months, the researchers used the optical fractionator probe in Stereo Investigator to estimate cell numbers in the regions of the brain where new neurons are known to be continuously generated after birth. Their results show that during juvenile life parts of the olfactory bulb increase in cell number by 40%, while parts of the hippocampus, a brain structure known to be important in short term memory, grew by 25%. Additionally, in parts of the brain where no postnatal neurogenesis is known to occur cell numbers decreased significantly during this same period of life in all the mice tested.

MBF Staff Scientist Dr. Jose Maldonado, who is a co-author of the study, spoke to us about his methods: “Using Stereo Investigator I was able to quantify cells with high enough precision that we were able to clearly see changes in cell numbers (both up and down) in different parts of the mouse brain across the life of the animal. These cell number estimates describe the dynamic nature of cell numbers in the postnatal brain— in some areas neurons are added and in some they are lost. This shows that the brain of mice and perhaps other mammals is not really ‘done’ being built until the organism is in adulthood.”

The researchers administered behavioral tests dealing with sound, smell, fear, and new environments to see how the mouse’s ability to learn and adapt to its environment may have changed due to the inability to add postnatally generated neurons.

According to the study’s co-author Dr. Jesse Cushman, several cognitive deficits were observed in mice where juvenile neurogenesis was prevented, and males and females were affected differently. Not surprisingly they found the importance of smell in learning reduced in the transgenic mice, and transgenic male mice were unable to remember new environments. Additionally, mice lacking juvenile neurogenesis who were trained to be afraid of a particular sound were excessively afraid of new sounds—a behavior observed in people with anxiety disorders.  Dr. Cushman explained that we see this behavior, “particularly in post-traumatic stress disorder, where for example, any loud sound may trigger an excessive fear response once a soldier returns home to civilian life,” he said.

Read the full paper in Frontiers in Behavioral Neuroscience.

 

Reference

Cushman JD, Maldonado J, Kwon EE, Garcia AD, Fan G, Imura T, Sofroniew MV and Fanselow MS (2012) Juvenile neurogenesis makes essential contributions to adult brain structure and plays a sex-dependent role in fear memories. Front. Behav. Neurosci. 6:3. doi: 10.3389/fnbeh.2012.00003

 

DHA Supplementation Prior to Brain Injury May Reduce Severity

Helmet, neck roll, shoulder pads, thigh pads, knee pads, mouth guard…  A football player’s list of protective gear goes on and on. New research suggests adding one more item to the list: DHA.

Formally known as docosahexaenoic acid, DHA is one of the human brain’s primary fatty acids. Essential for proper brain function, the omega-3 fatty acid is known to benefit patients with heart disease, cancer, and traumatic brain injuries. Researchers at the West Virginia University School of Medicine say DHA may also help lessen the blow to the brain when taken prior to a head injury.

In their study, the scientists examined the brains of a population of rats, which had received dietary supplementation of DHA for 30 days prior to a traumatic brain injury. They used the Optical Fractionator with Stereo Investigator to quantify the amyloid precursor protein-positive axons, a marker of injury in the brain. A stereological count of injured axons revealed a significantly decreased amount of APP-positive axons in the rats who had received DHA supplements.

In addition to stereological analysis, the researchers assessed the brain damage with immunohistochemistry and water maze testing. Each trial revealed evidence that supplemental DHA was beneficial in reducing the injury response.

“Our findings suggest that meaningful public health benefits are likely from increasing currently low dietary DHA omega-3 intakes in our population overall and, in particular, our at-risk populations,” say the authors.

Read the free abstract or access the full article in Neurosurgery.

Mills, J. D MD; Hadley, K. PhD; Bailes, J. E MD; “Dietary Supplementation With the Omega-3 Fatty Acid Docosahexaenoic Acid in Traumatic Brain Injury” Neurosurgery. 68(2):474-481, February 2011: doi: 10.1227/NEU.0b013e3181ff692b

{Image: Public Domain via Wikipedia}

Stereology: Avoiding Bias Using the Optical Fractionator

Optical Fractionator Stereo Investigator 8

by Dan Peruzzi, Ph.D.

Stereology allows us to estimate the amount and size of biological features that are impossible or prohibitive to measure exhaustively. If the stereological probe is designed well, and we can manage to
follow all the rules, bias due to sampling design and estimation methods will be eliminated, resulting in a more accurate estimate. For instance, when using the Optical Fractionator to estimate the number of cells in a particular region, we follow rules to make sure a given cell cannot be counted more than once. The reminders listed here will help you avoid some of the common sources of bias.

Section the Entire Region of Interest

Sampling should be systematic and random. Use the cryostat, vibratome, or microtome to take sections of the whole anatomical area; not just at a supposedly representative section or sections. Pick a section interval and randomly choose the starting section for each animal. This gives every section throughout
the entire region an equal chance of being picked for sampling.

Choose a “Point” on the Cell

If you apply the counting rules to the whole cell, large cells, and those oriented perpendicular to the plane of sectioning, will have a greater chance of being counted than smaller cells or cells with parallel orientation. To avoid size and orientation bias, do not use the whole volume of the cell as the basis of your counting decisions. Instead, dwindle the volume down to a point as much as possible. The finer the resolution in the optical plane (focus), the easier this is to do. The ‘point’ you choose must be uniquely identifiable on the particle. Some suggestions include the cell ‘top’ (i.e., the first part of the cell to come into focus) or the cell nucleolus, if there is one and only one per cell.

Use Thicker Sections

An Optical Fractionator probe typically requires approximately ten to fifteen optical planes through the z-axis of the disector. This is easier to achieve with better z-resolution and with thicker sections. Having many optical planes, instead of just a few, allows for a finer visual discrimination. The advantage is being able to distinguish more easily where the “point” on the cell is in order to determine if it falls inside the disector and should be counted. With fewer optical planes, observer bias can result and begin to reach significant levels.

Eliminating bias is desirable, but we must also do the proper amount of sampling, so the estimate will be precise. This makes it more likely that any given estimate will be closer to the mean of all possible estimates. In other words, eliminating bias will not be enough to efficiently get a good estimate. You must also do enough work for your required level of precision, but not so much that you are wasting time and valuable resources.

Learn more about using the Optical Fractionator at mbfbioscience.com.

Dan Peruzzi is a staff scientist at MBF Bioscience.

First published in The Scope, summer 2008.