The facial nucleus, present in vertebrate animals, is a group of neurons in the brainstem that receives instructions from neurons in the cortex to direct movement of the muscles of the face. For this paper, the authors characterized the facial nucleus of two similar species, African and Asian elephants, with muscular, dexterous trunks. These species’ faces share many similarities, but have distinctly different ear size and trunk morphology—areas controlled by the facial nucleus. The authors used varied methods, matched to the available elephant material, including Nissl staining, cell counting, axonal osmium tetroxide stains, somata drawings, cell fiber counting, and nerve tracing to examine the facial nucleus in specimens from African (n=4) and Asian elephants (n=4 elephants). Stereo Investigator^® software was used to acquire images of thin sections, conduct stereological procedures, and measure cell size and axon diameter.

Two methods were used to quantify cell populations in the elephant facial nucleus, an unbiased stereology approach and a model-based stereology strategy that consisted of complete counts of cell pieces in every tenth section were used. Using unbiased stereology, the researchers counted ~200-300 cells per specimen with the Stereo Investigator optical fractionator probe. To confirm their results, the research team next counted ~5000–8,000 cells and cell fragments per specimen, then corrected for double-counted cells. The results were equivalent; however, the unbiased stereology approach was much less time consuming.

The researchers found that the facial nucleus in elephants is much larger than in most mammals and it is comprised of approximately five-fold more neurons, but at significantly lower neuronal density. African elephants were found to have more neurons in the medial facial subnucleus than Asian elephants, consistent with their much larger and more expressive ears. Dorsal and lateral facial subnuclei, which control movement of the trunk, were elongated compared to other vertebrate mammals and contained many more neurons than land-based species. Interestingly, these regions had a distinct proximal-to-distal cells size increase. Comparison with other species and between newborn and adult elephants suggest that this increase in size is needed for to support the extreme axonal volumes associated with trunk innervation. These cell-size gradients were found to be a unique feature of the elephant facial nucleus. Finally, the research team identified a high-density motor fovea that they believe are associated with the tip of the trunk in African elephants. Asian and African elephants’ trunks differ in that Asian elephants have one dorsal trunk finger and they tend to engage much of their trunk in grasping objects by wrapping them in their trunks, whereas African elephants’ trunks have dorsal and ventral fingers that are often used to pinch objects. Their work suggests that African elephants have more neurons associated with the trunk tip than do Asian elephants and that control of African elephants’ trunk fingers resides in the motor foveae they identified.

The research described here relied heavily on cell-count data that was most efficiently obtained using the optical fractionator probe in Stereo Investigator^®. The authors found strong relationships between the number, density, and size of neurons and the position and function of elephant facial morphology. Their results pose interesting avenues for future research, including the role of ear movement in “auditory and infrasound perception” and follow-up studies on the cell-size differences found in the putative trunk representation in the facial nucleus and how these differences may be involved with elephants’ presumed need to compensate for inherent nerve conduction delays associated with their large size.

Learn more about industry-leading Stereo Investigator^® systems for image acquisition and stereological studies.

View our webinar that introduces Stereo Investigator^® and unbiased stereology.

Reference:

Kaufmann, L. V., Schneeweiß, U., Maier, E., Hildebrandt, T., & Brecht, M. (2022). Elephant Facial Motor Control. Science Advances, 8(43). https://doi.org/10.1126/sciadv.abq2789

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Babies with IUGR may develop health problems such as low resistance to infection. They may also have a hard time handling the stress of a vaginal birth. One possible cause of IUGR is that the fetus is not getting enough nutrients from the placenta.

In order to learn more about the structural differences in placentas in normal versus IUGR pregnancies, scientists at the Ludwig Maximilian University of Munich used Stereo Investigator to image tissue in both cases–finding that there are indeed quantifiable differences between the two.

One main difference is that the villi, the finger-like structures that allow nutrients and oxygen to flow from the mother to the baby, are smaller in volume in IUGR cases. Of the two types of villi present in a pregnancy, only one type—the contractile villi (the ones with muscle cells in their surrounding sheaths) were smaller. There was no difference in size between non-contractile villi in normal and IUGR placentas.

The figure shows Tukey plots of core clinical and gross anatomic data.

IUGRHowever, in both types of villi in IUGR placentas, the researchers observed reduced blood vessel volume, longer diffusion distances (distance from fetal to maternal blood), and less branching.

To achieve these results, the researchers extracted six tissue samples from 21 placentas (10 normal, 11 IUGR), and used an MBF Bioscience system comprising microscope, motorized stage, camera, z-encoder, and Stereo Investigator to image samples.

“This combination of software and hardware in the Stereo Investigator system makes it possible to image the tissue sections using systematic random sampling and perform unbiased stereology probes to estimate the percent by volume of different components of the placenta, the amount of branching of the villi, and the diffusion distance. The system allows for unbiased estimates in an efficient manner,” says MBF Bioscience Staff Scientist Dr. Dan Peruzzi.

In their study, the Munich researchers combined three Stereo Investigator probes, including the area fraction fractionator probe (used to estimate percentage by volume) and the probe used to measure diffusion distance, in a novel way.

Citation:

Barapatre, N., Kampfer, C., Henschen, S., Schmitz, C., Edler von Koch, F., Frank, H.G., Growth restricted placentas show severely reduced volume of villous components with perivascular myofibroblasts. Placenta, (109) 2021. https://doi.org/10.1016/j.placenta.2021.04.006

The post Differences Associated with Fetal Growth Restriction are Found Using Stereo Investigator appeared first on MBF Bioscience.

Set for release this spring, the new and improved Stereo Investigator will include a new imaging engine, display engine, automatic camera alignment, automatic lens calibration, the double disector, and live video zooming.  

“I’m excited to say that Stereo Investigator keeps getting better in so many ways. What is common among all these new features is increased functionality, more efficiency and better performance,” says MBF Bioscience Product Manager Nathan Liese.  

The new imaging engine will especially help working with large images, whether they are from the SRS image acquisition, confocal or light sheet microscopes. “Users will be amazed by how much faster they can work with their images”, says Nathan. 

The software’s new automatic alignment and calibration features will be game-changers for researchers with large turn over, especially for those working in core facilities and large labs with many users.  

These new features eliminate the time-consuming process of manually aligning the camera and calibrating lenses. The new automatic camera alignment and calibration functionality promises to convert a formerly complex process that previously could take five to 20-minutes, to a simple one that takes a few minutes. 

Two other new features: Double Disector and Live Video Zooming were specific requests from our users, and help make Stereo Investigator an even more comprehensive tool for stereology studies. Double Disector facilitates the counting process in cases where populations of multiple types need to be quantified within the same study, and Live Video Zooming offers the added convenience of digital zooming to examine hard to see objects.  

Overall Stereo Investigator’s new imaging engine and new tracing engine make the software more powerful than ever, with the ability to handle larger images, load data files much faster, open and save files faster, and more effectively use your computer’s resources. 

The post Stereo Investigator Microscope Edition Gets New Imaging Engine, Automatic Alignment, and More appeared first on MBF Bioscience.

DNA damage occurs in human cells at a constant rate. These cells are usually able to repair themselves, but sometimes deficiencies in certain genes cause the repair process to shut down. When damaged DNA isn’t fixed, mutations can occur that cause accelerated aging or cancerous tumors to form (Hoeijmakers, 2009). Scientists at Erasmus University Medical Center in Rotterdam have found a way to slow down the process – at least in mice.

In a study published in Nature, the researchers report that when mice deficient in the DNA-repair genes Ercc1 or Xpg are put on a restricted diet, they experience better overall health and increased lifespans compared to DNA-repair-deficient mice fed a normal diet. They also found significantly lower levels of neurodegeneration in the brains and spinal cords of diet restricted animals compared to controls.

“Here we report that a dietary restriction of 30 percent tripled the median and maximal remaining lifespans of these progeroid mice, strongly retarding numerous aspects of accelerated aging Mice undergoing dietary restriction retained 50 percent more neurons and maintained full motor function far beyond the lifespan of mice fed ad libitum,” (Vermeij, et al 2016).

Since the DNA-repair-deficient mice were already smaller and weaker than normal mice, the Rotterdam researchers wondered whether diet restriction would be beneficial or detrimental to their health. They found that gradually restricting the diets of DNA-repair-deficient mice starting at age seven weeks increased their median lifespans from 10 to 35 weeks in males and 13 to 39 weeks in females as compared to controls.

They also saw significant differences in the levels of neurodegeneration between these two populations. Using Stereo Investigator, they found 50 percent more neurons in the brains of diet-restricted mice compared to those fed a normal diet. They also saw lower levels of cells expressing p53 – a protein expressed in response to DNA damage – in diet-restricted mice.

According to the authors, dietary restriction may not fix defects in DNA repair mechanisms, but it may help to reduce the severity and speed at which the damage occurs.

“Our findings establish the Ercc1 mouse as a powerful model organism for health-sustaining interventions, reveal potential for reducing endogenous DNA damage, facilitate a better understanding of the molecular mechanism of dietary restriction and suggest a role for counterintuitive dietary-restriction-like therapy for human progeroid genome instability syndromes and possibly neurodegeneration in general,” (Vermeij, et al 2016).

Vermeij W.P., Dollé M.E.T., Reiling E., Jaarsma D., Payan-Gomez C, Bombardieri C.R., Wu H., Roks A.J.M., Botter S.M., van der Eerden B.C., Youssef S.A., Kuiper R.V., Nagarajah B., van Oostrom C.T., Brandt R.M.C., Barnhoorn S., Imholz S., Pennings J.L.A., de Bruin A., Gyenis Á., Pothof J, Vijg J, van Steeg H., and Hoeijmakers J.H.J. (2016) Restricted diet delays accelerated aging and genomic stress in DNA repair deficient mice. Nature 537, 427-431, doi:10.1038/nature19329

Hoeijmakers JH (2009) DNA Damage, aging, and cancer. N Engl J Med; 361:1475-1485, DOI: 10.1056/NEJMra0804615

Stock image of DNA used in accordance with the CC0 public domain license.

The post Diet Restriction Slows Neurodegeneration and Extends Lifespan of DNA-Repair-Deficient Mice appeared first on MBF Bioscience.

Representative images of Iba-1+ microglia in the postnatal day 10 rat hippocampus. Image courtesy of Anna Klintsova, PhD.

Children born with fetal alcohol spectrum disorders face a range of physical and cognitive impairments including long-term deficits in learning, behavior, and immune function. In a paper published in Neuroscience, Dr. Anna Klintsova and her lab at the University of Delaware report that activation of the brain’s immune response may contribute to some of the damage caused by fetal alcohol spectrum disorders.

In their study, the researchers used Stereo Investigator and Neurolucida to examine the hypothesis that exposure to alcohol while the brain is growing rapidly is associated with abnormal microglial activation and high levels of pro-inflammatory proteins which impair learning-related plasticity; leading to neuro-developmental and psychopathological disorders.

“My lab has been using both Stereo Investigator and Neurolucida for more than a decade in all quantitative neuroanatomical studies, including the featured one,” said Dr. Anna Klintsova. “We find this software to be user-friendly, reliable and essential for obtaining unbiased results.”

They used Stereo Investigator to quantify the number of microglia in the hippocampus of neonatal rats who were exposed to alcohol during the equivalent of the third trimester of a human pregnancy. The researchers expected to see an increased number of microglia in alcohol-exposed neonatal rats, however they found a decreased number of microglia. Despite the decrease in microglia number, there was a significant increase in pro-inflammatory proteins expressed by microglia and an increase in microglial activation.

To measure microglial activation, the researchers quantified the area of cell territory using Neurolucida. Activated microglia have a smaller cell territory than resting microglia, so the smaller cell territory found in alcohol exposed rats indicates a more active state.

This research supports the hypothesis that abnormal microglia activation plays a role in fetal alcohol spectrum disorders, however more research is needed to further understand the relationship.

Boschen, K., Ruggiero, M.J., Klintsova, A.Y., (2016) Neonatal binge alcohol exposure increases microglial activation in the developing rat hippocampus. Neuroscience 324: 355–366. DOI: 10.1016/j.neuroscience.2016.03.033

The post Uncovering the role of microglia in fetal alcohol spectrum disorders appeared first on MBF Bioscience.

The optical fractionator probe was used to quantify the number of neurons and glia in the dentate gyrus

Doctors have used lithium to treat patients with bipolar disorder since the 1970s. Known for its efficacy in stabilizing patients’ moods by regulating manic episodes, lithium is also associated with a decreased risk of suicide. But while this naturally occurring element is the most widely prescribed medication for those suffering from bipolar disorder, scientists still have much to learn about how lithium physically affects the brain.

A recent study published in the journal Bipolar Disorders adds to the growing body of evidence that says lithium contributes to cell proliferation in parts of the brain. Conducted by scientists at the University of Mississippi and the VU University Medical Center in Amsterdam, the study revealed an increased number of neurons and glia, and increased astrocyte density in the dentate gyrus of lithium-treated mice versus controls treated with a placebo.

Using the optical fractionator probe in Stereo Investigator, the researchers quantified the number of Nissl stained neurons and glial cells, and calculated astrocyte density. The results showed twenty-five percent more neurons and twenty-one percent more glia in the denate gyrus of lithium-treated mice. They also performed a stereological examination of another brain region – the medial prefrontal cortex (mPFC), but did not witness significant differences between lithium-treated and control mice in this area.

“In this study, particular cortical regions, ie. the fascia dentata in the hippocampus and the mPFC in the cerebral cortex needed to be selected in histological sections of the mice brains,” explained Dr. Harry B.M. Uylings, “therefore the stereological counting procedure applied was the best one. Stereo Investigator greatly assisted in the counting of cells, and the software’s excel data-output was especially beneficial.”

According to the paper, the findings present a more detailed picture of lithium-induced alterations in the dentate gyrus cellular phenotype than previously available, and provide the first evidence for lithium-induced increases in glia and astrocytes.

The authors also explain that while cell number increased in the dentate gyrus of lithium-treated mice, the region’s overall volume as well as that of the greater hippocampus was unaffected by the element. The volume of the dentate gyrus and the hippocampus as a whole was measured with the Cavalieri method in Stereo Investigator.  The researchers describe the dissociation between cell proliferation and volume as “an interesting observation that warrants further investigation.”

Rajkowska, G., Clarke, G., Mahajan, G., Licht, C.M., van de Werd, H.J., Yuan, P., Stockmeier, C.A., Maji, H.K., Uylings, H.B., Differential effect of lithium on cell number in the hippocampus and prefrontal cortex in adult mice: a stereological study. Bipolar Disord. 2016 Feb;18(1):41-51. doi: 10.1111/bdi.12364.

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An immunostained image of myelin basic protein in the cerebella of a mouse brain with an iron-sufficient diet compared with the brain of a mouse exposed to alcohol and fed an iron-insufficient diet. It shows the reduced cerebellar size due to the ID-alcohol combination. Green is MBP immunostain, blue is DAPI for nuclei. Image courtesy of Susan Smith, PhD.

If a pregnant woman drinks alcohol, she risks giving birth to a baby with physical and cognitive deficits – characteristics of fetal alcohol spectrum disorders. In a new study, researchers say that when the mother is low in iron, the consequences are even worse.

The scientists examined two groups of pregnant rats – one group was fed an iron sufficient diet while the other was fed a diet with insufficient iron levels. The offspring from both groups were exposed to alcohol from 4 to 9 days after birth – a time when their brains are going through a growth spurt and are particularly sensitive to alcohol. They were compared to offspring who received an iron-sufficient diet but were not exposed to alcohol. This growth spurt correlates to a growth spurt in humans that occurs during the third trimester of pregnancy.

The researchers used delay and trace eye blink classical conditioning methods to assess the offspring’s learning and memory. Learning impairments were reported in both alcohol-exposed groups regardless of their iron status, but more extreme impairments were seen in iron deficient rats compared to iron sufficient rats. After the behavioral tests were completed, the researchers studied the cerebellum and hippocampus – brain regions involved in learning and memory – at a cellular level.

Using the Optical Fractionator probe in Stereo Investigator, the research team quantified neurons in two different areas of the rat brain: the cerebellar interpositus nucleus and the CA1 region of the hippocampus. Their unbiased stereological analysis revealed significant neuronal loss in the alcohol-exposed iron deficient rat brains compared to the iron sufficient rat brains.

“The most important finding from this study is that maternal iron status strongly influences alcohol’s neurobehavioral damage in the developing offspring,” the authors say in their paper. These data endorse that the treatment of maternal [iron deficiency] through normalization of iron status should improve the child’s developmental outcome despite the gestational alcohol exposure.” (Huebner, et al.)

Huebner, S.M., Tran, T.D., Rufer, E.S., Crump P.M., Smith S.M., (2015) Maternal iron deficiency worsens the associative learning deficits and hippocampal and cerebellar losses in a rat model of fetal alcohol spectrum disorders. Alcoholism: Clinical and Experimental Research doi: 10.1111/acer.12876.

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Phosphorylated tau pS422 immunoreactive profiles (dark brown) in the cortex of P301S mice after repetitive mild TBI. Image courtesy of Dr. Leyan Xu, Department of Pathology, Johns Hopkins University.

Over the course of a football game or a boxing match, athletes may experience a series of mild concussions. Some of these athletes develop a condition known as chronic traumatic encephalopathy (CTE), a neurodegenerative disease characterized by the build-up of abnormal tau protein that eventually leads to dementia. But not every athlete develops CTE after repetitive mild traumatic brain injury, and scientists think genetic factors are involved.

In a recent study, researchers at the Johns Hopkins University School of Medicine found that the density of abnormal tau protein increased exponentially in mice that had a genetic mutation thought to cause neurodegenerative diseases. Their findings contrast with previous studies of mice without genetic mutation, where abnormal tau protein build-up did not occur. This evidence leads the scientists to infer that genetic factors play a role in the onset of CTE.

According to the paper, published in Experimental Neurology, scientists have identified 40 different types of mutations in the gene that makes the brain prone to tau aggregation. “Transgenic mice harboring these mutations can be used to ask whether repetitive mTBI can accelerate onset and course of tauopathy or worsen the outcomes of transgenic disease,” the authors say in their paper.

In this study, Dr. Leyan Xu and his team examined the P301S strain of transgenic mice which harbor a tau gene mutation. After exposing the animals to a series of impact acceleration (IA) injuries involving a weight drop technique, the researchers examined both the neocortex and the retina. The visual tract is a pathway strongly associated with TBI in this mouse model, which is why the researchers investigated the retina in addition to the neocortex.

Using the optical fractionator probe in Stereo Investigator, they analyzed neocortical neurons immunohistochemically labeled with a marker for hyperphosphorylated tau protein – in other words, tau gone awry. They did not witness significant differences in tau build-up between the neocortices of the experimental mice and control mice, however they did see differences between these groups when they examined their retinas. Their findings revealed a 20 fold increase in retinal ganglion cells with hyperphosphorylated tau after one mTBI hit in transgenic mice, compared to controls, a 50 fold increase after four hits, and a 60 fold increase after 12 hits. Both experimental and control mice displayed similar patterns of traumatic axonal injury.

“Our current findings demonstrate that repetitive mTBI accelerates tauopathy in mice predisposed to tau accumulation. A single mTBI event also appears to have an effect in the progression of tauopathy, but repetitive injury has an even greater effect,” (Xu, et al)

Xu, L., Ryu, J., Nguyen, J.V., Arena, J., Rha, E., Vranis, P., Hitt, D., Marsh-Armstrong, N., Koliatsos, V.E., (2015) Evidence for accelerated tauopathy in the retina of transgenic P301S tau mice exposed to repetitive mild traumatic brain injury. Experimental Neurology. doi: 10.1016/j.expneurol.2015.08.014

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Micrograph of cholinergic neurons in the nucleus basalis of Meynert. Image from Wikipedia.

Cholinergic neurons degenerate at devastating rates in Alzheimer’s disease, but Dr. Mark Tuszynski and his team at the University of California, San Diego may have found a way to slow the decline.

Their study, published in JAMA Neurology, reports that nerve growth factor gene therapy increased the size, axonal sprouting, and signaling of cholinergic neurons in 10 Alzheimer’s disease patients.

The patients were enrolled in a clinical trial between 2001 and 2012. Ex vivo and in vivo methods of gene therapy were used to deliver nerve growth factor – a protein that protects neurons and stimulates growth – to the patients. Eight received an implant of their own skin cells that were genetically modified to express nerve growth factor (ex vivo ) and two patients received injections that induced neurons already in the brain to express nerve growth factor (in vivo). In all 10 patients, gene therapy was delivered to the nucleus basalis of Meynert – part of the basal forebrain rich in cholinergic neurons that undergoes degeneration during Alzheimer’s disease. 

The patients’ survival time ranged from one to 10 years. After they had died, researchers analyzed the effects of nerve growth factor on cholinergic neurons.

The axons of cholinergic neurons, labeled with p75, grew toward the source of the nerve growth factor in all 10 patients. To determine if there was a change in cell size, researchers used the nucleator probe in Stereo Investigator to analyze cholinergic neurons of 3 patients who received gene therapy via the ex vivo method in one hemisphere – the other hemisphere was used as a control. Results from Stereo Investigator showed that cell bodies were larger in the treated hemisphere vs. the untreated hemisphere.

Finally, to find out if nerve growth factor induced signaling within cells, the researchers analyzed the amount of CREB and c-fos – markers for cell activation – in 2 patients who received nerve growth factor in vivo. An elevated amount of CREB and c-fos was found when compared to control regions. Neurons exhibiting tau pathology also expressed nerve growth factor, indicating that degenerating cells could respond to nerve growth factor gene therapy.

A phase 2 clinical study is currently under way to report cognitive outcomes in patients with Alzheimer’s disease.

“Collectively, these anatomical findings support the rationale for clinical trials to test the hypothesis that sustained growth factor delivery over time can reduce cell degeneration and stimulate cell function in chronic neurodegenerative disorders, thereby slowing functional decline,” Tuszynski, et al.

Tuszynski, M.H., Yang, J.H., Pay, M.M., Masliah, E., Barba, D., U, H.S., Conner, J.M., Kobalka, P., Roy, S., and Nagahara A.H. (2015). Nerve Growth Factor Gene Therapy: Activation of Neuronal Responses in Alzheimer Disease. JAMA Neurology, published online August 24, 2015. DOI: 10.1001/jamaneurol.2015.1807.

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1. it works with many different microscopes and imaging technologies
2. it provides unbiased data about neuron populations and regions of interest
3. it comes with strong technical and research support provided by MBF Bioscience

source: google scholar

Dr. Mark West, co-developer of the Optical Fractionator stereology probe and Professor of Medical Neurobiology at Aarhus University in Denmark, says “Stereo Investigator is the most reliable tool for collecting unbiased stereology data. It’s backed by excellent technical and research support teams at MBF – you can call to ask about stereology probes, tissue preparation, microscope hardware, basically anything regarding your stereology study.”

In 2014, more labs than ever before also used Stereo Investigator to obtain stereology data from image stacks. Researchers captured images with Stereo Investigator using widefield fluorescence, confocal, single-photon, and/or two-photon microscope to acquire image stacks at random sites throughout their tissue specimens. These image stacks, acquired at the microscope in a systematic and random manner, are suitable for stereological analysis.

Stereo Investigator is increasingly used with a Zeiss ApoTome structured-illumination device on a widefield fluorescence microscope. This technique gives clear, confocal-like images without having to purchase an expensive confocal microscope. Researchers can improve their images even more by running deconvolution software on the acquired images before performing stereology.

In 2014, researchers used Stereo Investigator to discover that postnatal neurogenesis erases memories. Read their paper in Science Magazine.

This past year also brought hints about the future of stereology. MBF Bioscience was awarded an NIH grant to develop a new method for automated cell counting. Soon researchers will no longer need to manually mark each cell to collect data – Stereo Investigator will automatically detect and quantify cells.

Stereo Investigator will continue to evolve to incorporate the latest technology for unbiased stereology, but it will always provide reliable, unbiased stereology data whether you use a brightfield microscope or the latest two-photon microscope.

Then again, the numbers below say it all, Stereo Investigator is the most trusted and productive system for stereology–not only in 2014–but since the advent of computerized stereology:

source: google scholar

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