Clearing, immunofluorescence, and confocal microscopy for the three-dimensional imaging of murine testes and study of testis biology.

Optical clearing techniques provide unprecedented opportunities to study large tissue samples at histological resolution, eliminating the need for physical sectioning while preserving the three-dimensional structure of intact biological systems. There is significant potential for applying optical clearing to reproductive tissues. In testicular biology, for example, the study of spermatogenesis and the use of spermatogonial stem cells offer high-impact applications in fertility medicine and reproductive biotechnology. The objective of our study is to apply optical clearing, immunofluorescence, and confocal microscopy to testicular tissue in order to reconstruct its three-dimensional microstructure in intact samples. We used Triton-X/DMSO clearing in combination with refractive index matching to achieve optical transparency of fixed mouse testes. An antibody against smooth muscle actin was used to label peritubular myoid cells of seminiferous tubules while an antibody against ubiquitin C-terminal hydrolase was used to label Sertoli cells and spermatogonia in the seminiferous epithelium. Specimens were then imaged using confocal fluorescence microscopy. We were able to successfully clear testicular tissue and utilize immunofluorescent probes. Additionally, we successfully visualized the histological compartments of testicular tissue in three-dimensional reconstructions. Optical clearing combined with immunofluorescence and confocal imaging offers a powerful new method to analyze the cytoarchitecture of testicular tissue at histological resolution while maintaining the macro-scale perspective of the intact system. Considering the importance of the murine model, our developed method represents a significant contribution to the field of male reproductive biology, enabling the study of testicular function.

Genistein diet does not modify crypt morphology in the ob/ob mouse jejunum: a comparison of cryostat and clearing techniques.

INTRODUCTION: Diabetes is commonly associated with gastrointestinal dysfunction. We have previously shown that transepithelial short circuit current, Isc (chloride secretion), is significantly reduced in the jejunum from ob/ob mice vs lean controls, and consumption of 600 mg genistein/kg of diet (600 G) for 4 weeks significantly rescues Isc. We aimed to evaluate whether morphological changes in the jejunal crypts contribute to the rescue of Isc. METHODS: Male mice (ob/ob and lean controls) were fed either a genistein-free diet or genistein-containing diet (600 G). Comparisons of crypt morphology were made for crypt depth, length, and numbers of proliferative cells. Assessments of crypt measures using DAPI and 5-ethynyl-2'-deoxyuridine (EdU) were performed using traditional cryostat sectioning and an innovative 3D optical clearing method. RESULTS: We found that crypt length in the ob/ob genistein-fed group was significantly greater when measured with cleared tissue (85.19±4.73 µm, P<0.05, n=8) compared to lengths measured with cryostat (65.42±3.48 µm, n=8). In addition, proliferative EdU+ counts were approximately fivefold greater with clearing, compared to counts obtained via single plane images from cryostat sections for all groups measured. The average length to EdU+ ratio was unchanged between groups. CONCLUSION: Thus, we conclude that genistein diet does not affect overall cellular proliferation or crypt morphology, other than for the modest increased crypt length measured via clearing in the ob/ob genistein group. The increase in crypt length is likely indicative of the greater accuracy of the 3D measures compared to single plane. Genistein diet-induced increases in the intestinal Isc are therefore likely not attributed to changes in intestinal crypt morphology.

Nature, Mind, and Medicine: A Model for Mind-Body Healing.

OBJECTIVE: The human mind-body possesses a remarkable innate ability to heal. Grounded in the evolutionarily conserved systems of the brain and body, nature appears to function as the fundamental source of wellness along the two vectors of attention and relaxation. Yet, our species is moving away from nature at a time when humanity is just beginning to rediscover its benefits. CONCLUSIONS: Exposure to natural environments may provide a "window" of healing that can be extended through a continuum of intervention through the use of guided meditation and ultimately hypnotic suggestion. The result may be an improved ability to promote greater executive functioning and more robust immune regulation. The time has come for a more holistic medicine guided by the hand of nature.

Optical clearing of small intestine for three-dimensional visualization of cellular proliferation within crypts.

New methods on optical clearing provide a valuable alternative to traditional physical section histology. Optical clearing allows investigation of relatively large tissue samples at histological resolution while maintaining the three-dimensional architecture of the intact system. There is significant potential for applying optical clearing to gastrointestinal tissues. In particular, intestinal crypts contain high concentrations of stem cells, making these structures especially important for research on cellular proliferation in the intestinal epithelium. The objective of our study is to demonstrate an optical clearing method that is easy to implement and is compatible with mitotic fluorescent labeling. The optical clearing method we present utilizes a Triton/DMSO delipidization step followed by refractive index matching, rendering the tissue nearly transparent. We use EdU click chemistry to fluorescently label mitotic cell nuclei. Our results demonstrate successful clearing of jejunal samples with readily visible EdU staining by means of confocal microscopy. Three-dimensional reconstruction of labeled samples reveals preservation of intestinal cytoarchitecture including muscular, submucosal, and mucosal layers. Additionally, the morphology of intestinal crypts and individual EdU-positive mitotic nuclei are visible in sharp detail within their intact three-dimensional organization. In summary, we present an optical clearing method that is easy to implement and has the potential to provide more accurate assessment of cellular proliferation within the gastrointestinal tract in both healthy and disease states.

Brain volume of the newly-discovered species Rhynchocyon udzungwensis (Mammalia: Afrotheria: Macroscelidea): implications for encephalization in sengis.

The Gray-faced Sengi (Rhynchocyon udzungwensis) is a newly-discovered species of sengi (elephant-shrew) and is the largest known extant representative of the order Macroscelidea. The discovery of R. udzungwensis provides an opportunity to investigate the scaling relationship between brain size and body size within Macroscelidea, and to compare this allometry among insectivorous species of Afrotheria and other eutherian insectivores. We performed a spin-echo magnetic resonance imaging (MRI) scan on a preserved adult specimen of R. udzungwensis using a 7-Tesla high-field MR imaging system. The brain was manually segmented and its volume was compiled into a dataset containing previously-published allometric data on 56 other species of insectivore-grade mammals including representatives of Afrotheria, Soricomorpha and Erinaceomorpha. Results of log-linear regression indicate that R. udzungwensis exhibits a brain size that is consistent with the allometric trend described by other members of its order. Inter-specific comparisons indicate that macroscelideans as a group have relatively large brains when compared with similarly-sized terrestrial mammals that also share a similar diet. This high degree of encephalization within sengis remains robust whether sengis are compared with closely-related insectivorous afrotheres, or with more-distantly-related insectivorous laurasiatheres.

Selective reduction of Von Economo neuron number in agenesis of the corpus callosum.

Von Economo neurons (VENs) are large spindle-shaped neurons localized to anterior cingulate cortex (ACC) and fronto-insular cortex (FI). VENs appear late in development in humans, are a recent phylogenetic specialization, and are selectively destroyed in frontotemporal dementia, a disease which profoundly disrupts social functioning and self-awareness. Agenesis of the corpus callosum (AgCC) is a congenital disorder that can have significant effects on social and emotional behaviors, including alexithymia, difficulty intuiting the emotional states of others, and deficits in self- and social-awareness that can impair humor, comprehension of non-literal or affective language, and social judgment. To test the hypothesis that VEN number is selectively reduced in AgCC, we used stereology to obtain unbiased estimates of total neuron number and VEN number in postmortem brain specimens of four normal adult controls, two adults with isolated callosal dysgenesis, and one adult whose corpus callosum and ACC were severely atrophied due to a non-fatal cerebral arterial infarction. The partial agenesis case had approximately half as many VENs as did the four normal controls, both in ACC and FI. In the complete agenesis case the VENs were almost entirely absent. The percentage of neurons in FI that are VENs was reduced in callosal agenesis, but was actually slightly above normal in the stroke patient. These results indicate that the VEN population is selectively reduced in AgCC, but that the VENs do not depend on having an intact corpus callosum. We conclude that in agenesis of the corpus callosum the reduction in the number of VENs is not the direct result of the failure of this structure to develop, but may instead be another consequence of the genetic disruption that caused the agenesis. The reduction of the VEN population could help to explain some of the social and emotional deficits that are seen in this disorder.

Anatomical analysis of an aye-aye brain (Daubentonia madagascariensis, primates: Prosimii) combining histology, structural magnetic resonance imaging, and diffusion-tensor imaging.

This report presents initial results of a multimodal analysis of tissue volume and microstructure in the brain of an aye-aye (Daubentonia madagascariensis). The left hemisphere of an aye-aye brain was scanned using T2-weighted structural magnetic resonance imaging (MRI) and diffusion-tensor imaging (DTI) prior to histological processing and staining for Nissl substance and myelinated fibers. The objectives of the experiment were to estimate the volume of gross brain regions for comparison with published data on other prosimians and to validate DTI data on fiber anisotropy with histological measurements of fiber spread. Measurements of brain structure volumes in the specimen are consistent with those reported in the literature: the aye-aye has a very large brain for its body size, a reduced volume of visual structures (V1 and LGN), and an increased volume of the olfactory lobe. This trade-off between visual and olfactory reliance is likely a reflection of the nocturnal extractive foraging behavior practiced by Daubentonia. Additionally, frontal cortex volume is large in the aye-aye, a feature that may also be related to its complex foraging behavior and sensorimotor demands. Analysis of DTI data in the anterior cingulum bundle demonstrates a strong correlation between fiber spread as measured from histological sections and fiber spread as measured from DTI. These results represent the first quantitative comparison of DTI data and fiber-stained histology in the brain.

Asymmetric regional cerebral blood flow in sedated baboons measured by positron emission tomography (PET).

The analysis of structural brain asymmetry has been a focal point in anthropological theories of human brain evolution and the development of lateralized behaviors. While physiological brain asymmetries have been documented for humans and animals presenting with pathological conditions or under certain activation tasks, published studies on baseline asymmetries in healthy individuals have produced conflicting results. We tested for the presence of cerebral blood flow asymmetries in 7 healthy, sedated baboons using positron emission tomography, a method of in vivo autoradiography. Five of the 7 baboons exhibited hemispheric asymmetries in which left-sided flow was significantly greater than right-sided flow. Furthermore, the degree of asymmetry in 8 of 24 brain regions was found to be significantly correlated with age; older individuals exhibited a higher degree of asymmetry than younger individuals. Cerebral blood flow itself was uncorrelated with age, and differences between males and females were not significant.