Tissue S100/calgranulin expression and blood neutrophil-to-lymphocyte ratio (NLR) in prostatic disorders in dogs.

BACKGROUND: Prostatic carcinoma (PCA) is a rare but severe condition in dogs that is similar to the androgen-independent form of PCA in men. In contrast to humans, PCA is difficult to diagnose in dogs as reliable biomarkers, available for PCA screening in human medicine, are currently lacking in small animal oncology. Calprotectin (S100A8/A9) and S100A12 are Ca2+-binding proteins of the innate immune system with promising potential to distinguish malignant from benign urogenital tract conditions, similar to the blood neutrophil-to-lymphocyte-ratio (NLR). However, both have not yet been extensively investigated in dogs with PCA. Thus, this study aimed to evaluate the expression of the S100/calgranulins (calprotectin, S100A12, and their ratio [Cal-ratio]) in prostatic biopsies from nine dogs with PCA and compare them to those in dogs with benign prostatic lesions (eight dogs with prostatitis and ten dogs with benign prostatic hyperplasia [BPH]) as well as five healthy controls. In addition, blood NLRs were investigated in twelve dogs with PCA and 22 dogs with benign prostatic conditions. RESULTS: Tissue S100A8/A9+ cell counts did not differ significantly between tissue from PCA and prostatitis cases (P = 0.0659) but were significantly higher in dogs with prostatitis than BPH (P = 0.0013) or controls (P = 0.0033). S100A12+ cell counts were significantly lower in PCA tissues than in prostatitis tissue (P = 0.0458) but did not differ compared to BPH tissue (P = 0.6499) or tissue from controls (P = 0.0622). Cal-ratios did not differ significantly among the groups but were highest in prostatitis tissues and significantly higher in those dogs with poor prostatitis outcomes than in patients that were still alive at the end of the study (P = 0.0455). Blood NLR strongly correlated with prostatic tissue S100A8/A9+ cell counts in dogs with PCA (ρ = 0.81, P = 0.0499) but did not differ among the disease groups of dogs. CONCLUSIONS: This study suggests that the S100/calgranulins play a role in malignant (PCA) and benign (prostatic inflammation) prostatic conditions and supports previous results in lower urinary tract conditions in dogs. These molecules might be linked to the inflammatory environment with potential effects on the inflammasome. The blood NLR does not appear to aid in distinguishing prostatic conditions in dogs. Further investigation of the S100/calgranulin pathways and their role in modulation of tumor development, progression, and metastasis in PCA is warranted.

Tissue S100/calgranulin expression and blood neutrophil-to-lymphocyte ratio (NLR) in dogs with lower urinary tract urothelial carcinoma.

BACKGROUND: Urothelial carcinoma (UC) is the most common neoplasm of the canine lower urinary tract, affecting approximately 2% of dogs. Elderly female patients of certain breeds are predisposed, and clinical signs of UC can easily be confused with urinary tract infection or urolithiasis. Diagnosis and treatment are challenging given the lack of disease-specific markers and treatments. The S100A8/A9 complex and S100A12 protein are Ca2+-binding proteins expressed by cells of the innate immune system and have shown promise as urinary screening markers for UC. The neutrophil-to-lymphocyte ratio (NLR) can also aid in distinguishing certain neoplastic from inflammatory conditions. Our study aimed to evaluate the tissue expression of S100/calgranulins and the blood NLR in dogs with UC. Urinary bladder and/or urethral tissue samples from dogs with UC (n = 10), non-neoplastic inflammatory lesions (NNUTD; n = 6), and no histologic changes (n = 11) were evaluated using immunohistochemistry. Blood NLRs were analyzed in dogs with UC (n = 22) or NNUTD (n = 26). RESULTS: Tissue S100A12-positive cell counts were significantly higher in dogs with lower urinary tract disease than healthy controls (P = 0.0267 for UC, P = 0.0049 for NNUTD), with no significant difference between UC and NNUTD patients. Tissue S100A8/A9-positivity appeared to be higher with NNUTD than UC, but this difference did not reach statistical significance. The S100A8/A9+-to-S100A12+ ratio was significantly decreased in neoplastic and inflamed lower urinary tract tissue compared to histologically normal specimens (P = 0.0062 for UC, P = 0.0030 for NNUTD). NLRs were significantly higher in dogs with UC than in dogs with NNUTD, and a cut-off NLR of ≤ 2.83 distinguished UC from NNUTD with 41% sensitivity and 100% specificity. Higher NLRs were also associated with a poor overall survival time (P = 0.0417). CONCLUSIONS: These results confirm that the S100/calgranulins play a role in the immune response to inflammatory and neoplastic lower urinary tract diseases in dogs, but the tissue expression of these proteins appears to differ from their concentrations reported in urine samples. Further investigations of the S100/calgranulin pathways in UC and their potential as diagnostic or prognostic tools and potential therapeutic targets are warranted. The NLR as a routinely available marker might be a useful surrogate to distinguish UC from inflammatory conditions.

Establishment and validation of a guinea pig model for human congenital toxoplasmosis.

BACKGROUND: Toxoplasma gondii is an obligate intracellular parasite with a worldwide distribution. Congenital infection in humans and animals may lead to severe symptoms in the offspring, especially in the brain. A suitable animal model for human congenital toxoplasmosis is currently lacking. The aim of this study is to establish and validate the guinea pig as a model for human congenital toxoplasmosis by investigating the impact of the T. gondii infection dose, the duration of infection and the gestational stage at infection on the seroconversion, survival rate of dams, fate of the offspring, T. gondii DNA loads in various offspring tissues and organs and the integrity of the offspring brain. METHODS: Pregnant guinea pigs were infected with three different doses (10, 100, 500 oocysts) of T. gondii strain ME49 at three different time points during gestation (15, 30, 48 days post-conception). Serum of dams was tested for the presence of T. gondii antibodies using immunoblotting. T. gondii DNA levels in the dam and offspring were determined by qPCR. Offspring brains were examined histologically. RESULTS: We found the survival rate of dams and fate of the offspring to be highly dependent on the T. gondii infection dose with an inoculation of 500 oocysts ending lethally for all respective offspring. Moreover, both parameters differ depending on the gestational stage at infection with infection in the first and third trimester of gestation resulting in a high offspring mortality rate. The duration of infection was found to substantially impact the seroconversion rate of dams with the probability of seroconversion exceeding 50% after day 20 post-infection. Furthermore, the infection duration of dams influenced the T. gondii DNA loads in the offspring and the integrity of offspring brain. Highest DNA levels were found in the offspring brain of dams infected for ≥ 34 days. CONCLUSION: This study contributes to establishing the guinea pig as a suitable model for human congenital toxoplasmosis and thus lays the foundation for using the guinea pig as a suitable animal model to study scientific questions of high topicality and clinical significance, which address the pathogenesis, diagnosis, therapy and prognosis of congenital toxoplasmosis.

Evaluation of Villus Synovium From Unaffected Metacarpophalangeal Joints of Adult and Juvenile Horses.

Horses are a widely accepted model for osteoarthritis (OA) research. Synovial tissue sampling is commonly used in studies to evaluate and grade the progress of OA or to assess treatment effects. Synovial explants play an important role in ex-vivo studies, increasingly replacing the use of living animals. To understand histomorphological changes in the process of joint-related diseases such as OA, detailed information about histomorphometric parameters of unaffected synovial villi is necessary. The objective of the present study was to evaluate the mean width of the intimal synovial lining and its cellularity as well as the vascularization of the subintimal layer in juvenile and adult horses not affected by a joint-related disease. One hundred synovial samples from both metacarpophalangeal joints from 25 horses (one day to 24 years old) were collected to evaluate the following parameters on digitalized hematoxylin-eosin stained samples: Width of intimal synovial lining measured by the distance from the inner joint surface to the subintimal layer; density of the cells making up the intimal synovial lining by counting cell nuclei; vascularization of the subintimal layer measured by the number and size of vessels in relation to the subintimal area. The median width of the intimal lining did not differ among juvenile (22.34 µm) and adult (23.34 µm) horses. The cellularity of the intimal lining was significantly lower in juvenile (one cell/143.8 µm2) than in adult (one cell /188.7µm2), (P < .001) horses as well as the density of blood vessels per mm2 within the subintimal layer (juveniles 1/mm2 vs. adults 0.05/mm2), (P < .001). This study provides morphometric data regarding synovial intimal width, intimal cellularity, and vascularization of equine synovial villi of unaffected horses. For future studies, age-related characteristics should be taken into consideration when synovial tissue samples are used for in-vivo and in-vitro studies.

An investigation into uterine capacity based on litter and placental characteristics in two sow lines with different prolificacy (Danish Landrace x Danish Yorkshire versus German Saddleback).

Litter size in modern so called hyperprolific pig (Sus scrofa Linnaeus) breeds such as of crossbred Danish Landrace x Danish Yorkshire (LY/YL) sows increased remarkably over recent years, however, commonly associated with reduced piglet birth weight and higher within litter birth weight variability likely due to a limited uterine capacity. Since investigation into this issue is patchy, the aim of this study was to investigate uterine capacity based on litter and placental characteristics in two sow lines with different prolificacy, that is crossbred Danish genetic (Danish Landrace x Danish Yorkshire; DG; n = 14) and purebred German Saddleback (GS) sows (n = 12). Parameters recorded were litter size, piglet birth weight and vitality, placental weight and surface area as well as placental vascularization. Litters of DG were on average larger than of GS (p < .001). Piglets of DG weighed on average less than GS (p < .001) and were less vital (p < .001-.142). Increasing litter size was associated with reduced piglet birth weight and increased within litter birth weight variability in GS, but not in DG. DG had on average a lower placental weight (p < .001) and smaller placentae (p < .001) than GS, but the placenta was on average more efficient than of GS (based on the quotient of piglet and corresponding placental weight; p < .001). Vascularization of placentae was on average not or only slightly different between breeds (p < .05 - .982). Remarkably, however, vascularization of the lateral and apical chorionic epithelium of the chorionic ridges as the immediate foetal/maternal interface was on average slightly higher in DG than GS (p < .05-.111). Results thus demonstrate that uterine capacity based on litter and placental characteristics is higher in DG than GS sows.

Under the skin: Ixodes ticks in the subcutaneous tissue of red foxes (Vulpes vulpes) from Germany.

BACKGROUND: Ixodes spp. are vectors of zoonotic pathogens. All three active life stages (larvae, nymphs, adults) need to feed on a host in order to develop. Usually ticks parasitize attached to the external surface of their hosts' skin. Interestingly, in some cases ticks can also be found in the subcutaneous tissue in a variety of hosts, such as red foxes (Vulpes vulpes), raccoon dogs (Nyctereutes procyonoides) and dogs. METHODS: The visceral side of 126 red fox-furs from Germany was examined visually searching for ticks. The localization of ticks was recorded and assigned to ten specific body parts. Morphological identification of ticks was performed according to standardized taxonomic protocols. Ticks which could not be further identified were examined genetically via conventional PCR targeting the 16S rRNA and cox1 gene. Hematoxylin and eosin (H&E) staining was used for histopathological examination. RESULTS: In 111 out of 126 (88.1%) examined coats, at least one tick was found in the subcutaneous tissue. A total of 1203 ticks were removed from the subcutaneous tissue. Well-preserved ticks could be identified based on morphological criteria, but most ticks were in a progressed state of decomposition. Here, morphological species identification was not successful. Also, PCR methods did not lead to a successful species identification. The following species and development stages were found by morphological identification: Ixodes ricinus (female, n = 289; male, n = 8; nymph, n = 1), I. hexagonus (female, n = 2), I. canisuga (female, n = 1). Male I. ricinus were found individually or copulating in pairs with females. Subcutaneous ticks were localized at three predominant affected body parts: ears, axillar and inguinal region. Histological examination of subcutaneous ticks revealed a granulomatous panniculitis. CONCLUSIONS: To the authors' knowledge, this is the first finding of highly prevalent subcutaneous ticks in red foxes from Germany. Subcutaneous location of ticks seems to be very common in red foxes and the rule rather than the exception. Deep embedment of longirostra and long feeding times of females seem to put the subcutaneous location in favor. Most foxes were infested in the inguinal area, where the skin is thin and less hairy.

Effects of Hypoxia and Acidosis on Cardiac Electrophysiology and Hemodynamics. Is NHE-Inhibition by Cariporide Still Advantageous?

Hypoxia often leads to severe cardiac malfunctions. It is assumed that intracellular calcium overload is -inter alia- responsible for left ventricular (LV) deterioration. Inhibition of the sodium-proton exchanger (NHE), which finally inhibits/slows calcium overload, may ameliorate cardiac function. Our aim was to evaluate cariporide, an inhibitor of NHE1 in a Langendorff-perfused heart model. To discriminate a potentially different impact of extracellular acidosis and hypoxia we examined 48 Chinchilla Bastard rabbits divided into 8 experimental groups: control group (pH = 7.4, O2 = 100%) without or with cariporide (1 µM), acidosis group (pH = 7.0, O2 = 100%) without or with cariporide (1 µM), hypoxia group (pH = 7.4, O2 = 40%) without or with cariporide (1 µM) and hypoxia+acidosis group (pH = 7.0, O2 = 40%) without or with cariporide (1 µM). Hearts were subjected to acidotic/hypoxic conditions for 90 min followed by 60 min of reperfusion. Hypoxia and hypoxia+acidosis led to a severe deterioration of LV function with a decrease in LV pressure by about 70% and an increase of end-diastolic pressure from 6.7 ± 0.6 to 36.8 ± 5.4 (hypoxia) or from 7.0 ± 0.2 to 18.6 ± 4.1 (hypoxia+acidosis). Moreover, maximum contraction velocity decreased from about 1,800 mmHg/s to 600 mmHg/s during hypoxia ± acidosis and maximum relaxation velocity deteriorated from -1,500 mmHg/s to about -600 mmHg/s. During reperfusion hearts subjected to hypoxia+acidosis recovered faster than hearts subjected to hypoxia alone, reaching control levels after 5 min of reperfusion. Electrophysiologic analysis revealed an 1.2 fold increase in both dispersion of activation-recovery interval and in total activation time in the hypoxia ± acidosis group. Cariporide application significantly improved LV hemodynamics and electrophysiology in the hypoxia group but not in the group subjected to hypoxia+acidosis. Immunohistologic analysis of cardiac specimen revealed a significant increase of factors involved in hypoxia/reperfusion injury like nitrotyrosine and poly-ADP-ribose as well as apoptosis-inducing factors like AIF or cleaved-caspase 3 in LV after hypoxia ± acidosis. ATP was reduced by hypoxia but not by acidosis. Again, cariporide mitigated these processes only in the hypoxia alone group, but not in the group with additional acidosis. Acidosis without hypoxia only marginally disturbed LV function and electrophysiology, and was not affected by cariporide. Thus, our study demonstrated that several detrimental effects of hypoxia were mitigated or abrogated by acidosis and that NHE-inhibition improved only hypoxia-induced cardiac dysfunction.

Anti-oxidative or anti-inflammatory additives reduce ischemia/reperfusions injury in an animal model of cardiopulmonary bypass.

Severe inborn cardiac malformations are typically corrected in cardioplegia, with a cardio-pulmonary bypass (CPB) taking over body circulation. During the operation the arrested hearts are subjected to a global ischemia/reperfusion injury. Although the applied cardioplegic solutions have a certain protective effect, application of additional substances to reduce cardiac damage are of interest.18 domestic piglets (10-15 kg) were subjected to a 90 min CPB and a 120 min reperfusion phase without or with the application of epigallocatechin-3-gallate (10 mg/kg body weight) or minocycline (4 mg/kg body weight), with both drugs given before and after CPB. 18 additional sham-operated piglets without or with epigallocatechin-3-gallate or minocycline served as controls. In total 36 piglets were analyzed (3 CPB-groups and 3 control groups without or with epigallocatechin-3-gallate or minocycline respectively; 6 piglets per group). Hemodynamic and blood parameters and ATP-measurements were assessed. Moreover, a histological evaluation of the heart muscle was performed. RESULTS: Piglets of the CPB-group needed more catecholamine support to achieve sufficient blood pressure. Ejection fraction and cardiac output were not different between the 6 groups. However, cardiac ATP-levels and blood lactate were significantly lower and creatine kinase was significantly higher in the three CPB-groups. Markers of apoptosis, hypoxia, nitrosative and oxidative stress were significantly elevated in hearts of the CPB-group. Nevertheless, addition of epigallocatechin-3-gallate or minocycline significantly reduced markers of myocardial damage. Noteworthy, EGCG was more effective in reducing markers of hypoxia, whereas minocycline more efficiently decreased inflammation. CONCLUSIONS: While epigallocatechin-3-gallate or minocycline did not improve cardiac hemodynamics, markers of myocardial damage were significantly lower in the CPB-groups with epigallocatechin-3-gallate or minocycline supplementation.

Developmental horizons in the pre-natal development of the Greater cane rat (Thryonomys swinderianus).

The Greater cane rat (GCR, Thyronomys swinderianus) is a precocial rodent predominantly found within Africa. Economic and scientific interests have led to several research efforts towards the domestication and better understanding of the biology and development of this rodent. Despite these efforts, information on the pre-natal development of this rodent is currently lacking. This study characterises distinct developmental milestones including skin pigmentation, emergence and distributions of hairs, calvarium consistency, teeth eruption, development of appendages, sensory organs and external genitalia in the pre-natal GCR and assesses quantitative body parameters, that is body weight, body and crown-rump lengths across its entire gestation length (gestation days [GDs] 10-140). Using these external features, we provide baseline reference ontogenetic scales for GCR embryos and fetuses, employable for stage, age and sex estimation of the pre-natal GCR in future studies. We observed that the first evidence of an embryo was not seen before the end of the first trimester (GD50) and that the late second trimester (GD80-GD100) marks the transition from embryogenesis to fetogenesis in the GCR. As both events occur at a much later developmental time point when compared to precocial non-rodents including human, sheep and pig and slightly later when compared to other precocial rodents such as guinea pig, our data provide first indication that the pre-natal GCR development might be associated with a reproductive delay. Together, this study expands our knowledge of the development and biology of the GCR, which will improve reproductive and breeding management, and native species conservation of this hystricomorph mammal.

Mucosal expression of S100A12 (calgranulin C) and S100A8/A9 (calprotectin) and correlation with serum and fecal concentrations in dogs with chronic inflammatory enteropathy.

S100A12 and S100A8/A9 (calprotectin) are released from activated mononuclear cells and belong to the group of damage associated molecular patterns. Fecal S100A12 and S100A8/A9 concentrations have been suggested as biomarkers of intestinal inflammation in dogs with chronic inflammatory enteropathies (CIE). However, the mucosal cellular infiltrate in dogs with CIE is primarily lymphocytic-plasmacytic. Whether fecal S100A12 and S100A8/A9 levels reflect the number and/or activity of intestinal mucosal mononuclear cells, or whether these proteins are also produced by other cells has not been investigated. Thus, the aim of this study was to evaluate intestinal mucosal S100A12 and S100A8/A9 positivity and a potential relationship with the respective protein concentrations in serum and fecal samples in dogs with CIE. Serum (single sample), fecal samples (from 3 consecutive days), and gastrointestinal tissue biopsies (i.e., stomach, duodenum, ileum, and colon) were evaluated from 21 dogs with CIE. Serum and fecal S100A12 and S100A8/A9 concentrations were measured by analytically validated in-house ELISAs. Tissue biopsies underwent routine histopathology and immunohistochemical evaluation for S100A12 and S100A8/A9 positivity (S100A12+ and S100A8/A9+, each recorded as positive cells/mm2). S100A12+ and S100A8/A9+ cells were identified in all segments of the gastrointestinal tract, but were predominantly localized in the lamina propria (LP). Duodenal LP S100A12 positivity correlated statistically significantly with that in the stomach and ileum (ρ = 0.66 and 0.69, both p < 0.01), but was inversely correlated with the severity of macrophage infiltration in the duodenum (ρ=-0.47, p = 0.042). Ileal LP S100A8/A9 positivity correlated positively with the extent of ileal neutrophil and macrophage infiltration (ρ=0.61, p = 0.047). Fecal S100A12 concentrations strongly correlated with the number of S100A12+ cells along the entire gastrointestinal tract (ρ = 0.76, p = 0.028), whereas serum S100A12 concentrations were inversely correlated to colonic S100A12+ cell counts (ρ=-0.50, p = 0.043). Mucosal S100A8/A9+ cell counts were not associated with the corresponding fecal or serum S100A8/A9 concentrations. These results suggest that the intestinal mucosa in dogs with CIE contains an increased number of activated (pro-inflammatory) phagocytes expressing and secreting the S100A12 protein, but the macrophage population seen on routine histopathology is predominantly mature (anti-inflammatory) with a reduced or absent expression of S100A12 and a normal or increased expression of S100A8/A9. However, the distribution of intestinal S100A8/A9 expression requires further study.

Iron concentrations in neurons and glial cells with estimates on ferritin concentrations.

BACKGROUND: Brain iron is an essential as well as a toxic redox active element. Physiological levels are not uniform among the different cell types. Besides the availability of quantitative methods, the knowledge about the brain iron lags behind. Thereby, disclosing the mechanisms of brain iron homeostasis helps to understand pathological iron-accumulations in diseased and aged brains. With our study we want to contribute closing the gap by providing quantitative data on the concentration and distribution of iron in neurons and glial cells in situ. Using a nuclear microprobe and scanning proton induced X-ray emission spectrometry we performed quantitative elemental imaging on rat brain sections to analyze the iron concentrations of neurons and glial cells. RESULTS: Neurons were analyzed in the neocortex, subiculum, substantia nigra and deep cerebellar nuclei revealing an iron level between [Formula: see text] and [Formula: see text]. The iron concentration of neocortical oligodendrocytes is fivefold higher, of microglia threefold higher and of astrocytes twofold higher compared to neurons. We also analyzed the distribution of subcellular iron concentrations in the cytoplasm, nucleus and nucleolus of neurons. The cytoplasm contains on average 73% of the total iron, the nucleolus-although a hot spot for iron-due to its small volume only 6% of total iron. Additionally, the iron level in subcellular fractions were measured revealing that the microsome fraction, which usually contains holo-ferritin, has the highest iron content. We also present an estimate of the cellular ferritin concentration calculating [Formula: see text] ferritin molecules per [Formula: see text] in rat neurons. CONCLUSION: Glial cells are the most iron-rich cells in the brain. Imbalances in iron homeostasis that lead to neurodegeneration may not only be originate from neurons but also from glial cells. It is feasible to estimate the ferritin concentration based on measured iron concentrations and a reasonable assumptions on iron load in the brain.

A New Genotype of Feline Morbillivirus Infects Primary Cells of the Lung, Kidney, Brain and Peripheral Blood.

Paramyxoviruses comprise a large number of diverse viruses which in part give rise to severe diseases in affected hosts. A new genotype of feline morbillivirus, tentatively named feline morbillivirus genotype 2 (FeMV-GT2), was isolated from urine of cats with urinary tract diseases. Whole genome sequencing showed about 78% nucleotide homology to known feline morbilliviruses. The virus was isolated in permanent cell lines of feline and simian origin. To investigate the cell tropism of FeMV-GT2 feline primary epithelial cells from the kidney, the urinary bladder and the lung, peripheral blood mononuclear cells (PBMC), as well as organotypic brain slice cultures were used for infection experiments. We demonstrate that FeMV-GT2 is able to infect renal and pulmonary epithelial cells, primary cells from the cerebrum and cerebellum, as well as immune cells in the blood, especially CD4⁺ T cells, CD20⁺ B cells and monocytes. The cats used for virus isolation shed FeMV-GT2 continuously for several months despite the presence of neutralizing antibodies in the blood. Our results point towards the necessity of increased awareness for this virus when clinical signs of the aforementioned organs are encountered in cats which cannot be explained by other etiologies.

A stereotaxic breed-averaged, symmetric T2w canine brain atlas including detailed morphological and volumetrical data sets.

Stereotaxic systems and automatic tissue segmentation routines enable neuronavigation as well as reproducible processing of neuroimage datasets. Such systems have been developed for humans, non-human-primates, sheep, and rodents, but not for dogs. Although dogs share important neurofunctional and -anatomical features with humans, and in spite of their importance in translational neuroscience, little is known about the variability of the canine brain morphology and, possibly related, function. Moreover, we lack templates, tissue probability maps (TPM), and stereotaxic brain labels for implementation in standard software utilities such as Statistical Parametric Mapping (SPM). Hence, objective and reproducible, image-based investigations are currently impeded in dogs. We have created a detailed stereotaxic reference frame for dogs including TPM and tissue labels, enabling inter-individual and cross-study neuroimage analysis. T2w datasets were acquired from 16 neurologically inconspicuous dogs of different breeds by 3T MRI. The datasets were averaged after initial preprocessing using linear and nonlinear registration algorithms as implemented in SPM8. TPM for gray (GM) and white matter (WM) as well as cerebrospinal fluid (CSF) were created. Different cortical, subcortical, medullary, and CSF regions were manually labeled to create a spatial binary atlas being aligned with the template. A proof-of-concept for automatic determination of morphological and volumetrical characteristics was performed using additional canine datasets (n = 64) including a subgroup of laboratory beagles (n = 24). Overall, 21 brain regions were labeled using the segmented tissue classes of the brain template. The proof-of-concept trial revealed excellent suitability of the created tools for image processing and subsequent analysis. There was high intra-breed variability in frontal lobe and hippocampus volumes, and noticeable inter-breed corpus callosum volume variation. The T2w brain template provides important, breed-averaged canine brain anatomy features in a spatial standard coordinate system. TPM allows automatic tissue segmentation using SPM and enables unbiased automatic image processing or morphological characterization in different canine breeds. The reported volumetric and morphometric results may serve as a starting point for further research aimed at in vivo analysis of canine brain anatomy and function.

In vitro drusen model - three-dimensional spheroid culture of retinal pigment epithelial cells.

Age-related macular degeneration (AMD) is a leading cause of blindness in people over 50 years of age in many developed countries. Drusen are yellowish extracellular deposits beneath retinal pigment epithelium (RPE) found in aging eyes and considered as a biomarker of AMD. However, the biogenesis of drusen has not been elucidated. We reported previously that multicellular spheroids of human RPE cells constructed a well-differentiated monolayer of RPE with a Bruch's membrane. We determined that RPE spheroids exhibited drusen formation between the RPE and Bruch's membrane with expression of many drusen-associated proteins, such as amyloid ß and complement components, the expression of which was altered by a challenge with oxidative stress. Artificial lipofuscin-loaded RPE spheroids yielded drusen more frequently. In the current study, we showed that drusen originates from the RPE. This culture system is an attractive tool for use as an in vitro drusen model, which might help elucidate the biogenesis of drusen and the pathogenesis of related diseases, such as AMD.

Neural Progenitors in the Developing Neocortex of the Northern Tree Shrew (Tupaia belangeri) Show a Closer Relationship to Gyrencephalic Primates Than to Lissencephalic Rodents.

The neocortex is the most complex part of the mammalian brain and as such it has undergone tremendous expansion during evolution, especially in primates. The majority of neocortical neurons originate from distinct neural stem and progenitor cells (NPCs) located in the ventricular and subventricular zone (SVZ). Previous studies revealed that the SVZ thickness as well as the abundance and distribution of NPCs, especially that of basal radial glia (bRG), differ markedly between the lissencephalic rodent and gyrencephalic primate neocortex. The northern tree shrew (Tupaia belangeri) is a rat-sized mammal with a high brain to body mass ratio, which stands phylogenetically mid-way between rodents and primates. Our study provides - for the first time - detailed data on the presence, abundance and distribution of bRG and other distinct NPCs in the developing neocortex of the northern tree shrew (Tupaia belangeri). We show that the developing tree shrew neocortex is characterized by an expanded SVZ, a high abundance of Pax6+ NPCs in the SVZ, and a relatively high percentage of bRG at peak of upper-layer neurogenesis. We further demonstrate that key features of tree shrew neocortex development, e.g., the presence, abundance and distribution of distinct NPCs, are closer related to those of gyrencephalic primates than to those of ferret and lissencephalic rodents. Together, our study provides novel insight into the evolution of bRG and other distinct NPCs in the neocortex development of Euarchontoglires and introduces the tree shrew as a potential novel model organism in the area of human brain development and developmental disorders.

Epigallocatechin Gallate Reduces Ischemia/Reperfusion Injury in Isolated Perfused Rabbit Hearts.

Cardioplegic arrest during heart operations is often used in cardiac surgery. During cardioplegia, the heart is subjected to a global ischemia/reperfusion-injury. (-)-epigallocatechin gallate (EGCG), one of the main ingredients of green tea, seems to be beneficial in various cardiac diseases. Therefore, the aim of our study was to evaluate EGCG in a rabbit model of cardioplegic arrest. Twenty four mature Chinchilla rabbits were examined. Rabbit hearts were isolated and perfused according to Langendorff. After induction of cardioplegia (without and with 20 µmol/L EGCG, n = 6 each) the hearts maintained arrested for 90-min. Thereafter, the hearts were re-perfused for 60 min. During the entire experiment hemodynamic and functional data were assessed. At the end of each experiment, left ventricular samples were processed for ATP measurements and for histological analysis. Directly after cessation of cardioplegia, all hearts showed the same decline in systolic and diastolic function. However, hearts of the EGCG-group showed a significantly faster and better hemodynamic recovery during reperfusion. In addition, tissue ATP-levels were significantly higher in the EGCG-treated hearts. Histological analysis revealed that markers of nitrosative and oxidative stress were significantly lower in the EGCG group. Thus, addition of EGCG significantly protected the cardiac muscle from ischemia/reperfusion injury.

The Basal Radial Glia Occurs in Marsupials and Underlies the Evolution of an Expanded Neocortex in Therian Mammals.

A hallmark of mammalian brain evolution is the emergence of the neocortex, which has expanded in all mammalian infraclasses (Eutheria, Marsupialia, Monotremata). In eutherians, neocortical neurons derive from distinct neural stem and progenitor cells (NPCs). However, precise data on the presence and abundance of the NPCs, especially of basal radial glia (bRG), in the neocortex of marsupials are lacking. This study characterized and quantified the NPCs in the developing neocortex of a marsupial, the tammar wallaby (Macropus eugenii). Our data demonstrate that its neocortex is characterized by high NPC diversity. Importantly, we show that bRG exist at high relative abundance in the tammar indicating that this cell type is not specific to the eutherian neocortex and that similar mechanisms may underlie the formation of an expanded neocortex in eutherian and marsupial mammals. We also show that bRG are likely to have been present in the therian ancestor, so did not emerge independently in the eutherian and marsupial lineages. Moreover, our data support the concept that changes in multiple parameters contribute to neocortex expansion and demonstrate the importance of bRG and other NPCs for the development and expansion of the mammalian neocortex.

Ischemic regulation of brain-derived neurotrophic factor-mediated cell volume and TrkB expression in glial (Müller) and bipolar cells of the rat retina.

BACKGROUND: Osmotic swelling of neurons and glial cells contributes to retinal edema and neurodegeneration. BDNF, a major neuroprotectant in the retina, was shown to inhibit osmotic swelling of glial (Müller) and bipolar cells in the rat retina; the effect of BDNF on the bipolar cell swelling is mediated by inducing a release of neuroprotective cytokines from Müller cells (Berk et al., Neuroscience 295:175-186, 2015). We determined whether BDNF-mediated cell volume regulation was altered after transient retinal ischemia. METHODS: Retinal slices from the eyes of rats that underwent a 1-h pressure-induced retinal ischemia and from control eyes were superfused with a hypoosmotic solution. RESULTS: Exogenous BDNF prevented osmotic swelling of Müller cells in both control and post-ischemic retinal slices. BDNF also prevented osmotic swelling of bipolar cells in the control retina, but not in the ischemic retina. On the other hand, exogenous bFGF prevented the swelling of both Müller and bipolar cells in the ischemic retina. Freshly isolated Müller cells of control retinas displayed immunoreactivity of truncated but not full-length TrkB. In contrast, Müller cells of post-ischemic retinas displayed immunoreactivity of both TrkB isoforms. Bipolar cells isolated from control and post-ischemic retinas were immunolabeled for both TrkB isoforms. CONCLUSIONS: The data may suggest that the ischemic abrogation of the BDNF effect in bipolar cells is related to altered BDNF receptor expression in Müller cells. Glial upregulation of full-length TrkB may support the survival of Müller cells in the ischemic retina, but may impair the BDNF-induced release of neuroprotective cytokines such as bFGF from Müller cells.

Detection and Quantification of ß-Amyloid, Pyroglutamyl Aß, and Tau in Aged Canines.

Canine cognitive dysfunction syndrome is an age-associated disorder that resembles many aspects of human Alzheimer disease. The characterization of canine cognitive dysfunction syndrome has been restricted to selected laboratory dogs and mongrels, thereby limiting our knowledge of potential breed-related and age-related differences. We examined the brains of 24 dogs from various breeds. The frontal cortex, hippocampus, and entorhinal cortex were investigated. Deposits of ß-amyloid (Aß) and tau were analyzed phenotypically and quantified stereologically. In all dogs aged 10 years or older, plaques containing pyroglutamyl Aß and Aß8-17 were detected. Within the ventral hippocampus, significantly more pyroglutamyl Aß plaques were deposited in small and medium dogs than in large dogs. Hyperphosphorylated tau with formation of neurofibrillary tangles was observed in 3 animals aged 13 to 15 years. This study provides the first investigation of pyroglutamyl Aß in comparison with total Aß (as shown by Aß8-17 immunoreactivity) in dogs of different breeds, sizes, and ages. Our results indicate that canine cognitive dysfunction syndrome is relatively common among aged canines, thereby emphasizing the relevance of such populations to translational Alzheimer disease research.

The extracellular matrix molecule brevican is an integral component of the machinery mediating fast synaptic transmission at the calyx of Held.

KEY POINTS: The proteoglycan brevican is a major component of the extracellular matrix of perineuronal nets and is highly enriched in the perisynaptic space suggesting a role for synaptic transmission. We have introduced the calyx of Held in the auditory brainstem as a model system to study the impact of brevican on dynamics and reliability of synaptic transmission. In vivo extracellular single-unit recordings at the calyx of Held in brevican-deficient mice yielded a significant increase in the action potential (AP) transmission delay and a prolongation of pre- and postsynaptic APs. The changes in dynamics of signal transmission were accompanied by the reduction of presynaptic vGlut1 and ultrastructural changes in the perisynaptic space. These data show that brevican is an important mediator of fast synaptic transmission at the calyx of Held. ABSTRACT: The extracellular matrix is an integral part of the neural tissue. Its most conspicuous manifestation in the brain are the perineuronal nets (PNs) which surround somata and proximal dendrites of distinct neuron types. The chondroitin sulfate proteoglycan brevican is a major component of PNs. In contrast to other PN-comprising proteoglycans (e.g. aggrecan and neurocan), brevican is mainly expressed in the perisynaptic space closely associated with both the pre- and postsynaptic membrane. This specific localization prompted the hypothesis that brevican might play a role in synaptic transmission. In the present study we specifically investigated the role of brevican in synaptic transmission at a central synapse, the calyx of Held in the medial nucleus of the trapezoid body, by the use of in vivo electrophysiology, immunohistochemistry, biochemistry and electron microscopy. In vivo extracellular single-unit recordings were acquired in brevican-deficient mice and the dynamics and reliability of synaptic transmission were compared to wild-type littermates. In knockout mice, the speed of pre-to-postsynaptic action potential (AP) transmission was reduced and the duration of the respective pre- and postsynaptic APs increased. The reliability of signal transmission, however, was not affected by the lack of brevican. The changes in dynamics of signal transmission were accompanied by the reduction of (i) presynaptic vGlut1 and (ii) the size of subsynaptic cavities. The present results suggest an essential role of brevican for the functionality of high-speed synaptic transmission at the calyx of Held.