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Abnormal tau inclusions are hallmarks of Alzheimer's disease and predictors of clinical decline. Several tau PET tracers are available for neurodegenerative disease research, opening avenues for molecular diagnosis in vivo. However, few have been approved for clinical use. Understanding the neurobiological basis of PET signal validation remains problematic because it requires a large-scale, voxel-to-voxel correlation between PET and (immuno) histological signals. Large dimensionality of whole human brains, tissue deformation impacting co-registration, and computing requirements to process terabytes of information preclude proper validation. We developed a computational pipeline to identify and segment particles of interest in billion-pixel digital pathology images to generate quantitative, 3D density maps. The proposed convolutional neural network for immunohistochemistry samples, IHCNet, is at the pipeline's core. We have successfully processed and immunostained over 500 slides from two whole human brains with three phospho-tau antibodies (AT100, AT8, and MC1), spanning several terabytes of images. Our artificial neural network estimated tau inclusion from brain images, which performs with ROC AUC of 0.87, 0.85, and 0.91 for AT100, AT8, and MC1, respectively. Introspection studies further assessed the ability of our trained model to learn tau-related features. We present an end-to-end pipeline to create terabytes-large 3D tau inclusion density maps co-registered to MRI as a means to facilitate validation of PET tracers.

Elodea canadensis Michx. (common waterweed) and Elodea nuttallii (Planch.) H. St. John (Nuttall’s waterweed), two invasive aquatic plants from North America, have coexisted in European water bodies since the early 20th century. New localities for both species in Croatia continued to be discovered during a study that ran from 2016 to 2023 as a part of the annual implementation of Water Framework Directive monitoring that covered the entire territory of Croatia (786 sampling points in total). Based on these data, the distribution and ecology of both species were analysed. Elodea canadensis was found at 30 sampling points, mostly in rivers, and E. nuttallii at 15 sampling points, mostly in artificial canals. Nearly three-quarters (72.5%) of all elodea sampling points were in the Pannonian Ecoregion. Elodea canadensis was discovered for the first time in the Continental—Dinaric and Mediterranean—Dinaric Subecoregions. To study the ecology of the species, for each sampling point, vegetation relevés were performed and monthly measurements of physico-chemical parameters were collected. The most common accompanying species for both elodeas are presented, and the difference in species assemblages between the sites with E. canadensis and E. nuttallii was confirmed with the ANOSIM test. Furthermore, Indicator Species Analysis revealed eight species characteristic of E. canadensis sites and eleven species characteristic of E. nuttallii sites. Fitting multivariate models (CCA and NPMR) to species abundance revealed the ecological reaction of E. canadensis and E. nuttallii to environmental descriptors. The most strongly contributing environmental descriptors that influence the distribution of both Elodea species are biochemical oxygen demand, electrical conductivity and total phosphorus. In Croatia, the replacement of E. canadensis with E. nuttallii was observed in several water bodies with high nutrient loads.

An extensive macrophyte field survey of running and standing waters was conducted from 2016 to 2021 at 786 sampling sites across Croatia as a part of the implementation of the Water Framework Directive. This survey is the first to present a comprehensive floristic catalogue of the freshwater bryoflora, along with an analysis of the distribution and diversity patterns on a national level. In all, 83 bryophyte species (68 mosses and 15 liverworts) were recorded in the 228 sites, with average species richness of 4.17 species per site. The most frequent species were Fontinalis antipyretica, Rhynchostegium riparioides, Leptodictyum riparium and Cratoneuron filicinum. The majority of the species encountered were rarely found, with over 70% of species recorded on less than 10 sampling sites and the majority of the species not being truly aquatic, rather being classified as facultative aquatics. The Dinaric Ecoregion, characterised by clean, cold, fast-flowing karstic rivers, especially in the Continental Subecoregion, supported higher freshwater bryophyte diversity than the lowland Pannonian Ecoregion, with mostly slow, eutrophic lowland watercourses with unstable sandy and gravelly alluvial sediments. Chorological comparison of Croatian eco- and subecoregions revealed the expected dominance of circumpolar and European elements, i.e. temperate chorotypes, as well as some biogeographical differences. The most frequent life forms were aquatic trailings and turfs. Amongst the recorded species, perennials and colonists were the most represented life strategies. The analysis of both the life-form and life-strategy spectra showed some differences amongst the Croatian regions, supporting the fact that the Dinaric Ecoregion provides more truly aquatic habitats and microhabitats suitable for the freshwater bryophytes, while in the Pannonian Ecoregion freshwater bryophytes dominantly inhabit the periodically submerged riparian zones, for example shaded lowland forest streams and rivulets or gently sloping margins of rivers and lakes.

Abstract Background The bryophytes are a plant group that is smaller than and not as well known as the vascular plants. They are less used and are almost completely neglected in ethnobotanical studies. Traditional nativity scenes depicting the birth of Christ are commonly decorated with both vascular plants and bryophytes. The aim of this study was to document the diversity of decorative bryophytes sold during the Advent season at farmers’ markets in Croatia (Southeastern Europe, Balkan Peninsula). Methods Twenty-eight farmers’ markets in the two largest Croatian cities (Zagreb in the continental part and Split in the Mediterranean part) were studied in the search for local vendors selling bryophytes during the pre-Christmas season. The bryophytes collected were identified and analysed with respect to families, growth type, life forms and threat status. Results Among 275 collected specimens, 43 moss and four liverwort species were identified. The mean number of species per vendor was 3.5. The most frequent species were Hypnum cupressiforme, Homalothecium sericeum and Ctenidium molluscum. Mats, wefts and tufts were the most common life-forms, while pleurocarpous prevailed over acrocarpous mosses, as they are usually pinnately branched and form large carpets, suitable for decorations. The overall selection of bryophytes and the decorations made of them were more diverse and abundant in inland Croatia, where 49 vendors at 15 farmers’ markets sold goods containing 43 species. In Mediterranean Croatia, at six farmers’ markets only 29 vendors sold goods, which contained 18 species. A considerable number of species that are less attractive to harvesters were collected non-intentionally, entangled in carpets of other, more frequent species. Among them, Rhodobryum ontariense and Loeskeobryum brevirostre are rare and insufficiently recorded in Croatia so far. Conclusions The present study provided a first perspective on the use of bryophytes in traditional nativity scenes in Croatia and Southeastern Europe, contributing to scarce ethnobotanical documentation of the decorative use of bryophytes in Christmas festivities in Europe and globally.

It has been widely documented that the complex structure of forest ecosystems supports considerable bryophyte species and functional diversity. In this study, we assessed the diversity, distribution and ecological and phytogeographical features of bryophytes across a gradient of temperate forest types on Mt. Papuk. This is the largest and highest mountain in the lowland, Pannonian part of Croatia, with high geological diversity and various temperate forests covering 95% of the mountain. According to the predominant tree species (oak vs. beech), geological bedrock (calcareous vs. siliceous) and soil reaction (alkaline vs. acidic), 21 study plots were classified into four distinct forest types. In all, 184 bryophyte species (35 liverworts and 149 mosses) were recorded. Although the forest types investigated did not differ significantly with respect to species richness, each was characterized by a considerable number of diagnostic bryophyte species. According to our results, one of the main ecological factors determining the variability of the forest bryophyte composition was geological bedrock and the associated soil reaction. Basiphilous forests developed on carbonate bedrock harbored more thermophilous and nitrophilous bryophytes and were characterized by southern-temperate and Mediterranean–Atlantic biogeographic elements. In contrast, acidophilous forests growing on silicate bedrock were characterized by wide-boreal and boreo-arctic–montane elements, i.e., bryophytes indicating cooler habitats and nitrogen-deficient soils. Based on the results, we hypothesized that the main latitudinal biogeographic distinction between southern and northern biogeographic elements is driven more by geological substrate than by the main tree species in forest communities. The present study confirmed previous findings that bryophytes are good and specific habitat indicators and show associations with different forest types, which can help to understand the complexity, ecological microconditions and biogeographic characteristics of forest communities.

Stereotaxy is based on the precise image-guided spatial localization of targets within the human brain. Even with the recent advances in MRI technology, histological examination renders different (and complementary) information of the nervous tissue. Although several maps have been selected as a basis for correlating imaging results with the anatomical locations of sub-cortical structures, technical limitations interfere in a point-to-point correlation between imaging and anatomy due to the lack of precise correction for post-mortem tissue deformations caused by tissue fixation and processing. We present an alternative method to parcellate human brain cytoarchitectural regions, minimizing deformations caused by post-mortem and tissue-processing artifacts and enhancing segmentation by means of modified high thickness histological techniques and registration with MRI of the same specimen and into MNI space (ICBM152). A three-dimensional (3D) histological atlas of the human thalamus, basal ganglia, and basal forebrain cholinergic system is displayed. Structure's segmentations were performed in high-resolution dark-field and light-field microscopy. Bidimensional non-linear registration of the histological slices was followed by 3D registration with in situ MRI of the same subject. Manual and automated registration procedures were adopted and compared. To evaluate the quality of the registration procedures, Dice similarity coefficient and normalized weighted spectral distance were calculated and the results indicate good overlap between registered volumes and a small shape difference between them in both manual and automated registration methods. High thickness high-resolution histological slices in combination with registration to in situ MRI of the same subject provide an effective alternative method to study nuclear boundaries in the human brain, enhancing segmentation and demanding less resources and time for tissue processing than traditional methods.

The pedunculopontine nucleus (PPN) has been proposed as target for deep brain stimulation (DBS) in patients with postural instability and gait disorders due to its involvement in muscle tonus adjustments and control of locomotion. However, it is a deep-seated brainstem nucleus without clear imaging or electrophysiological markers. Some studies suggested that diffusion tensor imaging (DTI) may help guiding electrode placement in the PPN by showing the surrounding fiber bundles, but none have provided a direct histological correlation. We investigated DTI fractional anisotropy (FA) maps from in vivo and in situ post-mortem magnetic resonance images (MRI) compared to histological evaluations for improving PPN targeting in humans. A post-mortem brain was scanned in a clinical 3T MR system in situ. Thereafter, the brain was processed with a special method ideally suited for cytoarchitectonic analyses. Also, nine volunteers had in vivo brain scanning using the same MRI protocol. Images from volunteers were compared to those obtained in the post-mortem study. FA values of the volunteers were obtained from PPN, inferior colliculus, cerebellar crossing fibers and medial lemniscus using histological data and atlas information. FA values in the PPN were significantly lower than in the surrounding white matter region and higher than in areas with predominantly gray matter. In Nissl-stained histologic sections, the PPN extended for more than 10 mm in the rostro-caudal axis being closely attached to the lateral parabrachial nucleus. Our DTI analyses and the spatial correlation with histological findings proposed a location for PPN that matched the position assigned to this nucleus in the literature. Coregistration of neuroimaging and cytoarchitectonic features can add value to help establishing functional architectonics of the PPN and facilitate neurosurgical targeting of this extended nucleus.

BackgroundImmunofluorescence (IF) plays a major role in quantifying protein expression in situ and understanding cell function. It is widely applied in assessing disease mechanisms and in drug discovery research. Automation of IF analysis can transform studies using experimental cell models. However, IF analysis of postmortem human tissue relies mostly on manual interaction, often subjected to low-throughput and prone to error, leading to low inter and intra-observer reproducibility. Human postmortem brain samples challenges neuroscientists because of the high level of autofluorescence caused by accumulation of lipofuscin pigment during aging, hindering systematic analyses. We propose a method for automating cell counting and classification in IF microscopy of human postmortem brains. Our algorithm speeds up the quantification task while improving reproducibility.New methodDictionary learning and sparse coding allow for constructing improved cell representations using IF images. These models are input for detection and segmentation methods. Classification occurs by means of color distances between cells and a learned set.ResultsOur method successfully detected and classified cells in 49 human brain images. We evaluated our results regarding true positive, false positive, false negative, precision, recall, false positive rate and F1 score metrics. We also measured user-experience and time saved compared to manual countings.Comparison with existing methodsWe compared our results to four open-access IF-based cell-counting tools available in the literature. Our method showed improved accuracy for all data samples.ConclusionThe proposed method satisfactorily detects and classifies cells from human postmortem brain IF images, with potential to be generalized for applications in other counting tasks.

Abnormal tau inclusions are hallmarks of Alzheimer's disease and predictors of clinical decline. Several tau PET tracers are available for neurodegenerative disease research, opening avenues for molecular diagnosis in vivo. However, few have been approved for clinical use. Understanding the neurobiological basis of PET signal validation remains problematic because it requires a large-scale, voxel-to-voxel correlation between PET and (immuno) histological signals. Large dimensionality of whole human brains, tissue deformation impacting co-registration, and computing requirements to process terabytes of information preclude proper validation. We developed a computational pipeline to identify and segment particles of interest in billion-pixel digital pathology images to generate quantitative, 3D density maps. The proposed convolutional neural network for immunohistochemistry samples, IHCNet, is at the pipeline's core. We have successfully processed and immunostained over 500 slides from two whole human brains with three phospho-tau antibodies (AT100, AT8, and MC1), spanning several terabytes of images. Our artificial neural network estimated tau inclusion from brain images, which performs with ROC AUC of 0.87, 0.85, and 0.91 for AT100, AT8, and MC1, respectively. Introspection studies further assessed the ability of our trained model to learn tau-related features. We present an end-to-end pipeline to create terabytes-large 3D tau inclusion density maps co-registered to MRI as a means to facilitate validation of PET tracers.

Three-dimensional brain imaging through cutting-edge MRI technology allows assessment of physical and chemical tissue properties at sub-millimeter resolution. In order to improve brain understanding as part of diagnostic tasks using MRI images, other imaging modalities to obtain deep cerebral structures and cytoarchitectural boundaries have been investigated. Under availability of postmortem samples, the fusion of MRI to brain histology supports more accurate description of neuroanatomical structures since it preserves microscopic entities and reveal fine anatomical details, unavailable otherwise. Nonetheless, histological processing causes severe tissue deformation and loss of the brain original 3D conformation, preventing direct comparisons between MRI and histology. This paper proposes an interactive computational pipeline designed to register multimodal brain data and enable direct histology-MRI correlation. Our main contribution is to develop schemes for brain data fusion, distortion corrections, using appropriate diffeomorphic mappings to align the 3D histological and MRI volumes. We describe our pipeline and preliminary developments of scalable processing schemes for highresolution images. Tests consider a postmortem human brain, and include qualitatively and quantitatively results, such as 3D visualizations and the Dice coefficient (DC) between brain structures. Preliminary results show promising DC values when comparing our scheme results to manually labeled neuroanatomical regions defined by a neurosurgeon on MRI and histology data sets. DC was computed for the left caudade gyrus (LC), right hippocampus (RH) and lateral ventricles (LV).

A comprehensive survey of Croatian watercourses covering the whole of the national territory and investigating inherent watercourse heterogeneity was conducted to explore the ecological responses of the most frequent freshwater bryophytes with respect to water chemistry variables and land use within the catchment area. Direct multivariate ordination (CCA) of vegetation data paired with 18 environmental variables revealed that freshwater bryophytes and their assemblages were segregated along the gradients of water chemistry and the proportion of natural and urban area within the catchment. Generalized additive models (GAM) were employed to explore the ecological responses of individual species. The results showed that most of the investigated species preferred natural, clean, well-oxygenated watercourses, with low nutrient and organic matter content, as well as with low electrical conductivity. Species such as Palustriella falcata, Eucladium vertcillatum, Dichodontium flavescens and Jungermannia atrovirens had narrow ecological niches and were restricted to pristine watercourses, while the most frequent and widely distributed species, such as Fontinalis antipyretica, Rhynchostegium riparioides, Cratoneuron filicinum, Fissidens crassipes, Cinclidotus fontinaloides and C. riparius, had a wide ecological tolerance. Riccia fluitans and Leptodyctium riparium had wide ecological ranges, but with optima in hypereutrophic waters with high nutrient and organic content, as well as high electrical conductivity. Furthermore, these two species were frequently associated with a high share of intensive agriculture and a low share of natural land within the catchment.

The genus Xerobdella contains three species of land leeches confined to the Palearctic region, one of which is X. anulata (Autrum, 1958), an exceptionally rare endemic of the Dinaric Alps. In the current study, we provide new data and a literature overview on this rarely encountered species, presenting its currently known distribution, providing additional data on its morphology and, for the first time, presenting comprehensive data on its habitat preferences and seasonal dynamics. Additionally, we provide novel DNA barcodes for the Dinaric land leech and compare the obtained sequences with the related X. lecomtei. Altogether, 22 specimens of X. anulata were collected using pitfall traps in three habitat types: managed forests with adjacent meadows and a primeval forest in the Dinaric Alps of Croatia. We report the first finding of X. anulata in open habitats, which harbored most of the specimens. Our findings show that X. anulata exhibits surface activity, highlighting the effectiveness of pitfall traps in sampling such elusive taxa, with perspectives for future morphological, phenological and even molecular research.

A comprehensive field survey of 527 sites on 293 watercourses across Croatia revealed 76 sites (14.42%) in which bryophytes were the dominant part of the macrophyte vegetation. Using classification and ordination analyses, we obtained five community types segregated across the gradients of several climatic, physiographic and water chemistry parameters. The Didymodon tophaceus–Apopellia endiviifolia and the Berula erecta-Cratoneuron filicinum communities were mostly confined to the clean and basic karstic rivers of the Dinaric Ecoregion under the influence of the Mediterranean climate, with the Didymodon tophaceus–Apopellia endiviifolia community being a tufa-forming community associated with the seasonally dry watercourses of small catchment areas and cascades along the larger karstic rivers, while the Berula erecta–Cratoneuton filicinum community was mostly associated with rivers with larger catchment areas and permanent flow. On the other hand, the Oxyrrhynchium hians–Chiloscyphus pallescens community and the Fissidens pusillus–Veronica beccabunga community were associated with eutrophic water restricted to small rivers of the Pannonian Ecoregion under the influence of the temperate climate and flowing over silicate bedrock. The most represented and widespread in Croatia was the Cinclidotus community, displaying the widest ecological range in the study. It was mostly associated with the relatively clean karstic rivers of large catchment areas belonging to the Dinaric Ecoregion, with the majority of the sites under the influence of a temperate climate with higher precipitation during the warm period of the year. The geographical patterns of the freshwater bryophyte communities showed that the relatively clean, fast and cold karstic rivers belonging to the Dinaric Ecoregion provide habitats that harbour a greater diversity of bryophyte communities than the watercourses of the Pannonian Ecoregion, where bryophyte-dominated communities are restricted to a small number of small lowland and semi-montane rivers and predominantly occupy periodically flooded microhabitats such as river margins.

Background and Purpose: The paper presents the forest vegetation of periodically flooded and wet forests of hardwood tree species along the Mirna River (Istria, Croatia). The main objective was to study the older and less influential stands, and to compare them among themselves and with related syntaxa of the Mediterranean and continental area. Materials and Methods: The research was conducted on the basis of 33 new phytocoenological relevés and 12 from previous studies, according to the principles of the standard Central European Phytocoenological School. Results and Conclusions: Based on 45 phytocoenological relevés two main vegetation types were found. In the lowest and periodically flooded habitats grow forests dominated by Fraxinus angustifolia and numerous hygrophilous species. In somewhat higher and drier localities, but with a high level of ground waters, grow mixed forests of Quercus robur, Fraxinus angustifolia, Ulmus minor and Carpinus betulus, with a greater presence of mesophilous species. The paper analyzes their mutual relationship, phytocoenological affiliation, as well as their position with regard to the related syntaxa of the Mediterranean and continental area. The results suggest isolation and a transitional character of the studied forests, which is a consequence of the biogeographical position in the north Mediterranean, of the ecological conditions, and to a lesser extent of anthropogenic influence.

Background: Canine transmissible venereal (TVT) is a transplantable tumor usually transmitted to genital organs during coitus. Tumor cell inoculation is also possible in extragenital sites via licking or sniffing of the vaginal or preputial discharge. Metastases occur predominantly in males involving mainly the regional lymph nodes. On rare occasions, TVT metastases may affect kidney, spleen, tonsils, pancreas, lung, eye, brain, pituitary gland and musculature. Primary ocular involvement of TVT is not commonly reported in dogs, with few cases cited in the literature. The purpose of this study is to describe the ocular manifestations, histological features and immunohistochemical analysis in a canine intraocular TVT. Case: A 8-year-old male dog of mixed breed weighing 14.3 kg, was presented because red protrusion from both eyes. After of clinical examination, the dog was submitted to bilateral enucleation. In the histopathological examination was observed tumor cells uniformly round, arranged in solid sheets by delicate connective-tissue stroma. The cells have centrally located round nuclei that contain 1 or 2 prominent nucleolus. The cytoplasm was scant, sometimes contain brown pigment. Immunohistochemical staining with commercially available antibodies such as lysozyme, CD3, CD45R, PAX5 and PNL2 was performed to obtain a more accurate diagnosis. Immunohistochemical staining revealed strong cytoplasmic reactivity to lysozyme in about 90% cells. Weak cytoplasmic immunoreactivity of CD45R was detected in 80-90% of cells and negative immunoreactivity was observed in the CD3, PAX-5, PNL2 antibodies. Discussion: The use of imunohistochemical markers excluded other types of round cell tumors, such as lymphomas, melanomas, amelanotic melanomas, poorly differentiated carcinomas and mast cell tumors. However, some authors described similar immunostaining in histiocitomas, but the specific staining for Lysozyme, histopathological analisys and immunohistochemical evaluation was fundamental for this diagnosis. As a differential diagnosis for ophthalmic lesions, there are malignant and benign and non-neoplastic neoplastic processes. As malignant neoplasms of intraocular location, there are melanomas, poorly differentiated carcinomas, mast cell tumors. In the case of benign variants, adenomas are highlighted. Round cell neoplasms are also important differential diagnoses, including mast cell tumor, histiocioma, and lymphoma. Non-neoplastic processes, such as granulomatous lesions caused by Leishmaniasis, should also be considered. Linked to this, the performance of immunohistochemistry, due to the range of possible tumor markers, becomes essential for the diagnostic conclusion by excluding other types of round cell tumors, such as lymphoma, melanoma, poorly differentiated carcinomas and mast cell tumors. The prognosis for ocular TVT is good in situations where enucleation occurs, as in this case. That the patient was healthy and in the absence of neoplastic cells due to being affected only in the eye region. Finally, histopathological and imunohistochemistry are examination are confirmatory are the definitive diagnosis. Despite the specific labeling for Lysozyme, it was present in 90% of the cells, and the non-sensitivity of imunoreactivity to CD3 and PAX-5, similar results already described. Thus, and due to the fact that the importance of immunohistochemistry is observed, it suggests - if the use of this technique can validate the confirmation of marking of primary cells of the tumor, in this case reported here. [ABSTRACT FROM AUTHOR]