Quantitative Morphometry Research Articles

Comparative specialization of intrinsic cardiac neurons in humans, mice and pigs.

Intrinsic cardiac neurons (ICNs) play a crucial role in the proper functioning of the heart; yet a paucity of data pertaining to human ICNs exist. We took a multidisciplinary approach to complete a detailed cellular comparison of the structure and function of ICNs from mice, pigs and humans. Immunohistochemistry of whole and sectioned ganglia, transmission electron microscopy, intracellular microelectrode recording and dye filling for quantitative morphometry were used to define the neurophysiology, histochemistry and ultrastructure of these neurons across species. The densely packed, smaller ICNs of mouse lacked dendrites, formed axosomatic connections and had high synaptic efficacy constituting an obligatory synapse. At pig ICNs, a convergence of subthreshold cholinergic inputs onto extensive dendritic arbors supported greater summation and integration of synaptic input. Human ICNs were tonically firing, with synaptic stimulation evoking large suprathreshold EPSPs like mouse, and subthreshold potentials like pig. Ultrastructural examination of synaptic terminals revealed conserved architecture, yet small clear vesicles were larger in pigs and humans. The presence and localization of ganglionic neuropeptides was distinct, with abundant vasoactive intestinal polypeptide observed in human but not pig or mouse ganglia, and little substance P or calcitonin gene-related peptide in pig ganglia. Action potential waveforms were similar, but human ICNs had larger after-hyperpolarizations. Intrinsic excitability differed; 95% of human neurons were tonic, all pig neurons were phasic, and both phasic and tonic phenotypes were observed in mouse. In combination, this publicly accessible, multimodal atlas of ICNs from mice, pigs and humans identifies similarities and differences in the evolution of ICNs. KEY POINTS: Intrinsic cardiac neurons (ICNs) are essential to the regulation of cardiac function. We investigated the neurochemistry, morphology, ultrastructure, membrane physiology and synaptic transmission of ICNs from donated human hearts in parallel with identical studies of ICNs from mice and pigs to create a publicly accessible cellular atlas detailing the structure and function of these neurons across species. In addition to presenting foundational data on human ICNs, this comparative study identifies both conserved and derived attributes of these neurons within mammals. The findings have significant implications for understanding the regulation of cardiac autonomic function in humans and may greatly influence strategies for neuromodulation in conditions such as atrial fibrillation and heart failure.

Unmanned Aerial Vehicle Technology for Quantitative Morphometry and Geomorphic Processes – Study Case in Rotational Landslide Deposited Areas

Unmanned Aerial Vehicle Technology for Quantitative Morphometry and Geomorphic Processes – Study Case in Rotational Landslide Deposited Areas

A Standardized Porcine Model for Partial-Thickness Wound Healing Studies: Design, Characterization, Model Validation, and Histological Insights.

Wound healing is a complex process that is still not fully understood despite extensive research. To address this, we aimed to design and characterize a standardized porcine model for the evaluation of wound healing, dressings, cell therapies, and pharmaceutical agents. Using a standardized approach, we examined the wound healing process in 1.2 mm-deep dermatome wounds at defined positions in 11 female pigs. Unlike previous studies that have only described/analyzed selected punch biopsies, we performed and described histological analyses along the complete wound length using quantitative morphometric methods. All animals remained fully healthy following surgery and showed no signs of infection. Our histopathological evaluation using a predetermined grading score and quantitative manual morphometry demonstrated the impact of different tissue sampling methods, sampling sites, and residual dermis thickness on wound healing. Our study presents a reproducible model for wound healing evaluation and demonstrates the usefulness of porcine models for assessing dermal and epidermal wound healing. The use of histological analyses over the complete wound length provides advantages over previous studies, leading to the possibility of a deeper understanding of the wound healing process. This model could potentially facilitate future research on novel wound dressings and local wound healing therapies.

CAS3D: 3D quantitative morphometry on Second Harmonic Generation image volumes from single skeletal muscle fibers

The CAS3D image processing method intuitively applies a combination of Fourier space and real space 3D analysis algorithms to volumetric images of single skeletal muscle fiber Myosin II Second Harmonic Generation (SHG) XYZ image data. Our developed tool automatically quantifies the myofibrillar orientation in muscle samples by determining the cosine angle sum of intensity gradients in 3D (CAS3D) while determining the mean sarcomere length (SL) and sample orientation. The expected CAS3D values could be reproduced from ideal artificial data sets. Applied random noise in artificial images lowers the detected CAS3D value, and for noise levels below 20%, the correlation can be approximated by a linear function with a slope of −0.006 CAS3D/noise%. The deviations in SL and orientation detection were determined on ideal and noisy artificial data sets and were statistically indistinguishable from 0 (null hypothesis t-test P > 0.1). The software was applied to a previously published data set of single skeletal muscle fiber volumetric SHG image data from a rat intensive care unit (ICU) model of ventilator-induced diaphragm dysfunction (VIDD) with treatment regimens involving the small anti-inflammatory molecules BGP-15, vamorolone, or prednisolone. Our method reliably reproduced the results of the previous work and improved the standard deviation of the cosine angle sum detection in all sample groups from a mean of 0.03 to 0.008. This improvement is achieved by applying analysis algorithms to the whole volumetric images in 3D in contrast to the previously common method of slice-wise XY analysis.

#2215 Early molecular and cellular features of aortic remodeling in a mild CKD-MBD model with low bone turnover

Abstract Background and Aims Vascular calcification associated with a maladaptive bone response is common in CKD and is a major cause of mortality (up to 60% depending on CKD stage). Alterations in several intracellular signaling pathways have been reported during the cellular phenotype transition and vascular calcification, however the initial phases of vascular remodeling in early CKD-MBD remain poorly understood. The aim of this study was to investigate the early molecular and cellular features of aortic remodeling prior to calcification in a model of mild CKD-MBD with low bone turnover. Method We induced CKD-MBD by 3/4 nephrectomy (Nx, n = 8) in adult male spontaneously hypertensive rats (SHR) with normal phosphate (Pi) intake (0.6%). Controls were sham-operated Wistar rats (n = 8) and SHR (n = 8). Chronic kidney injury and Pi exchange parameters, serum levels of calciprotein particles (CPPs), intact PTH, intact FGF23, dickkopf-1 and sclerostin, static bone histomorphometry, aortic morphology (H&E, Masson's trichrome, calcein, von Kossa stain), CD31, aSMA, nestin, Col1a1, PDGFR-beta, PiT1, LGR4 and intracellular signaling molecules—phospho-ERK1/2, active (non-phospho) beta-catenin, Hes1, Gli1 were analyzed by IHC with quantitative morphometry and IF after 4 months. Confocal microscopy was performed at the Centre for Collective Use of the Pavlov Institute of Physiology, Russian Academy of Sciences (Zeiss LSM 710). Results The study found that Nx rats with chronic kidney injury comparable to human CKD S2 exhibited lower bone turnover (Fig. 1). The Nx group had higher serum levels of Pi and CPPs vs. control group, but there were no differences in Pi-regulatory factors (Figs 1, 2b). Additionally, the Nx group showed aorta remodeling, which was characterized by an increase in intima thickness, cellularity, and collagen accumulation in the medial layer (Figs 1, 2a). The histological parameters of intimal remodeling and perivascular fibrosis were found to be directly correlated with serum levels of Pi and CPPs. In Nx, there was a decrease in medial αSMA expression accompanied by an increase in phospho-ERK1/2, PiT1, nestin, Gli1, beta-catenin, dickkopf-1, and an decrease in Hes1 immunomorphological staining (Figs 1, 2a, c). αSMA-negative cells demonstrated the expression of osteogenesis-related molecules, such as phospho-ERK1/2 (Fig. 2a, c), and occasionally, RunX2 (Fig. 2a). Phospho-ERK1/2 and PiT1 were found to co-localize in Nx (Fig. 2c). Intimal remodeling was observed, characterized by increased beta-catenin and nestin expression (Fig. 2c). Conclusion In mild CKD-MBD with low bone turnover, early intimal and medial aortic remodeling was associated with higher serum Pi and CPPs levels, and upregulation of PiT1, ERK1/2. In addition, other signals likely related to osteogenesis (RunX2, LGR4) and regulation of vascular progenitor cells (Nestin, Wnt, Notch, Hedgehog) may be implicated.

#1464 Alterations of podocytes in focal segmental glomerulosclerosis: a pilot study

Abstract Background and Aims The molecular features of idiopathic podocytopathies remain unexplored, preventing the development of effective therapies to control the mechanisms of disease initiation and progression. This includes idiopathic focal segmental glomerulosclerosis (FSGS), one of the most common forms of immune glomerulopathies (IG) and the cause of renal failure and replacement therapy. In FSGS, podocytes undergo pronounced cytoskeletal alterations. Along with a decrease in Wilms tumor protein (WT1) and α-SMA expression, there is evidence for increased expression of intermediate filament proteins (vimentin, desmin, nestin) and Wnt/β-catenin signaling upregulation in IG. Decrease in WT1 is associated with upregulation in Wnt/β-catenin and p53 to promote podocyte dysfunction and albuminuria. Upregulation desmin increases the mechanical stability of cells, allowing podocytes to undergo morphological changes on the tensile glomerular capillary wall. This pilot study was aimed to evaluate the significance of WT1, β-catenin, and desmin glomerular expression in early primary FSGS. Method We reviewed the histopathological and clinical data of patients with biopsy proven FSGS (N = 14, experimental group), IgA nephropathy (N = 12, positive control), without AKI, infectious diseases, heart failure, respiratory insufficiency and cancer pathology. Renal surgery specimens from patients without IG (N = 12) were studied as negative control. Glomerulosclerosis was assessed as percentage of all glomeruli. Mesangial lesions were measured semi-quantitatively in scores (0-3). Glomerular WT1, β-catenin, and desmin expression was assessed by IHC-P with quantitative morphometry of IHC-staining area (open software QuPath, Orbit Image Analysis). Molecule's co-expression was analyzed by IF-p and confocal microscopy (Zeiss LSM 710; study conducted in the Center for Collective Use “Confocal microscopy” by Pavlov Institute of Physiology Russian Academy of Sciences). Results We examined young and middle-aged patients with CKD stages 1-3 and moderate morphological changes in the glomeruli. Gender, age, eGFR and the percentage of glomeruli with segmental sclerosis were not significantly different between IG groups (all p for interaction >0.05). Patients with FSGS have lower levels of serum albumin (p < 0.001) and higher levels of proteinuria (p < 0.001) as compared to IgAN. Glomeruli from IgAN patients showed more mesangial cell proliferation (p = 0.001), expansion of the mesangial matrix (p = 0.05) and global glomerular sclerosis (p = 0.003) as compared to FSGS. An immunomorphological study showed lower WT1 expression in both IG groups compared to the negative control. In the IgAN, lower WT1 expression was associated with higher BC area (Fig. 1A, D). BC expression did not differ between FSGS and negative control (Fig. 1B,D). In the combined IG group, BC directly correlated with global (r = 0.63, p = 0.001) and segmental (r = 0.63, p = 0.001) glomerular sclerosis. In FSGS, a trend towards to increase in the area of desmin IHC-staining was detected, however, no significant differences between groups were found (Fig. 1C,D). BC and desmin were co-localized with the podocyte marker WT1 (Fig. 2). BC and desmin were also expressed in WT1-negative glomerular cells (Fig. 2). Differential molecular expression patterns in glomeruli have been identified. In IgAN, co-expressing WT1 and BC (orange) was dominate in glomerular cells (Fig. 2A); the proportion of IHC expression of WT1 and BC in glomerular cells was directly correlated (r = 0.63, p = 0.031). In FSGS, WT1+ cells predominantly co-expressed desmin (magenta) but not BC; some glomerular cells were only desmin-positive (Fig. 2B). BC was expressed to a lesser extent compared to IgAN, and in some cells it was co-expressed with desmin (Fig. 2B). Conclusion In FSGS, an increase in desmin expression could be an early feature of pathogenesis before significant decrease in the number of WT1+ podocytes and changes in β-catenin expression. Activation of desmin likely characterizes damage and decrease in the number of functioning podocytes.

Duodenal quantitative mucosal morphometry in children with environmental enteric dysfunction: a cross-sectional multicountry analysis

BackgroundEnvironmental enteric dysfunction (EED), a chronic inflammatory condition of the small intestine, is an important driver of childhood malnutrition globally. Quantifying intestinal morphology in EED allows for exploration of its association with functional and disease outcomes. ObjectivesWe sought to define morphometric characteristics of childhood EED and determine whether morphology features were associated with disease pathophysiology. MethodsMorphometric measurements and histology were assessed on duodenal biopsy slides for this cross-sectional study from children with EED in Bangladesh, Pakistan, and Zambia (n = 69), and those with no pathologic abnormality (NPA; n = 8) or celiac disease (n = 18) in North America. Immunohistochemistry was also conducted on 46, 8, and 18 biopsy slides, respectively. Linear mixed-effects regression models were used to reveal morphometric differences between EED compared with NPA or celiac disease and identify associations between morphometry and histology or immunohistochemistry among children with EED. ResultsIn duodenal biopsies, median EED villus height (248 μm), crypt depth (299 μm), and villus:crypt (V:C) ratio (0.9) values ranged between those of NPA (396 μm villus height; 246 μm crypt depth; 1.6 V:C ratio) and celiac disease (208 μm villus height; 365 μm crypt depth; 0.5 V:C ratio). Among EED biopsy slides, morphometric assessments were not associated with histologic parameters or immunohistochemical markers, other than pathologist-determined subjective semiquantitative villus architecture. ConclusionsMorphometric analysis of duodenal biopsy slides across geographies identified morphologic features of EED, specifically short villi, elongated crypts, and a smaller V:C ratio relative to NPA slides, although not as severe as in celiac slides. Morphometry did not explain other EED features, suggesting that EED histopathologic processes may be operating independently of morphology. Although acknowledging the challenges with obtaining relevant tissue, these data form the basis for further assessments of the role of morphometry in EED.

Local, Quantitative Morphometry of Fibroproliferative Lung Injury Using Laminin.

Investigations into the mechanisms of injury and repair in fibroproliferative disease require consideration of the spatial heterogeneity inherent in the disease. Most scoring of fibrotic remodeling in preclinical animal models relies on the modified Ashcroft score, which is an ordinal rubric of macroscopic resolution. The obvious limitations of manual histopathologic scoring have generated an unmet need for unbiased, repeatable scoring of fibroproliferative burden in tissue. Using computer vision approaches on immunofluorescence imaging of the extracellular matrix component laminin, we generated a robust and repeatable quantitative remodeling scorer. In the bleomycin lung injury model, the quantitative remodeling scorer shows significant agreement with the modified Ashcroft scale. This antibody-based approach is easily integrated into larger multiplex immunofluorescence experiments, which we demonstrate by testing the spatial apposition of tertiary lymphoid structures to fibroproliferative tissue, a poorly characterized phenomenon observed in both human interstitial lung diseases and preclinical models of lung fibrosis. The tool reported in this article is available as a stand-alone application that is usable without programming knowledge.

Применение методов морфометрии для оценки гистопатологии в доклинических исследованиях

Morphometry is one of the most important methods of preclinical studies. It is an integral part of microscopy analysis. The main problems of microscopic analysis are variability of microscopic findings, absence of uniformity of the terms and definitions, the subjectivity of the pathologist’s perception. Thus, the traditional histopathologic description does not can use for a full and objective assessment of experimental disease. The pathologist can evaluate the whole of microscopic findings and make a diagnosis. However, he can overlook small differences, that can be critical for comparative analysis. Application of morphometry methods helps to detect small differences in the degree of severity of microscopic findings. Counting of tissue’s elements, measurement their size or using semiquantitative scales allows to reduce or, even, exclude the subjectivity of the perception. This review describes methods of quantitative and qualitative morphometry based on commonly used types of measurement scales. The descriptive histology is the simplest means of analyzing. It is a search method and applies in every research for assessment of samples, detection target organs, determining the frequency of developing changes in animal’s groups. Pathologists use the most frequently semiquantitative scales. It is a simple method, that do not need special equipment. It allows to compare the degree of severity of the effects of a test sample on target organs, in contrast to descriptive analyze. Conversion qualitative data about the pathological findings to quantitative data allows significantly increase reliability and reproducibility of results of research. But, one must carefully choose a scoring scale according to modeled disease. The quantitative morphometry includes planimetry and stereology. It needs special measuring tools or software. The result of quantitative analisis allows to compare the degree of severity of a pathological process and to detect how many times the one effect is bigger, then another one. Also, it makes it possible to mathematically model the processes, calculating dose-response relationships for example. Planimetry based on linear measurements and counting of elements in a two-dimensional plane of tissue slice. The result of stereometric measurements is quantitative characteristics of elements in the whole volume of the object, in contrast to planimetry, where the result is quantitative characteristics of elements in part of the object. The method has maximum objectivity and reliability, but time-consuming and needs carefully a pre-planning and an applying rigorous sampling methods. Described methods are not completely interchangeable. They can be taken in combination, that increases the quality of research.

Expression of Placental Lipid Transporters in Pregnancies Complicated by Gestational and Type 1 Diabetes Mellitus.

The objective of the study was to assess the expression of proteins responsible for placental lipid transport in term pregnancies complicated by well-controlled gestational (GDM) and type 1 diabetes mellitus (PGDM). A total of 80 placental samples were obtained from patients diagnosed with PGDM (n = 20), GDM treated with diet (GDMG1, n = 20), GDM treated with diet and insulin (GDMG2, n = 20), and a non-diabetic control group (n = 20). Umbilical and uterine artery blood flows were assessed by means of ultrasound in the period prior to delivery and computer-assisted quantitative morphometry of immunostained placental sections was performed to determine the expression of selected proteins. The morphometric analysis performed for the vascular density-matched placental samples demonstrated a significant increase in the expression of fatty acid translocase (CD36), fatty acid binding proteins (FABP1, FABP4 and FABP5), as well as a decrease in the expression of endothelial lipase (EL) and fatty acid transport protein (FATP4) in the PGDM-complicated pregnancies as compared to the GDMG1 and control groups (p < 0.05). No significant differences with regard to the placental expression of lipoprotein lipase (LPL) and FATP6 protein between GDM/PGDM and non-diabetic patients were noted. Maternal pre-pregnancy weight, body mass index, placental weight as well as the expression of LPL and FABP4 were selected by the linear regression model as the strongest contributors to the fetal birth weight. To conclude, in placentas derived from pregnancies complicated by well-controlled PGDM, the expression of several lipid transporters, including EL, CD36, FATP4, FABP1, FABP4 and FABP5, is altered. Nonetheless, only LPL and FABP4 were significant predictors of the fetal birth weight.

Quantitative morphometry of compensated liver cirrhosis can aid to predict the outcomes of surgical treatment

Objective — to define quantitative morphometric characteristics of hepatic parenchyma lesions at compensated liver cirrhosis (CLC), which will allow more accurately to assess its functional reserves and predict the outcomes of surgical treatment. Materials and methods. Intraoperative marginal liver biopsy was performed for 74 patients with CLC, who underwent surgical treatment. Morphological examinations with quantitative morphometry of intraoperative liver biopsies were done with the use of cytological analyzer with software «Integral‑2MT» produced by the association «Kvant» (Kyiv). The connective tissue area (CTA), unchanged hepatocytes area (UHA), stromal‑parenchymatous ratio (SPR), the volume of hepatocytes in the state of necrosis and/or necrobiosis (VHSNN), the volume of hepatocytes in the state of division (VHSD) were determined. The statistical analysis was performed by use of Statistica 12 software package. Results. Three types of morphological pattern of CLC were isolated. Type I (type A) portal cirrhosis with weakly pronounced signs of parenchymatous and stromal reaction (mono‑multilobular type, portal cirrhosis). The CTA was 66.73±1.71 mm2, SPR was 0.285±0.019, UHA was 234.13±11.5 mm2, VHSNN was 11.21±0.74%, VHSD was 10.23±0.57%. Type II cirrhosis (type B) — cirrhosis of mono‑multilobular type with moderately expressed signs of parenchymatous and stromal reaction. The CTA was 126.69±12.5 mm2, SPR was 0.617±0.031, UHA was 205.34±13.8 mm2, VHSNN was 17.32±0.63%, VHSD was 15.43±0.48%. Type III cirrhosis (type C) — cirrhosis with pronounced signs of parenchymatous and stromal reaction, more often of multilobular type. The CTA was 240.16±13.4 mm2, SPR was 1.344±0.089, UHA was 178.69±18.7 mm2, VHSNN was 23.97±0.75%, VHSD was 11.07±0.58%. Analysis of immediate results of surgical treatment demonstrated, that no one patient with A‑type CLC died, while 9.5% of patients with B‑type and 25.0% of patients with C‑type CLC died in the early postoperative period. Conclusions. Quantitative morphometry demonstrated that pathological changes in the liver at CLC are heterogeneous and can be categorized in to 3 types (A, B, C). Three types of morphological pattern at CLC, which were isolated, are characterized by significant differences in CTA, UHA, SPR, VHSNN, VHSD parameters. At transition of A‑type cirrhosis into C‑type, volume of hepatic parenchyma becomes to be decreased, while volume of connective tissue becomes to be increased. This is accompanied by decrease in UHA, increase in CTA, SPR and VHSNN. Surgical intervention in patients with type C morphological pattern of cirrhosis is associated with a high risk (25%) of poor outcome, which indicates the need to limitations for indications to elective surgical operations in this category of patients. Thus, quantitative morphometry of liver biopsies in CLC patients can aid to predict the outcomes of surgical treatment.

Combined light and electron microscopy (CLEM) to quantify methamphetamine-induced alpha-synuclein-related pathology

Methamphetamine (METH) produces a cytopathology, which is rather specific within catecholamine neurons both in vitro and ex vivo, in animal models and chronic METH abusers. This led some authors to postulate a sort of parallelism between METH cytopathology and cell damage in Parkinson’s disease (PD). In fact, METH increases and aggregates alpha-syn proto-fibrils along with producing spreading of alpha-syn. Although alpha-syn is considered to be the major component of aggregates and inclusions developing within diseased catecholamine neurons including classic Lewy body (LB), at present, no study provided a quantitative assessment of this protein in situ, neither following METH nor in LB occurring in PD. Similarly, no study addressed the quantitative comparison between occurrence of alpha-syn and other key proteins and no investigation measured the protein compared with non-protein structure within catecholamine cytopathology. Therefore, the present study addresses these issues using an oversimplified model consisting of a catecholamine cell line where the novel approach of combined light and electron microscopy (CLEM) was used measuring the amount of alpha-syn, which is lower compared with p62 or poly-ubiquitin within pathological cell domains. The scenario provided by electron microscopy reveals unexpected findings, which are similar to those recently described in the pathology of PD featuring packing of autophagosome-like vesicles and key proteins shuttling autophagy substrates. Remarkably, small seed-like areas, densely packed with p62 molecules attached to poly-ubiquitin within wide vesicular domains occurred. The present data shed new light about quantitative morphometry of catecholamine cell damage in PD and within the addicted brain.

Quantitative brain morphometry identifies cerebellar, cortical, and subcortical gray and white matter atrophy in late-onset Tay-Sachs disease

Quantitative brain morphometry identifies cerebellar, cortical, and subcortical gray and white matter atrophy in late-onset Tay-Sachs disease

Distribution of neurovascular structures within the prostate gland and their relationship to complications after radical prostatectomy.

Radical prostatectomy remains the main choice of treatment for prostate cancer. However, despite improvements in surgical techniques and neurovascular sparing procedures, rates of erectile dysfunction, and urinary incontinence remain variable. This is due, at least in part, to an incomplete understanding of neurovascular structures associated with the prostate. The objective of this study was to provide a comprehensive, detailed histological overview of the distribution of nerves and blood vessels within the prostate, facilitating subsequent correlation of prostatic neurovascular structures with patients' clinical outcomes after radical prostatectomy. Neurovascular structures within the prostate were investigated in a total of 309 slides obtained from 15 patients who underwent non-nerve-sparing radical prostatectomy. Immunohistochemical staining was performed to identify and distinguish between parasympathetic and sympathetic nerves, whereas hematoxylin and eosin staining was used to identify blood vessels. The total number, density, and relative position of nerves and blood vessels were established using quantitative morphometry and illustrated using visualization approaches. Patient-specific outcome data were then used to establish whether the internal distribution of nerves and blood vessels within the prostate correlated with the nature and extent of complications after surgery. One-way analysis of variance tests and unpaired ttests were applied to establish statistically significant differences across the measured variables. Nerves and blood vessels were present across all prostatic levels and regions. However, their number and density varied considerably between regions. Assessment of the precise positioning of neurovascular structures revealed that the majority of nerve fibers were located within the dorsal and peripheral aspects of the gland. In contrast, blood vessels were predominantly located within ventral and dorsal midline regions. The number of intraprostatic nerves was found to be significantly lower in patients who recovered their continence within 12 months of surgery, compared to those whose recovery took 12 months or longer. We report an unexpected disconnect between the localization and positioning of nerve fibers and blood vessels within the prostate. Moreover, individual variability in the density of intraprostatic neurovascular structures appears to correlate with the successful recovery of urinary continence after radical prostatectomy, suggesting that differences in intrinsic neurovascular arrangements of the prostate influence postoperative outcomes.

Cone-Beam Computed Tomography to evaluate changes in trabecular lower jawbone microstructure caused by bone loss and antiresorptive treatment.

The aim of this preliminary report is to evaluate the usefulness of CBCT to assess trabecular mandible microstructural properties in normal ewes and to compare the quantitative changes associated with ovariectomy and antiresorptive treatment. Twelve adult Corriedale ewes (n=4/group) aged 3-4 years were divided into 3 groups and studied for 28 months. Eight ewes were ovariectomized (OVX) and divided into OVX and OVX+ZOL groups (n=4/group) which were treated as follows, by jugular injection: OVX received saline solution and OVX+ZOL received zoledronate (Zol) (Gador SA, CABA, Argentina) (4 mg/month). Another four ewes were subjected to sham surgery (SHAM group) and received saline solution. Densitometry showed that jaw mineral content (BMC) and density (BMD) were significantly lower in OVX than in SHAM and OVX+ZOL ewes; no difference was observed between OVX+ ZOL and SHAM groups. CBCT analysis showed that bone volume (BV/TV%); trabecular thickness (TbTh); connectivity density (CD) and anisotropy degree (AD) were significantly lower, and trabecular spacing (TbSp), significantly higher in OVX than in SHAM ewes. AD was significantly higher and TbSp significantly lower in OVX+ZOL than in OVX groups. BV/TV%, TbTh and CD showed a clear tendency to being higher in OVX+ZOL than in OVX groups. No statistical difference was observed between OVX+ZOL and SHAM ewes. CBCT in a nondestructive, fast, very precise procedure for measuring bone morphometric indices without biopsies, which are not indicated for morphometric evaluation in osteoporosis. The current study demonstrated the potential of the high-resolution CBCT imaging to assess in vivo quantitative bone morphometry and bone quality of lower jaw cancellous bone under normal conditions and to differentiate changes associated with excessive bone loss induced by estrogen withdrawal and antiresorptive intervention.

Quantitative brain morphometry identifies cerebellar, cortical, and subcortical gray and white matter atrophy in late-onset Tay-Sachs disease.

Cerebellar atrophy is a characteristic sign of late-onset Tay-Sachs disease (LOTS). Other structural neuroimaging abnormalities are inconsistently reported. Our study aimed to perform a detailed whole-brain analysis and quantitatively characterize morphometric changes in LOTS patients. Fourteen patients (8 M/6F) with LOTS from three centers were included in this retrospective study. For morphometric brain analyses, we used deformation-based morphometry, voxel-based morphometry, surface-based morphometry, and spatially unbiased cerebellar atlas template. The quantitative whole-brain morphometric analysis confirmed the finding of profound pontocerebellar atrophy with most affected cerebellar lobules V and VI in LOTS patients. Additionally, the atrophy of structures mainly involved in motor control, including bilateral ventral and lateral thalamic nuclei, primary motor and sensory cortex, supplementary motor area, and white matter regions containing corticospinal tract, was present. The atrophy of the right amygdala, hippocampus, and regions of occipital, parietal and temporal white matter was also observed in LOTS patients in contrast with controls (p < 0.05, FWE corrected). Patients with dysarthria and those initially presenting with ataxia had more severe cerebellar atrophy. Our results show predominant impairment of cerebellar regions responsible for speech and hand motor function in LOTS patients. Widespread morphological changes of motor cortical and subcortical regions and tracts in white matter indicate abnormalities in central motor circuits likely coresponsible for impaired speech and motor function.

Quantitative Analysis of NKX2-3 Expression in Human Colon: An Immunohistochemical Study.

In mice, Nkx2-3 homeodomain transcription factor defines the vascular specification of secondary and tertiary lymphoid tissues of the intestines. In human studies, polymorphisms in NKX2-3 have been identified as a susceptibility factor in inflammatory bowel diseases, whereas in mice, its absence is associated with protection against experimental colitis and enhanced intestinal epithelial proliferation. Here, we investigated the expression of NKX2-3 in normal, polyp, and adenocarcinoma human colon samples using immunohistochemistry and quantitative morphometry, correlating its expression with endothelial and mesenchymal stromal markers. Our results revealed that the expression of NKX2-3 is regionally confined to the lamina propria and lamina muscularis mucosae, and its production is restricted mostly to endothelial cells and smooth muscle cells with variable co-expression of CD34, alpha smooth muscle antigen (αSMA), and vascular adhesion protein-1 (VAP-1). The frequency of NKX2-3-positive cells and intensity of expression correlated inversely with aging. Furthermore, in most colorectal carcinoma samples, we observed a significant reduction of NKX2-3 expression. These findings indicate that the NKX2-3 transcription factor is produced by both endothelial and non-endothelial tissue constituents in the colon, and its expression changes during aging and in colorectal malignancies. (J Histochem Cytochem XX: XXX-XXX, XXXX).

Placental perfusion imaging on 3Tesla magnetic resonance imaging using pseudo-continuous arterial spin labelling: an initial experience

BackgroundDuring pregnancy, the placenta plays an important role in the development of the fetus by supplying it with oxygen and nutrition, eliminating waste, and acting as an immunological barrier. Pseudo-continuous arterial spin labelling (pCASL) MRI was recently shown to be a promising sequence for measuring perfusion in the placenta. The aim was to obtain the perfusion measurements of the placenta in normal pregnancies using 3 Tesla MRI from 19 to 38 weeks of gestational age.ResultsThis was a cohort observational study. One hundred and sixty (160) singleton pregnancies from 19 to 38 weeks of gestational age were included. A 3D pCASL sequence was performed in the axial plane for placental blood flow (PBF). Data post-processing was performed on a workstation using Ready View software for 3D ASL with automated generation of quantitative placental pattern-based morphometry (PBM). The mean values of placental perfusion were extracted by averaging the data obtained. A significant positive correlation was observed between the PBF and increasing GA using Karl Pearson’s coefficient of correlation values (r-value 0.77, p-value < 0.001), respectively. Average PBF values from 19 to 38 weeks were (89.4 ± 13.5 to 155.3 ± 2.8 ml/100 g/min). According to our intra-class correlation coefficient (ICC), inter-observer reproducibility of placental blood flow shows good agreement between observers (0.98).ConclusionsNormal PBF values using ASL in 3 T MRI from 19 to 38 weeks of gestational age were provided. Statistical analysis revealed a significant positive correlation between gestational age and placental blood flow. In the future, it may help to identify placental perfusion abnormalities like placental insufficiency, preeclampsia, and fetal growth restriction (FGR).

Analysis of the morphology of retinal vascular cells in zebrafish (Danio rerio).

Background: Zebrafish (Danio rerio) have been established in recent years as a model organism to study Diabetic Retinopathy (DR). Loss of endothelial cells and pericytes is an early hallmark sign of developing DR in the mammalian retina. However, morphology, numbers, ratios, and distributions of different vascular cells in the retinal compartment in zebrafish have not yet been analyzed and compared with the mammalian retina. Methods: The retinal trypsin digest protocol was established on the zebrafish retina. Cell types were identified using the Tg(nflk:EGFP)-reporter line. Cells were quantified using quantitative morphometry. Results: Vascular cells in the zebrafish retina have distinct morphologies and locations. Nuclei of vascular mural cells appear as long and flat nuclei located near the vessel wall. Round nuclei within the vessel walls can be identified as endothelial cells. The vessel diameter decreases from central to peripheral parts of the retina. Additionally, the numbers of vascular cells decrease from central to peripheral parts of the retina. Discussion: The retinal trypsin digest protocol, which can be applied to the zebrafish retina, provides novel insights into the zebrafish retinal vascular architecture. Quantification of the different cell types shows that, in comparison to the mammalian retina, zebrafish have higher numbers of mural cells and an increased mural cell to endothelial cell ratio. This protocol enables to quantify mural cell and endothelial cell numbers, is easily adaptable to different transgenic and mutant zebrafish lines and will enable investigators to compare novel models on a single cell level.

Particle profiling of EV-lipoprotein mixtures by AFM nanomechanical imaging.

The widely overlapping physicochemical properties of lipoproteins (LPs) and extracellular vesicles (EVs) represents one of the main obstacles for the isolation and characterization of these pervasive biogenic lipid nanoparticles. We herein present the application of an atomic force microscopy (AFM)-based quantitative morphometry assay to the rapid nanomechanical screening of mixed LPs and EVs samples. The method can determine the diameter and the mechanical stiffness of hundreds of individual nanometric objects within few hours. The obtained diameters are in quantitative accord with those measured via cryo-electron microscopy (cryo-EM); the assignment of specific nanomechanical readout to each object enables the simultaneous discrimination of co-isolated EVs and LPs even if they have overlapping size distributions. EVs and all classes of LPs are shown to be characterised by specific combinations of diameter and stiffness, thus making it possible to estimate their relative abundance in EV/LP mixed samples in terms of stoichiometric ratio, surface area and volume. As a side finding, we show how the mechanical behaviour of specific LP classes is correlated to distinctive structural features revealed by cryo-EM. The described approach is label-free, single-step and relatively quick to perform. Importantly, it can be used to analyse samples which prove very challenging to assess with several established techniques due to ensemble-averaging, low sensibility to small particles, or both, thus providing a very useful tool for quickly assessing the purity of EV/LP isolates including plasma- and serum-derived preparations.