Assessment of neurodegeneration and neuronal loss in aged 5XFAD mice.

Neuronal loss resulting from progressive neurodegeneration is a major pathological feature of Alzheimer's disease (AD). Here, we present a protocol to detect neurodegeneration, neuronal apoptosis, and neuronal loss in 5XFAD mouse strain, which is a well-established model for interrogating the molecular mechanism of neuronal death in AD. This protocol describes the use of the neurodegenerative marker Fluro-Jade C, cleaved caspase-3 immunofluorescent staining and Nissl staining for the analysis of neurodegeneration and neuronal loss in 5XFAD mice. For complete details on the use and execution of this protocol, please refer to Zhang et al. (2021).

Do pathologists agree with each other on the histological assessment of pT1b oesophageal adenocarcinoma?

Background: In early (T1) oesophageal adenocarcinoma (OAC), the histological profile of an endoscopic resection specimen plays a pivotal role in the prediction of lymph node metastasis and the potential need for oesophagectomy with lymphadenectomy. Objective: To evaluate the inter-observer agreement of the histological assessment of submucosal (pT1b) OAC. Methods: Surgical and endoscopic resection specimens with pT1b OAC were independently reviewed by three gastrointestinal pathologists. Agreement was determined by intraclass correlation coefficient for continuous variables, and Fleiss' kappa (κ) for categorical variables. Bland-Altman plots of the submucosal invasion depth were made. Results: Eighty-five resection specimens with pT1b OAC were evaluated. The agreement was good for differentiation grade (κ=0.77, 95% confidence interval (CI) 0.68-0.87), excellent for lymphovascular invasion (κ=0.88, 95% CI 0.76-1.00) and moderate for submucosal invasion depth using the Paris and Pragmatic classifications (κ=0.60, 95% CI 0.49-0.72 and κ=0.42, 95% CI 0.33-0.51, respectively). Systematic mean differences between pathologists were detected for the measurement of submucosal invasion depth, ranging from 297 µm to 602 µm. Conclusions: A substantial discordance was found between pathologists for the measurement of submucosal invasion depth in pT1b OAC. Differences may lead to an over- or underestimation of the lymph node metastasis risk, with grave implications for the treatment strategy. Review by a second gastrointestinal pathologist is recommended to improve differentiating between a favourable and an unfavourable histological profile.

In vivo assessment of increased oxidation of branched-chain amino acids in glioblastoma.

Altered branched-chain amino acids (BCAAs) metabolism is a distinctive feature of various cancers and plays an important role in sustaining tumor proliferation and aggressiveness. Despite the therapeutic and diagnostic potentials, the role of BCAA metabolism in cancer and the activities of associated enzymes remain unclear. Due to its pivotal role in BCAA metabolism and rapid cellular transport, hyperpolarized 13C-labeled α-ketoisocaproate (KIC), the α-keto acid corresponding to leucine, can assess both BCAA aminotransferase (BCAT) and branched-chain α-keto acid dehydrogenase complex (BCKDC) activities via production of [1-13C]leucine or 13CO2 (and thus H13CO3-), respectively. Here, we investigated BCAA metabolism of F98 rat glioma model in vivo using hyperpolarized 13C-KIC. In tumor regions, we observed a decrease in 13C-leucine production from injected hyperpolarized 13C-KIC via BCAT compared to the contralateral normal-appearing brain, and an increase in H13CO3-, a catabolic product of KIC through the mitochondrial BCKDC. A parallel ex vivo 13C NMR isotopomer analysis following steady-state infusion of [U-13C]leucine to glioma-bearing rats verified the increased oxidation of leucine in glioma tissue. Both the in vivo hyperpolarized KIC imaging and the leucine infusion study indicate that KIC catabolism is upregulated through BCAT/BCKDC and further oxidized via the citric acid cycle in F98 glioma.

Quantitative and histological assessment of maternal-fetal transmission of Trypanosoma cruzi in guinea pigs: An experimental model of congenital Chagas disease.

OBJECTIVE: We evaluated the effect of Trypanosoma cruzi infection on fertility, gestation outcome, and maternal-fetal transmission in guinea pigs (Cavia porcellus). METHODS: Animals were infected with T. cruzi H4 strain (TcI lineage) before gestation (IBG) or during gestation (IDG). Tissue and sera samples of dams and fetuses were obtained near parturition. RESULTS: All IBG and IDG dams were seropositive by two tests, and exhibited blood parasite load of 1.62±2.2 and 50.1±62 parasites/µl, respectively, by quantitative PCR. Histological evaluation showed muscle fiber degeneration and cellular necrosis in all infected dams. Parasite nests were not detected in infected dams by histology. However, qPCR analysis detected parasites-eq/g heart tissue of 153±104.7 and 169.3±129.4 in IBG and IDG dams, respectively. All fetuses of infected dams were positive for anti-parasite IgG antibodies and tissue parasites by qPCR, but presented a low level of tissue inflammatory infiltrate. Fetuses of IDG (vs. IBG) dams exhibited higher degree of muscle fiber degeneration and cellular necrosis in the heart and skeletal tissues. The placental tissue exhibited no inflammatory lesions and amastigote nests, yet parasites-eq/g of 381.2±34.3 and 79.2±84.9 were detected in IDG and IBG placentas, respectively. Fetal development was compromised, and evidenced by a decline in weight, crow-rump length, and abdominal width in both groups. CONCLUSIONS: T. cruzi TcI has a high capacity of congenital transmission even when it was inoculated at a very low dose before or during gestation. Tissue lesions, parasite load, and fetal under development provide evidence for high virulence of the parasite during pregnancy. Despite finding of high parasite burden by qPCR, placentas were protected from cellular damage. Our studies offer an experimental model to study the efficacy of vaccines and drugs against congenital transmission of T. cruzi. These results also call for T. cruzi screening in pregnant women and adequate follow up of the newborns in endemic areas.

Improved performance of quantitative collagen parameters versus standard histology in longitudinal assessment of nonadvanced liver fibrosis for chronic hepatitis B.

Monitoring longitudinal nonadvanced fibrosis is a more common scenario in management of chronic hepatitis B (CHB), for which, however, current evaluation methods generally lack sufficient performance. We conducted a proof-of-concept study to evaluate the performance of quantitative fibrous collagen parameters (q-FP) in the assessment. Data sets from a prior CHB trial (NCT00962533) with mostly mild-to-moderate fibrosis participants were used for this study. 301 subjects with paired liver biopsies were consecutively included. Of these, 139 subjects were used to establish the test and the rest for internal validation. Fibrosis change between baseline and week 104 of treatment was blindly assessed with q-FP and was compared with Ishak fibrosis staging. There were 70% and 93% subjects with Ishak F0-2 at baseline and week 104, respectively. For the test of the subjects, q-FP and Ishak staging showed no difference in determining the incidence of fibrosis regression (68% vs 67%; difference = 0.7%, P = 1.00). Q-FP demonstrated that the regression was independently associated with the antiviral efficacy endpoint (OR 3.0, 95% CI 1.4-6.5, P = .005), but Ishak failed the detection (OR 0.6, 95% CI 0.3-1.3, P = .24). Moreover, q-FP directly revealed a higher fibrosis-resistance to antiviral treatment in virus genotypes C vs B and in males vs females. These results were confirmed in the validation subjects. Additionally, a functional model built on the test subjects showed an accuracy of 82% in stratifying fibrosis reversibility of the validation subjects. In conclusion, q-FP could have improved efficiency and accuracy in the longitudinal assessment of mild-to-moderate CHB fibrosis, indicating a potential alternative to current evaluation methodologies.

Elastogram quality assessment score in vibration-controlled transient elastography: Diagnostic performance compared to digital morphometric analysis of liver biopsy in chronic hepatitis C.

Vibration-controlled transient elastography (VCTE) is widely used for noninvasive fibrosis staging in chronic hepatitis C. However, internal validation is based solely on variability and success rate and lacks reproducible quality indicators. We analysed the graphic representation of shear wave propagation in comparison with morphometric results of liver biopsy, eliminating observer variability bias. Individual elastograms were classified according to two morphologic criteria: extension of wave propagation (length of the graphic representation) and shear wave dispersal (level of parallelism displayed in the elastogram). Then, a score based on these criteria stratified the elastogram in classes I through III (highest to lowest technical quality). Liver stiffness results of each measurement were compared with collagen contents in liver biopsy by morphometric analysis. A total of 3243 elastograms were studied (316 patients). Digital morphometry in liver biopsy showed significant fibrosis in 66% of samples and advanced fibrosis in 31%. Elastogram quality analysis resulted in 1438 class I measurements (44%), 1070 class II (34%) and 735 class III. Area under the receiver operating curve (AUROC) for severe fibrosis according to class (I, II and III) was 0.941, 0.887 and 0.766, respectively. For advanced fibrosis, AUROCs were 0.977, 0.883 and 0.781, respectively. Spearman's correlation testing for all classes and levels of fibrosis demonstrated significant independent association (r2  = -.95, P < .01). Our study is the first to propose measurable quality criteria for VTCE and to validate them against objective assessment of liver biopsy through digital morphometric imaging analysis. We concluded that VCTE performance is significantly influenced by quality assessment of individual measurements. Considering these criteria in clinical practice may improve accuracy.

Ivermectin treatment of Loa loa hyper-microfilaraemic baboons (Papio anubis): Assessment of microfilarial load reduction, haematological and biochemical parameters and histopathological changes following treatment.

BACKGROUND: Individuals with high intensity of Loa loa are at risk of developing serious adverse events (SAEs) post treatment with ivermectin. These SAEs have remained unclear and a programmatic impediment to the advancement of community directed treatment with ivermectin. The pathogenesis of these SAEs following ivermectin has never been investigated experimentally. The Loa/baboon (Papio anubis) model can be used to investigate the pathogenesis of Loa-associated encephalopathy following ivermectin treatment in humans. METHODS: 12 baboons with microfilarial loads > 8,000mf/mL of blood were randomised into four groups: Group 1 (control group receiving no drug), Group 2 receiving ivermectin (IVM) alone, Group 3 receiving ivermectin plus aspirin (IVM + ASA), and Group 4 receiving ivermectin plus prednisone (IVM + PSE). Blood samples collected before treatment and at Day 5, 7 or 10 post treatment, were analysed for parasitological, hematological and biochemical parameters using standard techniques. Clinical monitoring of animals for side effects took place every 6 hours post treatment until autopsy. At autopsy free fluids and a large number of standard organs were collected, examined and tissues fixed in 10% buffered formalin and processed for standard haematoxylin-eosin staining and specific immunocytochemical staining. RESULTS: Mf counts dropped significantly (p<0.05) in all animals following ivermectin treatment with reductions as high as (89.9%) recorded; while no significant drop was observed in the control animals. Apart from haemoglobin (Hb) levels which recorded a significant (p = 0.028) drop post treatment, all other haematological and biochemical parameters did not show any significant changes (p>0.05). All animals became withdrawn 48 hours after IVM administration. All treated animals recorded clinical manifestations including rashes, itching, diarrhoea, conjunctival haemorrhages, lymph node enlargement, pinkish ears, swollen face and restlessness; one animal died 5 hours after IVM administration. Macroscopic changes in post-mortem tissues observed comprised haemorrhages in the brain, lungs, heart, which seen in all groups given ivermectin but not in the untreated animals. Microscopically, the major cellular changes seen, which were present in all the ivermectin treated animals included microfilariae in varying degrees of degeneration in small vessels. These were frequently associated with fibrin deposition, endothelial changes including damage to the integrity of the blood vessel and the presence of extravascular erythrocytes (haemorrhages). There was an increased presence of eosinophils and other chronic inflammatory types in certain tissues and organs, often in large numbers and associated with microfilarial destruction. Highly vascularized organs like the brain, heart, lungs and kidneys were observed to have more microfilariae in tissue sections. The number of mf seen in the brain and kidneys of animals administered IVM alone tripled that of control animals. Co-administration of IVM + PSE caused a greater increase in mf in the brain and kidneys while the reverse was noticed with the co-administration of IVM + ASA. CONCLUSIONS: The treatment of Loa hyper-microfilaraemic individuals with ivermectin produces a clinical spectrum that parallels that seen in Loa hyper-microfilaraemic humans treated with ivermectin. The utilization of this experimental model can contribute to the improved management of the adverse responses in humans.

Assessment of antigen presenting cell infiltration in lung tissues of patients with bronchiectasis.

BACKGROUND: Bronchiectasis is a chronic disease characterized by permanent dilatation of the conducting airways accompanied by sustained inflammation. AIMS: To assess whether chronic inflammation of lungs in bronchiectasis is associated with alterations in the numbers of infiltrating antigen presenting cell (APC). SETTING AND DESIGN: Lobectomy specimens from 12 nonsmoker, nonasthmatic patients with acquired (noncongenital) bronchiectasis and six control patients were included in the study. Histopathology slides were reviewed, and immunohistochemical markers for dendritic cells (DCs) macrophages and Langerhans cells have been applied and analyzed. MATERIALS AND METHODS: Tissue specimens were stained by immunohistochemistry using markers for DCs (CD83 and CD23), macrophages (CD68 and CD163), and Langerhans cells (CD1A and S-100 protein). The mean cell counts of stained cells in five high power microscopic fields were recorded. STATISTICAL ANALYSIS USED: Descriptive statistics, mean, standard deviation, median, and interquartile range were used. A nonparametric Mann-Whitney U-test was used to compare cell counts between bronchiectasis and control patients. P <0.05 was considered significant. RESULTS: The mean age of patients with bronchiectasis and controls was 36.7 ± 16.6 and 31.8 ± 22.6 years, respectively. The predominant cell type among the patients was macrophage (median 50.5) followed by DCs (median 44.85), histiocytes (median 32), and Langerhans cells (median 5%). Compared to the controls a significantly higher number of macrophages (P = 0.01), DCs (P = 0.001), and Langerhans cells (P = 0.014) were present. CONCLUSION: Chronic inflammatory response in acquired (noncongenital) bronchiectasis is most probably mediated by increased infiltration of APCs in lung tissues.

Histopathological assessment of the infection of maize leaves by Fusarium graminearum, F. proliferatum, and F. verticillioides.

Young maize plants were inoculated on unfolded mature leaves and on folded immature leaves with Fusarium graminearum, Fusarium proliferatum, and Fusarium verticillioides suspensions. Infection and symptom development of disease on these asymptomatic mature leaves and immature leaves were then documented. Subcuticular infection was found by the three Fusarium species on both symptomatic and symptomless leaves. The three Fusarium species penetrated the stomata of immature leaves by the formation of appressoria-like structures, infection cushions or by direct penetration. Infection by the three species of Fusarium via stomata is reported here for the first time. The superficial hyphae and re-emerging hyphae of the three species produced conidia. The macroconidia of F. graminearum produced secondary macroconidia and F. proliferatum formed microconidia inside the leaf tissues that sporulated through stomata and trichomes. The infection of maize leaves by the three species of Fusarium and their sporulation may contribute inoculum to cob and kernel infection.

New Magnetic Resonance Imaging Index for Renal Fibrosis Assessment: A Comparison between Diffusion-Weighted Imaging and T1 Mapping with Histological Validation.

A need exists to noninvasively assess renal interstitial fibrosis, a common process to all kidney diseases and predictive of renal prognosis. In this translational study, Magnetic Resonance Imaging (MRI) T1 mapping and a new segmented Diffusion-Weighted Imaging (DWI) technique, for Apparent Diffusion Coefficient (ADC), were first compared to renal fibrosis in two well-controlled animal models to assess detection limits. Validation against biopsy was then performed in 33 kidney allograft recipients (KARs). Predictive MRI indices, ΔT1 and ΔADC (defined as the cortico-medullary differences), were compared to histology. In rats, both T1 and ADC correlated well with fibrosis and inflammation showing a difference between normal and diseased kidneys. In KARs, MRI indices were not sensitive to interstitial inflammation. By contrast, ΔADC outperformed ΔT1 with a stronger negative correlation to fibrosis (R(2) = 0.64 against R(2) = 0.29 p < 0.001). ΔADC tends to negative values in KARs harboring cortical fibrosis of more than 40%. Using a discriminant analysis method, the ΔADC, as a marker to detect such level of fibrosis or higher, led to a specificity and sensitivity of 100% and 71%, respectively. This new index has potential for noninvasive assessment of fibrosis in the clinical setting.

Immunohisto- and Cytochemistry Analysis of Connexins.

Immunohistochemistry (IHC) is a ubiquitous used technique to identify and analyze protein expression in the context of tissue and cell morphology. In the connexin research field, IHC is applied to identify the subcellular location of connexin proteins, as this can be directly linked to their functionality. The present chapter describes a protocol for fluorescent IHC to detect connexin proteins in tissues slices and cells, with slight modifications depending on the nature of biological sample, histological processing, and/or protein expression level. Basically, fluorescent IHC is a short, simple, and cost-effective technique, which allows the visualization of proteins based on fluorescent-labeled antibody-antigen recognition.

Evaluation of cost effective diagnostic tools in characterisation of Acute Leukemia in Southern India.

The incidence of Acute Leukemia (AL) subtypes varies according to geographical distribution and more predominant in developing countries. The aim here was to evaluate the usefulness of cost effective diagnostic tools in characterization of Acute Lymphoblastic Leukemia (ALL) in resource poor population. One hundred and two AL cases were diagnosed. For diagnosis, cytochemical analysis and immunohistochemistry were performed. Among the children < 12 years, ALL was 64.3% while AML accounted for 30%. In patients > 12 years, ALL was 59.4% and AML was 31.3%. The B-ALL occurred most frequently than T-ALL in both the age groups while based on immunophenotyping in AML, CD13 was the most commonly expressed antigen. Hence, cost effective diagnostic tools namely the immunophenotyping and cytochemistry are useful and improve accuracy and rapidly risk-stratify patients that were diagnosed with acute leukemia.

In vivo imaging techniques: a new era for histochemical analysis.

In vivo imaging techniques can be integrated with classical histochemistry to create an actual histochemistry of water. In particular, Magnetic Resonance Imaging (MRI), an imaging technique primarily used as diagnostic tool in clinical/preclinical research, has excellent anatomical resolution, unlimited penetration depth and intrinsic soft tissue contrast. Thanks to the technological development, MRI is not only capable to provide morphological information but also and more interestingly functional, biophysical and molecular. In this paper we describe the main features of several advanced imaging techniques, such as MRI microscopy, Magnetic Resonance Spectroscopy, functional MRI, Diffusion Tensor Imaging and MRI with contrast agent as a useful support to classical histochemistry.

Overview and assessment of the histochemical methods and reagents for the detection of ß-galactosidase activity in transgenic animals.

Bacterial ß-galactosidase is one of the most widely used reporter genes in experiments involving transgenic and knockout animals. In this review we discuss the current histochemical methods and available reagents to detect ß-galactosidase activity. Different substrates are available, but the most commonly used is X-gal in combination with potassium ferri- and ferro-cyanide. The reaction produces a characteristic blue precipitate in the cells expressing ß-galactosidase, and despite its efficiency in staining whole embryos, its detection on thin tissue sections is difficult. Salmon-gal is another substrate, which in combination with ferric and ferrous ions gives a reddish-pink precipitate. Its sensitivity for staining tissue sections is similar to that of X-gal. Combining X-gal or Salmon-gal with tetrazolium salts provides a faster and more sensitive reaction than traditional ß-galactosidase histochemistry. Here, we compare the traditional ß-galactosidase assay and the combination of X-gal or Salmon-gal with three tetrazolium salts: nitroblue tetrazolium, tetranitroblue tetrazolium and iodonitrotetrazolium. Based on an assessment of the sensitivity and specificity of the different combinations of substrates, we are proposing an optimized and enhanced method for ß-galactosidase detection in histological sections of the transgenic mouse brain. Optimal staining was obtained with X-gal in combination with nitroblue tetrazolium, which provides a faster and more specific staining than the traditional X-gal combination with potassium ferri- and ferro-cyanide. We recommend the X-gal/nitroblue tetrazolium staining mixture as the first choice for the detection of ß-galactosidase activity on histological sections. When faster results are needed, Salmon-gal/nitroblue tetrazolium should be considered as an alternative, while maintaining acceptable levels of noise.

Wound Healing of the Laterally Positioned Flap: A Histomorphometric Assessment.

The aim of this study was to histomorphometrically assess the soft tissue anatomy in single gingival recessions (GR) treated with a laterally positioned flap (LPF). Five patients presenting maxillary first molars with GR to the apex of the buccal surface of the mesial-buccal root were invited to take part. The LPF-treated roots were removed en bloc (the root and the soft tissue covering the treated GR) 3 to 4 months postoperatively. Photomicrographs of Mallory trichrome stain sections were taken to allow reassessment of the specimens regarding the longitudinal dimensions of the crevicular/sulcular and junctional epithelia. The use of LPF resulted in new attachment with formation of crevicular epithelium, long junctional epithelium, and some connective tissue, re-establishing the normal anatomical characteristics of the soft tissues covering the previously exposed root.

External quality assessment for acid fast bacilli smear microscopy in eastern part of Ethiopia.

BACKGROUND: External quality assessment (EQA) of sputum smear microscopy is essential and indispensable component of any tuberculosis program. This study assessed the EQA of acid fast bacilli (AFB) smear microscopy through onsite evaluation, blinded rechecking and panel test. A one year study was conducted on eight health institution laboratories from December 2011 to December 2012. Onsite evaluation, blinded rechecking and panel tests were used to collect data. Data were analyzed using SPSS version 16. Sensitivity, specificity, predictive values, and proportions of false readings were calculated. The level of agreement was measured using Kappa (κ) value. RESULTS: Problems observed during onsite evaluation include shortages of materials, disinfectant, and poor storage and working condition. A total of 578 slides were collected for blinded rechecking, of which 102 (17.6%) were reported as positive by peripheral laboratories. The panel test revealed an overall error of 17 (25.25%) of which 14 (17.5%) were minor errors [low false negative 6 (7.5%) and low false positive 8 (10%)], and 3 (3.75%) were major errors (high false positive). The sensitivity, specificity, positive predictive values (PPV) and negative predictive values (NPV) of the peripheral laboratories were 83.5, 97.8, 91.7, and 95.7, respectively. The false readings at the peripheral laboratories were 32 (5.5%). Agreement on reading the slides was observed on 546 (94.5%) slides (K = 0.84, SE = 0.054). CONCLUSIONS: Lack of reagents, supplies, favorable working environment and AFB related technical problems were identified in the peripheral laboratories. High false negative error was found to be the predominant major error. A continuous and strong EQA scheme should be implemented to avoid reporting errors and produce quality sputum results.

Assessment of Inflammation in an Acute on Chronic Model of Inflammatory Bowel Disease with Ultrasound Molecular Imaging.

BACKGROUND: Ultrasound (US) molecular imaging has shown promise in assessing inflammation in preclinical, murine models of inflammatory bowel disease. These models, however, initiated acute inflammation on previously normal colons, in contrast to patients where acute exacerbations are often in chronically inflamed regions. In this study, we explored the potential of dual P- and E-selectin targeted US imaging for assessing acute inflammation on a murine quiescent chronic inflammatory background. METHODS: Chronic colitis was induced using three cycles of 4% DSS in male FVB mice. Acute inflammation was initiated 2 weeks after the final DSS cycle through rectal administration of 1% TNBS. Mice at different stages of inflammation were imaged using a small animal ultrasound system following i.v. injection of microbubbles targeted to P- and E-selectin. In vivo imaging results were correlated with ex vivo immunofluorescence and histology. RESULTS: Induction of acute inflammation resulted in an increase in the targeted US signal from 5.5 ± 5.1 arbitrary units (a.u.) at day 0 to 61.0 ± 45.2 a.u. (P < 0.0001) at day 1, 36.3 ± 33.1 a.u. at day 3, returning to levels similar to control at day 5. Immunofluorescence showed significant increase in the percentage of P- and E-selectin positive vessels at day 1 (P-selectin: 21.0 ± 7.1% of vessels; P < 0.05; E-selectin: 16.4 ±3.7%; P < 0.05) compared to day 0 (P-selectin: 10.3 ± 5.7%; E-selectin: 7.3 ± 7.0%). CONCLUSIONS: Acute inflammation can be accurately measured in a clinically relevant murine model of chronic IBD using ultrasound molecular imaging with a dual P- and E- selectin-targeted contrast agent.

In Vivo MRI Assessment of Hepatic and Splenic Disease in a Murine Model of Schistosomiasis [corrected].

BACKGROUND: Schistosomiasis (or bilharzia), a major parasitic disease, affects more than 260 million people worldwide. In chronic cases of intestinal schistosomiasis caused by trematodes of the Schistosoma genus, hepatic fibrosis develops as a host immune response to the helminth eggs, followed by potentially lethal portal hypertension. In this study, we characterized hepatic and splenic features of a murine model of intestinal schistosomiasis using in vivo magnetic resonance imaging (MRI) and evaluated the transverse relaxation time T2 as a non-invasive imaging biomarker for monitoring hepatic fibrogenesis. METHODOLOGY/PRINCIPAL FINDINGS: CBA/J mice were imaged at 11.75 T two, six and ten weeks after percutaneous infection with Schistosoma mansoni. In vivo imaging studies were completed with histology at the last two time points. Anatomical MRI allowed detection of typical manifestations of the intestinal disease such as significant hepato- and splenomegaly, and dilation of the portal vein as early as six weeks, with further aggravation at 10 weeks after infection. Liver multifocal lesions observed by MRI in infected animals at 10 weeks post infection corresponded to granulomatous inflammation and intergranulomatous fibrosis with METAVIR scores up to A2F2. While most healthy hepatic tissue showed T2 values below 14 ms, these lesions were characterized by a T2 greater than 16 ms. The area fraction of increased T2 correlated (rS = 0.83) with the area fraction of Sirius Red stained collagen in histological sections. A continuous liver T2* decrease was also measured while brown pigments in macrophages were detected at histology. These findings suggest accumulation of hematin in infected livers. CONCLUSIONS/SIGNIFICANCE: Our multiparametric MRI approach confirms that this murine model replicates hepatic and splenic manifestations of human intestinal schistosomiasis. Quantitative T2 mapping proved sensitive to assess liver fibrogenesis non-invasively and may therefore constitute an objective imaging biomarker for treatment monitoring in diseases involving hepatic fibrosis.

Use of eriochrome cyanine R in routine histology and histopathology: is it time to say goodbye to hematoxylin?

Eriochrome cyanine R (ECR) is a synthetic anionic dye that forms complexes with cations such as iron. We found that an iron-ECR (Fe-ECR) mixture provided either nuclear or myelin staining depending on the differentiator used. Selective nuclear staining was obtained by differentiation in an aqueous HCl solution, pH 0.95, followed by a wash in slightly alkaline tap water; the pH difference facilitated control of differentiation. When used with an eosin B counterstain, results were nearly indistinguishable from standard hematoxylin and eosin (H & E) staining. Nuclear staining with Fe-ECR provides tinctorial features similar to regressive aluminum-hemateins as well as resistance to acidic solutions such as those of iron hemateins. Fe-ECR also stained selectively intestinal cells of the diffuse neuroendocrine system (DNES). In addition to its use as an H & E substitute, acid differentiated Fe-ECR produced acid-resistant and selective nuclear counterstaining in combination with Alcian blue, and in the Papanicolaou and van Gieson techniques. With alkali differentiation, Fe-ECR produced selective myelin staining, which was compatible with neutral red counterstaining. Myelin sheaths were stained aqua blue. Fe-ECR could be used for both cytological and histological samples, and was suitable for use in automated tissue stainers. ECR also is less expensive than hematoxylin. Hematoxylin still may be preferred as a nuclear counterstain for some immunostaining methods for which Fe-ECR mixtures probably are too acidic.

Magnetic resonance imaging assessment of effective ablated volume following high intensity focused ultrasound.

Under magnetic resonance (MR) guidance, high intensity focused ultrasound (HIFU) is capable of precise and accurate delivery of thermal dose to tissues. Given the excellent soft tissue imaging capabilities of MRI, but the lack of data on the correlation of MRI findings to histology following HIFU, we sought to examine tumor response to HIFU ablation to determine whether there was a correlation between histological findings and common MR imaging protocols in the assessment of the extent of thermal damage. Female FVB mice (n = 34), bearing bilateral neu deletion tumors, were unilaterally insonated under MR guidance, with the contralateral tumor as a control. Between one and five spots (focal size 0.5 × 0.5 × 2.5 mm3) were insonated per tumor with each spot receiving approximately 74.2 J of acoustic energy over a period of 7 seconds. Animals were then imaged on a 7T MR scanner with several protocols. T1 weighted images (with and without gadolinium contrast) were collected in addition to a series of T2 weighted and diffusion weighted images (for later reconstruction into T2 and apparent diffusion coefficient maps), immediately following ablation and at 6, 24, and 48 hours post treatment. Animals were sacrificed at each time point and both insonated/treated and contralateral tumors removed and stained for NADH-diaphorase, caspase 3, or with hematoxylin and eosin (H&E). We found the area of non-enhancement on contrast enhanced T1 weighted imaging immediately post ablation correlated with the region of tissue receiving a thermal dose CEM43 ≥ 240 min. Moreover, while both tumor T2 and apparent diffusion coefficient values changed from pre-ablation values, contrast enhanced T1 weighted images appeared to be more senstive to changes in tissue viability following HIFU ablation.