Acute Toxicity Assessment: Macroscopic and Ultrastructural Effects in Mice Treated with Oral Tetrodotoxin.

Tetrodotoxin (TTX) is an extremely toxic marine compound produced by different genera of bacteria that can reach humans through ingestion mainly of pufferfish but also of other contaminated fish species, marine gastropods or bivalves. TTX blocks voltage-gated sodium channels inhibiting neurotransmission, which in severe cases triggers cardiorespiratory failure. Although TTX has been responsible for many human intoxications limited toxicological data are available. The recent expansion of TTX from Asian to European waters and diversification of TTX-bearing organisms entail an emerging risk of food poisoning. This study is focused on the acute toxicity assessment of TTX administered to mice by oral gavage following macroscopic and microscopic studies. Necropsy revealed that TTX induced stomach swelling 2 h after administration, even though no ultrastructural alterations were further detected. However, transmission electron microscopy images showed an increase of lipid droplets in hepatocytes, swollen mitochondria in spleens, and alterations of rough endoplasmic reticulum in intestines as hallmarks of the cellular damage. These findings suggested that gastrointestinal effects should be considered when evaluating human TTX poisoning.

Assessment of adverse outcome of Excel Mera 71 in Anabas testudineus by histological and ultrastructural alterations.

Present study was designed to evaluate the adverse effect of glyphosate-based herbicide, Excel Mera 71 in Anabas testudineus on comparative basis under field and laboratory conditions. Field (750 g/acre) and laboratory (17.2 mg/L) experiments were performed for a period of 30 days. For field experiment special type of cages were prepared. Fish gill, liver, and kidney were analyzed for histology and ultrastructural responses. A significant increment in morphometric indices (DTC) was observed in gill, liver and kidney of A. testudineus under laboratory condition (p < 0.05) and responses showed the degree of pathogenicity in the order of liver > kidney > gills. However, under field study significant increase in DTC value was observed in gill and liver (p < 0.05). Among the scanning electron microscopic (SEM) observations necrosis and loss of microridges, and damage in stratified epithelial cells were prominent in gill, although higher prevalence of alterations was observed under laboratory study than field study. Additionally, transmission electron microscopic (TEM) observations also depicted higher prevalence of pathological lesions under laboratory study compared with field observation. Among the TEM observations damage in chloride and pavement cells, degenerative mitochondria and nucleus (in gill); severe vacuolation, necrosed nucleus and vesiculated network in case of liver and degenerated epithelial cells, cytoplasmic vacuolation, and damage in proximal convoluted tubules (PCT) in case of kidney were prominent. Therefore, these findings demonstrated that Excel Mera 71 induces significant damage in tissues of A. testudineus and these responses might be considered as biomarkers for monitoring herbicidal toxicity on fish in aquatic body.

The diagnostic value of [18F]-FDG-PET/CT in assessment of radiation renal injury in Tibet minipigs model.

BACKGROUND: Radiation-induced kidney damage can severely affect renal function, and have a serious impact on glucose reabsorption. Fluoro-2-deoxyglucose positron emission tomography (FDG-PET) is routinely utilized for metabolic imaging of glucose utilization. In this study, we are trying to assess the diagnostic value of 18F-FDG-PET/CT on measuring hyperacute effect of total body irradiation (TBI) on the kidneys. METHODS: Forty-eight Tibet minipigs were treated by TBI of different dosages using an 8-MV X-ray linear accelerator. Whole-body 18F-FDG-PET/CT was performed at 6, 24 and 72 h followed by histologic examination, blood samples' and renal function analysis. RESULTS: The uptake of 18F-FDG was significantly different between 11/14 Gy dose groups and control group, the standard Uptake Values reached a maximal level at 72 h after 14-Gy TBI treatment. At doses over 8 Gy, histological observation showed formation of tube casts, degeneration, necrosis of tubular cells, inflammatory cell infiltration and dilatation of the mitochondria of tubule cells. Renal function analysis confirmed the changes in blood urea nitrogen and creatinine levels at various dosages and time intervals. Immunohistochemistry and western blot results indicate that the expression levels of IL-10 and TNF-α proteins were positively correlated with radiation dose up to 8 Gy. CONCLUSIONS: 18F-FDG PET/CT can reflect pathological changes in kidneys and it may be a useful tool for rapid and non-invasive assessment in cases of suspected radiation-induced kidney damage.

Rice bran protein hydrolysates attenuate diabetic nephropathy in diabetic animal model.

INTRODUCTION: Diabetic nephropathy (DN) is an important microvascular complication of uncontrolled diabetes. The features of DN include albuminuria, extracellular matrix alterations, and progressive renal insufficiency. Rice bran protein hydrolysates (RBPs) have been reported to have antihyperglycemic, lipid-lowering, and anti-inflammatory effects in diabetic rats. Our study was to investigate the renoprotective effects of RBP in diabetic animals and mesangial cultured cells. METHODS: Eight-week-old male db/m and db/db mice were orally treated with tap water or RBP (100 or 500 mg/kg/day) for 8 weeks. At the end of the experiment, diabetic nephropathy in kidney tissues was investigated for histological, ultrastructural, and clinical chemistry changes, and biomarkers of angiogenesis, fibrosis, inflammation, and antioxidant in kidney were analyzed by Western blotting. Protection against proangiogenic proteins and induction of cytoprotection by RBP in cultured mesangial cells was evaluated. RESULTS: RBP treatment improved insulin sensitivity, decreased elevated fasting serum glucose levels, and improved serum lipid levels and urinary albumin/creatinine ratios in diabetic mice. RBP ameliorated the decreases in podocyte slit pore numbers, thickening of glomerular basement membranes, and mesangial matrix expansion and suppressed elevation of MCP-1, ICAM-1, HIF-1α, VEGF, TGF-ß, p-Smad2/3, and type IV collagen expression. Moreover, RBP restored suppressed antioxidant Nrf2 and HO-1 expression. In cultured mesangial cells, RBP inhibited high glucose-induced angiogenic protein expression and induced the expression of Nrf2 and HO-1. CONCLUSION: RBP attenuates the progression of diabetic nephropathy and restored renal function by suppressing the expression of proangiogenic and profibrotic proteins, inhibiting proinflammatory mediators, and restoring the antioxidant and cytoprotective system.

The Application of Paraphenylenediamine Staining for Assessment of Phospholipidosis.

Drug-induced phospholipidosis is characterized by intracellular accumulation of phospholipids with lamellar bodies in cells exposed to xenobiotics. Demonstration of the lamellar bodies by transmission electron microscopy (TEM) is the hallmark for a definitive diagnosis of phospholipidosis. However, the preparation of tissue samples for TEM and their ultrastructural evaluation are technically challenging and time consuming. Paraphenylenediamine (PPD) is essentially a fat stain, and the staining mechanism is based upon the osmication of unsaturated lipids. Thus, the application of PPD staining to osmicated tissue samples is considered an optimal way to identify lipids. We evaluated the potential of PPD staining to localize phospholipid accumulations on osmium-fixed semi-thin tissue sections of the lung, kidney, and liver, which were affected with phospholipidosis, under a light microscope. PPD staining revealed the presence of PPD positive dark fine granular material in the cytoplasm for all affected tissues examined, which correlated ultrastructurally with lamellar bodies as well as a light microscopic finding of cytoplasmic vacuolation. The great advantage of PPD is that it can be incorporated into the protocol for standard TEM tissue preparation and significantly improve the efficiency of TEM work. In conclusion, PPD provides a simple, sensitive, and reliable method for visualizing phospholipid accumulation on light microscopy and represents an easy tool to study drug-induced phospholipidosis.

Morphological assessment of thin basement membrane disease.

Thin basement membrane disease is more common than IgA nephropathy or Alport syndrome, which are also associated with the presence of erythrocyturia. Very few reports on the disorder are available in the Polish literature. The objective of this work was to analyze the results from 83 patients with thin basement membrane syndrome as well as to formulate a proposal of strict morphological assessment criteria for the disorder. Attention was drawn to the requirement of thickness of the lamina densa rather than the entire basement membrane thickness and a sufficiently high number of loops featuring thinned lamina densa, namely at least 80% of loops, being taken into account. Occurrence of other morphological changes associated with the disorder and clinical symptoms other than erythrocyturia was also highlighted.

Langartech: A Custom-Made MALDI Matrix Sprayer for MALDI Imaging Mass Spectrometry.

Matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry (IMS) is a powerful tool for investigating the distribution of proteins and other molecules within biological systems through the in situ analysis of tissue sections, enabling molecular histology. MALDI IMS can determine the distribution of hundreds of unknown compounds in a single measurement while maintaining spatial and molecular integrity. The matrix spraying stage is a key factor in making this technique more sensitive and robust. In this article, we describe a custom-made matrix sprayer (Langartech), which is both inexpensive (estimated cost of about €3000, or $3500) and reliable compared with the alternatives present in the market today, with prices greater than €20,000 ($25,000). Several comparisons were made between our Langartech sprayer and one of the high-end matrix sprayers commercially available: ImagePrep (Bruker Daltonics). Focusing on lateral resolution and observed peak intensities, overall results show that our sprayer behaves in a very competitive fashion, especially when taking into account the huge difference in sophistication level and price.

MG132 ameliorates kidney lesions by inhibiting the degradation of Smad7 in streptozotocin-induced diabetic nephropathy.

BACKGROUND: Smad7 is the main negative regulatory protein in the transforming growth factor-ß (TGF-ß) downstream signaling pathway, which plays an important role in diabetic nephropathy (DN) and may be related to the ubiquitin proteasome pathway (UPP). AIM: We investigated the role of UPP in regulating TGF-ß/SMAD signaling and explored the therapeutic effect of the ubiquitin proteasome inhibitor MG132 on DN. METHODS: Wistar rats were randomly divided into a diabetes group and a normal control group. Rats in the diabetes group were injected intraperitoneally with streptozotocin. Diabetic rats were then randomly divided into a diabetic nephropathy group (DN group), an MG132 high concentration (MH) group, and an MG132 low concentration (ML) group. After 8 weeks of treatment, 24-hour urinary microalbumin (UAlb), urinary protein/urinary creatinine (Up/Ucr) values, ALT, AST, Bcr, kidney damage, TGF-ß, Smad7, fibronectin (FN), and Smurf2 were detected. RESULTS: The body mass and Smad7 protein expression decreased in DN group, but kidney weight, kidney weight index, UAlb, Up/Ucr, FN and Smurf2 mRNA expression, and TGF-ß protein expression increased. However, these changes diminished following treatment with MG132, and a more pronounced effect was evident in MH group compared to ML group. CONCLUSION: MG132 alleviates kidney damage by inhibiting Smad7 ubiquitin degradation and TGF-ß activation in DN.

Sonographic quantification of a hepato-renal index for the assessment of hepatic steatosis in comparison with 3T proton magnetic resonance spectroscopy.

CONTEXT: Nonalcoholic fatty liver disease is the most frequent hepatic disorder in the developed world. Currently, liver biopsy and proton magnetic resonance spectroscopy (H-MRS) are considered the gold standard methods for the quantification of liver fat deposits. OBJECTIVE: To determine whether a Sonographic Hepato-Renal Index (SHRI) calculated using a standard workstation, without a specifically designed software, is an adequate alternative to H-MRS for the quantification of fat liver content and diagnosis of steatosis in the general population. METHODS: A total of 121 volunteers (mean age=46 years, range=21-77 years) were recruited at three medical centers in Granada (Southern Spain) from among individuals attending routine general checkups. All participants were examined by ultrasound and by H-MRS 3T, which served as a reference for the diagnosis of steatosis. The SHRI was calculated as the ratio between the echogenicity of the liver and that of the right renal parenchyma. The validity of the methodology was assessed by receiver operating characteristic curves and correlation tests. RESULTS: The quantitative SHRI showed a strong correlation (Spearman's coefficient=0.89, P<0.001) with the H-MRS 3T. The optimal SHRI cutoff points of 1.21, 1.28, and 2.15 yielded 100% sensitivity for the diagnoses of steatosis greater than 5, 25, and 50%, respectively, with a specificity greater than 70%. CONCLUSION: This study shows that the SHRI is a valid, simple, reliable, and cost-effective screening tool for the identification, assessment, and quantification of hepatic steatosis in the general population.

[Ultrastructural assessment of the role of dysangiogenesis in congenital hydronephrosis].

OBJECTIVE: To confirm the key role of vascular malformation by ultrasound examination and to make a more detailed study of the manifestations of dysnephro- and angiogenesis. SUBJECTS AND METHODS: The study enrolled 34 children aged 3 days to 7 years with congenital hydronephrosis, who were divided into 3 groups in accordance with the degree of renal hemodynamic disorders, the criterion for which was a resistive index (RI). RESULTS: The performed electron microscopic study revealed the signs of malformed vessels of all diameters, as well as hypoplastic changes in the renal parenchyma in children of all ages in all the groups. The most significant ultrastructural signs demonstrating a close correlation between dysangio- and dysnephrogenetic processes are the uniformity of structural failure in the glomerular and arteriolar basement membrane, which shows up in the irregularity of its thickness and obliteration of its layers, as well as the immaturity of endothelial cells of both glomerular and arteriolar capillaries (large sizes and a round shape). The important factor confirming their relationship is a direct correlation between the increased RI in all branches of the renal artery as hypoplastic changes progress in the parenchyma of hydronephrotic kidneys. CONCLUSION: The investigation demonstrated the interdependence of dysangio- and dysnephrotic processes in children with congenital hydronephrosis.

Assessment of renal volume by three-dimensional ultrasonography in pregnant bitches: an experimental study using virtual organ computer-aided analysis.

BACKGROUND: To assess and to compare the renal volume evolution in bitches during pregnancy by two-dimensional (2D) ultrasonography using the ellipsoid technique (volume = length x width x depth x 0.523) and three-dimensional (3D) ultrasonography using the Virtual Organ Computer-aided AnaLysis (VOCAL) method. A longitudinal prospective study was performed with 17 normal Golden Retrievers bitches during pregnancy from heat to the last third of gestation. The ultrasound scans were performed by two veterinarians. The left and right kidneys were assessed in three moments (day 0 = non-pregnant bitches; days 1st to 20th of pregnancy and days 21st to 40th of pregnancy) by three techniques (ellipsoid; VOCAL 12° and VOCAL 30°). For reproducibility calculations, we used the intraclass correlation coefficient (ICC). RESULTS: The inferential result of the volumes in ANOVA revealed the interaction effect between side and moment (p = 0.009). The 3D techniques showed, in average, the same renal volumes (p = 0.137) regardless of the side and moment. Considering the right side, the renal volume in the day 0 was smaller than the day 21st to 40th (p = 0.029). Considering the left side, the renal volume at day 0 was smaller than the day 1st to 20th (p = 0.020) and day 21st to 40th (p = 0.007). It was found good intra observer reproducibility (ICC > 0.9) and none of the three techniques showed a good inter observer reproducibility (ICC < 0.7). CONCLUSION: The renal volume bitches by 3D ultrasonography using the VOCAL method (12° and 30°) had good correlation with the volume obtained by 2D ultrasonography method.

Dose-dependent in-vivo toxicity assessment of silver nanoparticle in Wistar rats.

This study aims to suggest the limits of silver nanoparticle (AgNP) uses for medicinal purpose and was performed to explore the effect of various doses of silver nanoparticle in rats. Four different doses of AgNP (4, 10, 20, and 40 mg/kg) were injected intravenously. For safety evaluation of injected AgNP, body weight, organ coefficient, whole blood count, and biochemistry panel assay for liver function enzyme (AST, ALT, ALP, and GGTP), comet assay, ROS, and histological parameter were performed; 10-12 week old animals were randomly divided into groups of six individuals each for control, and doses of 40, 20, 10, and 4 mg/kg AgNP injected. Significant changes were observed (p < 0.01) in hematological parameters (WBC count, platelets counts, haemoglobin, and RBC count) in the 40 and 20 mg/kg groups. The changes were non-significant in the other groups (4 and 10 mg/kg group). In the 40 mg/kg group, a significant increase was also found in liver function enzymes like ALT and AST (p < 0.01), ALP (p < 0.01), GGTP (p < 0.01), and bilirubin (p < 0.01). ROS in blood serum increased in the high dose group. Tail migration in single cell gel electrophoresis in the 40, 20, 10, 4 mg/kg, and control groups was 34.9, 29.5, 17.8, 5.8, and 0.0 µm, respectively, which indicated damage in the DNA strand in the high dose group. EDXRF showed a ∼ 10-times increase in silver concentration in the 40 mg/kg group and TEM image also showed particle deposition in the 40 mg/kg group. This study indicates that the AgNP in doses (< 10 mg/kg) is safe for biomedical application and has no side-effects, but its high dose (> 20 mg/kg) is toxic.

In vivo assessment of mitochondrial toxicity of metacavir in Rhesus monkeys after three months of intravenous administration.

AIM: To explore the potential mitochondrial toxicities and their severities of intravenously administered metacavir, a nucleoside analog, in rhesus monkeys. METHODS: Totally 21 rhesus monkeys were randomly divided into 4 groups: metacavir 120 mg/kg group, metacavir 40 mg/kg group, zidovudine(AZT) 50 mg/kg group, and blank control group. Animals were killed after the completion of dosing or further observed in a 4-week recovery phase. Changes of structure of mitochondria in liver, kidney, skeletal muscles, and cardiac muscles were observed under transmission electron microscope(TEM). Changes of the activities of mitochondrial respiratory chain complexes and mitochondrial DNA were also determined. RESULTS: In metacavir 120 mg/kg group, some mitochondrial injuries were found in skeletal muscle, cardiac muscle, and liver, including that some cristae was broken and became sparse in density in the skeletal muscle, the morphology and size of mitochondria remained unchanged. Metacavir decreased the activities of respiratory chain complexes I and II and the mtDNA contents in three tissues in a dose-dependent manner; however, the extent of such decrease was lower than that in AZT 50 mg/kg group. The mitochondrial injuries in metacavir 40 mg/kg group were mild in each tissue and no obvious change in mitochondrial function was noted. On week 4 in the recovery phase, results showed that all these injuries were reversible after drug withdrawal. CONCLUSION: These results suggest that metacavir has not a high risk for potential mitochondrial-related effects in rhesus monkeys.

An improved and cost-effective methodology for the reduction of autofluorescence in direct immunofluorescence studies on formalin-fixed paraffin-embedded tissues.

Interference by autofluorescence is one of the major shortcomes of immunofluorescence analysis by confocal laser scanning microscopy (CLSM). CLSM requires minimal tissue autofluorescence and reduced unspecific fluorescence background, requisites that become more critical when direct immunofluorescence studies are concerned. To control autofluorescence, different reagents and treatments can be used. Until now, the efficacy of the processes described depended on the tissue type and on the processing technique, no general recipe for the control of autofluorescence being available. Using paraffin sections of archival formalin-fixed murine liver, kidney and pancreas, we have found that previously described techniques were not able to reduce autofluorescence to levels that allowed direct immunofluorescence labelling. In this work, we aimed at improving currently described methodologies so that they would allow reduction of the autofluorescent background without affecting tissue integrity or direct immunofluorescence labelling. We have found that the combination of short-duration, high-intensity UV irradiation and Sudan Black B was the best approach to reduce autofluorescence in highly vascularised, high lipofuscins' content tissues, such as murine liver and kidney, and poorly vascularised, low lipofuscins' content tissues such as the pancreas. In addition, we herein show that this methodology is highly effective in reducing autofluorescent background to levels that allow detection of specific signals by direct immunofluorescence.

Ochratoxin A and citrinin nephrotoxicity in New Zealand White rabbits: an ultrastructural assessment.

In the present investigation, ochratoxin A (OTA) (0.75 mg/kg feed) and citrinin (CIT) (15 mg/kg feed) were fed alone and in combination to young growing New Zealand White rabbits for 60 days to evaluate renal ultrastructural alterations. The severity and intensity of renal ultrastructural changes varied with the type of the treatment, and predominant and consistent lesions were recorded in the proximal convoluted tubule (PCT) lining cells. The significant changes in mitochondria, the most affected cell organelle in all the treatment groups, included mitochondrial disintegration and distortion, pleomorphism, cluster formation and misshapen appearance such as signet ring, dumbbell, cup and U shapes. Intra-cisternal sequestrations of involuting mitochondria, and thickening of basal layer of PCT epithelial cells with partial detachment, were the characteristic features observed in OTA and combination treatments. CIT treatment revealed crenated nucleus, loss of nucleolus, depletion of cytoplasmic organelles, mitochondrial pleomorphism, nuclear fragmentation, uniform folding of cell membrane and cytoplasmic vacuolations in the PCTs. Focal thickening of the glomerular basement membrane and degeneration of endothelial cells were the prominent alterations in the glomeruli in OTA and combination treatments. Distal convoluted tubules were unaffected in CIT treatment, however, mild to moderate lesions were observed in OTA and combination treated rabbits. It may be concluded that on simultaneous exposure, CIT potentiated the toxic effects of OTA on renal ultrastructure.

North American opossum Didelphis virginiana as a fetal nephrotoxicity model: histologic and ultrastructural assessment of uranyl nitrate (UN)-induced damage.

We have used our opossum model of fetal nephrotoxicity to investigate uranyl nitrate-induced morphologic changes in the developing kidney. The present study establishes a renal dose response curve for the uranyl nitrate (UN). We find that pups treated with nonlethal doses of UN do not demonstrate growth retardation compared to saline-treated controls. The kidneys of UN-treated pups are heavier than the control animals, an effect less apparent the longer the pups are followed. A low dose of 60 mg/kg of UN administered to small pups causes slight histologic derangement but nevertheless more change than the same dose administered to larger more mature pups. Using a dose of 100 mg/kg of UN that effectively causes nonfatal renal disruption, we examined the kidneys from 4 to 42 days following injection. We find that tubular dilation and epithelial necrosis starts soon after treatment (day 4) and reaches its maximum during the second and third week (11 and 22 days). Architectural restoration appears complete by the end of the third week. By electron microscopy, UN induces sequential structural damage with loss of proximal tubule brush border, epithelial necrosis with intact basement membranes and regeneration at 4, 11, and 22 days. Residual tubular mitochondrial damage is present at 42 days in spite of histologically normal tubules. No apparent lesions are seen in glomeruli. Fibroblastic interstitial proliferation in UN-treated kidneys at 11 days is not followed by appreciable fibrosis when assessed at 22 and 42 days. As the structural changes caused by 100 mg/ml UN administration in fetal opossum kidneys are reversible, this is a useful model to study the molecular mediators responsible for this form of renal damage and repair.

Assessment of cell proliferation on porous microcarriers by means of image analysis.

Spherical porous microcarriers (PMCs) made from collagen-glycosaminoglycan crosslinked copolymers have exhibited considerable promise as growth surfaces for the proliferation of anchorage-dependent mammalian cell lines and have demonstrated the ability to entrap anchorage-independent cells. However, quantification of cell growth on PMCs has proved difficult. A method of measuring the proliferation of PMCs, based on image analysis, is presented. Using CV1 and CHO cell lines, samples of PMCs were removed from culture at various times, fixed, embedded and sectioned. The 2 microns sections were stained, photographed and digitized in three colors. A computer program was developed to evaluate digitized PMC cross-sections and to classify pixels as conforming to either background, cytoplasmic, matrix or nuclear parameters, based on a set of classification rules determined by statistical analysis. Growth curves were generated by relating the number of pixels occupied by cellular material to the total number of pixels in the PMC cross-section. The PMCs were found to foster cell proliferation, with cell densities approaching 100% occupancy.

Quantitative assessment of lysosomal size, number and enzyme activity in mouse kidney during maturational development.

Image and cytochemical analyses were undertaken to determine possible correlation between the number and size of acid phosphatase-positive granules (lysosomes), and variation in acid phosphatase (AcP) activity in the proximal tubule cells of mouse-kidney during growth and development. Eighteen ddY strain mice ages: 1 day, 1 and 2 weeks, and 1, 2 and 10 months were used. The lanthanide-based method for the ultrastructural localization of AcP-activity was employed. The number and size of AcP positive granules were quantitatively analyzed by image analysis, and AcP activity by X-ray microanalysis. Significance was evaluated by 2-tailed-Student's t-test for the difference between means. AcP activity was observed in the lysosomes and the reaction product appeared dense and heterogeneous. In some cells, it appeared apparently homogeneous. The results showed that the number and size of AcP Positive granules (lysosomes) increased significantly from the first day after birth, recorded a peak in one week time and thereafter, it gradually declined until the 10th month. The result of X-ray microanalysis demonstrated a variation in accordance with the degree of AcP activity at different ages of the animals studied. The AcP activity decreased significantly from day one and progressively until the 10th month. From the results of the present work, it could be inferred that the changes in size and number of AcP positive granules, at least, at the early stage, and/or the variation in AcP activity are related to the growth and development of the animal.

Nuclear shape analysis for assessment of prognosis in renal cell carcinoma.

Clinically localized renal cell carcinoma is cured by radical nephrectomy in 47% [stage T3a (II)] to 65% [stage T1, T2 (I)] of the patients. Local recurrence and metastatic disease probably result from undetectable microscopic metastases present at operation. Chemotherapy and immunotherapy may improve cure rates if administered adjuvantly. The outcome of individual patients who share surgical stage cannot be predicted reliably by tumor histology, pathological and/or nuclear deoxyribonucleic acid analysis. Two groups of 10 patients with clinically localized renal cell carcinoma were similar by sex distribution (5 men and 5 women), surgical stage (stages T1 in 1, T2 in 6 and T3a in 3 patients) and age (54.3 +/- 15.2 standard deviation versus 55.8 +/- 8.7 years). Group 1 had no recurrences with a minimum followup of 5 years and a mean followup of 10 years. Group 2 died of metastatic renal cell carcinoma after a mean of 5 years. All neoplastic areas of each paraffin-embedded operative specimen were randomly sampled and the nuclear perimeter of 150 cancerous cells was digitized. There were 25 shape descriptors calculated for each nucleus. All shape descriptors for each patient were described by 19 statistical tests. Nuclear perimeter and area as well as mean nuclear roundness factor failed to separate the 2 groups. Range median quartiles of ellipticities by Fourier analysis, coefficients of variation of chain code minimums and relative means of largest 10 convexity values produced greatest separation (Mann-Whitney-Wilcoxon test p less than 0.001, and variance normalized difference 3.21, 3.29 and 2.83, respectively). These descriptors normalized and summed provided near perfect separation (Mann-Whitney-Wilcoxon test p less than 0.001 and variance normalized difference 3.59). We developed a quantitative nuclear morphometric analysis system that permitted the correct assignment of outcome in 19 of 20 patients. Accurate prediction of prognosis in patients with clinically localized renal cell carcinoma by nuclear shape analysis may allow for selection of patients for adjuvant therapy who have clinically undetectable metastatic disease.

Effects of cooling rate and glycerol concentration on the structure of the frozen kidney: assessment by cryo-scanning electron microscopy.

An experimental technique, employing a directional solidification stage for controlled freezing of tissue samples and low-temperature scanning electron microscopy for observation of the structure of the frozen-hydrated samples, was used to study freezing processes in the kidney. Parametric studies in which the cooling rate during freezing and the concentration of glycerol in the tissue were varied confirmed the results of earlier freeze-substitution studies. The results suggest a mechanism for ice propagation in the kidney similar to that already proposed for the liver, in which ice originates in, and is subsequently propagated through, the peritubular vasculature. The ice front dehydrates the cells and tubular structures encountered in its path, thus preventing intraluminal freezing. At higher rates of cooling and increased concentrations of glycerol there is less dehydration of cortical structure and intraluminal freezing occurs.