Concentric Lamellar Bodies Research Articles

Myelin Ultrastructure Terminology in Disease and Recovery Processes.

Ultrastructural evaluation of myelin coat helps to understand the possible background of pathological changes leading to deterioration or complete loss of nerve functions. A number of terms were previously introduced to describe the fine structural changes in myelin under various conditions. We believe that using a common terminology will be helpful to interpret the structure/function relationship in neurological disorders empowering the diagnosis and possible therapeutical approaches. In this paper, we present examples of ultrastructural changes in myelin during myelination, demyelination, re-myelination and dysmyelination processes and we reviewed the terminology previously used.We tried to include all studies reporting ultrastructural findings with no limitation to the experimental conditions, the species used and the disorders. The terminology used to describe the structural findings included compacted myelin, partially compacted myelin, noncompacted myelin, redundancy (hypermyelination, tomacula, myelinosome), splitting, complete circular splitting, myelin degradation, concentric lamellar bodies (myelin figures), loss of myelin lamellae, polyaxonal Schwann cells and necrotic cell debris.Ultrastructural data described in this paper aimed to provide a guide for future studies. We concluded that the evaluation of ultrastructural changes in any neurological disorder is greatly valuable for a better understanding of pathological and physiological changes occured. We also believe that supporting the ultrastructural findings with quantitative methods in the future will be of great value.

Clinicopathological Features of Cardiac Glycolipid Storage Disease in an Adult Pug

A 12-year-old neutered male pug suffered cardiac arrest and died under general anaesthesia during diagnostic imaging for evaluation of exercise intolerance and respiratory crisis. Histopathological evaluation revealed two types of storage material, glycolipid and lipopigment, having differential distributions in multiple organs. The heart was most strikingly affected and other less affected tissues included the liver, brain, kidneys and skin. Cardiomyocytes were swollen with extensive sarcoplasmic vacuolation together with coalescing areas of myocardial fibrosis. Transmission electron microscopy revealed irregular myelin-like structures and complex concentric lamellar bodies dominating the sarcoplasm and displacing myofibrils. These findings were consistent with a lysosomal storage disease (LSD) as the cause of cardiac disease and death. The unique clinical presentation, histomorphology and ultrastructural features of the material suggested a glycolipid storage disease most closely resembling Anderson-Fabry (Fabry) disease in man. Fabry disease is a LSD that can present in later life and is characterized by loss of α-galactosidase A function and, often, accumulation of glycosphingolipids in tissues including the heart, kidneys, vascular endothelium and smooth muscle.

Molecular biomarkers of phospholipidosis in rat blood and heart after amiodarone treatment.

Phospholipidosis (PLD) is characterized by an intracellular accumulation of phospholipids in lysosomes and concurrent development of concentric lamellar bodies. It is induced in humans and in animals by drugs with a cationic amphiphilic structure. The purpose of the present study was to identify a set of molecular biomarkers of PLD in rat blood and heart, hypotheticallya pplicable in preclinical screens within the drug development process. A toxicological study was set up in rats orally treated up to 11 days with 300 mg kg(–1) per day(–1) amiodarone (AMD). Light and transmission electron microscopy investigations were performed to confirm the presence of lamellar bodies indicative of phospholipid accumulation. The effects of AMD upon the transcriptome of these tissues were estimated using DNA microarray technology. Microarray data analysis showed that a total of 545 and 8218 genes were modulated by AMD treatment in heart and blood, respectively. Some genes implicated in the phospholipid accumulation in cells, such as phospholipase A2, showed similar alterations of gene expression. After transcriptome criteria of analysis and target selection, including also the involvement in the onset of PLD, 7 genes (Pla2g2a, Pla2g7, Gal, Il1b, Cebpb, Fcgr2b, Acer 2) were selected as candidate biomarkers of PLD in heart and blood tissues, and their potential usefulness as a sensitive screening test was screened and confirmed by quantitative Real-Time PCR analysis. Collectively, these data underscore the importance of transcriptional profiling in drug discovery and development, and suggest blood as a surrogate tissue for possible phospholipid accumulation in cardiomyocytes.

Histological and electron micr oscopy findings in fabry disease cardiomyopathy: a case report

<h3>Aim</h3> To understand the features and role of histological and electron microscopy findings in a cardiac biopsy from patients with suspected Fabry disease. <h3>Methods</h3> Report of a recent case and review of current literature on the histological and ultrastructural findings in Fabry disease cardiomyopathy. <h3>Results</h3> Perinuclear vacuolisation of cardiac myocytes was seen on light microscopy and dense concentric lamellar bodies or my-elin figures were present on electron microscopy. <h3>Discussion</h3> The light microscopy findings are non-specific and can be seen in a range of metabolic and non-metabolic diseases. The diagnosis of Fabry disease must be complemented by electron microscopy, biochemical findings and ultimately genetic studies.

Structure Based Model for the Prediction of Phospholipidosis Induction Potential of Small Molecules

Drug-induced phospholipidosis (PLD), characterized by an intracellular accumulation of phospholipids and formation of concentric lamellar bodies, has raised concerns in the drug discovery community, due to its potential adverse effects. To evaluate the PLD induction potential, 4,161 nonredundant drug-like molecules from the National Institutes of Health Chemical Genomics Center (NCGC) Pharmaceutical Collection (NPC), the Library of Pharmacologically Active Compounds (LOPAC), and the Tocris Biosciences collection were screened in a quantitative high-throughput screening (qHTS) format. The potential of drug-lipid complex formation can be linked directly to the structures of drug molecules, and many PLD inducing drugs were found to share common structural features. Support vector machine (SVM) models were constructed by using customized atom types or Molecular Operating Environment (MOE) 2D descriptors as structural descriptors. Either the compounds from LOPAC or randomly selected from the entire data set were used as the training set. The impact of training data with biased structural features and the impact of molecule descriptors emphasizing whole-molecule properties or detailed functional groups at the atom level on model performance were analyzed and discussed. Rebalancing strategies were applied to improve the predictive power of the SVM models. Using the undersampling method, the consensus model using one-third of the compounds randomly selected from the data set as the training set achieved high accuracy of 0.90 in predicting the remaining two-thirds of the compounds constituting the test set, as measured by the area under the receiver operator characteristic curve (AUC-ROC).

Weka Machine Learning for Predicting the Phospholipidosis Inducing Potential

The drug discovery and development process is lengthy and expensive, and bringing a drug to market may take up to 18 years and may cost up to 2 billion $US. The extensive use of computer-assisted drug design techniques may considerably increase the chances of finding valuable drug candidates, thus decreasing the drug discovery time and costs. The most important computational approach is represented by structure-activity relationships that can discriminate between sets of chemicals that are active/inactive towards a certain biological receptor. An adverse effect of some cationic amphiphilic drugs is phospholipidosis that manifests as an intracellular accumulation of phospholipids and formation of concentric lamellar bodies. Here we present structure-activity relationships (SAR) computed with a wide variety of machine learning algorithms trained to identify drugs that have phospholipidosis inducing potential. All SAR models are developed with the machine learning software Weka, and include both classical algorithms, such as k-nearest neighbors and decision trees, as well as recently introduced methods, such as support vector machines and artificial immune systems. The best predictions are obtained with support vector machines, followed by perceptron artificial neural network, logistic regression, and k-nearest neighbors.

Progressive renal insufficiency associated with amiodarone-induced phospholipidosis

Progressive renal insufficiency associated with amiodarone-induced phospholipidosis

Determination of Phospholipidosis Potential Based on Gene Expression Analysis in HepG2 Cells

Phospholipidosis (PLD) is characterized by an intracellular accumulation of phospholipids in lysosomes and the concurrent development of concentric lamellar bodies. Recently, H. Sawada et al. (2005, Toxicol. Sci. 83, 282-292) identified 17 genes as potential biomarkers of PLD in HepG2 cells. The present study was undertaken to determine if this set of genes measured by quantitative PCR could be validated in the same cell line. The objective was also to investigate the dose-response relationship to further validate the assay and to select the concentrations to use for screening activities. In a first experiment (one concentration tested), out of the 17 genes, the best gene biomarkers of PLD (i.e., 11 genes) were selected for practical screening reasons. Based on these genes, 91.6% (i.e., 11 of 12) of the compounds known to induce PLD were identified as positive and all the negative compounds (i.e., five of five) were also confirmed. When the data obtained in the first experiment were compared to the data by Sawada et al., (2005) the coefficient of correlation calculated was slightly higher than 75%. In the second experiment (26 compounds [all 17 compounds from the first experiment plus 9 other compounds] tested at a minimum of three concentrations), 93.3% (14/15) of the compounds known to induce PLD were identified as such and all the negative controls (six compounds) were also confirmed. Three compounds likely to induce PLD were identified as positive in our assay. Finally, two compounds for which no data are available were also tested. When both experiments 1 and 2 were compared, the coefficient of correlation for 16 compounds tested at the same concentrations reached 87.7%. In conclusion, the present study further confirms the utility of gene expression in HepG2 cells to identify a potential to induce PLD. Finally, based on the data presented, researchers are encouraged to use a range of minimum three concentrations (e.g., 12.5, 25, and 50 microM) to screen for PLD in the human HepG2 cell line.

Histologic and electron microscopy findings in myocardium of treated Fabry disease

The well-described histologic and electron microscopic findings in Fabry disease cardiomyopathy are hypertrophic vacuolated cells with electron dense concentric lamellar bodies. We present altered findings in an endomyocardial biopsy from a patient with treated Fabry disease. A 51-year-old male with Fabry disease, treated with recombinant alpha-galactosidase enzyme replacement therapy for over 18 months, underwent an endomyocardial biopsy for heart failure. The histologic changes showed widespread hypertrophy and vacuolization with rare eosinophilic bodies. Electron microscopy failed to reveal the characteristic globotriaosylceramide concentric lamellar bodies (myelin figures) in the sarcoplasm. Instead, extensive aggregates and single tubular crystalline structures, giant secondary lysosomes as well as abnormal branched chain glycogen were present. This is the first histologic description of long-standing treated Fabry disease in cardiac myocytes.

Validation of an in vitro screen for phospholipidosis using a high-content biology platform

Several cationic amphiphilic drugs cause local or systemic phospholipidosis (PLD) after chronic exposure in preclinical species. PLD is characterized by the accumulation of drug, phospholipid, and concentric lamellar bodies in cellular lysosomes. We have developed a fluorescence-based in vitro screen that is predictive of PLD using the Cellomics ArrayScan high-content screening platform, which captures and analyzes images from 96-well cell culture microtiter plates using multichannel fluorescence microscopy. I-13.35 adherent mouse spleen macrophage cells were cultured with drug and a fluorescently tagged phospholipid, N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine (NBD-PE). Drug concentrations were used in a range from 1 to 100 micro mol/L. After 24 h incubations, the cells were fixed with formalin. NBD-PE uptake was quantified in controls and treated cells. Nuclei were identified by Hoechst 33258 staining and dead cells were identified using ethidium homodimer-2 incorporation. Thus, confounding accumulation of NBD-PE due to cytotoxicity that produces false-positive results at high concentrations was eliminated from quantitation by ethidium staining and employing cell gating (dead cell rejection). The assay was found to be both sensitive and selective in that 26 of 28 positive, phospholipidogenic controls and 8 of 8 negative, non-phospholipidogenic controls were correctly called.

Ultrastructure of tetrasporogenesis in the red alga Osmundea spectabilis var. spectabilis (Rhodomelaceae: Ceramiales: Rhodophyta)

Tetrasporogenesis in Osmundeaspectabilis (Postels and Ruprecht) K.W. Nam var. spectabilis proceeds through three developmental stages. A uninucleate tetraspore mother cell with synaptonemal complexes depicted during early prophase of meiosis I comprises the youngest stage. Subsequently, the nuclear envelope obtains a highly irregular profile and is surrounded by perinuclear endoplasmic reticulum (ER) and numerous mitochondria. Proplastids with peripheral thylakoid and numerous straight-profiled dictyosomes are present. Dictyosome vesicles discharge their contents, initiating tetraspore wall formation. A four-nucleate tetraspore mother cell is formed prior to tetrahedral cleavage. During the second stage of tetraspore formation the nuclei are very irregular in outline and move toward the central region where they fuse. The formation of fibrous vacuoles originates from the fibrous vacuole associated organelles (FVAOs). Thylakoid formation from concentric lamellar bodies (CLBs) seems to occur in plastids. Extremely hypertrophied dictyosomes with two tightly compressed and densely laminated mid-regions produce vesicles which contribute to enlargement of the fibrous vacuoles. During the second stage of tetraspore development striated vesicles are formed and starch granule formation increases. Mature tetraspores are characterized by numerous cored vesicles and abundant starch granules. Plastids have a fully developed system of internal thylakoids, and cleavage is usually complete at this stage. Mature tetraspores are surrounded by a bi-layered wall and the tetrasporangial wall. Unbranched, tubular structures connecting the plasmalemma with the peripheral sheets of ER are possibly involved in wall deformation. A distinct blebbing nuclear envelope appears to sequester vesicles into the cytoplasm.

Drug-induced phospholipidosis: are there functional consequences?

Phospholipidosis induced by drugs with a cationic amphiphilic structure is a generalized condition in humans and animals that is characterized by an intracellular accumulation of phospholipids and the concurrent development of concentric lamellar bodies. The primary mechanism responsible for the development of phospholipidosis is an inhibition of lysosomal phospholipase activity by the drugs. While the biochemical and ultrastructural features of the condition have been well characterized, much less effort has been directed toward understanding whether the condition has adverse effects on the organism. While there are a few cationic amphiphilic drugs that have been reported to cause phospholipidosis in humans, the principal concern with this condition is in the pharmaceutical industry during preclinical testing. While this class of drugs should technically be referred to as cationic lipophilic, the term cationic amphiphilic is widely used and recognized in this field, and for this reason, the terminology cationic amphiphilic drugs (CADs) will be employed in this Minireview. The aim of this Minireview is to provide an evaluation of the state of knowledge on the functional consequences of CAD-induced phospholipidosis.

Electron microscopy of oligodendroglia in severe mental illness

Qualitative electron microscopy was performed to verify whether brain pathology in schizophrenia and bipolar disorder is associated with alterations of oligodendroglial cells and myelinated fibers. Ultrastructural signs of apoptosis and necrosis of oligodendroglial cells were found in the prefrontal area 10 and the caudate nucleus in both schizophrenia and bipolar disorder. Damage of myelin sheath lamellae, with the formation of concentric lamellar bodies, were detected in both brain structures in schizophrenia. There was also a significant decrease in the area of the nucleus and the volume density of mitochondria in oligodendrogliocytes in the caudate nucleus and in the prefrontal cortex in schizophrenia, as compared to normal controls. Volume density of heterochromatin was significantly increased (+14%) in the caudate nucleus in schizophrenia. The density of concentric lamellar bodies (as an indicator of damage of myelinated fibers) was dramatically increased (4.5-fold) in the caudate nucleus in schizophrenia, as compared to controls, and was positively correlated with volume density of heterochromatin. Multiple regression analysis and analysis of covariance demonstrated that these changes could not be explained by the effects of postmortem delay, age, neuroleptic medication, or gender. Pathology of oligodendroglia might be an essential feature of severe mental disorders.

Pathological study of mice with total deficiency of sphingolipid activator proteins (SAP knockout mice).

Sphingolipid activator proteins (SAPs) A to D are lysosomal factors required in degradation of sphingolipids with short hydrophilic head groups and are derived from a precursor protein. Sap-B deficiency causes a variant of metachromatic leukodystrophy and sap-C deficiency causes a variant of Gaucher disease. Human total SAP deficiency has been reported in two patients in a single family. In these cases, various inclusions were described in the liver, skin, muscle and peripheral nerves ultrastructurally, but there was no report on the pathological study of the central nervous system (CNS). With targeted disruption of the precursor protein gene, we have generated mice with total SAP deficiency. These mice developed progressive neurological symptoms around day 20 and could not survive beyond day 40. Their cardinal pathology is extensive neurovisceral storage. Neuronal storage was already detected in the dorsal root ganglia as early as postnatal day 1 and diffuse neuronal storage was detected in the CNS after day 10. This storage was immunoreactive with anti-ubiquitin antibody and ultrastructurally appeared as inclusions consisting of numerous concentric lamellar and dense granular structures in the perikarya as well as in dendrites and axons. Axonal spheroids containing electron-dense concentric lamellar bodies and neurofilaments were also conspicuous. The extent of neuronal storage, numbers of storage neurons and axonal spheroids increased with age, accompanied with hypomyelination, astrogliosis and increase of macrophages. After day 30, argyrophilic tangle-like structures, which were immunoreactive with an antibody to phosphorylated neurofilaments, were found in the perikarya of many spinal and some neocortical neurons. Inclusions with various ultrastructural features were also noted in the glial cells, choroid plexus epithelial cells, vascular endothelial cells, Schwann cells, macrophages, fibroblasts, hepatocytes, and renal tubular epithelial cells. Some inclusions in the visceral organs were closely similar to those described in human cases of total SAP deficiency. The ultrastructural features of these inclusions in SAP knockout mice appeared unique and were different from those of other known sphingolipidoses.

Stimulation of Plastidogenesis Induced by 5‐Azacytidine in Euglena gracilis Klebs

Abstract:When etiolated Euglena gracilis was treated with 10 mM 5‐azacytidine (5‐azaC), an inhibitor of DNA methylation, stimulation of plastidogenesis in both dark and light conditions was observed. The phenomenon occurred in 10–15% of the cells possibly due to the asynchronicity of the cultures. The main features of this sub‐population, as evaluated by electron and fluorescence microscopy, were the following: 1. the presence in darkness of differentiating proplastids that were red fluorescent under UV, positive to TCNBT cytochemical reaction (specific for PSII) and negative to DAB (specific for PSI); 2. the acceleration of proplastid differentiation during the first 20–30 h of illumination; 3. the occurrence in both culture conditions of concentric lamellar bodies (LBS). These structures were considered to be proplastids blocked in the first step of evolution, since they emitted a red fluorescence, were contained within compartments limited by a triple‐layered envelope, were reactive to TCNBT in darkness and to both TCNBT and DAB in light conditions. Even if the action mechanism of 5‐azaC on plastidogenesis in Euglena remains to be defined, the induced stimulatory effect on plastid differentiation pointed to a relationship between DNA methylation and plastid development. Furthermore, the presence of LBS opens the possibility of studying early aspects of plastid development in Euglena.

A 26-week oral repeated dose toxicity study of (+/-)-4-diethylamino-1,1-dimethylbut-2-yn-1-yl 2-cyclohexyl-2-hydroxy-2-phenylacetate monohydrochloride monohydrate (NS-21), a novel drug for urinary frequency and incontinence, in rats followed by a 9-week recovery test

A 26-week oral repeated dose toxicity study of (+/-)-4-diethylamino-1, 1-dimethylbut-2-yn-1-yl 2-cyclohexyl-2-hydroxy-2-phenylacetate monohydrochloride monohydrate (NS-21), a new drug for the treatment of urinary frequency and incontinence, was conducted in Sprague-Dawley rats. Male and female rats were given the drug orally for 26 weeks at doses of 0 (control), 5, 50 and 500 mg/kg. After discontinuation of the treatment, a 9-week recovery test was also conducted. Two cases of death occurred in the 500 mg/kg group. Mydriasis, salivation and lacrimation were seen in the 50 and 500 mg/kg groups. Alopecia, a suppression of body weight gain and an increase in water consumption were seen in the 500 mg/kg group. Food consumption measurement showed no abnormalities attributable to the treatment. Ophthalmologic examination confirmed mydriasis in the 50 and 500 mg/kg groups. Urinalysis showed an increase in urine volume in the 50 and 500 mg/kg groups, and an increase in urinary protein and decreases in Na+, K+ and Cl- excretions in the 500 mg/kg group. Hematological examination showed decreases in hemoglobin, hematocrit, MCV, MCH, MCHC and lymphocytes in the 500 mg/kg group. Blood chemical examination showed increases in total cholesterol, phospholipid and total protein and decreases in glucose, triglyceride, free T3 and T4 in the 500 mg/kg group. Measurements of liver drug-metabolizing enzymes showed an increase in T4UDP-GT activity in the 50 and 500 mg/kg groups, and an increase in cytochrome P-450 in the 500 mg/kg group. Pathological examination disclosed hepatocellular hypertrophy caused by hyperplasia of smooth-ER in the 50 and 500 mg/kg groups, and a decrease in number of glycogen granules in the 500 mg/kg group. Stimulated thyroid follicles were seen in the 50 and 500 mg/kg groups. Increases in incidence and severity of chronic progressive nephropathy were also observed in the 500 mg/kg group. In this dose group, adrenocortical hypertrophy was also observed. The recovery test showed that the above-mentioned changes were satisfactorily reversible. The serum concentrations of NS-21 and its active metabolite, RCC-36, in the treated groups were increased in a dose-dependent manner. No treatment-related effects were seen in the 5 mg/kg group. These results show that the NOAEL (no observed adverse effect level) of NS-21 is 5 mg/kg for 26-week oral toxicity in rats.

A 13-week oral repeated dose toxicity study of (+/-)-4-diethylamino-1,1-dimethylbut-2-yn-1-yl 2-cyclohexyl-2-hydroxy-2-phenylacetate monohydrochloride monohydrate (NS-21), a novel drug for urinary frequency and incontinence, in dogs followed by a 5-week recovery test

A 13-week oral repeated dose toxicity study of (+/-)-4-diethylamino-1,1-dimethylbut-2-yn-1-yl 2-cyclohexyl-2-hydroxy-2-phenylacetate monohydrochloride monohydrate (NS-21), a new drug for the treatment of urinary frequency and incontinence, was conducted in beagle dogs. Male and female dogs were given the drug orally for 13 weeks at doses of 0 (control), 5, 25 and 125 mg/kg. After discontinuation of the treatment, a 5-week recovery test was also conducted. No effects related to the treatment were observed on survival. Mydriasis and a decrease in body weight or a suppression of its weight gain were seen in the 25 and 125 mg/kg groups. Vomiting, salivation and a decrease in food consumption were seen in the 125 mg/kg group. Ophthalmologic examination confirmed the mydriasis in the 125 mg/kg group. Electrocardiographic examination and urinalysis showed no abnormalities attributable to the treatment. Hematological examination showed an increase in number of platelets in the 125 mg/kg group. Blood chemical examination revealed increases in GPT and ALP and a decrease in albumin in the 25 and 125 mg/kg groups, and an increase in triglyceride in the 125 mg/kg group. Pathological examination disclosed hepatocellular hypertrophy in the 125 mg/kg group, hyperplasia of smooth-ER and concentric lamellar bodies derived from the smooth-ER, and bile pigments in the bile capillary, hepatocyte and stellate cells of Kupffer in the 25 and 125 mg/kg groups. Megakaryocytes in mesenteric lymph node were observed in the 25 and 125 mg/kg groups. The recovery test showed that the above-mentioned changes were satisfactorily reversible or the degree and frequency of these changes were lowered. No treatment-related effects were seen in the 5 mg/kg group. These results show that the NOAEL (no observed adverse effect level) of NS-21 is 5 mg/kg for 13-week oral toxicity in dogs.

A 12-month oral repeated dose toxicity study of (+/-)-4-diethylamino-1,1-dimethylbut-2-yn-1-yl 2-cyclohexyl-2-hydroxy-2-phenylacetate monohydrochloride monohydrate (NS-21), a novel drug for urinary frequency and incontinence, in dogs followed by a 2-month recovery test

A 12-month oral repeated dose toxicity study of (+/-)-4-diethylamino-1, 1-dimethylbut-2-yn-1-yl 2-cyclohexyl-2-hydroxy-2-phenylacetate monohydrochloride monohydrate (NS-21), a new drug for the treatment of urinary frequency and incontinence, was conducted in beagle dogs. Male and female dogs were given the drug orally for 12 months at doses of 0 (control), 3, 17.5 and 100 mg/kg. After discontinuation of the treatment, a 2-month recovery test was also conducted. No effects related to the treatment were observed on survival and water consumption. Mydriasis, vomiting and a decrease in body weight or a suppression of its weight gain were seen in the 17.5 and 100 mg/kg groups. Salivation and a decrease in food consumption were seen in the 100 mg/kg group. Ophthalmologic examination confirmed the mydriasis in the 17.5 and 100 mg/kg groups. Electrocardiographic and hematological examinations and urinalysis showed no abnormalities attributable to the treatment. Blood chemical examination revealed increases in GPT and ALP in the 17.5 and 100 mg/kg groups, increases in GOT and triglyceride and a decrease in total protein in the 100 mg/kg group. Pathological examination disclosed hepatocellular hypertrophy and concentric lamellar bodies derived from the smooth-ER in the 100 mg/kg group, and hyperplasia of smooth-ER, an increase in number of lysosomes and bile pigments in the bile capillary, hepatocyte and stellate cells of Kupffer in the 17.5 and 100 mg/kg groups. The recovery test showed that the above-mentioned changes were satisfactorily reversible or the degree and frequency of these changes were lowered. The serum concentrations of NS-21 and its active metabolite. RCC-36, in the treated groups were increased in a dose-dependent manner. No treatment-related effects were seen in the 3 mg/kg group. These results show that the NOAEL (no observed adverse effect level) of NS-21 is 3 mg/kg for 12-month oral toxicity in dogs.

Concentric Lamellar Bodies in Axon Terminals and Preterminal Axons of Cerebellar Purkinje Cells in Groggy Mutant Rat: Acid Phosphatase Cytochemistry

The process of formation and degeneration of concentric lamellar bodies (CLBs) in axon terminals and preterminal axons of Purkinje cells were examined by acid phosphatase (ACPase) cytochemistry. In myelinated axons, tubular structures measuring 20-50 nm in diameter contained ACPase reaction products. In altered axon terminals and preterminal axons of Purkinje cells, elongated saccular structures contained numerous reaction products. These saccular structures were arranged concentrically and enclosed cytoplasmic organelles. In the CLBs displaying degenerative profiles, ACPase reaction products were dispersed in the degenerating materials. The nature of the tubular structures and their roles in the transport of ACPase and the formation of CLBs are discussed.

Age-related and region-specific increase in number of concentric lamellar bodies in axon terminals and presynaptic axons in central nervous system of groggy mutant rat.

In some regions of the central nervous system (CNS) of the groggy rat, a mutant with a movement disorder, concentric lamellar bodies (CLBs) were formed in numerous axon terminals and presynaptic axons. These bodies were counted electron microscopically in the lateral cerebellar nuclei of Slc:Wistar and groggy mutant rats at 20 to 180 days of age. In the Slc:Wistar rat groups, the mean numbers of axonal CLBs were mostly under 1.0 per 100 microns2, except for the 30-day-old rat group which showed a value of 1.7. In the groggy rat groups, the numbers of axonal CLBs greatly increased from 40 days of age onward, reaching the maximum mean number of 23.7 per 100 microns2 in the 90-day-old rat group and subsequently decreasing; however, significant numbers were still present in the 180-day-old rat group. Since these bodies have been reported to contain an acid phosphatase (ACPase), the regional specificity of their appearance in the CNS was examined by light and electron microscopic ACPase histochemistry. In the 90- and 180-day-old groggy rats, numerous particulate deposits of ACPase reaction products were found in the neuropil of the lateral, interposed and medial cerebellar nuclei, the superior, lateral and spinal vestibular nuclei, and the spinal gracile nuclei. By electron microscopy, the particulate deposits in the cerebellar and vestibular nuclei were confirmed as the CLBs in axon terminals, while those in the gracile nuclei were revealed to be the dystrophic axons. Thus, it was suggested that the axon terminals and presynaptic axons, having a high capacity to form the CLBs in the groggy rats from 40 days of age onward, belong to the Purkinje cells.