Subcutaneous and intraperitoneal lipogranulomas following subcutaneous injection of olive oil in Sprague-Dawley rats.

Olive oil is commonly employed as a solubilizing agent for lipophilic materials in preclinical studies in rodents. Here we report that following subcutaneous (SC) injection of olive oil to Sprague-Dawley (SD) rats, local SC lipogranulomas formed, which were associated with an unusual location of the same changes in the peritoneum. Macroscopically, multifocal white spots were found over the liver and mesentery. Histologically, lipid granulomas were seen in the SC injection site, as well as on the capsular or serosal surface of the abdominal organs. No abnormal clinical signs were noted except for swelling at the injection site. The olive oil may have reached the peritoneal cavity from the SC tissue passively via the lymphatic vessels or actively after engulfment by antigen-presenting cells via the lymphatic or blood vessels. These findings are of particular importance for drug safety assessments, as the occurrence of lipogranulomas in locations distant from the site of administration may lead to misinterpretation of histological results. We suggest that these aberrations may be induced by the administration of olive oil as a vehicle.

The role of the lymphatic system in subcutaneous absorption of macromolecules in the rat model.

The purpose of this study was to assess the contribution of lymphatics to the systemic bioavailability of macromolecules following SC administration in a rat model. The rat model included continuous lymph collection from the thoracic lymph duct and concurrent serial blood sampling from freely moving animals. A thoracic lymph duct-jugular vein shunt produced by an implanted connective cannula, and maintained during the recovery period, enabled superior rat survival and prevented lymphatic cannula occlusion. The SC absorption of three macromolecules (bovine insulin, bovine serum albumin, and recombinant human erythropoietin alpha) was assessed in comparison to the non-lymph cannulated control group. For all tested molecules, only minimal amounts (less than 3%) of the SC administered dose were detected in the collected lymph. In the rat model, following SC administration, the macromolecules were absorbed mainly through the blood capillaries with minimal contribution of the lymphatic system to systemic bioavailability. The relatively small elevation in the lymphatic concentration, which occurred in all molecules, may be attributed to the redistribution of the molecules from the blood to the interstitial fluid compartment. These findings are important since rodents are commonly used in preclinical evaluation of macromolecular drugs.

The effect of general anesthesia on the intestinal lymphatic transport of lipophilic drugs: comparison between anesthetized and freely moving conscious rat models.

The purpose of this study was to evaluate the impact of general anesthesia on the lymphatic transport of orally administered drugs. Vitamin D(3) (0.5 mg/kg), a model lipophilic molecule with significant lymphatic transport, was administered to anesthetized rats in close proximity to the lymphatic cannulation procedure. The lymphatic and non-lymphatic absorption of the vitamin in this experimental model was compared to lymph-duct cannulated freely moving conscious rats. The amounts of vitamin D(3) transported via the lymph in the anesthetized animals throughout the time frame of this experimental model (8 h) were 25% lower as compared to the conscious animals, but showed similar absorption kinetics. However, the duration of the anesthesia is limited and thus failed to produce the complete picture of the absorption process. The cumulative percent of the vitamin dose that was recovered in the lymph as well as the vitamin plasma AUC values were both 25% lower in the anesthetized animals as compared to the conscious animals. Hence, the anesthesia did not influence the proportion of the vitamin fraction absorbed via the different pathways. The lymph flow rate was significantly decreased by the anesthesia (threefold), however, higher lymph vitamin concentrations in these animals led to lower differences in the vitamin lymphatic transport (25%) between the models. In conclusion, the anesthetized rat model is suitable for approximating the lymphatic transport. However, the conscious rat model is still required in order to have a more precise and complete measurement of lymphatic transport.

Compact MRI for the detection of teratoma development following intrathecal human embryonic stem cell injection in NOD-SCID mice.

Stem cells are emerging as a promising new treatment modality for a variety of central nervous system disorders. However, their use is hampered by the potential for the development of teratomas and other tumors. Therefore, there is a crucial need for the development of methods for detecting teratomas in preclinical safety studies. The aim of the current study is to assess the ability of a compact Magnetic Resonance Imaging (MRI) system to detect teratoma formation in mice. Five NOD-SCID mice were injected intrathecally with human embryonic stem cells (hESCs), with two mice serving as controls. In vivo MRI was performed on days 25 and 48, and ex vivo MRI was performed after scheduled euthanization (day 55). MRI results were compared to histopathology findings. Two animals injected with hESCs developed hind-limb paresis and paralysis, necessitating premature euthanization. MRI examination revealed abnormal pale areas in the spinal cord and brain, which correlated histopathologically with teratomas. This preliminary study shows the efficacy of compact MRI systems in the detection of small teratomas following intrathecal injection of hESCs in a highly sensitive manner. Although these results should be validated in larger studies, they provide further evidence that the use of MRI in longitudinal studies offers a new monitoring strategy for preclinical testing of stem cell applications.

Lens epithelial cell growth on the anterior optic of 2 hydrophobic intraocular lens models.

PURPOSE: To evaluate lens epithelial cell (LEC) proliferation on the anterior optic of 2 types of hydrophobic intraocular lenses (IOLs). SETTING: Harlan Biotech Israel and Kaplan Medical Center, Rehovot, Israel. DESIGN: Experimental study. METHODS: Ten New Zealand white rabbits had crystalline lens removal and implantation of an Acrysof IOL in 1 eye (Group 1) and a Tecnis IOL in the fellow eye (Group 2). A weekly biomicroscopy examination was performed during the 2-week study duration, after which the rabbits were humanely killed. The eyes were enucleated, and the complexes containing the IOL and the capsular bag were evaluated for the presence of LECs and large cells (macrophages and giant cells) on the IOL anterior optic and for proteinaceous matrix deposits along the capsulorhexis margin. RESULTS: Lens epithelial cell were detected on all of the IOLs in Group 1 and on none in Group 2 (P < .001). Areas of proteinaceous matrix deposits adjacent to the edge of the capsulorhexis were detected on 8 IOLs in Group 1 and on none in Group 2 (P < .001). Large cells were identified on all IOLs in Group 1 and on 9 IOLs in Group 2 (P = 1.0). No difference in the general inflammation markers was found between the 2 IOL types (P = .234). CONCLUSIONS: Lens epithelial cell with corresponding proteinaceous matrix deposits were significantly more abundant on the IOLs in Group 1 than on the IOLs in Group 2. There was no difference in the presence of large cells or in general inflammation markers between the 2 IOL types. FINANCIAL DISCLOSURE: No author has a financial or proprietary interest in any material or method mentioned.

A chemically induced rat model of hemolysis with disseminated thrombosis.

Although hemolytic anemia and thrombosis, which can be serious or even lethal, are often encountered in daily common practice, their pathogenesis has remained obscure, partially because of the absence of appropriate models. Here we present a unique chemically induced rat model of hemolytic anemia and disseminated thrombosis in which the organs developing infarction are comparable to those seen in humans. We exposed male and female Fischer F344 rats to two, three, or four daily doses of 2-butoxyethanol (BE) at 250 mg/kg body weight and examined for hemolysis and histopathological evidence of disseminated thrombosis on d 2, 3, 4, and 29. Time-course BErelated erythrocytic changes were statistically significant in both sexes. Evidence of thrombosis and infarction was seen mainly in females dosed more than once with widespread thrombotic crisis after two or three dosing, likely explicable by the more significant morphological changes in erythrocytes and hemolysis observed in this gender. We documented thrombosis and infarction in the heart, brain, lungs, eyes, and bones. Our model with its list of target organs similar to that observed in human diseases characterized by hemolysis and thrombosis [for example, thalassemia, sickle cell disease (SCD), paroxysmal nocturnal hemoglobinuria (PNF), disseminated intravascular coagulation (DIC), thrombotic thrombocytopenic purpura (TTP), and hemolytic uremic syndrome (HUS)] suggests that it can be an excellent tool to study the pathogenesis of such complications.

Ocular expression of vascular cell adhesion molecule (VCAM-1) in 2-butoxyethanol-induced hemolysis and thrombosis in female rats.

We demonstrated previously that exposure of rats to 2-butoxyethanol (BE) was associated with morphological changes in red blood cells, hemolytic anemia, and disseminated thrombosis and infarction in different organs including the eyes. In order to elucidate the mechanism of thrombosis formation, we examined in this study the histology and immunohistochemical expression of vascular cell adhesion molecule-1 (VCAM-1), endothelial intercellular adhesion molecule-1 (ICAM-1), and P-selectin in the eyes of the female F344 rat exposed to 2, 3, or 4 daily doses of BE/250 mg/kg body weight. In this BE hemolysis and thrombosis model, positive VCAM-1 expression occurred only in eyes of rats exposed to 3 and 4 doses and was localized in the iris (epithelium lining the posterior surface, anterior mesenchymal epithelium), ciliary processes (lining epithelium, stromal cells), and retina (hypertrophic retinal pigment epithelium). Only weak immunolabeling was seen in eyes exposed to 2 doses. The appearance of VCAM-1 immunostaining correlated with the development of thrombosis located in the same structures. No change in ICAM-1 or P-selectin expression was seen. This immunolabeling distribution suggests that VCAM-1 functions in the pathogenesis of BE-related thrombosis by promoting adhesion of erythrocytes to the endothelium.

Disseminated thrombosis-induced growth plate necrosis in rat: a unique model for growth plate arrest.

Exposure of rats to 2-butoxyethanol (BE) produces early hemolytic anemia and disseminated thrombosis. This leads to infarctions in multiple organs, including bones and cartilage. BE, administered for different durations of exposure in two separate experiments, produced metaphyseal vascular thrombosis, growth plate infarction, and partial or complete physeal growth arrest. This reproducible model may serve as a useful tool in the study of some conditions that manifest growth plate damage. The suitability of this model for investigating the pathogenesis of growth plate necrosis and as a model for potential therapy for various human growth plate disorders are discussed.

Electron microscopy of wet tissues: a case study in renal pathology.

In this report we introduce wet-tissue scanning electron microscopy, a novel technique for direct imaging of wet tissue samples using backscattered electrons. Samples placed in sealed capsules are imaged through a resilient, electron-transparent membrane. The contrast of the imaged samples may be enhanced by chemical staining. The samples several millimeters thick and imaged without sectioning, makes this technique suitable for rapid analysis of tissue specimens. We applied this technique to D-limonene-induced nephropathy where accumulation of hyaline protein droplets is induced in proximal and distal convoluted tubules of the kidney. Images obtained by scanning electron microscopy of hydrated kidney specimens exhibited superior resolution, contrast, and magnification compared with those obtained by conventional light microscopy of paraffin sections. The electron micrographs can be obtained within an hour of tissue removal, whereas preparation for light microscopy requires at least 1 day. These advantages of the wet scanning electron microscopy technique indicate its potential utility in a wide range of applications in histopathology and toxicology.

2-Butoxyethanol enhances the adherence of red blood cells.

We recently presented a unique, chemically-induced rat model of hemolytic anemia and disseminated thrombosis. In this 2-butoxyethanol (BE)-induced model the organs developing infarction are comparable to those seen in human diseases, characterized by hemolysis and thrombosis (e.g., thalassemia, sickle-cell disease, paroxysmal nocturnal hemoglobinuria, disseminated intravascular coagulation, thrombotic thrombocytopenic purpura, and hemolytic uremic syndrome). Red blood cells (RBCs) have special flow properties, namely, self-aggregability, deformability, and potential adherence to endothelial cells (ECs) of the blood vessel wall, which are essential for adequate blood flow and tissue perfusion; their alteration facilitates circulatory disorders. To examine the possible contribution of alterations in RBC flow properties to the observed thrombosis in the present investigation we determined the BE-induced changes in adherence, aggregability, and deformability of RBCs from male and female Fischer F344 rats exposed to two, three, or four daily doses of BE at 250 mg BE/kg body weight. Control animals were treated with the vehicle alone. Blood was taken on days 2, 3, 4, and 29. The administration of BE did not affect the RBCs aggregability but markedly enhanced their adherence to extracellular matrix; such enhancement was correlated with adherence to cultured ECs. RBC/EC interaction has been shown to be a potent catalyst of vascular occlusion in hemolytic hemoglobinopathies; thus the enhanced RBC adherence to EC is a likely mechanism by which thrombosis and organ infarct are induced in BE-treated rats.