Cutaneous mastocytosis with a mutation in the juxtamembrane domain of c-kit in a young laboratory beagle dog.

Cutaneous mastocytosis, which resembles a subset of urticaria pigmentosa in humans, is rare in dogs. We herein report unrepresentative neoplastic proliferation of mast cells in ventral skin removed routinely from a nine-month-old female laboratory beagle dog at necropsy. A histological examination revealed diffuse extensive cellular infiltration from the superficial to deep dermis in most parts of the skin around the fourth and fifth mammary papilla without nodule formation. Tumor cells were fairly monomorphic, well-differentiated mast cells with round nuclei of small distinct nucleoli and moderate to abundant, slightly eosinophilic and granular cytoplasm. A perivascular arrangement of mast cells was noted at the margin of the lesions. Infiltration of eosinophils and degeneration of collagen were not observed in the dermis. Cutaneous mastocytosis was diagnosed based on these features. A sequence analysis of lesions revealed the deletion of Gln555 to Ile570 within the juxtamembrane domain of c-kit (exon 11).

Imaging of reactive oxygen species using [(3)H]hydromethidine in mice with cisplatin-induced nephrotoxicity.

BACKGROUND: Reactive oxygen species (ROS) have been implicated in cisplatin-induced nephrotoxicity. The aim of this study was to investigate the potential of using [(3)H]-labeled N-methyl-2,3-diamino-6-phenyl-dihydrophenanthridine ([(3)H]hydromethidine) for ex vivo imaging of regional ROS overproduction in mouse kidney induced by cisplatin. METHODS: Male C57BL/6 J mice were intraperitoneally administered with a single dose of cisplatin (30 mg/kg). Renal function was assessed by measuring serum creatinine and blood urea nitrogen (BUN) levels and morphology by histological examination. Renal malondialdehyde levels were measured as a lipid peroxidation marker. Autoradiographic studies were performed with kidney sections from mice at 60 min after [(3)H]hydromethidine injection. RESULTS: Radioactivity accumulation after [(3)H]hydromethidine injection was observed in the renal corticomedullary area of cisplatin-treated mice and was attenuated by pretreatment with dimethylthiourea (DMTU), a hydroxyl radical scavenger. Cisplatin administration significantly elevated serum creatinine and BUN levels, caused renal tissue damage, and promoted renal lipid peroxidation. These changes were significantly suppressed by DMTU pretreatment. CONCLUSIONS: The present study showed that [(3)H]hydromethidine was rapidly distributed to the kidney after its injection and trapped there in the presence of ROS such as hydroxyl radicals, suggesting that [(3)H]hydromethidine is useful for assessment of the renal ROS amount in cisplatin-induced nephrotoxicity.

Involvement of neutrophil gelatinase-associated lipocalin and osteopontin in renal tubular regeneration and interstitial fibrosis after cisplatin-induced renal failure.

The kidney has a capacity to recover from ischemic or toxic insults that result in cell death, and timely tissue repair of affected renal tubules may arrest progression of injury, leading to regression of injury and paving the way for recovery. To investigate the roles of neutrophil gelatinase-associated lipocalin (NGAL/lcn2) and osteopontin (OPN/spp1) during renal regeneration, the expression patterns of NGAL and OPN in the cisplatin-induced rat renal failure model were examined. NGAL expression was increased from day 1 after injection; it was seen mainly in the completely regenerating proximal tubules of the cortico-medullary junction on days 3-35; however, the expression was not seen in abnormally dilated or atrophied renal tubules surrounded by fibrotic lesions. On the other hand, OPN expression was increased from day 5 and the increased expression developed exclusively in the abnormal renal tubules. NGAL expression level well correlated with the proliferating activity in the regenerating renal epithelial cells, whereas OPN significantly correlated with the α-smooth muscle actin-positive myofibroblast appearance, expression of transforming growth factor (TGF)-ß1, and the number of CD68-positive macrophages. Interestingly, rat renal epithelial cell line (NRK-52E) treated with TGF-ß1 decreased NGAL expression, but increased OPN expression in a dose-dependent manner. Because increases of TGF-ß1, myofibroblasts and macrophages contribute to progressive interstitial renal fibrosis, OPN may be involved in the pathogenesis of fibrosis; on the contrary, NGAL may play a role in tubular regeneration after injury. Expression analysis of NGAL and OPN would be useful to investigate the tubule damage in renal-toxicity.

Ectopic tissue consisting of a mixture of glandular gastric, intestinal, and exocrine pancreatic tissue in the forestomach of a rat.

The development of ectopic gastric, intestinal, or pancreatic tissue in the gastrointestinal tract is extremely rare in rats, although it is fairly common in humans. In this report, we describe an unusual case in which a mixture of different types of ectopic tissue was found in the forestomach of a rat. A solitary white nodular/polypoid structure, which measured 5 mm in size, was detected on the luminal surface of the greater curvature of the forestomach in an 8-week-old female Crl:CD(SD) rat. A histological examination revealed that the lesion contained ectopic glandular gastric tissue, including gastric surface mucous cells, parietal cells, and pyloric gland cells, which was confirmed by immunohistochemistry. Moreover, the lesion also contained villin-positive columnar intestinal absorptive cells and chymotrypsin-positive pancreatic exocrine tissue. To the best of our knowledge, this is the first study to detect a mixture of ectopic glandular gastric, intestinal, and exocrine pancreatic tissue in a rat.

L-type voltage-dependent calcium channel is involved in the snake venom group IA secretory phospholipase A2-induced neuronal apoptosis.

Snake venom group IA secretory phospholipase A2 (sPLA2-IA) is known as a neurotoxin. Snake venom sPLA2s are neurotoxic in vivo and in vitro, causing synergistic neurotoxicity to cortical cultures when applied with toxic concentrations of glutamate. However, it has not yet been cleared sufficiently how sPLA2-IA exerts neurotoxicity. Here, we found sPLA2-IA induced neuronal cell death in a concentration-dependent manner. This death was a delayed response requiring a latent time for 6h. sPLA2-IA-induced neuronal cell death was accompanied with apoptotic blebbing, condensed chromatin, and fragmented DNA, exhibiting apoptotic features. NMDA receptor blockers suppressed the neurotoxicity of sPLA2-IA, but an AMPA receptor blocker did not. Interestingly, L-type voltage-dependent Ca(2+) channel (L-VDCC) blocker significantly protected neurons from the sPLA2-IA-induced apoptosis. On the other hand, neither N-VDCC blockers nor P/Q-VDCC blocker did. In conclusion, we demonstrated that sPLA2-IA induced neuronal cell death via apoptosis. Furthermore, the present study suggests that not only NMDA receptor but also L-VDCC contributed to the neurotoxicity of snake venom sPLA2-IA.

Expression patterns of heat shock protein 25 in carbon tetrachloride-induced rat liver injury.

Heat shock protein 25 (Hsp25) is a molecular chaperone playing roles in cytoprotection. We investigated the distribution and localization of Hsp25 expression in CCl(4)-induced rat hepatic lesions; liver samples were obtained from 3 h to 10 days after a single oral administration of CCl(4). Immunohistochemically, Hsp25-positive hepatocytes started to appear in the perivenular area at 6 h after CCl(4) administration. Their number and strength increased till day 1. Expression of Hsp25 mRNA significantly increased after 3 h and proceeded to increase with time till day 1. Apoptotic hepatocytes were detected around the perivenular area after 6 h. The area where Hsp25-positive hepatocytes were observed till day 1 corresponded to the area where apoptotic hepatocytes were seen. On days 2 and 3, degenerative and/or necrotic hepatocytes in the perivenular area were replaced by macrophages reacting to ED1 (for CD68) and ED2 (for CD163); Hsp25 expression was seen in hepatocytes around the perivenular area and there was a close relationship of reactive macrophages with Hsp25-positive hepatocytes, suggesting a potential role for Hsp25 in suppressing injury by inflammation. The mRNA expression of tumor necrosis factor-α, monocyte chemoattractant protein-1 and osteopontin, which can be produced by infiltrating macrophages, corresponded to that of Hsp25 from day 1 to day 3; these factors might be related to the induction of Hsp25 expression. The shift of the Hsp25 expression pattern in the liver lesion might have depended on microenvironmental conditions evoked by interactions between necrobiotic hepatocytes and infiltrating macrophages. Thus, Hsp25 expression analyses should be beneficial for evaluations of hepatotoxicants.

Glaucoma in a New Zealand White Rabbit Fed High-cholesterol Diet.

Goniodysgenesis, malformation of the filtration angle, was observed in a New Zealand white rabbit supplied with 100 g/day rabbit chow containing 0.2% cholesterol for 10 months. Histopathology revealed cupping of the optic disc, atrophy of the retina and hyalinization of the ciliary body in the bilateral eyeballs. These findings corresponded with histopathological features caused by glaucoma. On the basis of these findings, we diagnosed this lesion as glaucoma, and classified it as primary glaucoma because of the presence of developmental defects of the filtration angle. In this case, hypercholesterolemia-induced changes, such as aggregation of lipid-laden macrophages and cholesterin clefts in the sclera or choroid, might cause deterioration of the lesions in glaucoma.

Effect of ultrafine zinc oxide (ZnO) nanoparticles on induction of oral tolerance in mice.

Ultrafine nanoparticles of zinc oxide (ZnO) recently became available as a substitute for larger-size fine ZnO particles. However, the biological activity of ultrafine ZnO currently remains undefined. In the present study, we investigated the effect of ultrafine ZnO on oral tolerance that plays an important role in the prevention of food allergy. Oral tolerance was induced in mice by a single oral administration (i.e., gavage) of 25 mg of ovalbumin (OVA) 5 days prior to a subcutaneous immunization with OVA (Day 0). Varying doses of ultrafine (diameter: approximately 21 nm) as well as fine (diameter: < 5 microm) ZnO particles were given orally at the same time during the OVA gavage. The results indicated that a single oral administration of OVA was followed by significant decreases in serum anti-OVA IgG, IgG(1), IgG(2a), and IgE antibodies and in the proliferative responses to the antigen by these hosts' spleen cells. The decreases in these immune responses to OVA were associated with a marked suppression of secretion of interferon (IFN)gamma, interleukin (IL)-5, and IL-17 by these lymphoid cells. Treatment with either ultrafine or fine ZnO failed to affect the oral OVA-induced suppression of antigen-specific IgG, IgG(1), IgG(2a), and IgE production or lymphoid cell proliferation. The suppression induced by the oral OVA upon secretion of IFN gamma, IL-5, and IL-17 was also unaffected by either size of ZnO. These results indicate that ultrafine particles of ZnO do not appear to modulate the induction of oral tolerance in mice.

Fibroblast growth factor 2 induces apoptosis in the early primary culture of rat cortical neurons.

In the central nervous system, fibroblast growth factor 2 (FGF2) is known to have important functions in cell survival and differentiation. In addition to its roles as a neurotrophic factor, we found that FGF2 caused cell death in the early primary culture of cortical neurons. FGF2-induced neuronal cell death showed apoptotic characters, e.g., chromatin condensation and DNA fragmentation. The ultrastructural morphology of FGF2-treated neurons indicated apoptotic features such as progressive cell shrinkage, blebbing of the plasma membrane, loss of cytosolic organelles, clumping of chromatin, and fragmentation of DNA. Tyrosine kinase inhibitors significantly rescued neurons from FGF2-induced apoptosis. FGF2 potentiated a marked influx of Ca(2+) into neurons before apoptosis. Both a calcium chelator and L-type voltage-sensitive Ca(2+) channel (L-VSCC) blockers attenuated FGF2-induced apoptosis, whereas other blockers of VSCCs such as N-type and P/Q-types did not. Blockers of L-VSCCs significantly suppressed FGF2-enhanced Ca(2+) influx into neurons. Moreover, FGF2 also generated reactive oxygen species (ROS) before apoptosis. Radical scavengers reduced not only the FGF2-generated ROS, but also the FGF2-induced Ca(2+) influx and apoptosis. In conclusion, we demonstrated that FGF2 caused apoptosis via L-VSCCs in the early neuronal culture.

Spontaneous Erythroid Leukemia in a 7-Week-Old Crl:CD (SD) Rat.

A young male Crl:CD (SD) rat with erythroid leukemia that presented with emaciation, abdominal distension and a pale visible mucosal membrane was euthanized at 7 weeks of age. At necropsy, enlargement of liver, spleen and pancreatic lymph node was noted. Analysis of blood smear samples revealed many mono- or binucleated erythroblasts that had PAS-positive vacuoles in the cytoplasm. Histopathologically, neoplastic proliferation of atypical cells was observed in the hepatic sinusoids, splenic red pulp, bone marrow, pancreatic lymph node, kidney and lung. Neoplastic cells showed a round to spindle shape, and some neoplastic cells had deeply stained small nuclei and small cytoplasms and resembled erythroblasts. Immunohistochemically, many neoplastic cells were positive for hemoglobin. To our knowledge, this is the first report of erythroid leukemia in a rat of this age. The observed features were similar to those of pure erythroid leukemia in humans.

Ultrastructural and morphometrical evaluation of the parathyroid gland in iron-lactate-overloaded rats.

Iron lactate was given to Sprague-Dawley rats intravenously at the dosage of 10 mg/kg/day and the early effects on the parathyroid gland were examined ultrastructurally along with the blood level of parathyroid hormone (PTH) after single, 3-day or 6-day administration. Blood levels of electrolytes and other parameters related to osteoclast dynamics were also measured by blood chemistry and histopathology. The plasma parathyroid hormone (PTH) level was elevated in the single and 3-day dosing group but was reduced in the 6-day dosing group. Histopathologically, an increase of osteoclasts in the primary spongiosa was observed in the 3- and 6-day dosing groups. Image analysis of the parathyroid gland revealed that the average area of the storage granule decreased during a experimental period, with the number of storage granules decreasing in the 3- and 6-day dosing group. The chief cells of the parathyroid gland were moderately atrophied in the 6-day dosing group. These results demonstrate that iron lactate immediately promotes discharge of PTH from the storage granules after the treatment and induces an increase of osteoclasts in the primary spongiosa. The findings collectively suggest a pathophysiological mechanism of iron lactate-induced osteopenia in rats.

Glomerular calcification induced by bolus injection with dibasic sodium phosphate solution in Sprague-Dawley rats.

To elucidate the nephrotoxicity of phosphate, dibasic sodium phosphate solution was given to Sprague-Dawley rats by daily bolus intravenous administration at concentrations of 0, 1, 25, 250, or 360 mM (0, 1, 28, 284, or 408 mg/kg Na2HPO4) for 14 days, and the kidneys were pathologically examined. There were no remarkable changes in blood chemistry values; however, urinalysis revealed mild to moderate proteinuria in the 250 and 360 mM groups. The kidneys from the 360 mM group were macroscopically pale. Histopathology revealed panglomerular deposition of basophilic dense granules, which were positive for von Kossa's staining, accompanied by dose-dependent degeneration of the glomerular epithelium and parietal epithelium in the 250 and 360 mM groups. Electron microscopic examination showed fusion of podocytes and increased microvilli, with large amounts of debris in the Bowman's space. Low-density lamellar structures were present not only in the glomerular epithelium, basement membrane, mesangial matrix and parietal epithelium but also within the Bowman's space depending on the severity of the glomerular lesion. Phosphorus and calcium were detected by X-ray microanalysis as fine particles admixed with lamellar structures. These results suggest that high-dose phosphate used in this study transiently overloads the glomerular epithelium during filtration through glomerular capillaries and produces insoluble calcium salt and glomerular lesions, resulting in proteinuria.

Novel binding sites of 15-deoxy-Delta12,14-prostaglandin J2 in plasma membranes from primary rat cortical neurons.

15-Deoxy-Delta12,14-prostaglandin J2 (15d-Delta12,14-PGJ2) is an endogenous ligand for a nuclear peroxysome proliferator activated receptor-gamma (PPAR). We found novel binding sites of 15d-Delta12,14-PGJ2 in the neuronal plasma membranes of the cerebral cortex. The binding sites of [3H]15d-Delta12,14-PGJ2 were displaced by 15d-Delta12,14-PGJ2 with a half-maximal concentration of 1.6 microM. PGD2 and its metabolites also inhibited the binding of [3H]15d-Delta12,14-PGJ2. Affinities for the novel binding sites were 15d-Delta12,14-PGJ2 > Delta12-PGJ2 > PGJ2 > PGD2. Other eicosanoids and PPAR agonists did not alter the binding of [3H]15d-Delta12,14-PGJ2. In primary cultures of rat cortical neurons, we examined the pathophysiologic roles of the novel binding sites. 15d-Delta12,14-PGJ2 triggered neuronal cell death in a concentration-dependent manner, with a half-maximal concentration of 1.1 microM. The neurotoxic potency of PGD2 and its metabolites was also 15d-Delta12,14-PGJ2 > Delta12-PGJ2 > PGJ2 > PGD2. The morphologic and ultrastructural characteristics of 15d-Delta12,14-PGJ2-induced neuronal cell death were apoptotic, as evidenced by condensed chromatin and fragmented DNA. On the other hand, we detected little neurotoxicity of other eicosanoids and PPAR agonists. In conclusion, we demonstrated that novel binding sites of 15d-Delta12,14-PGJ2 exist in the plasma membrane. The present study suggests that the novel binding sites might be involved in 15d-Delta12,14-PGJ2-induced neuronal apoptosis.

Group IB secretory phospholipase A2 induces neuronal cell death via apoptosis.

Group IB secretory phospholipase A2 (sPLA2-IB) mediates cell proliferation, cell migration, hormone release and eicosanoid production via its receptor in peripheral tissues. In the CNS, high-affinity binding sites of sPLA2-IB have been documented. However, it remains obscure whether sPLA2-IB causes biologic or pathologic response in the CNS. To this end, we examined effects of sPLA2-IB on neuronal survival in primary cultures of rat cortical neurons. sPLA2-IB induced neuronal cell death in a concentration-dependent manner. This death was a delayed response requiring a latent time for 6 h; sPLA2-IB-induced neuronal cell death was accompanied with apoptotic blebbing, condensed chromatin, and fragmented DNA, exhibiting apoptotic features. Before cell death, sPLA2-IB liberated arachidonic acid (AA) and generated prostaglandin D2 (PGD2) from neurons. PGD2 and its metabolite, Delta12-PGJ2, exhibited neurotoxicity. Inhibitors of sPLA2 and cyclooxygenase-2 (COX-2) significantly suppressed not only AA release, but also PGD2 generation. These inhibitors significantly prevented neurons from sPLA2-IB-induced neuronal cell death. In conclusion, we demonstrate a novel biological response, apoptosis, of sPLA2-IB in the CNS. Furthermore, the present study suggests that PGD2 metabolites, especially Delta12-PGJ2, might mediate sPLA2-IB-induced apoptosis.

Human group IIA secretory phospholipase A2 induces neuronal cell death via apoptosis.

Expression of group IIA secretory phospholipase A2 (sPLA2-IIA) is documented in the cerebral cortex (CTX) after ischemia, suggesting that sPLA2-IIA is associated with neurodegeneration. However, how sPLA2-IIA is involved in the neurodegeneration remains obscure. To clarify the pathologic role of sPLA2-IIA, we examined its neurotoxicity in rats that had the middle cerebral artery occluded and in primary cultures of cortical neurons. After occlusion, sPLA2 activity was increased in the CTX. An sPLA2 inhibitor, indoxam, significantly ameliorated not only the elevated activity of the sPLA2 but also the neurodegeneration in the CTX. The neuroprotective effect of indoxam was observed even when it was administered after occlusion. In primary cultures, sPLA2-IIA caused marked neuronal cell death. Morphologic and ultrastructural characteristics of neuronal cell death by sPLA2-IIA were apoptotic, as evidenced by condensed chromatin and fragmented DNA. Before apoptosis, sPLA2-IIA liberated arachidonic acid (AA) and generated prostaglandin D2 (PGD2), an AA metabolite, from neurons. Indoxam significantly suppressed not only AA release, but also PGD2 generation. Indoxam prevented neurons from sPLA2-IIA-induced neuronal cell death. The neuroprotective effect of indoxam was observed even when it was administered after sPLA2-IIA treatment. Furthermore, a cyclooxygenase-2 inhibitor significantly prevented neurons from sPLA2-IIA-induced PGD2 generation and neuronal cell death. In conclusion, sPLA2-IIA induces neuronal cell death via apoptosis, which might be associated with AA metabolites, especially PGD2. Furthermore, sPLA2 contributes to neurodegeneration in the ischemic brain, highlighting the therapeutic potential of sPLA2-IIA inhibitors for stroke.