Tetsuro Sugimoto

C-reactive protein (CRP) measurement in canine serum following experimentally-induced acute gastric mucosal injury

To establish the diagnostic significance of canine C-reactive protein (CRP) in gastrointestinal disorders, the serum canine CRP concentration was measured in dogs with experimentally-induced acute gastric mucosal injury. Gastric injury was induced in one male and one female beagle by a single dose oral administration of acetylsalicylic acid (200 mg/kg body weight) or indomethacin (60 mg/kg body weight), or sodium chloride (1000 mg/kg body weight). CRP was measured prior to dose, and 1, 3, 7, and 14 days after the administration of the drugs, together with the total leucocyte counts and serum iron. Changes in the serum CRP in dogs with gastric injury were similar for the three test compounds, and reflected by the endoscopic findings. CRP values increased from 87 to 390 mg/l within 1 to 3 days after the compound administration but returned nearly to the predose levels within 14 days. Endoscopy revealed haemorrhagic erosion of the gastric mucosa in all dogs one day after dosing, with no evidence of the erosions observed after 7 days in many of the dogs. Changes of the total leucocyte and serum iron also occurred following gastric injury, but these changes were not as marked as those observed for CRP. The results of this study suggest that serum CRP level may be a useful indicator of a gastrointestinal mucosal injury in dogs.

Subtotal Nephrectomy Stimulates Cyclooxygenase 2 Expression and Prostacyclin Synthesis in the Rat Remnant Kidney

Background: Recently, two isoforms of cyclooxygenase (COX) have been identified, a constitutive form (COX-1) and a mitogen-inducible form (COX-2). Several studies have suggested that COX is activated in renal insufficiency, but little is known about the relationship between progression of renal insufficiency and the COX isoforms. Methods: Five-sixths-nephrectomized (NX) rats were used. 4, 8, and 12 weeks after nephrectomy, the renal cortical prostaglandin contents and the expression levels of the two isoforms of COX were determined by enzyme immunoassay and Western-blotting, respectively. The localization of COX was examined by immunohistochemistry. Results: Renal cortical prostacyclin (PGI2) and COX-2 were significantly upregulated 8 and 12 weeks after NX, while COX-1 remained at the basal level. There was a high correlation between COX-2 and creatinine clearance (r = –0.845). There was also a high correlation between COX-2 and PGI2 (r = 0.816). Immunohistochemistry revealed the expression of COX-2 to be enhanced in the macula densa in NX rats. Conclusions: Renal cortical COX-2 and prostacyclin were upregulated corresponding to the progression of renal insufficiency in NX rats. These results suggest enhancement of COX-2 expression in the macula densa, perhaps stimulated by a decrease in renal blood flow which upregulates PGI2 synthesis to protect the kidney from ischemia in renal insufficiency.

PTHrP and PTH/PTHrP Receptor 1 Expression in Odontogenic Cells of Normal and HHM Model Rat Incisors

Parathyroid hormone related peptide (PTHrP) was discovered as a causative factor of humoral hypercalcemia of malignancy (HHM). We examined PTHrP and its receptor (PTHR1) expression patterns in odontogenic cells in normal and HHM model rat incisors. Nontreated nude rats serving as the normal control and HHM model rats produced by implantation of PTHrP-expressing tumor (LC-6) cells were prepared. HHM rats fractured its incisor, and histopathologically, restrict population of odontoblasts showed findings classified as “shortening of high columnar odontoblasts” and “dentin niche.” The incisors were immunostained against PTHrP and PTHR1. In normal rats, PTHrP and PTHR1 colocalized in ameloblasts, cementoblasts, and odontoblastic cells from mesenchymal cells to columnar odontoblasts. In high columnar odontoblasts, PTHrP solely expressed. In the HHM animals, although the expression patterns were identical to those of the normal rats in normal area, the shortened high columnar odontoblasts maintained PTHR1 expression and dentin niche comprising odontoblastic cells expressed both proteins. In the HHM model, the protein expression patterns changed in the odontoblastic cells with histological anomalies, and thus direct relations between the anomalies and PTHrP/PTHR1 axis are suggested.

Histopathological study on the PTHrP-induced incisor lesions in rats.

Parathyroid hormone related peptide (PTHrP) was discovered as a causative factor of humoral hypercalcemia of malignancy (HHM). The present study elucidates the histopathological characters of incisor lesions in the HHM rat model. Nude rats were implanted with PTHrP-expressing tumor (LC-6) cells, maintained for 12 weeks, after which the mandibular incisors were collected. Incisor fractures were observed grossly. Microscopically, hypercalcified dentin, dentin niche with osteodentin, and thinning of dentin were observed. Hypercalcified dentin was observed as a basophilic line of calcified dentin without associated odontoblastic changes, whereas dentin niche and thinning of dentin occurred with osteodentin and loss of cell height, respectively. In contrast with hypercalcified dentin, which was distributed throughout the dentin, dentin niche and thinning of dentin were localized to the labial area of the apical and middle region, and to the labial and lingual areas of the middle and incisal region, respectively. These results suggest that hypercalcemia affected the entire calcification process resulting in hypercalcified dentin, and that high PTHrP concentrations affected selective populations of odontoblasts resulting in formation of dentin niche and thinning of dentin. The localization of dentin niche and thinning of dentin also suggest that PTHrP may also be involved odontoblastic development in the rat.

Characterization of Multinuclear Hepatocytes Induced in Rats by Mitemcinal (GM-611), an Erythromycin Derivative

Mitemcinal is an erythromycin derivative with motilin agonistic action, developed as a gastrointestinal motor-activating agent. The characteristics of mitemcinal-induced multinuclear hepatocytes (MNHs, hepatocytes with three or more nuclei per cell) from detailed morphological observations together with the results of a study on the mechanisms of MNH formation by combining cytocentrifuge preparations with 5-bromo-2’-deoxyuridine cumulative labeling are reported. MNHs were observed only in rats in the high-dose groups of the subchronic study, with a higher incidence in females and reversibility after twenty-eight days of drug withdrawal, but not observed in dogs. In the chronic study, the incidence increased relative to the dose. Histopathologically, MNHs were preferentially observed in the centrilobular zone, without nuclear atypia or mitotic figures. In the cell kinetic study, the labeling pattern of MNHs included all-positive, all-negative, and mixed labeling patterns of nuclei. The all-negative pattern indicated that the cells were formed by fusion of nondividing cells. The current results indicate that the cell kinetic approach effectively demonstrated the mechanism of mitemcinal-induced MNHs as fusion of hepatocytes and that drug-induced disturbance of mitosis is not involved in the multinucleation of MNHs by mitemcinal.

Histopathological Study of Time Course Changes in PTHrP-Induced Incisor Lesions of Rats

Parathyroid hormone related peptide (PTHrP) was discovered as a causative factor of humoral hypercalcemia of malignancy (HHM). In the present study using HHM model rats, the time course of odontoblastic response to PTHrP and its relation to incisal fracture were elicited. Nude rats were implanted with PTHrP-expressing tumor (LC-6) cells, mandibular incisors were collected at several time points. Microscopically 3 distinctive types of odontoblastic/dentin lesions were observed. Hypercalcfied dentin, which was reported as hypercalcemia-induced lesion in previous reports, observed in all areas of the dentin from week 5–10 samplings. Dentin niche, observed solely in week-10 sampling point, exhibited a nature identical to that of reparative odontoblast reported in the literatures of various cytotoxic agents. Since cytotoxicites were neither observed prior to the lesions nor reported as a role of PTHrP, the reparative response may have derived from highly sustained levels of PTHrP. Loss of columnar odontoblasts height was initially observed at week-5 time point in the middle section of the incisor. This primary loss of cell height prior to incisor fracture was considered to be the earliest response to the increased PTHrP levels of this model.

Stimulation of adrenal chromaffin cell proliferation by hypercalcemia induced by intravenous infusion of calcium gluconate in rats.

Increased incidence of adrenal pheochromocytoma is frequently encountered in rat carcinogenicity studies. In some of the studies, the finding is judged to be due to a rat-specific mechanism of carcinogenesis caused by a disturbance of calcium homeostasis. However, direct evidence that the proliferation of chromaffin cells in the adrenal medulla is induced solely by hypercalcemia is not available. In this study, calcium gluconate was intravenously infused for 7 days to rat chromaffin cells by a tail cuff method, and cumulative labeling with bromodeoxyuridine (BrdU) was carried out to evaluate the proliferative activity. The serum calcium concentration was dose-dependently increased, and a high calcium concentration was stably sustained from day 2 to 7. In the adrenal medulla, BrdU-positive chromaffin cells increased in the calcium gluconate-treated animals, and the BrdU-labeling index increased in a dose-dependent manner. In addition, an increased BrdU-labeling index of chromaffin cells was shown to correlate with the serum calcium concentration. Our results demonstrate that hypercalcemia directly enhances the proliferative activity of chromaffin cells and that the proliferative activity is correlated with the serum calcium concentration.

Expression of bone marrow stromal cell specific antigen during murine development:Its expression in embryonic hematopoietic tissues as well as in other developing tissues

Monoclonal antibody R4‐A9 demonstrated specificity for a cell surface antigen of stromal cells in murine bone marrow and spleen. In order to Identify patterns of expression that may elucidate the potential role of R4‐A9 antigen, the developmental expression of this antigen in mouse embryos from 8 days post‐coitum to 5 days post‐partum was investigated by immunohistochemistry. At an early developmental stage, weak staining for R4‐A9 antigen could be detected in the yolk sac. At later stages, strong staining of this antigen was detected predominantly in the embryonic liver, the main site of embryonic hematopoiesis. However, concomitant with the decreased staining in the liver, increased expression of this antigen was observed in bone marrow and spleen. Therefore, the changes in expression in those hematopofetic tissues suggest that its expression is coordinately regulated during the developmental stage of the sites of embryonic hematopoiesis. Compared with the distribution of R4‐A9 antigen in adult tissues as previously reported, the expression of this antigen in fetal tissues was more widespread during the period of organogenesis, and was most abundant in other developing tissues, including the heart, skin, and lung. In contrast, fetal expression detected in hematopoietic and other developing tissues was lost after birth. These results taken together show a marked gradient of R4‐A9 antigen expression, with the highest level at the peak of organ development, raising the possibility that this molecule may act as a growth/differentiation factor both in hematopoietic and other developing tissues in a fetus.