Histological Properties of Main and Accessory Olfactory Bulbs in the Common Hippopotamus.

The olfactory system of mammals comprises a main olfactory system that detects hundreds of odorants and a vomeronasal system that detects specific chemicals such as pheromones. The main (MOB) and accessory (AOB) olfactory bulbs are the respective primary centers of the main olfactory and vomeronasal systems. Most mammals including artiodactyls possess a large MOB and a comparatively small AOB, whereas most cetaceans lack olfactory bulbs. The common hippopotamus (Hippopotamus amphibius) is semiaquatic and belongs to the order Cetartiodactyla, family Hippopotamidae, which seems to be the closest extant family to cetaceans. The present study evaluates the significance of the olfactory system in the hippopotamus by histologically analyzing the MOB and AOB of a male common hippopotamus. The MOB comprised six layers (olfactory nerve, glomerular, external plexiform, mitral cell, internal plexiform, and granule cell), and the AOB comprised vomeronasal nerve, glomerular, plexiform, and granule cell layers. The MOB contained mitral cells and tufted cells, and the AOB possessed mitral/tufted cells. These histological features of the MOB and the AOB were similar to those in most artiodactyls. All glomeruli in the AOB were positive for anti-Gαi2, but weakly positive for anti-Gαo, suggesting that the hippopotamus vomeronasal system expresses vomeronasal type 1 receptors with a high affinity for volatile compounds. These findings suggest that the olfactory system of the hippopotamus is as well developed as that of other artiodactyl species and that the hippopotamus might depend on its olfactory system for terrestrial social communication.

Hazard characterization of an anti-human tissue factor antibody by combining results of tissue cross-reactivity studies and distribution of hemorrhagic lesions in monkey toxicity studies.

Tissue cross-reactivity (TCR) studies are conducted when developing therapeutic antibodies, but their value is sometimes questioned because the positive organs often do not match the target organs of toxicity. We conducted TCR studies in human and cynomolgus monkey tissues for the development of an anti-human tissue factor antibody (TFAb) and also for a commercially available antibody, to clarify the true distribution of the target antigen. Tissue factor (TF) was found to be distributed in a wide variety of organs and tissues, including the heart and urinary bladder, in human and monkey. Administration of the TFAb to cynomolgus monkey caused hemorrhagic lesions mainly in the heart and urinary bladder in an incidental manner. This was thought to show the physiological role of TF in regulating hemostasis in these organs. Because the distribution of antigen in human and monkey was similar, the possibility that the TFAb would have similar effects in human was judged to be high, and because of the incidental nature of the effects, that they would be difficult to avoid. Thus it was possible to prospectively characterize the hazardous potential of a therapeutic antibody by accurately evaluating the tissue distribution of the target antigen and understanding its biological nature.

Experimental investigation of bone mineral density in Thoroughbreds using quantitative computed tomography.

Bone mineral density (BMD) is one of the indications of the strength and health. BMD measured by quantitative computed tomography (QCT) was compared with that measured by dual energy X-ray absorptiometry (DXA) and radiographic bone aluminum equivalence (RBAE). Limbs were removed from horses that had been euthanized for reasons not associated with this study. Sixteen limbs (left and right metacarpals and metatarsals) from 4 horses were used to compare BMD as measured by QCT with those measured by DXA and RBAE. There was a strong correlation between BMD values measured by QCT and those measured by DXA (R(2)=0.85); correlation was also observed between values obtained by QCT and those obtained by RBAE (R(2)=0.61). To investigate changes in BMD with age, 37 right metacarpal bones, including 7 from horses euthanized because of fracture were examined by QCT. The BMD value of samples from horses dramatically increased until 2 years of age and then plateaued, a pattern similar to the growth curve. The BMD values of bone samples from horses euthanized because of fracture were within the population range, and samples of morbid fracture were not included. The relationship between BMD and age provides a reference for further quantitative studies of bone development and remodeling. Quantitative measurement of BMD using QCT may have great potential for the evaluation of bone biology for breeding and rearing management.

Class II lupus nephritis with podocyte injury in imiquimod-induced lupus-prone mice.

Lupus nephritis (LN) is a renal disease presented in systemic lupus erythematosus (SLE) and is divided into 6 classes based on histopathological criteria set by the International Society of Nephrology/Renal Pathology Society. Major mouse models of SLE usually develop class III/IV LN, which are characterized by subendothelial deposits and endocapillary hypercellularity. We examined the pathological features of kidneys in a mouse model of SLE induced by a toll-like receptor 7 agonist, imiquimod (IMQ). In experiment 1, eleven-female FVB/NJcl wild type mice were treated with IMQ on their ear skin 3 times per week. Plasma anti-dsDNA increased from 2 weeks after first IMQ treatment and 2 mice exhibited nephrotic syndrome from 6 weeks. Histopathology revealed eosinophilic mesangial deposits in all mice from 4 weeks. Subsequently, podocytes showed enlargement with vacuolation and their numbers decreased in 6 mice. There was no infiltration of inflammatory cells, subendothelial deposits in glomeruli, or subepithelial deposits in glomeruli. In experiment 2 using 10 mice at 8 weeks after IMQ treatment, the mesangial deposits were observed in all mice and confirmed to be IgG, IgA, IgM, C1q and C3 by immunofluorescence, which corresponds to class II LN. Foot process effacement was detected by transmission electron microscopy and was considered to lead to proteinuria. These mice exhibited pathological characteristics corresponding to class II LN and podocyte injury, which make it distinct from other mouse models of SLE.

Digital workflows for pathological assessment of rat estrous cycle stage using images of uterine horn and vaginal tissue.

Assessment of the estrous cycle of mature female mammals is an important component of verifying the efficacy and safety of drug candidates. The common pathological approach of relying on expert observation has several drawbacks, including laborious work and inter-viewer variability. The recent advent of image recognition technologies using deep learning is expected to bring substantial benefits to such pathological assessments. We herein propose 2 distinct deep learning-based workflows to classify the estrous cycle stage from tissue images of the uterine horn and vagina, respectively. These constructed models were able to classify the estrous cycle stages with accuracy comparable with that of expert pathologists. Our digital workflows allow efficient pathological assessments of the estrous cycle stage in rats and are thus expected to accelerate drug research and development.

Distribution and sequence of pyknotic cells in rat fetuses exposed to busulfan.

Busulfan, an antineoplastic bifunctional-alkylating agent, is known to induce developmental anomalies. In the present study, we examined the distribution and sequence of pyknotic cells in rat fetal tissues exposed to busulfan. Pregnant rats on gestation day 13 were administered intraperitoneally 30 mg/kg of busulfan, and fetal tissues were examined at 6, 12, 24, 36, 48, 72 and 96 hours after treatment (HAT). Pyknosis of component cells was observed markedly in the brain, moderately in the eyes and spinal cord and mildly in the craniofacial tissue, mandible, limb buds, tail bud, ganglions, alimentary tract, lungs, kidneys, pancreas and liver. In the brain, mitotic inhibition was also detected. Most of the pyknotic cells were considered to be apoptotic cells judging from the results of TUNEL staining and electron microscopic examination. Commonly in the above-mentioned tissues, pyknotic cells began to increase at 24 HAT, peaked at 36 or 48 HAT and disappeared at 96 HAT, which is when the histological picture returned to normal in most tissues except for the brain, spinal cord and eyes. The present study clarified the outline of busulfan-induced apoptosis in rat fetuses.

Crucial Role of Rapgef2 and Rapgef6, a Family of Guanine Nucleotide Exchange Factors for Rap1 Small GTPase, in Formation of Apical Surface Adherens Junctions and Neural Progenitor Development in the Mouse Cerebral Cortex.

Cerebral neocortex development in mammals requires highly orchestrated events involving proliferation, differentiation, and migration of neural progenitors and neurons. Rapgef2 and Rapgef6 constitute a unique family of guanine nucleotide exchange factors for Rap1 small GTPase, which is known to play crucial roles in migration of postmitotic neurons. We previously reported that conditional knockout of Rapgef2 in dorsal telencephalon (Rapgef2-cKO) resulted in the formation of an ectopic cortical mass (ECM) resembling that of subcortical band heterotopia. Here we show that double knockout of Rapgef6 in Rapgef2-cKO mice (Rapgef2/6-dKO) results in marked enlargement of the ECM. While Rapgef2-cKO affects late-born neurons only, Rapgef2/6-dKO affects both early-born and late-born neurons. The Rapgef2-cKO cortex at embryonic day (E) 15.5, and the Rapgef2/6-dKO cortex at E13.5 and E15.5 show disruption of the adherens junctions (AJs) on the apical surface, detachment of radial glial cells (RGCs) from the apical surface and disorganization of the radial glial fiber system, which are accompanied by aberrant distribution of RGCs and intermediate progenitors, normally located in the ventricular zone and the subventricular zone, respectively, over the entire cerebral cortex. Moreover, intrauterine transduction of Cre recombinase into the Rapgef2(flox/flox) brains also results in the apical surface AJ disruption and the RGC detachment from the apical surface, both of which are effectively suppressed by cotransduction of the constitutively active Rap1 mutant Rap1(G12V). These results demonstrate a cell-autonomous role of the Rapgef2/6-Rap1 pathway in maintaining the apical surface AJ structures, which is necessary for the proper development of neural progenitor cells.

Sequence of busulfan-induced neural progenitor cell damage in the fetal rat brain.

The sequence of neural progenitor cell (NPC) damage induced in fetal rat brain by transplacental exposure to busulfan, an antineoplastic bifunctional-alkylating agent, on gestational day 13 was examined by immunohistochemical and real-time RT-PCR analyses. Following busulfan treatment, pyknotic NPCs first appeared in the medial layer and then extended to the dorsal layer of the ventricular zone (VZ) of the telencephalon. Pyknotic NPCs that were immunohistochemically positive for cleaved caspase-3, i.e. apoptotic NPCs, began to increase at 24 h after treatment, peaked at 48 h, and returned to the control levels at 96 h. On the other hand, the index (%) of phospho-histone H3-positive NPCs, i.e. mitotic NPCs, and that of BrdU-positive NPCs, i.e. S-phase cells, decreased in accordance with the increase in the index of apoptotic NPCs. Prior to the peak time of apoptotic NPCs, the indices of p53- and p21-positive NPCs peaked at 36 h. In addition, the expression levels of p21 and Puma (p53-target genes) mRNAs were elevated in real-time RT-PCR analysis. These findings indicated that busulfan not only induced apoptosis through the p53-mediated intrinsic pathway but also inhibited cell proliferation in NPCs, resulting in a reduction of the width of the telencephalon. On the other hand, in spite of up-regulation of p21 expression, the expression of cyclin D1, part of the cell cycle machinery of the G1/S transition, and the expression levels of Cdc20 and cyclin B1 which are involved in G2/M transition, showed no changes, giving no possible information of busulfan-induced cell cycle arrest in NPCs.