Kazuro Miyahara

Effects of Chondroitinase ABC on Intradiscal Pressure in Sheep : An In Vivo Study

Study Design. In vivo intradiscal measurements of pressure in lumbar discs treated with chondroitinase ABC were performed. Objective. To determine the decrease in lumbar intradiscal pressure after chemonucleolysis by chondroitinase ABC in sheep. Summary of Background Data. No previous study has assessed in vivo intradiscal pressure after chemonucleolysis. This study investigated the effect of chondroitinase ABC on intradiscal pressure in terms of a dose and time relation. It also included roentgenographic observations and evaluation of the correlation between disc space narrowing and decrease in intradiscal pressure. Methods. Chondroitinase ABC was injected in the lumbar intervertebral discs of sheep at doses of 1, 5, and 50 U. Phosphate buffered saline also was injected as a negative control measure. One week before injection, then 1 and 4 weeks afterward, intradiscal pressure was measured using a catheter microtip pressure transducer. Simultaneously, standard lateral roentgenographs were taken, and the disc height index was calculated. Results. The intradiscal pressure clearly was decreased 1 week after chondroitinase ABC injection. A further decrease was observed up to 4 weeks. This pressure decrease was dose dependent. The disc height indexes also decreased with time, but the state of the change was different from that of the changes in intradiscal pressure. No clear quantitative correlation was found between intradiscal pressure and disc height index. Conclusions. Chondroitinase ABC can induce the reduction of intradiscal pressure in the lumbar spine.

Cryopreservation of Canine Embryos

The assisted reproductive techniques (ARTs) such as in vitro fertilization, embryo transfer, and cryopreservation of gametes have contributed considerably to the development of biomedical sciences in addition to improving infertility treatments in humans as well as the breeding of domestic animals. However, ARTs used in canine species have strictly limited utility when compared with other mammalian species, including humans. Although successful somatic cell cloning has been reported, artificial insemination by frozen semen to date is only available for the improved breeding and reproduction for companion and working dogs as well as guide dogs for the blind. We describe here the successful cryopreservation of embryos and subsequent embryo transfer in dogs. Canine embryos were collected from excised reproductive organs after artificial insemination and subsequently cryopreserved by a vitrification method. When the 4-cell to morula stage of cryopreserved embryos were nonsurgically transferred into the uteri of nine recipient bitches using a cystoscope, five recipients became pregnant and four of them delivered a total of seven pups. The cryopreservation of embryos in canine species will facilitate the transportation and storage of genetic materials and will aid in the elimination of vertically transmitted diseases in dogs. In addition, this technique will contribute to the improved breeding of companion and working dogs such as guide dogs, drug-detecting dogs, and quarantine dogs.

Evidence that the dominant follicle of the first wave is more active than that of the second wave in terms of its growth rate, blood flow supply and steroidogenic capacity in cows

To clarify the different characteristics of the dominant follicle (DF), the DF in first ovarian follicular wave (DF-1) after spontaneous ovulation and DF in second follicular wave (DF-2) and after induced ovulation of the first-wave DF by GnRH were examined in non-lactating Holstein cows. Follicular maturation of DF-1 and DF-2 were induced by PGF2α and GnRH treatment on Day 6 and 8 (Day 0=Day of follicular wave emergence), respectively. Follicular growth and blood flow (BF) in the follicular wall of DF-1 and DF-2 were examined. To analyze sex steroids in follicular fluid (FF) and amount of mRNA in granulosa cells, DF-1 and DF-2 were aspirated on Day 8 or 9 in different estrous cycle. Diameter in DF-1 was larger than DF-2 on Day 8 and 9. From Day 8 to 9, BF area (BFA) and percentage of the follicular wall with BF, which represents the degree of distribution of BF, increased in DF-1 but not in DF-2. BFA per length of follicle circumference with BF, which represents the thickness of BF, was not different between DF-1 and DF-2. Concentrations of 17β-estradiol (E2) in plasma, E2 and androstendione in FF and amounts of LH receptor mRNA were greater in the DF-1 on Day 8. Gene expression for steroidogenesis, prostaglandin synthesis and angiogenesis did not differ between DF-1 and DF-2. These results indicated that DF-1 were more active than DF-2 in growth, BF supply and steroidogenesis. The greater BFA observed in the DF-1 may be derived from as a result of the greater vascularity in the follicular wall.

Changes of lymphocyte subpopulations and natural killer cells in mice sensitized with Toxoplasma lysate antigen before and after Babesia infection.

When 8-week-old BALB/c mice were sensitized with two intramuscular injections of Toxoplasma lysate antigen (TLA) at 2 week interval, the numbers of sIg(+), Thy-1,2(+), Lyt-1,2(+) Lyt-2,2(+), and Asialo GM1(ASGM1)(+) cells in the spleen, liver and peripheral blood increased by 2 to 4 times over those found in unsensitized mice of the same age. When TLA-sensitized and unsensitized mice were infected with Babesia, 4 of 10 (40%) of the TLA-sensitized mice survived infection, while none of the unsensitized control mice lived longer than 14 days after Babesia infection. By contrast, sensitization of nude mice with TLA had no effect on survival, and mice did not live more than 12 days. The number of thymic Thy-1,2(+) cells decreased in TLA-sensitized and unsensitized BALB/c mice by almost 80% within 10 days after infection (AI). During the same time, the numbers of B cells, T cells, and NK cells increased in the spleen, liver and peripheral blood of both sensitized and unsensitized mice. Especially notable were increases in numbers of Lyt-2,2(+) cells in the spleen and blood and increases in numbers of NK cells in the spleen, liver and blood in both TLA-sensitized and unsensitized mice. When spleen cells from TLA-sensitized and unsensitized mice were cultured in the presence or absence of TLA for 6 days, assays for cytotoxicity using NK-insensitive P-815 target cells and NK-sensitive YAC-1 target cells demonstrated higher rates of cytotoxicity in cultures of TLA-sensitized spleen cells.

Extracranial meningioma in a dairy cow

MENINGIOMA is one of the most common primary intracranial tumours in cats and dogs, but is rarely reported in cattle (Koestner and Higgins 2002). The meningiomas that have been found in cattle have been primary intracranial tumours (Bwangamoi and Sachs 1969, Yadgirker and others 1970, Josephson and Little 1990). There are six recognised histological types of meningiomas: meningiothelial, transitional, angioblastic, microcystic, psammomatous and fibroblastic (Koestner and Higgins 2002). All previously reported cases of bovine meningioma have been fibroblastic, except for one which was psammomatous (Eroksuz and others 1999). Koestner and Higgins (2002) reported that in dogs, 82 per cent of all meningiomas are intracranial, 15 per cent are intraspinal and 3 per cent are retrobulbar. Even in human beings, who have a comparatively high rate of meningioma, extracranial meningioma is a very rare presentation (Trotter and others 2001, Nadkarni and others 2002). This short communication describes a case of extracranial meningioma in a dairy cow. A five-year-old Holstein cow was admitted to the Veterinary Medical Teaching Hospital of Obihiro University. The cow presented with a non-painful swelling on the right side of the forehead and exophthalmos of the right eye (Fig 1), which was reported as having been progressively worsening over a period of nine months. Despite this condition, the cow had been eating well, appeared healthy and showed no neurological signs. Before admission, chronic frontal sinusitis had been tentatively diagnosed and a treatment of antibiotics prescribed; however, the treatment was unsuccessful and the cow’s deformity continued to worsen. On admission, the cow’s rectal temperature, heart rate, respiratory rate and the results of haematological examination were all within reference ranges. Blood chemistry revealed an elevated level of lactate dehydrogenase (2330 iu/l, reference range 1700 to 2300 iu/l); the levels of alkaline/phosphatase, calcium and phosphorus appeared normal. Radiographs of the skull revealed a soft tissue opacity at the right aspect of the frontal bone, parietal bone and frontal sinus. Signs of fluid were not observed. The initial differential diagnosis of the abnormal head conformation was chronic frontal sinusitis, abscess, osteoma or malignant neoplasia. However, the haematological examination and radiographs did not indicate an infection. The decision was therefore made to trephine the forehead over the mass. This revealed no fluid or abscess, but a solid, yellowish-white mass was found in the frontal sinus. Histopathological examination of a biopsy specimen revealed a neoplastic proliferation of spindle cells arranged in interfacing fascicles; storiform patterns and whorl formations were also seen. The neoplastic cells within the mass had basophilic, ovoid or polygonal nuclei with dispersed chromatin, and mitotic figures were frequently observed. The spindle cells were separated by mild to moderate amounts of collagen. Multifocal areas of necrosis and calcification were observed. The tumour cells did not appear to invade the vessels. On the basis of these findings, a fibroblastic tumour or meningioma was suspected, but the origin of the tumour could not be identified. The animal was euthanased at the owner’s request. Before gross dissection, the cow’s head was examined using computed tomography (CT) and magnetic resonance imaging (MRI), with the purpose of establishing the internal anatomy of the lesion. Images were obtained with a helical

Zygomycotic Mediastinal Lymphadenitis in Beef Cattle with Ruminal Tympany

ABSTRACT A 9-month-old steer was autopsied due to recurrent ruminal tympany. A macroscopic examination found an enlarged caudal mediastinal lymph node, and a section of the lymph node revealed necrosis with marked calcification, similar to tuberculous lymphadenitis. Histopathologically, the lesion consisted of multiple coagulative necrotic foci and fibrosis with macrophage, lymphocyte, eosinophil and multinucleated giant cell infiltration. Non-uniform width hyphae were detected in the necrotic area and within the cytoplasm of the multinucleated giant cells, and they were found to be anti-Rhizopus arrhizus antibody positive in an immunohistochemical examination. Therefore, the steer was diagnosed with necrotic caudal mediastinal lymphadenitis due to zygomycetes infection, and inhibition of eructation by the enlarged lymph node was the likely cause of the ruminal tympany.

Influence of transfusion of lymphokine-activated T killer cells on inflammatory responses in dogs after laparotomy

The influence of transfusion of lymphokine-activated T killer cells (T-LAK) on inflammatory responses was examined in dogs after laparotomy. Plasma C-reactive protein (CRP) level, cell numbers of peripheral blood lymphocytes (PBLs) and T lymphocyte subsets (CD3+, CD4+ and CD8+) and mRNA expression levels of cytokines including interleukin (IL)-2, IL-12, IL-4, IL-10 and transforming growth factor (TGF)-β in peripheral blood mononuclear cells (PBMCs) were measured in dogs with (T-LAK group) or without (control group) a single T-LAK administration immediately after laparotomy. The plasma CRP level initially increased and then decreased to the normal range at 7 days after laparotomy in the T-LAK group, which was earlier than in the control group. The expression level of IL-10 mRNA showed a marked postoperative increase and was significantly higher than the preoperative level on day 7 (P<0.05), whereas the level in the control group showed no clear change after laparotomy. A significant increase in IL-2 mRNA expression level in the T-LAK group was observed on day 14, which was two weeks earlier than in the control group (P<0.05). These results suggest that T-LAK therapy in dogs after laparotomy leads to earlier resolution of postoperative inflammation by production of an anti-inflammatory cytokine (IL-10) in the early phase of the postoperative period and earlier restoration of cell-mediated immunity related to cytokine production by PBMCs.

Body surface area measurement in laboratory miniature pigs using a computed tomography scanner.

The body surface area (BSA) of an organism is an important parameter for evaluating physiological functions. In drug development, normalization by BSA is an appropriate method for extrapolating doses between species. The BSA of animals has generally been estimated by multiplying a constant by the power of the body weight (BW). Recently, the use of miniature pigs in non-clinical studies for medical drugs or devices has gradually been increasing. However, verification of their BSA is not as yet sufficient. In this study, we measured the BSAs of 40 laboratory miniature pigs (11 males and 9 females of Göttingen minipig and 14 males and 6 females of Nippon Institute for Biological Science [NIBS] miniature pig) by analyzing computed tomography (CT) images, since measurements using a CT scanner were expected to more precisely determine BSA than classical measuring techniques. The measurement results showed the BSAs of the 20 Göttingen minipigs to range from 0.4358 to 0.8356 m(2) (the working BW range: 12.7-37.0 kg) and 20 NIBS miniature pigs to range from 0.2906 to 0.8675 m(2) (the working BW range: 7.9-41.5 kg). Since accuracy and reproducibility were confirmed by measuring the surface area of an acrylic cuboid, we concluded the measurement method employed in this study to be very reliable. We propose the following estimating formula for BSA of laboratory miniature pigs: 100 × BSA [m(2)] = 7.98 × BW [kg](2/3).

Evaluation of costimulatory molecules in dogs with B cell high grade lymphoma

B cell high grade lymphoma is the most common hematopoietic malignancy in dogs. Although the immune checkpoint molecules, programmed death-1 (PD-1) and cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4), and immune checkpoint inhibitors have been evaluated for the treatment of various human lymphoid malignancies, the expression of those molecules and their relationship with prognosis remain unknown in canine lymphoma. The objective of this study was to evaluate the expression of costimulatory molecules on peripheral blood lymphocytes and tumor infiltrating lymphocytes, in addition to associated ligand expression in the lymph nodes of patients with B cell multicentric high grade lymphoma. Eighteen patients diagnosed with B cell high grade lymphoma and nine healthy control dogs were enrolled. Flow cytometric analysis revealed that the expression of PD-1 on CD4+ peripheral and tumor infiltrating lymphocytes and CTLA-4 on CD4+ peripheral lymphocytes was significantly higher in the lymphoma group than in the control group. The expression level of CD80 mRNA was significantly lower in the lymphoma group than in the control group. In contrast, there were no significant differences in PD-L1, PD-L2, and CD86 expression between the groups. Dogs with CTLA-4 levels below the cutoff values, which were determined based on receiver operating characteristic curves, on peripheral CD4+, CD8+, and tumor infiltrating CD4+ lymphocytes had significantly longer survival than dogs with values above the cutoff. Although it is uncertain whether the expression of immune checkpoint molecules affect the biological behavior of canine lymphoma, one possible explanation is that PD-1 and CTLA-4 might be associated with the suppression of antitumor immunity in dogs with B cell high grade lymphoma, particularly through CD4+ T cells.

MRI of lobar holoprosencephaly in a cat with hypodipsic hypernatraemia

Case summary A 2-year-old neutered female domestic shorthair cat presented with a history of hypodipsia, recurrent hypernatraemia, pelvic limb ataxia and tremor. The serum arginine vasopressin level was low for the serum osmolality. MRI of the brain revealed a failure of separation of the cerebrum, which manifested as absence of the rostral part of the corpus callosum, fornix and septum pellucidum, thus resulting in a single fused ventricle. The diagnosis was lobar holoprosencephaly with hypodipsic hypernatraemia. Relevance and novel information To our knowledge, this is the first description of the MRI characteristics of lobar holoprosencephaly in a cat. This report suggests that MRI examination should be considered for precise diagnosis of hypodipsic hypernatraemia in young cats.