Search

For pituitary regenerative medicine, the creation of a hypophyseal model in monkeys is necessary to conduct future preclinical studies; however, previous studies reported that hypophysectomy in monkeys is not always safe or satisfactory. This study aimed to create a hypophyseal dysfunction model in a cynomolgus monkey using a safer surgical technique and establish the protocol of pituitary hormone replacement therapy for this model. Surgical resection of the pituitary gland of a 7.8-year-old healthy adult cynomolgus male monkey weighing 5.45 kg was performed to create a hypophyseal dysfunction model for future regenerative studies. Endoscopic transoral transsphenoidal surgery was used to perform hypophysectomy under navigation support. These procedures were useful for confirming total removal of the pituitary gland without additional bone removal and preventing complications such as cerebrospinal fluid leakage. Total removal was confirmed by pathological examination and computed tomography. Hypopituitarism was verified with endocrinological examinations including stimulation tests. Postoperatively, the monkey’s general condition of hypopituitarism was treated with hormone replacement therapy, resulting in long-term survival. The success of a minimally invasive and safe surgical method and long-term survival indicate the creation of a hypophyseal dysfunction model in a cynomolgus monkey; hence, this protocol can be employed in the future.

Uterus transplantation (UTx) is now a treatment for women with uterine factor infertility to have a child. However, UTx is still largely at the experimental stage, and many medical issues remain unsolved. Therefore, adequate studies in large animals including non-human primates are required for validation of these issues. UTx research, especially in non-human primates, can provide important information for its full establishment in humans due to the anatomical and physiological similarities between the two. We accumulated data from UTx studies using cynomolgus macaques since 2009 and established autologous and allogeneic UTx models which led to deliveries after performing the procedure. In this paper, we summarized key points to develop UTx models in cynomolgus macaques based on our experience. UTx models in non-human primates can surely contribute new and beneficial knowledge in this field and can be useful for the further development of UTx in humans.

There are few effective antimicrobial agents against Balantidium coli infection. The effect of paromomycin sulfate (PS) against B. coli was confirmed in this study of 596 captive cynomolgus monkeys. In several trials, the minimum dose and duration of oral administration of PS were 25 mg/day for 5 + 5 days, with a 2-day withdrawal interval. To facilitate daily PS administration, pumpkin cakes supplemented with PS were made, which not only resulted in precise effects but also increased the efficiency of preparation and administration of PS by the animal care staff. No cysts or trophozoites were detected at 14 or 16 days after the last treatments. There were no obvious differences in blood and biochemical parameters between before and after administration of PS. These results indicate that PS is effective for elimination of B. coli without hematological side effects. These data could contribute to the control of microbiological pathogens during veterinary care and colony management in primate facilities.

Delivery following uterus transplantation (UTx)&mdash, an approach for treating uterine factor infertility&mdash, has not been reported in nonhuman primate models. Here, six female major histocompatibility complex (MHC)-defined cynomolgus macaques that underwent allogeneic UTx were evaluated. Antithymocyte globulin and rituximab were administered to induce immunosuppression and a triple maintenance regimen was used. Menstruation resumed in all animals with long-term survival, except one, which was euthanized due to infusion associated adverse reaction to antithymocyte globulin. Donor-specific antibodies (DSA) were detected in cases 2, 4, and 5, while humoral rejection occurred in cases 4 and 5. Post-transplant lymphoproliferative disorder (PTLD) developed in cases 2 and 3. Pregnancy was attempted in cases 1, 2, and 3 but was achieved only in case 2, which had haploidentical donor and recipient MHCs. Pregnancy was achieved in case 2 after recovery from graft rejection coincident with DSA and PTLD. A cesarean section was performed at full-term. This is the first report of a successful livebirth following allogeneic UTx in nonhuman primates, although the delivery was achieved via UTx between a pair carrying haploidentical MHCs. Experimental data from nonhuman primates may provide important scientific knowledge needed to resolve unsolved clinical issues in UTx.

Uterus transplantation (UTx) is a potential option for women with uterine factor infertility to have a child. The clinical features indicating irreversible rejection of the uterus are unknown. In our experimental series of allogeneic UTx in cynomolgus macaques, six female macaques were retrospectively examined, which were unresponsive to treatment with immunosuppressants (i.e. irreversible rejection). Clinical features including general condition, hematology, uterine size, indocyanine green (ICG) fluorescence imaging by laparotomy, and histopathological findings of the removed uterus were evaluated. In all cases, general condition was good at the time of diagnosis of irreversible rejection and thereafter. Laboratory evaluation showed temporary increases in white blood cells, lactate dehydrogenase and C-reactive protein, then these levels tended to decrease gradually. In transabdominal ultrasonography, the uterus showed time-dependent shrinkage after transient swelling at the time of diagnosis of irreversible rejection. In laparotomy, a whitish transplanted uterus was observed and enhancement of the transplanted uterus was absent in ICG fluorescence imaging. Histopathological findings in each removed uterus showed hyalinized fibrosis, endometrial deficit, lymphocytic infiltration and vasculitis. These findings suggest that uterine transplantation rejection is not fatal, in contrast to rejection of life-supporting organs. Since the transplanted uterus with irreversible rejection atrophies naturally, hysterectomy may be unnecessary.

Study question How long is the allowable warm ischemic time of the uterus and what morphological and biochemical changes are caused by uterine ischemia/reperfusion injury in cynomolgus macaques? Summary answer Warm ischemia in the uterus of cynomolgus macaques is tolerated for up to 4 h and reperfusion after uterine ischemia caused no further morphological and biochemical changes. What is known already Uterus transplantation is a potential option for women with uterine factor infertility. The allowable warm ischemic time and ischemia/reperfusion injury of the uterus in humans and non-human primates is unknown. Study design, size, duration This experimental study included 18 female cynomolgus macaques with periodic menstruation. Participants/materials, setting, methods Animals were divided into six groups of three monkeys each: a control group and groups with uterine ischemia for 0.5, 1, 2, 4 and 8 h. Biopsies of uterine tissues were performed before blood flow blockage, after each blockage time, and after reperfusion for 3 h. Blood sampling was performed after each blockage time, and after reperfusion for 5, 15 and 30 min for measurement of biochemical data. Resumption of menstruation was monitored after the surgical procedure. Morphological, physiological and biochemical changes after ischemia and reperfusion were evaluated. Main results and the role of chance Mild muscle degeneration and zonal degeneration were observed in all animals subjected to warm ischemia for 4 or 8 h, but there were no marked differences in the appearance of specimens immediately after ischemia and after reperfusion for 3 h in animals subjected to 4 or 8 h of warm ischemia. There were no significant changes in any biochemical parameters at any time point in each group. Periodical menstruation resumed in all animals with warm ischemia up to 4 h, but did not recover in animals with warm ischemia for 8 h with atrophic uteri. Limitations, reason for caution Warm ischemia in actual transplantation was not exactly mimicked in this study because uteri were not perfused, cooled, transplanted or reanastomosed with vessels. Results in non-human primates cannot always be extrapolated to humans. Wider implications of the findings The findings suggest that the tolerable warm ischemia time in the uterus is expected to be longer than that in other vital organs. Study funding/competing interest(s) This study was supported by the Japan Society for the Promotion of Science (JSPS) KAKENHI Grant Number 26713050. None of the authors has a conflict of interest to declare.

Two radiated tortoises (Astrochelys radiata) exhibited anorexia and hypokinesia. In both cases, hematological and serum biochemical examinations revealed high alkaline phosphatase levels, moderately high aspartate aminotransferase levels and white blood cell counts approximately within the normal range. Despite being treated, the tortoises died 9 and 43 days after the first clinical examination. Gross pathological examinations revealed that the livers of both animals were extremely swollen and contained pale yellow necrotic tissue. Histopathological assessment revealed that the livers contained a massive area of hepatic necrosis surrounded by migration of macrophages and multinucleated giant cells. In one of the cases, severe fibrosis was observed. The present study provides reference information for similar cases in the future.

Uterus transplantation (UTx) is an option for women with uterine factor infertility to have a child, but is still in the experimental stage. Therefore, allogeneic animal models of UTx are required for resolution of clinical issues. In this study, long-term outcomes were evaluated in four recipients (cases 1&ndash, 4) after allogeneic UTx in cynomolgus macaques. Immunosuppression with antithymocyte globulin induction and a triple maintenance regimen was used. Postoperative ultrasonography and biopsy of the transplanted uterus and immunoserological examinations were performed. All four recipients survived for &gt, 3 months after surgery, but continuous menstruation did not resume, although temporary menstruation occurred (cases 1 and 2). All animals were euthanized due to irreversible rejection and no uterine blood flow (cases 1, 2 and 4) and post-transplant lymphoproliferative disorder (case 3). Donor-specific antibodies against MHC class I and II were detected in cases 1, 2 and 4, but not in case 3. Peripheral lymphocyte counts tended to elevate for CD3+, CD20+ and NK cells in conjunction with uterine rejection, and all animals had elevated stimulation indexes of mixed lymphocyte reaction after surgery. Establishment of allogeneic UTx in cynomolgus macaque requires further exploration of immunosuppression, but the clinicopathological features of uterine rejection are useful for development of human UTx.

Introduction The objective of this study was to examine the allowable warm ischemic time and pathological changes due to ischemia and reperfusion injury in the uterus of the cynomolgus monkey as a model for uterus transplantation. Material and methods Six female cynomolgus monkeys were used in the study. The uterus was resected from the vaginal canal and connected through the bilateral ovarian and uterine arteries and veins only. One animal was used as a control. In the other five animals, the bilateral uterine and ovarian vessels were clamped for 0.5, 1, 2, 4 and 8 h, respectively. Biopsy of the smooth muscle tissue of corpus uteri was performed after each ischemic time and after subsequent reperfusion for 3 h. Biopsy samples were observed by light and electron microscopy. Menstruation recovery was monitored. Results There were no particular findings in both light and electron microscopy after ischemia for up to 2 h and after subsequent reperfusion. There were no marked changes after ischemia for 4 h, but dilated nuclear pores and rough endoplasmic reticulum swelling were found after reperfusion. These changes also occurred, along with mitochondrial swelling and cristae loss after ischemia for 8 h, and plasma membrane loss, nuclear fragmentation and chromatin condensation were found after reperfusion. Periodical menstruation restarted in all animals with ischemia up to 4 h, but the animal with ischemia for 8 h had amenorrhea and uterine atrophy. Conclusions The uterus of the cynomolgus monkey tolerates warm ischemia for up to 4 h.

Autosomal dominant polycystic kidney disease (ADPKD) caused by PKD1 mutations is one of the most common hereditary disorders. However, the key pathological processes underlying cyst development and exacerbation in pre-symptomatic stages remain unknown, because rodent models do not recapitulate critical disease phenotypes, including disease onset in heterozygotes. Here, using CRISPR/Cas9, we generate ADPKD models with PKD1 mutations in cynomolgus monkeys. As in humans and mice, near-complete PKD1 depletion induces severe cyst formation mainly in collecting ducts. Importantly, unlike in mice, PKD1 heterozygote monkeys exhibit cyst formation perinatally in distal tubules, possibly reflecting the initial pathology in humans. Many monkeys in these models survive after cyst formation, and cysts progress with age. Furthermore, we succeed in generating selective heterozygous mutations using allele-specific targeting. We propose that our models elucidate the onset and progression of ADPKD, which will serve as a critical basis for establishing new therapeutic strategies, including drug treatments., ゲノム編集技術を用いてカニクイザルモデルにおいて常染色体優性多発性嚢胞腎(ADPKD)の病態再現に成功 --小動物では病態再現できない難病の研究に新たな道--. 京都大学プレスリリース. 2019-12-13.

No study has reported an animal model of uterus transplantation (UTx) using cynomolgus macaques. We aimed to establish a surgical technique of allogeneic UTx assuming the recovery of a uterus from a deceased donor in cynomolgus macaques. Four allogeneic UTxs were performed in female cynomolgus macaques. Donor surgeries comprised en bloc recovery of organs with iliac vessels on both sides, and/or abdominal aorta/vena cava after sufficient perfusion from one femoral artery or external iliac artery. Before perfusion, 150 mL of whole blood was obtained from the donor for subsequent blood transfusion to the recipient. Four uterine grafts were orthotopically transplanted to recipients. End-to-side anastomosis was performed to the iliac vessels on one side in case 1 and iliac vessels on both sides in case 2; aorto-aorto/cavo-caval anastomosis was performed in cases 3 and 4. Arterial blood flow of the uterine grafts was determined by intraoperative indocyanine green (ICG) angiography. ICG angiography results showed sufficient blood flow to all uterine grafts, and anaemia did not progress. Under appropriate immune suppression, all recipients survived for more than 90 days post-transplantation, without any surgical complications. We describe a surgical technique for allogeneic UTx in cynomolgus macaques.

Two radiated tortoises (Astrochelys radiata) exhibited anorexia and hypokinesia. In both cases, hematological and serum biochemical examinations revealed high alkaline phosphatase levels, moderately high aspartate aminotransferase levels and white blood cell counts approximately within the normal range. Despite being treated, the tortoises died 9 and 43 days after the first clinical examination. Gross pathological examinations revealed that the livers of both animals were extremely swollen and contained pale yellow necrotic tissue. Histopathological assessment revealed that the livers contained a massive area of hepatic necrosis surrounded by migration of macrophages and multinucleated giant cells. In one of the cases, severe fibrosis was observed. The present study provides reference information for similar cases in the future.

It has long been debated whether watershed infarcts are caused by hemodynamic or embolic mechanisms. In the present study, we investigated microembolic roles in the pathogenesis of watershed infarcts by examining MRI in a macaque monkey model of multiple microinfarcts. 50 μm microbeads were injected into each internal carotid artery twice with a month interval. Monkeys (n=4) injected with 2250-2800 microbeads per unilateral side showed both cortical and internal watershed infarcts in the acute phase and atrophic changes with microbleeds in the chronic phase. These results suggest embolic pathogenesis can contribute to the genesis of both cortical and internal watershed infarcts in primates.

In order to clarify the possible contribution of the abnormal polyol pathway to the development of diabetic nephropathy, the effect of aldose reductase inhibitor on renal function and morphology was examined in streptozotocin (STZ)-induced diabetic rats. Six months after STZ injection, glomerular filtration rate and renal plasma flow showed marked decline with significant increase in nuclear-free mesangial area (MA) and relative mesangial area (RMA; MA per glomerular area) in diabetic rats. Oral administration of an aldose reductase inhibitor, Epalrestat, prevented renal hypofunction and mesangial expansion in diabetic rats without influencing the levels of blood glucose. These results suggest that the abnormal polyol pathway in diabetic rats is closely related to the development of mesangial expansion, a morphologic representative of diabetic glomerulopathy, and renal hypofunction.

Four primary histiogenic tumors of the epididymis were discovered in 110 male B6C3F1 mice used in a carcinogenicity study in our laboratory. From the results, all these tumors were considered to be spontaneous. Histologically, the tumors were characterized by compact growth of spindle to oval shaped histiocytic cells with or without a cleaved nucleus. Focal hemorrhage was present in three cases, and erythrophagocytosis occurred in such lesions. The tumors had some similar histological features of histiocytic sarcomas, which had been observed with high malignancy in other male and female animals. However, systemic growth and atypism of neoplastic cells found in the histiocytic sarcomas were not observed in these epididymal tumors. On the other hand, four histiogenic tumors of the uterus, with features characteristic of epididymal tumors, were observed in 106 female mice. The uterine histiogenic tumors were regarded as benign or precursor lesions of histiocytic sarcoma. Furthermore, it is suspected that histiocytic sarcoma arises from the epididymis.

Chordoma occurred in the sacrococcygeal region of the vertebral column of a 97-week old male control F344 rat. Macroscopically, it was a mass (20 mm in diameter) protruding into the retroperitoneum and the cut surface was white to dark-reddish and showed a gelatinous appearance. Histologically, the tumor was composed of physaliphorous and stellate cells growing in nests or cords and divided into lobules by a thin fibrous stroma. The tumor cells locally invaded adjacent tissues, but no metastasis to other organs was noted. Centrally, the tumor often had islands of bone that entrapped physaliphorous cells and the bone tissue consisted of well-differentiated osteocytes. Based on these findings, this tumor was diagnosed as chordoma with osseous element.

In order to elucidate the relationship between cellular injury and lipid peroxidation induced by hexavalent chromium (CrVI), isolated rat hepatocytes treated with any one of scavengers of active oxygen species, antioxidants or antichromium agent were incubated with K2Cr2O7 as CrVI (1 mM Cr). After the incubation, the development of lipid peroxidation was determined as thiobarbituric acid (TBA)-reacting materials in total lipid extracts from the incubated hepatocytes. Cellular injury was observed as a leakage of lactate dehydrogenase (LDH) from hepatocytes into incubation medium. The contents of reduced glutathione (GSH) in hepatocytes were also assessed. Results obtained were as follows: (1) CrVI facilitated lipid peroxidation in isolated hepatocytes after 20 min of incubation. On the other hand, the cellular injury induced by CrVI was barely observed even after 60 min of incubation. (2) The CrVI-induced lipid peroxidation was inhibited by catalase and mannitol as scavengers of active oxygen species, or N,N'-diphenyl-p-phenylenediamine and alpha-tocopherol as antioxidants. However the cytotoxicity of CrVI could not be prevented by these chemicals. (3) CrVI depleted the contents of intracellular GSH and diminished the activities of glutathione reductase (GR) and glutathione-S-transferase (GST) except glutathione peroxidase. (4) The scavengers of active oxygen species and the antioxidants could not prevent the depletion of intracellular GSH induced by CrVI. (6) Ascorbic acid, antichromium agent, prevented all of the lipid peroxidation, the cellular injury and intracellular GSH depletion induced by CrVI.(ABSTRACT TRUNCATED AT 250 WORDS)

To study the effects of hexavalent and trivalent chromium on lipid peroxidation, isolated rat hepatocytes were incubated with different concentrations of chromium compounds at 37°C for 60min. Lipid peroxidation was determined as thiobarbituric acid (TBA)-reacting materials. Cellular injury was observed as a leakage of lactate dehydrogenase (LDH) from hepatocytes into incubation medium. The contents of reduced glutathione(GSH) in hepatocytes were also assessed. Results obtained were as follows: (1) Lipid peroxidation of isolated rat hepatocytes which was expressed as TBA-reactant formation was inhibited by trivalent chromium at the range of concentrations tested in this experiment (125∼1000μM). Hexavalent one inhibited lipid peroxidation at low concentration (125μM), but facilitated that at high concentration (1000μM). (2) LDH-leakage was facilitated by the addition of hexavalent chromium (K2Cr2O7, 125∼1000μM), on the other hand trivalent one (Cr(NO3)3) inhibited it significantly at concentrations more than 250μM. (3) The hexavalent chromium-induced lipid peroxidation was inhibited by antioxidants such as N, N'-dipheyl-p-phenylenediamine (DPPD), α-tocopherol and diethyl dithiocarbamate (DDTC). However, LDH-leakage from hepatocytes was not inhibited by these antioxidants. (4) After the pretreatment with GSH depleting agent such as diethyl maleate (DEM) on isolated hepatocytes, the lipid peroxidation induced by hexavalent chromium was significantly enhanced and was dependent on the chromium concentrations in the incubation medium. Furthermore this enhancement of lipid peroxidation was canceled by the addition of GSH (10mM). (5) The lipid peroxidation induced by ascorbate was significantly inhibited by the addition of hexavalent chromium as well as trivalent one. This effect of hexavalent chromium might be essentially due to the reduction of hexavalent chromium to trivalent one by ascorbate. These results suggest that chromium in trivalent form inhibits lipid peroxidation in isolated hepatocytes, while hexavalent one facilitates it but is not necessarily correlated to cellular injury.