Neurovascular description in the South American plains vizcacha, Lagostomus maximus (Chinchilloidea, Caviomorpha). A study involving evolutionarily related species of Caviomorpha and Muroidea.

Oxygenated blood is required for the adequate metabolic activity of the brain. This is supplied by the circle of Willis (CoW) and the vertebrobasilar and carotid systems. The CoW ensures blood flow in case of arterial stenosis or occlusion. Different animal models have been explored for the CoW morphological and functional study. This work aims to characterize the vascular architecture of the CoW of the plains vizcacha, Lagostomus maximus (Suborder: Hystricomorpha), and to compare it with evolutionarily related species of Caviomorpha and Muroidea. The blood supply in adult plains vizcachas was studied using latex cerebrovascular casts and angiography. A caudo-rostral flow direction was determined, beginning in the spinal and vertebral arteries and converging in the basilar artery which bifurcates in the carotid-basilar communication in the caudal communicating arteries. In the first third of its course, the caudal cerebral arteries project laterally, and the middle and rostral cerebral arteries bifurcate from their rostral terminal segment, supplying the temporo-parietal and frontal cortex. The CoW architecture is mainly conserved between rodent species. Likewise, the small neurovascular variations observed could be considered phylogenetic morphological variations more than evolutionary adaptations. The absence of the rostral communicating artery that generates the rostral open architecture of the CoW in the vizcacha as in the other analyzed species, supports the need for a revision of the CoW classical function as a security system. Finally, this work supports the importance of expanding our understanding of brain anatomy among species, which may contribute to a better understanding of functional neuroanatomy.

Therapy for feline secondary hypertriglyceridemia with fenofibrate.

OBJECTIVES: The aim of this study was to assess the short-term safety and efficacy of fenofibrate in controlling secondary hypertriglyceridemia in cats. METHODS: This was a prospective cohort study. Seventeen adult cats with hypertriglyceridemia (serum triglycerides [TG] >160 mg/dl) were enrolled. Cats received a median dose of 5 mg/kg (range 3.2-6) fenofibrate (q24h PO) for 1 month. Serum TG, total cholesterol (TC), creatine kinase and liver enzymes (alanine aminotransferase, aspartate aminotransferase and alkaline phosphatase) were evaluated before (t0) and after 1 month (t1) of fenofibrate treatment. RESULTS: The causes of secondary hypertriglyceridemia were diabetes mellitus (DM; 29.4%), obesity (29.4%), hyperadrenocorticism (HAC) and DM (11.7%), HAC without DM (5.9%), hypersomatotropism (HST) and DM (5.9%), hypothyroidism (5.9%), long-term treatment with glucocorticoids (5.9%) and chylothorax (5.9%). Serum TG (t0 median 920 mg/dl [range 237-1780]; t1 median 51 mg/dl [range 21-1001]; P = 0.0002) and TC (t0 median 278 mg/dl [range 103-502]; t1 median 156 mg/dl [range 66-244]; P = 0.0001) concentrations showed a significant decrease after 1 month of fenofibrate treatment. Fifteen cats normalized their TG concentration at t1 (88.2%). Of the eight cats that were hypercholesterolemic at t0, six (75%) normalized their TC concentrations at t1. One of 17 cats (5.9 %) presented with diarrhea; the remaining 16 did not show any adverse effects. CONCLUSIONS AND RELEVANCE: DM and obesity are the most common endocrine causes of secondary hyperlipidemia, although it can also be found in cats with HAC, HST or hypothyroidism. This study suggests that fenofibrate treatment was associated with reduction and normalization of TG and TC concentrations in cats with moderate and severe hypertriglyceridemia, regardless of the cause of secondary hypertriglyceridemia. Further work should focus on controlled studies with a greater number of cases.

Cabergoline treatment in cats with diabetes mellitus and hypersomatotropism.

OBJECTIVES: The aim of this study was to evaluate the safety and efficacy of cabergoline to control hypersomatotropism (HST) and diabetes mellitus (DM) in cats. METHODS: This was a prospective cohort study. Twenty-three cats with HST and concurrent DM were enrolled. Cats received a dose of 10 µg/kg cabergoline q48h PO for 6 months. Serum insulin-like growth factor 1 (IGF-1) and fructosamine concentrations, insulin dose and Insulin Resistance Index (IRI) were measured at the time of diagnosis of HST and at the start of cabergoline treatment (t0), and 3 months (t1) and 6 months (t2) during cabergoline treatment. RESULTS: A decrease and normalization of serum IGF-1 concentration was observed in 35% and 26% of cats, respectively. Median IGF-1 (t0: 1350 ng/ml [range 832-1501]; t1: 1284 ng/ml [range 365-1501]; t2: 1240 ng/ml [range 263-1501]; P = 0.016) decreased significantly. Twelve cats underwent diagnostic imaging of the pituitary area. The median pituitary height at t0 of cats that experienced an IGF-1 reduction (n = 5/12) was significantly lower compared with those that did not experience an IGF-1 reduction (n = 7/12) (3.2 mm [range 3.1-3.7] vs 6 mm [range 3.5-9.5]; P = 0.011). Median fructosamine (t0: 628 µmol/l [range 400-963]; t1: 404 µmol/l [range 249-780]; t2: 400 µmol/l [range 260-815]; P <0.0001), insulin dose (t0: 1.3 IU/kg [range 0.5-4.6]; t0: 0.5 IU/kg [range 0-2.3]; t2: 0.4 IU/kg [range 0-2.1]; P <0.0001) and IRI (t0: 800 µmolIU/kgl [range 257-2700]; t1: 300 µmolIU/kgl [range 0-1498]; t2: 250 µmolIU/kgl [range 0-1498]; P <0.0001) decreased significantly during cabergoline treatment. Eight cats achieved diabetic remission between months 1 and 6 of cabergoline treatment (median time to achieve remission: 3 months [range 1-6]). Three cats experienced asymptomatic hypoglycemia. CONCLUSIONS AND RELEVANCE: Cabergoline was effective in normalizing IGF-1 concentration in 26% of cats. Cabergoline improved diabetes control and was associated with remission of DM in 35% of cases. Cabergoline could be a treatment option for cats with HST and DM, especially in those cases with a relatively small pituitary tumor.

Findings suggestive of coronary microvascular dysfunction in cats with myocardial ischemia.

Background: Myocardial infarction (MI) is an important cause of death and disability among humans worldwide. Few studies have reported the occurrence of MI in small animals as well. Reports in human medicine indicate that up to 30% of patients with clinical signs compatible with myocardial ischemia suggestive of coronary disease exhibit normal epicardial arteries at angiography. These symptoms have been associated with a syndrome characterized by alterations in cardiac microvasculature, known as coronary microvascular dysfunction (CMD). Aim: This study aimed to describe the necropsy findings and clinical-pathological characterization (when available) of cats with histopathological findings suggesting CMD. Methods: Necropsy records of cats presenting histopathological diagnosis compatible with acute and/or chronic MI, with normal epicardial arteries and microvascular disorders were evaluated. Results: Twenty animals met the inclusion criteria. Eight cats (40%) exhibited findings compatible with mild hypertrophic cardiomyopathy (HCM) without left atrial enlargement, one (5%) presented restrictive cardiomyopathy, and another one (5%) had lesions consistent with histiocytoid cardiomyopathy. The remaining cats (50%) showed alterations compatible with severe HCM with left atrial enlargement. In all cases, epicardial arteries were normal (without obstruction). All the evaluated hearts exhibited myocardial multifocal fibrosis along with replacement of cardiomyocytes by adipose tissue and blood vessels with hyperplasia and hypertrophy of the muscular layer with protrusion of the nuclei of the endothelial cells. Conclusion: These findings suggest the presence of microvascular dysplasia of the coronary arteries. Further studies are necessary to confirm and clinically characterize these results.

Diabetes mellitus remission in a cat with hyperadrenocorticism after cabergoline treatment.

CASE SUMMARY: A 7-year-old spayed female domestic shorthair cat weighing 5 kg was referred with polyuria, polydipsia, lethargy, abdominal distension and dermatologic abnormalities. Diabetes mellitus was diagnosed and treatment was started with a diet for diabetic cats and insulin glargine (1 IU q12h SC). Hyperadrenocorticism (HAC) was suspected and diagnosed based on clinical signs, increased urinary cortisol:creatinine ratio, lack of suppression on low-dose dexamethasone suppression test and abdominal ultrasonography demonstrating bilateral adrenal enlargement. Oral cabergoline (10 µg/kg every other day) was initiated. After the second administration of cabergoline, the cat suffered from clinical hypoglycemia and no longer required insulin. One month after insulin withdrawal, blood work and urine analysis results showed normoglycemia, a normal serum fructosamine concentration (244 µmol/l) and normal urine analysis without glycosuria. Diabetic remission persisted until its death 7 months later. In addition, cabergoline treatment was associated with improvement in clinical signs such as lethargy, seborrhea, alopecia and abdominal distension. RELEVANCE AND NOVEL INFORMATION: To our knowledge, this is the first reported case of the use of cabergoline in a cat with HAC, as well as the first reported case of diabetic remission in a cat with HAC after cabergoline treatment. Cabergoline could be an alternative treatment for diabetic cats with pituitary-dependent HAC. Further work should focus on different protocols with greater number of cases.

A plasmid encoding the extracellular domain of CD40 ligand and Montanide™ GEL01 as adjuvants enhance the immunogenicity and the protection induced by a DNA vaccine against BoHV-1.

DNA vaccines are capable of inducing humoral and cellular immunity, and are important to control bovine herpesvirus 1 (BoHV-1), an agent of the bovine respiratory disease complex. In previous work, a DNA plasmid that encodes a secreted form of BoHV-1 glycoprotein D (pCIgD) together with commercial adjuvants provided partial protection against viral challenge of bovines. In this work, we evaluate new molecules that could potentiate the DNA vaccine. We show that a plasmid encoding a soluble CD40 ligand (CD40L) and the adjuvant Montanide™ GEL01 (GEL01) activate in vitro bovine afferent lymph dendritic cells (ALDCs). CD40L is a co-stimulating molecule, expressed transiently on activated CD4+ T cells and, to a lesser extent, on activated B cells and platelets. The interaction with its receptor, CD40, exerts effects on the presenting cells, triggering responses in the immune system. GEL01 was designed to improve transfection of DNA vaccines. We vaccinated cattle with: pCIgD; pCIgD-GEL01; pCIgD with GEL01 and CD40L plasmid (named pCIgD-CD40L-GEL01) or with pCIneo vaccines. The results show that CD40L plasmid with GEL01 improved the pCIgD DNA vaccine, increasing anti-BoHV-1 total IgGs, IgG1, IgG2 subclasses, and neutralizing antibodies in serum. After viral challenge, bovines vaccinated with pCIgD-GEL01-CD40L showed a significant decrease in viral excretion and clinical score. On the other hand, 80% of animals in group pCIgD-GEL01-CD40L presented specific anti-BoHV-1 IgG1 antibodies in nasal swabs. In addition, PBMCs from pCIgD-CD40L-GEL01 had the highest percentage of animals with a positive lymphoproliferative response against the virus and significant differences in the secretion of IFNγ and IL-4 by mononuclear cells, indicating the stimulation of the cellular immune response. Overall, the results demonstrate that a plasmid expressing CD40L associated with the adjuvant GEL01 improves the efficacy of a DNA vaccine against BoHV-1.

Bovine Dendritic Cell Activation, T Cell Proliferation and Antibody Responses to Foot-And-Mouth Disease, Is Similar With Inactivated Virus and Virus Like Particles.

Foot-and-mouth disease (FMD) is a highly contagious disease of cloven-hoofed animals that causes severe economic losses in the livestock industry. Currently available vaccines are based on the inactivated FMD virus (FMDV). Although inactivated vaccines have been effective in controlling the disease, they have some disadvantages. Because of these disadvantages, investigations are being made to produce vaccines in low containment facilities. The use of recombinant empty capsids (also referred as Virus Like Particles, VLPs) has been reported to be a promising candidate as a subunit vaccine because it avoids the use of virus in the vaccine production and conserves the conformational epitopes of the virus. Mignaqui and collaborators have produced recombinant FMDV empty capsids from serotype A/ARG/2001 using a scalable technology in mammalian cells that elicited a protective immunity against viral challenge in a mouse model. However, further evaluation of the immune response elicited by these VLPs in cattle is required. In the present work we compare the effect that VLPs or inactivated FMDV has on bovine dendritic cells and the humoral response elicited in cattle after a single vaccination.

Nutritional deficiency and placenta calcification underlie constitutive, selective embryo loss in pregnant South American plains vizcacha, Lagostomus maximus (Rodentia, Caviomorpha).

Plains vizcacha females are able to ovulate up to 800 oocytes per estrus cycle. However, just 10-12 embryos are implanted and only two of them, those located nearest the cervix, are gestated to term. Between 26 and 70 days post-coitum, a constitutive resorption occurs from the embryos located proximal to the ovary, extending progressively toward those distally implanted. Our previous studies on the dynamics of gestation in L. maximus, led us to hypothesize some kind of placental and nutritional insufficiency as the basis for the resorption process. We analyzed histology and arterial architecture of the reproductive tract in pregnant and non-pregnant females. Uterine horns are irrigated through the uterine artery, a branch of the internal iliac artery, in an ascending way from the cervix; segmental arteries irrigating the embryo vesicles become thinner as they approach the ovary. Contrast solution administered during angiographies accumulated in the placenta of embryos closest to cervix. Thus, blood stream favors the embryos nearest the cervix, indicating a gradual nutritional deficiency of those closest to the ovary. Besides, placenta becomes calcified early, at mid-gestation, during the resorption process. Finally, the detection of specialized endothelial venules and inflammatory cells suggest the concurrent participation of immunological processes in embryo vesicles undergoing resorption.

CRISPR-on for activation of endogenous SMARCA4 and TFAP2C expression in bovine embryos.

CRISPR-mediated transcriptional activation, also known as CRISPR-on, has proven efficient for activation of individual or multiple endogenous gene expression in cultured cells from several species. However, the potential of CRISPR-on technology in preimplantation mammalian embryos remains to be explored. Here, we report for the first time the successful modulation of endogenous gene expression in bovine embryos by using the CRISPR-on system. As a proof of principle, we targeted the promoter region of either SMARCA4 or TFAP2C genes, transcription factors implicated in trophoblast lineage commitment during embryo development. We demonstrate that CRISPR-on provides temporal control of endogenous gene expression in bovine embryos, by simple cytoplasmic injection of CRISPR RNA components into one cell embryos. dCas9VP160 activator was efficiently delivered and accurately translated into protein, being detected in the nucleus of all microinjected blastomeres. Our approach resulted in the activation of SMARCA expression shortly after microinjection, with a consequent effect on downstream differentiation promoting factors, such as TFAP2C and CDX2. Although targeting of TFAP2C gene did not result in a significant increase in TFAP2C expression, there was a profound induction in CDX2 expression on day 2 of development. Finally, we demonstrate that CRISPR-on system is suitable for gene expression modulation during the preimplantation period, since no detrimental effect was observed on microinjected embryo development. This study constitutes a first step toward the application of the CRISPR-on system for the study of early embryo cell fate decisions in cattle and other mammalian embryos, as well as to design novel strategies that may lead to an improved trophectoderm development.

Endoscopy, histology and electron microscopy analysis of foetal membranes in pregnant South American plains vizcacha reveal unusual excrescences on the yolk sac.

The South American hystricognathe Lagostomus maximus is a fossorial rodent whose females show unique reproductive characteristics. They have a 155-day long gestation, show massive polyovulation and a selective process of embryonic resorption in the first half of gestation. In order to explore and perform an in-situ characterization of the reproductive tract, we visualized internal structures through ultrasonography and video-endoscopy in pregnant and non-pregnant females. We describe the finding of protruding structures that lie on the yolk sac and their histological and ultrastructural characterization. The placenta was covered with whitish, small pearl-shaped structures. These structures were also seen on the extra-embryonic space, being the amnion and the umbilical cord free of them. Pearl-shaped structures were composed with loose connective tissue, lacked blood vessels, and showed collagen fibers organized in a spiral form. They were anchored by pedicles to the villous surface of the extraembryonic membrane. We discuss the biological and evolutionary meaning of the pearl-shaped structures that relate L. maximus to the African origin of the South American hystricognathe fauna.