The specific inhibition of the cardiac electrogenic sodium/bicarbonate cotransporter leads to cardiac hypertrophy.

Two alkalinizing mechanisms coexist in cardiac myocytes to maintain intracellular pH: sodium/bicarbonate cotransporter (electroneutral isoform NBCn1 and electrogenic isoform NBCe1) and sodium/proton exchanger (NHE1). Dysfunction of these transporters has previously been reported to be responsible for the development of cardiovascular diseases. The aim of this study was to evaluate the contribution of the downregulation of the NBCe1 to the development of cardiac hypertrophy. To specifically reduce NBCe1 expression, we cloned shRNA into a cardiotropic adeno-associated vector (AAV9-shNBCe1). After 28 days of being injected with AAV9-shNBCe1, the expression and the activity of NBCe1 in the rat heart were reduced. Strikingly, downregulation of NBCe1 causes significant hypertrophic heart growth, lengthening of the action potential in isolated myocytes, an increase in the duration of the QT interval and an increase in the frequency of Ca2+ waves without any significant changes in Ca2+ transients. An increased compensatory expression of NBCn1 and NHE1 was also observed. We conclude that reduction of NBCe1 is sufficient to induce cardiac hypertrophy and modify the electrical features of the rat heart.

Comparative Anatomy of the Forearm and Hand of Wildcat (Leopardus geoffroyi), Ocelot (Leopardus pardalis) and Jaguar (Panthera onca)

Introduction The thoracic limbs of cats facilitate jumping and represent one of their main ways for pursuing and capturing prey. The main muscles and nerves involved in these activities are present in the region of the forearm and of the hand. The scant anatomical reference availabe on South American cats species justifies the present comparative study. Materials and Methods The forelimbs of wildcat, ocelot and jaguar wild felines were fixed. Images of the dissected limbs were captured using a digital camera. Measurements were made using a caliper. Results The long and short heads of the extensor carpi radialis muscle of the ocelot and of the jaguar showed a great development in comparison with those of the wildcat. The flexor digitorum profundus muscle in the three felines is formed by five heads. In the jaguar, the radial or deep head presented two sesamoid bones. The brachioradialis muscle of the jaguar and of the ocelot is insertedmedially at the distal end of the radius and at the proximal row of the carpus by a thick and flattened tendon. The pronator teres muscle of the jaguar extended to the carpal region. In the wildcat and in the ocelot, this muscle was less developed Conclusions The main variations observed between the forearms and the hands of these South American cats were found between the supinator and the pronator muscles, presenting a variation in the size of their bellies and tendons. Our study of the muscular characteristics represents a contribution to the homologies and differences of the forearm and hand muscles of each of these species of felines.

Association between Degree of Anaplasia and Degree of Inflammation with the Expression of COX-2 in Feline Injection Site Sarcomas.

Feline injection site sarcomas (FISSs) are mesenchymal neoplasms that develop at the sites of delivery of vaccines or other injectable products. Vaccine adjuvants can trigger an intense and persistent inflammatory response that may lead to neoplastic transformation. The proinflammatory role of cyclo-oxygenase (COX)-2 is well known and its overexpression has prognostic value in multiple neoplastic processes. One hundred and seventeen FISSs were evaluated for the degree of inflammation and anaplasia. Immunohistochemistry was used to determine the expression of COX-2 in these sarcomas. There was a significant association between the degree of inflammation and the expression of COX-2 by neoplastic cells. COX-2 expression was lower in tumours with higher degrees of anaplasia. These findings may be useful in predicting the sensitivity of FISSs to treatment with COX-2 inhibitors. The potential therapeutic use of such agents could then be restricted to tumours with lower degrees of anaplasia.

Investigation of apoptosis in cultured cells infected with equine herpesvirus 1.

Many viruses alter different stages of apoptosis of infected cells as a strategy for successful infection. Few studies have addressed mechanisms of equine herpesvirus 1 (EHV-1) strain-induced cell death. We investigated the effect of an abortigenic strain (AR8 strain) on heterologous Madin-Darby bovine kidney cells and homologous equine dermis (ED) cells cell lines. We compared morphologic and biochemical features of early and late apoptosis at different postinfection times. We investigated translocation of phosphatidylserine to the cell surface, nuclear fragmentation and changes in the cytoskeleton using flow cytometry and annexin V/propidium iodide staining, DNA laddering, terminal deoxynucleotidyl transferase UTP nick-end labeling assay and immunofluorescence staining of cytokeratin 18 cleavage. AR8 EVH-1 strain interfered with apoptosis in both cell lines, particularly during the middle stage of the replication cycle; this was more evident in ED cells. Although this antiapoptotic effect has been reported for other alpha herpesviruses, our findings may help elucidate how EHV-1 improves its infectivity during its cycle.

Canine mammary carcinomas: influence of histological grade, vascular invasion, proliferation, microvessel density and VEGFR2 expression on lymph node status and survival time.

Spontaneous invasive non-inflammatory canine mammary carcinomas (CMC) and their regional lymph nodes (LN) were analysed (n = 136). Histological grade (HG) and vascular invasion (VI) in the tumours and lymph node status were recorded. Proliferation index (PI), microvessel density (MVD) and vascular endothelial growth factor receptor 2 (VEGFR2) expression were estimated using anti-proliferating cell nuclear antigen (PCNA), anti-von Willebrand factor and anti-Flk-1, respectively. Eighteen months follow-up was performed (34 bitches). Tumours of different grades showed differences regarding PI, Flk-1/integrated optical density (Flk-1/IOD) and MVD. Every feature showed significant association with LN status through bivariate analyses. From multivariate analyses, VI and Flk-1/IOD were selected to predict LN status. Data revealed that the probability of a CMC-bearing bitch to remain alive at 1, 4, 5 and 14-18 months was 0.91, 0.87, 0.81 and 0.77, respectively. Besides LN status, VI was the only feature positively correlated with survival time, although a trend to shorter survival of animal patients bearing high expressing VEGFR2 CMC was noted.

Microvascular lesions and changes in cell proliferation and death, and cytokine expression in the placentas of mice experimentally infected with Equid Herpesvirus 1.

This study describes the changes observed in the placentas of mice experimentally infected with an abortigenic strain of EHV-1 at mid-pregnancy and euthanized at days 3 and 4 post-infection. We analyzed microscopic vascular alterations, cell proliferation and death by immunohistochemistry, and the expression of IFN-γ, TNF-α and the IL-10 by qPCR and flow cytometry. Infected mice showed slight respiratory signs and ruffled fur during the first two days post-infection. Virus isolation and DNA detection were positive only in the lungs of the infected mice. Vascular congestion, increase in the labyrinth area, and a significant reduction in fetal capillary endothelium surface of infected placentas were found. Cell proliferation was significantly reduced in the infected placentas, whereas the apoptosis was significantly increased. IL10, TNF and IFN-γ showed different expression in the infected placentas and uteri. The effects of EHV-1 during pregnancy depend on different pathogenic mechanisms in which vascular alterations, and cell death and proliferation and local cytokine changes are compromised.

Protective effects of intranasal immunization with recombinant glycoprotein d in pregnant BALB/c mice challenged with different strains of equine herpesvirus 1.

Equine herpesvirus (EHV)-1 induces respiratory infection, neurological disorders and abortion in horses. Most of the currently available attenuated or inactivated vaccines against this infection are administered intramuscularly and only provide partial protection against the respiratory disease. The present study examines the effect of intranasal immunization with purified EHV-1 recombinant glycoprotein D (gD) in BALB/c mice followed by challenge with three different EHV-1 strains during early to mid-pregnancy. The induced viral infection was evaluated by virus isolation, DNA detection by polymerase chain reaction, histopathology and immunohistochemical localization of antigen in the lung, placenta and uterus. Non-immunized mice showed clinical signs of infection, positive virus isolation from lungs and uteri, and abortion induced by one of the virus strains. Endometrial lesions developed in some of these animals that have been described previously only in horses. Immunized mice and their offspring had no viral infection or typical lesions. Intranasally administered gD therefore induced partial or complete protection against three different EHV-1 strains in BALB/c mice.

Presence of c-kit positive cells in fetal and adult bovine forestomachs.

The interstitial cells of Cajal (ICC) have been reported to regulate gastrointestinal motility. We investigated the distribution and the morphological and morphometric characteristics of the immunohistochemical reaction against c-kit in the forestomachs of fetal, newborn and adult cows. The anti-c-kit reaction revealed different populations of ICC among age groups and organs. ICC were more numerous and smaller in fetuses. Larger ICC were identified in newborns, except for those in the rumen. During the earliest stages of development, ICC were abundant in the inner layer of the muscularis and were consistently associated with this layer. In all samples, ICC were found in the outer layer of the tunica muscularis. ICC were found between the two muscle layers in the omasum at all ages; however, they were identified only in the rumen of the adult. Our study demonstrated that ICC are present in the forestomach of bovines.

Intermediate filament proteins expression and carbohydrate moieties in trophoblast and decidual cells of mature cat placenta.

The aim of this study was to characterize cytoskeletal intermediate filament proteins and glycoconjugates of syncytiotrophoblast, cytotrophoblast and decidual cells of feline endotheliochorial placenta. Samples from 12 normal pregnant female cats, after 45 ± 5 days of gestation, were obtained removing the uterine horns by hysterectomy. Sections were processed for routine observation and for immunohistochemistry using anticytokeratin, antivimentin and antidesmin antibodies. In addition, lectin histochemistry was performed using a panel of several biotinylated lectins to characterize glycosides expression profile. Cytotrophoblast and syncytiotrophoblast showed immunoreactivity only with acidic and basic cytokeratins. Decidual cells were only positive to vimentin, consistent with their origin from endometrial fibroblasts. Trophoblast expressed a broad population of glycans, highly exposing terminal N-acetyl glucosamine residues and non-sialylated galactose and N-acetyl galactosamine oligomers. Oligosaccharides bound by Phaseolus vulgaris erythroagglutinin were the only highly branched N-linked residues evidenced in cats, and they were restricted to the syncytium. Unlike results reported on humans, mice and rats on lectin affinity of decidual cells, sialid acids and complex N-linked oligosaccharides were not demonstrated in cats. Glycosylation of proteins determines many of their final properties, thus becoming essential for the embryo-maternal dialogue during implantation and placentation. Changes in glycosylation pattern have been related to pathological pregnancies in other species. Hence, the knowledge about glycosylation profile of the normal cat placenta may lead to a better understanding of both normal and pathological reproductive events.

Anatomical study of the forearm and hand nerves of the domestic cat ( Felis catus), puma ( Puma concolor) and jaguar ( Panthera onca).

The innervation of the forearm and hand regions of cats has not been well described despite its importance for any surgery or any neurological disorder. It is probably the main area where disorders of peripheral nerves in this species are observed. In felines, the forelimbs facilitate the jump and represent the most important way for capturing prey. The main muscles and nerves involved in this activity are located in the region of the forearm and hand. The aim of the present study was to provide a detailed description of the innervation of the forearm and hand regions of the jaguar and puma, in comparison with that of the domestic cat, contributing thus with the anatomical knowledge of the area for applying it to surgery and pathology. The forearms of three pumas and two jaguars (all of them fixed in formalin) and of six domestic cats (fresh) were dissected. The nerves path and their forearm distribution patterns of all three species were described. The analysed results indicate that the observed variations between species are minimal; thus, the anatomy described for domestic cats can be widely applied to American wild felids.

Food odor, visual danger stimulus, and retrieval of an aversive memory trigger heat shock protein HSP70 expression in the olfactory lobe of the crab Chasmagnathus granulatus.

Although some of the neuronal substrates that support memory process have been shown in optic ganglia, the brain areas activated by memory process are still unknown in crustaceans. Heat shock proteins (HSPs) are synthesized in the CNS not only in response to traumas but also after changes in metabolic activity triggered by the processing of different types of sensory information. Indeed, the expression of citosolic/nuclear forms of HSP70 (HSC/HSP70) has been repeatedly used as a marker for increases in neural metabolic activity in several processes, including psychophysiological stress, fear conditioning, and spatial learning in vertebrates. Previously, we have shown that, in the crab Chasmagnathus, two different environmental challenges, water deprivation and heat shock, trigger a rise in the number of glomeruli of the olfactory lobes (OLs) expressing HSC/HSP70. In this study, we initially performed a morphometric analysis and identified a total of 154 glomeruli in each OL of Chasmagnathus. Here, we found that crabs exposed to food odor stimuli also showed a significant rise in the number of olfactory glomeruli expressing HSC/HSP70. In the crab Chasmagnathus, a powerful memory paradigm based on a change in its defensive strategy against a visual danger stimulus (VDS) has been extensively studied. Remarkably, the iterative presentation of a VDS caused an increase as well. This increase was triggered in animals visually stimulated using protocols that either build up a long-term memory or generate only short-term habituation. Besides, memory reactivation was sufficient to trigger the increase in HSC/HSP70 expression in the OL. Present and previous results strongly suggest that, directly or indirectly, an increase in arousal is a sufficient condition to bring about an increase in HSC/HSP70 expression in the OL of Chasmagnathus.

Morphology, morphometry, histochemistry and lectin histochemistry of the vagina of the plains viscacha (Lagostomus maximus).

The aim of the present work was to describe the morphology of the vagina in Lagostomus maximus and to characterize its epithelial cells using morphometric and histochemical techniques (variations of PAS, Alcian blue and lectin histochemistry). Thirty-five sexually mature adult females were captured in their natural environment during four periods of the year and their genital organs were dissected. The vaginal wall of the viscacha has three tunics: mucosa, muscularis and adventitia or serosa according to the region. The epithelium is stratified in both cranial and caudal regions, but its characteristics vary depending on the physiological state. In anestrous, nonpregnant females have a stratified epithelium of two to three cellular layers with columnar PAS-positive superficial mucous cells. During the follicular phase, the epithelium of the vagina is stratified squamous and cornified. Females at early, middle and term pregnancy have a columnar stratified epithelium with mucous cells. Glycoproteins in the mucous cells were detected using PAS, PA*S, KOH/PA*/BH/PAS; and Alcian blue, pH 0.5, pH 1, pH 2.5 and 0.006 M). Lectin histochemistry showed that UEA-I and RCA-1 lectins reacted strongly or moderately with epithelial cells in all stages analyzed. These results indicate the presence of L-fucose and ß-galactose. Binding with other lectins was variable.

Effects of Solanum glaucophyllum toxicity on cell proliferation and apoptosis in the small and large intestine of rabbits.

Vitamin D regulates mineral homeostases and enterocyte proliferation and differentiation. Hypervitaminosis D generates changes in cell proliferation, differentiation and apoptosis in several organs. We analysed morphometric parameters and proliferative and apoptotic indices in the intestinal epithelium of rabbits with hypervitaminosis D induced by the chronic treatment with the calcinogenic plant Solanum glaucophyllum. Rabbits were treated for 15 or 30 days. A group was treated for 15 days and led to possible recovery for 30 days. Another group was nutritionally restricted for 30 days. Morphological, morphometric, proliferative and apoptotic changes were found in the treated animals. Mild atrophy and reduced proliferation was found in the jejunum and ileum. Apoptosis increased in the crypts of the ileum and in the superficial epithelium and crypts of the rectum. Most of the alterations were partially recovered. The possible involvement in these changes of the hypervitaminosis D-like state induced by S. glaucophyllum is discussed.

Chorioallantoic and yolk sac placentation in the plains viscacha (Lagostomus maximus) - a caviomorph rodent with natural polyovulation.

OBJECTIVES: Reproduction in the plains viscacha is characterized by the polyovulation of hundreds of oocytes, the loss of implantation and the development of 1-3 offspring. Our goal was to determine whether placental development was affected by these specializations. STUDY DESIGN: Thirteen placentas from early pregnancy to near-term pregnancy were analyzed using histological, immunohistochemical and transmission electron microscopy. RESULTS: An inverted, villous yolk sac was present. Placentas were formed by the trophospongium, labyrinth and subplacenta. A lobulated structure with a hemomonochorial barrier was established early in pregnancy. Proliferating trophoblast that was clustered at the outer border and inside the labyrinth was responsible for placental growth. Trophoblast invasion resulted from the cellular trophoblast and syncytial streamers derived from the subplacenta. Different from other caviomorphs, numerous giant cells were observed. CONCLUSIONS: The principle processes of placentation in caviomorphs follow an extraordinarily stable pattern that is independent of specializations, such as polyovulation.

Restorative effect of intracerebroventricular insulin-like growth factor-I gene therapy on motor performance in aging rats.

Insulin-like growth factor-I (IGF-I) is a powerful neuroprotective molecule in the brain and spinal cord. We have previously shown that intracerebroventricular (i.c.v.) IGF-I gene therapy is an effective strategy to increase IGF-I levels in the cerebrospinal fluid (CSF). Since aging in rats is associated with severe motor function deterioration, we implemented i.c.v. IGF-I gene therapy in very old rats (30-31 months) and assessed the beneficial impact on motor performance. We used recombinant adenovectors (RAds) expressing either green fluorescent protein (GFP) or rat IGF-I. Injection in the lateral or fourth ventricle led to high transgene expression in the ependymal cell layer in the brain and cervical spinal cord. RAd-IGF-I-injected rats but not RAd-GFP-injected controls, showed significantly increased levels of CSF IGF-I. Motor tests showed the expected age-related decline in aged rats. Seventeen-day IGF-I gene therapy induced a significant improvement in motor performance in the aged but not in the young animals. These results show that IGF-I is an effective restorative molecule in the aging brain and spinal cord. The data also reveal that the ependymal route constitutes a promising approach for implementing protective IGF-I gene therapy in the aging CNS.

Glycoconjugate histochemistry in the small and large intestine of normal and Solanum glaucophyllum-intoxicated rabbits.

Vitamin D participates in mineral homeostasis, immunomodulation, cell growth and differentiation. The leaves of Solanum glaucophyllum contain high levels of 1,25-dihydroxyvitamin D3 as glycoside derivatives and their chronic ingestion generates a hypervitaminosis D-like state. We analyzed changes on carbohydrate expression as a cell differentiation indicator on samples of the small and large intestine of S. glaucophyllum-intoxicated rabbits, using conventional and lectin histochemistry. Male New Zealand white rabbits were intoxicated with S. glaucophyllum during two or four weeks and killed the day after. A group of animals ("possibly recovered group") were intoxicated during 15 days and killed at day 45 of the beginning of the experiment. We found changes in the lectin binding pattern in the small and large intestine of the intoxicated rabbits. Some of these changes were reverted in the possibly recovered group. Vitamin D could be a new regulator factor of the intestinal glycosylation process.

Calbindin D28k expression and the absence of apoptosis in the cerebellum of Solanum bonariense L-intoxicated bovines.

Solanum bonariense intoxication is characterized by cerebellar neuronal vacuolation, degeneration, and necrosis. Cerebellar Purkinje cells seem especially susceptible, but more research is needed to determine the pathogenesis of neuronal necrosis and the mechanism of Purkinje cell susceptibility. Calbindin D28k (CbD28k) is highly expressed in Purkinje cells and has been used as a marker for normal and degenerative Purkinje cells. The goal of this study was to describe S bonariense-induced disease by ascertaining Purkinje cell-specific degenerative changes using CbD28k expression and to correlate this with apoptosis in Purkinje cells, as determined using TUNEL (transferase-mediated dUTP-biotin nick end-labeling) and ultrastructural changes. In all cases, an increase in both dose and duration of S bonariense intoxication resulted in a decrease in the number of Purkinje cells. CbD28k immunohistochemistry was an excellent marker for Purkinje cells because immunoreactivity did not change in normal or degenerative tissues. This finding suggests that excessive calcium excitatory stimulation does not induce rapid neuronal degeneration and death. As found in previous studies, TUNEL tests and electron microscopy suggest that Purkinje cell degeneration and death are not occurring via an apoptotic process. These findings suggest that S bonariense poisoning induces progressive Purkinje cell death that is not mediated by excitotoxicity or apoptotic activation.

The ependymal route for insulin-like growth factor-1 gene therapy in the brain.

I.c.v. administration of the peptide insulin-like growth factor-1 (IGF-1) has been shown to be an effective neuroprotective strategy in the brain of different animal models, a major advantage being the achievement of high concentrations of IGF-1 in the brain without altering serum levels of the peptide. In order to exploit this therapeutic approach further, we used high performance recombinant adenoviral (RAd) vectors expressing their transgene under the control of the potent mouse cytomegalovirus immediate early (mCMV) promoter, to transduce brain ependymal cells with high efficiency and to achieve effective release of transgenic IGF-1 into the cerebrospinal fluid (CSF). We constructed RAd vectors expressing either a chimeric green fluorescent protein fused to HSV-1 thymidine kinase (TK/GFP)(fus), or the cDNA encoding rat IGF-1, both driven by the mCMV promoter. The vectors were injected into the lateral ventricles of young rats and chimeric GFP expression in brain sections was assessed by fluorescence microscopy. The ependymal cell marker vimentin was detected by immunofluorescence and nuclei were labeled with the DNA dye 4',6-diamidino-2-phenylindole. Blood and CSF samples were drawn at different times post-vector injection. In all cerebral ventricles, vimentin immunoreactive cells of the ependyma were predominantly transduced by RAd-(TK/GFP)(fus), showing nuclear and cytoplasmic expression of the transgene. For tanycytes (TK/GFP)(fus) expression was evident in their cytoplasmic processes as they penetrated deep into the hypothalamic parenchyma. I.c.v. injection of RAd-IGF-1 induced high levels of IGF-1 in the CSF but not in serum. We conclude that the ependymal route constitutes an effective approach for implementing experimental IGF-1 gene therapy in the brain.

Morphological and histochemical characteristics of the epithelium of ovarian lamellae of Genypterus blacodes (Schneider, 1801).

The physiological significance of the glycoproteins (GPs) secreted by the epithelium of ovarian lamellae is discussed in reference to the reproductive biology of G. blacodes. Histochemical procedures for localising and characterising GPs were used to determine the cytoplasmic components of cells of the epithelium that covers the ovarian lamellae of pink cuskeel, Genypterus blacodes (Schneider, 1801) (Pisces, Ophidiidae), during spawning. This species is one of the most valuable demersal fish resources in the Argentine Sea, mainly due its large size and flesh quality. GPs with oxidizable vicinal diol groups, sialic acid with or without O-acyl substituents, O-acyl sugars, neutral sugars and GPs with carboxyl and sulphate groups were detected. Light microscope examination showed morphological changes in the epithelium of ovarian lamellae during the spawning season, associated with a secretory activity of mucus. Optical density studies revealed the presence of polyploid cells encompassing those morphological changes. Results of the present study suggest that the epithelium of ovarian lamellae of G. blacodes performs a secretory role, which is intensified during ovarian maturity, suggesting that G. blacodes could release masses of eggs enveloped in mucus.

Paneth cells: histochemical and morphometric study in control and Solanum glaucophyllum intoxicated rabbits.

The intestinal epithelium has a critical roll in host defence. One specialised cell type involved in this function is the Paneth cell, which secretes many substances with antimicrobial properties in response to different stimuli. Under pathological conditions, changes in the Paneth cell number, morphology and location as well as in granule number, morphology and composition have been reported. In the normal animal, 1,25-dihydroxyvitamin D3 participates in the maintenance of mineral homeostasis, immunomodulation and cell proliferation and differentiation. Solanum glaucophyllum, a calcinogenic plant containing high levels of 1,25-dihydroxyvitamin D3, is responsible for a condition known as enzootic calcinosis in ruminants, characterised by loss of body condition and mineralization of soft tissues. Using and established rabbit model, this study analyses the changes that rabbit Paneth cells undergo during intoxication with S. glaucophyllum. Male New Zealand white rabbits were experimentally intoxicated with S. glaucophyllum for 15 or 30 days. Lectin, immunohistochemical and morphometric studies were carried out on Paneth cells from samples of jejunum. SBA, DBA and WGA lectins bound to Paneth cells-granules in both normal and intoxicated rabbits, with more heterogenity in the labelling of granules from intoxicated rabbits. Paneth cells in both groups were immunonegative for lysosyme. A time and dose-dependent increase in the size and number of Paneth cells was found in both intoxicated groups. We suggest that the changes described in these cells may be directly or indirectly induced by S. glaucophyllum intoxication.