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.

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.

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.

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.

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.

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.

Histological and lectin binding changes in the genital tract of mice infected with Tritrichomonas foetus.

An experimental murine model of bovine genital tritrichomonosis is described. Female mice were inoculated per vaginam with Tritrichomonas foetus and a sample of the study population was killed every 3 days up to 60 days post-infection. Microscopical changes in the reproductive organs were assessed and immunohistochemistry was used to detect T. foetus within these tissues. Lectin histochemistry was used to determine changes in the expression of carbohydrates within the reproductive mucosa. A range of microscopical changes were detected in the uterine endometrium by 10 days post-inoculation and these were associated with the presence of the protozoan. The endometrial changes included endometritis and ulceration, mucosal atrophy and glandular metaplasia, and were similar to those reported in naturally infected cows. Changes in lectin binding were recognized first in the vagina where there was increased binding of Ulex europaeus agglutinin-1 (UEA-1) which was maximal on day 16 post-inoculation. Within the uterus, there was increased binding of soy bean agglutinin (SBA) which was maximal on day 19 post-inoculation, and of peanut agglutinin (PNA) which was maximal on day 16 post-inoculation. These changes in carbohydrate expression parallel the infection kinetics, since they appeared first in the vagina and later in the uterus. The changes may reflect either a host reaction against the infection or the production of enzymes by T. foetus, which act to enhance adhesion and colonization of the genital organs by the organism. The kinetics and pathogenesis of this murine infection are similar to those of the natural bovine disease, suggesting that this model system may be valuable for further studies of this disease.

Restorative effect of insulin-like growth factor-I gene therapy in the hypothalamus of senile rats with dopaminergic dysfunction.

Insulin-like growth factor-I (IGF-I) is emerging as a powerful neuroprotective molecule that is strongly induced in the central nervous system after different insults. We constructed a recombinant adenoviral vector (RAd-IGFI) harboring the gene for rat IGF-I and used it to implement IGF-I gene therapy in the hypothalamus of senile female rats, which display hypothalamic dopaminergic (DA) neurodegeneration and as a consequence, chronic hyperprolactinemia. Restorative IGF-I gene therapy was implemented in young (5 months) and senile (28 months) female rats, which received a single intrahypothalamic injection of 3 x 10(9) plaque-forming units of RAd-betagal (a control adenoviral vector expressing beta-galactosidase) or RAd-IGFI and were killed 17 days post-injection. In the young animals, neither vector modified serum prolactin levels, but in the RAd-IGFI-injected senile rats a nearly full reversion of their hyperprolactinemic status was recorded. Morphometric analysis revealed a significant increase in the total number of tyrosine hydroxylase-positive cells in the hypothalamus of experimental as compared with control senile animals (5874+/-486 and 3390+/-498, respectively). Our results indicate that IGF-I gene therapy in senile female rats is highly effective for restoring their hypothalamic DA dysfunction and thus reversing their chronic hyperprolactinemia.

Characterization of interstitial cells of Cajal in bowel of cattle (Bos taurus).

Interstitial cells of Cajal (ICC) have been described in the gastrointestinal tract of different mammals including humans, horses, pigs, rats, dogs, mice and guinea-pigs. In the present study, ICC were identified in the jejunum of Bos taurus using polyclonal anti-c-Kit antibodies in immunohistochemical assays. Vimentin and desmin intermediate filaments were also determined using monoclonal antibodies. ICC were found in the tunica muscularis either in a palisade distribution pattern between the outer longitudinal and the inner circular layers (ICC-MP) or freely distributed in clusters in the longitudinal layer (ICC-LM). Morphometric studies determined that ICC have a fusiform shape presenting cytoplasmic prolongations. ICC were positive to c-Kit and vimentin antigens but negative to desmin. We have observed and described for the first time the presence of ICC in a ruminant. As observed in the aforementioned mammals, bovine ICC were associated with the myenteric plexus. Nevertheless, the presence of widespread ICC in the longitudinal muscular layer of the jejunum differs from previously described studies of other mammals.

Lectin binding patterns in hyperplastic and metaplastic bullock prostate tissues after diethylstilbestrol administration.

Hyperplasia and squamous metaplasia of the prostatic epithelium are conditions induced by oestrogens. Diethylstilbestrol (DES) has been banned from cattle used for beef production because of the health risks. The potential use of molecular markers for the detection of illegal oestrogen administration was evaluated by taking samples of prostatic tissue from control bullocks, bullocks which had been treated with oestrogens, and bullocks sacrificed 21 and 90 days after a single dose of DES. The expression of the glycoconjugates was examined by lectinhistochemistry and the lectin binding pattern was characterised in epithelium and connective tissue. In the animals sacrificed after 21 days there was an increase in the binding of one lectin (JAC) and there was an increase in the binding of one of the other lectins (DBA) in the animals sacrificed after 90 days. An increase in SWGA lectin staining was observed in the bullocks that had probably been treated with oestrogen and in the animals sacrificed 90 days after the inoculation with DES. There were also differences between the binding of SWGA in the control bullocks and the other groups.

Cutaneous lymphangiosarcoma in a young dog: clinical, anatomopathological and lectinhistochemical description.

Lymphangiosarcoma is a rare tumour in domestic animals arising from lymphatic endothelial cells. Occasionally, microscopic differentiation with haemangiosarcoma may be difficult. The aim of the present study was to describe a lymphangiosarcoma in a 1-year-old female Doberman Pinscher dog and to characterize its lectinhistochemical binding pattern as compared with that of haemangiosarcoma. The dog was presented because of a cutaneous painful swelling located in the left axilla. Histological diagnosis confirmed lymphangiosarcoma. The dog was killed. Necropsy revealed mediastinal lymph nodes' involvement. Twenty lectins were tested in tissue sections of this case as well as in four haemangiosarcomas from other dogs. Staining intensity was issued upon optical density determinations. Percentage of lectinhistochemical staining area was also conducted. RCA-I showed the most intense and wide distributed labelling pattern for lymphangiosarcoma. PHA-E was the counterpart for haemangiosarcoma. Should similar results be obtained in further studies, such differences could aid in the differential diagnosis between lymphangiosarcoma and haemangiosarcoma when histological pictures were not conclusive.

Normal phagocytic capacity of macrophages isolated from different canine organs.

Conventional methods used in laboratory animals for collection of resident macrophages (MAC) proved suitable for isolation of hepatic, splenic, dermic, peritoneal and alveolar MAC of healthy dogs. Phagocytic assays of IgG opsonised SRBC were performed with resident MAC obtained from these organs. Results showed the existence of statistically significant differences among phagocytic capacity of the investigated MAC. According to this, three groups of MAC could be defined: a first group comprising dermic, splenic and alveolar MAC; a second group comprising hepatic MAC and a third group comprising peritoneal MAC.