Carolina Natalia Zanuzzi

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

Abstract 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 30days. A group was treated for 15days and led to possible recovery for 30days. Another group was nutritionally restricted for 30days. 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.

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.

Inflammation Controls Sensitivity of Human and Mouse Intestinal Epithelial Cells to Galectin-1

Galectins play key roles in the inflammatory cascade. In this study, we aimed to analyze the effect of galectin‐1 (Gal‐1) in the function of intestinal epithelial cells (IECs) isolated from healthy and inflamed mucosa. IECs isolated from mice or patients with inflammatory bowel diseases (IBD) were incubated with different pro‐inflammatory cytokines, and Gal‐1 binding, secretion of homeostatic factors and viability were assessed. Experimental models of food allergy and colitis were used to evaluate the in vivo influence of inflammation on Gal‐1 binding and modulation of IECs. We found an enhanced binding of Gal‐1 to: (a) murine IECs exposed to IL‐1β, TNF, and IL‐13; (b) IECs from inflamed areas in intestinal tissue from IBD patients; (c) small bowel of allergic mice; and (d) colon from mice with experimental colitis. Our results showed that low concentrations of Gal‐1 favored a tolerogenic micro‐environment, whereas high concentrations of this lectin modulated viability of IECs through mechanisms involving activation of caspase‐9 and modulation of Bcl‐2 protein family members. Our results showed that, when added in the presence of diverse pro‐inflammatory cytokines such as tumor necrosis factor (TNF), IL‐13 and IL‐5, Gal‐1 differentially promoted the secretion of growth factors including thymic stromal lymphopoietin (TSLP), epidermal growth factor (EGF), IL‐10, IL‐25, and transforming growth factor (TGF‐β1). In conclusion, we found an augmented binding of Gal‐1 to IECs when exposed in vitro or in vivo to inflammatory stimuli, showing different effects depending on Gal‐1 concentration. These findings highlight the importance of the inflammatory micro‐environment of mucosal tissues in modulating IECs susceptibility to the immunoregulatory lectin Gal‐1 and its role in epithelial cell homeostasis. J. Cell. Physiol. 231: 1575–1585, 2016.

Calcium-calmodulin dependent protein kinase mediates the intracellular signaling pathways of cardiac apoptosis in mice with impaired glucose tolerance

Spontaneous sarcoplasmic reticulum (SR) Ca2+ release events increased in fructose‐rich diet mouse (FRD) myocytes vs. control diet (CD) mice, in the absence of significant changes in SR Ca2+ load. In HEK293 cells, hyperglycaemia significantly enhanced [3H]ryanodine binding and Ca2+/calmodulin‐dependent protein kinase II (CaMKII) phosphorylation of RyR2‐S2814 residue vs. normoglycaemia. These increases were prevented by CaMKII inhibition. FRD significantly augmented cardiac apoptosis in WT vs. CD‐WT mice, which was prevented by co‐treatment with the reactive oxygen species scavenger Tempol. Oxidative stress was also increased in FRD‐SR‐autocamide inhibitory peptide (AIP) mice, expressing the SR‐targeted CaMKII inhibitor AIP, without any significant enhancement of apoptosis vs. CD‐SR‐AIP mice. FRD produced mitochondrial swelling and membrane depolarization in FRD‐WT mice but not in FRD‐S2814A mice, in which the CaMKII site on ryanodine receptor 2 was ablated. FRD decreased mitochondrial area, mean Feret diameter and the mean distance between SR and the outer mitochondrial membrane vs. CD hearts. This remodelling was prevented in AC3I mice, with cardiac‐targeted CaMKII inhibition.

Effects of Different Anesthetics in the Murine Model of EHV-1 Infection

Mice are commonly used as an experimental model to investigate the Equid herpesvirus 1 (EHV-1) infection. This model easily reproduces the disease, and the clinical signs are more or less similar to those observed in the horse, the natural host. During natural infection, the acute course of respiratory infection is mandatory for the development of adaptive immune response. Since interactions between EHV-1 and anesthetics are possible, the study investigated whether the early events of murine pulmonary immune response could be affected by different anesthetics. Therefore, mice were experimentally infected with a unique EHV-1 strain under the effects of ether, ketamine/xylazine, or isoflurane. Clinical signs and histopathological lesions in the lungs were described, and the cell death and proliferation rates of sham-inoculated or infected animals were quantified using immunohistochemistry. Clinical signs were more severe in animals anesthetized with ether. Qualitative differences in the recruited inflammatory cells were observed following application of anesthesia. The level of infection between the infected groups was not statistically significant. However, lungs from ketamine/xylazine-anesthetized animals showed the highest cell death rates, whereas those from isoflurane-anesthetized animals showed the highest proliferation rates. It has been emphasized that anesthetics alone or their interactions with EHV-1 modify the response against the infection. An appropriate selection of the anesthetic during experimental studies is relevant to minimize wrong conclusions.

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.

Paneth Cell Identification in the Small Intestine of Guinea Pig Offsprings (Cavia porcellus)

The aim of this study was to determine the presence, number, and morphometrical characteristics of Paneth cells (PC) in the small intestine of guinea pigs during lactation. We used 48 pups from 0 to 15 days old. Samples from small intestine were fixed in 10% buffered formaldehyde (pH 7.4) and processed for histological and morphometrical studies using hematoxylin and eosin (HE), Phloxine tartrazine or Massons Trichome staining, or immunohistochemistry for lysozyme. PC were morphologically identified at day 2 using Massons Trichome or Phloxine tartrazine stainings, and at day 4 using HE, whereas using immunohistochemistry they were recognized from birth. Morphometrical differences were found between the intestinal sections at each age studied, and within each section during the first weeks of life. In all developmental stage, the highest number of PC was observed in the duodenum of 13 days old guinea pigs. Our results confirm the presence of PC in the small intestine of guinea pigs from birth. Anat Rec, 297:856–863, 2014.

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.

Gut Permeability and Glucose Absorption Are Affected at Early Stages of Graft Rejection in a Small Bowel Transplant Rat Model

Background Intestinal transplantation (ITx) faces many challenges due to the complexity of surgery and to the multiple immunological reactions that lead to the necessity of rigorous follow-up for early detection of acute cellular rejection (ACR). Our aim was to determine the kinetics of ACR using an experimental ITx model, with emphasis in the characterization of the process using different approaches, including the use of functional assays of absorptive and barrier function. Methods ITx in rats conducting serial sampling was performed. Clinical monitoring, graft histology, proinflammatory gene expression, and nitrosative stress determination were performed. Also, glucose absorption, barrier function using ovalbumin translocation, and contractile function were analyzed. Results The model used reproduced the different stages of ACR. Allogeneic ITx recipients showed signs of rejection from postoperative day (POD) 5, with increasing severity until 12 POD. Histological evaluation showed mild rejection in early sampling and severe rejection at late stages, with alterations in all graft layers. IL-6, CXCL 10, IFNg, and nitrite plasmas levels showed behavior coincident with histopathology. Remarkably, allogeneic grafts showed a marked alteration of glucose absorptive capacity from POD 5 that was sustained until endpoint. Coincidently, barrier function alteration was evidenced by luminal ovalbumin translocation to serum. Contractile function was progressively impaired along ACR. Conclusions Glucose absorption and barrier function are altered at early stages of ACR when histological alterations or gene expression changes were much subtle. This observation may provide simple evaluation tools that could be eventually translated to the clinics to contribute to early ACR diagnosis.

Neurons of the rat cervical spinal cord express vimentin and neurofilament after intraparenchymal injection of kainic acid

Intermediate filaments (IF) can be altered under disorders such as neurodegenerative diseases. Kainic acid (KA) induce behavioral changes and histopathological alterations of the spinal cord of injected rats. Our goal was to evaluate the IF expression in neurons during this injury model. Animals were injected with KA at the C5 segment of the cervical spinal cord and euthanized at 1, 3 and 7 post injection (pi) days. Neuronal cell counting showed a significant loss of neurons at the injection site when compared with those of sham and non-operated animals. Immunohistochemistry for vimentin and neurofilament showed positive labeling of perikarya in sham and KA-injected animals since day 1 pi that lasted for the remaining experimental days. Colocalization analysis between enolase and vimentin or neurofilament confirmed a high index of colocalization in both experimental groups at day 1 pi. This index decreased in sham animals by day 3 pi whereas that of KA-injected animals remained high throughout the experiment. These results may suggest that perikarya initiate an unconventional IF expression, which may respond to the neuronal damage induced by the mechanical injury and the excitotoxic effect of KA. It seems that vimentin and neurofilament expression may be a necessary change to promote recovery of the damaged tissue.