Eduardo Juan Gimeno

Lysosomal Storage Disease Caused by Sida carpinifolia Poisoning in Goats

A neurologic disease characterized by ataxia, hypermetria, hyperesthesia, and muscle tremors of the head and neck was observed for 2 years in a flock of 28 Anglo-Nubian and Saanen goats on a farm with 5 ha of pasture. Six newborns died during the first week of life, and five abortions were recorded. The predominant plant in the pasture was Sida carpinifolia. The disease was reproduced experimentally in two goats by administration of this plant. Three goats with spontaneous disease and the two experimental animals were euthanatized and necropsied. No significant gross lesions were observed. Fragments of several organs, including the central nervous system, were processed for histopathology. Small fragments of the cerebellar cortex, liver, and pancreas of two spontaneously poisoned goats and two experimentally poisoned goats were processed for electron microscopy. Multiple cytoplasm vacuoles in hepatocytes, acinar pancreatic cells, and neurons, especially Purkinje cells, were the most striking microscopic lesions in the five animals. Ultrastructural changes included membrane-bound vacuoles in hepatocytes, Kupffer cells, acinar pancreatic cells, Purkinje cells, and the small neurons of the granular cell layer of the cerebellum. Paraffin-embedded sections of the cerebellum and pancreas were submitted for lectin histochemical analysis. The vacuoles in different cerebellar and acinar pancreatic cells reacted strongly to the following lectins: Concanavalia ensiformis, Triticum vulgaris, and succinylated Triticum vulgaris. The pattern of staining, analyzed in Purkinje cells and acinar pancreatic cells coincides with results reported for both swainsonine toxicosis and inherited mannosidosis.

Lysosomal storage disease in Sida carpinifolia toxicosis: an induced mannosidosis in horses

REASONS FOR PERFORMING STUDY This study reports a neurological disease unrecognised until now in ponies in southern Brazil. HYPOTHESIS Epidemiological data strongly suggests that the ingestion of Sida carpinifolia is involved in the aetiology. We tested the hypothesis that it is an acquired lyosomal storage disease. METHODS Following the death of 3 ponies, all ponies from the premises were closely monitored; epidemiological data and clinical findings carefully recorded. Fragments of several organs, including CNS, were fixed in neutral formalin and embedded in paraffin-wax. Sections were stained with haematoxylin and eosin. Representative sections of the cerebellum and trigeminal ganglia were submitted to lectin histochemical procedures. RESULTS The neurological disorder, characterised by stiff gait, muscle tremors, abdominal pain and death, was observed on a farm with 3 hectares of pasture. Three of 11 ponies died 15-20 days after they had been introduced into a new paddock heavily infested by the plant Sida carpinifolia. No significant gross lesions were observed. The main histological findings included multiple cytoplasmatic vacuoles in swollen neurones in the brain, cerebellum, spinal cord, autonomic ganglia (trigeminal and celiac ganglia), and submucosal and myenteric plexus of the intestines. In the kidneys, there was marked vacuolation of the proximal convoluted tubular cells. Sections of cerebellum and trigeminal ganglion were submitted to lectin histochemistry. The vacuoles in different cerebellar and ganglion cells reacted strongly to the following lectins: Concanavalia ensiformis, Triticum vulgaris and succinylated-Triticum vulgaris. CONCLUSIONS The pattern of staining coincides with that of both swainsonine toxicosis and inherited mannosidosis reports. The histopathological changes were similar to those described in S. carpinifolia spontaneous and experimental poisoning in goats. This disease seems to be similar to Swainsona, Oxytropis and Astragalus toxicosis. POTENTIAL RELEVANCE S. carpinifolia should be evaluated as a possible cause in the diagnosis of equine neuropathies.

An imbalance between apoptosis and proliferation contributes to follicular persistence in polycystic ovaries in rats

BackgroundCystic ovarian disease is an important cause of infertility that affects bovine, ovine, caprine and porcine species and even human beings. Alterations in the ovarian micro-environment of females with follicular cysts could alter the normal processes of proliferation and programmed cell death in ovarian cells. Thus, our objective was to evaluate apoptosis and proliferation in ovarian cystic follicles in rats in order to investigate the cause of cystic follicle formation and persistence.MethodsWe compared the number of in situ apoptotic cells by TUNEL assay, expression of active caspase-3 and members of Bcl-2 family by immunohistochemistry; and cell proliferation by the expression of the proliferation markers: PCNA and Ki-67.ResultsThe proliferation index was low in granulosa of tertiary and cystic follicles of light exposed rats when compared with tertiary follicles of control animals, while in theca interna only cystic follicles presented low proliferation index when compared with tertiary follicles (p < 0.05). The granulosa of cysts exhibited a similar cell DNA fragmentation to early atretic follicles. In the granulosa and theca interna, active caspase-3 shown similar immunostaining levels in tertiary and cystic follicles (p < 0.05). The granulosa cells presented high expression of Bcl-2, Bcl-xL and Bcl-w in the tertiary and cystic follicles with diminishing intensity in the atretic follicles, except with Bcl-w where the intensity was maintained in the atretic follicles (p < 0.05). The expression of Bax was weak in the healthy and cystic follicles. In the theca interna, Bcl-2 expression was the same as the pattern found in the granulosa; no differences were found between tertiary and cystic follicles from both groups for Bcl-xL and Bcl-w. The expression of Bax in this layer was higher in the tertiary follicles of the treated animals (p < 0.05) while the values for cystic follicles were similar to those in the tertiary follicles of controls. The theca externa showed low expression of the pro and anti-apoptotic proteins.ConclusionThese results show that the combination of weak proliferation indices and low apoptosis observed in follicular cysts, could explain the cause of the slow growth of cystic follicles and the maintenance of a static condition without degeneration, which leads to their persistence. These alterations may be due to structural and functional modifications that take place in these cells and could be related to hormonal changes in animals with this condition.

Estrogen Receptors α and β and Progesterone Receptors in Normal Bovine Ovarian Follicles and Cystic Ovarian Disease

The purpose of this study was to determine by immunohistochemistry the expression of estrogen and progesterone receptors in ovarian follicular structures from cows with cystic ovarian disease (COD) and to compare these with normal ovarian structures. Secondary, tertiary, atretic, and cystic follicles were evaluated. The follicular cysts of animals with COD presented a significantly higher expression of estrogen receptor α in all follicular layers than secondary, tertiary, and atretic follicles in both groups (P < .05). The intensity of estrogen receptor β in the granulosa cell layer was stronger in tertiary than in secondary and atretic follicles in normal animals (P < .05) and in growing and cystic follicles in animals with COD (P < .05). Theca cells were scarcely stained in the 2 groups. Growing follicles and cysts from COD animals were less stained than tertiary follicles from normal animals (P < .05). Differences did not exist between the 2 groups with regard to the progesterone receptor. Ovaries of animals with COD exhibited altered estrogen receptors expression compared with that in normal animals.

Expression of Cytoskeletal Proteins in the Follicular Wall of Induced Ovarian Cysts

Several experimental models have been developed for the study of the polycystic ovarian syndrome in the rat. In the present study, the syndrome was induced by exposure to constant light, and the expression of cytoskeletal proteins in the follicular wall was evaluated by immunohistochemistry. We analyzed the immunohistochemically stained area (IHCSA) by image analysis to evaluate the expression of intermediate filaments (vimentin, desmin, cytokeratins, gliofibrillary acidic protein and neurofilaments) and α-smooth muscle actin (α-SMA) in cystic ovaries in relation to normal ovaries. The granulosa cell layer of cystic follicles had a significantly greater IHCSA for vimentin than the normal antral follicles. This difference was also significant between atretic and antral follicles. Cytokeratins showed a very low expression in the granulosa cells of antral follicles of control ovaries while in granulosa cells of atretic and cystic follicles they showed a significantly higher IHCSA. Immunohistochemical localization of desmin and α-SMA was restricted to the theca externa. Immunoreactivity for gliofibrillary acidic protein and neurofilament was negative. The highest intensity in the staining with vimentin and cytokeratins observed in the granulosa cells of the cystic follicles is probably due to structural and functional changes that occur during the process of cystogenesis and they could be associated with intense changes in the expression of cytoskeletal proteins that may be essential to the proper cellular functioning.

Aspectos clínicos e patológicos da intoxicação por Sida carpinifolia (Malvaceae) em caprinos no Rio Grande do Sul

This report includes the clinical and pathological studies of a lysosomal storage disease which spontaneously occurred in three flocs of goats e after consumption of Sida carpinifolia, the predominant plant in the paddocks where the animals were grazing. In the outbreaks a total of 25 out of 51 animals were affected. Post-mortem examination was performed on 11 goats. The disease was experimentally induced by dosing goats with Sida carpinifolia. The plant was administered in natura or dried to 3 animals. No clinical or pathological changes were observed in one goat dosed with Sida rhombifolia ad libidum during 40 days. Clinical signs of the poisoning were ataxia, hypermetria, muscle tremors in the head and neck and disorders of deglutition. The clinical signs were exacerbated by movement. After the surviving animals had been moved to other pastures and stopped eating the plant, clinical signs were still observed during 24 months. At necropsy, no significant gross lesions were observed. Microscopic lesions included various degrees of vacuolization in the cytoplasm of neurons and glial cells. Similar lesions were observed in the acinar pancreatic cells, hepatocytes, proximal convoluted tubular cells, follicular epithelial cells of the thyroid gland and macrophages of lymph nodes. In the surviving animals, mild neuronal cytoplasmic vacuolization was observed, and few cells were eosinophilic and shrunken. In these cases neurons, especially Purkinje cells, had disappeared. Through the histochemical study of the cerebellar sections, the lysosomal storage disease was characterized as an alpha-mannosidosis. The vacuoles within the Purkinje cells strongly reacted with lectins of Concanavalia ensiformis, Triticum vulgaris and succinylated Triticum vulgaris. The pattern observed in this investigation is similar to those seen in other poisonings by swainsonine-containing plants.

Morphological characterization of the female prostate (Skene's gland or paraurethral gland) of Lagostomus maximus maximus

The Skenes (paraurethral) gland is the histologic homologue to the male prostate. Much experimental work has been done on women and on various rodents. In this study we describe for the first time the anatomical and histological characteristics of the paraurethral gland in the plain viscacha (Lagostomus maximus maximus). This gland is formed by tubuloalveolar adenomers surrounded by connective tissue and smooth muscle fibers. The adenomers are lined by cuboidal or columnar epithelium. In the lumen of the adenomers, PAS positive secretions could be detected. The conduits are lined by bi-stratified epithelium. In some aspects this gland is similar to that of other mammals.

First egg protein with a neurotoxic effect on mice.

While many invertebrates sequester toxic compounds to endow eggs with chemical defences, here we show, for the first time to our knowledge, the identification of a neurotoxin of proteinaceous nature localized inside an egg. Egg extracts from the freshwater apple snail Pomacea canaliculata displayed a neurotoxic effect in mice upon intraperitoneal injection (i.p.) (LD50, 96h 2.3mg/kg). Egg protein and total lipids were analysed separately and the only fraction displaying a highly toxic effect (LD50, 96h 0.25mg/kg, i.p.) was further purified to homogeneity as an oligomeric glyco-lipoprotein of 400kDa and two subunits biochemically and immunologically indistinguishable from the previously described perivitellin PV2. The neurotoxin was heat sensitive and there was evidence of circulating antibody response to sublethal i.p. doses on mice. Clinical signs, histopathological and immunocytochemical studies revealed damage mostly in mice spinal cord. Experiments showed chromatolysis and a decreased response to calbindin D-28K associated with a significant increase of TUNEL-positive cells in the dorsal horn neurons. These results suggest that calcium buffering and apoptosis may play a role in the neurological disorders induced by the toxin in mammalian central nervous system. This is the first report of a mollusc neurotoxin genetically encoded outside the cone-snail species.

Loss of NeuN immunoreactivity in rat spinal cord neurons during aging

A morphologic study of the impact of aging on neuron marker expression was performed in different segments of the rat spinal cord. Spinal cord specimens from young (5 months), middle-aged (12 months) and senile (32 months) female rats were assessed. We found a complete loss of neuron-specific nuclear protein (NeuN) immunoreactivity in cervical, thoracic and lumbar segments of the senile animals whereas neuron-specific enolase (NSE) immunoreactivity was comparable in young and senile rats. These findings in otherwise morphologically well preserved spinal cord neurons are of interest and reveal that NeuN may not be a reliable marker to identify neurons in the spinal cord of aging rats.

Polycystic ovarian syndrome: temporal characterization of the induction and reversion process in an experimental model

Numerosos modelos experimentais tem sido desenvolvimos para o estudo da sindrome do ovario policistico em ratos. No presente estudo, a sindrome foi inducida por exposicao a luz constante. O processo foi avaliado durante sua inducao e inclusive durante sua reversao. O ciclo estral foi analisado atraves de citologia vaginal; parametros reprodutivos foram avaliados por acasalamento, bem como a morfologia ovariana. Todos animais desenvolveram a sindrome depois de 13 semanas de luz permanente. As caracteristicas histologicas dos ovarios, na semana 15, foram similares aquelas observadas na sindrome do ovario policistico em humanos e outras especies. Apos a regressao da sindrome, nao houve diferenta em nenhum dos parametros reprodutivos avaliados, quando comparados com o grupo controle.