Extensive nonhomologous meiotic synapsis between normal chromosome axes of an rcp(3;6)(p14;q21) translocation in a hairless Mexican boar.

Due to its low fertility, expressed as small litter size, a Mexican hairless boar was subjected to cytogenetic investigation. Analysis of G-banded mitotic chromosomes revealed a reciprocal chromosome translocation, rcp(3;6) (p14;q21). Synaptonemal complex analysis showed a regular pairing behavior of the translocation chromosome axes, always resulting in a quadrivalent configuration. However, due to extensive nonhomologous pairing between the axes of nonderivative chromosomes 3 and 6, the quadrivalent mostly had an asymmetrical cross-shaped morphology. The nonhomologous pairing occurred not only at mid and late pachytene, but also at the earliest stage of pachytene. It seems that early pachytene heterosynapsis is a common phenomenon in the pairing behavior of pig reciprocal translocations. Therefore, heterosynapsis may reduce apoptosis of germ cells due to partial absence of homologous synapsis during the pairing phase of meiosis. The frequency of spermatocytes showing quadrivalent configurations with unpaired axial segments apparently did not affect germ cell progression in the boar, since fairly normal testicular histology was noticed.

Ultrastructural analysis of guided nerve regeneration using progesterone- and pregnenolone-loaded chitosan prostheses.

Recently, numerous guide chambers for the treatment of injured nerves made up of different biomaterials have been designed, capable of hosting living cells or carrying neurotrophic or neuroactive substances to be directly released to the injured tissue. In this study, chitosan prostheses containing neurosteroids (progesterone and pregnenolone) were used for bridging a 10-mm gap in the rabbit facial nerve. Gas chromatography was used to quantify neurosteroid content in the prostheses prior to and after subcutaneous implantation at different periods of up to 60 days. The regeneration of the nerve fibers were evaluated at 15 and 45 days after axotomy by means of ultrastructural morphometric analysis. Different nerve fibers regenerative patterns were seen depending the groups studied and the analyzed stages. At 15 days after axotomy, the newly regenerating tissue revealed Schwann cells holding nonmyelinated nerve fiber bundles in an incipient and organized regenerative pattern. At 45 days, the regenerating tissue showed myelinated nerve fibers of different sizes, shapes, and myelin sheath thickness. Although the regeneration of the nerve fibers under neurosteroid treatment showed statistically significant differences in comparison with vehicle regenerated tissue, progesterone-loaded chitosan prostheses produced the best guided nerve regeneration response. These findings indicate that chitosan prostheses allowed regeneration of nerve fibers in their lumen, and when containing neurosteroids produced a faster guided nerve regeneration acting as a long-lasting release delivery vehicle.

Facial nerve regeneration through progesterone-loaded chitosan prosthesis. A preliminary report.

Biodegradable nerve guides have represented new treatment alternatives for nerve repairing. They are gradually biodegradable, exert biological effects directly to the injured nerve, and act as drug- or cell-delivery devices. Furthermore, progesterone (PROG) has been demonstrated to promote injured peripheral nerve regeneration. In this study, it was hypothesized that PROG delivered from chitosan prostheses provides better facial nerve regenerative response than chitosan prostheses with no PROG. As there are no reports on the use of the former as nerve-guide material in the regeneration of injured nerves, this is the main objective of the present work. Chitosan prostheses containing PROG were used to bridge 10-mm gaps in rabbit facial nerves. The regenerated nerves were evaluated 45 days after implantation in animals with the use of light microscopy and morphometric analysis. Gas chromatography was used in order to quantify PROG content in prosthesis prior to and after implantation in subcutaneous tissue at different periods of up to 60 days. In addition, the prosthesis walls were evaluated with histological techniques in order to assess their integrity and the surrounding tissue reaction. Chitosan prostheses allowed PROG release during the time needed for nerve regeneration. At 45 days myelinated nerve fibers were observed in both the proximal and distal stumps. This parameter and the N ratio were higher in the progesterone-treated group when compared to that of the vehicle control. Findings indicate that chitosan prostheses were useful in nerve regeneration, acting as a long-lasting PROG delivery device a faster nerve regeneration.

Utilización de prótesis de quitosana y silicona en la regeneración del nervio ciático axotomizado de ratas / Quitosan and silicon protesis of the axiotomized cyatic nerve in rats

Para la tubulización de nervios lesionados se ha utilizado silicona con buenos resultados en defectos menores de 3 cm. La silicona es considerada como un material inerte, pero tiene como inconveniente que no es absorbible y es necesaria una segunda cirugía para retirarla. Recientemente, se ha centrado el interés en la utilización de compuestos bioactivos, tales como, la quitosana, homopolímero de estructura lineal con enlaces1-4, N acetilglucosamina, obtenida de la desacetilación de la quitina. La quitosana es absorbible, hipoalergénica, inmunoestimulante y puede actuar como vehículo para liberación prolongada de compuestos. En este trabajo, fue usada para tubulizar el nervio ciático de ratas, para ello se compararon los efectos de ambas prótesis (silicona y quitosana), se analizó la supervivencia neuronal en el dominio medular del nervio ciático y la recuperación locomotriz por medio del índice funcional del nervio ciático. En ninguno de los grupos de animales tubulizados con quitosana o silicona se encontraron indicios de degeneración neuronal en el dominio medular correspondiente. Los animales tubulizados con prótesis de quitosana presentaron una mejor recuperación funcional, esto indica que las prótesis de quitosana produjeron efectos similares a los que resultaron con silicona. Una de las ventajas inmediatas por el uso de quitosana fue evitar una segunda cirugía para retirar la prótesis; sin embargo, este biomaterial posee muchas otras cualidades que facilitan la recuperación de nervios seccionados, mismas que deberán estudiarse utilizando otras técnicas.

Electrophysiological and morphological alterations in peripheral nerves by the pig paramyxovirus of blue eye disease in neonatal pigs.

The pig paramyxovirus of blue eye disease (PPBED) produces central nervous system (CNS) damage leading to death in piglets. However, when PPBED was injected into the muscle and came into contact with hind limb peripheral nerves and was transported to the CNS, it did not cause death and could be a mechanism by which to induce protection. This study analyses whether PPBED causes electrophysiological and morphological alterations in infected hind limb peripheral nerves. It also studies, whether PPBED induces the onset of haemagglutination inhibitory antibodies (HIA) when it is transported to the spinal cord after medial gastrocnemius (MG) intramuscular injection. PPBED was detected by an immunohistochemical method and nerve morphology was studied using electron microscopy. The physiological status of the nerve was evaluated with electrophysiological techniques. The electrical threshold of the infected MG nerve increased four- or five fold compared to that in the ipsilateral lateral gastrocnemius or in the MG nerve on the control side. The infected nerve fibres underwent myelin sheet disarrangement and their internal fibre diameter decreased. PPBED induced the onset of HIA.

Congo red effect on cyst viability and cell wall structure of encysting. Entamoeba invadens.

BACKGROUND: The cell wall of Entamoeba invadens cysts is composed of chitin microfibrils as the main structural component. It has been demonstrated in yeast that the chitin cell wall assembly is altered by dyes such as Congo red (CR) and Calcofluor. METHODS: The purpose of this work was to study the cell wall assembly under the effect of CR dye on encysting E. invadens by means of light and electron microscopy, after the amebas were subjected to the effect of 100-2,000 micrograms CR/mL. Experiments were performed either in BI-S-33 or in mLG media. RESULTS: Trophozoite growth was not inhibited by 100-1,000 micrograms/mL CR after 8 days of incubation in BI-S-33 medium. However, low levels of growth were observed with 2,000 micrograms/mL of dye. No significant differences in morphologically viable (hyaline) cyst production occurred after 24-48 h, when 100 micrograms CR/mL was used, while the highest concentration of CR (2,000 micrograms/mL) resulted in a significant decrease of hyaline cyst yield; dead cysts prevailed in cultures, particularly at 72 h of CR treatment. Differentiation of amebas incubated in the presence of 500-2,000 micrograms/mL CR produced abnormal chitin deposits, rendering irregularly thick or double cell walls, as shown by transmission and scanning electron microscopy. Cyst cultures obtained under 100 micrograms/mL CR produced as many trophozoites as did the control when they were incubated in BI-S-33, but only low numbers of trophozoites were found in culture cysts obtained under higher CR doses. CONCLUSION: Our results suggest that CR affects E. invadens encystment, alters the cell wall formation, and also affects the cyst viability.

Pathogenic action of Entamoeba invadens: intestinal epithelium invasion by trophozoites in vitro.

Interactions between live Entamoeba invadens trophozoites and guinea-pig caecal explants were studied. A high percentage of amoebae adhering to the apical surface of epithelial cells was observed 10-20 min after infection, but no histopathological changes were observed. After 30 min, mild oedema at the base of the interglandular epithelium and death of some epithelial cells were evident. The epithelial barrier was invaded by amoebae at desquamating zones and phagocytosis of epithelial cells or cellular debris was occasionally observed. Invasion of the mucosa and tissue necrosis became more severe with increased time of incubation. The continuity of epithelial lining was severely compromised after 2 h of infection and erosive lesions were prominent in the mucosa. These results demonstrate that E. invadens is able to invade the intestinal epithelium although it reportedly lacks the powerful cytotoxic and cytolytic elements described for E. histolytica.

Simultaneous growth and mass encystation of Entamoeba invadens under axenic conditions.

Encystation of Entamoeba invadens IP-1 strain trophozoites was induced in low glucose medium (LG). The numbers of trophozoites, cysts and nuclei per cyst were determined; excystation of detergent-resistant axenic cysts was induced in both BI-S-33 and LG media. It was found that after 48 and 72 h of incubation in LG, cyst production was higher than trophozoites inoculated, 90 and 65% of those cysts being morphologically viable, respectively, as differentiation proceeded cysts became tri- and tetranucleated.E. invadens cysts were able to excyst either in BI-S-33 or LG media. There were no differences in growth kinetics when amebic cultures from cysts excysted in BI-S-33 were compared with parent strain. On the contrary, lower yields of trophozoites were achieved with amebas excysted and further cultured in LG medium, but they were able to grow and simultaneously undergo mass encystation. This, as well as other evidence, suggests that E. invadens trophozoites are able to modulate their physiology according to the nutrients and other factors available in the medium, in order to accomplish, growth, encystation or simultaneous growth and mass encystation. Induction of life cycle of pathogenic amebas under axenic conditions can provide answers to inhibit encystment and/or excystment.

Intracellular digestion of human erythrocytes by Entamoeba histolytica: a kinetic study in vitro.

The objective of this investigation was to quantitatively evaluate, by using light and transmission electron microscopy, the intracellular digestion of human erythrocytes (HE), ingested by Entamoeba histolytica trophozoites in vitro. Amebas fed 10 min with HE were postincubated 3-12 h at 36.5 degrees C without HE, and fixed with glutaraldehyde. After staining by the benzidine reaction, HE per ameba, and percent of amebas containing at least one HE were determined by light microscopy. Trophozoites ingested an average of eight HE per ameba and 92% of them contained HE after the 10 min pulse. Erythrocytes were clearly observed as reddish-brown corpuscles in the amebic cytoplasm. During postphagocytosis incubations progressive loss of HE staining, as well as changes in size of food vacuoles, were observed, thus proving the intracellular digestion of HE. The hydrolysis of HE was corroborated with the electron microscope, the HE cytoplasmic matrix being the first structure catabolized and later the plasma membrane. Quantitative analysis demonstrated decrease of 56% of ameba containing HE, and 1.35 HE per ameba on average, after 3 h postphagocytosis. Afterwards, both parameters decreased at a slow rate until HE disappeared. The t1/2 of HE within amebas was 2 h.

[In vitro model for the analysis of the interaction between Shigella flexneri and the intestinal epithelium]. / Un modelo in vitro para el análisis de la interacción entre Shigella flexneri y el epitelio intestinal.

An in vitro study of the adhesion and invasion of Shigella flexneri was implemented, by means of incubation of laminary cuts of cecal mucosa of Dunkin-Hartley guinea pigs in a suspension of Shigella flexneri, which was isolated from a patient with bacillary dysentery. The laminae were placed in plastic chambers for two hours at 37%C. After this the bacterial suspension was discarded so as to eliminate the bacilli which were not adhered. The epithelium was washed with saline and was processed for analysis with scanning electron microscope. The topology of the mucosa incubated with Shigella flexneri was similar to that of the witnesses. The bacteria which adhered to the mucosa were dispersed individually or in clumps of varied numbers. The main alteration observed upon the epithelial surface were depressions due to a lateral separation of the microvilli which may have originated the endocytic stomas containing bacterias. The results of this study allow the proposition of the use of explants, so as to study the interaction between Shigella flexneri and the intestinal epithelium, with the possibility of modifying different experimental variables.

Elimination of epimural microflora from guinea pig cecal epithelium by antimicrobial drugs.

The effect of antimicrobial drugs on epimural bacteria of guinea pig cecum was assessed by scanning electron microscopy. Metronidazole (500 hg/ml), neomycin (50 hg/ml) or both combined were given orally in drinking water ad libitum for five consecutive days, and representative fragments of cecum were processed for analysis. In untreated animals large areas of mucosa were lined by spiral-shaped bacteria were eliminated by treatment with neomycin, only remaining fusiform bacteria at openings of crypts. metronidazole was effective for eliminating all bacterial populations; the same effect was achieved with a combination of neomycin and metronidazole, rendering the cecum free of epimural bacteria, the health of guinea pig was unaffected by these treatments. The cecal epithelium of antimicrobial-treated animals can be used for experimental studies without interference of epimural bacteria.

[Massive production of Entamoeba histolytica cysts under axenic conditions]. / Producción masiva de quistes de Entamoeba histolytica en condiciones axénicas.

After an exponential phase of growth, HK9 strain amebas, kept in the axenic medium PEHPS, spontaneously acquire a form morphologically similar to various natural cysts, as well as a resistance to hypotonic shock, due to the effect of a wall, partially composed of polysaccharides. The number of differentiated amebas increases gradually, although their viability diminishes, in function of the incubation time. On the ninth day, 96% of the population is made up of these cells, although only 6% are viable. The ultramicroscopic structure of the great majority of differentiated amebas corresponds to that of immature cysts. These, and the PEHPS medium, constitute a good model for a characterization of the start of the differentiation of E. histolytica, and open the opportunity of obtaining in axenic conditions, massive cultures of mature cysts.

Quantitative evaluation of intracellular degradation in Entamoeba invadens.

A quantitative study on digestion of erythrocytes by Entamoeba invadens was attempted. Trophozoites of the IP-1 strain were fed red blood cells for 30 min, and subsequently phagocytosis was stopped by means of osmotic shock; post-phagocytosis incubations for up to 15 h were made in order to evaluate intracellular digestion, after staining the red blood cells with benzidine. Eighty-two per cent of trophozoites were capable of phagocytosing erythrocytes, containing an average of 5.5 erythrocytes per amoeba. Erythrocyte digestion within amoebae was shown by loss of benzidine-stainable material and proceeded with a first-order kinetics, with a t1/2 approximately 7 h. Within 15 h there were no amoebae containing erythrocytes. The procedure described may be useful for the evaluation of intracellular digestion in other Entamoeba species.