Development of an animal model to study meshes used in genital prolapse surgery.

OBJECTIVE: The objective was to develop an animal model using bacterial inoculation to evaluate tissue integration and tolerance to meshes used in genital prolapse surgery. STUDY DESIGN: We placed three different meshes under the abdominal skin of 120 Wistar rats: a polypropylene monofilament non-coated mesh (Parietene), a polypropylene monofilament collagen-coated mesh (Ugytex) and a polyethylene terephthalate mesh (Mersuture). We performed bacterial inoculation just after implantation with 1 ml of 10(7) colonies forming unit (CFU) of Staphylococcus epidermidis or Escherichia coli. Rats were sacrificed 7, 14, 60, and 90 days after intervention. We used polarised light microscopy to analyse the collagen deposition and organisation. We quantified the inflammation cells. Bacterial analysis and quantification of the explanted meshes were performed. The exact Fisher's test and Kruskal-Wallis test were used for statistics. RESULTS: We did not find any significant difference between inoculated or non-inoculated meshes in terms of collagen deposition. The scarring process seemed stable at day 90. Tissue integration was best with the polypropylene meshes, which allowed the development of a well-organised, mature connective tissue. Inflammatory reaction was higher in inoculated meshes, but only at day 7. At day 90, we found a high number of macrophages and multinuclear cells around all the meshes. There was no significant difference between prostheses that had been inoculated and those that had not with regard to positive bacterial culture. Quantification of bacterial colonies decreased with time. CONCLUSION: In this animal model, we did not find any clinically related difference in infection and tissue integration between the meshes used in genital prolapse. Such experimental studies must be carried out whenever new prostheses become available before their use is validated in common practice.

Optimal management of extreme oligozoospermia by an appropriate cryopreservation programme.

BACKGROUND: Severe oligozoospermia is characterized by sperm count fluctuations that may result in insufficient quantities of motile sperm for ICSI on the day of oocyte retrieval, thus necessitating testicular biopsy. To avoid this, we proposed that patients, with transient azoospermia or repeatedly low sperm counts, make a safety pool of frozen spermatozoa before ICSI attempts. METHODS: Seventy cryptozoospermic (<10(3) spermatozoa/ml) and 46 oligozoospermic patients (10(3)-10(5)/ml) were included. Although all oligozoospermic patients succeeded in sperm banking, only 44 of 70 cryptozoospermic patients were successful. Others underwent testicular extraction of spermatozoa. The ICSI results for frozen sperm from cryptozoospermic patients were compared with those obtained with fresh sperm from a group of normal patients (>10(5) spermatozoa/ml). RESULTS: In this prospective matched, controlled study, five cryptozoospermic, but no oligozoospermic, patients failed to produce sperm on the ICSI day, and frozen sperm was used instead. Although fertilization and pregnancy rates (per attempt) using fresh (49% and 5/44, respectively) and frozen sperm (54% and one-fifth, respectively) were similar for this cryptozoospermic group, the results for fresh sperm were significantly lower when compared with the control group (66% and 16/43, P < 0.0001, P < 0.001, respectively). In contrast, results for the oligospermic and control groups were similar. CONCLUSIONS: Banking of ejaculated sperm is helpful for cryptozoospermic patients.

Seminal haploid cell detection by flow cytometry in non-obstructive azoospermia: a good predictive parameter for testicular sperm extraction.

BACKGROUND: Testicular sperm extraction (TESE) associated with ICSI gives patients suffering from non-obstructive azoospermia (NOA) the possibility of becoming a father. The success rate of TESE based on sperm recovery is approximately 50%, and the commonly used non-invasive parameters are not predictive enough. Only the invasive testis biopsy has a good prognostic value. The aim of this study was to evaluate the prognostic value of the detection of seminal haploid cells by flow cytometry (FCM) in order to avoid unnecessary testicular biopsy. METHODS: For 37 NOA patients undergoing testicular biopsy, we measured testis size, serum FSH and inhibin B levels and carried out seminal cytology, seminal FCM analysis and histological examination. RESULTS: Sperm were found in 18 biopsies. These results were correlated with cytology, FCM analysis and the histological examination. FCM was more sensitive than cytology (100 versus 59%) but less specific (67 versus 83.5%) whereas the histological observation of complete spermatogenesis appeared to be less sensitive (50%) but more specific (100%). CONCLUSION: Detection of seminal haploid cells by FCM appears to be an interesting non-invasive technique which can predict TESE results and improve the management of NOA patients.

Immunolocalization of inhibin and activin alpha and betaB subunits and expression of corresponding messenger RNAs in the human adult testis.

Inhibin B is a testicular peptide hormone that regulates FSH secretion in a negative feedback loop. Inhibin B is a dimer of an alpha and a beta(B) subunit. In adult testes, the cellular site of production is still controversial, and it was hypothesized that germ cells contribute to inhibin B production. To determine which cell types in the testes may produce inhibin B, the immunohistochemical localization of the two subunits of inhibin B were examined in adult testicular biopsies with normal spermatogenesis, spermatogenic arrest, or Sertoli cell only (SCO) tubules. Moreover, using in situ hybridization with mRNA probes, the mRNA expression patterns of inhibin alpha and inhibin/activin beta(B) subunits have been investigated. In all testes, Sertoli cells and Leydig cells showed positive immunostaining for inhibin alpha subunit and expressed inhibin alpha subunit mRNA. Using inhibin beta(B) subunit immunoserum on testes with normal spermatogenesis and with spermatogenic arrest, intense labeling was located in germ cells from pachytene spermatocytes to round spermatids but not in Sertoli cells. Inhibin beta(B) subunit mRNA expression was intense in germ cells from spermatogonia to round spermatids and in Sertoli cells in these testes. In testes with SCO, high inhibin beta(B) subunit mRNA labeling density was observed in both Sertoli cells and Leydig cells, whereas beta(B) subunit immunostaining was negative for Sertoli cells and faintly positive for Leydig cells. These results agree with the recent opinion that inhibin B in adult men is possibly a joint product of Sertoli cells and germ cells.

Conductivity of underdoped YBa(2)Cu(3)O(7-delta): evidence for incoherent pair correlations in the pseudogap regime.

A two-channel scenario for the conductivity of underdoped YBa 2Cu 3O (7-delta) is proposed. One is the single-particle excitations channel, which dominates in the optimally doped material, whose resistivity is linear as a function of temperature. The other one gives a contribution which merges the 3D Aslamazov-Larkin fluctuation conductivity at low temperature and obeys a power law at high temperature, depending on two superconductive parameters (T(c) and the zero temperature coherence length xi(c0)) and an energy scale Delta(*). This allows one to address the nature of the pseudogap in favor of incoherent pairing.

Immunohistochemical and ultrastructural study of adult porcine endocrine pancreas during the different steps of islet isolation.

Treatment of diabetes mellitus by transplantation of isolated pancreatic islets could constitute an alternative to human pancreas allograft. Before transplantation, porcine islets are submitted to a procedure of isolation and purification. The quality of islets through these different steps may be assessed by morphological and functional studies. The aim of this work was the histological characterization of the four main cell types of porcine adult endocrine islets during the different steps of the isolation procedure using immunohistochemistry (IHC) applied in light (LM) and electron microscopy (EM). In fresh pancreas, islets were various sizes and shapes in LM. The number was not found different between the different portions of the pancreas. In IHC, insulin (Ins)-secreting cells accounted for the majority of the islet cells, while glucagon(Glu)-somatostatin (Som)- and polypeptide(PP)-immunoreactive cells, in decreasing number, were found in the mantle around the core of Ins-cells. In EM, B-cells contained poly-hedric granules with a dense central core and clear halo. Glu granules were spherical and very dense. D-cells and PP-cells were characterized by numerous granules, rather spherical and of inequal density for Som and more ellipsoidal for PP granules. After purification in Euroficoll, in EM, the four cellular types remained recognizable, but underwent vacuolization, mitochondrial swelling, and enlargement of intercellular spaces. After 3 days of culture on plastic dishes, as on Biopore membranes in a Millicell insert, microvilli appeared and vacuolization increased in EM. At the seventh day of culture, in EM, most of the cells were lysed in contrast to LM where at the same time, the four cell types were clearly identified by IHC but only in collagen matrix. Important discrepancies were noticed between LM and EM. This fact emphasizes the complementarity of morphological and functional studies in assessment of the quality of an islet isolation.

Immunogold labelling of paired helical filaments and amyloid fibrils by specific monoclonal and polyclonal antibodies.

Senile plaque and paired helical filament (PHF) formation are characteristic of Alzheimer's disease, but the mechanisms leading to these lesions still remain unclear. To understand them better, we have performed different immunolabellings of amyloid protein and PHF. We describe a very specific immunodetection of PHF with AD2, a monoclonal antibody directed against a hyperphosphorylated epitope of PHF-tau, and use double immunolabelling to show that PHF and plaque amyloid are discretely labelled by different antibodies. We also discuss different mechanisms of PHF maturation.

[Multiple cerebral hemorrhage and amyloid angiopathy of the white matter in a case of Alzheimer's disease]. / Hémorragies cérébrales multiples et angiopathie amyloïde de la substance blanche dans un cas de maladie d'Alzheimer.

Amyloid angiopathy is a common pathological finding in Alzheimer's disease. It usually involves leptomeningeal and cortical vessels but spares the white matter. It may cause lobar cerebral hemorrhages at a late stage of the disease. We report a case of Alzheimer's disease at an early stage with diffuse lesions of amyloid angiopathy including some within the white matter, apparently responsible for 2 deep and 1 superficial cerebral hemorrhages.

The immunohistochemical evidence of amyloid diffuse deposits as a pathological hallmark in Alzheimer's disease.

We performed an immunocytochemical study of cerebral cortex from cases of Alzheimer's disease and from aged nondemented controls, using periodic acid pretreatment and polyclonal beta-protein antibodies. In addition to senile plaques (SP) and amyloid angiopathy (AA), the beta-protein antibodies detected band-like deposits present throughout the cortical layers. Moreover, large plaque-like infiltrations with diffuse and amorphous characteristics were observed in the cortical gray and white matter, and these deposits were often associated with capillaries. Our results suggest that an abundance of these lesions, which were detected only with this immunostaining procedure in Alzheimer cortex, may be characteristic of Alzheimer's disease.

Neuronal-specific expression of human copper-zinc superoxide dismutase gene in transgenic mice: animal model of gene dosage effects in Down's syndrome.

It has been suggested that copper-zinc superoxide dismutase (CuZn SOD) increment, by accelerating hydrogen peroxide formation, might promote oxidative damage within trisomy 21 cells and might be involved in the various neurobiological abnormalities found in Down's syndrome such as premature aging and Alzheimer-type neurological lesions. In order to test this hypothesis, we have developed strains of transgenic mice carrying the human CuZn SOD gene. The human transgene expression resulted in increased CuZn SOD activity predominantly in the brain (1.93 fold). Immunohistochemical and in situ hybridization analysis of brain sections revealed that human CuZn SOD protein and mRNA was preferentially expressed in neurons, particularly in pyramidal cells of Ammon's horn and granule cells of gyrus dentate. The amount of thiobarbituric acid (TBA)-reactive material was significantly higher in transgenic brains compared to controls, strongly suggesting an increased level of peroxidation in vivo. These results support the notion that CuZn SOD gene dosage effect could play a role in the pathogenesis of rapid aging features in the brain of Down's syndrome patients.

Could Wallerian degeneration contribute to "leuko-araiosis" in subjects free of any vascular disorder?

To determine the possible role of Wallerian degeneration secondary to the grey matter neuronal loss in the pathogenesis of "leuko-araiosis", computerised tomography (CT) of the brain was studied in 98 normotensive and non diabetic subjects free of cardiac diseases: 32 with Alzheimer's disease, 36 with Parkinson's disease, eight with progressive supranuclear palsy, and 22 controls. In Alzheimer's disease, leuko-araiosis scores were greater than in control subjects. Leuko-araiosis was more prominent in anterior periventricular areas in Parkinson's disease and progressive supranuclear palsy, and in posterior periventricular areas in Alzheimer's disease. In two patients with Alzheimer's disease and leuko-araiosis, necropsy revealed diffuse white matter pallor, mild fibrillary astrocytosis, and in one patient limited hyaline thickening of small white matter vessels, without any infarction or hypertensive change. Changes were more severe in white matter close to cortical areas with a great density of neurofibrillary tangles. Leuko-araiosis was more severe or more widespread in Alzheimer's disease than in Parkinson's disease, progressive supranuclear palsy and normal ageing. Differences in the location of leuko-araiosis between the four groups might be due to differences in the location of the grey matter disorder and Wallerian degeneration rather than amyloid in Alzheimer's disease, Parkinson's disease, progressive supranuclear palsy and normal ageing. Wallerian degeneration might be another cause of leuko-araiosis in neuro-degenerative disorders beside previously reported extra-cerebral predisposing factors and amyloid angiopathy.

Neuronal localization of copper-zinc superoxide dismutase protein and mRNA within the human hippocampus from control and Alzheimer's disease brains.

The distribution of cells containing copper-zinc superoxide dismutase (CuZn SOD) protein and mRNA was studied in hippocampi from normal humans and patients with Alzheimer's disease (AD) by using immunohistochemistry and in situ hybridization. Using antisera against native and denatured CuZn SOD protein, we have determined that immunostaining was intense in pyramidal neurons of the cornu ammonis, in granule cells of the dentate gyrus and very weak in other cells. In the hippocampus of an Alzheimer's patient, successive immunostaining of the same tissue section by antiCuZn SOD and antipaired helical filaments antisera show that both normal and degenerating cells were labeled by the antiCuZn SOD antiserum. Thus, large pyramidal neurons which are susceptible to degenerative processes in AD have the property to contain high amount of CuZn SOD protein. In situ hybridization was performed on paraformaldehyde-fixed hippocampus sections of normal human brains and AD brains with a 35 S labeled DNA probe homologous to human CuZn SOD mRNA. Our results show that CuZn SOD transcripts are present at high abundance in pyramidal neurons of the CA1-CA4 fields, subiculum, and in granule cells of the dentate gyrus. This cellular distribution is similar to that obtained with the antiCuZn SOD antiserum. This might indicate that biochemical pathways leading to superoxide radicals generation are specially active in these neurons, requiring an active transcription of CuZn-SOD gene.

[Towards the development of an in vivo study model of Alzheimer-type neurofibrillary degeneration]. / Vers la mise au point d'un modèle d'étude in vitro de la dégénérescence neurofibrillaire de type Alzheimer.

Progress in the search for the cause of Alzheimer's disease is considerably hampered by the lack of animal or in vitro model. We have shown that in Alzheimer's disease two pathological variants of Tau proteins, called Tau 64 and Tau 69, are regularly present in neural tissue undergoing neurofibrillary degeneration. Beside their diagnostic value, Tau 64 and Tau 69 might enable such a model to be devised at long last. It now seems possible to investigate for biochemical disorders capable of inducing the emergence of these two Tau proteins in neuron cultures or among transgenic animals. The innumerable pathogenetic tracks of Alzheimer's disease (aluminium, zinc, superoxide dismutase and free radicals, proteases and antiproteases, beta protein A4 precursor, etc.) should then be opened to exploration.

Pathological proteins Tau 64 and 69 are specifically expressed in the somatodendritic domain of the degenerating cortical neurons during Alzheimer's disease. Demonstration with a panel of antibodies against Tau proteins.

Bundles of paired helical filaments (PHF) accumulate in the pyramidal neurons that degenerate during Alzheimer's disease. This neurofibrillary degeneration is highly correlated with clinical signs of dementia. During the degenerating process, Tau proteins, which are the major antigenic components of PHF, are abnormally phosphorylated and two pathological isoforms named Tau 64 and 69 are expressed. We have studied their immunoblot distribution in the cortical gray and white matter from different regions of normal and Alzheimer brains, to determine if the degenerating process preferentially affects the somatodendritic or the axonal domain. Two categories of antibodies were used. The first category consisted of anti-human native Tau, anti-Tau proteins from different vertebrates, anti-PHF, monoclonal antibody Alz-50 and an anti-C terminal repeated region of Tau. In control brains, these antibodies strongly detected normal Tau proteins in the gray matter while Tau immunodetection was weak in the white matter. In Alzheimer brain cortices, each antibody detected Tau 64 and 69 in gray matter extracts but not at all in white matter extracts. The second category of anti-Tau consisted of the anti-PHF saturated with normal brain protein extracts. This antiserum only probed the abnormally phosphorylated Tau proteins. It detected Tau 64 and 69 exclusively in the cortical gray matter of Alzheimer brains. Moreover, a 55-kDa Tau protein was also immunolabelled, which might be an intermediary form between normal Tau and Tau 64 and 69. Our results demonstrate that Tau proteins are normal and major components of the somatodendritic domain and that Tau pathology, reflected by the presence of Tau 64 and 69, affects preferentially this domain during Alzheimer's disease.

[Alzheimer's disease: the beta amyloid protein A4 is also present in the cortical white matter]. / Maladie d'Alzheimer: la bêta protéine amyloïde A4 est aussi présente dans la substance blanche corticale.

Amyloid deposits constituted with the beta amyloid protein A4 (beta PA4) have been recently immunodetected in skin and intestine wall of Alzheimer's patients. These findings support the hypothesis of an extraneuronal origin of the beta PA4. Until now, these amyloid deposits had not been observed in the white matter of Alzheimer's brains. Using an antiserum against the 1-10 N Ter subsequence of the beta PA4, we immunodetected amyloid deposits in white matter sections of Alzheimer's brains, pretreated with periodic acid. The immunolabelled amyloid substance was associated with capillaries. These original findings are in good agreement with the vascular origin of the beta PA4.