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.

Alzheimer's disease: microtubule-associated proteins 2 (MAP 2) are not components of paired helical filaments.

In Alzheimer's disease, the most characteristic neuropathological changes are the formation of neurofibrillary tangles (NFT) and neuritic plaques (NP) characterized by the presence of bundles of paired helical filaments (PHF) that accumulate in the degenerating neurites and neuronal cell bodies. Although the protein composition of the PHF is ill-defined, a number of microtubule-associated proteins have been implicated in these lesions. Here we report results with an antiserum monospecific for the microtubule-associated protein MAP 2 which does not cross-react with any other microtubular protein. Immunostaining with this antibody of sections from an Alzheimer's brain show a strong reactivity with NFT but no reactivity at the level of the NP. On the other hand, immunostaining of Alzheimer's brain sections with another antibody specific for the microtubule-associated protein tau shows strong staining of PHF on both NFT and NP. These findings confirm the presence of the tau proteins in the PHF and strongly suggest that MAP 2 may not be a main structural component of the PHF. Labelling of NFT with the anti-MAP 2 antiserum suggests a non-specific binding of MAP 2 to the PHF during the process of NFT formation.

Expression of ETS proto-oncogenes in astrocytes in human cortex.

In order to investigate a possible function of ETS proto-oncogenes in human brain, we incubated a polyclonal antibody raised against the viral region of E26 homologous to ETS1 and ETS2 with human brain frontal cortex sections. Our results show that this antibody decorates astrocytes but not neurons. By using astrocytomas of different grades as a source of astrocytes, we demonstrate the presence of ETS1 and ETS2 messenger RNAs and proteins. This leads to the idea that ETS genes are expressed in cells with dividing potentialities in human cortex and that they could provide a new marker for glial cells. Recently, a microduplication on chromosome 21 including ETS2 locus was described in karyotypically 'normal' Down's syndrome and suspected in Alzheimer's disease; when testing Alzheimer's disease-affected brain cortex sections, no obvious difference was observed with the technique used.

Dystrophic peptidergic neurites in senile plaques of Alzheimer's disease hippocampus precede formation of paired helical filaments.

The relationship between peptidergic dystrophic neurites and paired helical filament (PHF)-positive neurites in Alzheimer's disease (AD) senile plaques (SPs) was studied using combined fluorescence and bright-field optics. Cryostat sections of AD hippocampi were first stained with thioflavine-S and immunolabelled with antisera raised against different neuropeptides: somatostatin-28(1-12), somatostatin-14, neuropeptide Y, cholecystokinin (CCK) and substance P. Secondly, using the elution-restaining procedure, sections were immunolabelled with anti-tau/PHF. In immature SPs, clusters of abnormal, swollen neurites were found. The dystrophic, strongly peptidic-positive neurites contained fewer PHFs than the poorly positive ones. Cell bodies, exhibiting a peptidic content, could be found within SPs without any alteration. These results suggest the following sequence of events: an extracellular poisoning mechanism, perhaps the amyloid substance, first changes the structure of presynaptic endings and causes the formation of ballooning dystrophic neurites filled with their normal peptidic content. Subsequently, intracellular degradation occurs with formation of the PHFs. Then the other structures such as dendrites and perikarya are damaged by the same mechanism. Therefore, this phenomenon seems to precede any formation of PHFs in SPs.

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.

Preferential localization of copper zinc superoxide dismutase in the vulnerable cortical neurons in Alzheimer's disease.

The distribution of cells containing CuZn superoxide dismutase (CuZn SOD) was determined in hippocampi and associative cortex from normal and Alzheimer's individuals by using antisera against native and denatured CuZn SOD proteins. Immunostaining was intense in large pyramidal neurons, moderate in hippocampal granule cells and very weak in other cells. In the hippocampus of an Alzheimer's patient, successive immunostaining of the same tissue section by anti CuZn SOD and anti paired helical filaments antisera show that both normal and degenerating cells are labelled by the anti CuZn SOD antiserum. Thus, large pyramidal neurons which are potentially susceptible to degenerative processes in AD have the property to contain higher amounts of CuZn SOD than other brain cells.

Alzheimer's disease: Tau proteins, the promoting factors of microtubule assembly, are major components of paired helical filaments.

A rabbit antiserum was raised against paired helical filaments (PHF). This antiserum stains specifically neurofibrillary tangles (NFT) at the light-microscopic level and PHF at the electron-microscopic level in sections of Alzheimer neocortex and hippocampus. We studied the nature of the antigens recognized by this antiserum by immunocytochemistry, immunoblots and immunoadsorption. These approaches showed that the anti-PHF specifically labels a set of low molecular weight 65-50 kDa microtubule-associated proteins, named Tau proteins, which are promoting factors of microtubule assembly. Furthermore, antisera against Tau proteins stained NFT. It is concluded that neurofibrillary tangles are very likely composed of aggregated Tau proteins. This process might be due to an abnormal Tau protein synthesis or to an unknown lesion of certain pyramidal neurons leading to an aggregation of Tau proteins.

Transformation of degenerating neurofibrils into amyloid substance in Alzheimer's disease: histochemical and immunohistochemical studies.

Degenerating neurofibrils (DNF), which are composed of paired helical filaments (PHF) and amyloid fibrils (AF), are the 2 characteristic pathological fibrillar deposits in Alzheimer cortex. These fibrils were simultaneously studied by 2 techniques: The immunolabelling with a specific antiserum raised against PHF and elective thioflavine S staining of AF. In neuronal perikaryons, neurofibrillary tangles (NFT) consist of 3 populations: firstly, strongly immunolabelled tangles were weakly thioflavine-stained. Secondly, less dense tangles were weakly immunolabelled but strongly thioflavine-stained. Thirdly, ghost tangles which correspond to extracellular NFT were exclusively thioflavine-stained. Thus, it is likely that NFT are degraded to form extracellular AF. Around neuritic plaques and some vessels with amyloid angiopathy, immunolabelled neurites, thioflavine-stained neurites and transition figures were also observed. On the other hand, the central core of plaques and pathological vessel walls were strongly thioflavine-stained but were never immunoreactive. In conclusion, these observations favour catabolism of PHF bundles found in NFT and in degenerating neurites into an amyloid substance. This amyloid substance seems different from other amyloid deposits found in the central core of neuritic plaques and vessel walls.

Characterization of two pathological tau protein, variants in Alzheimer brain cortices.

Tau proteins were detected in brain tissue homogenates from 10 patients with Alzheimer's disease versus 10 age-matched controls using the immunoblot technique and 2 polyclonal antibodies: anti-paired helical filaments (PHF) and anti-human native Tau proteins. In control brains, both antisera detected identically the normal set of Tau proteins, with molecular weight (MW) ranging from 45 to 62 kDa. Moreover, in association areas of neocortex from Alzheimer brains, the antisera detected 2 additional Tau variants of 64 and 69 kDa. Tau 64 and 69 were not found in regions of Alzheimer brains where the Alzheimer pathology was absent (caudate nucleus or cerebellum for example). The heavy MW of Tau 64 and 69 is due to their phosphorylation state as shown by the decrease of their MW after alkaline phosphatase treatment. Therefore, Tau 64 and Tau 69 are specific markers of the Alzheimer's disease neuronal degenerating process and their characterization demonstrates that an abnormal phosphorylation of Tau really occurs during the disease. Tau 64 and 69 were isolated with normal Tau proteins while the PHF were insoluble. Therefore, Tau proteins are likely to be abnormally phosphorylated prior to their incorporation in the PHF structure. Consequently, they might appear before the lesions and might be instrumental for the search of biochemical deregulations that precede the neurofibrillary degeneration.

Immunocytochemical profile of neurofibrillary tangles in Down's syndrome patients of different ages.

Brains were obtained at autopsy from 24 patients with Down's syndrome, ranging in age from 13 to 71 years. Neurofibrillary tangle containing neurones of the hippocampus were stained using a Palmgren silver method and immunocytochemically (PAP) using antisera to paired helical filament protein, human tau protein and ubiquitin, as primary antibody. Counts of cells stained by each method were compared. In patients under 50 years of age, in whom only a limited number of tangle bearing cells were present, the number of profiles visualized with silver, anti-paired helical filament and anti-tau methods were similar. However, in patients over 50 years of age (and in certain of those under 50), in whom numerous tangles were present, the number of cell profiles visualized with silver and anti-paired helical filament methods were still similar though anti-tau detected fewer positive cells. This was because of the increased presence, in such patients, of extracellular tangles which had "lost" anti-tau immunoreactivity. Such data suggest that although tau protein forms a major antigenic determinant of neurofibrillary tangles in Down's syndrome (as it does in Alzheimer's disease) this protein may only decorate the basic paired helical filament protein skeleton, and is removed by macrophagic activity upon neuronal death. In all patients, anti-ubiquitin revealed fewer tangles than any other method. It is possible that ubiquitin may be present only transiently, within tangles perhaps following initial formation and lasting only as long as the normal protein degradation processes remain viable within the diseased neurone.

Dystrophic neuropeptidergic neurites in senile plaques of Alzheimer's disease precede formation of paired helical filaments.

The relationship between peptidergic neurites and paired helical filaments (PHF)-positive neurites in Alzheimer's disease (AD) senile plaques (SP) was studied using combined fluorescence and bright field optics. Cryostat sections of AD hippocampi were first stained by thioflavine-S and immunolabeled with antisera raised against different neuropeptides: somatostatin 28(1-12) (som 28(1-12)), somatostatin 14 (som 14), neuropeptide Y (NPY), cholecystokinin (CCK) and substance P (sP). Secondly, using the elution-restaining procedure, sections were immunolabeled with anti-tau/PHF. In immature SP, clusters of abnormal, swollen neurites were found. The dystrophic, strongly peptidic-positive neurites contained less PHF than the poorly positive ones. Cell bodies, exhibiting a peptidic content, could be found within SP without any alteration. These results suggest the following sequence of events: an extracellular poisoning mechanism, perhaps the amyloid substance, first changes the structure of presynaptic endings and causes the formation of ballooning dystrophic neurites filled with their normal peptidic content. Subsequently, intracellular degradation occurs with formation of the PHF. Then the other structures such as dendrites and perikarya are damaged by the same mechanism. Therefore this phenomenon seems to precede any formation of PHF in SP.

[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.

[Immunohistochemical study of the basic lesions of Alzheimer's disease]. / Etude immunohistochimique des lésions élémentaires de la maladie d'Alzheimer.

Alzheimer's disease is characterized by the presence of two argentophilic histopathologic brain lesions: neurofibrillary tangles (NFT) and senile (neuritic) plaques (SP). NFT consist of large perikaryal masses of abnormal cytoplasmic fibers, most of which have the ultrastructural appearance of pairs of intermediate-sized (10 nm) filaments wound into a double helix and named Paired Helical Filaments (PHF). PHF also occur within the degenerating neurites of SP. The insolubility of PHF in strong detergents is turned to account for their isolation. We have isolated these structures from an Alzheimer brain and raised antibodies against PHF. The anti-PHF antibodies detected specifically NFT and SP on nervous tissue sections of Alzheimer brains, and also NFT in the hippocampus of normal aged brains. The stainings of NFT and SP by the anti-PHF or the classical Bodian silver staining technique were compared. The immunohistochemical method is more precise, more reproducible, more specific and will be of great interest for the quantification of these structures, specially when they are in minor quantities particularly in the atypical disease. Furthermore, the anti-PHF antibodies did not visualize neurofilament containing structures, and did not react with neurofilament protein subunits on immunoblots. These results are compared to those reported in the literature.

[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.

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.