Stephan R. Lolov

Changes in NADPH-diaphorase neurons of the rat laterodorsal and pedunculopontine tegmental nuclei in aging

The aim of the present study was to compare the morphological pattern and the quantitative parameters of nitric oxide (NO)-containing neurons in the laterodorsal (LTD) and pedunculopontine (PPN) tegmental nuclei of 3-, 12- and 26-month-old rats. NADPH-diaphorase (NADPH-d) histochemical reaction, as a marker of the cholinergic neurons in the two mesopontine nuclei, and computer-assisted image analysis were used. The relationships between the neurons stained for NADPH-d and choline acetyltransferase (ChAT) were examined using a double-labelling procedure. The results demonstrated only occasional ChAT positive somata that did not exhibit NADPH-d staining. The volume of the LTD and PPN and the number of NADPH-d neurons remained unaltered with advancing age. However, ANOVA demonstrated a significant effect of age and level on the cross-sectional areas, maximum diameters and staining intensity of NADPH-d somata in the LTD and PPN. The three parameters were reduced in 26-month-old rats compared to 3-month-old rats. The changes in the morphological appearance of NADPH-d somata and processes as well as the quantitative analysis pointed to age-related neuronal atrophy. It was accompanied by hypertrophy of some neighbouring neurons, suggesting a compensatory mechanism which would counteract the degenerative changes. The age-dependent alterations in the LTD and PPN were rather similar.

Antimeasles immunoglobulin G in sera of patients with otosclerosis is lower than that in healthy people.

Background There is some evidence for an inflammatory process as a driving force in otosclerosis. Two popular hypotheses for the induction of this chronic inflammation have been proposed: an autoimmune phenomenon induced by an otic capsule specific antigen and measles virus infection. Methods Antibodies against measles virus hemagglutinin, polymerase, nucleocapsid, and matrix proteins were evaluated in sera from otosclerotic patients and in sera from healthy age-and sex-matched controls by use of the Western blot analyses. Results Significant differences were not detected between healthy men and women or between otosclerotic men and women. There were significantly stronger reactions against all viral proteins in the group of healthy women as compared with otosclerotic women despite a high standard deviation. The group of healthy male blood donors demonstrated significantly stronger reactions against polymerase and nucleocapsid proteins. Healthy blood donors again demonstrated stronger reaction compared with respective otosclerotic patients in a separate reaction for viral matrix protein. Conclusion Our observation is consistent with viral participation in otosclerotic pathogenesis, but it is difficult to say if the diminished antimeasles humoral response is a consequence or the cause for a local measles infection. In light of the present data, we can discuss autoantibodies in otosclerosis as a sign of autoimmunity triggered by measles virus.

Aging and the dendritic morphology of the rat laterodorsal and pedunculopontine tegmental nuclei

The morphological appearance and quantitative parameters characterizing the dendrites of NADPH-diaphorase-stained neurons in the laterodorsal (LDT) and pedunculopontine (PPN) tegmental nuclei of 3-, 12- and 26-month-old rats were studied. All dendritic segments were classified according to the number of terminal and link segments they drain and the vertex analysis was used to quantify the dendritic tree and to determine its configuration. Morphological aberrations of the dendrites as local swelling, nodulation, thinning, shrinkage, folding and even the appearance of stumps were observed with advancing age. The quantitative analysis demonstrated a significant reduction (one-way ANOVA) of the total dendritic length, mean terminal path length, maximal segment length, total segment number and number of terminal segments at the rostral two thirds of the LDT and in the PPN. The mean vertex path length and the mean segment length significantly decreased only at the rostral level of the LDT. Plotting of the segment length against equivalent orders showed a decrease in all generations of the dendritic segments. The vertex ratios remained constant and indicated that the configuration of the dendritic tree remained unchanged during aging. The alterations in the dendrites mainly developed after 12 months of age. The age-related changes in the morphology and quantitative parameters of the dendrites in the rostral two thirds of the LDT and PPN were rather similar, which could be explained by the common anatomical, neurochemical and electrophysiological features. Thus, the present results suggest a mild, but continuous regression of the dendritic tree of the rat LDT and PPN in normal aging.

Age-related changes in the NADPH-diaphorase-positive neuronal perikarya of the dorsolateral column of the periaqueductal gray in the rat.

The distribution of reduced nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) positive neuronal perikarya in the rostral, middle and caudal parts of the dorsolateral periaqueductal gray (DLPAG) in 3-, 12- and 26-month-old rats was compared by means of histochemistry and computer assisted image analysis. The total number, the maximum diameter, the cross-sectional area and the optical density (OD) of the NADPH-d positive neurons were analyzed. The results demonstrate that there are no significant differences in any of the investigated parameters between the left and the right parts of the same age and of the same level. The total cell number in the DLPAG of 26-month-old rats was significantly decreased in comparison with 3- and 12-month-old rats. The cross-sectional area increased between 3 and 12 months of age, and decreased in 26-month-old rats in comparison with 12-month-old rats. The OD increased significantly between 3 and 12 months of age, and decreased significantly between 12 and 26 months of age only at caudal level. The observed mild changes could lead to alterations in the brain physiology of aging.

Conformation-sensitive immunoassay: optimisation, validation and evaluation.

A conformation-sensitive immunoassay (CSI) has recently been developed (Pfund and Bourdage, 1990). This paper describes the optimal laboratory protocol which makes this method useful as an early screening procedure for conformation-sensitive monoclonal antibodies. The method was validated with a panel of 12 monoclonal antibodies. Results from our experiments confirm that this variant of CSI can be applied in the analysis of purified proteins and glycoproteins, as well as complex mixtures of antigens.

The Bulgarian Hearing in Noise Test

Bulgaria is a small country (111 001.9 sq. km; population 7 761 000) in southeastern Europe. Bulgarian, the mother language of 84.8% of the population, is an Indo-European language and a member of the southern branch of the Slavic languages. Currently, there are approximately 10 million people speaking Bulgarian, including 1.5 to 2 million second-language speakers. Some communities in Canada, Greece, Hungary, Israel, Moldova, Romania, Serbia, Montenegro, Turkey (Europe), Ukraine, and the USA also speak Bulgarian (Wikipedia, 2006). The aim of this report is to present the current state of the development of the Bulgarian HINT.

Decreased Measles Virus-Neutralizing Activity in Sera from Otosclerotic Patients

Hypothesis: Any kind of depressed systemic anti-measles reaction can lead to the induction of a local immune response in the inner ear and possibly to reactivation of bone turnover in this region. Methods: Different dilutions of sera were tested for neutralizing activity against a constant viral concentration. The ability of measles virus to infect and replicate in the cell monolayer was detected by enumeration of living and growing cells with a colored reaction. Results: Virus-neutralizing activity in the sera of patients with confirmed otosclerosis was significantly weaker than in that of healthy controls. When age- and sex-matched pairs were compared, the neutralizing activity in the healthy counterpart was higher in 5 cases. Nearly complete viral neutralization was achieved with samples containing inactivated complement and in IgG-containing fractions, but not in immunoglobulin-depleted samples. Conclusion: The present study is consistent with measles virus participation at least in the initiation of some cases of otosclerosis.

Antimeasles Immunoglobulin G and Virus-Neutralizing Activity in Sera of Patients with Otosclerosis

Otosclerosis is considered as an organ-specific measles virus (MV)-induced disease. The majority of people are infected with MV during childhood, and the immune activation is characterized by a lifelong persistence. Any kind of depressed systemic antimeasles reaction can lead to induction of a local immune response in the inner ear. MV proteins were resolved by conventional polyacrylamide gel electrophoresis in the presence of a denaturing detergent. In subsequent Western blot, healthy blood donors demonstrated a stronger reaction against most proteins than age- and sex-matched otosclerotic patients. Different serum dilutions were tested for neutralizing activity against constant MV concentration. Nearly complete viral neutralization was achieved with samples containing inactivated complement and in immunoglobulin-G-containing fractions, and activity in sera from patients with confirmed otosclerosis was significantly weaker than in healthy individuals. Our observations are consistent with viral participation in otosclerotic pathogenesis, but it is difficult to say if the diminished antimeasles humoral response is a consequence of or the cause for a local measles infection.

Age-related changes in the NADPH-diaphorase-positive dendrites in the dorsolateral neuronal column of the periaqueductal gray in rat.

The effects of age on the dendritic tree of reduced nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d)-positive neurons in the dorsolateral neuronal column of the periaqueductal gray (DLPAG) in the rat were examined, using NADPH-d histochemistry and computer-assisted analysis. In 26-month-old rats some of the neurons exhibited considerably branched dendrites, while others show amputation stumps, or local swellings in the initial dendritic portion. Quantitative analysis demonstrated that the number of dendritic segments in DLPAG increased with advancing age. The mean segment length and the mean length of the terminal segment significantly increased in 12-month-old rats compared to 3-month-old rats and decreased in 26-month-old rats in comparison to 12-month-old, as the reduction was significant for the terminal segments only. The total dendritic length of NADPH-d-stained neurons increased with aging. The increase was more pronounced in 12-month-old rats compared to 3-month-old rats and the difference was significant in 26-month-old rats. The number of the nodal vertices, Va and Vb, significantly increased between 12- and 26-month of age. The dendritic reorganization was considered a sign of compensatory mechanism counteracting the degenerative changes.

An express immunological method for detection of human seminal plasma

Monoclonal antibodies (Mabs) against human seminal plasma (HSP) were produced and during screening procedures dissociation constants of the antigen/antibody complexes were determined. Mab 1E5 was selected for further studies because of its high reactivity in an enzyme-linked immunoassay (ELISA) and high affinity for its corresponding antigen. The specificity of Mab 1E5 was checked in absorption ELISA with human organ extracts and some biological secretions. It was established that the 1E5-corresponding epitope was a thermostable peptide moiety which could be detected in HSP, only. This monoclonal antibody was used for the development of an express method for detection of human semen. The assay was applied for screening of 57 cases of suspected rape. A complete correlation was found between the results obtained by the proposed test and by routine microscopic methods. The newly designed immunoassay is reliable, it is easily performed and it is less time-consuming.