Co-localization of vasoactive intestinal polypeptide, γ-aminobutyric acid and choline acetyltransferase in neocortical interneurons of the adult rat

Interneurons immunoreactive for vasoactive intestinal polypeptide (VIP) are integral elements of columnar organization patterns in the rat cerebral cortex. By application of the sensitive mirror technique, the co-localization of VIP with the classical inhibitory neurotransmitter γ-aminobutyric acid (GABA) and the acetylcholine-synthesizing enzyme, choline acetyltransferase (ChAT), was investigated in neocortical neurons. Furthermore, the frequency of co-localization of ChAT with GABA was determined. In a sample of 118 VIP-immunoreactive neurons, mostly from the primary somatosensory cortex, it was demonstrated that virtually all of them reveal immunoreactivity for GABA and, therefore, are to be GABAergic. Moreover, 34% of mostly bipolar, VIP-positive neurons contained ChAT and are, thus, supposedly cholinergic as well. Co-localization of VIP and ChAT varied according to cortical laminae. Finally, 88% of a total of 60 ChAT-immunoreactive neurons were also immunostained for GABA. It is concluded that almost all VIP-immunoreactive neurons and most of the cholinergic neurons in rat neocortex represent partly overlapping subpopulations of inhibitory interneurons utilizing GABA.     

The synthesis and localization of alternatively spliced fibronectin EIIIB in resting and thrombin-treated megakaryocytes

There are several species of alternatively spliced fibronectin (FN). One of these, FN EIIIB, is primarily present in embryonic and in proliferating and migrating cells and is believed to be important for cell maturation. We have studied the synthesis, localization, and secretion of this FN isoform in isolated guinea pig megakaryocytes, nonmegakaryocytic bone marrow cells, and platelets. There was 7.5 times more general FN in megakaryocytes than in nonmegakaryocytic cells based on the analysis of equivalent amounts of protein. FN EIIIB was detected by Western blotting in megakaryocytes but not in nonmegakaryocytic cells present in bone marrow. Neither megakaryocytes nor platelets secreted FN EIIIB, while megakaryocytes secreted 25.3% +/- 4.6% general FN and platelets secreted about 61% general FN in response to thrombin. Analysis of immunostained cells by confocal microscopy revealed that FN EIIIB had been redistributed to the surface of megakaryocytes in response to thrombin. Synthesis was studied by metabolic labeling, and megakaryocytes were shown to synthesize FN and FN EIIIB. Thus, megakaryocytes and platelets are among a small number of adult cells and tissues that synthesize and contain FN EIIIB. The expression of FN EIIIB on the megakaryocyte surface may influence migration and maturation.     

Three-dimensional in vitro model of adipogenesis: comparison of culture conditions

In vivo and in vitro studies have demonstrated both promise and current limitations in tissue engineering of fat. Herein, we report the establishment of a well-defined three-dimensional (3-D) in vitro model useful for systematic investigations of 3-D adipogenesis. Polyglycolic acid fiber meshes were dynamically seeded with 3T3-L1 preadipocytes; subsequently, cell-polymer constructs were hormonally induced and cultivation under three different conditions was evaluated. Regarding tissue coherence and intracellular lipid content, culture of cell-polymer constructs either dynamically in well plates or in stirred bioreactors yielded similar results, which were distinctly improved compared with static conditions in well plates. At the protein and mRNA levels, significantly increased expression of genes characteristic for a mature adipose phenotype was demonstrated for constructs dynamically cultured in well plates, as compared with static conditions. Furthermore, investigation of lipolysis under stimulating and inhibiting conditions demonstrated functionality of the dynamically differentiated constructs. Using dynamic culture conditions, the presented in vitro model system is suggested as a valuable tool serving both fat tissue engineering and basic research by facilitating investigations of tissue-inherent features not possible under conventional 2-D culture conditions.     

Comparison of viable cell yield from excised versus aspirated adipose tissue

The correction of soft-tissue defects by adipose tissue transplantation often produces poor and unpredictable results. The implantation of isolated and cultured preadipocytes offers a solution to this problem since these cells differentiate into adipocytes when implanted in vivo. A field of major interest is to maximize the yield of preadipocytes isolated from adipose tissue showing only low contamination with other cell types. Aspiration and excision are two concurrent clinical ways of harvesting adipose tissue for the isolation of preadipocytes. This tissue is usually discarded after surgery. In this study, the yield of preadipocytes obtained from liposuction material was compared to that of excised adipose tissue. Furthermore, we determined the loss of precursor cells if isolation of preadipocytes was delayed for 24 h. Preadipocytes were isolated from the stromal cell fraction of human subcutaneous adipose tissue samples. Harvesting of adipose tissue by suction was performed according to the Coleman procedure (manually applied negative pressure using a 10-ml syringe with a blunt tip cannula). Isolation was either carried out within 60 min after extraction or after storage for 24 h in culture medium at 4 degrees C. Isolated preadipocytes were cultured for 24 h, trypsinized and counted in a Neubauer chamber. Our results show clearly that the yield of preadipocytes isolated from liposuction material (within 60 min after extraction and after 24 h of storage) is higher than the cell yield from excised adipose tissue. Overnight storage for 24 h leads to a significant loss of preadipocytes in excised tissue but not in liposuction material. The high yield of cells isolated from liposuction material proves that extraction by suction does not damage the stromal cell fraction in the adipose tissue. If cell isolation is not performed immediately after the operation, liposuction material is clearly the better alternative for storage.     

VIRAL ENCEPHALITIS PRESENTING AS CATATONIC SCHIZOPHRENIA (A Case Report)

A rare case of viral encephalitis in an 18 year old recruit who presented with signs of catatonic schizophrenia is reported.KEY WORDS: Viral encephalitis, Catatonic schizophrenia     

ROLE OF EPIDURAL MEDICATION IN LUMBOSCIATIC SYNDROME

Role of epidural medication through caudal route was studied in 109 patients having lumbago with or without sciatica to highlight the value of this mode of treatment which relieved symptoms in more than 70% of cases without hospitalisation and without being off work for long periods as in usual methods of conservative treatment.KEY WORDS: Epidural medication, Backache, Lumbago, Sciatica